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Sample records for clinical ffpe samples

  1. Optimized Clinical Use of RNALater and FFPE Samples for Quantitative Proteomics

    DEFF Research Database (Denmark)

    Bennike, Tue Bjerg; Kastaniegaard, Kenneth; Padurariu, Simona;

    we compare to FFPE and frozen samples being the control. Methods From the sigmoideum of two healthy participants’ twenty-four biopsies were extracted using endoscopy. The biopsies was stabilized either by being directly frozen, RNAlater, FFPE or incubated for 30 min at room temperature prior to FFPE....... Furthermore, human mastoid bone and human peripheral blood mononuclear cells were stabilized in a similar manner. The characterization of the protein content was analysed by high throughput gel free quantitative proteomics, followed by an analysis of post-translational modifications and label free...... quantification, using ProteinPilot and MaxQuant respectively. Results and Discussion We were able to identify a similar high number of proteins regardless of sample stabilization method, as well the abundance in RNAlater and frozen was close to identical. The five most abundant post-translational modification...

  2. Random DNA fragmentation allows detection of single-copy, single-exon alterations of copy number by oligonucleotide array CGH in clinical FFPE samples.

    Science.gov (United States)

    Hostetter, Galen; Kim, Su Young; Savage, Stephanie; Gooden, Gerald C; Barrett, Michael; Zhang, Jian; Alla, Lalitamba; Watanabe, April; Einspahr, Janine; Prasad, Anil; Nickoloff, Brian J; Carpten, John; Trent, Jeffrey; Alberts, David; Bittner, Michael

    2010-01-01

    Genomic technologies, such as array comparative genomic hybridization (aCGH), increasingly offer definitive gene dosage profiles in clinical samples. Historically, copy number profiling was limited to large fresh-frozen tumors where intact DNA could be readily extracted. Genomic analyses of pre-neoplastic tumors and diagnostic biopsies are often limited to DNA processed by formalin-fixation and paraffin-embedding (FFPE). We present specialized protocols for DNA extraction and processing from FFPE tissues utilizing DNase processing to generate randomly fragmented DNA. The protocols are applied to FFPE clinical samples of varied tumor types, from multiple institutions and of varied block age. Direct comparative analyses with regression coefficient were calculated on split-sample (portion fresh/portion FFPE) of colorectal tumor samples. We show equal detection of a homozygous loss of SMAD4 at the exon-level in the SW480 cell line and gene-specific alterations in the split tumor samples. aCGH application to a set of archival FFPE samples of skin squamous cell carcinomas detected a novel hemizygous deletion in INPP5A on 10q26.3. Finally we present data on derivative of log ratio, a particular sensitive detector of measurement variance, for 216 sequential hybridizations to assess protocol reliability over a wide range of FFPE samples.

  3. High quality copy number and genotype data from FFPE samples using Molecular Inversion Probe (MIP microarrays

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    Bondy Melissa

    2009-02-01

    Full Text Available Abstract Background A major challenge facing DNA copy number (CN studies of tumors is that most banked samples with extensive clinical follow-up information are Formalin-Fixed Paraffin Embedded (FFPE. DNA from FFPE samples generally underperforms or suffers high failure rates compared to fresh frozen samples because of DNA degradation and cross-linking during FFPE fixation and processing. As FFPE protocols may vary widely between labs and samples may be stored for decades at room temperature, an ideal FFPE CN technology should work on diverse sample sets. Molecular Inversion Probe (MIP technology has been applied successfully to obtain high quality CN and genotype data from cell line and frozen tumor DNA. Since the MIP probes require only a small (~40 bp target binding site, we reasoned they may be well suited to assess degraded FFPE DNA. We assessed CN with a MIP panel of 50,000 markers in 93 FFPE tumor samples from 7 diverse collections. For 38 FFPE samples from three collections we were also able to asses CN in matched fresh frozen tumor tissue. Results Using an input of 37 ng genomic DNA, we generated high quality CN data with MIP technology in 88% of FFPE samples from seven diverse collections. When matched fresh frozen tissue was available, the performance of FFPE DNA was comparable to that of DNA obtained from matched frozen tumor (genotype concordance averaged 99.9%, with only a modest loss in performance in FFPE. Conclusion MIP technology can be used to generate high quality CN and genotype data in FFPE as well as fresh frozen samples.

  4. High quality copy number and genotype data from FFPE samples using Molecular Inversion Probe (MIP) microarrays

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    Wang, Yuker; Carlton, Victoria E.H.; Karlin-Neumann, George; Sapolsky, Ronald; Zhang, Li; Moorhead, Martin; Wang, Zhigang C.; Richardson, Andrea L.; Warren, Robert; Walther, Axel; Bondy, Melissa; Sahin, Aysegul; Krahe, Ralf; Tuna, Musaffe; Thompson, Patricia A.; Spellman, Paul T.; Gray, Joe W.; Mills, Gordon B.; Faham, Malek

    2009-02-24

    A major challenge facing DNA copy number (CN) studies of tumors is that most banked samples with extensive clinical follow-up information are Formalin-Fixed Paraffin Embedded (FFPE). DNA from FFPE samples generally underperforms or suffers high failure rates compared to fresh frozen samples because of DNA degradation and cross-linking during FFPE fixation and processing. As FFPE protocols may vary widely between labs and samples may be stored for decades at room temperature, an ideal FFPE CN technology should work on diverse sample sets. Molecular Inversion Probe (MIP) technology has been applied successfully to obtain high quality CN and genotype data from cell line and frozen tumor DNA. Since the MIP probes require only a small ({approx}40 bp) target binding site, we reasoned they may be well suited to assess degraded FFPE DNA. We assessed CN with a MIP panel of 50,000 markers in 93 FFPE tumor samples from 7 diverse collections. For 38 FFPE samples from three collections we were also able to asses CN in matched fresh frozen tumor tissue. Using an input of 37 ng genomic DNA, we generated high quality CN data with MIP technology in 88% of FFPE samples from seven diverse collections. When matched fresh frozen tissue was available, the performance of FFPE DNA was comparable to that of DNA obtained from matched frozen tumor (genotype concordance averaged 99.9%), with only a modest loss in performance in FFPE. MIP technology can be used to generate high quality CN and genotype data in FFPE as well as fresh frozen samples.

  5. Evaluation of frozen tissue-derived prognostic gene expression signatures in FFPE colorectal cancer samples.

    Science.gov (United States)

    Zhu, Jing; Deane, Natasha G; Lewis, Keeli B; Padmanabhan, Chandrasekhar; Washington, M Kay; Ciombor, Kristen K; Timmers, Cynthia; Goldberg, Richard M; Beauchamp, R Daniel; Chen, Xi

    2016-01-01

    Defining molecular features that can predict the recurrence of colorectal cancer (CRC) for stage II-III patients remains challenging in cancer research. Most available clinical samples are Formalin-Fixed, Paraffin-Embedded (FFPE). NanoString nCounter® and Affymetrix GeneChip® Human Transcriptome Array 2.0 (HTA) are the two platforms marketed for high-throughput gene expression profiling for FFPE samples. In this study, to evaluate the gene expression of frozen tissue-derived prognostic signatures in FFPE CRC samples, we evaluated the expression of 516 genes from published frozen tissue-derived prognostic signatures in 42 FFPE CRC samples measured by both platforms. Based on HTA platform-derived data, we identified both gene (99 individual genes, FDR FFPE tumor tissues to detect frozen tissue-derived prognostic gene expression signatures for CRC patients. PMID:27623752

  6. Accurate data processing improves the reliability of Affymetrix gene expression profiles from FFPE samples.

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    Maurizio Callari

    Full Text Available Formalin fixed paraffin-embedded (FFPE tumor specimens are the conventionally archived material in clinical practice, representing an invaluable tissue source for biomarkers development, validation and routine implementation. For many prospective clinical trials, this material has been collected allowing for a prospective-retrospective study design which represents a successful strategy to define clinical utility for candidate markers. Gene expression data can be obtained even from FFPE specimens with the broadly used Affymetrix HG-U133 Plus 2.0 microarray platform. Nevertheless, important major discrepancies remain in expression data obtained from FFPE compared to fresh-frozen samples, prompting the need for appropriate data processing which could help to obtain more consistent results in downstream analyses. In a publicly available dataset of matched frozen and FFPE expression data, the performances of different normalization methods and specifically designed Chip Description Files (CDFs were compared. The use of an alternative CDFs together with fRMA normalization significantly improved frozen-FFPE sample correlations, frozen-FFPE probeset correlations and agreement of differential analysis between different tumor subtypes. The relevance of our optimized data processing was assessed and validated using two independent datasets. In this study we demonstrated that an appropriate data processing can significantly improve the reliability of gene expression data derived from FFPE tissues using the standard Affymetrix platform. Tools for the implementation of our data processing algorithm are made publicly available at http://www.biocut.unito.it/cdf-ffpe/.

  7. MALDI direct analysis and imaging of frozen versus FFPE tissues: what strategy for which sample?

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    Wisztorski, Maxence; Franck, Julien; Salzet, Michel; Fournier, Isabelle

    2010-01-01

    Significant advances have been made in the past decade in the field of mass spectrometry imaging with MALDI ion sources (MALDI-MSI). While MALDI-MSI has high potential in the field of biology and in the clinic, a challenge for MALDI-MSI has been to adapt itself to a greater range of sample types. In particular, much of the biological archived materials for pathology studies are tissue biopsies fixed with paraformaldehyde and embedded in paraffin (FFPE tissues) because of the high stability of such samples. Thus, there has been a need to develop strategies for analyzing FFPE samples as this would allow retrospective studies of past clinical cases on large cohorts of existing samples. Obviously, PAF fixation, by inducing protein cross-linking, causes problems for molecular analysis by MS. We developed on tissue digestion strategies for overcoming these difficulties and allowing molecular data to be retrieved from FFPE samples no matter how long they have been stored. These digestion strategies preserve localization from digested proteins making MALDI-MSI of proteins possible by monitoring the resulting peptides. We present methods and protocols for FFPE samples. These strategies have proven to be valuable for all tested FFPE samples and have opened archived tissues from hospital banks to MALDI-MSI.

  8. MicroRNA Stability in FFPE Tissue Samples: Dependence on GC Content.

    Science.gov (United States)

    Kakimoto, Yu; Tanaka, Masayuki; Kamiguchi, Hiroshi; Ochiai, Eriko; Osawa, Motoki

    2016-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs responsible for fine-tuning of gene expression at post-transcriptional level. The alterations in miRNA expression levels profoundly affect human health and often lead to the development of severe diseases. Currently, high throughput analyses, such as microarray and deep sequencing, are performed in order to identify miRNA biomarkers, using archival patient tissue samples. MiRNAs are more robust than longer RNAs, and resistant to extreme temperatures, pH, and formalin-fixed paraffin-embedding (FFPE) process. Here, we have compared the stability of miRNAs in FFPE cardiac tissues using next-generation sequencing. The mode read length in FFPE samples was 11 nucleotides (nt), while that in the matched frozen samples was 22 nt. Although the read counts were increased 1.7-fold in FFPE samples, compared with those in the frozen samples, the average miRNA mapping rate decreased from 32.0% to 9.4%. These results indicate that, in addition to the fragmentation of longer RNAs, miRNAs are to some extent degraded in FFPE tissues as well. The expression profiles of total miRNAs in two groups were highly correlated (0.88 FFPE cardiac tissues instead of miR-1, which was predominant before fixation. Subsequent quantitative PCR (qPCR) analyses revealed that miRNAs with GC content of less than 40% are more degraded than GC-rich miRNAs (pFFPE samples cannot be directly compared with that of fresh frozen samples. The combination of miRNA deep sequencing and other quantitative analyses, such as qPCR, may improve the utility of archival FFPE tissue samples. PMID:27649415

  9. Use of Sequenom sample ID Plus® SNP genotyping in identification of FFPE tumor samples.

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    Jessica K Miller

    Full Text Available Short tandem repeat (STR analysis, such as the AmpFlSTR® Identifiler® Plus kit, is a standard, PCR-based human genotyping method used in the field of forensics. Misidentification of cell line and tissue DNA can be costly if not detected early; therefore it is necessary to have quality control measures such as STR profiling in place. A major issue in large-scale research studies involving archival formalin-fixed paraffin embedded (FFPE tissues is that varying levels of DNA degradation can result in failure to correctly identify samples using STR genotyping. PCR amplification of STRs of several hundred base pairs is not always possible when DNA is degraded. The Sample ID Plus® panel from Sequenom allows for human DNA identification and authentication using SNP genotyping. In comparison to lengthy STR amplicons, this multiplexing PCR assay requires amplification of only 76-139 base pairs, and utilizes 47 SNPs to discriminate between individual samples. In this study, we evaluated both STR and SNP genotyping methods of sample identification, with a focus on paired FFPE tumor/normal DNA samples intended for next-generation sequencing (NGS. The ability to successfully validate the identity of FFPE samples can enable cost savings by reducing rework.

  10. Dose-Response Analysis of RNA-Seq Profiles in Archival Formalin-fixed paraffin-embedded (FFPE) Samples

    Science.gov (United States)

    Formalin-fixed paraffin-embedded (FFPE) samples provide a vast untapped resource for chemical safety and translational science. To date, genomic profiling of FFPE samples has been limited by poor RNA quality and inconsistent results with limited utility in dose-response assessmen...

  11. Multiplex bisulfite PCR resequencing of clinical FFPE DNA

    OpenAIRE

    Korbie, Darren; Lin, Erica; Wall, David; Nair, Shalima S; Stirzaker, Clare; Clark, Sue J; Trau, Matt

    2015-01-01

    Background The clinical utility of DNA methylation as a predictive or prognostic biomarker requires scalable resequencing protocols for bisulfite-converted DNA. Key features of any validation method should be adaptability for low- or high-throughput needs and high reproducibility, and should only require minimal amounts of precious clinical sample as input material. Critically, this method should also deliver robust results when working with bisulfite-converted DNA extracted from formalin-fix...

  12. Discrepancies between VEGF −1154 G>A Polymorphism Analysis Performed in Peripheral Blood Samples and FFPE Tissue

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    Giorgia Marisi

    2014-07-01

    Full Text Available Single nucleotide polymorphisms (SNPs may be associated with the response or toxicity to different types of treatment. Although SNP analysis is usually performed on DNA from peripheral blood, formalin fixed paraffin-embedded (FFPE tissue is often used for retrospective studies. We analyzed VEGF (−2578C>A, −1498C>T, −1154G>A, −634C>G, +936C>T and eNOS (+894G>T, −786T>C, VNTR (variable number of tandem repeats 27bp intron 4 polymorphisms by direct sequencing or Real Time PCR in 237 patients with advanced colorectal cancer. Peripheral blood was used for 153 patients, whereas only FFPE tumor tissue was available for 84 patients. All SNP frequencies were in Hardy-Weinberg Equilibrium (HWE, with the exception of VEGF −1154, which was only in HWE in peripheral blood specimens. We therefore analyzed this SNP in DNA extracted from FFPE tumor tissue compared to FFPE healthy tissue and peripheral blood from 20 patients. Numerous heterozygous patients in peripheral blood DNA were homozygous for the A-allele in both tumor and healthy FFPE tissues. Our findings indicate that, although FFPE tissue might be a suitable specimen for genotyping, VEGF −1154 does not give reliable results on this type of material. As other SNPs may also have this limitation, genotype concordance should first be confirmed by comparing results obtained from FFPE and fresh sample analyses.

  13. Improved reproducibility in genome-wide DNA methylation analysis for PAXgene® fixed samples compared to restored FFPE DNA

    DEFF Research Database (Denmark)

    Andersen, Gitte Brinch; Hager, Henrik; Hansen, Lise Lotte;

    2014-01-01

    , precluding the use of the valuable archives of specimens with long-term follow-up data. Therefore, restoration protocols for DNA from formalin-fixed and paraffin-embedded (FFPE) samples have been developed, although they are cost-intensive and time-consuming. An alternative to FFPE and snap......Chip. Quantitative DNA methylation analysis demonstrated that the methylation profile in PAXgene-fixed tissues showed, in comparison with restored FFPE samples, a higher concordance with the profile detected in frozen samples. We demonstrate, for the first time, that DNA from PAXgene conserved tissue performs better...... compared with restored FFPE DNA in genome-wide DNA methylation analysis. In addition, DNA from PAXgene tissue can be directly used on the array without prior restoration, rendering the analytical process significantly more time- and cost-effective....

  14. Pre-Analytical Considerations for Successful Next-Generation Sequencing (NGS: Challenges and Opportunities for Formalin-Fixed and Paraffin-Embedded Tumor Tissue (FFPE Samples

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    Gladys Arreaza

    2016-09-01

    Full Text Available In cancer drug discovery, it is important to investigate the genetic determinants of response or resistance to cancer therapy as well as factors that contribute to adverse events in the course of clinical trials. Despite the emergence of new technologies and the ability to measure more diverse analytes (e.g., circulating tumor cell (CTC, circulating tumor DNA (ctDNA, etc., tumor tissue is still the most common and reliable source for biomarker investigation. Because of its worldwide use and ability to preserve samples for many decades at ambient temperature, formalin-fixed, paraffin-embedded tumor tissue (FFPE is likely to be the preferred choice for tissue preservation in clinical practice for the foreseeable future. Multiple analyses are routinely performed on the same FFPE samples (such as Immunohistochemistry (IHC, in situ hybridization, RNAseq, DNAseq, TILseq, Methyl-Seq, etc.. Thus, specimen prioritization and optimization of the isolation of analytes is critical to ensure successful completion of each assay. FFPE is notorious for producing suboptimal DNA quality and low DNA yield. However, commercial vendors tend to request higher DNA sample mass than what is actually required for downstream assays, which restricts the breadth of biomarker work that can be performed. We evaluated multiple genomics service laboratories to assess the current state of NGS pre-analytical processing of FFPE. Significant differences in pre-analytical capabilities were observed. Key aspects are highlighted and recommendations are made to improve the current practice in translational research.

  15. Pre-Analytical Considerations for Successful Next-Generation Sequencing (NGS): Challenges and Opportunities for Formalin-Fixed and Paraffin-Embedded Tumor Tissue (FFPE) Samples

    Science.gov (United States)

    Arreaza, Gladys; Qiu, Ping; Pang, Ling; Albright, Andrew; Hong, Lewis Z.; Marton, Matthew J.; Levitan, Diane

    2016-01-01

    In cancer drug discovery, it is important to investigate the genetic determinants of response or resistance to cancer therapy as well as factors that contribute to adverse events in the course of clinical trials. Despite the emergence of new technologies and the ability to measure more diverse analytes (e.g., circulating tumor cell (CTC), circulating tumor DNA (ctDNA), etc.), tumor tissue is still the most common and reliable source for biomarker investigation. Because of its worldwide use and ability to preserve samples for many decades at ambient temperature, formalin-fixed, paraffin-embedded tumor tissue (FFPE) is likely to be the preferred choice for tissue preservation in clinical practice for the foreseeable future. Multiple analyses are routinely performed on the same FFPE samples (such as Immunohistochemistry (IHC), in situ hybridization, RNAseq, DNAseq, TILseq, Methyl-Seq, etc.). Thus, specimen prioritization and optimization of the isolation of analytes is critical to ensure successful completion of each assay. FFPE is notorious for producing suboptimal DNA quality and low DNA yield. However, commercial vendors tend to request higher DNA sample mass than what is actually required for downstream assays, which restricts the breadth of biomarker work that can be performed. We evaluated multiple genomics service laboratories to assess the current state of NGS pre-analytical processing of FFPE. Significant differences in pre-analytical capabilities were observed. Key aspects are highlighted and recommendations are made to improve the current practice in translational research. PMID:27657050

  16. Comparison of Nanostring nCounter® Data on FFPE Colon Cancer Samples and Affymetrix Microarray Data on Matched Frozen Tissues.

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    Xi Chen

    Full Text Available The prognosis of colorectal cancer (CRC stage II and III patients remains a challenge due to the difficulties of finding robust biomarkers suitable for testing clinical samples. The majority of published gene signatures of CRC have been generated on fresh frozen colorectal tissues. Because collection of frozen tissue is not practical for routine surgical pathology practice, a clinical test that improves prognostic capabilities beyond standard pathological staging of colon cancer will need to be designed for formalin-fixed paraffin-embedded (FFPE tissues. The NanoString nCounter® platform is a gene expression analysis tool developed for use with FFPE-derived samples. We designed a custom nCounter® codeset based on elements from multiple published fresh frozen tissue microarray-based prognostic gene signatures for colon cancer, and we used this platform to systematically compare gene expression data from FFPE with matched microarray array data from frozen tissues. Our results show moderate correlation of gene expression between two platforms and discovery of a small subset of genes as candidate biomarkers for colon cancer prognosis that are detectable and quantifiable in FFPE tissue sections.

  17. Comparison of Nanostring nCounter® Data on FFPE Colon Cancer Samples and Affymetrix Microarray Data on Matched Frozen Tissues.

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    Chen, Xi; Deane, Natasha G; Lewis, Keeli B; Li, Jiang; Zhu, Jing; Washington, M Kay; Beauchamp, R Daniel

    2016-01-01

    The prognosis of colorectal cancer (CRC) stage II and III patients remains a challenge due to the difficulties of finding robust biomarkers suitable for testing clinical samples. The majority of published gene signatures of CRC have been generated on fresh frozen colorectal tissues. Because collection of frozen tissue is not practical for routine surgical pathology practice, a clinical test that improves prognostic capabilities beyond standard pathological staging of colon cancer will need to be designed for formalin-fixed paraffin-embedded (FFPE) tissues. The NanoString nCounter® platform is a gene expression analysis tool developed for use with FFPE-derived samples. We designed a custom nCounter® codeset based on elements from multiple published fresh frozen tissue microarray-based prognostic gene signatures for colon cancer, and we used this platform to systematically compare gene expression data from FFPE with matched microarray array data from frozen tissues. Our results show moderate correlation of gene expression between two platforms and discovery of a small subset of genes as candidate biomarkers for colon cancer prognosis that are detectable and quantifiable in FFPE tissue sections. PMID:27176004

  18. A laser microdissection-based workflow for FFPE tissue microproteomics: Important considerations for small sample processing.

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    Longuespée, Rémi; Alberts, Deborah; Pottier, Charles; Smargiasso, Nicolas; Mazzucchelli, Gabriel; Baiwir, Dominique; Kriegsmann, Mark; Herfs, Michael; Kriegsmann, Jörg; Delvenne, Philippe; De Pauw, Edwin

    2016-07-15

    Proteomic methods are today widely applied to formalin-fixed paraffin-embedded (FFPE) tissue samples for several applications in research, especially in molecular pathology. To date, there is an unmet need for the analysis of small tissue samples, such as for early cancerous lesions. Indeed, no method has yet been proposed for the reproducible processing of small FFPE tissue samples to allow biomarker discovery. In this work, we tested several procedures to process laser microdissected tissue pieces bearing less than 3000 cells. Combined with appropriate settings for liquid chromatography mass spectrometry-mass spectrometry (LC-MS/MS) analysis, a citric acid antigen retrieval (CAAR)-based procedure was established, allowing to identify more than 1400 proteins from a single microdissected breast cancer tissue biopsy. This work demonstrates important considerations concerning the handling and processing of laser microdissected tissue samples of extremely limited size, in the process opening new perspectives in molecular pathology. A proof of the proposed method for biomarker discovery, with respect to these specific handling considerations, is illustrated using the differential proteomic analysis of invasive breast carcinoma of no special type and invasive lobular triple-negative breast cancer tissues. This work will be of utmost importance for early biomarker discovery or in support of matrix-assisted laser desorption/ionization (MALDI) imaging for microproteomics from small regions of interest. PMID:26690073

  19. An approach to optimize sample preparation for MALDI imaging MS of FFPE sections using fractional factorial design of experiments.

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    Oetjen, Janina; Lachmund, Delf; Palmer, Andrew; Alexandrov, Theodore; Becker, Michael; Boskamp, Tobias; Maass, Peter

    2016-09-01

    A standardized workflow for matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI imaging MS) is a prerequisite for the routine use of this promising technology in clinical applications. We present an approach to develop standard operating procedures for MALDI imaging MS sample preparation of formalin-fixed and paraffin-embedded (FFPE) tissue sections based on a novel quantitative measure of dataset quality. To cover many parts of the complex workflow and simultaneously test several parameters, experiments were planned according to a fractional factorial design of experiments (DoE). The effect of ten different experiment parameters was investigated in two distinct DoE sets, each consisting of eight experiments. FFPE rat brain sections were used as standard material because of low biological variance. The mean peak intensity and a recently proposed spatial complexity measure were calculated for a list of 26 predefined peptides obtained by in silico digestion of five different proteins and served as quality criteria. A five-way analysis of variance (ANOVA) was applied on the final scores to retrieve a ranking of experiment parameters with increasing impact on data variance. Graphical abstract MALDI imaging experiments were planned according to fractional factorial design of experiments for the parameters under study. Selected peptide images were evaluated by the chosen quality metric (structure and intensity for a given peak list), and the calculated values were used as an input for the ANOVA. The parameters with the highest impact on the quality were deduced and SOPs recommended. PMID:27485623

  20. Use of Sequenom Sample ID Plus® SNP Genotyping in Identification of FFPE Tumor Samples

    OpenAIRE

    Jessica K Miller; Nicholas Buchner; Lee Timms; Shirley Tam; Xuemei Luo; Brown, Andrew M. K.; Danielle Pasternack; Robert G Bristow; Michael Fraser; Boutros, Paul C; McPherson, John D.

    2014-01-01

    Short tandem repeat (STR) analysis, such as the AmpFlSTR® Identifiler® Plus kit, is a standard, PCR-based human genotyping method used in the field of forensics. Misidentification of cell line and tissue DNA can be costly if not detected early; therefore it is necessary to have quality control measures such as STR profiling in place. A major issue in large-scale research studies involving archival formalin-fixed paraffin embedded (FFPE) tissues is that varying levels of DNA degradation can re...

  1. A multiplex PCR predictor for aCGH success of FFPE samples.

    NARCIS (Netherlands)

    Beers, E.H. van; Joosse, S.A.; Ligtenberg, M.J.L.; Fles, R.; Hogervorst, F.B.L.; Verhoef, S.; Nederlof, P.M.

    2006-01-01

    Formalin-fixed, paraffin-embedded (FFPE) tissue archives are the largest and longest time-spanning collections of patient material in pathology archives. Methods to disclose information with molecular techniques, such as array comparative genomic hybridisation (aCGH) have rapidly developed but are s

  2. Comparison of pre-analytical FFPE sample preparation methods and their impact on massively parallel sequencing in routine diagnostics.

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    Carina Heydt

    Full Text Available Over the last years, massively parallel sequencing has rapidly evolved and has now transitioned into molecular pathology routine laboratories. It is an attractive platform for analysing multiple genes at the same time with very little input material. Therefore, the need for high quality DNA obtained from automated DNA extraction systems has increased, especially to those laboratories which are dealing with formalin-fixed paraffin-embedded (FFPE material and high sample throughput. This study evaluated five automated FFPE DNA extraction systems as well as five DNA quantification systems using the three most common techniques, UV spectrophotometry, fluorescent dye-based quantification and quantitative PCR, on 26 FFPE tissue samples. Additionally, the effects on downstream applications were analysed to find the most suitable pre-analytical methods for massively parallel sequencing in routine diagnostics. The results revealed that the Maxwell 16 from Promega (Mannheim, Germany seems to be the superior system for DNA extraction from FFPE material. The extracts had a 1.3-24.6-fold higher DNA concentration in comparison to the other extraction systems, a higher quality and were most suitable for downstream applications. The comparison of the five quantification methods showed intermethod variations but all methods could be used to estimate the right amount for PCR amplification and for massively parallel sequencing. Interestingly, the best results in massively parallel sequencing were obtained with a DNA input of 15 ng determined by the NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA. No difference could be detected in mutation analysis based on the results of the quantification methods. These findings emphasise, that it is particularly important to choose the most reliable and constant DNA extraction system, especially when using small biopsies and low elution volumes, and that all common DNA quantification techniques can

  3. A microRNA isolation method from clinical samples

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    Sepideh Zununi Vahed

    2016-03-01

    Conclusion: The current isolation method can be applied for most clinical samples including cells, formalin-fixed and paraffin-embedded (FFPE tissues and even body fluids with a wide applicability in molecular biology investigations.

  4. Targeted high throughput sequencing in clinical cancer Settings: formaldehyde fixed-paraffin embedded (FFPE tumor tissues, input amount and tumor heterogeneity

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    Schaefer Georg

    2011-09-01

    Full Text Available Abstract Background Massively parallel sequencing technologies have brought an enormous increase in sequencing throughput. However, these technologies need to be further improved with regard to reproducibility and applicability to clinical samples and settings. Methods Using identification of genetic variations in prostate cancer as an example we address three crucial challenges in the field of targeted re-sequencing: Small nucleotide variation (SNV detection in samples of formalin-fixed paraffin embedded (FFPE tissue material, minimal amount of input sample and sampling in view of tissue heterogeneity. Results We show that FFPE tissue material can supplement for fresh frozen tissues for the detection of SNVs and that solution-based enrichment experiments can be accomplished with small amounts of DNA with only minimal effects on enrichment uniformity and data variance. Finally, we address the question whether the heterogeneity of a tumor is reflected by different genetic alterations, e.g. different foci of a tumor display different genomic patterns. We show that the tumor heterogeneity plays an important role for the detection of copy number variations. Conclusions The application of high throughput sequencing technologies in cancer genomics opens up a new dimension for the identification of disease mechanisms. In particular the ability to use small amounts of FFPE samples available from surgical tumor resections and histopathological examinations facilitates the collection of precious tissue materials. However, care needs to be taken in regard to the locations of the biopsies, which can have an influence on the prediction of copy number variations. Bearing these technological challenges in mind will significantly improve many large-scale sequencing studies and will - in the long term - result in a more reliable prediction of individual cancer therapies.

  5. The effects of age-in-block on RNA-seq analysis of archival formalin-fixed paraffin-embedded (FFPE) samples

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    Archival samples represent a vast resource for identification of chemical and pharmaceutical targets. Previous use of formalin-fixed paraffin-embedded (FFPE) samples has been limited due to changes in RNA introduced by fixation and embedding procedures. Recent advances in RNA-seq...

  6. [Use of archival formalin-fixed, paraffin-embedded (FFPE) tissue samples for molecular genetic analysis in diffuse large B-cell lymphoma (DLBCL)].

    Science.gov (United States)

    Jarošová, Marie; Kučerová, Jana; Flodr, Patrik; Mikešová, Michaela; Procházka, Vít; Papajík, Tomáš

    2014-04-01

    The currently valid molecular genetic subclassification of patients with diffuse large B-cell lymphoma (DLBCL) into three prognostic subgroups based on expression profiling has been the objective of numerous genetic studies. In routine clinical practice, however, expression profiling technology remains unavailable for the most of centers. Apart from the technology, in some cases molecular genetic laboratories have problems obtaining high-quality material, i.e. fresh tissues, for RNA isolation to determine gene expression. One possibility is to determine the gene expression from RNA obtained by isolation from formalin-fixed, paraffin-embedded (FFPE) tissue. This pilot study aimed at isolating RNA from FFPE in patients diagnosed with DLBCL and verifying the potential use of such RNA for the expression analysis of 7 selected genes. Although the study showed that it is possible to isolate RNA and determine the expression of the selected genes from archival material, the values of relative expression of some genes in the set were too variable to be used for unambiguous prognostic classification. It was confirmed that retrospective analyses of selected genes may be performed with sufficient material obtained, and that properly archived blocks may be used for molecular biology analyses even after 8 years.

  7. Nucleic acids from long-term preserved FFPE tissues are suitable for downstream analyses.

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    Ludyga, Natalie; Grünwald, Barbara; Azimzadeh, Omid; Englert, Sonja; Höfler, Heinz; Tapio, Soile; Aubele, Michaela

    2012-02-01

    Tissues used for clinical diagnostics are mostly formalin-fixed and paraffin-embedded (FFPE) which provides many advantages. However, the quality of the obtained nucleic acids (NA) is reduced and this turns out to be a challenge for further molecular analyses. Although the spectrum of analyses of NA extracted from FFPE tissue has increased, the standard operating procedures for NA isolation from old tissue blocks still need to be improved. Here, we compared the efficiency of different NA extraction methods, using FFPE tissues of variable age and origin, with respect to downstream analyses. Our study showed that the phenol-chloroform isoamyl alcohol (PCI) and the commercial Qiagen protocol yielded samples with highest purity. The PCI protocol delivered the longest amplicons even from samples from the 1970s. We developed a short (1 h) tissue lysis procedure that turned out to be highly time- and cost-effective when DNA quality was tested using single and multiplex PCR. Compared to a 1-day lysis-protocol, the amplicons were only 100 bp shorter. In addition, single-copy genes used in daily routine were successfully amplified from long-term stored FFPE samples following 1-h tissue-lysis. The RNA integrity numbers (RIN) determined on RNA isolated from FFPE tissues indicated degraded RNA; however, all RINs were above the generally agreed threshold of 1.4. We showed that, depending on the purpose of the analysis, NA retrieved from FFPE tissues older than 40 years may be successfully used for molecular analysis.

  8. MicroRNA Stability in Postmortem FFPE Tissues: Quantitative Analysis Using Autoptic Samples from Acute Myocardial Infarction Patients.

    Directory of Open Access Journals (Sweden)

    Yu Kakimoto

    Full Text Available MicroRNAs (miRNAs are very short (18-24 nucleotides nucleic acids that are expressed in a number of biological tissues and have been shown to be more resistant to extreme temperatures and pH compared to longer RNA molecules, like mRNAs. As miRNAs contribute to diverse biological process and respond to various kinds of cellular stress, their utility as diagnostic biomarkers and/or therapeutic targets has recently been explored. Here, we have evaluated the usefulness of miRNA quantification during postmortem examination of cardiac tissue from acute myocardial infarction (AMI patients. Cardiac tissue was collected within one week of the patient's death and either frozen (19 samples or fixed in formalin for up to three years (36 samples. RNA integrity was evaluated with an electropherogram, and it appears that longer RNAs are fragmented after death in the long-term fixed samples. Quantitative PCR was also performed for seven miRNAs and three other small RNAs in order to determine the appropriate controls for our postmortem analysis. Our data indicate that miR-191 and miR-26b are more suitable than the other types of small RNA molecules as they are stably detected after death and long-term fixation. Further, we also applied our quantitation method, using these endogenous controls, to evaluate the expression of three previously identified miRNA biomarkers, miR-1, miR-208b, and miR-499a, in formalin-fixed tissues from AMI patients. Although miR-1 and miR-208b decreased (1.4-fold and increased (1.2-fold, respectively, in the AMI samples compared to the controls, the significance of these changes was limited by our sample size. In contrast, the relative level of miR-499a was significantly decreased in the AMI samples (2.1-fold. This study highlights the stability of miRNAs after death and long-term fixation, validating their use as reliable biomarkers for AMI during postmortem examination.

  9. Absolute quantitation of Met using mass spectrometry for clinical application: assay precision, stability, and correlation with MET gene amplification in FFPE tumor tissue.

    Directory of Open Access Journals (Sweden)

    Daniel V T Catenacci

    Full Text Available Overexpression of Met tyrosine kinase receptor is associated with poor prognosis. Overexpression, and particularly MET amplification, are predictive of response to Met-specific therapy in preclinical models. Immunohistochemistry (IHC of formalin-fixed paraffin-embedded (FFPE tissues is currently used to select for 'high Met' expressing tumors for Met inhibitor trials. IHC suffers from antibody non-specificity, lack of quantitative resolution, and, when quantifying multiple proteins, inefficient use of scarce tissue.After describing the development of the Liquid-Tissue-Selected Reaction Monitoring-mass spectrometry (LT-SRM-MS Met assay, we evaluated the expression level of Met in 130 FFPE gastroesophageal cancer (GEC tissues. We assessed the correlation of SRM Met expression to IHC and mean MET gene copy number (GCN/nucleus or MET/CEP7 ratio by fluorescence in situ hybridization (FISH.Proteomic mapping of recombinant Met identified 418TEFTTALQR426 as the optimal SRM peptide. Limits of detection (LOD and quantitation (LOQ for this peptide were 150 and 200 amol/µg tumor protein, respectively. The assay demonstrated excellent precision and temporal stability of measurements in serial sections analyzed one year apart. Expression levels of 130 GEC tissues ranged (<150 amol/µg to 4669.5 amol/µg. High correlation was observed between SRM Met expression and both MET GCN and MET/CEP7 ratio as determined by FISH (n = 30; R2 = 0.898. IHC did not correlate well with SRM (n = 44; R2 = 0.537 nor FISH GCN (n = 31; R2 = 0.509. A Met SRM level of ≥1500 amol/µg was 100% sensitive (95% CI 0.69-1 and 100% specific (95% CI 0.92-1 for MET amplification.The Met SRM assay measured the absolute Met levels in clinical tissues with high precision. Compared to IHC, SRM provided a quantitative and linear measurement of Met expression, reliably distinguishing between non-amplified and amplified MET tumors. These results demonstrate a novel

  10. Nucleic Acid Purification from FFPE on the Maxwell® 16 Instrument

    OpenAIRE

    Hooper, K; English, J; Rekke, A.; Owles, C.; Mandrekar, M.; Mandrekar, P.; Hite, D.; Bellevue, S.; Horejsh, D; Urh, M.; Brazas, Robert

    2013-01-01

    Formalin fixed paraffin embedded (FFPE) samples are commonly used for archiving pathological samples for molecular oncology labs and researchers. Traditional methods for the purification of nucleic acids from FFPE tissue samples are often labor intensive, include the use of hazardous organic reagents, and involve difficult pre-processing steps. Here, we describe an automated method for the purification of DNA and RNA from FFPE tissue sections using the Maxwell® 16 instrument that simplifies p...

  11. Formalin-fixed, paraffin-embedded (FFPE) tissue epigenomics using Infinium HumanMethylation450 BeadChip assays.

    Science.gov (United States)

    de Ruijter, Tim C; de Hoon, Joep P J; Slaats, Jeroen; de Vries, Bart; Janssen, Marjolein J F W; van Wezel, Tom; Aarts, Maureen J B; van Engeland, Manon; Tjan-Heijnen, Vivianne C G; Van Neste, Leander; Veeck, Jürgen

    2015-07-01

    Current genome-wide methods to detect DNA-methylation in healthy and diseased tissue require high-quality DNA from fresh-frozen (FF) samples. However, well-annotated clinical samples are mostly available as formalin-fixed, paraffin-embedded (FFPE) tissues containing poor-quality DNA. To overcome this limitation, we here aimed to evaluate a DNA restoration protocol for usage with the genome-wide Infinium HumanMethylation450 BeadChip assay (HM-450K). Sixty-six DNA samples from normal colon (n=9) and breast cancer (n=11) were interrogated separately using HM-450K. Analyses included matched FF/FFPE samples and technical duplicates. FFPE DNA was processed with (FFPEr) or without a DNA restoration protocol (Illumina). Differentially methylated genes were finally validated in 24 additional FFPE tissues using nested methylation-specific PCR (MSP). In summary, β-values correlation between FFPEr duplicates was high (ρ=0.9927 (s.d. ±0.0015)). Matched FF/FFPEr correlation was also high (ρ=0.9590 (s.d. ±0.0184)) compared with matched FF/FFPE (ρ=0.8051 (s.d. ±0.1028). Probe detection rate in FFPEr samples (98.37%, s.d. ±0.66) was comparable to FF samples (99.98%, s.d. ±0.019) and substantially lower in FFPE samples (82.31%, s.d. ±18.65). Assay robustness was not decreased by sample archival age up to 10 years. We could also demonstrate no decrease in assay robustness when using 100 ng of DNA input only. Four out of the five selected differentially methylated genes could be validated by MSP. The gene failing validation by PCR showed high variation of CpG β-values in primer-binding sites. In conclusion, by using the FFPE DNA restoration protocol, HM-450K assays provide robust, accurate and reproducible results with FFPE tissue-derived DNA, which are comparable to those obtained with FF tissue. Most importantly, differentially methylated genes can be validated using more sensitive techniques, such as nested MSP, altogether providing an epigenomics platform for

  12. Evaluation of five DNA extraction methods for detection of H. pylori in formalin-fixed paraffin-embedded (FFPE) liver tissue from patients with hepatocellular carcinoma.

    Science.gov (United States)

    Rabelo-Gonçalves, Elizabeth; Roesler, Bruna; Guardia, Ana Carolina; Milan, Arlete; Hara, Natalicia; Escanhoela, Cecília; Almeida, Jazon; Boin, Ilka; Zeitune, José Murilo

    2014-03-01

    Since Helicobacter spp. DNA was identified in liver tissue resected from patients with hepatocelullar carcinoma (HCC), researchers have suggested a role of this bacterium in hepatic carcinogenesis. Archives of formalin-fixed, paraffin-embedded (FFPE) tissues represent an extraordinary source for clinical studies providing many advantages. However, DNA extraction from FFPE tissues is laborious, time-consuming and still remains a challenge. The aim of this study was to evaluate five protocols for DNA extraction from FFPE liver obtained from patients with HCC in order to detect Helicobacter pylori DNA. These methods were: (1) QIAamp FFPE Tissue Kit, (2) QIAamp DNA Mini Kit, (3) Wizard SV Genomic DNA Purification System, (4) RealiaPrep FFPE gDNA Miniprep System and (5) phenol-chloroform. H. pylori detection was performed using 16S rRNA gene amplification by PCR. The highest total amount of DNA was obtained using the phenol-chloroform method. Analyses of 16S rRNA gene amplification did not show statistically significant differences among the methods (p=0.466), although the highest percentage of positive cases (70%) was found in samples extracted with phenol-chloroform. We suggest that of the five methods evaluated, phenol/chloroform is the most suitable for detection of H. pylori in FFPE liver from patients with HCC.

  13. Next-generation sequencing of RNA and DNA isolated from paired fresh-frozen and formalin-fixed paraffin-embedded samples of human cancer and normal tissue.

    Directory of Open Access Journals (Sweden)

    Jakob Hedegaard

    Full Text Available Formalin-fixed, paraffin-embedded (FFPE tissues are an invaluable resource for clinical research. However, nucleic acids extracted from FFPE tissues are fragmented and chemically modified making them challenging to use in molecular studies. We analysed 23 fresh-frozen (FF, 35 FFPE and 38 paired FF/FFPE specimens, representing six different human tissue types (bladder, prostate and colon carcinoma; liver and colon normal tissue; reactive tonsil in order to examine the potential use of FFPE samples in next-generation sequencing (NGS based retrospective and prospective clinical studies. Two methods for DNA and three methods for RNA extraction from FFPE tissues were compared and were found to affect nucleic acid quantity and quality. DNA and RNA from selected FFPE and paired FF/FFPE specimens were used for exome and transcriptome analysis. Preparations of DNA Exome-Seq libraries was more challenging (29.5% success than that of RNA-Seq libraries, presumably because of modifications to FFPE tissue-derived DNA. Libraries could still be prepared from RNA isolated from two-decade old FFPE tissues. Data were analysed using the CLC Bio Genomics Workbench and revealed systematic differences between FF and FFPE tissue-derived nucleic acid libraries. In spite of this, pairwise analysis of DNA Exome-Seq data showed concordance for 70-80% of variants in FF and FFPE samples stored for fewer than three years. RNA-Seq data showed high correlation of expression profiles in FF/FFPE pairs (Pearson Correlations of 0.90 +/- 0.05, irrespective of storage time (up to 244 months and tissue type. A common set of 1,494 genes was identified with expression profiles that were significantly different between paired FF and FFPE samples irrespective of tissue type. Our results are promising and suggest that NGS can be used to study FFPE specimens in both prospective and retrospective archive-based studies in which FF specimens are not available.

  14. Toward deciphering proteomes of formalin-fixed paraffin-embedded (FFPE) tissues

    OpenAIRE

    Magdeldin, Sameh; Yamamoto, Tadashi

    2012-01-01

    Formalin-fixed paraffin-embedded (FFPE) tissue specimens comprise a potentially valuable resource for both prospective and retrospective biomarker discovery. Unlocking the proteomic profile of clinicopathological FFPE tissues is a critically essential step for annotating clinical findings and predicting biomarkers for ultimate disease prognosis and therapeutic follow-up.

  15. HaloPlex Targeted Resequencing for Mutation Detection in Clinical Formalin-Fixed, Paraffin-Embedded Tumor Samples.

    Science.gov (United States)

    Moens, Lotte N J; Falk-Sörqvist, Elin; Ljungström, Viktor; Mattsson, Johanna; Sundström, Magnus; La Fleur, Linnéa; Mathot, Lucy; Micke, Patrick; Nilsson, Mats; Botling, Johan

    2015-11-01

    In recent years, the advent of massively parallel next-generation sequencing technologies has enabled substantial advances in the study of human diseases. Combined with targeted DNA enrichment methods, high sequence coverage can be obtained for different genes simultaneously at a reduced cost per sample, creating unique opportunities for clinical cancer diagnostics. However, the formalin-fixed, paraffin-embedded (FFPE) process of tissue samples, routinely used in pathology departments, results in DNA fragmentation and nucleotide modifications that introduce a number of technical challenges for downstream biomolecular analyses. We evaluated the HaloPlex target enrichment system for somatic mutation detection in 80 tissue fractions derived from 20 clinical cancer cases with paired tumor and normal tissue available in both FFPE and fresh-frozen format. Several modifications to the standard method were introduced, including a reduced target fragment length and two strand capturing. We found that FFPE material can be used for HaloPlex-based target enrichment and next-generation sequencing, even when starting from small amounts of DNA. By specifically capturing both strands for each target fragment, we were able to reduce the number of false-positive errors caused by FFPE-induced artifacts and lower the detection limit for somatic mutations. We believe that the HaloPlex method presented here will be broadly applicable as a tool for somatic mutation detection in clinical cancer settings.

  16. Improved RNA quality and TaqMan® Pre-amplification method (PreAmp to enhance expression analysis from formalin fixed paraffin embedded (FFPE materials

    Directory of Open Access Journals (Sweden)

    Pirotta Marco

    2008-02-01

    Full Text Available Abstract Background Archival formalin-fixed paraffin-embedded (FFPE tissues represent an abundant source of clinical specimens; however their use is limited in applications involving analysis of gene expression due to RNA degradation and modification during fixation and processing. This study improved the quality of RNA extracted from FFPE by introducing a heating step into the selected extraction protocols. Further, it evaluated a novel pre-amplification system (PreAmp designed to enhance expression analysis from tissue samples using assays with a range of amplicon size (62–164 bp. Results Results from the Bioanalyzer and TaqMan® data showed improvement of RNA quality extracted using the modified protocols from FFPE. Incubation at 70°C for 20 minutes was determined to be the best condition of those tested to disrupt cross-links while not compromising RNA integrity. TaqMan® detection was influenced by master mix, amplicon size and the incorporation of a pre-amplification step. TaqMan® PreAmp consistently achieved decreased CT values in both snap frozen and FFPE aliquots compared with no pre-amplification. Conclusion Modification to extraction protocols has facilitated procurement of RNA that may be successfully amplified using QRT-PCR. TaqMan® PreAmp system is a robust and practical solution to limited quantities of RNA from FFPE extracts.

  17. Multicentre validation study of nucleic acids extraction from FFPE tissues.

    NARCIS (Netherlands)

    Bonin, S.; Hlubek, F.; Benhattar, J.; Denkert, C.; Dietel, M.; Fernandez, P.L.; Hofler, G.; Kothmaier, H.; Kruslin, B.; Mazzanti, C.M.; Perren, A.; Popper, H.; Scarpa, A.; Soares, P.; Stanta, G.; Groenen, P.J.T.A.

    2010-01-01

    In most pathology laboratories worldwide, formalin-fixed paraffin embedded (FFPE) samples are the only tissue specimens available for routine diagnostics. Although commercial kits for diagnostic molecular pathology testing are becoming available, most of the current diagnostic tests are laboratory-b

  18. Intraparenchymal mesenchymal chondrosarcoma of the frontal lobe--a case report and molecular detection of specific gene fusions from archival FFPE sample.

    Science.gov (United States)

    Sajjad, Emir Ahmed; Sikora, Katarzyna; Paciejewski, Tomasz; Garbicz, Filip; Paskal, Wiktor; Szacht, Milena; Grajkowska, Wieslawa; Włodarski, Pawel Krzysztof

    2015-01-01

    Mesenchymal chondrosarcoma is a rare tumor of cartilaginous origin characterized by its bimorphic pattern composed of highly undifferentiated small round cells separated by islands of well-differentiated hyaline cartilage. It exhibits higher malignancy and earlier occurrence in comparison to classic chondrosarcomas. Recently identified HEY1-NCOA2 and IRF2BP2-CDX1 gene fusions confirm their distinct molecular origin and pose a promising diagnostic marker. The majority of cases arise from craniofacial bones. In this study, we present a rare case of mesenchymal chondrosarcoma encompassed within the brain parenchyma of the frontal lobe without any dural or bone attachment. We demonstrate histopathological findings and confirm the HEY1-NCOA2 gene fusion in a formalin-fixed paraffin-embedded archival sample using simple reverse transcription polymerase chain reaction (RT-PCR) method. IRF2BP2-CDX1 gene fusion was absent in the analyzed sample. The clinical follow-up is also presented with a review of treatment modalities for this entity.

  19. Expression of EBV Encoded viral RNA 1, 2 and anti-inflammatory Cytokine (interleukin-10 in FFPE lymphoma specimens: a preliminary study for diagnostic implication in Pakistan

    Directory of Open Access Journals (Sweden)

    Qadri Ishtiaq

    2011-07-01

    Full Text Available Abstract Background Epstein Barr Virus (EBV plays a significant role as a cofactor in the process of tumorigenesis and has consistently been associated with a variety of malignancies. EBV encoded RNAs (EBER1 and EBER2 are the most abundant viral transcripts in latently EBV-infected cells and their role in viral infection is still unclear. Formalin Fixed Paraffin Embedded (FFPE tissues of surgically removed carcinoma biopsies are widely available form but have never been exploited for expressional studies previously in Pakistan. Immunohistochemistry (IHC and in situ hybridization (ISH in FFPE biopsy tissues remains the gold standard for proving EBV relationship in a histopathological lesion but their reagents associated limitations confines their reliability in some applications. Recently introduced targeted drug delivery systems induce viral lytic gene expression and therefore require more sensitive method to quantify viral as well as cellular gene expression. Methods Eight (8 lymphoma samples were screened to detect the EBV genome. Qualitative and quantitative expression of EBV Encoded RNAs (EBER1, EBER2 and anti-inflammatory cytokine (interleukin-10 in FFPE EBV positive lymphoma tissue samples were then analysed by using Reverse transcriptase Polymerase Chain Reaction (RT-PCR and Real Time Polymerase Chain Reaction (qRT-PCR, respectively. Results In this study we have successfully quantified elevated expressional levels of both cellular and viral transcripts, namely EBER1, EBER2 and anti-inflammatory cytokine (IL-10 in the FFPE Burkitt's lymphoma (BL specimens of Pakistani origin. Conclusions These results indicate that FFPE samples may retain viral as well as cellular RNA expression information at detectable level. To our knowledge, this is first study which represents elevated expressional levels of EBER1, EBER2 and IL-10 in FFPE tissue samples of Burkitt's lymphoma in Pakistan. These observations will potentially improve current lacunas in

  20. Cross-laboratory validation of the OncoScan® FFPE Assay, a multiplex tool for whole genome tumour profiling

    OpenAIRE

    Foster, Joseph M.; Oumie, Assa; Togneri, Fiona S.; Vasques, Fabiana Ramos; Hau, Debra; Taylor, Morag; Tinkler-Hundal, Emma; Southward, Katie; Medlow, Paul; McGreeghan-Crosby, Keith; Halfpenny, Iris; McMullan, Dominic J.; Quirke, Phil; Keating, Katherine E; Griffiths, Mike

    2015-01-01

    Background Adoption of new technology in both basic research and clinical settings requires rigorous validation of analytical performance. The OncoScan® FFPE Assay is a multiplexing tool that offers genome-wide copy number and loss of heterozygosity detection, as well as identification of frequently tested somatic mutations. Methods In this study, 162 formalin fixed paraffin embedded samples, representing six different tumour types, were profiled in triplicate across three independent laborat...

  1. Two Methods for High-Throughput NGS Template Preparation for Small and Degraded Clinical Samples Without Automation

    OpenAIRE

    Kamberov, E.; Tesmer, T.; Mastronardi, M.; Langmore, John

    2012-01-01

    Clinical samples are difficult to prepare for NGS, because of the small amounts or degraded states of formalin-fixed tissue, plasma, urine, and single-cell DNA. Conventional whole genome amplification methods are too biased for NGS applications, and the existing NGS preparation kits require intermediate purifications and excessive time to prepare hundreds of samples in a day without expensive automation. We have tested two 96-well manual methods to make NGS templates from FFPE tissue, plasma,...

  2. Sample processing considerations for detecting copy number changes in formalin-fixed, paraffin-embedded tissues.

    Science.gov (United States)

    Jacobs, Sharoni

    2012-11-01

    The Whole Genome Sampling Analysis (WGSA) assay in combination with Affymetrix GeneChip Mapping Arrays is used for copy number analysis of high-quality DNA samples (i.e., samples that have been collected from blood, fresh or frozen tissue, or cell lines). Formalin-fixed, paraffin-embedded (FFPE) samples, however, represent the most prevalent form of archived clinical samples, but they provide additional challenges for molecular assays. FFPE processing usually results in the degradation of FFPE DNA and in the contamination and chemical modification of these DNA samples. Because of these issues, FFPE DNA is not suitable for all molecular assays designed for high-quality DNA samples. Strategies recommended for processing FFPE DNA samples through WGSA and to the Mapping arrays are described here. PMID:23118355

  3. Sample processing considerations for detecting copy number changes in formalin-fixed, paraffin-embedded tissues.

    Science.gov (United States)

    Jacobs, Sharoni

    2012-11-01

    The Whole Genome Sampling Analysis (WGSA) assay in combination with Affymetrix GeneChip Mapping Arrays is used for copy number analysis of high-quality DNA samples (i.e., samples that have been collected from blood, fresh or frozen tissue, or cell lines). Formalin-fixed, paraffin-embedded (FFPE) samples, however, represent the most prevalent form of archived clinical samples, but they provide additional challenges for molecular assays. FFPE processing usually results in the degradation of FFPE DNA and in the contamination and chemical modification of these DNA samples. Because of these issues, FFPE DNA is not suitable for all molecular assays designed for high-quality DNA samples. Strategies recommended for processing FFPE DNA samples through WGSA and to the Mapping arrays are described here.

  4. Exome Enrichment and SOLiD Sequencing of Formalin Fixed Paraffin Embedded (FFPE Prostate Cancer Tissue

    Directory of Open Access Journals (Sweden)

    Saskia Biskup

    2012-07-01

    Full Text Available Next generation sequencing (NGS technologies have revolutionized cancer research allowing the comprehensive study of cancer using high throughput deep sequencing methodologies. These methods detect genomic alterations, nucleotide substitutions, insertions, deletions and copy number alterations. SOLiD (Sequencing by Oligonucleotide Ligation and Detection, Life Technologies is a promising technology generating billions of 50 bp sequencing reads. This robust technique, successfully applied in gene identification, might be helpful in detecting novel genes associated with cancer initiation and progression using formalin fixed paraffin embedded (FFPE tissue. This study’s aim was to compare the validity of whole exome sequencing of fresh-frozen vs. FFPE tumor tissue by normalization to normal prostatic FFPE tissue, obtained from the same patient. One primary fresh-frozen sample, corresponding FFPE prostate cancer sample and matched adjacent normal prostatic tissue was subjected to exome sequencing. The sequenced reads were mapped and compared. Our study was the first to show comparable exome sequencing results between FFPE and corresponding fresh-frozen cancer tissues using SOLiD sequencing. A prior study has been conducted comparing the validity of sequencing of FFPE vs. fresh frozen samples using other NGS platforms. Our validation further proves that FFPE material is a reliable source of material for whole exome sequencing.

  5. The influence of DNA degradation in formalin-fixed, paraffin-embedded (FFPE) tissue on locus-specific methylation assessment by MS-HRM.

    Science.gov (United States)

    Daugaard, Iben; Kjeldsen, Tina E; Hager, Henrik; Hansen, Lise Lotte; Wojdacz, Tomasz K

    2015-12-01

    Readily accessible formalin-fixed paraffin embedded (FFPE) tissues are a highly valuable source of genetic material for molecular analyses in both research and in vitro diagnostics but frequently genetic material in those samples is highly degraded. With locus-specific methylation changes being widely investigated for use as biomarkers in various aspects of clinical disease management, we aimed to evaluate to what extent standard laboratory procedures can approximate the quality of the DNA extracted from FFPE samples prior to methylation analyses. DNA quality in 107 FFPE non-small cell lung cancer (NSCLC) samples was evaluated using spectrophotometry and gel electrophoresis. Subsequently, the quality assessment results were correlated with the results of locus specific methylation assessment with methylation sensitive high resolution melting (MS-HRM). The correlation of template quality with PCR amplification performance and HRM based methylation detection indicated a significant influence of DNA quality on PCR amplification but not on methylation assessment. In conclusion, standard laboratory procedures fairly well approximate DNA degradation of FFPE samples and DNA degradation does not seem to considerably affect locus-specific methylation assessment by MS-HRM.

  6. Post-mortem testing; germline BRCA1/2 variant detection using archival FFPE non-tumor tissue. A new paradigm in genetic counseling.

    Science.gov (United States)

    Petersen, Annabeth Høgh; Aagaard, Mads Malik; Nielsen, Henriette Roed; Steffensen, Karina Dahl; Waldstrøm, Marianne; Bojesen, Anders

    2016-08-01

    Accurate estimation of cancer risk in HBOC families often requires BRCA1/2 testing, but this may be impossible in deceased family members. Previous, testing archival formalin-fixed, paraffin-embedded (FFPE) tissue for germline BRCA1/2 variants was unsuccessful, except for the Jewish founder mutations. A high-throughput method to systematically test for variants in all coding regions of BRCA1/2 in archival FFPE samples of non-tumor tissue is described, using HaloPlex target enrichment and next-generation sequencing. In a validation study, correct identification of variants or wild-type was possible in 25 out of 30 (83%) FFPE samples (age range 1-14 years), with a known variant status in BRCA1/2. No false positive was found. Unsuccessful identification was due to highly degraded DNA or presence of large intragenic deletions. In clinical use, a total of 201 FFPE samples (aged 0-43 years) were processed. Thirty-six samples were rejected because of highly degraded DNA or failed library preparation. Fifteen samples were investigated to search for a known variant. In the remaining 150 samples (aged 0-38 years), three variants known to affect function and one variant likely to affect function in BRCA1, six variants known to affect function and one variant likely to affect function in BRCA2, as well as four variants of unknown significance (VUS) in BRCA1 and three VUS in BRCA2 were discovered. It is now possible to test for germline BRCA1/2 variants in deceased persons, using archival FFPE samples from non-tumor tissue. Accurate genetic counseling is achievable in families where variant testing would otherwise be impossible.

  7. Post-mortem testing; germline BRCA1/2 variant detection using archival FFPE non-tumor tissue. A new paradigm in genetic counseling.

    Science.gov (United States)

    Petersen, Annabeth Høgh; Aagaard, Mads Malik; Nielsen, Henriette Roed; Steffensen, Karina Dahl; Waldstrøm, Marianne; Bojesen, Anders

    2016-08-01

    Accurate estimation of cancer risk in HBOC families often requires BRCA1/2 testing, but this may be impossible in deceased family members. Previous, testing archival formalin-fixed, paraffin-embedded (FFPE) tissue for germline BRCA1/2 variants was unsuccessful, except for the Jewish founder mutations. A high-throughput method to systematically test for variants in all coding regions of BRCA1/2 in archival FFPE samples of non-tumor tissue is described, using HaloPlex target enrichment and next-generation sequencing. In a validation study, correct identification of variants or wild-type was possible in 25 out of 30 (83%) FFPE samples (age range 1-14 years), with a known variant status in BRCA1/2. No false positive was found. Unsuccessful identification was due to highly degraded DNA or presence of large intragenic deletions. In clinical use, a total of 201 FFPE samples (aged 0-43 years) were processed. Thirty-six samples were rejected because of highly degraded DNA or failed library preparation. Fifteen samples were investigated to search for a known variant. In the remaining 150 samples (aged 0-38 years), three variants known to affect function and one variant likely to affect function in BRCA1, six variants known to affect function and one variant likely to affect function in BRCA2, as well as four variants of unknown significance (VUS) in BRCA1 and three VUS in BRCA2 were discovered. It is now possible to test for germline BRCA1/2 variants in deceased persons, using archival FFPE samples from non-tumor tissue. Accurate genetic counseling is achievable in families where variant testing would otherwise be impossible. PMID:26733283

  8. Formalin-fixed paraffin-embedded clinical tissues show spurious copy number changes in array-CGH profiles.

    Science.gov (United States)

    Mc Sherry, E A; Mc Goldrick, A; Kay, E W; Hopkins, A M; Gallagher, W M; Dervan, P A

    2007-11-01

    Formalin-fixed paraffin-embedded (FFPE) archival clinical specimens are invaluable in discovery of prognostic and therapeutic targets for diseases such as cancer. However, the suitability of FFPE-derived genetic material for array-based comparative genomic hybridization (array-CGH) studies is underexplored. In this study, genetic profiles of matched FFPE and fresh-frozen specimens were examined to investigate DNA integrity differences between these sample types and determine the impact this may have on genetic profiles. Genomic DNA was extracted from three patient-matched FFPE and fresh-frozen clinical tissue samples. T47D breast cancer control cells were also grown in culture and processed to yield a fresh T47D sample, a fresh-frozen T47D sample and a FFPE T47D sample. DNA was extracted from all the samples; array-CGH conducted and genetic profiles of matched samples were then compared. A loss of high molecular weight DNA was observed in the FFPE clinical tissues and FFPE T47D samples. A dramatic increase in absolute number of genetic alterations was observed in all FFPE tissues relative to matched fresh-frozen counterparts. In future, alternative fixation and tissue-processing procedures, and/or new DNA extraction and CGH profiling protocols, may be implemented, enabling identification of changes involved in disease progression using stored clinical specimens.

  9. The use of laser microdissection in the identification of suitable reference genes for normalization of quantitative real-time PCR in human FFPE epithelial ovarian tissue samples.

    Directory of Open Access Journals (Sweden)

    Jing Cai

    Full Text Available Quantitative real-time PCR (qPCR is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, GAPDH, 18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLP0, RPL13A, and B2M were analyzed in 50 ovarian samples from normal, benign, borderline, and malignant tissues. For reliable results, laser microdissection (LMD, an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA and nonparametric (Kruskal-Wallis tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes. Results showed that epithelial cells occupied a small percentage of the normal ovary indeed. The expression of ACTB, PPIA, RPL13A, RPLP0, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLP0, and TBP and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. Our results highlight the use of homogeneous ovarian tissues and multiple-reference normalization strategy, e.g. the combination of ACTB, PPIA, RPLP0, and TBP, for qPCR in epithelial ovarian tissues, whereas GAPDH, the most commonly used reference gene, is not recommended, especially as a single reference gene.

  10. 肝癌石蜡包埋组织miRNA提取方法的优化%Optimization of MicroRNA extraction method from HCC FFPE tissues

    Institute of Scientific and Technical Information of China (English)

    党裔武; 容敏华; 陈罡

    2012-01-01

    Objective To modify and optimize the microRNA(miRNA) extraction and detection approach from hepatocellular carcinoma(HCC) formalin fixed paraffin embedded(FFPE) tissues. Methods miRNeasy FFPE kit was performed to investigate the optimizational thickness oi sections from HCC FFPE tissues and. time of protea.se K. treatment. Afterwards,expression of miR. 221, miR 146a,let7 a,miR 191,miR 103 and RNU6B was detected by real time RT PCR. The difference of the miRNA expression be tween fresh cells and FFPE tissues from the same cell line was also compared by real time RT PCR. Results 30 μm thickness and 48 h of proteinase K treatment were the best parameters to isolate miRNA. Different expression levels of all the miRNAs could be detected in both the HCC cells samples and clinic FFPE samples. There was no significant variation of the miRNA expression be tween fresh cells and FFPE samples from the same cell lines. Conclusion The optimizational miRNeasy FFPE method is applicable for miRNA extraction from FFPE tissues. The method is productive and can be popularized for the miRNA isolation for different FFPE tissues.%目的 改进并优化从肝癌(HCC)甲醛固定石蜡包埋组织(FFPE)提取并检测微小RNA(miRNA)的方法.方法 使用miRNeasy FFPE试剂盒探讨提取HCC石蜡组织miRNA最佳切片厚度及蛋白酶K作用时间.实时定量RT-PCR检测miR-221、miR-146a、let7-a、miR-191、miR-103及RNU6B的表达量.将HCC细胞制成石蜡组织,对比同一细胞系新鲜细胞及石蜡包埋细胞的各个miRNA表达量差异.结果 切片厚度30 μm,蛋白酶K作用时间48 h时提取miRNA效果最佳.各HCC细胞系及临床HCC石蜡组织均有不同水平的miRNA-221、miR-146a、let7-a、miR-191、miR-103及RNU6B表达.同一细胞系新鲜细胞与石蜡包埋后的各miRNA表达无明显差别.结论 改良后的miRNeasy FFPE 提取方法能提取石蜡组织中的miRNA,可重复性强,可推广用于临床各种石蜡包埋组织的miRNA提取.

  11. Gene expression profiling of RNA extracted from FFPE tissues: NuGEN technologies' whole-transcriptome amplification system.

    Science.gov (United States)

    Turner, Leah; Heath, Joe Don; Kurn, Nurith

    2011-01-01

    Gene expression profiling of RNA isolated from formalin fixed, paraffin-embedded (FFPE) tissue samples has been historically challenging. Yet FFPE samples are sought-after because of the in-depth retrospective records typically associated with them rendering these samples a valuable resource for translational medicine studies. Extensive degradation, chemical modifications, and cross-linking have made it difficult to isolate RNA of sufficient quality required for large-scale gene expression profiling studies. NuGEN Technologies' WT-Ovation™ FFPE System linearly amplifies RNA from FFPE samples through a robust and simple whole-transcriptome approach using as little as 50 ng total RNA isolated from FFPE samples. The amplified material may be labeled with validated kits and/or protocols from NuGEN for analysis on any of the major gene expression microarray platforms, including: Affymetrix, Agilent, and Illumina gene expression arrays. Results compare well with those obtained using RNA from fresh-frozen samples. RNA quality from FFPE samples varies significantly and neither sample age nor sample size analysis via gel electrophoresis or the Agilent Bioanalyzer system accurately predict materials suitable for amplification. Therefore, NuGEN has validated a correlative qPCR-based analytical method for the RNA derived from FFPE samples which effectively predicts array results. The NuGEN approach enables fast and successful analysis of samples previously thought to be too degraded for gene expression analysis.

  12. MALDI mass spectrometry imaging of formalin-fixed paraffin-embedded tissues in clinical research.

    Science.gov (United States)

    Gorzolka, Karin; Walch, Axel

    2014-11-01

    The molecular investigation of archived formalin-fixed, paraffin-embedded (FFPE) tissue samples provides the chance to obtain molecular patterns as indicatives for treatment and clinical end points. MALDI mass spectrometry imaging is capable of localizing molecules like proteins and peptides in tissue sections and became a favorite platform for the targeted and non-targeted approaches, especially in clinical investigations for biomarker research. In FFPE tissues the recovery of proteomic information is constrained by fixation-induced cross-links of proteins. The promising new insights obtained from FFPE in combination with the comprehensive patients' data caused much progress in the optimization of MS imaging protocols to investigate FFPE samples. This review presents the past and current research in MALDI MS imaging of FFPE tissues, demonstrating the improvement of analyses, their actual limitations, but also the promising future perspectives for histopathological and tissue-based research.

  13. Detection of alpha human papillomaviruses in archival formalin-fixed, paraffin-embedded (FFPE) tissue specimens.

    Science.gov (United States)

    Kocjan, Boštjan J; Hošnjak, Lea; Poljak, Mario

    2016-03-01

    Formalin-fixed, paraffin-embedded (FFPE) tissue specimens stored in pathology departments worldwide are an invaluable source for diagnostic purposes when fresh clinical material is unavailable as well as for retrospective molecular and epidemiological studies, especially when dealing with rare clinical conditions for which prospective collection is not feasible. Accurate detection of HPV infection in these specimens is particularly challenging because nucleic acids are often degraded and therefore, not suitable for amplification of larger fragments of the viral genome or viral gene transcripts. This review provides a brief summary of molecular methods for detecting alpha-HPV DNA/RNA in FFPE tissue specimens. We specifically address the key procedural and environmental factors that have the greatest impact on the quality of nucleic acids extracted from FFPE tissue specimens, and describe some solutions that can be used to increase their integrity and/or amplifiability. Moreover, commonly used methods for HPV DNA/RNA detection in FFPE tissue specimens are presented and discussed, focusing on studies using polymerase chain reaction as an HPV detection method and published after 1999. Finally, we briefly summarize our 22 years of experience with HPV detection in FFPE tissue specimens.

  14. Multicentre validation study of nucleic acids extraction from FFPE tissues.

    Science.gov (United States)

    Bonin, Serena; Hlubek, Falk; Benhattar, Jean; Denkert, Carsten; Dietel, Manfred; Fernandez, Pedro L; Höfler, Gerald; Kothmaier, Hannelore; Kruslin, Bozo; Mazzanti, Chiara Maria; Perren, Aurel; Popper, Helmuth; Scarpa, Aldo; Soares, Paula; Stanta, Giorgio; Groenen, Patricia J T A

    2010-09-01

    In most pathology laboratories worldwide, formalin-fixed paraffin embedded (FFPE) samples are the only tissue specimens available for routine diagnostics. Although commercial kits for diagnostic molecular pathology testing are becoming available, most of the current diagnostic tests are laboratory-based assays. Thus, there is a need for standardized procedures in molecular pathology, starting from the extraction of nucleic acids. To evaluate the current methods for extracting nucleic acids from FFPE tissues, 13 European laboratories, participating to the European FP6 program IMPACTS (www.impactsnetwork.eu), isolated nucleic acids from four diagnostic FFPE tissues using their routine methods, followed by quality assessment. The DNA-extraction protocols ranged from homemade protocols to commercial kits. Except for one homemade protocol, the majority gave comparable results in terms of the quality of the extracted DNA measured by the ability to amplify differently sized control gene fragments by PCR. For array-applications or tests that require an accurately determined DNA-input, we recommend using silica based adsorption columns for DNA recovery. For RNA extractions, the best results were obtained using chromatography column based commercial kits, which resulted in the highest quantity and best assayable RNA. Quality testing using RT-PCR gave successful amplification of 200 bp-250 bp PCR products from most tested tissues. Modifications of the proteinase-K digestion time led to better results, even when commercial kits were applied. The results of the study emphasize the need for quality control of the nucleic acid extracts with standardised methods to prevent false negative results and to allow data comparison among different diagnostic laboratories.

  15. Analysis of formalin-fixed, paraffin-embedded (FFPE) tissue via proteomic techniques and misconceptions of antigen retrieval.

    Science.gov (United States)

    O'Rourke, Matthew B; Padula, Matthew P

    2016-01-01

    Since emerging in the late 19(th) century, formaldehyde fixation has become a standard method for preservation of tissues from clinical samples. The advantage of formaldehyde fixation is that fixed tissues can be stored at room temperature for decades without concern for degradation. This has led to the generation of huge tissue banks containing thousands of clinically significant samples. Here we review techniques for proteomic analysis of formalin-fixed, paraffin-embedded (FFPE) tissue samples with a specific focus on the methods used to extract and break formaldehyde crosslinks. We also discuss an error-of-interpretation associated with the technique known as "antigen retrieval." We have discovered that this term has been mistakenly applied to two disparate molecular techniques; therefore, we argue that a terminology change is needed to ensure accurate reporting of experimental results. Finally, we suggest that more investigation is required to fully understand the process of formaldehyde fixation and its subsequent reversal. PMID:27177815

  16. Analytical Validation of AmpliChip p53 Research Test for Archival Human Ovarian FFPE Sections.

    Directory of Open Access Journals (Sweden)

    Matthew J Marton

    Full Text Available The p53 tumor suppressor gene (TP53 is reported to be mutated in nearly half of all tumors and plays a central role in genome integrity. Detection of mutations in p53 can be accomplished by many assays, including the AmpliChip p53 Research Test. The AmpliChip p53 Research Test has been successfully used to determine p53 status in hematologic malignancies and fresh frozen solid tissues but there are few reports of using the assay with formalin fixed, paraffin-embedded (FFPE tissue. The objective of this study was to describe analytical performance characterization of the AmpliChip p53 Research Test to detect p53 mutations in genomic DNA isolated from archival FFPE human ovarian tumor tissues. Method correlation with sequencing showed 96% mutation-wise agreement and 99% chip-wise agreement. We furthermore observed 100% agreement (113/113 of the most prevalent TP53 mutations. Workflow reproducibility was 96.8% across 8 samples, with 2 operators, 2 reagent lots and 2 instruments. Section-to-section reproducibility was 100% for each sample across a 60 μm region of the FFPE block from ovarian tumors. These data indicate that the AmpliChip p53 Research Test is an accurate and reproducible method for detecting mutations in TP53 from archival FFPE human ovarian specimens.

  17. Formalin-Fixed, Paraffin-Embedded Tissues (FFPE) as a Robust Source for the Profiling of Native and Protease-Generated Protein Amino Termini.

    Science.gov (United States)

    Lai, Zon Weng; Weisser, Juliane; Nilse, Lars; Costa, Fabrizio; Keller, Eva; Tholen, Martina; Kizhakkedathu, Jayachandran N; Biniossek, Martin; Bronsert, Peter; Schilling, Oliver

    2016-06-01

    Dysregulated proteolysis represents a hallmark of numerous diseases. In recent years, increasing number of studies has begun looking at the protein termini in hope to unveil the physiological and pathological functions of proteases in clinical research. However, the availability of cryopreserved tissue specimens is often limited. Alternatively, formalin-fixed, paraffin-embedded (FFPE) tissues offer an invaluable resource for clinical research. Pathologically relevant tissues are often stored as FFPE, which represent the most abundant resource of archived human specimens. In this study, we established a robust workflow to investigate native and protease-generated protein N termini from FFPE specimens. We demonstrate comparable N-terminomes of cryopreserved and formalin-fixed tissue, thereby showing that formalin fixation/paraffin embedment does not proteolytically damage proteins. Accordingly, FFPE specimens are fully amenable to N-terminal analysis. Moreover, we demonstrate feasibility of FFPE-degradomics in a quantitative N-terminomic study of FFPE liver specimens from cathepsin L deficient or wild-type mice. Using a machine learning approach in combination with the previously determined cathepsin L specificity, we successfully identify a number of potential cathepsin L cleavage sites. Our study establishes FFPE specimens as a valuable alternative to cryopreserved tissues for degradomic studies. PMID:27087653

  18. Profiling critical cancer gene mutations in clinical tumor samples.

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    Laura E MacConaill

    Full Text Available BACKGROUND: Detection of critical cancer gene mutations in clinical tumor specimens may predict patient outcomes and inform treatment options; however, high-throughput mutation profiling remains underdeveloped as a diagnostic approach. We report the implementation of a genotyping and validation algorithm that enables robust tumor mutation profiling in the clinical setting. METHODOLOGY: We developed and implemented an optimized mutation profiling platform ("OncoMap" to interrogate approximately 400 mutations in 33 known oncogenes and tumor suppressors, many of which are known to predict response or resistance to targeted therapies. The performance of OncoMap was analyzed using DNA derived from both frozen and FFPE clinical material in a diverse set of cancer types. A subsequent in-depth analysis was conducted on histologically and clinically annotated pediatric gliomas. The sensitivity and specificity of OncoMap were 93.8% and 100% in fresh frozen tissue; and 89.3% and 99.4% in FFPE-derived DNA. We detected known mutations at the expected frequencies in common cancers, as well as novel mutations in adult and pediatric cancers that are likely to predict heightened response or resistance to existing or developmental cancer therapies. OncoMap profiles also support a new molecular stratification of pediatric low-grade gliomas based on BRAF mutations that may have immediate clinical impact. CONCLUSIONS: Our results demonstrate the clinical feasibility of high-throughput mutation profiling to query a large panel of "actionable" cancer gene mutations. In the future, this type of approach may be incorporated into both cancer epidemiologic studies and clinical decision making to specify the use of many targeted anticancer agents.

  19. Role of deregulated microRNAs in breast cancer progression using FFPE tissue.

    Directory of Open Access Journals (Sweden)

    Liang Chen

    Full Text Available MicroRNAs (miRNAs contribute to cancer initiation and progression by silencing the expression of their target genes, causing either mRNA molecule degradation or translational inhibition. Intraductal epithelial proliferations of the breast are histologically and clinically classified into normal, atypical ductal hyperplasia (ADH, ductal carcinoma in situ (DCIS and invasive ductal carcinoma (IDC. To better understand the progression of ductal breast cancer development, we attempt to identify deregulated miRNAs in this process using Formalin-Fixed, Paraffin-Embedded (FFPE tissues from breast cancer patients. Following tissue microdissection, we obtained 8 normal, 4 ADH, 6 DCIS and 7 IDC samples, which were subject to RNA isolation and miRNA expression profiling analysis. We found that miR-21, miR-200b/c, miR-141, and miR-183 were consistently up-regulated in ADH, DCIS and IDC compared to normal, while miR-557 was uniquely down-regulated in DCIS. Interestingly, the most significant miRNA deregulations occurred during the transition from normal to ADH. However, the data did not reveal a step-wise miRNA alteration among discrete steps along tumor progression, which is in accordance with previous reports of mRNA profiling of different stages of breast cancer. Furthermore, the expression of MSH2 and SMAD7, two important molecules involving TGF-β pathway, was restored following miR-21 knockdown in both MCF-7 and Hs578T breast cancer cells. In this study, we have not only identified a number of potential candidate miRNAs for breast cancer, but also found that deregulation of miRNA expression during breast tumorigenesis might be an early event since it occurred significantly during normal to ADH transition. Consequently, we have demonstrated the feasibility of miRNA expression profiling analysis using archived FFPE tissues, typically with rich clinical information, as a means of miRNA biomarker discovery.

  20. Post-mortem testing; germline BRCA1/2 variant detection using archival FFPE non-tumor tissue

    DEFF Research Database (Denmark)

    Petersen, Annabeth Høgh; Jørgensen, Mads Malik Aagaard; Nielsen, Henriette Roed;

    2016-01-01

    Accurate estimation of cancer risk in HBOC families often requires BRCA1/2 testing, but this may be impossible in deceased family members. Previous, testing archival formalin-fixed, paraffin-embedded (FFPE) tissue for germline BRCA1/2 variants was unsuccessful, except for the Jewish founder...... mutations. A high-throughput method to systematically test for variants in all coding regions of BRCA1/2 in archival FFPE samples of non-tumor tissue is described, using HaloPlex target enrichment and next-generation sequencing. In a validation study, correct identification of variants or wild......1, six variants known to affect function and one variant likely to affect function in BRCA2, as well as four variants of unknown significance (VUS) in BRCA1 and three VUS in BRCA2 were discovered. It is now possible to test for germline BRCA1/2 variants in deceased persons, using archival FFPE...

  1. Validation of DNA methylation profiling in formalin-fixed paraffin-embedded samples using the Infinium HumanMethylation450 Microarray

    OpenAIRE

    Moran, Sebastián; Vizoso, Miguel; Martinez-Cardús, Anna; Gomez, Antonio; Matías-Guiu, Xavier; Chiavenna, Sebastián M; Fernandez, Andrés G; Esteller, Manel

    2014-01-01

    A formalin-fixed paraffin-embedded (FFPE) sample usually yields highly degraded DNA, which limits the use of techniques requiring high-quality DNA, such as Infinium Methylation microarrays. To overcome this restriction, we have applied an FFPE restoration procedure consisting of DNA repair and ligation processes in a set of paired fresh-frozen (FF) and FFPE samples. We validated the FFPE results in comparison with matched FF samples, enabling us to use FFPE samples on the Infinium HumanMethyl...

  2. Targeted or whole genome sequencing of formalin fixed tissue samples: potential applications in cancer genomics.

    Science.gov (United States)

    Munchel, Sarah; Hoang, Yen; Zhao, Yue; Cottrell, Joseph; Klotzle, Brandy; Godwin, Andrew K; Koestler, Devin; Beyerlein, Peter; Fan, Jian-Bing; Bibikova, Marina; Chien, Jeremy

    2015-09-22

    Current genomic studies are limited by the poor availability of fresh-frozen tissue samples. Although formalin-fixed diagnostic samples are in abundance, they are seldom used in current genomic studies because of the concern of formalin-fixation artifacts. Better characterization of these artifacts will allow the use of archived clinical specimens in translational and clinical research studies. To provide a systematic analysis of formalin-fixation artifacts on Illumina sequencing, we generated 26 DNA sequencing data sets from 13 pairs of matched formalin-fixed paraffin-embedded (FFPE) and fresh-frozen (FF) tissue samples. The results indicate high rate of concordant calls between matched FF/FFPE pairs at reference and variant positions in three commonly used sequencing approaches (whole genome, whole exome, and targeted exon sequencing). Global mismatch rates and C · G > T · A substitutions were comparable between matched FF/FFPE samples, and discordant rates were low (<0.26%) in all samples. Finally, low-pass whole genome sequencing produces similar pattern of copy number alterations between FF/FFPE pairs. The results from our studies suggest the potential use of diagnostic FFPE samples for cancer genomic studies to characterize and catalog variations in cancer genomes. PMID:26305677

  3. Comparison of whole-exome sequencing of matched fresh and formalin fixed paraffin embedded melanoma tumours: implications for clinical decision making.

    Science.gov (United States)

    De Paoli-Iseppi, Ricardo; Johansson, Peter A; Menzies, Alexander M; Dias, Kerith-Rae; Pupo, Gulietta M; Kakavand, Hojabr; Wilmott, James S; Mann, Graham J; Hayward, Nicholas K; Dinger, Marcel E; Long, Georgina V; Scolyer, Richard A

    2016-04-01

    The identification of recurrent driver mutations by whole-exome sequencing (WES) of fresh-frozen human cancers and the subsequent development of novel targeted therapies have recently transformed the treatment of many cancers including melanoma. In routine clinical practice, fresh-frozen tissue is rarely available and mutation testing usually needs to be carried out on archival formalin fixed, paraffin embedded (FFPE) tissue, from which DNA is typically fragmented, cross-linked and of lower quality. In this study we aimed to determine whether WES data generated from genomic DNA (gDNA) extracted from FFPE tissues can be produced reliably and of clinically-actionable standard. In this study of ten melanoma patients, we compared WES data produced from analysis of gDNA isolated from FFPE tumour tissue with that isolated from fresh-frozen tumour tissue from the same specimen. FFPE samples were sequenced using both Illumina's Nextera and NimbleGen SeqCap exome capture kits. To examine mutations between the two tissue sources and platforms, somatic mutations in the FFPE exomes were called using the matched fresh tissue sequence as a reference. Of the 10 FFPE DNA samples, seven Nextera and four SeqCap samples passed library preparation. On average, there were 5341 and 2246 variants lost in FFPE compared to matched fresh tissue utilising Nextera and SeqCap kits, respectively. In order to explore the feasibility of future clinical implementation of WES, FFPE variants in 27 genes of important clinical relevance in melanoma were assessed. The average concordance rate was 43.2% over a total of 1299 calls for the chosen genes in the FFPE DNA. For the current clinically most important melanoma mutations, 0/3 BRAF and 6/8 (75%) NRAS FFPE calls were concordant with the fresh tissue result, which was confirmed using a Sequenom OncoCarta Panel. The poor performance of FFPE WES indicates that specialised library construction to account for low quality DNA and further refinements will

  4. Comparison of whole-exome sequencing of matched fresh and formalin fixed paraffin embedded melanoma tumours: implications for clinical decision making.

    Science.gov (United States)

    De Paoli-Iseppi, Ricardo; Johansson, Peter A; Menzies, Alexander M; Dias, Kerith-Rae; Pupo, Gulietta M; Kakavand, Hojabr; Wilmott, James S; Mann, Graham J; Hayward, Nicholas K; Dinger, Marcel E; Long, Georgina V; Scolyer, Richard A

    2016-04-01

    The identification of recurrent driver mutations by whole-exome sequencing (WES) of fresh-frozen human cancers and the subsequent development of novel targeted therapies have recently transformed the treatment of many cancers including melanoma. In routine clinical practice, fresh-frozen tissue is rarely available and mutation testing usually needs to be carried out on archival formalin fixed, paraffin embedded (FFPE) tissue, from which DNA is typically fragmented, cross-linked and of lower quality. In this study we aimed to determine whether WES data generated from genomic DNA (gDNA) extracted from FFPE tissues can be produced reliably and of clinically-actionable standard. In this study of ten melanoma patients, we compared WES data produced from analysis of gDNA isolated from FFPE tumour tissue with that isolated from fresh-frozen tumour tissue from the same specimen. FFPE samples were sequenced using both Illumina's Nextera and NimbleGen SeqCap exome capture kits. To examine mutations between the two tissue sources and platforms, somatic mutations in the FFPE exomes were called using the matched fresh tissue sequence as a reference. Of the 10 FFPE DNA samples, seven Nextera and four SeqCap samples passed library preparation. On average, there were 5341 and 2246 variants lost in FFPE compared to matched fresh tissue utilising Nextera and SeqCap kits, respectively. In order to explore the feasibility of future clinical implementation of WES, FFPE variants in 27 genes of important clinical relevance in melanoma were assessed. The average concordance rate was 43.2% over a total of 1299 calls for the chosen genes in the FFPE DNA. For the current clinically most important melanoma mutations, 0/3 BRAF and 6/8 (75%) NRAS FFPE calls were concordant with the fresh tissue result, which was confirmed using a Sequenom OncoCarta Panel. The poor performance of FFPE WES indicates that specialised library construction to account for low quality DNA and further refinements will

  5. Assessment of the 2-d gel-based proteomics application of clinically archived formalin-fixed paraffin embedded tissues.

    Science.gov (United States)

    Davalieva, Katarina; Kiprijanovska, Sanja; Polenakovic, Momir

    2014-04-01

    Hospital tissue repositories possess a vast and valuable supply of disease samples with matched retrospective clinical information. Detection and characterization of disease biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues will greatly aid the understanding of the diseases mechanisms and help in the development of diagnostic and prognostic markers. In this study, the possibility of using full-length proteins extracted from clinically archived FFPE tissues in two-dimensional (2-D) gel-based proteomics was evaluated. The evaluation was done based on two types of tumor tissues (breast and prostate) and two extraction protocols. The comparison of the 2-D patterns of FFPE extracts obtained by two extraction protocols with the matching frozen tissue extracts showed that only 7-10% of proteins from frozen tissues can be matched to proteins from FFPE tissues. Most of the spots in the 2-D FFPE's maps had pl 4-6, while the percentages of proteins with pl above 6 were 3-5 times lower in comparison to the fresh/frozen tissue. Despite the three-fold lower number of the detected spots in FFPE maps compared to matched fresh/frozen maps, 67-78% of protein spots in FFPE could not be matched to the corresponding spots in the fresh/frozen tissue maps indicating irreversible protein modifications. In conclusion, the inability to completely reverse the cross-linked complexes and overcome protein fragmentation with the present day FFPE extraction methods stands in the way of effective use of these samples in 2-D gel based proteomics studies.

  6. Multiplexed color-coded probe-based gene expression assessment for clinical molecular diagnostics in formalin-fixed paraffin-embedded human renal allograft tissue.

    Science.gov (United States)

    Adam, Benjamin; Afzali, Bahman; Dominy, Katherine M; Chapman, Erin; Gill, Reeda; Hidalgo, Luis G; Roufosse, Candice; Sis, Banu; Mengel, Michael

    2016-03-01

    Histopathologic diagnoses in transplantation can be improved with molecular testing. Preferably, molecular diagnostics should fit into standard-of-care workflows for transplant biopsies, that is, formalin-fixed paraffin-embedded (FFPE) processing. The NanoString(®) gene expression platform has recently been shown to work with FFPE samples. We aimed to evaluate its methodological robustness and feasibility for gene expression studies in human FFPE renal allograft samples. A literature-derived antibody-mediated rejection (ABMR) 34-gene set, comprised of endothelial, NK cell, and inflammation transcripts, was analyzed in different retrospective biopsy cohorts and showed potential to molecularly discriminate ABMR cases, including FFPE samples. NanoString(®) results were reproducible across a range of RNA input quantities (r = 0.998), with different operators (r = 0.998), and between different reagent lots (r = 0.983). There was moderate correlation between NanoString(®) with FFPE tissue and quantitative reverse transcription polymerase chain reaction (qRT-PCR) with corresponding dedicated fresh-stabilized tissue (r = 0.487). Better overall correlation with histology was observed with NanoString(®) (r = 0.354) than with qRT-PCR (r = 0.146). Our results demonstrate the feasibility of multiplexed gene expression quantification from FFPE renal allograft tissue. This represents a method for prospective and retrospective validation of molecular diagnostics and its adoption in clinical transplantation pathology.

  7. Legionella detection from clinical and environmental samples

    Directory of Open Access Journals (Sweden)

    Maria Luisa Ricci

    2004-12-01

    Full Text Available

    Several methods are used for diagnosis of legionellosis: culture, urinary antigen detection, serologic tests, PCR etc., characterised by different level of specificity, sensitivity and rapidity. Microbiological diagnosis is an essential tool to for the prompt adoption of an adequate antimicrobial therapy and to understand the real diffusion of legionellosis.

    Likewise, the environmental samples analysis allows us to know the distribution of the Legionella in the environment and to detect the origin of the infection during an outbreak by comparing clinical and environmental strains.

  8. Three dimensional imaging of paraffin embedded human lung tissue samples by micro-computed tomography.

    Directory of Open Access Journals (Sweden)

    Anna E Scott

    Full Text Available Understanding the three-dimensional (3-D micro-architecture of lung tissue can provide insights into the pathology of lung disease. Micro computed tomography (µCT has previously been used to elucidate lung 3D histology and morphometry in fixed samples that have been stained with contrast agents or air inflated and dried. However, non-destructive microstructural 3D imaging of formalin-fixed paraffin embedded (FFPE tissues would facilitate retrospective analysis of extensive tissue archives of lung FFPE lung samples with linked clinical data.FFPE human lung tissue samples (n = 4 were scanned using a Nikon metrology µCT scanner. Semi-automatic techniques were used to segment the 3D structure of airways and blood vessels. Airspace size (mean linear intercept, Lm was measured on µCT images and on matched histological sections from the same FFPE samples imaged by light microscopy to validate µCT imaging.The µCT imaging protocol provided contrast between tissue and paraffin in FFPE samples (15 mm x 7 mm. Resolution (voxel size 6.7 µm in the reconstructed images was sufficient for semi-automatic image segmentation of airways and blood vessels as well as quantitative airspace analysis. The scans were also used to scout for regions of interest, enabling time-efficient preparation of conventional histological sections. The Lm measurements from µCT images were not significantly different to those from matched histological sections.We demonstrated how non-destructive imaging of routinely prepared FFPE samples by laboratory µCT can be used to visualize and assess the 3D morphology of the lung including by morphometric analysis.

  9. Regulatory and ethical issues on the utilization of FFPE tissues in research.

    Science.gov (United States)

    With, Catherine M; Evers, David L; Mason, Jeffrey T

    2011-01-01

    Formalin-fixed, paraffin-embedded (FFPE) archival tissues and their associated diagnostic records represent an invaluable source of information on diseases where the patient outcomes are already known. Older archives contain many unique FFPE tissue specimens that would be impossible to replicate today due to changes in medical practice and technology. Unfortunately, there is no single regulatory or bioethical standard that covers research with FFPE tissue specimens. This makes it difficult for researchers to prepare protocols involving FFPE tissues and equally difficult for Institutional Review Boards to evaluate them. In this review, focused on US regulatory policy, the application of the Common Rule and the Privacy Rule of the Health Insurance Portability and Accountability Act to research involving FFPE tissue specimens will be discussed. It will be shown that the difficulty in applying regulatory and ethical standards to FFPE tissues results not from the tissues themselves, but from the personally identifiable health information associated with the tissue specimens.

  10. Quantification of covalently closed circular DNA of hepatitis B virus in FFPE liver tissues of chronic hepatitis B patients

    Directory of Open Access Journals (Sweden)

    Jia-qi HAN

    2011-05-01

    Full Text Available Objective To establish a method of detecting HBV covalently closed circular DNA(cccDNA in micro-formalin fixed paraffin imbedding(FFPE liver biopsy samples.Methods FFPE liver biopsies from 37 patients with chronic hepatitis B were studied.The intrahepatic HBV DNA was extracted and pre-treated with plasmid-safe ATP-dependent DNAse(PSAD,and then amplified by rolling circular amplification(RCA.The HBV cccDNA was quantitatively detected by Taqman real-time PCR with primers located on both sides of the gap of HBV DNA.The human β-actin gene served as the internal control.The sensitivity was tested by serially diluting the DNA templates with known concentrations.The repeatability and stability were evaluated with inter-assay and intra-assay.The level of intrahepatic HBV cccDNA,HBV total DNA,serum HBV DNA and ALT were also compared to find the relations between them.Results The quantitative detection method of cccDNA in micro-FFPE liver samples was successfully set up with considerable sensitivity,stability and specificity.The intrahepatic cccDNA level was significantly higher in the HBeAg-positive patients than in the HBeAg-negative patients(P < 0.05.The intrahepatic HBV cccDNA level was positively correlated with the serum and intra-hepatic HBV DNA level(r=0.539,P=0.001.Conclusion The assay established by present study is fit for the detection of HBV cccDNA in micro-FFPE liver biopsies.

  11. Whole-genome gene expression profiling of formalin-fixed, paraffin-embedded tissue samples.

    Directory of Open Access Journals (Sweden)

    Craig April

    Full Text Available BACKGROUND: We have developed a gene expression assay (Whole-Genome DASL, capable of generating whole-genome gene expression profiles from degraded samples such as formalin-fixed, paraffin-embedded (FFPE specimens. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated a similar level of sensitivity in gene detection between matched fresh-frozen (FF and FFPE samples, with the number and overlap of probes detected in the FFPE samples being approximately 88% and 95% of that in the corresponding FF samples, respectively; 74% of the differentially expressed probes overlapped between the FF and FFPE pairs. The WG-DASL assay is also able to detect 1.3-1.5 and 1.5-2 -fold changes in intact and FFPE samples, respectively. The dynamic range for the assay is approximately 3 logs. Comparing the WG-DASL assay with an in vitro transcription-based labeling method yielded fold-change correlations of R(2 approximately 0.83, while fold-change comparisons with quantitative RT-PCR assays yielded R(2 approximately 0.86 and R(2 approximately 0.55 for intact and FFPE samples, respectively. Additionally, the WG-DASL assay yielded high self-correlations (R(2>0.98 with low intact RNA inputs ranging from 1 ng to 100 ng; reproducible expression profiles were also obtained with 250 pg total RNA (R(2 approximately 0.92, with approximately 71% of the probes detected in 100 ng total RNA also detected at the 250 pg level. When FFPE samples were assayed, 1 ng total RNA yielded self-correlations of R(2 approximately 0.80, while still maintaining a correlation of R(2 approximately 0.75 with standard FFPE inputs (200 ng. CONCLUSIONS/SIGNIFICANCE: Taken together, these results show that WG-DASL assay provides a reliable platform for genome-wide expression profiling in archived materials. It also possesses utility within clinical settings where only limited quantities of samples may be available (e.g. microdissected material or when minimally invasive procedures are performed (e

  12. Clinical relevance of DNA microarray analyses using archival formalin-fixed paraffin-embedded breast cancer specimens

    International Nuclear Information System (INIS)

    The ability of gene profiling to predict treatment response and prognosis in breast cancers has been demonstrated in many studies using DNA microarray analyses on RNA from fresh frozen tumor specimens. In certain clinical and research situations, performing such analyses on archival formalin fixed paraffin-embedded (FFPE) surgical specimens would be advantageous as large libraries of such specimens with long-term follow-up data are widely available. However, FFPE tissue processing can cause fragmentation and chemical modifications of the RNA. A number of recent technical advances have been reported to overcome these issues. Our current study evaluates whether or not the technology is ready for clinical applications. A modified RNA extraction method and a recent DNA microarray technique, cDNA-mediated annealing, selection, extension and ligation (DASL, Illumina Inc) were evaluated. The gene profiles generated from FFPE specimens were compared to those obtained from paired fresh fine needle aspiration biopsies (FNAB) of 25 breast cancers of different clinical subtypes (based on ER and Her2/neu status). Selected RNA levels were validated using RT-qPCR, and two public databases were used to demonstrate the prognostic significance of the gene profiles generated from FFPE specimens. Compared to FNAB, RNA isolated from FFPE samples was relatively more degraded, nonetheless, over 80% of the RNA samples were deemed suitable for subsequent DASL assay. Despite a higher noise level, a set of genes from FFPE specimens correlated very well with the gene profiles obtained from FNAB, and could differentiate breast cancer subtypes. Expression levels of these genes were validated using RT-qPCR. Finally, for the first time we correlated gene expression profiles from FFPE samples to survival using two independent microarray databases. Specifically, over-expression of ANLN and KIF2C, and under-expression of MAPT strongly correlated with poor outcomes in breast cancer patients. We

  13. Clinical relevance of DNA microarray analyses using archival formalin-fixed paraffin-embedded breast cancer specimens

    Directory of Open Access Journals (Sweden)

    Done Susan J

    2011-06-01

    Full Text Available Abstract Background The ability of gene profiling to predict treatment response and prognosis in breast cancers has been demonstrated in many studies using DNA microarray analyses on RNA from fresh frozen tumor specimens. In certain clinical and research situations, performing such analyses on archival formalin fixed paraffin-embedded (FFPE surgical specimens would be advantageous as large libraries of such specimens with long-term follow-up data are widely available. However, FFPE tissue processing can cause fragmentation and chemical modifications of the RNA. A number of recent technical advances have been reported to overcome these issues. Our current study evaluates whether or not the technology is ready for clinical applications. Methods A modified RNA extraction method and a recent DNA microarray technique, cDNA-mediated annealing, selection, extension and ligation (DASL, Illumina Inc were evaluated. The gene profiles generated from FFPE specimens were compared to those obtained from paired fresh fine needle aspiration biopsies (FNAB of 25 breast cancers of different clinical subtypes (based on ER and Her2/neu status. Selected RNA levels were validated using RT-qPCR, and two public databases were used to demonstrate the prognostic significance of the gene profiles generated from FFPE specimens. Results Compared to FNAB, RNA isolated from FFPE samples was relatively more degraded, nonetheless, over 80% of the RNA samples were deemed suitable for subsequent DASL assay. Despite a higher noise level, a set of genes from FFPE specimens correlated very well with the gene profiles obtained from FNAB, and could differentiate breast cancer subtypes. Expression levels of these genes were validated using RT-qPCR. Finally, for the first time we correlated gene expression profiles from FFPE samples to survival using two independent microarray databases. Specifically, over-expression of ANLN and KIF2C, and under-expression of MAPT strongly correlated

  14. Oligonucleotide array outperforms SNP array on formalin-fixed paraffin-embedded clinical samples.

    Science.gov (United States)

    Nasri, Soroush; Anjomshoaa, Ahmad; Song, Sarah; Guilford, Parry; McNoe, Les; Black, Michael; Phillips, Vicky; Reeve, Anthony; Humar, Bostjan

    2010-04-01

    Compromised quality of formalin-fixed paraffin-embedded (FFPE)-derived DNA has compounded the use of archival specimens for array-based genomic studies. Recent technological advances have led to first successes in this field; however, there is currently no general agreement on the most suitable platform for the array-based analysis of FFPE DNA. In this study, FFPE and matched fresh-frozen (FF) specimens were separately analyzed with Affymetrix single nucleotide polymorphism (SNP) 6.0 and Agilent 4x44K oligonucleotide arrays to compare the genomic profiles from the two tissue sources and to assess the relative performance of the two platforms on FFPE material. Genomic DNA was extracted from matched FFPE-FF pairs of normal intestinal epithelium from four patients and were applied to the SNP and oligonucleotide platforms according to the manufacturer-recommended protocols. On the Affymetrix platform, a substantial increase in apparent copy number alterations was observed in all FFPE tissues relative to their matched FF counterparts. In contrast, FFPE and matched FF genomic profiles obtained via the Agilent platform were very similar. Both the SNP and the oligonucleotide platform performed comparably on FF material. This study demonstrates that Agilent oligonucleotide array comparative genomic hybridization generates reliable results from FFPE extracted DNA, whereas the Affymetrix SNP-based array seems less suitable for the analysis of FFPE material.

  15. Sample size determination in clinical trials with multiple endpoints

    CERN Document Server

    Sozu, Takashi; Hamasaki, Toshimitsu; Evans, Scott R

    2015-01-01

    This book integrates recent methodological developments for calculating the sample size and power in trials with more than one endpoint considered as multiple primary or co-primary, offering an important reference work for statisticians working in this area. The determination of sample size and the evaluation of power are fundamental and critical elements in the design of clinical trials. If the sample size is too small, important effects may go unnoticed; if the sample size is too large, it represents a waste of resources and unethically puts more participants at risk than necessary. Recently many clinical trials have been designed with more than one endpoint considered as multiple primary or co-primary, creating a need for new approaches to the design and analysis of these clinical trials. The book focuses on the evaluation of power and sample size determination when comparing the effects of two interventions in superiority clinical trials with multiple endpoints. Methods for sample size calculation in clin...

  16. Next-Generation Sequencing Workflow for NSCLC Critical Samples Using a Targeted Sequencing Approach by Ion Torrent PGM™ Platform

    Directory of Open Access Journals (Sweden)

    Irene Vanni

    2015-12-01

    Full Text Available Next-generation sequencing (NGS is a cost-effective technology capable of screening several genes simultaneously; however, its application in a clinical context requires an established workflow to acquire reliable sequencing results. Here, we report an optimized NGS workflow analyzing 22 lung cancer-related genes to sequence critical samples such as DNA from formalin-fixed paraffin-embedded (FFPE blocks and circulating free DNA (cfDNA. Snap frozen and matched FFPE gDNA from 12 non-small cell lung cancer (NSCLC patients, whose gDNA fragmentation status was previously evaluated using a multiplex PCR-based quality control, were successfully sequenced with Ion Torrent PGM™. The robust bioinformatic pipeline allowed us to correctly call both Single Nucleotide Variants (SNVs and indels with a detection limit of 5%, achieving 100% specificity and 96% sensitivity. This workflow was also validated in 13 FFPE NSCLC biopsies. Furthermore, a specific protocol for low input gDNA capable of producing good sequencing data with high coverage, high uniformity, and a low error rate was also optimized. In conclusion, we demonstrate the feasibility of obtaining gDNA from FFPE samples suitable for NGS by performing appropriate quality controls. The optimized workflow, capable of screening low input gDNA, highlights NGS as a potential tool in the detection, disease monitoring, and treatment of NSCLC.

  17. Quality analysis and clinical application of RNA extracted from formalin-fixed paraffin-embedded samples%福尔马林固定石蜡包埋样本提取的RNA质量分析及其临床应用

    Institute of Scientific and Technical Information of China (English)

    薛洋; 曾富春; 舒骏; 丛伟

    2015-01-01

    Objective To evaluate the quality and clinical application of RNA extracted from formalin-fixed paraffin-embed-ded samples. Methods A total of 994 FFPE samples were collected and RNA was extracted. The quality of RNA was evaluated and the RNA was used for detection of the gene mutation status of EML4-ALK and ROS1 gene. Sanger sequencing was used for assessing the accuracy of gene mutation detection by using RNA samples. Results The A260/A280 and A260/A230 of RNA from 994 cas-es of FFPE samples were not less than 1.8, the average concentration of RNA was 204.23ng/μl and all of 992 RNA samples could be effectively amplified. The results of EML4-ALK and ROS1 gene fusion detection were consistent with that of Sanger sequenc-ing. Conclusions The RNA extracted from FFPE samples is of high quality and suitable for subsequent gene mutation detection by qRT-PCR, which can provide scientific and reliable evidence for selection of tumor targeting drugs.%目的:评估国内福尔马林固定石蜡包埋(formalin-fixed paraffin-embedded,FFPE)样本抽提的RNA质量及其临床应用。方法收集FFPE样本994例,采用RNA分离纯化试剂盒提取纯化RNA,检测提取RNA的质量,并检测RNA样本中的EML4-ALK和ROS1基因突变状态,同时采用Sanger测序法验证使用RNA样本进行基因突变检测的准确性。结果从994例FFPE样本中分离提取的RNA的A260/A280和A260/A230均能达到1.8以上,平均浓度为204.23ng/μl,992例样本均能进行有效扩增。 RNA样本的EML4-ALK和ROS1基因融合检测结果与Sanger测序结果一致。结论从FFPE样本中分离得到的RNA质量良好,适用于后续的基因突变检测,可为肿瘤靶向药物的选择提供科学、可靠的依据。

  18. Bacteriophages in clinical samples can interfere with microbiological diagnostic tools.

    Science.gov (United States)

    Brown-Jaque, Maryury; Muniesa, Maite; Navarro, Ferran

    2016-09-09

    Bacteriophages are viruses that infect bacteria, and they are found everywhere their bacterial hosts are present, including the human body. To explore the presence of phages in clinical samples, we assessed 65 clinical samples (blood, ascitic fluid, urine, cerebrospinal fluid, and serum). Infectious tailed phages were detected in >45% of ascitic fluid and urine samples. Three examples of phage interference with bacterial isolation were observed. Phages prevented the confluent bacterial growth required for an antibiogram assay when the inoculum was taken from an agar plate containing lysis plaques, but not when taken from a single colony in a phage-free area. In addition, bacteria were isolated directly from ascitic fluid, but not after liquid enrichment culture of the same samples, since phage propagation lysed the bacteria. Lastly, Gram-negative bacilli observed in a urine sample did not grow on agar plates due to the high densities of infectious phages in the sample.

  19. Bacteriophages in clinical samples can interfere with microbiological diagnostic tools

    Science.gov (United States)

    Brown-Jaque, Maryury; Muniesa, Maite; Navarro, Ferran

    2016-01-01

    Bacteriophages are viruses that infect bacteria, and they are found everywhere their bacterial hosts are present, including the human body. To explore the presence of phages in clinical samples, we assessed 65 clinical samples (blood, ascitic fluid, urine, cerebrospinal fluid, and serum). Infectious tailed phages were detected in >45% of ascitic fluid and urine samples. Three examples of phage interference with bacterial isolation were observed. Phages prevented the confluent bacterial growth required for an antibiogram assay when the inoculum was taken from an agar plate containing lysis plaques, but not when taken from a single colony in a phage-free area. In addition, bacteria were isolated directly from ascitic fluid, but not after liquid enrichment culture of the same samples, since phage propagation lysed the bacteria. Lastly, Gram-negative bacilli observed in a urine sample did not grow on agar plates due to the high densities of infectious phages in the sample. PMID:27609086

  20. Improved reproducibility in genome-wide DNA methylation analysis for PAXgene-fixed samples compared with restored formalin-fixed and paraffin-embedded DNA.

    Science.gov (United States)

    Andersen, Gitte Brinch; Hager, Henrik; Hansen, Lise Lotte; Tost, Jörg

    2015-01-01

    Formalin fixation has been the standard method for conservation of clinical specimens for decades. However, a major drawback is the high degradation of nucleic acids, which complicates its use in genome-wide analyses. Unbiased identification of biomarkers, however, requires genome-wide studies, precluding the use of the valuable archives of specimens with long-term follow-up data. Therefore, restoration protocols for DNA from formalin-fixed and paraffin-embedded (FFPE) samples have been developed, although they are cost-intensive and time-consuming. An alternative to FFPE and snap-freezing is the PAXgene Tissue System, developed for simultaneous preservation of morphology, proteins, and nucleic acids. In the current study, we compared the performance of DNA from either PAXgene or formalin-fixed tissues to snap-frozen material for genome-wide DNA methylation analysis using the Illumina 450K BeadChip. Quantitative DNA methylation analysis demonstrated that the methylation profile in PAXgene-fixed tissues showed, in comparison with restored FFPE samples, a higher concordance with the profile detected in frozen samples. We demonstrate, for the first time, that DNA from PAXgene conserved tissue performs better compared with restored FFPE DNA in genome-wide DNA methylation analysis. In addition, DNA from PAXgene tissue can be directly used on the array without prior restoration, rendering the analytical process significantly more time- and cost-effective.

  1. miRNA expression profiling of formalin-fixed paraffin-embedded (FFPE hereditary breast tumors

    Directory of Open Access Journals (Sweden)

    Miljana Tanić

    2015-03-01

    Full Text Available Hereditary breast cancer constitutes only 5–10% of all breast cancer cases and is characterized by strong family history of breast and/or other associated cancer types. Only ~25% of hereditary breast cancer cases carry a mutation in BRCA1 or BRCA2 gene, while mutations in other rare high and moderate-risk genes and common low penetrance variants may account for additional 20% of the cases. Thus the majority of cases are still unaccounted for and designated as BRCAX tumors. MicroRNAs are small non-coding RNAs that play important roles as regulators of gene expression and are deregulated in cancer. To characterize hereditary breast tumors based on their miRNA expression profiles we performed global microarray miRNA expression profiling on a retrospective cohort of 80 FFPE breast tissues, including 66 hereditary breast tumors (13 BRCA1, 10 BRCA2 and 43 BRCAX, 10 sporadic breast carcinomas and 4 normal breast tissues, using Exiqon miRCURY LNA™ microRNA Array v.11.0. Here we describe in detail the miRNA microarray expression data and tumor samples used for the study of BRCAX tumor heterogeneity (Tanic et al., 2013 and biomarkers associated with positive BRCA1/2 mutation status (Tanic et al., 2014. Additionally, we provide the R code for data preprocessing and quality control.

  2. Comparison of Accuracy of Whole-Exome Sequencing with Formalin-Fixed Paraffin-Embedded and Fresh Frozen Tissue Samples

    OpenAIRE

    Ensel Oh; Yoon-La Choi; Mi Jeong Kwon; Ryong Nam Kim; Yu Jin Kim; Ji-Young Song; Kyung Soo Jung; Young Kee Shin

    2015-01-01

    Formalin fixing with paraffin embedding (FFPE) has been a standard sample preparation method for decades, and archival FFPE samples are still very useful resources. Nonetheless, the use of FFPE samples in cancer genome analysis using next-generation sequencing, which is a powerful technique for the identification of genomic alterations at the nucleotide level, has been challenging due to poor DNA quality and artificial sequence alterations. In this study, we performed whole-exome sequencing o...

  3. Yeasts isolated from clinical samples of AIDS patients

    Directory of Open Access Journals (Sweden)

    Neves Rejane Pereira

    2002-01-01

    Full Text Available In order to investigate yeasts in oropharyngeal secretion, urine, sputum and inguinal scales from AIDS patients, clinical samples were collected from one hundred patients interned in the Infectious and Parasitic Diseases Sector of the Hospital das Clínicas of the Universidade Federal de Pernambuco and in Hospital Universitário Osvaldo Cruz of the Universidade de Pernambuco. Yeasts were isolated from seventy-two out of one hundred and eight clinical samples. The isolated yeasts were: Candida albicans (sixty-two isolates, Candida tropicalis (four isolates, Candida glabrata (two isolates, Candida parapsilosis (two isolates, Candida krusei (one isolate and Trichosporon pullulans (one isolate.

  4. On an Approach to Bayesian Sample Sizing in Clinical Trials

    CERN Document Server

    Muirhead, Robb J

    2012-01-01

    This paper explores an approach to Bayesian sample size determination in clinical trials. The approach falls into the category of what is often called "proper Bayesian", in that it does not mix frequentist concepts with Bayesian ones. A criterion for a "successful trial" is defined in terms of a posterior probability, its probability is assessed using the marginal distribution of the data, and this probability forms the basis for choosing sample sizes. We illustrate with a standard problem in clinical trials, that of establishing superiority of a new drug over a control.

  5. Use of formalin-fixed and paraffin-embedded tissues for diagnosis and therapy in routine clinical settings.

    Science.gov (United States)

    Berg, Daniela; Malinowsky, Katharina; Reischauer, Bilge; Wolff, Claudia; Becker, Karl-Friedrich

    2011-01-01

    Formalin-fixed and paraffin-embedded (FFPE) tissues are used routinely everyday in hospitals world-wide for histopathological diagnosis of diseases like cancer. Due to formalin-induced cross-linking of proteins, FFPE tissues present a particular challenge for proteomic analysis. Nevertheless, there has been recent progress for extraction-based protein analysis in these tissues. Novel tools developed in the last few years are urgently needed because precise protein biomarker quantification in clinical FFPE tissues will be crucial for treatment decisions and to assess success or failure of current and future personalized molecular therapies. Furthermore, they will help to conceive why only a subset of patients responds to individualized treatments. Reverse phase protein array (RPPA) is a very promising new technology for quick and simultaneous analysis of many patient samples allowing relative and absolute protein quantifications. In this chapter, we show how protein extraction from FFPE tissues might facilitate the implementation of RPPA for therapy decisions and discuss challenges for application of RPPA in clinical trials and routine settings.

  6. Combining electrochemical sensors with miniaturized sample preparation for rapid detection in clinical samples.

    Science.gov (United States)

    Bunyakul, Natinan; Baeumner, Antje J

    2015-01-01

    Clinical analyses benefit world-wide from rapid and reliable diagnostics tests. New tests are sought with greatest demand not only for new analytes, but also to reduce costs, complexity and lengthy analysis times of current techniques. Among the myriad of possibilities available today to develop new test systems, amperometric biosensors are prominent players-best represented by the ubiquitous amperometric-based glucose sensors. Electrochemical approaches in general require little and often enough only simple hardware components, are rugged and yet provide low limits of detection. They thus offer many of the desirable attributes for point-of-care/point-of-need tests. This review focuses on investigating the important integration of sample preparation with (primarily electrochemical) biosensors. Sample clean up requirements, miniaturized sample preparation strategies, and their potential integration with sensors will be discussed, focusing on clinical sample analyses. PMID:25558994

  7. Learning Adaptive Forecasting Models from Irregularly Sampled Multivariate Clinical Data

    Science.gov (United States)

    Liu, Zitao; Hauskrecht, Milos

    2016-01-01

    Building accurate predictive models of clinical multivariate time series is crucial for understanding of the patient condition, the dynamics of a disease, and clinical decision making. A challenging aspect of this process is that the model should be flexible and adaptive to reflect well patient-specific temporal behaviors and this also in the case when the available patient-specific data are sparse and short span. To address this problem we propose and develop an adaptive two-stage forecasting approach for modeling multivariate, irregularly sampled clinical time series of varying lengths. The proposed model (1) learns the population trend from a collection of time series for past patients; (2) captures individual-specific short-term multivariate variability; and (3) adapts by automatically adjusting its predictions based on new observations. The proposed forecasting model is evaluated on a real-world clinical time series dataset. The results demonstrate the benefits of our approach on the prediction tasks for multivariate, irregularly sampled clinical time series, and show that it can outperform both the population based and patient-specific time series prediction models in terms of prediction accuracy.

  8. Comparison of Accuracy of Whole-Exome Sequencing with Formalin-Fixed Paraffin-Embedded and Fresh Frozen Tissue Samples.

    Directory of Open Access Journals (Sweden)

    Ensel Oh

    Full Text Available Formalin fixing with paraffin embedding (FFPE has been a standard sample preparation method for decades, and archival FFPE samples are still very useful resources. Nonetheless, the use of FFPE samples in cancer genome analysis using next-generation sequencing, which is a powerful technique for the identification of genomic alterations at the nucleotide level, has been challenging due to poor DNA quality and artificial sequence alterations. In this study, we performed whole-exome sequencing of matched frozen samples and FFPE samples of tissues from 4 cancer patients and compared the next-generation sequencing data obtained from these samples. The major differences between data obtained from the 2 types of sample were the shorter insert size and artificial base alterations in the FFPE samples. A high proportion of short inserts in the FFPE samples resulted in overlapping paired reads, which could lead to overestimation of certain variants; >20% of the inserts in the FFPE samples were double sequenced. A large number of soft clipped reads was found in the sequencing data of the FFPE samples, and about 30% of total bases were soft clipped. The artificial base alterations, C>T and G>A, were observed in FFPE samples only, and the alteration rate ranged from 200 to 1,200 per 1M bases when sequencing errors were removed. Although high-confidence mutation calls in the FFPE samples were compatible to that in the frozen samples, caution should be exercised in terms of the artifacts, especially for low-confidence calls. Despite the clearly observed artifacts, archival FFPE samples can be a good resource for discovery or validation of biomarkers in cancer research based on whole-exome sequencing.

  9. Antibiotic Susceptibilities of Acinetobacter Baumanii Strains Isolated from Clinical Samples

    OpenAIRE

    Harun Aðca

    2013-01-01

         Aim :  In this study it was aimed to investigate the antibiotic susceptibilities of Acinetobacter baumanii strains isolated from various clinical samples sent to Tavsanli State Hospital Microbiology Laboratory retrospectively. Material and Method: All of the cultures were examined for the agent and antibiotic susceptibilities. For the identification of bacteria, various chemical tests and BBL Crystal E/NF (Beckton Dickinson, ABD) system was used. Antibiotic susce...

  10. Vancomycin Resistance Pattern of Staphylococcus Aureus among Clinical Samples

    OpenAIRE

    S Saadat; K Solhjoo; A. Kazemi; Erfanian, S. (MSc); Ashrafian, F. (MSc)

    2014-01-01

    Background and Objective: Vancomycin is used for treatment of methicillin-resistant S. Aureus (MRSA) infections; therefore, resistance to this antibiotic is increasing. We aimed to determine the antibiotic resistance pattern and frequency of vancomycin resistant S. Areas (VRSA) strains isolated from clinical samples. Material and Methods: In this cross-sectional study, 100 S. Aureus isolates collected from hospitals in Shiraz during six months, 2012, were identified by biochemical, microbiolo...

  11. PCR Detection of Helicobacter pylori in Clinical Samples

    OpenAIRE

    Rimbara, Emiko; Sasatsu, Masanori; David Y Graham

    2013-01-01

    Helicobacter pylori is an important pathogen whose primary niche is the human stomach. H. pylori is etio-logically associated with gastric inflammation (gastritis), peptic ulcer disease, and gastric cancer. Both noninvasive (e.g., urea breath and stool antigen tests) and invasive (gastric biopsy for histology, culture, or PCR) tests are used for diagnosis. PCR detection of H. pylori has been reported using a variety of clinical samples including gastric biopsy, gastric juice, saliva, dental p...

  12. PCR detection of Helicobacter pylori in clinical samples.

    Science.gov (United States)

    Rimbara, Emiko; Sasatsu, Masanori; Graham, David Y

    2013-01-01

    Helicobacter pylori is an important pathogen whose primary niche is the human stomach. H. pylori is etiologically associated with gastric inflammation (gastritis), peptic ulcer disease, and gastric cancer. Both noninvasive (e.g., urea breath and stool antigen tests) and invasive (gastric biopsy for histology, culture, or PCR) tests are used for diagnosis. PCR detection of H. pylori has been reported using a variety of clinical samples including gastric biopsy, gastric juice, saliva, dental plaque, and stools as well as environmental samples. Whenever possibly, noninvasive tests are preferred over invasive tests. H. pylori are excreted in the stool. Culture from stool is variable whereas stool antigen testing is widely used. Stool consists of a complicated mixture of commensal bacteria and chemicals and often includes inhibitors of PCR. Nevertheless, simple extraction methods are available to efficiently extract DNA from human stools and nested-PCR targeting the 23S rRNA gene have proven to be highly sensitive for the detection of H. pylori. Detection of clarithromycin susceptibility/resistance is important clinically and the mutation of the 23S rRNA gene responsible for resistance can also be detected using stool. This described method can be modified for other clinical samples such as gastric juice or biopsy material. PMID:23104297

  13. Evaluation of different real time PCRs for the detection of Pneumocystis jirovecii DNA in formalin-fixed paraffin-embedded bronchoalveolar lavage samples.

    Science.gov (United States)

    de Leeuw, Bertie H C G M; Voskuil, W Sebastiaan; Maraha, Boulos; van der Zee, Anneke; Westenend, Pieter J; Kusters, Johannes G

    2015-06-01

    The presence of Pneumocystis jirovecii in fresh clinical materials can be detected by PCR with high sensitivity and is thus preferred over microscopic methods. However, fresh materials are not always available, and on formalin-fixed paraffin-embedded materials, PCR may result in reduced detection rates. In this study the diagnostic sensitivity of P. jirovecii real time PCR on DNA isolated from fresh bronchoalveolar lavage (BAL) samples versus that from matched FFPE derived DNA is analyzed. Our results indicate that when targeting a small DNA fragment P. jirovecii PCR can be performed on FFPE BAL samples with acceptable sensitivity (up to 83.3%). This is considerably higher than the 33.3% positives observed by classical staining of these samples.

  14. Cladosporium Species Recovered from Clinical Samples in the United States.

    Science.gov (United States)

    Sandoval-Denis, Marcelo; Sutton, Deanna A; Martin-Vicente, Adela; Cano-Lira, José F; Wiederhold, Nathan; Guarro, Josep; Gené, Josepa

    2015-09-01

    Cladosporium species are ubiquitous, saprobic, dematiaceous fungi, only infrequently associated with human and animal opportunistic infections. We have studied a large set of Cladosporium isolates recovered from clinical samples in the United States to ascertain the predominant species there in light of recent taxonomic changes in this genus and to determine whether some could possibly be rare potential pathogens. A total of 92 isolates were identified using phenotypic and molecular methods, which included sequence analysis of the internal transcribed spacer (ITS) region and a fragment of the large subunit (LSU) of the nuclear ribosomal DNA (rDNA), as well as fragments of the translation elongation factor 1 alpha (EF-1α) and actin (Act) genes. The most frequent species was Cladosporium halotolerans (14.8%), followed by C. tenuissimum (10.2%), C. subuliforme (5.7%), and C. pseudocladosporioides (4.5%). However, 39.8% of the isolates did not correspond to any known species and were deemed to comprise at least 17 new lineages for Cladosporium. The most frequent anatomic site of isolation was the respiratory tract (54.5%), followed by superficial (28.4%) and deep tissues and fluids (14.7%). Species of the two recently described cladosporiumlike genera Toxicocladosporium and Penidiella are reported for the first time from clinical samples. In vitro susceptibility testing of 92 isolates against nine antifungal drugs showed a variety of results but high activity overall for the azoles, echinocandins, and terbinafine.

  15. TOP1 gene copy number and TOP1/CEN-20 ratio in stage III colorectal cancer samples

    DEFF Research Database (Denmark)

    Rømer, Maria Unni Koefoed; Nygård, Sune Boris; Christensen, Ib Jarle;

    fixed paraffin embedded (FFPE) primary tumor tissue has been suggested as a predictive biomarker of the effect of irinotecan in the treatment of metastatic CRC. Quantification of TOP1 protein levels in FFPE tissue may be difficult and calls for alternative methods.We have recently reported on TOP1 FISH...... analyses on 50 FFPE primary CRC tissues. When compared with results from normal colorectal mucosa, 80 % of the tumors showed increased TOP1 gene copy number and 2/3 had increased TOP1/CEN-20 ratio. MATERIALS AND METHODS FFPE samples from 154 stage III CRC patients not receiving adjuvant chemotherapy were...

  16. Social Cognition in a Clinical Sample of Personality Disorder Patients

    Directory of Open Access Journals (Sweden)

    Amparo eRuiz-Tagle

    2015-05-01

    Full Text Available Social cognition was assessed in a clinical sample of Personality Disorder (PD stable patients receiving ambulatory treatment (N=17 and healthy matched controls (N=17 using tests of recognition of emotions in faces and eyes, in a test of social faux pas and in theory of mind stories. Results indicated that when compared with healthy controls, individuals with PD showed a clear tendency to obtain lower scoring in tasks assessing recognition of emotion in faces (T=-2,602, p=0,014, eyes (T=-3,593, p=0,001, TOM stories (T=-4,706, p=0,000 and Faux pas (T=-2,227, p=0,035. In the present pilot study, PD individuals with a normal cognitive efficiency showed an impaired performance at social cognition assessment including emotion recognition and theory of mind.

  17. Non-invasive prenatal chromosomal aneuploidy testing--clinical experience: 100,000 clinical samples.

    Directory of Open Access Journals (Sweden)

    Ron M McCullough

    Full Text Available OBJECTIVE: As the first laboratory to offer massively parallel sequencing-based noninvasive prenatal testing (NIPT for fetal aneuploidies, Sequenom Laboratories has been able to collect the largest clinical population experience data to date, including >100,000 clinical samples from all 50 U.S. states and 13 other countries. The objective of this study is to give a robust clinical picture of the current laboratory performance of the MaterniT21 PLUS LDT. STUDY DESIGN: The study includes plasma samples collected from patients with high-risk pregnancies in our CLIA-licensed, CAP-accredited laboratory between August 2012 to June 2013. Samples were assessed for trisomies 13, 18, 21 and for the presence of chromosome Y-specific DNA. Sample data and ad hoc outcome information provided by the clinician was compiled and reviewed to determine the characteristics of this patient population, as well as estimate the assay performance in a clinical setting. RESULTS: NIPT patients most commonly undergo testing at an average of 15 weeks, 3 days gestation; and average 35.1 years of age. The average turnaround time is 4.54 business days and an overall 1.3% not reportable rate. The positivity rate for Trisomy 21 was 1.51%, followed by 0.45% and 0.21% rate for Trisomies 18 and 13, respectively. NIPT positivity rates are similar to previous large clinical studies of aneuploidy in women of maternal age ≥ 35 undergoing amniocentesis. In this population 3519 patients had multifetal gestations (3.5% with 2.61% yielding a positive NIPT result. CONCLUSION: NIPT has been commercially offered for just over 2 years and the clinical use by patients and clinicians has increased significantly. The risks associated with invasive testing have been substantially reduced by providing another assessment of aneuploidy status in high-risk patients. The accuracy and NIPT assay positivity rate are as predicted by clinical validations and the test demonstrates improvement in the

  18. Antibiotic Susceptibilities of Acinetobacter Baumanii Strains Isolated from Clinical Samples

    Directory of Open Access Journals (Sweden)

    Harun Aðca

    2013-01-01

    Full Text Available      Aim :  In this study it was aimed to investigate the antibiotic susceptibilities of Acinetobacter baumanii strains isolated from various clinical samples sent to Tavsanli State Hospital Microbiology Laboratory retrospectively. Material and Method: All of the cultures were examined for the agent and antibiotic susceptibilities. For the identification of bacteria, various chemical tests and BBL Crystal E/NF (Beckton Dickinson, ABD system was used. Antibiotic susceptibilities were investigated according to CLSI criteria on Mueller Hinton agar by disc diffusion method. Results: There were 74 strains isolated and identified as Acinetobacter baumanii. Most of the strains were isolated from  tracheal aspirate specimens (46 % Most of the strains were isolated from nosocomial infections. Antibiotic resistance was high among strains. The most susceptible antibiotic was gentamicin (30%. Discussion: To prevent the development of resistance, antibiotics should be used carefully in appropriate doses and time, empirical  antibiotherapy should be determined for each centre according to resistance rates of the centre and should be regulated according to the antibiogram results. Increasing resistance rates in Acinetobacter strains leads to the usage of new alternative antibiotics.  

  19. Clinical audit for occupational therapy intervention for children with autism spectrum disorder: sampling steps and sample size calculation

    OpenAIRE

    Weeks, Scott; Atlas, Alvin

    2015-01-01

    A priori sample size calculations are used to determine the adequate sample size to estimate the prevalence of the target population with good precision. However, published audits rarely report a priori calculations for their sample size. This article discusses a process in health services delivery mapping to generate a comprehensive sampling frame, which was used to calculate an a priori sample size for a targeted clinical record audit. We describe how we approached methodological and defini...

  20. Improving the Prediction of Prostate Cancer Overall Survival by Supplementing Readily Available Clinical Data with Gene Expression Levels of IGFBP3 and F3 in Formalin-Fixed Paraffin Embedded Core Needle Biopsy Material.

    Directory of Open Access Journals (Sweden)

    Zhuochun Peng

    Full Text Available A previously reported expression signature of three genes (IGFBP3, F3 and VGLL3 was shown to have potential prognostic value in estimating overall and cancer-specific survivals at diagnosis of prostate cancer in a pilot cohort study using freshly frozen Fine Needle Aspiration (FNA samples.We carried out a new cohort study with 241 prostate cancer patients diagnosed from 2004-2007 with a follow-up exceeding 6 years in order to verify the prognostic value of gene expression signature in formalin fixed paraffin embedded (FFPE prostate core needle biopsy tissue samples. The cohort consisted of four patient groups with different survival times and death causes. A four multiplex one-step RT-qPCR test kit, designed and optimized for measuring the expression signature in FFPE core needle biopsy samples, was used. In archive FFPE biopsy samples the expression differences of two genes (IGFBP3 and F3 were measured. The survival time predictions using the current clinical parameters only, such as age at diagnosis, Gleason score, PSA value and tumor stage, and clinical parameters supplemented with the expression levels of IGFBP3 and F3, were compared.When combined with currently used clinical parameters, the gene expression levels of IGFBP3 and F3 are improving the prediction of survival time as compared to using clinical parameters alone.The assessment of IGFBP3 and F3 gene expression levels in FFPE prostate cancer tissue would provide an improved survival prediction for prostate cancer patients at the time of diagnosis.

  1. MicroRNA profiling of gastric cancer patients from formalin-fixed paraffin-embedded samples

    OpenAIRE

    OSAWA, SOSHI; Shimada, Yutaka; Sekine, Shinichi; Okumura, Tomoyuki; NAGATA, TAKUYA; Fukuoka, Junya; Tsukada, Kazuhiro

    2011-01-01

    MicroRNA (miRNA) is a small non-coding RNA that targets specific mRNA. Recent progress in the extraction of RNA from formalin-fixed paraffin-embedded (FFPE) tissues has facilitated miRNA profiling using samples stored in laboratories worldwide. In the present study, miRNA profiling of gastric cancer patients is determined using FFPE samples. First, criteria were established for determining evaluable RNA from the FFPE samples. miRNA profiling was then undertaken using miRNA oligo chips with 88...

  2. From clinical sites to biorepositories: effectiveness in blood sample management.

    Science.gov (United States)

    Lefebvre, Céline; Tremblay, Nancy; Iverson, Bonnie; Wong, David; McWeeny, Kerri; Saghbini, Michael; Martinez, Heather; Hogan, Michael; Gaudet, Daniel; Arsenault, Steve

    2010-12-01

    Today's biobanks must work to take full advantage of collected samples, while maximizing sample quality and minimizing costs to sustain operations for a long period of time. This is a tall order that will require collaboration and compromise for both end-users and collection sites. This article discusses the efforts of the Génome Québec-Centre Hospitalier Affilié Universitaire Régional de Chicoutimi Biobank to fractionate blood samples for the simultaneous preservation of plasma and DNA-containing layers while minimizing resources required for shipping and transport. This article also describes methods for successful reproducible application of the plasma-depleted blood sample to GenPlates (GenVault, Carlsbad, CA).

  3. Formalin-fixed paraffin-embedded tissue: The holy grail of clinical proteomics.

    Science.gov (United States)

    Broeckx, Valérie; Peeters, Lise; Maes, Evelyne; Pringels, Lentel; Verjans, Eddy-Tim; Landuyt, Bart

    2014-10-01

    Tissue is the most relevant biological material to gather insight in disease mechanisms by means of omics technologies. However, fresh frozen tissue, which is generally regarded as the best imaginable source for such studies, is often not available. In case it is available, the different ways of storage (e.g. -20°C, -80°C, liquid nitrogen, etc.) hamper the conduction of reproducible multicenter studies because of different protein degradation rates. Formalin-fixed paraffin-embedded (FFPE) tissue on the contrary is considered as a valuable alternative for fresh frozen tissue, because only a few standard operation procedures are applied worldwide for the preparation of these tissues and because they are all stored in the same way. However, a study on the impact of the different preparation protocols for FFPE tissue was still lacking. Therefore, Bronsert et al. in this issue [Bronsert, P., Weißer, J., Biniossek, M. L., Kuehs, M. et al., Proteomics Clin. Appl. 2014, 8 786-804] conducted such a study that provides proof that there is no significant effect between these sample preparations procedures, and thereby they further open the gate for FFPE tissues to enter the field of clinical proteomics.

  4. Sample size considerations for clinical research studies in nuclear cardiology.

    Science.gov (United States)

    Chiuzan, Cody; West, Erin A; Duong, Jimmy; Cheung, Ken Y K; Einstein, Andrew J

    2015-12-01

    Sample size calculation is an important element of research design that investigators need to consider in the planning stage of the study. Funding agencies and research review panels request a power analysis, for example, to determine the minimum number of subjects needed for an experiment to be informative. Calculating the right sample size is crucial to gaining accurate information and ensures that research resources are used efficiently and ethically. The simple question "How many subjects do I need?" does not always have a simple answer. Before calculating the sample size requirements, a researcher must address several aspects, such as purpose of the research (descriptive or comparative), type of samples (one or more groups), and data being collected (continuous or categorical). In this article, we describe some of the most frequent methods for calculating the sample size with examples from nuclear cardiology research, including for t tests, analysis of variance (ANOVA), non-parametric tests, correlation, Chi-squared tests, and survival analysis. For the ease of implementation, several examples are also illustrated via user-friendly free statistical software.

  5. Clinical audit for occupational therapy intervention for children with autism spectrum disorder: sampling steps and sample size calculation.

    Science.gov (United States)

    Weeks, Scott; Atlas, Alvin

    2015-01-01

    A priori sample size calculations are used to determine the adequate sample size to estimate the prevalence of the target population with good precision. However, published audits rarely report a priori calculations for their sample size. This article discusses a process in health services delivery mapping to generate a comprehensive sampling frame, which was used to calculate an a priori sample size for a targeted clinical record audit. We describe how we approached methodological and definitional issues in the following steps: (1) target population definition, (2) sampling frame construction, and (3) a priori sample size calculation. We recommend this process for clinicians, researchers, or policy makers when detailed information on a reference population is unavailable. PMID:26122044

  6. Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples: insights from liver tissue

    OpenAIRE

    Tanca, Alessandro; Abbondio, Marcello; Pisanu, Salvatore; Pagnozzi, Daniela; Uzzau, Sergio; Addis, Maria Filippa

    2014-01-01

    Background The growing field of formalin-fixed paraffin-embedded (FFPE) tissue proteomics holds promise for improving translational research. Direct tissue trypsinization (DT) and protein extraction followed by in solution digestion (ISD) or filter-aided sample preparation (FASP) are the most common workflows for shotgun analysis of FFPE samples, but a critical comparison of the different methods is currently lacking. Experimental design DT, FASP and ISD workflows were compared by subjecting ...

  7. Analytical artefacts in the speciation of arsenic in clinical samples

    Energy Technology Data Exchange (ETDEWEB)

    Slejkovec, Zdenka [Jozef Stefan Institute, Jamova 39, 1000 Ljubljana (Slovenia)], E-mail: zdenka.slejkovec@ijs.si; Falnoga, Ingrid [Jozef Stefan Institute, Jamova 39, 1000 Ljubljana (Slovenia); Goessler, Walter [Institute of Chemistry - Analytical Chemistry, Karl-Franzens University Graz, Universitaetsplatz 1, Graz (Austria); Elteren, Johannes T. van [National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana (Slovenia); Raml, Reingard [Institute of Chemistry - Analytical Chemistry, Karl-Franzens University Graz, Universitaetsplatz 1, Graz (Austria); Podgornik, Helena; Cernelc, Peter [University Medical Centre Ljubljana, Zaloska 7, 1000 Ljubljana (Slovenia)

    2008-01-21

    Urine and blood samples of cancer patients, treated with high doses of arsenic trioxide were analysed for arsenic species using HPLC-HGAFS and, in some cases, HPLC-ICPMS. Total arsenic was determined with either flow injection-HGAFS in urine or radiochemical neutron activation analysis in blood fractions (in serum/plasma, blood cells). The total arsenic concentrations (during prolonged, daily/weekly arsenic trioxide therapy) were in the {mu}g mL{sup -1} range for urine and in the ng g{sup -1} range for blood fractions. The main arsenic species found in urine were As(III), MA and DMA and in blood As(V), MA and DMA. With proper sample preparation and storage of urine (no preservation agents/storage in liquid nitrogen) no analytical artefacts were observed and absence of significant amounts of alleged trivalent metabolites was proven. On the contrary, in blood samples a certain amount of arsenic can get lost in the speciation procedure what was especially noticeable for the blood cells although also plasma/serum gave rise to some disappearance of arsenic. The latter losses may be attributed to precipitation of As(III)-containing proteins/peptides during the methanol/water extraction procedure whereas the former losses were due to loss of specific As(III)-complexing proteins/peptides (e.g. cysteine, metallothionein, reduced GSH, ferritin) on the column (Hamilton PRP-X100) during the separation procedure. Contemporary analytical protocols are not able to completely avoid artefacts due to losses from the sampling to the detection stage so that it is recommended to be careful with the explanation of results, particularly regarding metabolic and pharmacokinetic interpretations, and always aim to compare the sum of species with the total arsenic concentration determined independently.

  8. A confirmatory factor analysis of the WMS-III in a clinical sample with crossvalidation in the standardization sample.

    Science.gov (United States)

    Bradley Burton, D; Ryan, Joseph J; Axelrod, Bradley N; Schellenberger, Tony; Richards, Heather M

    2003-08-01

    A maximum likelihood confirmatory factor analysis (CFA) of the Wechsler Memory Scale-III (WMS-III) was performed by applying LISREL 8 to a general clinical sample (n=281). Analyses were designed to determine which of seven hypothesized oblique factor solutions could best explain memory as measured by the WMS-III. Competing latent variable models were identified in previous studies. Results in the clinical sample were crossvalidated by testing all models in the WMS-III standardization samples (combined n=1,250). Findings in both the clinical and standardization samples supported a four-factor model containing auditory memory, visual memory, working memory, and learning factors. Our analysis differed from that presented in the WMS-III manual and by other authors. We tested our models in a clinical sample and included selected word list subtests in order to test the viability of a learning dimension. Consistent with prior research, we were also unable to empirically support the viability of the immediate and delayed memory indices, despite allowing the error terms between the immediate and delayed memory subtests to correlate.

  9. A Short Version of the Revised ‘Experience of Close Relationships Questionnaire’: Investigating Non-Clinical and Clinical Samples

    OpenAIRE

    Wongpakaran, Tinakon; Wongpakaran, Nahathai

    2012-01-01

    Aim: This study seeks to investigate the psychometric properties of the short version of the revised ‘Experience of Close Relationships’ questionnaire, comparing non-clinical and clinical samples. Methods: In total 702 subjects participated in this study, of whom 531 were non-clinical participants and 171 were psychiatric patients. They completed the short version of the revised ‘Experience of Close Relationships’ questionnaire (ECR-R-18), the Perceived Stress Scale-10(PSS-10), the Rosenberg ...

  10. Equivalence of MammaPrint array types in clinical trials and diagnostics.

    Science.gov (United States)

    Beumer, Inès; Witteveen, Anke; Delahaye, Leonie; Wehkamp, Diederik; Snel, Mireille; Dreezen, Christa; Zheng, John; Floore, Arno; Brink, Guido; Chan, Bob; Linn, Sabine; Bernards, Rene; van 't Veer, Laura; Glas, Annuska

    2016-04-01

    MammaPrint is an FDA-cleared microarray-based test that uses expression levels of the 70 MammaPrint genes to assess distant recurrence risk in early-stage breast cancer. The prospective RASTER study proved that MammaPrint Low Risk patients can safely forgo chemotherapy, which is further subject of the prospective randomized MINDACT trial. While MammaPrint diagnostic results are obtained from mini-arrays, clinical trials may be performed on whole-genome arrays. Here we demonstrate the equivalence and reproducibility of the MammaPrint test. MammaPrint indices were collected for breast cancer samples: (i) on both customized certified array types (n = 1,897 sample pairs), (ii) with matched fresh and FFPE tissues (n = 552 sample pairs), iii) for control samples replicated over a period of 10 years (n = 11,333), and iv) repeated measurements (n = 280). The array type indicated a near perfect Pearson correlation of 0.99 (95 % CI: 0.989-0.991). Paired fresh and FFPE samples showed an excellent Pearson correlation of 0.93 (95 % CI 0.92-0.94), in spite of the variability introduced by intratumoral tissue heterogeneity. Control samples showed high consistency over 10 year's time (overall reproducibility of 97.4 %). Precision and repeatability are overall 98.2 and 98.3 %, respectively. Results confirm that the combination of the near perfect correlation between array types, excellent equivalence between tissue types, and a very high stability, precision, and repeatability demonstrate that results from clinical trials (such as MINDACT and I-SPY 2) are equivalent to current MammaPrint FFPE and fresh diagnostics, and can be used interchangeably. PMID:27002507

  11. Reliable single cell array CGH for clinical samples.

    Directory of Open Access Journals (Sweden)

    Zbigniew T Czyż

    Full Text Available BACKGROUND: Disseminated cancer cells (DCCs and circulating tumor cells (CTCs are extremely rare, but comprise the precursors cells of distant metastases or therapy resistant cells. The detailed molecular analysis of these cells may help to identify key events of cancer cell dissemination, metastatic colony formation and systemic therapy escape. METHODOLOGY/PRINCIPAL FINDINGS: Using the Ampli1™ whole genome amplification (WGA technology and high-resolution oligonucleotide aCGH microarrays we optimized conditions for the analysis of structural copy number changes. The protocol presented here enables reliable detection of numerical genomic alterations as small as 0.1 Mb in a single cell. Analysis of single cells from well-characterized cell lines and single normal cells confirmed the stringent quantitative nature of the amplification and hybridization protocol. Importantly, fixation and staining procedures used to detect DCCs showed no significant impact on the outcome of the analysis, proving the clinical usability of our method. In a proof-of-principle study we tracked the chromosomal changes of single DCCs over a full course of high-dose chemotherapy treatment by isolating and analyzing DCCs of an individual breast cancer patient at four different time points. CONCLUSIONS/SIGNIFICANCE: The protocol enables detailed genome analysis of DCCs and thereby assessment of the clonal evolution during the natural course of the disease and under selection pressures. The results from an exemplary patient provide evidence that DCCs surviving selective therapeutic conditions may be recruited from a pool of genomically less advanced cells, which display a stable subset of specific genomic alterations.

  12. Prevalence of dermatophytes and other fungal agents isolated from clinical samples

    OpenAIRE

    Kannan P.; Janaki C; Selvi G

    2006-01-01

    The common cause of skin infections are dermatophytes and opportunistic fungi. Aim of this study was to isolate and identify the fungal agents from clinical samples from patients with different mycoses. Clinical samples from 165 patients were subjected to potassium hydroxide (KOH) examination and culture isolation; causative agents were identified macroscopically and microscopically. All the 165 specimens were KOH positive and 110/165 (66.7%) samples were culture positive. Of these, hi...

  13. The Reliability and Validity of the Clinical Perfectionism Questionnaire in Eating Disorder and Community Samples

    OpenAIRE

    Egan, Sarah J.; Shafran, Roz; Lee, Michelle; Fairburn, Christopher G.; Cooper, Zafra; Doll, Helen A.; Palmer, Robert L.; Watson, Hunna J.

    2015-01-01

    Background: Clinical perfectionism is a risk and maintaining factor for anxiety disorders, depression and eating disorders. Aims: The aim was to examine the psychometric properties of the 12-item Clinical Perfectionism Questionnaire (CPQ). Method: The research involved two samples. Study 1 comprised a nonclinical sample (n = 206) recruited via the internet. Study 2 comprised individuals in treatment for an eating disorder (n = 129) and a community sample (n = 80). Results: Study 1 factor anal...

  14. miRNA Expression Analyses in Prostate Cancer Clinical Tissues.

    Science.gov (United States)

    Bucay, Nathan; Shahryari, Varahram; Majid, Shahana; Yamamura, Soichiro; Mitsui, Yozo; Tabatabai, Z Laura; Greene, Kirsten; Deng, Guoren; Dahiya, Rajvir; Tanaka, Yuichiro; Saini, Sharanjot

    2015-01-01

    A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA). PMID:26382040

  15. miRNA Expression Analyses in Prostate Cancer Clinical Tissues.

    Science.gov (United States)

    Bucay, Nathan; Shahryari, Varahram; Majid, Shahana; Yamamura, Soichiro; Mitsui, Yozo; Tabatabai, Z Laura; Greene, Kirsten; Deng, Guoren; Dahiya, Rajvir; Tanaka, Yuichiro; Saini, Sharanjot

    2015-09-08

    A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).

  16. RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies

    Directory of Open Access Journals (Sweden)

    Zhang Haiyu

    2007-12-01

    Full Text Available Abstract Background The development of protocols for RNA extraction from paraffin-embedded samples facilitates gene expression studies on archival samples with known clinical outcome. Older samples are particularly valuable because they are associated with longer clinical follow up. RNA extracted from formalin-fixed paraffin-embedded (FFPE tissue is problematic due to chemical modifications and continued degradation over time. We compared quantity and quality of RNA extracted by four different protocols from 14 ten year old and 14 recently archived (three to ten months old FFPE breast cancer tissues. Using three spin column purification-based protocols and one magnetic bead-based protocol, total RNA was extracted in triplicate, generating 336 RNA extraction experiments. RNA fragment size was assayed by reverse transcription-polymerase chain reaction (RT-PCR for the housekeeping gene glucose-6-phosphate dehydrogenase (G6PD, testing primer sets designed to target RNA fragment sizes of 67 bp, 151 bp, and 242 bp. Results Biologically useful RNA (minimum RNA integrity number, RIN, 1.4 was extracted in at least one of three attempts of each protocol in 86–100% of older and 100% of recently archived ("months old" samples. Short RNA fragments up to 151 bp were assayable by RT-PCR for G6PD in all ten year old and months old tissues tested, but none of the ten year old and only 43% of months old samples showed amplification if the targeted fragment was 242 bp. Conclusion All protocols extracted RNA from ten year old FFPE samples with a minimum RIN of 1.4. Gene expression of G6PD could be measured in all samples, old and recent, using RT-PCR primers designed for RNA fragments up to 151 bp. RNA quality from ten year old FFPE samples was similar to that extracted from months old samples, but quantity and success rate were generally higher for the months old group. We preferred the magnetic bead-based protocol because of its speed and higher quantity of

  17. More than a (negative feeling: Validity of the perceived stress scale in Serbian clinical and non-clinical samples

    Directory of Open Access Journals (Sweden)

    Jovanović Veljko

    2015-01-01

    Full Text Available The goal of the present study was to test the validity of a Serbian version of the Perceived Stress Scale. The PSS was administered to 157 psychiatric outpatients, 165 adults from the non-clinical population, and 283 university students. The results of the confirmatory factor analysis supported a bifactor model of the PSS with one general factor and two specific factors reflecting perceived distress and perceived self-efficacy. Internal consistencies of the scale and its two subscales were adequate across clinical and non-clinical samples. Results supported the ability of the scale to discriminate between clinical and non-clinical samples. The PSS evidenced good convergent validity, showing moderate to high positive correlations with measures of unpleasant emotional states and moderate negative correlations with positive affect and life satisfaction. All but one correlation remained significant after controlling for the measures of emotional distress. The results of the present research support validity of the PSS and its use in both clinical and non-clinical samples. [Projekat Ministarstva nauke Republike Srbije, br. 179006

  18. Chromatin immunoprecipitation from fixed clinical tissues reveals tumor-specific enhancer profiles.

    Science.gov (United States)

    Cejas, Paloma; Li, Lewyn; O'Neill, Nicholas K; Duarte, Melissa; Rao, Prakash; Bowden, Michaela; Zhou, Chensheng W; Mendiola, Marta; Burgos, Emilio; Feliu, Jaime; Moreno-Rubio, Juan; Guadalajara, Héctor; Moreno, Víctor; García-Olmo, Damián; Bellmunt, Joaquim; Mullane, Stephanie; Hirsch, Michelle; Sweeney, Christopher J; Richardson, Andrea; Liu, X Shirley; Brown, Myles; Shivdasani, Ramesh A; Long, Henry W

    2016-06-01

    Extensive cross-linking introduced during routine tissue fixation of clinical pathology specimens severely hampers chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) analysis from archived tissue samples. This limits the ability to study the epigenomes of valuable, clinically annotated tissue resources. Here we describe fixed-tissue chromatin immunoprecipitation sequencing (FiT-seq), a method that enables reliable extraction of soluble chromatin from formalin-fixed paraffin-embedded (FFPE) tissue samples for accurate detection of histone marks. We demonstrate that FiT-seq data from FFPE specimens are concordant with ChIP-seq data from fresh-frozen samples of the same tumors. By using multiple histone marks, we generate chromatin-state maps and identify cis-regulatory elements in clinical samples from various tumor types that can readily allow us to distinguish between cancers by the tissue of origin. Tumor-specific enhancers and superenhancers that are elucidated by FiT-seq analysis correlate with known oncogenic drivers in different tissues and can assist in the understanding of how chromatin states affect gene regulation.

  19. Chromatin immunoprecipitation from fixed clinical tissues reveals tumor-specific enhancer profiles.

    Science.gov (United States)

    Cejas, Paloma; Li, Lewyn; O'Neill, Nicholas K; Duarte, Melissa; Rao, Prakash; Bowden, Michaela; Zhou, Chensheng W; Mendiola, Marta; Burgos, Emilio; Feliu, Jaime; Moreno-Rubio, Juan; Guadalajara, Héctor; Moreno, Víctor; García-Olmo, Damián; Bellmunt, Joaquim; Mullane, Stephanie; Hirsch, Michelle; Sweeney, Christopher J; Richardson, Andrea; Liu, X Shirley; Brown, Myles; Shivdasani, Ramesh A; Long, Henry W

    2016-06-01

    Extensive cross-linking introduced during routine tissue fixation of clinical pathology specimens severely hampers chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) analysis from archived tissue samples. This limits the ability to study the epigenomes of valuable, clinically annotated tissue resources. Here we describe fixed-tissue chromatin immunoprecipitation sequencing (FiT-seq), a method that enables reliable extraction of soluble chromatin from formalin-fixed paraffin-embedded (FFPE) tissue samples for accurate detection of histone marks. We demonstrate that FiT-seq data from FFPE specimens are concordant with ChIP-seq data from fresh-frozen samples of the same tumors. By using multiple histone marks, we generate chromatin-state maps and identify cis-regulatory elements in clinical samples from various tumor types that can readily allow us to distinguish between cancers by the tissue of origin. Tumor-specific enhancers and superenhancers that are elucidated by FiT-seq analysis correlate with known oncogenic drivers in different tissues and can assist in the understanding of how chromatin states affect gene regulation. PMID:27111282

  20. KRAS Mutation Detection in Paired Frozen and Formalin-Fixed Paraffin-Embedded (FFPE) Colorectal Cancer Tissues

    OpenAIRE

    Solassol, Jérome; Ramos, Jeanne; Crapez, Evelyne; SAIFI, MAJDA; Mangé, Alain; Vianès, Evelyne; Lamy, Pierre-Jean; Costes, Valérie; Maudelonde, Thierry

    2011-01-01

    KRAS mutation has been unambiguously identified as a marker of resistance to cetuximab-based treatment in metastatic colorectal cancer (mCRC) patients. However, most studies of KRAS mutation analysis have been performed using homogenously archived CRC specimens, and studies that compare freshly frozen specimens and formalin-fixed paraffin-embedded (FFPE) specimens of CRC are lacking. The aim of the present study was to evaluate the impact of tissue preservation on the determination of KRAS mu...

  1. Identification of fungal pathogens in Formalin-fixed, Paraffin-embedded tissue samples by molecular methods.

    Science.gov (United States)

    Rickerts, Volker

    2016-02-01

    The etiology of invasive fungal infections (IFI) is incompletely understood due to diagnostic limitations including insensitivity of cultures and failure of histopathology to discriminate between different species. This diagnostic gap precludes the optimal use of antifungals, leading to adverse patient outcomes. The identification of fungal pathogens from Formalin-fixed, Paraffin-embedded tissue (FFPE) blocks by molecular methods is emerging as an alternative approach to study the etiology of IFI. PCR assays, including species specific- and broadrange fungal tests are used with FFPE samples from patients with proven IFI. Fungal species identification is achieved in 15-90% of the samples. This heterogeneity may be explained by the samples studied. However, comparison of different studies is impaired, as controls ruling out false positive-, false negative test results or PCR inhibition are frequently not reported. Studies using in situ hybridization also vary in the clinical samples included and the targeted fungi. In addition, target sequences, the probe chemistry and the detection of hybridization signals also account for the differences in diagnostic sensitivity. Using both approaches in parallel yields additive insights, potentially leading to a superior identification of fungal etiology and awareness of the limitations of both molecular diagnostic approaches.

  2. Aberrant microRNA expression in endometrial carcinoma using formalin-fixed paraffin-embedded (FFPE tissues.

    Directory of Open Access Journals (Sweden)

    Taek Sang Lee

    Full Text Available This study aimed to identify the candidate miRNAs in the carcinogenesis of endometrial carcinoma, and to explore whether FFPE material would be suitable for miRNA profiling. We identified the differences between miRNA expression profiles using human miRNA microarray in endometrioid endometrial adenocarcinomas (EECs and normal endometria. Of those tested, miR-200a*, miR-200b*, miR-141, miR-182, and miR-205 were greatly enriched. The expressions of these five miRNAs were validated using quantitative real-time reverse transcription-PCR (qRT-PCR. We then performed qRT-PCR miR expression profiling in 30 FFPE specimens (20 EECs, 10 normal endometria and re-confirmed the results of differential expression between cancer and normal tissue. Following this, we tested whether the specific inhibition of overexpressed miRNAs would alter chemosensitivity. In the in vitro cell viability assay, anti-miR200b* showed a trend toward enhanced cytotoxicity slightly in cisplatin compared to the negative control (p = 0.07. This information provided the candidate miRNAs for further confirmation of the role of miRNAs in the carcinogenesis of EECs, potentially serving as a diagnostic or therapeutic tool. FFPE specimens of endometrial tissues are suitable as a source for miRNA microarray profiling.

  3. Quality Control and Pre-Qualification of NGS Libraries Made from Clinical Samples

    OpenAIRE

    Langmore, John

    2013-01-01

    We have compared sequencing metrics from different types of clinical samples and different methods of making NGS libraries, for purposes of quality control of the samples and sample preps. We have performed the metrics using the HiSeq and MiSeq, however the same QC metrics could be measured on other platforms. By choosing metrics that can be measured from small amounts of data (e.g., 300,000 reads), we can measure the quality of the clinical samples and NGS libraries in a highly multiplexed f...

  4. Clinical, radiological and molecular diagnosis correlation in serum samples from patients with osteoarticular tuberculosis

    Directory of Open Access Journals (Sweden)

    Guadalupe García-Elorriaga

    2014-07-01

    Conclusions: Nested PCR in serum samples is a rapid, highly sensitive and specific modality for OTB detection. PCR should be performed in addition to clinical evaluation, imaging studies, acid-fast bacilli staining, culture and histopathology diagnosis, if possible.

  5. Psychotic experiences in a mental health clinic sample : implications for suicidality, multimorbidity and functioning

    NARCIS (Netherlands)

    Kelleher, I.; Devlin, N.; Wigman, J. T. W.; Kehoe, A.; Murtagh, A.; Fitzpatrick, C.; Cannon, M.

    2014-01-01

    Background Recent community-based research has suggested that psychotic experiences act as markers of severity of psychopathology. There has, however, been a lack of clinic-based research. We wished to investigate, in a clinical sample of adolescents referred to a state-funded mental health service,

  6. Validity of the Aberrant Behavior Checklist in a Clinical Sample of Toddlers

    Science.gov (United States)

    Karabekiroglu, Koray; Aman, Michael G.

    2009-01-01

    We investigated the congruent and criterion validity of the Aberrant Behavior Checklist (ABC) in a clinical sample of toddlers seen over 1 year in Turkey. All consecutive patients (N = 93), 14-43 months old (mean, 30.6 mos.), in a child psychiatry outpatient clinic were included. The ABC, Autism Behavior Checklist (AuBC), and Child Behavior…

  7. The Dutch version of the Child Posttraumatic Cognitions Inventory: validation in a clinical sample and a school sample

    Directory of Open Access Journals (Sweden)

    Julia Diehle

    2015-02-01

    Full Text Available Background: With the inclusion of trauma-related cognitions in the DSM-5 criteria for posttraumatic stress disorder (PTSD, the assessment of these cognitions has become essential. Therefore, valid tools for the assessment of these cognitions are warranted. Objective: The current study aimed at validating the Dutch version of the Child Posttraumatic Cognitions Inventory (CPTCI. Method: We included children aged 8–19 years in our study and assessed the factor structure, reliability and validity of the CPTCI in a clinical sample (n=184 and a school sample (n=318. Results: Our results supported the two-factor structure of the CPTCI and showed good internal consistency for the total scale and the two subscales. We found significant positive correlations between the CPTCI and measures of PTSD, depression, and anxiety disorder. The CPTCI correlated negatively with a measure of quality of life. Furthermore, we found significantly higher scores in the clinical sample than in the school sample. For children who received treatment, we found that a decrease in CPTCI scores was accompanied by a decrease in posttraumatic stress symptoms and comorbid problems indicating that the CPTCI is able to detect treatment effects. Conclusion: Overall, our results suggest that the Dutch CPTCI is a reliable and valid instrument.

  8. The Stice model of overeating: Tests in clinical and non-clinical samples

    NARCIS (Netherlands)

    Strien, T. van; Engels, R.C.M.E.; Leeuwe, J.F.J. van; Snoek, H.M.

    2005-01-01

    The present study tested the dual pathway model of Stice [Stice, E (1994). A review of the evidence for a sociocultural model of bulimia nervosa and an exploration of the mechanisms of action. Clinical Psychology Review, 14, 633-661 and Stice, E. (2001). A prospective test of the dual-pathway model

  9. mRNA transcript quantification in archival samples using multiplexed, color-coded probes

    Directory of Open Access Journals (Sweden)

    Gullane Patrick

    2011-05-01

    Full Text Available Abstract Background A recently developed probe-based technology, the NanoString nCounter™ gene expression system, has been shown to allow accurate mRNA transcript quantification using low amounts of total RNA. We assessed the ability of this technology for mRNA expression quantification in archived formalin-fixed, paraffin-embedded (FFPE oral carcinoma samples. Results We measured the mRNA transcript abundance of 20 genes (COL3A1, COL4A1, COL5A1, COL5A2, CTHRC1, CXCL1, CXCL13, MMP1, P4HA2, PDPN, PLOD2, POSTN, SDHA, SERPINE1, SERPINE2, SERPINH1, THBS2, TNC, GAPDH, RPS18 in 38 samples (19 paired fresh-frozen and FFPE oral carcinoma tissues, archived from 1997-2008 by both NanoString and SYBR Green I fluorescent dye-based quantitative real-time PCR (RQ-PCR. We compared gene expression data obtained by NanoString vs. RQ-PCR in both fresh-frozen and FFPE samples. Fresh-frozen samples showed a good overall Pearson correlation of 0.78, and FFPE samples showed a lower overall correlation coefficient of 0.59, which is likely due to sample quality. We found a higher correlation coefficient between fresh-frozen and FFPE samples analyzed by NanoString (r = 0.90 compared to fresh-frozen and FFPE samples analyzed by RQ-PCR (r = 0.50. In addition, NanoString data showed a higher mean correlation (r = 0.94 between individual fresh-frozen and FFPE sample pairs compared to RQ-PCR (r = 0.53. Conclusions Based on our results, we conclude that both technologies are useful for gene expression quantification in fresh-frozen or FFPE tissues; however, the probe-based NanoString method achieved superior gene expression quantification results when compared to RQ-PCR in archived FFPE samples. We believe that this newly developed technique is optimal for large-scale validation studies using total RNA isolated from archived, FFPE samples.

  10. Detection of classical swine fever virus (CSFV) in clinical samples by RT-PCR assay in clinical samples by RT-PCR assay using different pairs of primers

    International Nuclear Information System (INIS)

    The aim was to compare the efficiency of RT-PCT assays using four pairs of primers selected from different regions of the CSFV genome for the detection of CSFV in clinical samples of swine and wild boars. The four RT-PCR assays were able to detect CSFV in all 20 clinical samples which had been collected from dead swine and wild boars during the outbreaks of CSF in Slovakia in 1993 and 1994. The quality of the selected RT-PCR primers was determined as follows: gp55L/gp55U (E2), 324/326 (5'-NC), S1/S2 (NS5B) and gp54L/gp54U (NS2 genomic region). We conclude that gp55L/gp55U primers are the most suitable for direct detection of CSFV by RT-PCR in tissue homogenates of diseased animals

  11. Unreliability of Results of PCR Detection of Helicobacter pylori in Clinical or Environmental Samples ▿ †

    OpenAIRE

    Sugimoto, Mitsushige; Wu, Jeng-Yih; Abudayyeh, Suhaib; Hoffman, Jill; Brahem, Hajer; Al-Khatib, Khaldun; Yamaoka, Yoshio; David Y Graham

    2009-01-01

    The aim of this study was to compare published Helicobacter pylori primer pairs for their ability to reliably detect H. pylori in gastric biopsy specimens and salivary samples. Detection limits of the 26 PCR primer pairs previously described for detection of H. pylori DNA in clinical samples were determined. Sensitivity and specificity were determined using primers with detection limits of

  12. Rapid whole genome sequencing for the detection and characterization of microorganisms directly from clinical samples

    DEFF Research Database (Denmark)

    Hasman, Henrik; Saputra, Dhany; Sicheritz-Pontén, Thomas;

    2014-01-01

    Whole genome sequencing (WGS) is becoming available as a routine tool for clinical microbiology. If applied directly on clinical samples this could further reduce diagnostic time and thereby improve control and treatment. A major bottle-neck is the availability of fast and reliable bioinformatics...... information and drastically reduce diagnostic time. This may prove very useful, but the need for data analysis is still a hurdle to clinical implementation. To overcome this problem a publicly available bioinformatics tool was developed in this study....... tools. This study was conducted to evaluate the applicability of WGS directly on clinical samples and to develop easy-to-use bioinformatics tools for analysis of the sequencing data. Thirty-five random urine samples from patients with suspected urinary tract infections were examined using conventional...

  13. A Method to Correlate mRNA Expression Datasets Obtained from Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Tissue Samples: A Matter of Thresholds.

    Directory of Open Access Journals (Sweden)

    Dana A M Mustafa

    Full Text Available Gene expression profiling of tumors is a successful tool for the discovery of new cancer biomarkers and potential targets for the development of new therapeutic strategies. Reliable profiling is preferably performed on fresh frozen (FF tissues in which the quality of nucleic acids is better preserved than in formalin-fixed paraffin-embedded (FFPE material. However, since snap-freezing of biopsy materials is often not part of daily routine in pathology laboratories, one may have to rely on archival FFPE material. Procedures to retrieve the RNAs from FFPE materials have been developed and therefore, datasets obtained from FFPE and FF materials need to be made compatible to ensure reliable comparisons are possible.To develop an efficient method to compare gene expression profiles obtained from FFPE and FF samples using the same platform.Twenty-six FFPE-FF sample pairs of the same tumors representing various cancer types, and two FFPE-FF sample pairs of breast cancer cell lines, were included. Total RNA was extracted and gene expression profiling was carried out using Illumina's Whole-Genome cDNA-mediated Annealing, Selection, extension and Ligation (WG-DASL V3 arrays, enabling the simultaneous detection of 24,526 mRNA transcripts. A sample exclusion criterion was created based on the expression of 11 stably expressed reference genes. Pearson correlation at the probe level was calculated for paired FFPE-FF, and three cut-off values were chosen. Spearman correlation coefficients between the matched FFPE and FF samples were calculated for three probe lists with varying levels of significance and compared to the correlation based on all measured probes. Unsupervised hierarchical cluster analysis was performed to verify performance of the included probe lists to compare matched FPPE-FF samples.Twenty-seven FFPE-FF pairs passed the sample exclusion criterion. From the profiles of 27 FFPE and FF matched samples, the best correlating probes were identified

  14. Sample size and power for comparing two or more treatment groups in clinical trials.

    OpenAIRE

    Day, S. J.; Graham, D F

    1989-01-01

    Methods for determining sample size and power when comparing two groups in clinical trials are widely available. Studies comparing three or more treatments are not uncommon but are more difficult to analyse. A linear nomogram was devised to help calculate the sample size required when comparing up to five parallel groups. It may also be used retrospectively to determine the power of a study of given sample size. In two worked examples the nomogram was efficient. Although the nomogram offers o...

  15. DNA qualification workflow for next generation sequencing of histopathological samples.

    Science.gov (United States)

    Simbolo, Michele; Gottardi, Marisa; Corbo, Vincenzo; Fassan, Matteo; Mafficini, Andrea; Malpeli, Giorgio; Lawlor, Rita T; Scarpa, Aldo

    2013-01-01

    Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA) and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR) was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF) tissues, 6 formalin-fixed paraffin-embedded (FFPE) tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for

  16. DNA qualification workflow for next generation sequencing of histopathological samples.

    Directory of Open Access Journals (Sweden)

    Michele Simbolo

    Full Text Available Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF tissues, 6 formalin-fixed paraffin-embedded (FFPE tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard

  17. DNA qualification workflow for next generation sequencing of histopathological samples.

    Science.gov (United States)

    Simbolo, Michele; Gottardi, Marisa; Corbo, Vincenzo; Fassan, Matteo; Mafficini, Andrea; Malpeli, Giorgio; Lawlor, Rita T; Scarpa, Aldo

    2013-01-01

    Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA) and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR) was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF) tissues, 6 formalin-fixed paraffin-embedded (FFPE) tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for

  18. The Frequency of the Accidental Contamination with Laboratory Samples in Yazd Clinical Laboratories’ personnel in 2011

    Directory of Open Access Journals (Sweden)

    Jafari, AA. (PhD

    2014-05-01

    Full Text Available Background and Objective: laboratory personnel have always accidental exposure to clinical samples, which can cause the transmission of infection. This threat can be prevented and controlled by education for the use of safety instruments. The purpose was to determine the frequency of accidental exposure to laboratory samples among Yazd laboratory personnel in 2011. Material and Methods: This descriptive cross-sectional study was conducted on 100 of Yazd clinical laboratory personnel. The data was collected, using a valid and reliable questioner, via interview and analyzed by means of SPSS software. Results: Eighty-six percent of the subjects reported an experience of accidental exposure to clinical samples, such as blood, serum and urine. The causes were carelessness (41% and work overload (29%. Needle- stick was the most prevalent injury (52% particularly in sampler workers (51% and in their hands (69%. There wasn’t significant relationship between accidental exposure to laboratory samples and the variables such as private and governmental laboratories (p=0.517, kind of employment (p=0.411, record of services (p=0.439 and academic degree (p=0.454. The subjects aged 20-29 (p=0.034 and worked in sampling unit had the highest accidental exposure. Conclusion: based on the results, inexperience of the personnel especially in sampling room, overload at work and ignorance of applying safety instruments are known as the most important reasons for accidental exposure to clinical samples. Keywords: Contamination; accidental Exposure; Infectious agents; laboratory; personnel

  19. Genetic diversity of multidrug resistant Staphylococcus aureus isolated from clinical and non clinical samples in Egypt.

    Science.gov (United States)

    Bendary, M M; Solyman, S M; Azab, M M; Mahmoud, N F; Hanora, A M

    2016-01-01

    In recent years, the increasing incidence of diseases caused by Staphylococcus aureus (S. aureus) has been noted in the university hospitals of El-Sharkia and Assuit governorates - Egypt. Therefore, we studied the genetic relatedness of multidrug resistant S. aureus isolates from different sources in the above mentioned governorates. One hundred and fifty six S. aureus isolates were divided into 5 different groups, 1 non clinical isolates from different food products and 4 different clinical isolates of human and animal sources in the 2 different governorates. Epidemiological characteristics of 156 S. aureus isolates were determined by phenotypic methods including quantitative antibiogram typing and biofilm production. Genetic typing of 35 multidrug resistant (MDR) isolates (7 from each group) based on 16S rRNA gene sequence, virulence and antimicrobial resistance gene profiles was done. The genetic relatedness of the highest virulent strain from each group was detected based on different single locus sequence typing and multi-locus sequence typing (MLST). S. aureus strains isolated from different sources and geographical areas showed high diversity. The genetic typing revealed different sequence types and different sequences of coa and spa genes. S. aureus isolates were found highly diverse in Egypt. PMID:27609475

  20. Transcriptional profiling of degraded RNA in cryopreserved and fixed tissue samples obtained at autopsy

    Directory of Open Access Journals (Sweden)

    Alhasan Samir

    2006-12-01

    Full Text Available Abstract Background Traditional multiplexed gene expression methods require well preserved, intact RNA. Such specimens are difficult to acquire in clinical practice where formalin fixation is the standard procedure for processing tissue. Even when special handling methods are used to obtain frozen tissue, there may be RNA degradation; for example autopsy samples where degradation occurs both pre-mortem and during the interval between death and cryopreservation. Although specimens with partially degraded RNA can be analyzed by qRT-PCR, these analyses can only be done individually or at low levels of multiplexing and are laborious and expensive to run for large numbers of RNA targets. Methods We evaluated the ability of the cDNA-mediated Annealing, Selection, extension, and Ligation (DASL assay to provide highly multiplexed analyses of cryopreserved and formalin fixed, paraffin embedded (FFPE tissues obtained at autopsy. Each assay provides data on 1536 targets, and can be performed on specimens with RNA fragments as small as 60 bp. Results The DASL performed accurately and consistently with cryopreserved RNA obtained at autopsy as well as with RNA extracted from formalin-fixed paraffin embedded tissue that had a cryopreserved mirror image specimen with high quality RNA. In FFPE tissue where the cryopreserved mirror image specimen was of low quality the assay performed reproducibly on some but not all specimens. Conclusion The DASL assay provides reproducible results from cryopreserved specimens and many FFPE specimens obtained at autopsy. Gene expression analyses of these specimens may be especially valuable for the study of non-cancer endpoints, where surgical specimens are rarely available.

  1. Symptoms of Children with Autism Spectrum Disorder,a clinical sample

    Directory of Open Access Journals (Sweden)

    Ali Alavi Shooshtari

    2009-12-01

    Full Text Available "n Objective: "n "nThe aim of this report was to study the gender role on autismsymptoms distribution and severity in a clinical sample from Iran. Then, the results were compared with the published study from the same community population sample, Iran. "nMethod: The subjects of this retrospective study were a convenient clinical sample of the referrals of children with pervasive developmental disorders. The diagnosis was made according to DSM-IV diagnostic criteria. "nResults: "nMost of the subjects were boys. Boys were referred for evaluation more frequently than girls. The sample included 61 children and adolescents aged 2.1 to 15 years; of whom, 49 had autism. The mean age of children with autism was 7.2(SD=3.2 years. The mean of age, the diagnosis and severity of the symptoms were not related to gender . "n "n "nConclusion: Usually, those with severe cases of autism refer to clinics for treatment. Therefore, the clinical sample of children with autism is just the tip of the iceberg and they may not be the actual representative of community sample of children with autism. Preventive programs should be more focused on the screening and referring of inflected girls for service utilization .

  2. Evaluation of a nonradiometric system (BACTEC 9000 MB) for detection of mycobacteria in human clinical samples.

    OpenAIRE

    ZANETTI, S.; Ardito, F; SECHI, L.; Sanguinetti, M.; Molicotti, P.; Delogu, G.; Pinna, M P; NACCI, A.; Fadda, G.

    1997-01-01

    This study was carried out to evaluate the rate of recovery and time required for detection of mycobacteria from pulmonary and extrapulmonary human clinical samples, by using a fluorescence-quenching-based oxygen sensor (BACTEC 9000 MB; Becton Dickinson Microbiology Systems, Sparks, Md.). The results were compared with those obtained by microscopy, conventional culture in Lowenstein-Jensen (LJ) medium, and a BACTEC radiometric system (BACTEC 460 TB; Becton Dickinson). Of the 779 clinical samp...

  3. Indications, results, and clinical impact of endoscopic ultrasound (EUS)-guided sampling in gastroenterology

    DEFF Research Database (Denmark)

    Dumonceau, J-M; Polkowski, M; Larghi, A;

    2011-01-01

    This article is part of a combined publication that expresses the current view of the European Society of Gastrointestinal Endoscopy (ESGE) about endoscopic ultrasound (EUS)-guided sampling in gastroenterology, including EUS-guided fine needle aspiration (EUS-FNA) and EUS-guided trucut biopsy (EUS......-positive cytopathological results and needle tract seeding are also discussed. The present Clinical Guideline describes the results of EUS-guided sampling in the different clinical settings, considers the role of this technique in patient management, and makes recommendations on circumstances that warrant its use. A two...

  4. Validation of the Novaco Anger Scale-Provocation Inventory (Danish) With Nonclinical, Clinical, and Offender Samples

    DEFF Research Database (Denmark)

    Moeller, Stine Bjerrum; Novaco, Raymond; Heinola-Nielsen, Vivian;

    2015-01-01

    Anger has high prevalence in clinical and forensic settings, and it is associated with aggressive behavior and ward atmosphere on psychiatric units. Dysregulated anger is a clinical problem in Danish mental health care systems, but no anger assessment instruments have been validated in Danish....... Because the Novaco Anger Scale and Provocation Inventory (NAS-PI) has been extensively validated with different clinical populations and lends itself to clinical case formulation, it was selected for translation and evaluation in the present multistudy project. Psychometric properties of the NAS-PI were...... investigated with samples of 477 nonclinical, 250 clinical, 167 male prisoner, and 64 male forensic participants. Anger prevalence and its relationship with other anger measures, anxiety/depression, and aggression were examined. NAS-PI was found to have high reliability, concurrent validity, and discriminant...

  5. Indications, results, and clinical impact of endoscopic ultrasound (EUS)-guided sampling in gastroenterology

    DEFF Research Database (Denmark)

    Dumonceau, J-M; Polkowski, M; Larghi, A;

    2011-01-01

    This article is part of a combined publication that expresses the current view of the European Society of Gastrointestinal Endoscopy (ESGE) about endoscopic ultrasound (EUS)-guided sampling in gastroenterology, including EUS-guided fine needle aspiration (EUS-FNA) and EUS-guided trucut biopsy (EUS......-positive cytopathological results and needle tract seeding are also discussed. The present Clinical Guideline describes the results of EUS-guided sampling in the different clinical settings, considers the role of this technique in patient management, and makes recommendations on circumstances that warrant its use. A two......-page executive summary of evidence statements and recommendations is provided. A separate Technical Guideline describes the general technique of EUS-guided sampling, particular techniques to maximize the diagnostic yield depending on the nature of the target lesion, and sample processing. The target readership...

  6. High-quality genomic DNA extraction from formalin-fixed and paraffin-embedded samples deparaffinized using mineral oil

    OpenAIRE

    Lin, Jianghai; Kennedy, Stephen H.; Svarovsky, Therese; Rogers, Jeffrey; Kemnitz, Joseph W.; Xu, Anlong; Zondervan, Krina T

    2009-01-01

    Extracting DNA from formalin-fixed and paraffin-embedded (FFPE) tissue remains a challenge, despite numerous attempts to develop a more effective method. Polymerase chain reaction (PCR) success rates with DNA extracted using current methods remain low. We extracted DNA from 140 long-term archived FFPE samples using a simple but effective deparaffinization method, removing the wax with mineral oil, and a commercially available DNA extraction kit. DNA quality was subsequently tested in a genoty...

  7. Ruminations on rumination: anger and sadness rumination in a normative and clinical sample

    OpenAIRE

    Peled, Maya

    2006-01-01

    Anger rumination and sadness rumination were examined concurrently in a normative sample of adults (Study 1) and a clinical adolescent sample (Study 2). The purpose of this research was to assess if rumination on anger and sadness have distinct emotional and behavioural associations, and whether it is warranted to conceptualize them as separate constructs. In both studies, factor analysis indicated that items from analogous anger rumination and sadness rumination measures loaded onto two fact...

  8. Reverse Phase Protein Arrays—Quantitative Assessment of Multiple Biomarkers in Biopsies for Clinical Use

    Directory of Open Access Journals (Sweden)

    Stefanie Boellner

    2015-03-01

    Full Text Available Reverse Phase Protein Arrays (RPPA represent a very promising sensitive and precise high-throughput technology for the quantitative measurement of hundreds of signaling proteins in biological and clinical samples. This array format allows quantification of one protein or phosphoprotein in multiple samples under the same experimental conditions at the same time. Moreover, it is suited for signal transduction profiling of small numbers of cultured cells or cells isolated from human biopsies, including formalin fixed and paraffin embedded (FFPE tissues. Owing to the much easier sample preparation, as compared to mass spectrometry based technologies, and the extraordinary sensitivity for the detection of low-abundance signaling proteins over a large linear range, RPPA have the potential for characterization of deregulated interconnecting protein pathways and networks in limited amounts of sample material in clinical routine settings. Current aspects of RPPA technology, including dilution curves, spotting, controls, signal detection, antibody validation, and calculation of protein levels are addressed.

  9. Automated Broad-Range Molecular Detection of Bacteria in Clinical Samples.

    Science.gov (United States)

    Budding, Andries E; Hoogewerf, Martine; Vandenbroucke-Grauls, Christina M J E; Savelkoul, Paul H M

    2016-04-01

    Molecular detection methods, such as quantitative PCR (qPCR), have found their way into clinical microbiology laboratories for the detection of an array of pathogens. Most routinely used methods, however, are directed at specific species. Thus, anything that is not explicitly searched for will be missed. This greatly limits the flexibility and universal application of these techniques. We investigated the application of a rapid universal bacterial molecular identification method, IS-pro, to routine patient samples received in a clinical microbiology laboratory. IS-pro is a eubacterial technique based on the detection and categorization of 16S-23S rRNA gene interspace regions with lengths that are specific for each microbial species. As this is an open technique, clinicians do not need to decide in advance what to look for. We compared routine culture to IS-pro using 66 samples sent in for routine bacterial diagnostic testing. The samples were obtained from patients with infections in normally sterile sites (without a resident microbiota). The results were identical in 20 (30%) samples, IS-pro detected more bacterial species than culture in 31 (47%) samples, and five of the 10 culture-negative samples were positive with IS-pro. The case histories of the five patients from whom these culture-negative/IS-pro-positive samples were obtained suggest that the IS-pro findings are highly clinically relevant. Our findings indicate that an open molecular approach, such as IS-pro, may have a high added value for clinical practice. PMID:26763956

  10. Maternal Drug Abuse History, Maltreatment, and Functioning in a Clinical Sample of Urban Children

    Science.gov (United States)

    Onigu-Otite, Edore C.; Belcher, Harolyn M. E.

    2012-01-01

    Objective: This study examined the association between maternal drug abuse history, maltreatment exposure, and functioning, in a clinical sample of young children seeking therapy for maltreatment. Methods: Data were collected on 91 children, mean age 5.3 years (SD 1.0). The Preschool and Early Childhood Functional Assessment Scales (PECFAS) was…

  11. A Mediation Model of Interparental Collaboration, Parenting Practices, and Child Externalizing Behavior in a Clinical Sample

    Science.gov (United States)

    Kjobli, John; Hagen, Kristine Amlund

    2009-01-01

    The present study examined maternal and paternal parenting practices as mediators of the link between interparental collaboration and children's externalizing behavior. Parent gender was tested as a moderator of the associations. A clinical sample consisting of 136 children with externalizing problems and their families participated in the study.…

  12. Protein Profile study of clinical samples using Laser Induced Fluorescence as the detection method

    DEFF Research Database (Denmark)

    Karemore, Gopal Raghunath; Raja, Sujatha N.; Rai, Lavanya;

    2009-01-01

    by using hard and Fuzzy clustering methods. The study was performed to test the utility of the HPLC-LIF protein profiling method for classification of tissue samples as well as to establish a complementary method for histopathology for clinical diagnosis of the tissue as normal or malignant.  ...

  13. Radioimmunoassay in situ used to detect products of cloning mycoplasma pneumoniae DNA and clinical micro samples

    International Nuclear Information System (INIS)

    From the recombinants, which constructed in the laboratory, using plasmids as vector, one strain which express antigen of Mycoplasma pneumoniae by detecting in situ radioimmunoassay was obtained. The authors detected 7 children samples which were throat washing liquids of children suspected of atypical pneumoniae, 4 of them positive by using above method. This data indicated that it was suitable for clinical diagnosis directly

  14. Intrusions, avoidance and overgeneral memory in a non-clinical sample

    NARCIS (Netherlands)

    Hauer, Beatrijs J. A.; Wessel, I.; Merckelbach, H.

    2006-01-01

    Previous studies have shown a positive relationship between intrusions, effortful avoidance and overgeneral memory in people suffering from (mild) depression or PTSD. The purpose of the present study was to investigate these relationships in a non-clinical sample. As part of a mass testing session,

  15. Development and blind clinical validation of a microRNA based predictor of response to treatment with R-CHO(E)P in DLBCL

    DEFF Research Database (Denmark)

    Knudsen, Steen; Hother, Christoffer; Grønbæk, Kirsten;

    2015-01-01

    MicroRNAs (miRNA) are a group of short noncoding RNAs that regulate gene expression at the posttranscriptional level. It has been shown that microRNAs are independent predictors of outcome in patients with diffuse large B-cell lymphoma (DLBCL) treated with the drug combination R-CHOP. Based...... microRNAs was blindly validated in a cohort of 116 de novo DLBCL patients treated with R-CHOP or R-CHOEP as first line treatment. The predicted sensitivity based on diagnostic FFPE samples matched the clinical response, with decreasing sensitivity in poor responders (P = 0.03). When the International...

  16. Use of the experience sampling method in the context of clinical trials

    Science.gov (United States)

    Verhagen, Simone J W; Hasmi, Laila; Drukker, Marjan; van Os, J; Delespaul, Philippe A E G

    2016-01-01

    Objective The experience sampling method (ESM) is a structured diary technique to appraise subjective experiences in daily life. It is applied in psychiatric patients, as well as in patients with somatic illness. Despite the potential of ESM assessment, the improved logistics and its increased administration in research, its use in clinical trials remains limited. This paper introduces ESM for clinical trials in psychiatry and beyond. Methods ESM is an ecologically valid method that yields a comprehensive view of an individual's daily life. It allows the assessment of various constructs (eg, quality of life, psychopathology) and psychological mechanisms (eg, stress-sensitivity, coping). These constructs are difficult to assess using cross-sectional questionnaires. ESM can be applied in treatment monitoring, as an ecological momentary intervention, in clinical trials, or in single case clinical trials. Technological advances (eg, smartphone applications) make its implementation easier. Results Advantages of ESM are highlighted and disadvantages are discussed. Furthermore, the ecological nature of ESM data and its consequences are explored, including the potential pitfalls of ambiguously formulated research questions and the specificities of ESM in statistical analyses. The last section focuses on ESM in relation to clinical trials and discusses its future use in optimising clinical decision-making. Conclusions ESM can be a valuable asset in clinical trial research and should be used more often to study the benefits of treatment in psychiatry and somatic health. PMID:27443678

  17. Buffer AVL Alone Does Not Inactivate Ebola Virus in a Representative Clinical Sample Type.

    Science.gov (United States)

    Smither, Sophie J; Weller, Simon A; Phelps, Amanda; Eastaugh, Lin; Ngugi, Sarah; O'Brien, Lyn M; Steward, Jackie; Lonsdale, Steve G; Lever, Mark S

    2015-10-01

    Rapid inactivation of Ebola virus (EBOV) is crucial for high-throughput testing of clinical samples in low-resource, outbreak scenarios. The EBOV inactivation efficacy of Buffer AVL (Qiagen) was tested against marmoset serum (EBOV concentration of 1 × 10(8) 50% tissue culture infective dose per milliliter [TCID50 · ml(-1)]) and murine blood (EBOV concentration of 1 × 10(7) TCID50 · ml(-1)) at 4:1 vol/vol buffer/sample ratios. Posttreatment cell culture and enzyme-linked immunosorbent assay (ELISA) analysis indicated that treatment with Buffer AVL did not inactivate EBOV in 67% of samples, indicating that Buffer AVL, which is designed for RNA extraction and not virus inactivation, cannot be guaranteed to inactivate EBOV in diagnostic samples. Murine blood samples treated with ethanol (4:1 [vol/vol] ethanol/sample) or heat (60°C for 15 min) also showed no viral inactivation in 67% or 100% of samples, respectively. However, combined Buffer AVL and ethanol or Buffer AVL and heat treatments showed total viral inactivation in 100% of samples tested. The Buffer AVL plus ethanol and Buffer AVL plus heat treatments were also shown not to affect the extraction of PCR quality RNA from EBOV-spiked murine blood samples. PMID:26179307

  18. Buffer AVL Alone Does Not Inactivate Ebola Virus in a Representative Clinical Sample Type.

    Science.gov (United States)

    Smither, Sophie J; Weller, Simon A; Phelps, Amanda; Eastaugh, Lin; Ngugi, Sarah; O'Brien, Lyn M; Steward, Jackie; Lonsdale, Steve G; Lever, Mark S

    2015-10-01

    Rapid inactivation of Ebola virus (EBOV) is crucial for high-throughput testing of clinical samples in low-resource, outbreak scenarios. The EBOV inactivation efficacy of Buffer AVL (Qiagen) was tested against marmoset serum (EBOV concentration of 1 × 10(8) 50% tissue culture infective dose per milliliter [TCID50 · ml(-1)]) and murine blood (EBOV concentration of 1 × 10(7) TCID50 · ml(-1)) at 4:1 vol/vol buffer/sample ratios. Posttreatment cell culture and enzyme-linked immunosorbent assay (ELISA) analysis indicated that treatment with Buffer AVL did not inactivate EBOV in 67% of samples, indicating that Buffer AVL, which is designed for RNA extraction and not virus inactivation, cannot be guaranteed to inactivate EBOV in diagnostic samples. Murine blood samples treated with ethanol (4:1 [vol/vol] ethanol/sample) or heat (60°C for 15 min) also showed no viral inactivation in 67% or 100% of samples, respectively. However, combined Buffer AVL and ethanol or Buffer AVL and heat treatments showed total viral inactivation in 100% of samples tested. The Buffer AVL plus ethanol and Buffer AVL plus heat treatments were also shown not to affect the extraction of PCR quality RNA from EBOV-spiked murine blood samples.

  19. Clinical, radiological and molecular diagnosis correlation in serum samples from patients with osteoarticular tuberculosis

    Institute of Scientific and Technical Information of China (English)

    Guadalupe Garca-Elorriaga; Olga Martnez-Elizondo; Guillermo del Rey-Pineda; Csar Gonzlez-Bonilla

    2014-01-01

    Objective: To assess the role of polymerase chain reaction (PCR) in serum samples, in the diagnosis of osteoarticular tuberculosis (OTB) in a setting where only clinical and imaging diagnoses determine the treatment.Methods:A total of 44 consecutive serum specimens were collected from clinically suspected OTB patients, based on clinical and radiological [X-ray or magnetic resonance imaging/computed tomography] features. They were screened by in-house nested PCR. In addition, a few specimens were examined by Gram stain, acid-fast bacilli stain, histopathology and routine bacterial culture. A total of 39 specimens were collected from patients suffering from other bone diseases of nontuberculous origin and included as negative controls.Results:Of the 44 clinically suspected OTB patients, in-house nested PCR was positive in 40 (91%) cases; PCR was negative in 38 (97%) negative controls. Sensitivity and specificity of our in-house nested PCR was 90.9% and 97.4%, respectively. The PCR report was available within 48 h. It was possible to standardize serum PCR technique and in positive cases, a good correlation was observed in terms of an adequate treatment response.Conclusions:Nested PCR in serum samples is a rapid, highly sensitive and specific modality for OTB detection. PCR should be performed in addition to clinical evaluation, imaging studies, acid-fast bacilli staining, culture and histopathology diagnosis, if possible.

  20. Routine application of the polymerase chain reaction for detection of Mycobacterium tuberculosis in clinical samples.

    Science.gov (United States)

    Noordhoek, G T; Kaan, J A; Mulder, S; Wilke, H; Kolk, A H

    1995-01-01

    AIM--To investigate the use of the polymerase chain reaction (PCR) in the routine laboratory for the detection of Mycobacterium tuberculosis in clinical samples. METHODS--Samples were divided and processed separately for the detection of M tuberculosis by microscopy, culture and PCR. After DNA extraction, PCR was performed with primers specific for the insertion element IS6110 and the product was analysed by agarose gel electrophoresis, Southern blotting or dot blotting and hybridisation with a digoxigenin labelled internal probe. Each sample was tested for inhibitors of Taq polymerase with the aid of an internal control. Multiple negative and positive controls were used to monitor each step of the procedure. RESULTS--The data from two laboratories, using the same operating procedures, were combined. Of 1957 specimens, 79 (4%) were culture and PCR positive, while 1839 (93.9%) were negative in both tests. Thirty specimens (1.5%) were PCR positive only and nine (0.5%) were culture positive but PCR negative. CONCLUSION--Using culture and clinical history as the gold standard, sensitivity and specificity for PCR were 92.1% and 99.8%, respectively. With elaborate precautions, PCR is a suitable and reliable method for the detection of M tuberculosis in clinical samples in a routine microbiology laboratory. Images PMID:7490312

  1. Comparison of clinical samples for visceral Leishmaniasis diagnosis in asymptomatic dogs by PCR hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Ferreira, Sidney A.; Ituassu, Leonardo T.; Melo, Maria N. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Parasitologia], e-mail: saninoalmeida@gmail.com, e-mail: Itituassu@yahoo.com.br, e-mail: melo@icb.ufmg.br; Leite, Rodrigo S.; Andrade, Antero S.R. [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN-CNEN/MG), Belo Horizonte, MG (Brazil)], e-mail: rleite2005@gmail.com, e-mail: antero@cdtn.br

    2009-07-01

    The canine visceral leishmaniasis (CVL) diagnosis still represents a challenge because of complexity of this disease. The aim of present study was to compare different clinical samples for diagnosis of CVL by Polymerase Chain Reaction (PCR) combined with hybridization of {sup 32}P labeled probes. Bone marrow (BM), skin biopsy (SB), peripheral blood (PB) and conjunctival swab (CS) were used in this work. With this purpose 40 asymptomatic dogs, all positive by parasitological test, were obtained. From each animal were collected SB with sterile punches from ear internal surface, 1.0 mL of PB, BM aspirates from sternum and CS from both lower eyelid. Each clinical sample was submitted to suitable DNA purification process and PCR-hybridization assays. The positive results obtained with PCR were 55%, 25%, 30% and 22.5% for CS, BM, SB and PB respectively while the PCR followed by hybridization showed a positivity of 87.5%, 50%, 45% and 27.5% respectively. The hybridization assay was able to increase the PCR positivity in all kinds of clinical samples. The best performance was obtained using CS samples. We concluded that the PCR associated with DNA radioactive probes was a very sensitive tool for diagnosis of CVL in asymptomatic dogs and the CS has an important potential for regular screening of dogs. (author)

  2. Comparison of clinical samples for visceral Leishmaniasis diagnosis in asymptomatic dogs by PCR hybridization

    International Nuclear Information System (INIS)

    The canine visceral leishmaniasis (CVL) diagnosis still represents a challenge because of complexity of this disease. The aim of present study was to compare different clinical samples for diagnosis of CVL by Polymerase Chain Reaction (PCR) combined with hybridization of 32P labeled probes. Bone marrow (BM), skin biopsy (SB), peripheral blood (PB) and conjunctival swab (CS) were used in this work. With this purpose 40 asymptomatic dogs, all positive by parasitological test, were obtained. From each animal were collected SB with sterile punches from ear internal surface, 1.0 mL of PB, BM aspirates from sternum and CS from both lower eyelid. Each clinical sample was submitted to suitable DNA purification process and PCR-hybridization assays. The positive results obtained with PCR were 55%, 25%, 30% and 22.5% for CS, BM, SB and PB respectively while the PCR followed by hybridization showed a positivity of 87.5%, 50%, 45% and 27.5% respectively. The hybridization assay was able to increase the PCR positivity in all kinds of clinical samples. The best performance was obtained using CS samples. We concluded that the PCR associated with DNA radioactive probes was a very sensitive tool for diagnosis of CVL in asymptomatic dogs and the CS has an important potential for regular screening of dogs. (author)

  3. Lack of concordance in microarray gene expression responses to Phenobarbital in companion aged FFPE and Frozen liver samples

    Science.gov (United States)

    Despite the immense potential value of public and private biorepositories, direct utilization of archival tissues for molecular profiling has been limited. A major reason for this limited use is the difficulty in obtaining reliable transcriptomic profiles from formalin-fixed par...

  4. [Isolation of Sporothrix pallida complex in clinical and environmental samples from Chile].

    Science.gov (United States)

    Cruz Choappa, Rodrigo M; Vieille Oyarzo, Peggy I; Carvajal Silva, Laura C

    2014-01-01

    The isolation of S. pallida complex from medical samples and home garden soil of a patient in Chile is here in reported. Fungi of the Sporothrix schenckii complex can cause various infections. In Chile, the medical and environmental isolates of these this complex are rare. The aim of this study was to identify an unusual agent in a case of onychomycosis and to detect its presence in the patient's home garden. For this purpose, clinical samples were obtained by scraping the patient's subungueal first right toe nail as well as by taking soil samples from different areas of her home garden. Species identification was performed by morphophysiology and one of the strains isolated from the patient's toe nail was sent to CBS for molecular confirmation (14.062). S. pallida complex was identified both from the patient's toe nail and samples taken from her home garden.

  5. Sampling

    CERN Document Server

    Thompson, Steven K

    2012-01-01

    Praise for the Second Edition "This book has never had a competitor. It is the only book that takes a broad approach to sampling . . . any good personal statistics library should include a copy of this book." —Technometrics "Well-written . . . an excellent book on an important subject. Highly recommended." —Choice "An ideal reference for scientific researchers and other professionals who use sampling." —Zentralblatt Math Features new developments in the field combined with all aspects of obtaining, interpreting, and using sample data Sampling provides an up-to-date treat

  6. Evaluation of Mutational Testing of Preneoplastic Barrett's Mucosa by Next-Generation Sequencing of Formalin-Fixed, Paraffin-Embedded Endoscopic Samples for Detection of Concurrent Dysplasia and Adenocarcinoma in Barrett's Esophagus.

    Science.gov (United States)

    Del Portillo, Armando; Lagana, Stephen M; Yao, Yuan; Uehara, Takeshi; Jhala, Nirag; Ganguly, Tapan; Nagy, Peter; Gutierrez, Jorge; Luna, Aesis; Abrams, Julian; Liu, Yang; Brand, Randall; Sepulveda, Jorge L; Falk, Gary W; Sepulveda, Antonia R

    2015-07-01

    Barrett's intestinal metaplasia (BIM) may harbor genomic mutations before the histologic appearance of dysplasia and cancer and requires frequent surveillance. We explored next-generation sequencing to detect mutations with the analytical sensitivity required to predict concurrent high-grade dysplasia (HGD) and esophageal adenocarcinoma (EAC) in patients with Barrett's esophagus by testing nonneoplastic BIM. Formalin-fixed, paraffin-embedded (FFPE) routine biopsy or endoscopic mucosal resection samples from 32 patients were tested: nonprogressors to HGD or EAC (BIM-NP) with BIM, who never had a diagnosis of dysplasia or EAC (N = 13); progressors to HGD or EAC (BIM-P) with BIM and a worse diagnosis of HGD or EAC (N = 15); and four BIM-negative samples. No mutations were detected in the BIM-NP (0 of 13) or BIM-negative samples, whereas the BIM-P samples had mutations in 6 (75%) of 8 cases in TP53, APC, and CDKN2A (P = 0.0005), detected in samples with as low as 20% BIM. We found that next-generation sequencing from routine FFPE nonneoplastic Barrett's esophagus samples can detect multiple mutations in minute areas of BIM with high analytical sensitivity. Next-generation sequencing panels for detection of TP53 and possibly combined mutations in other genes, such as APC and CDKN2A, may be useful in the clinical setting to improve dysplasia and cancer surveillance in patients with Barrett's esophagus.

  7. Diversity of Bipolaris species in clinical samples in the United States and their antifungal susceptibility profiles.

    Science.gov (United States)

    da Cunha, K C; Sutton, D A; Fothergill, A W; Cano, J; Gené, J; Madrid, H; De Hoog, S; Crous, P W; Guarro, J

    2012-12-01

    A set of 104 isolates from human clinical samples from the United States, morphologically compatible with Bipolaris, were morphologically and molecularly identified through the sequence analysis of the internal transcribed space (ITS) region of the nuclear ribosomal DNA (rDNA). The predominant species was Bipolaris spicifera (67.3%), followed by B. hawaiiensis (18.2%), B. cynodontis (8.6%), B. micropus (2.9%), B. australiensis (2%), and B. setariae (1%). Bipolaris cynodontis, B. micropus, and B. setariae represent new records from clinical samples. The most common anatomical sites where isolates were recovered were the nasal region (30.7%), skin (19.2%), lungs (14.4%), and eyes (12.5%). The antifungal susceptibilities of 5 species of Bipolaris to 9 drugs are provided. With the exception of fluconazole and flucytosine, the antifungals tested showed good activity. PMID:23052310

  8. Comparison of Clinical Targeted Next-Generation Sequence Data from Formalin-Fixed and Fresh-Frozen Tissue Specimens

    OpenAIRE

    Spencer, David H.; Sehn, Jennifer K.; Abel, Haley J.; Watson, Mark A.; Pfeifer, John D; Duncavage, Eric J.

    2013-01-01

    Next-generation sequencing (NGS) has emerged as a powerful technique for the detection of genetic variants in the clinical laboratory. NGS can be performed using DNA from FFPE tissue, but it is unknown whether such specimens are truly equivalent to unfixed tissue for NGS applications. To address this question, we performed hybridization-capture enrichment and multiplexed Illumina NGS for 27 cancer-related genes using DNA from 16 paired fresh-frozen and routine FFPE lung adenocarcinoma specime...

  9. Validity Evidences for the Dimensional Clinical Personality Inventory in Outpatient Psychiatric Sample

    Directory of Open Access Journals (Sweden)

    Roberta Katz Abela

    2015-08-01

    Full Text Available The Dimensional Clinical Personality Inventory (IDCP was developed in Brazil for the assessment of pathological personality traits. This study aimed to seek validity evidence for the dimensions of IDCP based on external criteria, psychiatric diagnosis. We examined the profile in IDCP of 105 psychotherapy outpatients, previously diagnosed with personality disorders. The profiles were compared with the profile of the normative non-clinical sample and we conducted the repeated measures analysis to investigate whether the IDCP is able to discriminate consistent profiles for different diagnoses and compared the general population. The results suggest validity evidence based on external criteria for the IDCP dimensions and points to the clinical effectiveness of the instrument.

  10. A Simplified Method to Recover Urinary Vesicles for Clinical Applications, and Sample Banking

    OpenAIRE

    Musante, Luca; Tataruch, Dorota; Gu, Dongfeng; Benito-Martin, Alberto; Calzaferri, Giulio; Aherne, Sinead; Holthofer, Harry

    2014-01-01

    Urinary extracellular vesicles provide a novel source for valuable biomarkers for kidney and urogenital diseases: Current isolation protocols include laborious, sequential centrifugation steps which hampers their widespread research and clinical use. Furthermore, large individual urine sample volumes or sizable target cohorts are to be processed (e.g. for biobanking), the storage capacity is an additional problem. Thus, alternative methods are necessary to overcome such limitations. We have d...

  11. Vibrio parahaemolyticus Strains of Pandemic Serotypes Identified from Clinical and Environmental Samples from Jiangsu, China

    OpenAIRE

    Jingjiao eLi; Feng eXue; Zhenquan eYang; Xiaoping eZhang; Dexin eZeng; Guoxiang eChao; Yuan eJiang; Baoguang eLi

    2016-01-01

    Vibrio parahaemolyticus has emerged as a major foodborne pathogen in China, Japan, Thailand and other Asian countries. In this study, 72 strains of V. parahaemolyticus were isolated from clinical and environmental samples between 2006 and 2014 in Jiangsu, China. The serotypes and six virulence genes including thermostable direct hemolysin (TDR) and TDR-related hemolysin (TRH) genes were assessed among the isolates. Twenty five serotypes were identified and O3:K6 was one of the dominant seroty...

  12. Diversity of Bipolaris species in clinical samples in the United States and their antifungal susceptibility profiles

    OpenAIRE

    Cunha, DA; Sutton, D.A.; Fothergill, A. W.; Cano, J.; Gene, J.; Madrid, H.; Hoog, de, G.S.; Crous, P.W.; Guarro, J

    2012-01-01

    A set of 104 isolates from human clinical samples from the United States, morphologically compatible with Bipolaris, were morphologically and molecularly identified through the sequence analysis of the internal transcribed space (ITS) region of the nuclear ribosomal DNA (rDNA). The predominant species was Bipolaris spicifera (67.3%), followed by B. hawaiiensis (18.2%), B. cynodontis (8.6%), B. micropus (2.9%), B. australiensis (2%), and B. setariae (1%). Bipolaris cynodontis, B. micropus, and...

  13. A systems approach to clinical oncology: Focus on breast cancer

    Directory of Open Access Journals (Sweden)

    Leyland-Jones Brian

    2006-04-01

    Full Text Available Abstract During the past decade, genomic microarrays have been applied with some success to the molecular profiling of breast tumours, which has resulted in a much more detailed classification scheme as well as in the identification of potential gene signature sets. These gene sets have been applied to both the prognosis and prediction of outcome to treatment and have performed better than the current clinical criteria. One of the main limitations of microarray analysis, however, is that frozen tumour samples are required for the assay. This imposes severe limitations on access to samples and precludes large scale validation studies from being conducted. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR, on the other hand, can be used with degraded RNAs derived from formalin-fixed paraffin-embedded (FFPE tumour samples, the most important and abundant source of clinical material available. More recently, the novel DASL (cDNA-mediated Annealing, Selection, extension and Ligation assay has been developed as a high throughput gene expression profiling system specifically designed for use with FFPE tumour tissue samples. However, we do not believe that genomics is adequate as a sole prognostic and predictive platform in breast cancer. The key proteins driving oncogenesis, for example, can undergo post-translational modifications; moreover, if we are ever to move individualization of therapy into the practical world of blood-based assays, serum proteomics becomes critical. Proteomic platforms, including tissue micro-arrays (TMA and protein chip arrays, in conjunction with surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF/MS, have been the technologies most widely applied to the characterization of tumours and serum from breast cancer patients, with still limited but encouraging results. This review will focus on these genomic and proteomic platforms, with an emphasis placed on the utilization

  14. Clinical features and prognosis of a sample of patients with trisomy 13 (Patau syndrome) from Brazil.

    Science.gov (United States)

    Petry, Patrícia; Polli, Janaina B; Mattos, Vinícius F; Rosa, Rosana C M; Zen, Paulo R G; Graziadio, Carla; Paskulin, Giorgio A; Rosa, Rafael F M

    2013-06-01

    Trisomy 13 or Patau syndrome (PS) is a chromosomal disorder characterized by a well known presentation of multiple congenital anomalies. Our objective was to determine the clinical features and prognosis observed in a sample of patients with PS. The series was composed of patients with diagnosis of PS consecutively evaluated by a Clinical Genetics Service from a reference hospital of southern Brazil, in the period between 1975 and 2012. Statistical analysis was performed using PEPI program (version 4.0), with two-tailed Fisher's exact test for comparison of frequencies (P<0.05). The sample consisted of 30 patients, 60% male, median age at first evaluation of 9 days. Full trisomy of chromosome 13 was the main cytogenetic alteration (73%). The major clinical findings included: cryptorchidism (78%), abnormal auricles (77%), congenital heart defects (76%), polydactyly (63%), microphthalmia (60%) and micrognathia (50%). Four patients (13%) simultaneously had micro/anophthalmia, oral clefts and polydactyly. Some findings were only observed in our sample and included, among others, preauricular tags (10%), duplication of the hallux (3%) and spots following the lines of Blaschko (3%). Mosaicism (20% of cases) had a statistically significant association only with absence of cryptorchidism. The median of survival was 26 days. Patients with and without mosaicism had similar median of survival. Our findings, in agreement with the literature, show that the anomalies in patients with PS can be quite variable, sometimes even atypical. There is no pathognomonic finding, which may make the early identification of these patients challenging. PMID:23613355

  15. Significant Decline in Galactomannan Signal during Storage of Clinical Serum Samples

    Directory of Open Access Journals (Sweden)

    Samir G. Agrawal

    2013-06-01

    Full Text Available Galactomannan (GM is widely used for detection of invasive aspergillosis in high-risk haemato-oncology patients. Recent publications have reported a lack of repeatability of GM detection. The objective of this retrospective study was to assess the repeatability of GM levels during storage of clinical samples. In a GM screening strategy, positive sera were repeat tested as per manufacturer’s recommendations. Short-term (ST storage of samples was at +4 °C while long-term (LT storage was at −80 °C. Bronchoalveolar (BAL fluid was also repeating tested after ST storage and LT storage. Wilcoxon Signed Ranks Test was employed to assess the repeatability of GM levels. In a subset of 14 GM positive sera, repeat testing was performed on both the original serum and ethylenediaminetetraacetic acid (EDTA pre-treated sample. There was a significant reduction in GM signals on repeat testing following ST storage (median GM index: 0.65 vs. 0.19; p < 0.001 and LT storage (median GM index: 0.56 vs. 0.10; p < 0.001 of serum samples. Of samples that were initially GM positive, an average GM index reduction of 50% was seen, with approximately two-thirds becoming GM negative on repeat testing of the same sample. In contrast, GM signal loss was not seen on repeat testing of BAL fluid following ST or LT storage. When GM positive serum samples were repeat tested using EDTA pre-treated serum from the first step of the testing protocol, all samples remained GM positive. In contrast, when the same samples were repeat tested from the original collected serum, 9 samples (64% became GM negative. The significant reduction in GM signals during ST and LT storage of serum samples has implications for clinical management. Although the reasons for GM decline are unknown, they occur prior to the EDTA pre-treatment stage, indicating that the time from phlebotomy to testing should be minimized. BAL fluid GM index values remain stable.

  16. Clinical,radiological and molecular diagnosis correlation in serum samples from patients with osteoarticular tuberculosis

    Institute of Scientific and Technical Information of China (English)

    Guadalupe; Garcia-Elorriaga; Olga; Martinez-Elizondo; Guillermo; del; Rey-Pineda; Cesar; Gonzalez-Bonilla

    2014-01-01

    Objective:To assess the role of polymerase chain reaction(PCR)in serum sauples,in the diagnosis of osteoarticular tuberculosis(OTB)in a setting where only clinical and imaging diagnoses determine the treatment.Methods:A total of 44 consecutive serum specimens were collected from clinically suspected OTB patients,based on clinical and radiological[X-ray or magnetic resonance imagng/computecl tomography]features.They were scrcened by in-house nested PCR.In addition,a few specimens were examined by Gram stain,acid-fast bacilli stain,histand routine bacterial culture.A total of 39 specimens were collected from patients suffering from other bone diseases of nontuberculous origin and included as negative controls.Results:of the 44 clinically suspected OTB patients,in-house nested PCR was positive in 40(91%)cases;PCR was negative in 38(97%)negative controls.Sensitivity and specificity of our in—house nested PCR was 90.3%and 97.4%,respectively.The PCR report was available within 48 h.It was possible to standardize serum PCR technique and in positive cases,a good n was observed in terms of an adequate treatment response.Conclusions:Nested PCR in serum samples is a rapid,highly sensitive and specific modality for OTB detection,PCR should be performed in addition to clinical evaluation,imaging studies,acidfast bacilli staining,culture and histopathology diagnosis,if possible.

  17. Prevalence of Aspergillus species in clinical samples isolated in an Indian tertiary care hospital

    Directory of Open Access Journals (Sweden)

    Xess Immaculata

    2004-12-01

    Full Text Available CONTEXT (BACKGROUND: In recent times, it has become important to determine the prevalence of different Aspergillus species in clinical samples in view of difference in antifungal susceptibility noted in some species. AIMS: To determine the species prevalence of Aspergillus isolates in various clinical samples received in the Mycology Laboratory at our institute. METHOD: Over a period of 4-years, a total of 18,731 samples were processed, and species identification carried out by standard microbiological methods. RESULTS: Four hundred and fifty six samples (2.43% were culture positive for Aspergillus species. A.flavus (46.93% was the most common isolate, followed by A.fumigatus (37.72% and A.niger (15.35%. It was observed that A.fumigatus was the predominant species isolated from blood and respiratory specimens, A.flavus was predominantly isolated from nasal polyps whereas A.niger predominated in nail specimens. Culture positivity was highest in the age group 12-65 years and in males. Sixty-nine patients (15.13% were admitted to the intensive care unit. CONCLUSIONS: The study highlights the diverse manifestations caused by Aspergillus species in human beings and also throws light on the different species prevalent locally. The knowledge would prove useful in selecting empirical antifungal therapy and formulating prophylactic and pre-emptive strategies.

  18. Vibrio parahaemolyticus in shellfish and clinical samples during two large epidemics of diarrhoea in southern Chile.

    Science.gov (United States)

    Fuenzalida, Loreto; Hernández, Cristina; Toro, Jessica; Rioseco, M Luisa; Romero, Jaime; Espejo, Romilio T

    2006-04-01

    Large epidemics of diarrhoea associated with seafood consumption and Vibrio parahaemolyticus occurred during the austral summers of 2004 and 2005 in the environs of Puerto Montt, Chile (41 degrees 29'S 72 degrees 24'W). There are no reports of V. parahaemolyticus infections before 2004 in this region, their absence being explained by the low ocean temperatures which seldom reach 16 degrees C. We analysed V. parahaemolyticus obtained from shellfish and clinical samples during epidemics. Isolates were examined using conventional protocols and an improved method for restriction enzyme analysis using total bacterial DNA which permits direct genome restriction enzyme analysis by conventional gel electrophoresis (DGREA) with a similar discrimination index as restriction fragment length polymorphism-pulsed field gel electrophoresis (RFLP-PFGE). Analysis of clinical samples showed that the epidemics were caused by the V. parahaemolyticus O3:K6 pandemic clonal group. On the other hand, analysis of shellfish samples during both epidemics showed that 53% contained V. parahaemolyticus (3-93 g(-1)). Detailed analysis of 50 positive shellfish samples showed that only three contained detectable levels of the pandemic clone. Most V. parahaemolyticus isolates obtained from shellfish corresponded to non-pandemic clones differentiated into 14 groups by DGREA. In summary, the causative agent during epidemics was only a minor component of a small but diverse population of V. parahaemolyticus in shellfish. PMID:16584479

  19. [Sample size calculation in clinical post-marketing evaluation of traditional Chinese medicine].

    Science.gov (United States)

    Fu, Yingkun; Xie, Yanming

    2011-10-01

    In recent years, as the Chinese government and people pay more attention on the post-marketing research of Chinese Medicine, part of traditional Chinese medicine breed has or is about to begin after the listing of post-marketing evaluation study. In the post-marketing evaluation design, sample size calculation plays a decisive role. It not only ensures the accuracy and reliability of post-marketing evaluation. but also assures that the intended trials will have a desired power for correctly detecting a clinically meaningful difference of different medicine under study if such a difference truly exists. Up to now, there is no systemic method of sample size calculation in view of the traditional Chinese medicine. In this paper, according to the basic method of sample size calculation and the characteristic of the traditional Chinese medicine clinical evaluation, the sample size calculation methods of the Chinese medicine efficacy and safety are discussed respectively. We hope the paper would be beneficial to medical researchers, and pharmaceutical scientists who are engaged in the areas of Chinese medicine research. PMID:22292397

  20. Genotypic characterization, invasion index and antimicrobial resistance pattern in Listeria monocytogenes strains isolated from clinical samples

    Institute of Scientific and Technical Information of China (English)

    Behrooz Sadeghi Kalani; Abazar Pournajaf; Mansour Sedighi; Abbas Bahador; Gholamreza Irajian; Firuzeh Valian

    2015-01-01

    Objective: To evaluate antimicrobial resistance, invasion index and genetic profile in Listeriamonocytogenes isolated from clinical samples. Methods: At all, 170 clinical samples were collected from patients with spontaneous abortions hospitalized in Shariati hospital in Tehran during June 2010 to August 2013. Invasion index was determined using HeLa cells. The multiple-locus variable-number tandem-repeats analysis (MLVA) was used for evaluation of genetic relatedness. Results: Out of 14 L. monocytogenes isolates, 4 (28.57%), 2 (14.28%), 0 (0%), 5 (35.71%) and 3 (21.42%) were isolated from placental tissue, urine, blood, vaginal and rectal swabs, respectively. High resistance to penicillin and multidrug resistant were found amongst isolates. The invasion index was in the range of 0.001-0.007. Seven different types were obtained by MLVA assay and type 2 and 3 with 4 strains were the most frequent type. Strains isolated from the vagina and the placenta of the same type were also more resistant to penicillin. Conclusions: Since MLVA is a high-throughput screening method that is fairly inexpensive, easy to accomplish, rapid, and trustworthy, it is well suited to interlaboratory comparisons during epidemiological investigations. Also further studies of larger samples from a variety of sources such as food and animal specimens recommended comparing by MLVA method.

  1. Prevalence and Reasons for Tooth Loss in a Sample from a Dental Clinic in Brazil

    Directory of Open Access Journals (Sweden)

    Andréia Affonso Barretto Montandon

    2012-01-01

    Full Text Available Purpose. To evaluate the prevalence and reasons for teeth extractions in a sample from a dental clinic in Brazil. Methods. The prevalence of teeth mortality was analyzed by gender, age, tooth type and reasons for extraction on 800 teeth of 439 subjects, whose data was collected in clinical records in a convenience sample. Results. The groups with range from 35 to 44 years, 45 to 54 years and 55 to 64 years revealed significantly greater number of teeth extractions than other age groups (P<0.0001. The anterior teeth loss increased significantly with aging, while the tooth mortality of premolar and molar were higher in younger people. The caries was the more prevalent reason for tooth mortality among young and adults up to 44 years old, while the periodontal disease was the main reason for extractions from 45 years old until range of 81 years (P<0.0001. Conclusions. It can be suggested that some reasons for tooth loss were age-dependent, but the caries and the periodontal diseases were the main reasons for tooth mortality in this Brazilian sample.

  2. Gender Differences in Borderline Personality Disorder: Results From a Multinational, Clinical Trial Sample.

    Science.gov (United States)

    Silberschmidt, Amy; Lee, Susanne; Zanarini, Mary; Schulz, S Charles

    2015-12-01

    This study aims to extend previous research by considering gender differences in borderline personality (BPD) using both dimensional self-reported and clinical measures of symptomatology. Drawing from a cross-cultural, clinical trial sample, the authors compared female and male BPD subjects (N = 770; 211 male) between the ages of 18 and 65 using diagnostic and self-report data. The authors found that women with BPD have greater hostility and relationship disruption than men. Gender differences in eating disorders, particularly bulimia, are more divergent than in the general population. Generally, gender differences in BPD in this sample are consistent with known general population differences. Women show greater overall symptomatology, including depressive, anxious, and somatic symptoms. Men have higher rates of antisocial personality disorder and a trend toward higher rates of narcissistic personality disorder. However, several gender differences consistently found in the general population are not present in this BPD sample. There are no differences in aggression, suicidality, substance abuse, panic disorder, or obsessive-compulsive disorder. Gender differences in major depression and posttraumatic stress disorder are attenuated. These findings support the conclusion that BPD may diminish normal gender differences. PMID:25562535

  3. The impact of drug samples on clinical recommendations in dental education.

    Science.gov (United States)

    Hujoel, Philippe P; Gillette, Jane

    2011-10-01

    The distribution of branded drug samples in an educational setting may obscure the line between an evidence-based and a marketing-based education. The aim of this study was to evaluate the impact of branded drug samples on clinical recommendations in a dental education setting. Survey respondents exposed (n=95) and unexposed (n=80) to a specific branded drug sample program containing non-patented ingredients responded to a brief questionnare on recommendations for dentinal hypersensitivity. The results showed that an exposure to the branded drug sample was associated with twelve-fold increased odds for recommending the brand name (95 percent confidence interval [CI]: 5.8-24.5; precommending the therapeutic class to which the branded drug sample belonged (95 percent CI: 1.1-6.0; p=0.02); a 66 percent reduction in odds for considering other therapeutic classes (95 percent CI: 0.18-0.63; p=0.00015); and an 84 percent reduction in considering etiology (95 percent CI: 0.08-0.35; precommendation, narrowing therapeutic thinking, and decreased consideration for removing causes. PMID:22012775

  4. Quantitation of sulfate and thiosulfate in clinical samples by ion chromatography.

    Science.gov (United States)

    Cole, D E; Evrovski, J

    1997-11-21

    For assay of serum sulfate, quantitation by ion conductimetry after separation by anion-exchange chromatography is the method of choice. In comparison to classical barium precipitation methods, chromatographic methods demonstrate increased precision, specificity and sensitivity, and they may be superior to spectrophotometric methods that rely on organic cation precipitation of sulfate. The increased sensitivity and specificity, as well as the inherent capacity of chromatographic methods for simultaneous determination of other anions, has led to its increasing use in the determination of excreted sulfate in clinical profiles of urinary anion composition. Ion chromatography can also be used to quantitate free sulfate in other clinical samples, including cerebrospinal fluid, sweat, saliva, breast milk and human tissues. Finally, ion chromatography shows promise as a more precise and sensitive method for measurement of total acid-labile sulfoesters and thiosulfate.

  5. Survey and Rapid detection of Bordetella pertussis in clinical samples targeting the BP485 in China

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    Wei eLiu

    2015-03-01

    Full Text Available Bordetella pertussis is an important human respiratory pathogen. Here, we describe a loop-mediated isothermal amplification (LAMP method for the rapid detection of B. pertussis in clinical samples based on a visual test. The LAMP assay detected the BP485 target sequence within 60 min with a detection limit of 1.3 pg/µl, a 10-fold increase in sensitivity compared with conventional PCR. All 31 non-pertussis respiratory pathogens tested were negative for LAMP detection, indicating the high specificity of the primers for B. pertussis. To evaluate the application of the LAMP assay to clinical diagnosis, of 105 sputum and nasopharyngeal samples collected from the patients with suspected respiratory infections in China, a total of 12 Bordetella pertussis isolates were identified from 33 positive samples detected by LAMP-based surveillance targeting BP485. Strikingly, a 4.5 months old baby and her mother were found to be infected with B. pertussis at the same time. All isolates belonged to different B. pertussis multilocus sequence typing (MLST groups with different alleles of the virulence-related genes including 4 alleles of ptxA, 6 of prn, 4 of tcfA, 2 of fim2 and 3 of fim3. The diversity of B. pertussis carrying toxin genes in clinical strains indicates a rapid and continuing evolution of B. pertussis. This combined with its high prevalence will make it difficult to control. In conclusion, we have developed a visual detection LAMP assay, which could be a useful tool for rapid B. pertussis detection, especially in situations where resources are poor and in point-of-care tests.

  6. Vibrio parahaemolyticus Strains of Pandemic Serotypes Identified from Clinical and Environmental Samples from Jiangsu, China

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    Jingjiao eLi

    2016-05-01

    Full Text Available Vibrio parahaemolyticus has emerged as a major foodborne pathogen in China, Japan, Thailand and other Asian countries. In this study, 72 strains of V. parahaemolyticus were isolated from clinical and environmental samples between 2006 and 2014 in Jiangsu, China. The serotypes and six virulence genes including thermostable direct hemolysin (TDR and TDR-related hemolysin (TRH genes were assessed among the isolates. Twenty five serotypes were identified and O3:K6 was one of the dominant serotypes. The genetic diversity was assessed by multilocus sequence typing (MLST analysis, and 48 sequence types (STs were found, suggesting this V. parahaemolyticus group is widely dispersed and undergoing rapid evolution. A total of 25 strains of pandemic serotypes such as O3:K6, O5:K17 and O1:KUT were identified. It is worth noting that the pandemic serotypes were not exclusively identified from clinical samples, rather, nine strains were also isolated from environmental samples; and some of these strains harbored several virulence genes, which may render those strains pathogenicity potential. Therefore, the emergence of these environmental pandemic V. parahaemolyticus strains may poses a new threat to the public health in China. Furthermore, six novel serotypes and 34 novel STs were identified among the 72 isolates, indicating that V. parahaemolyticus were widely distributed and fast evolving in the environment in Jiangsu, China. The findings of this study provide new insight into the phylogenic relationship between V. parahaemolyticus strains of pandemic serotypes from clinical and environmental sources and enhance the MLST database; and our proposed possible O- and K- antigen evolving paths of V. parahaemolyticus may help understand how the serotypes of this dispersed bacterial population evolve.

  7. Rapid detection of Candida albicans by polymerase spiral reaction assay in clinical blood samples

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    Xiaoqun eJiang

    2016-06-01

    Full Text Available Candida albicans is the most common human yeast pathogen which causes mucosal infections and invasive fungal diseases. Early detection of this pathogen is needed to guide preventative and therapeutic treatment. The aim of this study was to establish a polymerase spiral reaction (PSR assay that rapidly and accurately detects C. albicans and to assess the clinical applicability of PSR-based diagnostic testing. Internal transcribed spacer 2 (ITS2, a region between 5.8S and 28S fungal ribosomal DNA, was used as the target sequence. Four primers were designed for amplification of ITS2 with the PSR method, which was evaluated using real time turbidity monitoring and visual detection using a pH indicator. Fourteen non- C. albicans yeast strains were negative for detection, which indicated the specificity of PSR assay was 100%. A 10-fold serial dilution of C. albicans genomic DNA was subjected to PSR and conventional PCR to compare their sensitivities. The detection limit of PSR was 6.9 pg/µl within 1 h, 10-fold higher than that of PCR (69.0 pg/µl. Blood samples (n=122 were collected from intensive care unit and hematological patients with proven or suspected C. albicans infection at two hospitals in Beijing, China. Both PSR assay and the culture method were used to analyze the samples. Of the 122 clinical samples, 34 were identified as positive by PSR. The result was consistent with those obtained by the culture method. In conclusion, a novel and effective C. albicans detection assay was developed that has a great potential for clinical screening and point-of-care testing.

  8. Statistical methods for detecting differentially abundant features in clinical metagenomic samples.

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    James Robert White

    2009-04-01

    Full Text Available Numerous studies are currently underway to characterize the microbial communities inhabiting our world. These studies aim to dramatically expand our understanding of the microbial biosphere and, more importantly, hope to reveal the secrets of the complex symbiotic relationship between us and our commensal bacterial microflora. An important prerequisite for such discoveries are computational tools that are able to rapidly and accurately compare large datasets generated from complex bacterial communities to identify features that distinguish them.We present a statistical method for comparing clinical metagenomic samples from two treatment populations on the basis of count data (e.g. as obtained through sequencing to detect differentially abundant features. Our method, Metastats, employs the false discovery rate to improve specificity in high-complexity environments, and separately handles sparsely-sampled features using Fisher's exact test. Under a variety of simulations, we show that Metastats performs well compared to previously used methods, and significantly outperforms other methods for features with sparse counts. We demonstrate the utility of our method on several datasets including a 16S rRNA survey of obese and lean human gut microbiomes, COG functional profiles of infant and mature gut microbiomes, and bacterial and viral metabolic subsystem data inferred from random sequencing of 85 metagenomes. The application of our method to the obesity dataset reveals differences between obese and lean subjects not reported in the original study. For the COG and subsystem datasets, we provide the first statistically rigorous assessment of the differences between these populations. The methods described in this paper are the first to address clinical metagenomic datasets comprising samples from multiple subjects. Our methods are robust across datasets of varied complexity and sampling level. While designed for metagenomic applications, our software

  9. Unreliability of results of PCR detection of Helicobacter pylori in clinical or environmental samples.

    Science.gov (United States)

    Sugimoto, Mitsushige; Wu, Jeng-Yih; Abudayyeh, Suhaib; Hoffman, Jill; Brahem, Hajer; Al-Khatib, Khaldun; Yamaoka, Yoshio; Graham, David Y

    2009-03-01

    The aim of this study was to compare published Helicobacter pylori primer pairs for their ability to reliably detect H. pylori in gastric biopsy specimens and salivary samples. Detection limits of the 26 PCR primer pairs previously described for detection of H. pylori DNA in clinical samples were determined. Sensitivity and specificity were determined using primers with detection limits of pylori-positive and -negative (by concordance by culture and histology) coded gastric biopsy specimens. These results were then confirmed with gastric biopsy specimens and saliva from patients with confirmed H. pylori status. Five of the twenty-six previously reported primer pairs (HP64-f/HP64-r, HP1/HP2, EHC-U/EHC-L, VAG-F/VAG-R, and ICT37/ICT38) had detection limits of 90% with gastric biopsy specimens. No combinations of primer pairs improved the results. Using these five primer pairs, 54% of the positive saliva samples were determined to be false positive; both the HP64-f/HP64-r and the HP1/HP2 sets produced false positives with saliva. We conclude that clinicians should not rely on results using current PCR primers alone to decide the H. pylori status of an individual patient or as a basis for treatment decisions. The results of studies based on PCR identification of H. pylori in environmental samples should be viewed with caution. Possibly, specific primers sets can be identified based on the presence of multiple putative virulence factor genes. PMID:19129407

  10. Clinical utility and performance of sock sampling in weaner pig diarrhoea

    DEFF Research Database (Denmark)

    Pedersen, Ken Steen; Okholm, Elisabeth; Johansen, Markku;

    2015-01-01

    , agreement between three consecutive herd examinations from the same herd and agreement between quantitative PCR results from pooled faecal samples and sock samples.Twenty-four veterinarians submitted faecal and sock samples for quantitative PCR testing from outbreaks of diarrhoea in nursery pigs (n=38 herds......’ clinical aetiological diagnosis and the pooled faecal sample was 0.18 (95% CL: 0.08–0.34), and Cohen’s Kappa was 0.03 (95% CL: −0.08 to 0.14). Antibiotic treatment or prevention strategies were changed in 0.63 (95% CL: 0.46–0.78) of the herds, and the veterinarians indicated that, for 0.32 (95% CL: 0......, and Cohen’s Kappa was 0.61 (95% CI: 0.26–0.95). In relation to detection of the individual infections, agreement was 0.63 (95% CI: 0.46–0.78), and Cohen’s Kappa was 0.53 (95% CI: 0.34–0.71).In conclusion, low pathogen diarrhoea is a common finding amongst diarrhoea outbreaks that are subjected to antibiotic...

  11. Coping behavior in normal and clinical samples: more similarities than differences?

    Science.gov (United States)

    Seiffge-Krenke, I

    1993-09-01

    In our studies we tried to integrate a developmental and a clinical perspective on coping and adaptation in adolescence. Starting with a review of the author's own research, involving over 3000 12- to 20-year-olds from various cultures, the problems typical of this developmental phase and the ways of coping with these normative demands are presented. The results show that coping skills of young people in dealing with age-specific problems have so far been considerably underestimated. Their response to problems stemming from different developmental fields such as parents, peers, school or future involved three main modes of coping: Active Coping, Internal Coping and Withdrawal. Withdrawal was employed very rarely and only for certain types of problems. Age, gender and problem-specific effects in coping were found. Whereas normal adolescents most frequently choose to cope with difficulties actively by means of social resources and to think out possible solutions, risk populations appear to have a more ambivalent pattern of coping strategies with high functionality and high dysfunctionality. Even their appraisal of problems is already disturbed; they feel more readily threatened by everyday problem situations and respond more uniformly with withdrawal. Finally, similarities between the female coping style in normal samples and the more pronounced ambivalent pattern in clinical samples were discussed and related to psychopathology. PMID:8282899

  12. Cognitive Predictors of Verbal Memory in a Mixed Clinical Pediatric Sample

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    Shelley C. Heaton

    2013-08-01

    Full Text Available Verbal memory problems, along with other cognitive difficulties, are common in children diagnosed with neurological and/or psychological disorders. Historically, these “memory problems” have been poorly characterized and often present with a heterogeneous pattern of performance across memory processes, even within a specific diagnostic group. The current study examined archival neuropsychological data from a large mixed clinical pediatric sample in order to understand whether functioning in other cognitive areas (i.e., verbal knowledge, attention, working memory, executive functioning may explain some of the performance variability seen across verbal memory tasks of the Children’s Memory Scale (CMS. Multivariate analyses revealed that among the cognitive functions examined, only verbal knowledge explained a significant amount of variance in overall verbal memory performance. Further univariate analyses examining the component processes of verbal memory indicated that verbal knowledge is specifically related to encoding, but not the retention or retrieval stages. Future research is needed to replicate these findings in other clinical samples, to examine whether verbal knowledge predicts performance on other verbal memory tasks and to explore whether these findings also hold true for visual memory tasks. Successful replication of the current study findings would indicate that interventions targeting verbal encoding deficits should include efforts to improve verbal knowledge.

  13. Childhood trauma associates with clinical features of schizophrenia in a sample of Chinese inpatients.

    Science.gov (United States)

    Li, Xian-Bin; Li, Qi-Yong; Liu, Jin-Tong; Zhang, Liang; Tang, Yi-Lang; Wang, Chuan-Yue

    2015-08-30

    This study examined the association between childhood trauma and clinical features, comorbid anxiety and post-traumatic stress disorder (PTSD) symptoms, and suicidal and aggressive behaviors in Chinese patients with schizophrenia. The Childhood Trauma Questionnaire - Short Form (CTQ-SF), the Impact of Events Scale - Revised (IES-R), and the State-Trait Anxiety Inventory (STAI) were administered to 182 Chinese inpatients with schizophrenia. The relationship between the severity and the number of traumic experiences and clinical features were analyzed. Physical neglect (PN) in childhood was reported in 71.7% of this sample, followed by emotional neglect (EN, 58.6%), sexual abuse (SA, 39.9%), emotional abuse (EA, 31.7%) and physical abuse (PA, 22.2%). Significant negative correlations existed between age of onset and the EA scores. Significant positive correlations were found between the subscores of IES-R, STAI and CTQ-SF. Patients with history of suicidal or aggressive behaviors had significantly higher trauma scores than patients without such behaviors. Exposure to childhood trauma is associated with early age of onset, more PTSD and anxiety symptoms, and history of suicidal and aggressive behaviors. A dose-effect may exist between severity, number of trauma experiences, and clinical features. PMID:26096662

  14. A review on the use of NEO-PI-R validity scales in normative, job selection, and clinical samples

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    Angel Blanch

    2009-06-01

    Full Text Available Background and Objectives: In this study we review the use of the Positive Presentation Management (PPM and Negative Presentation Management (NPM scales, two NEO-PI-R derived measures originally devised to control for biased and distorted responses. These scales have been used with normative, job selection and clinical samples, in cross-sectional and experimental studies. Methods: Web-based and manual searches in personality and psychological assessment journals were conducted, and information on the PPM and NPM scales was systematically recorded. Means, standard deviations and reliability coefficients were summarized and compared between three types of samples: normative, job selection and clinical. Results: Five studies were performed with normative samples (33%, 3 with employment samples (20% and 7 with clinical samples (47%. Cross-sectional designs were most common (60%, although there were also experimental studies (40%. Reported reliability coefficients were lower than usually accepted. There were differences in mean PPM and NPM scores in regard to the study sample background. Conclusions: There were some discrepancies when reporting PPM and NPM results across the reviewed studies. Normative and employment samples scored higher in PPM than clinical samples. Clinical samples scored higher in NPM than normative and employment samples The PPM and NPM scales could be useful in applied situations, although parallel sources of information should be taken into account to detect distorted responses to the questionnaire. However, the results on these scales should be systematically reported in future studies.

  15. Clinical illnesses associated with isolation of dysgonic fermenter 3 from stool samples.

    Science.gov (United States)

    Blum, R N; Berry, C D; Phillips, M G; Hamilos, D L; Koneman, E W

    1992-02-01

    The clinical significance of the fastidious organism DF-3 isolated from stool cultures is unclear. We sought to improve our understanding of this organism and to further define its association with human disease. Stool cultures for DF-3 were obtained from three sources: an ongoing study of enteric pathogens in patients infected with the human immunodeficiency virus, a screening procedure in which all stool samples submitted for Clostridium difficile toxin assay were cultured for DF-3, and stool samples submitted specifically for DF-3 culture. Retrospective clinical data were obtained from chart reviews of patients with positive cultures. Antimicrobial susceptibility testing and cell wall fatty acid analysis were performed for each DF-3 isolated. Eight isolates of DF-3 were obtained over a period of 8 months. All patients either had severe underlying disease or were immunocompromised, including three patients coinfected with human immunodeficiency virus and two patients with inflammatory bowel disease. The spectrum of clinical disease ranged from chronic diarrhea with a well-defined response to therapy for DF-3 to an asymptomatic carrier state. Cell wall fatty acid analysis of these isolates demonstrated a consistent pattern with a large peak of 12-methyltetradecanoate. DF-3, a fastidious gram-negative coccobacillus, can be recovered from stool cultures of immunocompromised patients by using selective media. The presence of 12-methyltetradecanoate in cell wall fatty acid analysis assists in identification. The increased use of a selective medium-(cefoperazone-vancomycin-amphotericin B) in the evaluation of diarrhea in immunocompromised hosts, including persons with inflammatory bowel disease, may better define the association of DF-3 with human gastrointestinal disease.

  16. Evaluation of dengue NS1 antigen rapid tests and ELISA kits using clinical samples.

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    Subhamoy Pal

    Full Text Available Early diagnosis of dengue virus (DENV infection can improve clinical outcomes by ensuring close follow-up, initiating appropriate supportive therapies and raising awareness to the potential of hemorrhage or shock. Non-structural glycoprotein-1 (NS1 has proven to be a useful biomarker for early diagnosis of dengue. A number of rapid diagnostic tests (RDTs and enzyme-linked immunosorbent assays (ELISAs targeting NS1 antigen (Ag are now commercially available. Here we evaluated these tests using a well-characterized panel of clinical samples to determine their effectiveness for early diagnosis.Retrospective samples from South America were used to evaluate the following tests: (i "Dengue NS1 Ag STRIP" and (ii "Platelia Dengue NS1 Ag ELISA" (Bio-Rad, France, (iii "Dengue NS1 Detect Rapid Test (1st Generation" and (iv "DENV Detect NS1 ELISA" (InBios International, United States, (v "Panbio Dengue Early Rapid (1st generation" (vi "Panbio Dengue Early ELISA (2nd generation" and (vii "SD Bioline Dengue NS1 Ag Rapid Test" (Alere, United States. Overall, the sensitivity of the RDTs ranged from 71.9%-79.1% while the sensitivity of the ELISAs varied between 85.6-95.9%, using virus isolation as the reference method. Most tests had lower sensitivity for DENV-4 relative to the other three serotypes, were less sensitive in detecting secondary infections, and appeared to be most sensitive on Day 3-4 post symptom onset. The specificity of all evaluated tests ranged from 95%-100%.ELISAs had greater overall sensitivity than RDTs. In conjunction with other parameters, the performance data can help determine which dengue diagnostics should be used during the first few days of illness, when the patients are most likely to present to a clinic seeking care.

  17. Prevalence of Extended –Spectrum-Beta-Lactamase-Producing Klebsiella Pneumonia Isolates from Clinical Samples

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    Alizade, H. (MSc

    2014-06-01

    Full Text Available Background and Objective: Klebsiella pneumonia (K.pneumonia is one of the common causes of nosocomial infections. The aim of this research was to determine the prevalence of beta-lactamase genes and phenotypic confirmation of extended–spectrum-beta-lactamase (ESBL producing K.pneumonia isolates from clinical samples. Material and Methods: In this study, 122 K.pneumonia were isolated from clinical specimens of Khoramabad city and were confirmed by standard bacteriological tests. The presence of ESBL enzymes was detected by combined disk diffusion method. PCR assay with specific primers was used to determine blaSHV, blaTEM, blaCTX-15 and blaCTX-M genes in the confirmed isolates. Results: of 122 K.pneumonia isolates, 78 (64.18% were positive for ESBL, using disk diffusion method. According to antibiogram results, 10.65% of isolates were resistant to cefotaxime, 3.27% to ceftazidime and 68.03% to both antibiotics. Ninety isolates (64.18% considered as ESBLs isolates, at the same time, with being resistant to cefotaxime and ceftazidime were also sensitive to cefotaxime-clavulanic acid and ceftazidime-clavulanic acid. In PCR assays, blaCTX-15, blaSHV, blaCTX-M and blaTEM genes were detected in 78.68%, 40.16%, 26.22% and 22.13% of isolates, respectively. Ten resistant patterns of genes were detected. Conclusion: The significance percentage of antibiotic resistant genes of K.pneumonia isolates from clinical samples in Khoramabad city had ESBLs genes; CTX-M category was the most prevalent encoding genes of these enzymes. Keywords: Klebsiella Pneumonia, Extended-Spectrum Beta-Lactamase, Antibiotic Resistance

  18. Efficient adaptive designs with mid-course sample size adjustment in clinical trials

    CERN Document Server

    Bartroff, Jay

    2011-01-01

    Adaptive designs have been proposed for clinical trials in which the nuisance parameters or alternative of interest are unknown or likely to be misspecified before the trial. Whereas most previous works on adaptive designs and mid-course sample size re-estimation have focused on two-stage or group sequential designs in the normal case, we consider here a new approach that involves at most three stages and is developed in the general framework of multiparameter exponential families. Not only does this approach maintain the prescribed type I error probability, but it also provides a simple but asymptotically efficient sequential test whose finite-sample performance, measured in terms of the expected sample size and power functions, is shown to be comparable to the optimal sequential design, determined by dynamic programming, in the simplified normal mean case with known variance and prespecified alternative, and superior to the existing two-stage designs and also to adaptive group sequential designs when the al...

  19. The frequency of Listeria monocytogenes strains recovered from clinical and non-clinical samples using phenotypic methods and confirmed by PCR

    OpenAIRE

    abazar pournajaf; lida lotfollahi; gholamreza irajian; abdollah ardebili; Behrooz Sadeghi kalani; mojtabata Taghizadeh armaki

    2013-01-01

    Background: Listeria monocytogenes is a facultative intracellular pathogen that causes listeriosis which has extensive clinical manifestations. Infections with L. monocytogenes are a serious threat to immunocompromised persons. The aim of this study was to determine the frequency of L. monocytogenes strains recovered from clinical and non-clinical samples using phenotypic methods and confirmed by PCR. Materials and Methods: In this study, 617 specimens were analyzed. All specimens were cu...

  20. Psychometric properties of the child and parent versions of Spence children's anxiety scale in a Danish community and clinical sample.

    Science.gov (United States)

    Arendt, Kristian; Hougaard, Esben; Thastum, Mikael

    2014-12-01

    This study examined the psychometric properties and norms of the Spence Children's Anxiety Scale (SCAS) and the associated parent version (SCAS-P) in a Danish community and a clinical sample. The total sample consisted of 1240 children (972 from community sample), age 7-17 years, and 805 parents (537 from community sample). Results indicated that SCAS and SCAS-P had good internal consistency on the total scale and all subscales, with exception of the subscale for fear of physical injury. Both scales showed satisfactory 2-week and 3-month retest stability. All subscales and total scales of the SCAS and SCAS-P discriminated between the clinical and community sample. A comparison with the Beck Youth Inventories and the Strength and Difficulty Questionnaire in the clinical sample supported the scales' convergent and divergent validity. Results of confirmatory factor analyses for SCAS and SCAS-P were in favor of the original model with six correlated factors.

  1. Evaluation of Inhibitor-Resistant Real-Time PCR Methods for Diagnostics in Clinical and Environmental Samples

    OpenAIRE

    Adrienne Trombley Hall; Ashley McKay Zovanyi; Deanna Rose Christensen; Jeffrey William Koehler; Timothy Devins Minogue

    2013-01-01

    Polymerase chain reaction (PCR) is commonly used for pathogen detection in clinical and environmental samples. These sample matrices often contain inhibitors of PCR, which is a primary reason for sample processing; however, the purification process is highly inefficient, becoming unacceptable at lower signature concentrations. One potential solution is direct PCR assessment without sample processing. Here, we evaluated nine inhibitor-resistant PCR reagents for direct detection of Francisella ...

  2. The phenomenology of the first panic attack in clinical and community-based samples.

    Science.gov (United States)

    Pané-Farré, Christiane A; Stender, Jan P; Fenske, Kristin; Deckert, Jürgen; Reif, Andreas; John, Ulrich; Schmidt, Carsten Oliver; Schulz, Andrea; Lang, Thomas; Alpers, Georg W; Kircher, Tilo; Vossbeck-Elsebusch, Anna N; Grabe, Hans J; Hamm, Alfons O

    2014-08-01

    The purpose of the study was to contrast first panic attacks (PAs) of patients with panic disorder (PD) with vs. without agoraphobia and to explore differences between first PAs leading to the development of PD and those that remain isolated. Data were drawn from a community survey (N=2259 including 88 isolated PAs and 75 PD cases). An additional sample of 234 PD patients was recruited in a clinical setting. A standardized interview assessed the symptoms of the first PA, context of its occurrence and subsequent coping attempts. Persons who developed PD reported more severe first PAs, more medical service utilization and exposure-limiting coping attempts than those with isolated PAs. The context of the first PA did not differ between PD and isolated PAs. PD with agoraphobia was specifically associated with greater symptom severity and occurrence of first attacks in public. Future research should validate these findings using a longitudinal approach. PMID:24973697

  3. Factor structure of the SOCRATES in a clinical sample of adolescents.

    Science.gov (United States)

    Maisto, Stephen A; Chung, Tammy A; Cornelius, Jack R; Martin, Christopher S

    2003-06-01

    This study investigated the Stages of Change Readiness and Treatment Eagerness Scale (SOCRATES; W. R. Miller & J. S. Tonigan, 1996) in adolescents presenting for treatment of alcohol use disorder (AUD). The participants were 80 males and 43 females (mean age = 16.8 years) who presented for AUD treatment (95.1% outpatient, 4.9% inpatient). Participants completed assessments at baseline and 1 year and provided information on alcohol use and related variables monthly between these 2 assessments. Principal-components and confirmatory factor analyses of the baseline SOCRATES identified 2 factors, Taking Steps and Recognition, which showed good internal consistency and concurrent and predictive evidence of validity. The results were interpreted as supporting the use of the SOCRATES with clinical samples of adolescents.

  4. Self-esteem in a clinical sample of morbidly obese children and adolescents

    DEFF Research Database (Denmark)

    Nowicka, P; Höglund, P; Birgerstam, P;

    2009-01-01

    AIM: To study self-esteem in clinical sample of obese children and adolescents. METHODS: Obese children and adolescents aged 8-19 years (n = 107, mean age 13.2 years, mean BMI 32.5 [range 22.3-50.6], mean BMI z-score 3.22 [range 2.19-4.79]; 50 boys and 57 girls) were referred for treatment...... of primary obesity. Self-esteem was measured with a validated psychological test with five subscales: physical characteristics, talents and skills, psychological well-being, relations with the family and relations with others. A linear mixed effect model used the factors gender and adolescence group......, and the continuous covariates: BMI z-scores, and BMI for the parents as fixed effects and subjects as random effects. RESULTS: Age and gender, but neither the child's BMI z-score nor the BMI of the parents were significant covariates. Self-esteem decreased (p

  5. PNA-based fluorescence in situ hybridization for identification of bacteria in clinical samples

    DEFF Research Database (Denmark)

    Fazli, Mustafa; Bjarnsholt, Thomas; Høiby, Niels;

    2014-01-01

    Fluorescence in situ hybridization with PNA probes (PNA-FISH) that target specific bacterial ribosomal RNA sequences is a powerful and rapid tool for identification of bacteria in clinical samples. PNA can diffuse readily through the bacterial cell wall due to its uncharged backbone, and PNA......-FISH can be performed with high specificity due to the extraordinary thermal stability of RNA-PNA hybrid complexes. We describe a PNA-FISH procedure and provide examples of the application of PNA-FISH for the identification of bacteria in chronic wounds, cystic fibrosis lungs, and soft tissue fillers....... In all these cases, bacteria can be identified in biofilm aggregates, which may explain their recalcitrance to antibiotic treatment....

  6. Evaluation of Microarray Preprocessing Algorithms Based on Concordance with RT-PCR in Clinical Samples

    DEFF Research Database (Denmark)

    Hansen, Kasper Lage; Szallasi, Zoltan Imre; Eklund, Aron Charles;

    2009-01-01

    Several preprocessing algorithms for Affymetrix gene expression microarrays have been developed, and their performance on spike-in data sets has been evaluated previously. However, a comprehensive comparison of preprocessing algorithms on samples taken under research conditions has not been...... that were most consistent with RT-PCR measurements, although the difference in performance between most of the algorithms was not statistically significant. CONCLUSIONS/SIGNIFICANCE: Our results support the choice of PLIER+16 for the preprocessing of clinical Affymetrix microarray data. However, other...... performed. METHODOLOGY/PRINCIPAL FINDINGS: We used TaqMan RT-PCR arrays as a reference to evaluate the accuracy of expression values from Affymetrix microarrays in two experimental data sets: one comprising 84 genes in 36 colon biopsies, and the other comprising 75 genes in 29 cancer cell lines. We...

  7. Preliminary Examination of the Interpersonal Psychological Theory of Suicide in an Adolescent Clinical Sample.

    Science.gov (United States)

    Horton, Sarah E; Hughes, Jennifer L; King, Jessica D; Kennard, Betsy D; Westers, Nicholas J; Mayes, Taryn L; Stewart, Sunita M

    2016-08-01

    This study offers a preliminary examination of the Interpersonal-Psychological Theory of Suicide (IPTS; Joiner 2005) in an adolescent clinical sample. The IPTS offers a nuanced framework that has many conceptual and practical merits. Although this theory has a growing base of evidence among adults, it has yet to be tested in adolescents using direct measures of its central constructs. Participants were 147 adolescents (76.2 % girls) on an inpatient psychiatric unit, who completed measures of key IPTS constructs of thwarted belongingness, perceived burdensomeness, acquired capability for suicide, as well as depression severity, hopelessness, and severity of suicidal symptoms. Our findings were largely consistent with hypotheses derived from the IPTS: perceived burdensomeness, and at a marginal level, thwarted belongingness, were independently associated with current suicidal ideation. The thwarted belongingness by perceived burdensomeness interaction marginally distinguished between adolescents with passive and active suicidal ideation. Acquired capability for suicide was associated with recent suicidal intent. Examination of all three IPTS constructs simultaneously revealed main effects of each construct (with a marginal effect of thwarted belongingness), and interaction effects for thwarted belongingness by perceived burdensomeness, and thwarted belongingness by perceived burdensomeness by acquired capability for suicide in association with suicidal symptom severity. Sex, age, depression severity, and hopelessness were controlled in all analyses. This study offers strong, albeit preliminary, support of the IPTS in a clinical adolescent sample. Assessment of IPTS constructs may be useful in determining persistent risk for suicide attempt. Prospective tests of the theory, and extensions to intervention and prevention should be considered in future IPTS research. PMID:26667025

  8. [Histological view of ethics in medicine and handling of residual samples in clinical laboratories].

    Science.gov (United States)

    Yoshida, Hiroshi

    2004-03-01

    One of the important ethical issues in clinical laboratory medicine is whether organs and/or specimens should belong to the examinees. Tracing back to ancient Greece, an episode of the death of Asklepios, killed by Zeus to revive the dead, and the great contribution of Hippocrates to medicine including the vow and ethics of medicine, have been described. In the relationship between doctors and patients, the former had been superior to the latter for more than 2400 years, however, the situation has been changing from that to the same position since 1960th, along with the development of bioethics from medical ethics. For the promotion of bioethics, world medical associations have contributed declarations and continuous discussion. The declarations are based on the avoidance of actions detrimental to the life, health, privacy or dignity of examinees. On the medical use of human organs and specimens in relation to human rights, the mind and the body, discussion has continued, however, a consensus on the details has not been reached. A view on the use of residual samples for methodological study, teaching and research in the clinical laboratory was proposed by the Japanese Society of Laboratory Medicine in 2002. Briefly, it included confidentiality of the laboratory staff, responsibility of the laboratory director, the absence of a necessity to obtain consent for the use of residual samples for methodological study when they are made anonymous or pooled, and the recommendation to obtain a judgement by an ethics committee for research use. The background and discussion for the proposal and the current situation on how to obtain consent from patients in Japan are mentioned.

  9. [Occurrence and antimicrobial susceptibility of Morganella morganii strains isolated from clinical samples].

    Science.gov (United States)

    Zalas-Wiecek, Patrycja; Gospodarek, Eugenia; Wróblewska, Joanna

    2012-01-01

    The aim of this study was the evaluation of occurrence and antimicrobial susceptibility of M morganii rods isolated from clinical samples. This study included 201 strains isolated in the Clinical Microbiology Department of Dr. A. Jurasz University Hospital in 2008-2010. Identification to species was carried out on the basis of the results of biochemical reactions included in the tests ID 32E and VITEK2 GN. Antimicrobial susceptibility of M. morganii rods was determined by the disk-diffusion method on Mueller-Hinton II Agar. Strains of M morganii most commonly isolated from skin and soft tissue, and material taken from the urinary tract, mainly from patients of Anesthesiology and Intensive Care Unit, Department of General and Vascular Surgery and Department of General Surgery and Endocrinology. All of M morganii strains isolated during the three years were susceptible to carbapenems. We reported decrease of strains susceptible to piperacillin and chloramphenicol. In 2010 we showed a higher percentage of strains intermediate to tigecycline, compared with 2009. We observed increase in the percentage of strains resistant to cefoperazone with sulbactam and reported decrease in the percentage of strains resistant and intermediate to aminoglycosides. Extended Spectrum Beta-Lactamases were produced by 13 (6,5%) of M morganii strains.

  10. IDENTIFICATION OF CANDIDA SPECIES FROM CLINICAL SAMPLES AND THEIR ANTIFUNGAL SUSCEPTIBILITY PATTERNS

    Directory of Open Access Journals (Sweden)

    Bhaskar

    2015-09-01

    Full Text Available OBJECTIVE AND BACKGROUND : The incidence of Candida infections has increased dramatically over the past few decades due to increase in the number of population susceptible to fungal infections. With multiple antifungal ag ents that are available and recovery of clinical isolates that exhibit inherent or developed resistance to commonly used antifungal agents, it has become imperative to do susceptibility testing routinely. The study was done to determine the predisposing fa ctors, species incidence and susceptibility pattern of Candida isolates to commonly use d antifungal agents. METHODS: A total of 108 Candida species were recovered from symptomatic clinical cases. Candida isolates were speciated by germ tube test, chlamydospore formation on corn meal agar and color produced on chromogenic media. Antifungal susceptibility test was done by disk diffusion method for nystatin, fluconazole, itraconazole, voriconazole and amphotericin - B. RESULTS: Candida albicans is the m ost frequently isolated species. However, non - albicans Candida species, taken as a group has predominated in clinical samples. Chromogenic agar medium showed good correlation in species identification in comparison with conventional germ tube test and chla mydospore formation on corn meal agar. C. albicans (41, C. tropicalis (33, C. krusei (30 and C. glabrata (04 were isolated. Candida species showed 95.4% susceptibility to amphotericin - B, 77.8% to voriconazol e, 69.4% to nystatin, 64.1% to f luconazole an d 63.9% to itraconazole. CONCLUSION : Increasing incidence of non - albicans species infection. Chromogenic medium can be used for species identification. Increasing resistance of Candida species to commonly used antifungal agents.

  11. A Multitrait-Multimethod Analysis of the Construct Validity of Child Anxiety Disorders in a Clinical Sample

    Science.gov (United States)

    Langer, David A.; Wood, Jeffrey J.; Bergman, R. Lindsey; Piacentini, John C.

    2010-01-01

    The present study examines the construct validity of separation anxiety disorder (SAD), social phobia (SoP), panic disorder (PD), and generalized anxiety disorder (GAD) in a clinical sample of children. Participants were 174 children, 6 to 17 years old (94 boys) who had undergone a diagnostic evaluation at a university hospital based clinic.…

  12. Intra-tumoral Heterogeneity of KRAS and BRAF Mutation Status in Patients with Advanced Colorectal Cancer (aCRC and Cost-Effectiveness of Multiple Sample Testing

    Directory of Open Access Journals (Sweden)

    Susan D. Richman

    2011-01-01

    Full Text Available KRAS mutation status is established as a predictive biomarker of benefit from anti-EGFr therapies. Mutations are normally assessed using DNA extracted from one formalin-fixed, paraffin-embedded (FFPE tumor block. We assessed heterogeneity of KRAS and BRAF mutation status intra-tumorally (multiple blocks from the same primary tumor. We also investigated the utility and efficiency of genotyping a ‘DNA cocktail’ prepared from multiple blocks. We studied 68 consenting patients in two randomized clinical trials. DNA was extracted, from ≥2 primary tumor FFPE blocks per patient. DNA was genotyped by pyrosequencing for KRAS codons 12, 13 and 61 and BRAF codon 600. In patients with heterogeneous mutation status, DNA cocktails were prepared and genotyped. Among 69 primary tumors in 68 patients, 7 (10.1% showed intratumoral heterogeneity; 5 (7.2% at KRAS codons 12, 13 and 2 (2.9% at BRAF codon 600. In patients displaying heterogeneity, the relevant KRAS or BRAF mutation was also identified in ‘DNA cocktail’ samples when including DNA from mutant and wild-type blocks. Heterogeneity is uncommon but not insignificant. Testing DNA from a single block will wrongly assign wild-type status to 10% patients. Testing more than one block, or preferably preparation of a ‘DNA cocktail’ from two or more tumor blocks, improves mutation detection at minimal extra cost.

  13. Rapid Isolation of Staphylococcus aureus Pathogens from Infected Clinical Samples Using Magnetic Beads Coated with Fc-Mannose Binding Lectin.

    Science.gov (United States)

    Bicart-See, A; Rottman, M; Cartwright, M; Seiler, B; Gamini, N; Rodas, M; Penary, M; Giordano, G; Oswald, E; Super, M; Ingber, D E

    2016-01-01

    Here we describe how Staphylococcus aureus bacteria can be rapidly isolated from clinical samples of articular fluid and synovial tissue using magnetic beads coated with the engineered chimeric human opsonin protein, Fc-mannose-binding lectin (FcMBL). The FcMBL-beads were used to capture and magnetically remove bacteria from purified cultures of 12 S. aureus strains, and from 8 articular fluid samples and 4 synovial tissue samples collected from patients with osteoarthritis or periprosthetic infections previously documented by positive S. aureus cultures. While the capture efficiency was high (85%) with purified S. aureus strains grown in vitro, direct FcMBL-bead capture from the clinical samples was initially disappointing (viscosity of these complex biological samples. Importantly, capture of pathogens using the FcMBL-beads was increased to 76% efficiency by pretreating clinical specimens with hypotonic washes, hyaluronidase and a protease cocktail. Using this approach, S. aureus bacteria could be isolated from infected osteoarthritic tissues within 2 hours after sample collection. This FcMBL-enabled magnetic method for rapid capture and concentration of pathogens from clinical samples could be integrated upstream of current processes used in clinical microbiology laboratories to identify pathogens and perform antibiotic sensitivity testing when bacterial culture is not possible or before colonies can be detected. PMID:27275840

  14. Rapid Isolation of Staphylococcus aureus Pathogens from Infected Clinical Samples Using Magnetic Beads Coated with Fc-Mannose Binding Lectin.

    Directory of Open Access Journals (Sweden)

    A Bicart-See

    Full Text Available Here we describe how Staphylococcus aureus bacteria can be rapidly isolated from clinical samples of articular fluid and synovial tissue using magnetic beads coated with the engineered chimeric human opsonin protein, Fc-mannose-binding lectin (FcMBL. The FcMBL-beads were used to capture and magnetically remove bacteria from purified cultures of 12 S. aureus strains, and from 8 articular fluid samples and 4 synovial tissue samples collected from patients with osteoarthritis or periprosthetic infections previously documented by positive S. aureus cultures. While the capture efficiency was high (85% with purified S. aureus strains grown in vitro, direct FcMBL-bead capture from the clinical samples was initially disappointing (< 5% efficiency. Further analysis revealed that inhibition of FcMBL binding was due to coating of the bacteria by immunoglobulins and immune cells that masked FcMBL binding sites, and to the high viscosity of these complex biological samples. Importantly, capture of pathogens using the FcMBL-beads was increased to 76% efficiency by pretreating clinical specimens with hypotonic washes, hyaluronidase and a protease cocktail. Using this approach, S. aureus bacteria could be isolated from infected osteoarthritic tissues within 2 hours after sample collection. This FcMBL-enabled magnetic method for rapid capture and concentration of pathogens from clinical samples could be integrated upstream of current processes used in clinical microbiology laboratories to identify pathogens and perform antibiotic sensitivity testing when bacterial culture is not possible or before colonies can be detected.

  15. Complete Genome Sequences of Three African Foot-and-Mouth Disease Viruses from Clinical Samples Isolated in 2009 and 2010.

    Science.gov (United States)

    Van Borm, Steven; Rosseel, Toon; Haegeman, Andy; Fana, Mpolokang Elliot; Seoke, Latoa; Hyera, Joseph; Matlho, George; Vandenbussche, Frank; De Clercq, Kris

    2016-01-01

    The complete genome sequences of three foot-and-mouth disease viruses (one virus of each serotype SAT1, SAT2 and O) were directly sequenced from RNA extracted from clinical bovine samples, demonstrating the feasibility of full-genome sequencing from strong positive samples taken from symptomatic animals. PMID:27151795

  16. The Appraisal of Social Concerns Scale: Psychometric Validation with a Clinical Sample of Patients with Social Anxiety Disorder

    Science.gov (United States)

    Schultz, Luke T.; Heimberg, Richard G.; Rodebaugh, Thomas L.; Schneier, Franklin R.; Liebowitz, Michael R.; Telch, Michael J.

    2006-01-01

    The Appraisal of Social Concerns (ASC) Scale was created by Telch et al. (2004) to improve upon existing self-report measures of social anxiety-related cognition. In a largely nonclinical sample, the ASC was found to possess three factors and was psychometrically sound. In a smaller clinical sample, the ASC demonstrated sensitivity to the effects…

  17. How Suitable is Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight for Metabolite Imaging from Clinical Formalin-Fixed and Paraffin-Embedded Tissue Samples in Comparison to Matrix-Assisted Laser Desorption/Ionization-Fourier Transform Ion Cyclotron Resonance Mass Spectrometry?

    Science.gov (United States)

    Buck, Achim; Balluff, Benjamin; Voss, Andreas; Langer, Rupert; Zitzelsberger, Horst; Aichler, Michaela; Walch, Axel

    2016-05-17

    In research and clinical settings, formalin-fixed and paraffin-embedded (FFPE) tissue specimens are collected routinely and therefore this material constitutes a highly valuable source to gather insight in metabolic changes of diseases. Among mass spectrometry techniques to examine the molecular content of FFPE tissue, mass spectrometry imaging (MSI) is the most appropriate when morphological and histological features are to be related to metabolic information. Currently, high-resolution mass spectrometers are widely used for metabolomics studies. However, with regards to matrix-assisted laser desorption/ionization (MALDI) MSI, no study has so far addressed the necessity of instrumental mass resolving power in terms of clinical diagnosis and prognosis using archived FFPE tissue. For this matter we performed for the first time a comprehensive comparison between a high mass resolution Fourier-transform ion cyclotron resonance (FTICR) mass spectrometer and a time-of-flight (TOF) instrument with lower mass resolving power. Spectra analysis revealed that about one-third of the detected peaks remained unresolved by MALDI-TOF, which led to a 3-5 times lower number of m/z features compared to FTICR measurements. Overlaid peak information and background noise in TOF images made a precise assignment of molecular attributes to morphological features more difficult and limited classification approaches. This clearly demonstrates the need for high-mass resolution capabilities for metabolite imaging. Nevertheless, MALDI-TOF allowed reproducing and verifying individual markers identified previously by MALDI-FTICR MSI. The systematic comparison gives rise to a synergistic combination of the different MSI platforms for high-throughput discovery and validation of biomarkers.

  18. Evaluation of the DSM-5 severity indicator for binge eating disorder in a clinical sample

    Science.gov (United States)

    Grilo, Carlos M.; Ivezaj, Valentina; White, Marney A.

    2015-01-01

    Objective This study tested the new DSM-5 severity criterion for binge eating disorder (BED) based on frequency of binge-eating in a clinical sample. This study also tested overvaluation of shape/weight as an alternative severity specifier. Method Participants were 834 treatment-seeking adults diagnosed with DSM-5 BED using semistructured diagnostic and eating-disorder interviews. Participants sub-grouped based on DSM-5 severity levels and on overvaluation of shape/weight were compared on demographic and clinical variables. Results Based on DSM-5 severity definitions, 331 (39.7%) participants were categorized as mild, 395 (47.5%) as moderate, 83 (10.0%) as severe, and 25 (3.0%) as extreme. Analyses comparing three (mild, moderate, and severe/extreme) severity groups revealed no significant differences in demographic variables or body mass index (BMI). Analyses revealed significantly higher eating-disorder psychopathology in the severe/extreme than moderate and mild groups and higher depression in moderate and severe/extreme groups than the mild group; effect sizes were small. Participants characterized with overvaluation (N = 449; 54%) versus without overvaluation (N = 384; 46%) did not differ significantly in age, sex, BMI, or binge-eating frequency, but had significantly greater eating-disorder psychopathology and depression. The robustly greater eating-disorder psychopathology and depression levels (medium-to-large effect sizes) in the overvaluation group was observed without attenuation of effect sizes after adjusting for ethnicity/race and binge-eating severity/frequency. Conclusions Our findings provide support for overvaluation of shape/weight as a severity specifier for BED as it provides stronger information about the severity of homogeneous groupings of patients than the DSM-5 rating based on binge-eating. PMID:26114779

  19. Functionalized graphene oxide for clinical glucose biosensing in urine and serum samples

    Directory of Open Access Journals (Sweden)

    Veerapandian M

    2012-12-01

    Full Text Available Murugan Veerapandian,1 Yeong-Tai Seo,2 Hyunkyung Shin,3 Kyusik Yun,1 Min-Ho Lee41Department of Bionanotechnology, Gachon University, Gyeonggi-Do, 2School of Electrical Engineering and Computer Science, Seoul National University, Seoul, 3Department of Mathematics and Information, Gachon University, Gyeonggi-Do, 4Korea Electronics Technology Institute, Medical IT Technology, Gyeonggi-Do, Republic of KoreaAbstract: A novel clinical glucose biosensor fabricated using functionalized metalloid-polymer (silver-silica coated with polyethylene glycol hybrid nanoparticles on the surface of a graphene oxide nanosheet is reported. The cyclic voltammetric response of glucose oxidase modification on the surface of a functionalized graphene oxide electrode showed a surface-confined reaction and an effective redox potential near zero volts, with a wide linearity of 0.1–20 mM and a sensitivity of 7.66 µA mM-1 cm-2. The functionalized graphene oxide electrode showed a better electrocatalytic response toward oxidation of H2O2 and reduction of oxygen. The practical applicability of the functionalized graphene oxide electrode was demonstrated by measuring the peak current against multiple urine and serum samples from diabetic patients. This new hybrid nanoarchitecture combining a three-dimensional metalloid-polymer hybrid and two-dimensional graphene oxide provided a thin solid laminate on the electrode surface. The easy fabrication process and retention of bioactive immobilized enzymes on the functionalized graphene oxide electrode could potentially be extended to detection of other biomolecules, and have broad applications in electrochemical biosensing.Keywords: clinical diagnostics, glucose biosensor, metalloid-polymer nanoparticles, glucose oxidase, cyclic voltammetry

  20. Levels and types of alcohol biomarkers in DUI and clinic samples for estimating workplace alcohol problems.

    Science.gov (United States)

    Marques, Paul R

    2012-02-01

    Widespread concern about illicit drugs as an aspect of workplace performance potentially diminishes attention on employee alcohol use. Alcohol is the dominant drug contributing to poor job performance; it also accounts for a third of the worldwide public health burden. Evidence from public roadways--a workplace for many--provides an example of work-related risk exposure and performance lapses. In most developed countries, alcohol is involved in 20-35% of fatal crashes; drugs other than alcohol are less prominently involved in fatalities. Alcohol biomarkers can improve detection by extending the timeframe for estimating problematic exposure levels and thereby provide better information for managers. But what levels and which markers are right for the workplace? In this paper, an established high-sensitivity proxy for alcohol-driving risk proclivity is used: an average eight months of failed blood alcohol concentration (BAC) breath tests from alcohol ignition interlock devices. Higher BAC test fail rates are known to presage higher rates of future impaired-driving convictions (driving under the influence; DUI). Drivers in alcohol interlock programmes log 5-7 daily BAC tests; in 12 months, this yields thousands of samples. Also, higher programme entry levels of alcohol biomarkers predict a higher likelihood of failed interlock BAC tests during subsequent months. This paper summarizes the potential of selected biomarkers for workplace screening. Markers include phosphatidylethanol (PEth), percent carbohydrate deficient transferrin (%CDT), gammaglutamyltransferase (GGT), gamma %CDT (γ%CDT), and ethylglucuronide (EtG) in hair. Clinical cut-off levels and median/mean levels of these markers in abstinent people, the general population, DUI drivers, and rehabilitation clinics are summarized for context. PMID:22311827

  1. Levels and Types of Alcohol Biomarkers in DUI and Clinic Samples for Estimating Workplace Alcohol Problemsa

    Science.gov (United States)

    Marques, Paul R

    2013-01-01

    Widespread concern about illicit drugs as an aspect of workplace performance potentially diminishes attention on employee alcohol use. Alcohol is the dominant drug contributing to poor job performance; it also accounts for a third of the worldwide public health burden. Evidence from public roadways – a workplace for many – provides an example for work-related risk exposure and performance lapses. In most developed countries, alcohol is involved in 20-35% of fatal crashes; drugs other than alcohol are less prominently involved in fatalities. Alcohol biomarkers can improve detection by extending the timeframe for estimating problematic exposure levels and thereby provide better information for managers. But what levels and which markers are right for the workplace? In this report, an established high-sensitivity proxy for alcohol-driving risk proclivity is used: an average 8 months of failed blood alcohol concentration (BAC) breath tests from alcohol ignition interlock devices. Higher BAC test fail rates are known to presage higher rates of future impaired-driving convictions (DUI). Drivers in alcohol interlock programs log 5-7 daily BAC tests; in 12 months, this yields thousands of samples. Also, higher program entry levels of alcohol biomarkers predict a higher likelihood of failed interlock BAC tests during subsequent months. This report summarizes selected biomarkers’ potential for workplace screening. Markers include phosphatidylethanol (PEth), percent carbohydrate deficient transferrin (%CDT), gammaglutamyltransferase (GGT), gamma %CDT (γ%CDT), and ethylglucuronide (EtG) in hair. Clinical cutoff levels and median/mean levels of these markers in abstinent people, the general population, DUI drivers, and rehabilitation clinics are summarized for context. PMID:22311827

  2. Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections.

    Science.gov (United States)

    Prost, Sandrine; Kishen, Ria E B; Kluth, David C; Bellamy, Christopher O C

    2016-01-01

    The recent availability of novel dyes and alternative light sources to facilitate complex tissue immunofluorescence studies such as multiplex labelling has not been matched by reports critically evaluating the considerations and relative benefits of these new tools, particularly in combination. Product information is often limited to wavelengths used for older fluorophores (FITC, TRITC & corresponding Alexa dyes family). Consequently, novel agents such as Quantum dots are not widely appreciated or used, despite highly favourable properties including extremely bright emission, stability and potentially reduced tissue autofluorescence at the excitation wavelength. Using spectral analysis, we report here a detailed critical appraisal and comparative evaluation of different light sources and fluorophores in multiplex immunofluorescence of clinical biopsy sections. The comparison includes mercury light, metal halide and 3 different LED-based systems, using 7 Qdots (525, 565, 585, 605, 625, 705), Cy3 and Cy5. We discuss the considerations relevant to achieving the best combination of light source and fluorophore for accurate multiplex fluorescence quantitation. We highlight practical limitations and confounders to quantitation with filter-based approaches. PMID:27632367

  3. Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections

    Science.gov (United States)

    Kishen, Ria E. B.; Kluth, David C.; Bellamy, Christopher O. C.

    2016-01-01

    The recent availability of novel dyes and alternative light sources to facilitate complex tissue immunofluorescence studies such as multiplex labelling has not been matched by reports critically evaluating the considerations and relative benefits of these new tools, particularly in combination. Product information is often limited to wavelengths used for older fluorophores (FITC, TRITC & corresponding Alexa dyes family). Consequently, novel agents such as Quantum dots are not widely appreciated or used, despite highly favourable properties including extremely bright emission, stability and potentially reduced tissue autofluorescence at the excitation wavelength. Using spectral analysis, we report here a detailed critical appraisal and comparative evaluation of different light sources and fluorophores in multiplex immunofluorescence of clinical biopsy sections. The comparison includes mercury light, metal halide and 3 different LED-based systems, using 7 Qdots (525, 565, 585, 605, 625, 705), Cy3 and Cy5. We discuss the considerations relevant to achieving the best combination of light source and fluorophore for accurate multiplex fluorescence quantitation. We highlight practical limitations and confounders to quantitation with filter-based approaches. PMID:27632367

  4. Structured Interview of Personality Organization (STIPO): preliminary psychometrics in a clinical sample.

    Science.gov (United States)

    Stern, Barry L; Caligor, Eve; Clarkin, John F; Critchfield, Kenneth L; Horz, Susanne; MacCornack, Verna; Lenzenweger, Mark F; Kernberg, Otto F

    2010-01-01

    In this article, we describe the development and preliminary psychometric properties of the Structured Interview of Personality Organization (STIPO), a semistructured interview designed for the dimensional assessment of identity, primitive defenses, and reality testing, the three primary content domains in the model of personality health and disorder elaborated by Kernberg (1984; Kernberg & Caligor, 2005). Results of this investigation, conducted in a clinical sample representing a broad range of personality pathology, indicate that identity and primitive defenses as operationalized in the STIPO are internally consistent and that interrater reliability for all 3 content domains is adequate. Validity findings suggest that the assessment of one's sense of self and significant others (Identity) is predictive of measures of positive and negative affect, whereas the maladaptive ways in which the subject uses his or her objects for purposes of regulating one's self experience (Primitive Defenses) is predictive of measures of aggression and personality disorder traits associated with cluster B personality disorders. We discuss implications of these findings in terms of the theory-driven and trait-based assessment of personality pathology. PMID:20013454

  5. Functionalized graphene oxide for clinical glucose biosensing in urine and serum samples.

    Science.gov (United States)

    Veerapandian, Murugan; Seo, Yeong-Tai; Shin, Hyunkyung; Yun, Kyusik; Lee, Min-Ho

    2012-01-01

    A novel clinical glucose biosensor fabricated using functionalized metalloid-polymer (silver-silica coated with polyethylene glycol) hybrid nanoparticles on the surface of a graphene oxide nanosheet is reported. The cyclic voltammetric response of glucose oxidase modification on the surface of a functionalized graphene oxide electrode showed a surface-confined reaction and an effective redox potential near zero volts, with a wide linearity of 0.1-20 mM and a sensitivity of 7.66 μA mM(-1) cm(-2). The functionalized graphene oxide electrode showed a better electrocatalytic response toward oxidation of H(2)O(2) and reduction of oxygen. The practical applicability of the functionalized graphene oxide electrode was demonstrated by measuring the peak current against multiple urine and serum samples from diabetic patients. This new hybrid nanoarchitecture combining a three-dimensional metalloid-polymer hybrid and two-dimensional graphene oxide provided a thin solid laminate on the electrode surface. The easy fabrication process and retention of bioactive immobilized enzymes on the functionalized graphene oxide electrode could potentially be extended to detection of other biomolecules, and have broad applications in electrochemical biosensing. PMID:23269871

  6. Turkish adaptation of the Fear of Spiders Questionnaire: Reliability and validity in non-clinical samples

    Directory of Open Access Journals (Sweden)

    Robert W. Booth

    2016-12-01

    Full Text Available The rapid, objective measurement of spider fear is important for clinicians, and for researchers studying fear. To facilitate this, we adapted the Fear of Spiders Questionnaire (FSQ into Turkish. The FSQ is quick to complete and easy to understand. Compared to the commonly used Spider Phobia Questionnaire, it has shown superior test-retest reliability and better discrimination of lower levels of spider fear, facilitating fear research in non-clinical samples. In two studies, with 137 and 105 undergraduates and unselected volunteers, our adapted FSQ showed excellent internal consistency (Cronbach’s α = .95 and .96 and test-retest reliability (r = .90, and good discriminant validity against the State–Trait Anxiety Inventory—Trait (r = .23 and Beck Anxiety Inventory—Trait (r = .07. Most importantly, our adapted FSQ significantly predicted 26 students’ self-reported discomfort upon approaching a caged tarantula; however, a measure of behavioural avoidance of the tarantula yielded little variability, so a more sensitive task will be required for future behavioural testing. Based on this initial testing, we recommend our adapted FSQ for research use. Further research is required to verify that our adapted FSQ discriminates individuals with and without phobia effectively. A Turkish-language report of the studies is included as supplementary material.

  7. Emotional processing in a non-clinical psychosis-prone sample.

    Science.gov (United States)

    van 't Wout, Mascha; Aleman, André; Kessels, Roy P C; Larøi, Frank; Kahn, René S

    2004-06-01

    Symptoms of psychosis have been proposed to form part of a continuous distribution of experiences in the general population rather than being an all-or-nothing phenomenon. Indeed, schizotypal signs have been reported in subjects from non-clinical samples. Emotional processing has been documented to be deficient in schizophrenia. In the present study, we tested the hypothesis whether putatively psychosis-prone subjects would show abnormalities in emotion processing. Based on the extremes of Launay-Slade Hallucination Scale (LSHS) ratings of 200 undergraduate students, two groups of subjects (total N=40) were selected. All 40 participants filled in the Schizotypal Personality Questionnaire (SPQ). We compared both groups on an alexithymia questionnaire and on four behavioral emotional information processing tasks. Hallucination-proneness was associated with an increased subjective emotional arousal and fantasy-proneness. Although no differences between the high and low group were observed on three behavioral emotion processing tasks, on the affective word-priming task presentation of emotional stimuli was associated with longer reactions times to neutral words in high schizotypal subjects. Also, SPQ scores correlated with several emotion processing tasks. We conclude that these findings lend partial support to the hypothesis of continuity between symptoms characteristic of schizophrenia and psychosis-related phenomena in the normal population. PMID:15099609

  8. Optimization and analysis of a quantitative real-time PCR-based technique to determine microRNA expression in formalin-fixed paraffin-embedded samples

    Directory of Open Access Journals (Sweden)

    Reis Patricia P

    2010-06-01

    Full Text Available Abstract Background MicroRNAs (miRs are non-coding RNA molecules involved in post-transcriptional regulation, with diverse functions in tissue development, differentiation, cell proliferation and apoptosis. miRs may be less prone to degradation during formalin fixation, facilitating miR expression studies in formalin-fixed paraffin-embedded (FFPE tissue. Results Our study demonstrates that the TaqMan Human MicroRNA Array v1.0 (Early Access platform is suitable for miR expression analysis in FFPE tissue with a high reproducibility (correlation coefficients of 0.95 between duplicates, p 35, we show that reproducibility between technical replicates, equivalent dilutions, and FFPE vs. frozen samples is best in the high abundance stratum. We also demonstrate that the miR expression profiles of FFPE samples are comparable to those of fresh-frozen samples, with a correlation of up to 0.87 (p Conclusion Our study thus demonstrates the utility, reproducibility, and optimization steps needed in miR expression studies using FFPE samples on a high-throughput quantitative PCR-based miR platform, opening up a realm of research possibilities for retrospective studies.

  9. A Multitrait–Multimethod Analysis of the Construct Validity of Child Anxiety Disorders in a Clinical Sample

    OpenAIRE

    Wood, Jeffrey J.; Bergman, R. Lindsey; Piacentini, John C.; Langer, David Adam

    2010-01-01

    The present study examines the construct validity of separation anxiety disorder (SAD), social phobia (SoP), panic disorder (PD), and generalized anxiety disorder (GAD) in a clinical sample of children. Participants were 174 children, 6 to 17 years old (94 boys) who had undergone a diagnostic evaluation at a university hospital based clinic. Parent and child ratings of symptom severity were assessed using the Multidimensional Anxiety Scale for Children (MASC). Diagnostician ratings were obtai...

  10. Detection of Shiga toxins genes by Multiplex PCR in clinical samples

    Directory of Open Access Journals (Sweden)

    2013-09-01

    Full Text Available Background: Different methods have been used for detection of shiga toxins; such as,  cell culture, ELISA, and RFPLA. However, all of these methods suffer from high cost, time-consumption and relatively low sensitivity. In this study we used Multiplex PCR method for detection of genes encoding shiga toxins. Material and Methods: In this study, 63 clinical samples were obtained from positive cultures of Shigella and E. coli O157, from Bahman 1391 until Ordibehesht 1392 in Mazandaran province. Initial confirmation of shiga toxins producing bacteria was performed by biochemical and serological methods. After DNA extraction, detection of stx1 and stx2 genes was accomplished by multiplex PCR.  For confirmation of the PCR amplicon, DNA sequencing was used. Antibiotic sensitivity tests were performed by disk diffusion method. Results:  Among the positive strains, 13 strains contained stx2 genes, 4 strains contained Stx/Stx1 genes and 4 strains harbored both Stx/Stx1 and Stx2. The DNA extracted from other Gram-negative bacteria was not protected by the relevant parts of these toxins. Sequencing of the amplified fragments indicated the correct toxin sequences.  The sensitivity for identification of Stx/Stx1 gene was 1.56 pg/ µl and for Stx2 was 1.08 pg/µl. The toxin positive strains were all sensitive to Cefixime, Gentamicin, Amikacin, Ceftriaxone, and Nitrofurantoin. Conclusion: This method is fast and accurate for detection of bacteria producing shiga toxin and can be used to identify different types of shiga toxin.

  11. Clinical validation of three short forms of the Dutch Wechsler Memory Scale – Fourth Edition (WMS-IV-NL) in a mixed clinical sample

    NARCIS (Netherlands)

    Bouman, Z.; Hendriks, M.P.H.; Veld, W.M. van der; Aldenkamp, A.P.; Kessels, R.P.C.

    2016-01-01

    The reliability and validity of three short forms of the Dutch version of the Wechsler Memory Scale–Fourth Edition (WMS-IV-NL) were evaluated in a mixed clinical sample of 235 patients. The short forms were based on the WMS-IV Flexible Approach, that is, a 3-subtest combination (Older Adult Battery

  12. The Structured Clinical Interview for DSM-IV Childhood Diagnoses (Kid-SCID): first psychometric evaluation in a Dutch sample of clinically referred youths

    NARCIS (Netherlands)

    J. Roelofs; P. Muris; C. Braet; A. Arntz; I. Beelen

    2014-01-01

    The Structured Clinical Interview for DSM-IV Childhood Disorders (Kid-SCID) is a semi-structured interview for the classification of psychiatric disorders in children and adolescents. This study presents a first evaluation of the psychometric properties of the Kid-SCID in a Dutch sample of children

  13. More than a (negative) feeling: Validity of the perceived stress scale in Serbian clinical and non-clinical samples

    OpenAIRE

    Jovanović Veljko; Gavrilov-Jerković Vesna

    2015-01-01

    The goal of the present study was to test the validity of a Serbian version of the Perceived Stress Scale. The PSS was administered to 157 psychiatric outpatients, 165 adults from the non-clinical population, and 283 university students. The results of the confirmatory factor analysis supported a bifactor model of the PSS with one general factor and two specific factors reflecting perceived distress and perceived self-efficacy. Internal consistencies of the...

  14. Detection and genetic characterization of foot‐and‐mouth disease viruses in samples from clinically healthy animals in endemic settings

    DEFF Research Database (Denmark)

    Jamal, Syed Muhammad; Ferrari, G.; Hussain, M.;

    2012-01-01

    in Pakistan (n = 245), one (of three) live animal market in Afghanistan (n = 61) and both the live animal markets in Tajikistan (n = 120) all tested negative. However, 2 of 129 (∼2%) samples from Gondal and 11 of 123 (9%) from Chichawatni markets in Pakistan were positive for FMDV RNA. Similarly, 12 of 81 (15......A total of 1501 oral swab samples from Pakistan, Afghanistan and Tajikistan were collected from clinically healthy animals between July 2008 and August 2009 and assayed for the presence of foot‐and‐mouth disease virus (FMDV) RNA. The oral swab samples from two (of four) live animal markets......%) samples from Kabul and 10 of 20 (50%) from Badakhshan in Afghanistan were found to be positive. Serotypes A and O of FMDV were identified within these samples. Oral swab samples were also collected from dairy colonies in Harbanspura, Lahore (n = 232) and Nagori, Karachi (n = 136), but all tested negative...

  15. P25-S Gene Expression Profiling from Formalin-Fixed, Paraffin-Embedded Tissues Using the QuantiGene Branched DNA Assay

    OpenAIRE

    Davies, J.; Maqsodi, B.; Yang, W.; Ma, Y.; Luo, Y.; McMaster, G.

    2007-01-01

    Large numbers of formalin-fixed, paraffin-embedded (FFPE) human tissue specimens with known clinical outcome are archived worldwide, representing a vast resource for biomarker and gene-disease association studies. However, RNA quality in FFPE tissues is compromised by chemical modifications and extensive fragmentation caused by formalin fixation. As a result, quantifying RNA in FFPE samples can be problematic.

  16. Parental Behavior and Adolescent Self-Esteem in Clinical and Nonclinical Samples.

    Science.gov (United States)

    Nielson, David M.; Metha, Arlene

    1994-01-01

    Investigated relationships between self-esteem and adolescents' perceptions of parental behaviors using nonclinical (n=119) and clinical (n=30) adolescents. Nonclinical adolescents scored higher than clinical adolescents on all self-esteem dimensions. Males scored higher than females only on dimension of Self-Esteem Competence. Perceptions of…

  17. Characterization of a clinical unit for digital radiography based on irradiation side sampling technology

    Energy Technology Data Exchange (ETDEWEB)

    Rivetti, Stefano [Fisica Medica, Ospedale di Sassuolo S.p.A., 41049 Sassuolo (Italy); Lanconelli, Nico [Alma Mater Studiorum, Physics Department, University of Bologna, 40127 Bologna (Italy); Bertolini, Marco; Nitrosi, Andrea [Medical Physics Unit, Azienda Ospedaliera ASMN, Istituto di Ricovero e Cura a Carattere Scientifico, 42123 Reggio Emilia (Italy); Burani, Aldo [Ospedale di Sassuolo S.p.A., 41049 Sassuolo (Italy)

    2013-10-15

    Purpose: A characterization of a clinical unit for digital radiography (FUJIFILM FDR D-EVO) is presented. This system is based on the irradiation side sampling (ISS) technology and can be equipped with two different scintillators: one traditional gadolinium-oxysulphide phosphor (GOS) and a needle structured cesium iodide (CsI) phosphor panel.Methods: The characterization was achieved in terms of response curve, modulation transfer function (MTF), noise power spectra (NPS), detective quantum efficiency (DQE), and psychophysical parameters (contrast-detail analysis with an automatic reading of CDRAD images). For both scintillation screens the authors accomplished the measurements with four standard beam conditions: RAQ3, RQA5, RQA7, and RQA9.Results: At the Nyquist frequency (3.33 lp/mm) the MTF is about 35% and 25% for CsI and GOS detectors, respectively. The CsI scintillator has better noise properties than the GOS screen in almost all the conditions. This is particularly true for low-energy beams, where the noise for the GOS system can go up to a factor 2 greater than that found for CsI. The DQE of the CsI detector reaches a peak of 60%, 60%, 58%, and 50% for the RQA3, RQA5, RQA7, and RQA9 beams, respectively, whereas for the GOS screen the maximum DQE is 40%, 44%, 44%, and 35%. The contrast-detail analysis confirms that in the majority of cases the CsI scintillator is able to provide improved outcomes to those obtained with the GOS screen.Conclusions: The limited diffusion of light produced by the ISS reading makes possible the achievement of very good spatial resolution. In fact, the MTF of the unit with the CsI panel is only slightly lower to that achieved with direct conversion detectors. The combination of very good spatial resolution, together with the good noise properties reached with the CsI screen, allows achieving DQE on average about 1.5 times greater than that obtained with GOS. In fact, the DQE of unit equipped with CsI is comparable to the best

  18. Occurrence of ADHD in parents of ADHD children in a clinical sample

    Directory of Open Access Journals (Sweden)

    Starck M

    2016-03-01

    Full Text Available Martina Starck,1 Julia Grünwald,1 Angelika A Schlarb1,21Faculty of Science, Department of Psychology, University of Tuebingen, Tuebingen, 2Department of Psychology, Faculty for Psychology and Sport Science, University of Bielefeld, Bielefeld, GermanyBackground: Despite the fact that there is a large amount of research on childhood attention deficit hyperactivity disorder (ADHD treatment and an increasing amount of research on adult ADHD, little is known about the prevalence and influence of parental ADHD. Therefore, this study examined the frequency of parental ADHD in a clinical sample of German children suffering from ADHD. We also tried to find different levels of symptom severity for prognostic relevance. Furthermore, the association between subtypes of ADHD in children and their parents was investigated.Method: In this study, parents of 79 ADHD children were screened for ADHD according to the Diagnostic and Statistical Manual of Mental Disorders, 5th edition and International Classification of Diseases, 10th edition. The Wender Utah Rating Scale and the ADHS-Self-Report were given to 75 mothers and 49 fathers for retrospective and current symptoms. Frequency of ADHD symptoms and severity groups was calculated and relationship between parental and children’s ADHD was tested.Results: ADHD occurrence for mothers of children with ADHD was 41.3%, for fathers 51.0%. About 16.0% of the mothers had a mixed type, 9.3% had a hyperactive-impulsive subtype, and 16.0% had an inattentive subtype. Of the fathers, 18.4% had a mixed type, 10.2% had a hyperactive-impulsive subtype, and 22.4% had an inattentive subtype; 61% of the mothers and 46.9% of the fathers had low symptom severity. Medium symptom severity was reported by 37.7% mothers and 46.9% fathers, while 1.3% of the mothers and 6.2% of the fathers showed severe symptoms. No significant correlation between parental and child diagnoses was observed.Conclusion: As nearly half of the parents

  19. Comparison of Two Assays for Molecular Determination of Rifampin Resistance in Clinical Samples from Patients with Buruli Ulcer Disease

    OpenAIRE

    Jansson, M.; Beissner, M.; Phillips, R. O.; Badziklou, K.; Piten, E.; Maman, I.; Sarfo, F S; Huber, K. L.; Rhomberg, A.; Symank, D.; Wagner, M.; Wiedemann, F; Nitschke, J.; Banla Kere, A.; Herbinger, K.-H.

    2014-01-01

    This study evaluates a novel assay for detecting rifampin resistance in clinical Mycobacterium ulcerans isolates. Although highly susceptible for PCR inhibitors in 50% of the samples tested, the assay was 100% M. ulcerans specific and yielded >98% analyzable sequences with a lower limit of detection of 100 to 200 copies of the target sequence.

  20. Childhood Trauma in Substance Use Disorder and Depression: An Analysis by Gender among a Brazilian Clinical Sample

    Science.gov (United States)

    Tucci, Adriana M.; Kerr-Correa, Florence; Souza-Formigoni, Maria Lucia O.

    2010-01-01

    Objective: In this study, we compared the frequency and intensity of childhood traumas in alcohol- or other drug-dependent patients, in patients with depression, and in a control group without psychiatric diagnoses. Methods: The study had a retrospective design of a clinical sample of men and women from the groups listed above. They were evaluated…

  1. Bottom–up protein identifications from microliter quantities of individual human tear samples. Important steps towards clinical relevance.

    Directory of Open Access Journals (Sweden)

    Peter Raus

    2015-12-01

    With 375 confidently identified proteins in the healthy adult tear, the obtained results are comprehensive and in large agreement with previously published observations on pooled samples of multiple patients. We conclude that, to a limited extent, bottom–up tear protein identifications from individual patients may have clinical relevance.

  2. A Comparison of Three Self-Report Measures of the Broader Autism Phenotype in a Non-Clinical Sample

    Science.gov (United States)

    Ingersoll, Brooke; Hopwood, Christopher J.; Wainer, Allison; Donnellan, M. Brent

    2011-01-01

    Three self-report measures of the broader autism phenotype (BAP) were evaluated in terms of their internal consistency, distribution of scores, factor structure, and criterion-related validity in a non-clinical sample. All measures showed a continuous distribution. The SRS-A and BAPQ showed expected sex differences and were superior to the AQ in…

  3. Detection and identification of Trichophyton tonsurans from clinical isolates and hairbrush samples by loop-mediated isothermal amplification system.

    Science.gov (United States)

    Yo, Ayaka; Yamamoto, Mikachi; Nakayama, Takako; Ishikawa, Jun; Makimura, Koichi

    2016-09-01

    Since the 1990s, there have been reports of the spread of dermatophytosis caused by Trichophyton tonsurans among contact sports athletes in several countries, including Japan. This study was performed to develop a loop-mediated isothermal amplification (LAMP) system for rapid and accurate detection and identification of T. tonsurans from clinical isolates or hairbrush samples for diagnosis and to prevent the spread of infection. A specific primer set was prepared by comparing the whole genome sequence of T. tonsurans with those of six other closely related dermatophytes. After confirming the sensitivity and specificity of this system, LAMP assay was performed using 37 clinical samples obtained from three healthy volunteers and 24 judo athletes. A total of 155 fungal isolates (56 strains of various standard fungi, 96 identified T. tonsurans isolates, three hairbrush-cultured isolates from judo athletes) and 37 hairbrush samples (34 samples from 24 judo athletes, and three samples from three healthy volunteers) were used for culture and LAMP assay, respectively. The assay showed no cross-reactivity to standard strains other than T. tonsurans. The detection limit was 100 copies of DNA template per tube. All of the 96 T. tonsurans isolates were amplified, and all samples from healthy volunteers showed negative results. Four of the 34 hairbrush samples obtained from judo athletes showed positive results in LAMP assay, and two of the four were positive in both culture and LAMP assay. We developed a rapid LAMP system with high specificity and sensitivity for diagnosis of T. tonsurans infection. PMID:26892741

  4. Detection and identification of Trichophyton tonsurans from clinical isolates and hairbrush samples by loop-mediated isothermal amplification system.

    Science.gov (United States)

    Yo, Ayaka; Yamamoto, Mikachi; Nakayama, Takako; Ishikawa, Jun; Makimura, Koichi

    2016-09-01

    Since the 1990s, there have been reports of the spread of dermatophytosis caused by Trichophyton tonsurans among contact sports athletes in several countries, including Japan. This study was performed to develop a loop-mediated isothermal amplification (LAMP) system for rapid and accurate detection and identification of T. tonsurans from clinical isolates or hairbrush samples for diagnosis and to prevent the spread of infection. A specific primer set was prepared by comparing the whole genome sequence of T. tonsurans with those of six other closely related dermatophytes. After confirming the sensitivity and specificity of this system, LAMP assay was performed using 37 clinical samples obtained from three healthy volunteers and 24 judo athletes. A total of 155 fungal isolates (56 strains of various standard fungi, 96 identified T. tonsurans isolates, three hairbrush-cultured isolates from judo athletes) and 37 hairbrush samples (34 samples from 24 judo athletes, and three samples from three healthy volunteers) were used for culture and LAMP assay, respectively. The assay showed no cross-reactivity to standard strains other than T. tonsurans. The detection limit was 100 copies of DNA template per tube. All of the 96 T. tonsurans isolates were amplified, and all samples from healthy volunteers showed negative results. Four of the 34 hairbrush samples obtained from judo athletes showed positive results in LAMP assay, and two of the four were positive in both culture and LAMP assay. We developed a rapid LAMP system with high specificity and sensitivity for diagnosis of T. tonsurans infection.

  5. "ISA-Lation" of Single-Stranded Positive-Sense RNA Viruses from Non-Infectious Clinical/Animal Samples.

    Directory of Open Access Journals (Sweden)

    Fabien Aubry

    Full Text Available Isolation of viral pathogens from clinical and/or animal samples has traditionally relied on either cell cultures or laboratory animal model systems. However, virus viability is notoriously susceptible to adverse conditions that may include inappropriate procedures for sample collection, storage temperature, support media and transportation. Using our recently described ISA method, we have developed a novel procedure to isolate infectious single-stranded positive-sense RNA viruses from clinical or animal samples. This approach, that we have now called "ISA-lation", exploits the capacity of viral cDNA subgenomic fragments to re-assemble and produce infectious viral RNA in susceptible cells. Here, it was successfully used to rescue enterovirus, Chikungunya and Tick-borne encephalitis viruses from a variety of inactivated animal and human samples. ISA-lation represents an effective option to rescue infectious virus from clinical and/or animal samples that may have deteriorated during the collection and storage period, but also potentially overcomes logistic and administrative difficulties generated when complying with current health and safety and biosecurity guidelines associated with shipment of infectious viral material.

  6. Chemistry Testing on Plasma Versus Serum Samples in Dialysis Patients: Clinical and Quality Improvement Implications.

    Science.gov (United States)

    Carey, Roger Neill; Jani, Chinu; Johnson, Curtis; Pearce, Jim; Hui-Ng, Patricia; Lacson, Eduardo

    2016-09-01

    Plasma samples collected in tubes containing separator gels have replaced serum samples for most chemistry tests in many hospital and commercial laboratories. Use of plasma samples for blood tests in the dialysis population eliminates delays in sample processing while waiting for clotting to complete, laboratory technical issues associated with fibrin formation, repeat sample collection, and patient care issues caused by delay of results because of incompletely clotted specimens. Additionally, a larger volume of plasma is produced than serum for the same amount of blood collected. Plasma samples are also acceptable for most chemical tests involved in the care of patients with ESRD. This information becomes very important when United States regulatory requirements for ESRD inadvertently limit the type of sample that can be used for government reporting, quality assessment, and value-based payment initiatives. In this narrative, we summarize the renal community experience and how the subsequent resolution of the acceptability of phosphorus levels measured from serum and plasma samples may have significant implications in the country's continued development of a value-based Medicare ESRD Quality Incentive Program.

  7. WISC-III and CAS: Which Correlates Higher with Achievement for a Clinical Sample?

    Science.gov (United States)

    Naglieri, Jack A.; De Lauder, Brianna Y.; Goldstein, Sam; Schwebech, Adam

    2006-01-01

    The relationships between Wechsler Intelligence Scale for Children-Third Edition (WISC-III) and the Cognitive Assessment System (CAS) with the Woodcock-Johnson Tests of Achievement (WJ-III) were examined for a sample of 119 children (87 males and 32 females) ages 6 to 16. The sample was comprised of children who were referred to a specialty clinic…

  8. Evaluation of inhibitor-resistant real-time PCR methods for diagnostics in clinical and environmental samples.

    Science.gov (United States)

    Trombley Hall, Adrienne; McKay Zovanyi, Ashley; Christensen, Deanna Rose; Koehler, Jeffrey William; Devins Minogue, Timothy

    2013-01-01

    Polymerase chain reaction (PCR) is commonly used for pathogen detection in clinical and environmental samples. These sample matrices often contain inhibitors of PCR, which is a primary reason for sample processing; however, the purification process is highly inefficient, becoming unacceptable at lower signature concentrations. One potential solution is direct PCR assessment without sample processing. Here, we evaluated nine inhibitor-resistant PCR reagents for direct detection of Francisella tularensis in seven different clinical and environmental samples using an established real-time PCR assay to assess ability to overcome PCR inhibition. While several of these reagents were designed for standard PCR, the described inhibitor resistant properties (ex. Omni Klentaq can amplify target DNA samples of up to 20% whole blood or soil) led to our evaluation with real-time PCR. A preliminary limit of detection (LOD) was determined for each chemistry in whole blood and buffer, and LODs (20 replicates) were determined for the top five chemistries in each matrix (buffer, whole blood, sputum, stool, swab, soil, and sand). Not surprisingly, no single chemistry performed the best across all of the different matrices evaluated. For instance, Phusion Blood Direct PCR Kit, Phire Hot Start DNA polymerase, and Phire Hot Start DNA polymerase with STR Boost performed best for direct detection in whole blood while Phire Hot Start DNA polymerase with STR Boost were the only reagents to yield an LOD in the femtogram range for soil. Although not the best performer across all matrices, KAPA Blood PCR kit produced the most consistent results among the various conditions assessed. Overall, while these inhibitor resistant reagents show promise for direct amplification of complex samples by real-time PCR, the amount of template required for detection would not be in a clinically relevant range for most matrices. PMID:24040090

  9. Evaluation of inhibitor-resistant real-time PCR methods for diagnostics in clinical and environmental samples.

    Directory of Open Access Journals (Sweden)

    Adrienne Trombley Hall

    Full Text Available Polymerase chain reaction (PCR is commonly used for pathogen detection in clinical and environmental samples. These sample matrices often contain inhibitors of PCR, which is a primary reason for sample processing; however, the purification process is highly inefficient, becoming unacceptable at lower signature concentrations. One potential solution is direct PCR assessment without sample processing. Here, we evaluated nine inhibitor-resistant PCR reagents for direct detection of Francisella tularensis in seven different clinical and environmental samples using an established real-time PCR assay to assess ability to overcome PCR inhibition. While several of these reagents were designed for standard PCR, the described inhibitor resistant properties (ex. Omni Klentaq can amplify target DNA samples of up to 20% whole blood or soil led to our evaluation with real-time PCR. A preliminary limit of detection (LOD was determined for each chemistry in whole blood and buffer, and LODs (20 replicates were determined for the top five chemistries in each matrix (buffer, whole blood, sputum, stool, swab, soil, and sand. Not surprisingly, no single chemistry performed the best across all of the different matrices evaluated. For instance, Phusion Blood Direct PCR Kit, Phire Hot Start DNA polymerase, and Phire Hot Start DNA polymerase with STR Boost performed best for direct detection in whole blood while Phire Hot Start DNA polymerase with STR Boost were the only reagents to yield an LOD in the femtogram range for soil. Although not the best performer across all matrices, KAPA Blood PCR kit produced the most consistent results among the various conditions assessed. Overall, while these inhibitor resistant reagents show promise for direct amplification of complex samples by real-time PCR, the amount of template required for detection would not be in a clinically relevant range for most matrices.

  10. ISOLATION AND SPECIATION OF CANDIDA FROM CLINICAL SAMPLES IN A TERTIARY CARE HOSPITAL AT KURNOOL, ANDHRAPRADESH, INDIA

    Directory of Open Access Journals (Sweden)

    Dasari

    2014-12-01

    Full Text Available : Candida is one of the most frequently encountered opportunistic fungi that cause infection in humans. The pathogenesis of Candida is complex and probably varies with each infection. This study was conducted to understand the prevalence of Candida from various clinical specimens of patients and to show the emergence of Non albicans Candida in clinical samples. This study also focused on the antifungal susceptibility which guides the clinicians to treat the infection effectively. METHODS: Clinical samples were collected from outpatients and inpatients of Government General Hospital, Kurnool over a period of one year from March2008 to June2009. Isolation, culture, speciation of Candida was done by using standard methods. Antifungal susceptibility testing was done by disc diffusion technique against amphotericin B, nystatin, fluconazole and clotrimazole. RESULTS: Candida manifests in various sites depending on the predisposing factors and immune status of the person. In this study we found the association of Candida with various predisposing factors (Pregnancy, Oral contraceptive pills’s, Immune suppression, Diabetes. This study observed the dominance of non-albicans Candida (51% in the clinical samples over Candida albicans (49%. The maximum antifungal susceptibility was observed against amphotericin B in both the albicans and non-albicans Candia, but non-albicans Candida showed maximum resistance to azoles. CONCLUSION: Candida albicans was the most predominant species (49% isolated in various clinical samples. There was an increase in the prevalence of non albicans Candida in this study. Among the nonalbicans Candida (51% Candid tropicalis was the commonest species isolated. Candida albicans showed maximum susceptibility to amphotericin B and maximum resistance to azoles was seen in nonalbicans Candida.

  11. [Clinical characteristics and social determinants in a sample of non-homebound elderly].

    Science.gov (United States)

    Morsch, Patricia; Pereira, Gustavo Nunes; Navarro, Joel Hirtz do Nascimento; Trevisan, Margarete Diprat; Lopes, Diene Gomes Colvara; Bós, Ângelo José Gonçalves

    2015-05-01

    This study aimed to assess social and clinical factors associated with the fact that older adults (≥ 60 years) go out of their homes. The study interviewed 5,898 older adults identified through home visits, randomly selected in 59 cities in the State of Rio Grande do Sul, Brazil. Multivariate logistic regression was used to assess the association between the outcome and independent variables. Factors associated with going out were being men, younger and married, presence of arthrosis, ease in performing specific activities, and good self-rated health. Heart disease was a negative factor for going out. Given the importance of social activity for quality of life and the World Health Organization policy for active aging, it is extremely important to consider clinical conditions that allow the older adults to remain active in the community. Studies like this can help to adjust public policies for the elderly, especially acting on modifiable clinical and functional conditions. PMID:26083177

  12. Methods for Studying MicroRNA Expression and Their Targets in Formalin-Fixed, Paraffin-Embedded (FFPE) Breast Cancer Tissues.

    Science.gov (United States)

    Gomes, Bruno Costa; Santos, Bruno; Rueff, José; Rodrigues, António Sebastião

    2016-01-01

    Drug resistance remains a burden in cancer treatment. In the past few years molecular genetics brought a new hope with personalized therapy. This individual approach allows the identification of genetic profiles that will respond better to a given treatment and consequently get a better outcome. Recently, physicians received an extra aid with the approval of molecular tools based on gene expression signatures. With these tools, physicians have the capacity to identify the probability of disease recurrence in the first 5 years following diagnosis, a fact that is essential for a more effective adjuvant therapy administration. However, some patients still relapse and acquire drug resistance and aggressive tumors. For that reason, a comprehensive understanding of the molecular players in drug resistance is of extreme importance. MicroRNAs have been described as regulators of various cellular pathways and as predictive and prognostic factors. As broad regulators, microRNAs also interfere with drug metabolism and drug targets. Thus it is of paramount importance to understand which microRNAs are deregulated in breast cancer and try to relate this misexpression with resistance to therapeutics, poor outcomes, and survival. Here, we describe a possible approach to study microRNA expression and respective targets from formalin-fixed, paraffin-embedded (FFPE) breast cancer tissues. FFPE tissues are regularly archived for long periods in pathology departments, and microRNAs are well conserved in these tissues. PMID:26910075

  13. Preservation of nucleic acids and tissue morphology in paraffin-embedded clinical samples: comparison of five molecular fixatives.

    Science.gov (United States)

    Staff, Synnöve; Kujala, Paula; Karhu, Ritva; Rökman, Annika; Ilvesaro, Joanna; Kares, Saara; Isola, Jorma

    2013-09-01

    Formalin fixation preserves tissue morphology at the expense of macromolecule integrity. Freshly frozen samples are the golden standard for DNA and RNA analyses but require laborious deep-freezing and frozen sectioning for morphological studies. Alternative tissue stabilisation methods are therefore needed. We analysed the preservation of nucleic acids, immunohistochemical staining properties and tissue morphology in paraffin-embedded clinical tissue samples fixed with Z7, RCL2, PAXgene, Allprotect and RNAlater. Formalin-fixed and deep-frozen samples were used as controls. Immunohistochemical analyses showed good preservation of antigenicity in all except Allprotect and RNAlater-fixed samples. RNA quality, based on RNA integrity number value by Bioanalyzer, was comparable with freshly frozen samples only in PAXgene-fixed samples. According to quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses, RNA from PAXgene samples yielded results similar to freshly frozen samples. No difference between fixatives was seen in DNA analyses (PCR and real-time PCR). In conclusion, PAXgene seems to be superior to other molecular fixatives and formaldehyde.

  14. Total Protein Profile and Drug Resistance in Candida albicans Isolated from Clinical Samples

    Directory of Open Access Journals (Sweden)

    Kamal Uddin Zaidi

    2016-01-01

    Full Text Available This study was done to assess the antifungal susceptibility of clinical isolates of Candida albicans and to evaluate its total protein profile based on morphological difference on drug resistance. Hundred and twenty clinical isolates of C. albicans from various clinical specimens were tested for susceptibility against four antifungal agents, namely, fluconazole, itraconazole, amphotericin B, and ketoconazole. A significant increase of drug resistance in clinical isolates of C. albicans was observed. The study showed 50% fluconazole and itraconazole resistance at 32 μg mL−1 with a MIC50 and MIC90 values at 34 and 47 and 36 and 49 μg mL−1, respectively. All isolates were sensitive to amphotericin B and ketoconazole. The SDS-PAGE protein profile showed a prevalent band of ~52.5 kDa, indicating overexpression of gene in 72% strains with fluconazole resistance. Since the opportunistic infections of Candida spp. are increasing along with drug resistance, the total protein profile will help in understanding the evolutionary changes in drug resistance and also to characterize them.

  15. Social and Emotional Outcomes of Child Sexual Abuse: A Clinical Sample in Turkey

    Science.gov (United States)

    Ozbaran, Burcu; Erermis, Serpil; Bukusoglu, Nagehan; Bildik, Tezan; Tamar, Muge; Ercan, Eyyup Sabri; Aydin, Cahide; Cetin, Saniye Korkmaz

    2009-01-01

    Childhood sexual abuse is a traumatic life event that may cause psychiatric disorders such as posttraumatic stress disorder and depression. During 2003-2004, 20 sexually abused children were referred to the Child and Adolescent Psychiatry Clinic of Ege University in Izmir, Turkey. Two years later, the psychological adjustment of these children (M…

  16. Clinical and Laboratory Data in a Sample of Greek Children with Autism Spectrum Disorders

    Science.gov (United States)

    Ververi, Athina; Vargiami, Efthymia; Papadopoulou, Vassiliki; Tryfonas, Dimitrios; Zafeiriou, Dimitrios I.

    2012-01-01

    The purpose of this study is to describe clinical and laboratory data, as well as comorbid disorders in Greek children with autism spectrum disorders (ASD). Data were retrospectively collected for 222 children aged 1.5-9 years. The mean age at diagnosis was 43.7 [plus or minus] 17.6 months. Significantly earlier diagnoses were noted in children…

  17. Evaluation of constitutive and inducible resistance to clindamycin in clinical samples of Staphylococcus aureus from a tertiary hospital

    Directory of Open Access Journals (Sweden)

    Angelita Bottega

    2014-10-01

    Full Text Available Introduction Infections caused by methicillin-resistant Staphylococcus aureus (MRSA have become common in hospitals and the community environment, and this wide resistance has limited patient treatment. Clindamycin (CL represents an important alternative therapy for infections caused by S. aureus. Antimicrobial susceptibility testing using standard methods may not detect inducible CL resistance. This study was performed to detect the phenotypes of resistance to macrolides-lincosamides-streptogramin B (MLSB antibiotics, including CL, in clinical samples of S. aureus from patients at a tertiary hospital in Santa Maria, State of Rio Grande do Sul, Brazil. Methods One hundred and forty clinical isolates were submitted to the disk diffusion induction test (D-test with an erythromycin (ER disk positioned at a distance of 20mm from a CL disk. The results were interpreted according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI. Results In this study, 29 (20.7% of the 140 S. aureus samples were resistant to methicillin (MRSA, and 111 (79.3% were susceptible to methicillin (MSSA. The constitutive resistance phenotype (cMLSB was observed in 20 (14.3% MRSA samples and in 5 (3.6% MSSA samples, whereas the inducible resistance phenotype (iMLSB was observed in 3 (2.1% MRSA samples and in 8 (5.8% MSSA samples. Conclusions The D-test is essential for detecting the iMLSB phenotype because the early identification of this phenotype allows clinicians to choose an appropriate treatment for patients. Furthermore, this test is simple, easy to perform and inexpensive.

  18. Multiple stage MS in analysis of plasma, serum, urine and in vitro samples relevant to clinical and forensic toxicology.

    Science.gov (United States)

    Meyer, Golo M; Maurer, Hans H; Meyer, Markus R

    2016-01-01

    This paper reviews MS approaches applied to metabolism studies, structure elucidation and qualitative or quantitative screening of drugs (of abuse) and/or their metabolites. Applications in clinical and forensic toxicology were included using blood plasma or serum, urine, in vitro samples, liquids, solids or plant material. Techniques covered are liquid chromatography coupled to low-resolution and high-resolution multiple stage mass analyzers. Only PubMed listed studies published in English between January 2008 and January 2015 were considered. Approaches are discussed focusing on sample preparation and mass spectral settings. Comments on advantages and limitations of these techniques complete the review.

  19. Establishment of an AlphaLISA assay for detection of hepatitis B virus X protein in FFPE from hepatocellular carcinoma patients%AlphaLISA法检测乙型肝炎病毒X蛋白方法学的建立

    Institute of Scientific and Technical Information of China (English)

    刘颖娟; 夏维; 刘松梅; 沈璠; 周新; 张红

    2014-01-01

    目的:建立新型的均相检测技术AlphaLISA(Amplifed Luminescent Proximity Homogeneous Assay)法,检测原发性肝细胞癌(Hepatocelluar carcinoma,HCC)病人石蜡包埋切片组织(Formalin-fixed and paraffin-embedded tis-sue,FFPE)中乙型肝炎病毒 X蛋白(HBx)。方法采用针对 HBx蛋白不同表位的两种单克隆抗体 X36C和3F6-G10,将其中一种抗体 X36C与受体微珠(Acceptor Beads)连接,另一种抗体3F6-G10生物素化,再将 HCC病人 FFPE总蛋白提取液、供体微珠(Donor Beads)及上述两种处理后的抗体在23℃共同孵育90 min后,用 EnSpire2300多功能酶标仪检测信号值。结果 FFPE 蛋白提取液检测信号值为4667,HBx蛋白标准品检测信号值为3606;FFPE蛋白提取液样本对照、试剂对照1(Bio-3F6-G10+Acceptor-X36C+Donor Beads)、试剂对照2(buffer)的信号值分别为5、823、79;当生物素化抗体浓度为0.1625nM时,HBX检测信号达到最大峰值,即 AlphaLISA出现 Hooking Effect;Al-phaLISA法检测 HBX阈值下限为5pg。结论本实验成功建立了 AlphaLISA法,用于检测 HCC病人 FFPE中的HBX。实验操作过程简单快捷,敏感性高于传统的 ELISA技术。%Objective To establish an AlphaLISA (Amplifed Luminescent Proximity Homogeneous Assay)for de-tection of hepatitis B virus (HBV)X protein (HBx)in FFPE from hepatocellular carcinoma (HCC)patients.Methods Two monoclonal antibodies of anti-HBx proteins,including X36C and 3F6-G10,are able to bind to the HBx protein different epitopes.X36C Antibody is connected to the acceptor beads and 3F6-G10 antibody is biotinylated using com-mercial kits.Then,streptavidin donor beads,X36C binding acceptor beads,biotinylated 3F6-G10,and HBx proteins (standards or FFPE samples)are incubated together at 23℃ for 90 minutes.After incubation,the signal values are de-tected by EnSpire 2300 Multimode Plate Reader.Results The signal values of HBx proteins in FFPEs

  20. Development of standardized methodology for identifying toxins in clinical samples and fish species associated with tetrodotoxin-borne poisoning incidents

    Directory of Open Access Journals (Sweden)

    Tai-Yuan Chen

    2016-01-01

    Full Text Available Tetrodotoxin (TTX is a naturally occurring toxin in food, especially in puffer fish. TTX poisoning is observed frequently in South East Asian regions. In TTX-derived food poisoning outbreaks, the amount of TTX recovered from suspicious fish samples or leftovers, and residual levels from biological fluids of victims are typically trace. However, liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry methods have been demonstrated to qualitatively and quantitatively determine TTX in clinical samples from victims. Identification and validation of the TTX-originating seafood species responsible for a food poisoning incident is needed. A polymerase chain reaction-based method on mitochondrial DNA analysis is useful for identification of fish species. This review aims to collect pertinent information available on TTX-borne food poisoning incidents with a special emphasis on the analytical methods employed for TTX detection in clinical laboratories as well as for the identification of TTX-bearing species.

  1. Matrix Metalloproteinase-9/Neutrophil Gelatinase-Associated Lipocalin Complex Activity in Human Glioma Samples Predicts Tumor Presence and Clinical Prognosis

    Directory of Open Access Journals (Sweden)

    Ming-Fa Liu

    2015-01-01

    Full Text Available Matrix metalloproteinase-9/neutrophil gelatinase-associated lipocalin (MMP-9/NGAL complex activity is elevated in brain tumors and may serve as a molecular marker for brain tumors. However, the relationship between MMP-9/NGAL activity in brain tumors and patient prognosis and treatment response remains unclear. Here, we compared the clinical characteristics of glioma patients with the MMP-9/NGAL activity measured in their respective tumor and urine samples. Using gelatin zymography assays, we found that MMP-9/NGAL activity was significantly increased in tumor tissues (TT and preoperative urine samples (Preop-1d urine. Activity was reduced by seven days after surgery (Postop-1w urine and elevated again in cases of tumor recurrence. The MMP-9/NGAL status correlated well with MRI-based tumor assessments. These findings suggest that MMP-9/NGAL activity could be a novel marker to detect gliomas and predict the clinical outcome of patients.

  2. Clinically significant fatigue: prevalence and associated factors in an international sample of adults with multiple sclerosis recruited via the internet.

    Directory of Open Access Journals (Sweden)

    Tracey J Weiland

    Full Text Available Fatigue contributes a significant burden of disease for people with multiple sclerosis (PwMS. Modifiable lifestyle factors have been recognized as having a role in a range of morbidity outcomes in PwMS. There is significant potential to prevent and treat fatigue in PwMS by addressing modifiable risk factors.To explore the associations between clinically significant fatigue and demographic factors, clinical factors (health-related quality of life, disability and relapse rate and modifiable lifestyle, disease-modifying drugs (DMD and supplement use in a large international sample of PwMS.PwMS were recruited to the study via Web 2.0 platforms and completed a comprehensive survey measuring demographic, lifestyle and clinical characteristics, including health-related quality of life, disability, and relapse rate.Of 2469 participants with confirmed MS, 2138 (86.6% completed a validated measure of clinically significant fatigue, the Fatigue Severity Scale. Participants were predominantly female from English speaking countries, with relatively high levels of education, and due to recruitment methods may have been highly pro-active about engaging in lifestyle management and self-help. Approximately two thirds of our sample (1402/2138; 65.6% (95% CI 63.7-67.7 screened positive for clinically significant fatigue. Bivariate associations were present between clinically significant fatigue and several demographic, clinical, lifestyle, and medication variables. After controlling for level of disability and a range of stable socio-demographic variables, we found increased odds of fatigue associated with obesity, DMD use, poor diet, and reduced odds of fatigue with exercise, fish consumption, moderate alcohol use, and supplementation with vitamin D and flaxseed oil.This study supports strong and significant associations between clinically significant fatigue and modifiable lifestyle factors. Longitudinal follow-up of this sample may help clarify the contribution

  3. Antimicrobial resistance trends among canine Escherichia coli isolates obtained from clinical samples in the northeastern USA, 2004–2011

    OpenAIRE

    Cummings, Kevin J.; Aprea, Victor A.; Altier, Craig

    2015-01-01

    Our objectives were to describe the antimicrobial susceptibility of Escherichia coli isolates from dogs in the northeastern USA and to identify temporal trends in resistance to selected antimicrobial agents. Data were collected retrospectively for all canine E. coli isolates from clinical samples submitted to Cornell University’s Animal Health Diagnostic Center between January 1, 2004 and December 31, 2011. Antimicrobial susceptibility testing was performed on 3519 canine E. coli isolates; fr...

  4. Rapid Detection/pathotyping of Newcastle disease virus isolates in clinical samples using real time polymerase chain reaction assay

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Abdul Wajid, Muhammad Wasim, Tahir Yaqub, Shafqat F Rehmani, Tasra Bibi, Nadia Mukhtar, Javed Muhammad, Umar Bacha, Suliman Qadir Afridi, Muhammad Nauman Zahid, Zia u ddin, Muhammad Zubair Shabbir, Kamran Abbas & Muneer Ahmad ### Abstract In the present protocol we describe the real time reverse transcription polymerase chain reaction (rRT-PCR) assay for the rapid detection/pathotyping of Newcastle disease virus (NDV) isoaltes in clinical samples. Fusion gene and matrix ...

  5. Birth Order and Sibling Gender Ratio of a Clinical Sample of Children and Adolescents Diagnosed with Attention Deficit Hyperactivity Disorder

    OpenAIRE

    Ahmad Ghanizadeh; Marzie Abotorabi-Zarchi; Mohammad Reza Mohammadi; Ali Firoozabadi

    2012-01-01

    Objective: It is not clear whether sibling’s gender ratio is associated with attention deficit hyperactivity disorder (ADHD). This study examines whether inattentiveness severity and hyperactivity/impulsivity severity are associated with birth order of children with ADHD.Method: Participants are a clinical sample of 173 children and adolescents with ADHD and 43 ones without ADHD. Diagnoses were made using Diagnostic and Statistical Manual of Mental Disorders forth edition-Text Revision (DSM-I...

  6. Anxiety and depression in parents of a Brazilian non-clinical sample of attention-deficit/ hyperactivity disorder (ADHD) students

    OpenAIRE

    D. Segenreich; D. Fortes; G. Coutinho; G. Pastura; Mattos, P

    2009-01-01

    Higher prevalence rates of anxiety and depression have been reported in parents of children with attention-deficit/hyperactivity disorder (ADHD). The interaction between the burden of ADHD in offspring, a higher prevalence rate of this highly inherited disorder in parents, and comorbidities may explain this finding. Our objective was to investigate levels of ADHD, anxious and depressive symptomatology, and their relationship in parents of ADHD children from a non-clinical sample using a dimen...

  7. Adult ADHD Symptoms and Five Factor Model Traits in a Clinical Sample: A Structural Equation Modeling Approach

    OpenAIRE

    Knouse, Laura E.; Traeger, Lara; O’Cleirigh, Conall; Safren, Steven A.

    2013-01-01

    Relationships among Attention-Deficit/Hyperactivity Disorder (ADHD) symptoms and adult personality traits have not been examined in larger clinically diagnosed samples. We collected multi-source ADHD symptom and self-report NEO Five-Factor Inventory (Costa & McCrae, 1992a) data from 117 adults with ADHD and tested symptom-trait associations using structural equation modeling. The final model fit the data. Inattention was positively associated with Neuroticism and negatively associated with Co...

  8. Functionalized graphene oxide for clinical glucose biosensing in urine and serum samples

    OpenAIRE

    Veerapandian M; Seo YT; Shin H; Yun K; Lee MH

    2012-01-01

    Murugan Veerapandian,1 Yeong-Tai Seo,2 Hyunkyung Shin,3 Kyusik Yun,1 Min-Ho Lee41Department of Bionanotechnology, Gachon University, Gyeonggi-Do, 2School of Electrical Engineering and Computer Science, Seoul National University, Seoul, 3Department of Mathematics and Information, Gachon University, Gyeonggi-Do, 4Korea Electronics Technology Institute, Medical IT Technology, Gyeonggi-Do, Republic of KoreaAbstract: A novel clinical glucose biosensor fabricated using functionalized metalloid-poly...

  9. Simultaneous Detection of Four Human Pathogenic Microsporidian Species from Clinical Samples by Oligonucleotide Microarray

    OpenAIRE

    Wang, Zheng; Orlandi, Palmer A.; David A Stenger

    2005-01-01

    Microsporidian species have been rapidly emerging as human enteric pathogens in immunocompromised and immunocompetent individuals in recent years. Routine diagnostic techniques for microsporidia in clinical laboratories are laborious and insensitive and tend to underestimate their presence. In most instances, they are unable to differentiate species of spores due to their small sizes and similar morphologies. In this study, we report the development of another protozoan oligonucleotide microa...

  10. Evolution and Diversity of Listeria monocytogenes from Clinical and Food Samples in Shanghai, China.

    Science.gov (United States)

    Zhang, Jianmin; Cao, Guojie; Xu, Xuebin; Allard, Marc; Li, Peng; Brown, Eric; Yang, Xiaowei; Pan, Haijian; Meng, Jianghong

    2016-01-01

    Listeria monocytogenes is a significant foodborne pathogen causing severe systemic infections in humans with high mortality rates. The objectives of this work were to establish a phylogenetic framework of L. monocytogenes from China and to investigate sequence diversity among different serotypes. We selected 17 L. monocytogenes strains recovered from patients and foods in China representing serotypes 1/2a, 1/2b, and 1/2c. Draft genome sequences were determined using Illumina MiSeq technique and associated protocols. Open reading frames were assigned using prokaryotic genome annotation pipeline by NCBI. Twenty-four published genomes were included for comparative genomic and phylogenetic analysis. More than 154,000 single nucleotide polymorphisms (SNPs) were identified from multiple genome alignment and used to reconstruct maximum likelihood phylogenetic tree. The 41 genomes were differentiated into lineages I and II, which consisted of 4 and 11 subgroups, respectively. A clinical strain from China (SHL009) contained significant SNP differences compared to the rest genomes, whereas clinical strain SHL001 shared most recent common ancestor with strain SHL017 from food. Moreover, clinical strains SHL004 and SHL015 clustered together with two strains (08-5578 and 08-5923) recovered from an outbreak in Canada. Partial sequences of a plasmid found in the Canadian strain were also present in SHL004. We investigated the presence of various genes and gene clusters associated with virulence and subgroup-specific genes, including internalins, L. monocytogenes pathogenicity islands (LIPIs), L. monocytogenes genomic islands (LGIs), stress survival islet 1 (SSI-1), and clustered regularly interspaced short palindromic repeats (CRISPR)/cas system. A novel genomic island, denoted as LGI-2 was identified. Comparative sequence analysis revealed differences among the L. monocytogenes strains related to virulence, survival abilities, and attributes against foreign genetic elements. L

  11. Clinical Diagnosis by Whole-Genome Sequencing of a Prenatal Sample

    OpenAIRE

    Talkowski, Michael E.; Ordulu, Zehra; Pillalamarri, Vamsee; Benson, Carol B.; Blumenthal, Ian; Connolly, Susan; Hanscom, Carrie; Hussain, Naveed; Pereira, Shahrin; Picker, Jonathan; Rosenfeld, Jill A.; Shaffer, Lisa G.; Wilkins-Haug, Louise E.; Gusella, James F.; Morton, Cynthia C.

    2012-01-01

    Conventional cytogenetic testing offers low-resolution detection of balanced karyotypic abnormalities but cannot provide the precise, gene-level knowledge required to predict outcomes. The use of high-resolution whole-genome deep sequencing is currently impractical for the purpose of routine clinical care. We show here that whole-genome “jumping libraries” can offer an immediately applicable, nucleotide-level complement to conventional genetic diagnostics within a time frame that allows for c...

  12. Genetic diversity of Cryptosporidium identified in clinical samples from cities in Brazil and Argentina

    Science.gov (United States)

    Peralta, Regina Helena Saramago; Velásquez, Jorge Néstor; Cunha, Flavia de Souza; Pantano, María Laura; Sodré, Fernando Campos; da Silva, Sidnei; Astudillo, Osvaldo Germán; Peralta, José Mauro; Carnevale, Silvana

    2016-01-01

    The identification and characterisation of Cryptosporidiumgenotypes and subtypes are fundamental to the study of cryptosporidiosis epidemiology, aiding in prevention and control strategies. The objective was to determine the genetic diversity ofCryptosporidium in samples obtained from hospitals of Rio de Janeiro, Brazil, and Buenos Aires, Argentina. Samples were analysed by microscopy and TaqMan polymerase chain reaction (PCR) assays forCryptosporidium detection, genotyped by nested-PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene and subtyped by DNA sequencing of the gp60 gene. Among the 89 samples from Rio de Janeiro, Cryptosporidium spp were detected in 26 by microscopy/TaqMan PCR. In samples from Buenos Aires,Cryptosporidium was diagnosed in 15 patients of the 132 studied. The TaqMan PCR and the nested-PCR-RFLP detected Cryptosporidium parvum, Cryptosporidium hominis, and co-infections of both species. In Brazilian samples, the subtypes IbA10G2 and IIcA5G3 were observed. The subtypes found in Argentinean samples were IbA10G2, IaA10G1R4, IaA11G1R4, and IeA11G3T3, and mixed subtypes of Ia and IIa families were detected in the co-infections. C. hominis was the species more frequently detected, and subtype family Ib was reported in both countries. Subtype diversity was higher in Buenos Aires than in Rio de Janeiro and two new subtypes were described for the first time. PMID:26814641

  13. Conceptual Model of Clinical Governance Information System for Statistical Indicators by Using UML in Two Sample Hospitals

    Science.gov (United States)

    Jeddi, Fatemeh Rangraz; Farzandipoor, Mehrdad; Arabfard, Masoud; Hosseini, Azam Haj Mohammad

    2016-01-01

    Objective: The purpose of this study was investigating situation and presenting a conceptual model for clinical governance information system by using UML in two sample hospitals. Background: However, use of information is one of the fundamental components of clinical governance; but unfortunately, it does not pay much attention to information management. Material and Methods: A cross sectional study was conducted in October 2012- May 2013. Data were gathered through questionnaires and interviews in two sample hospitals. Face and content validity of the questionnaire has been confirmed by experts. Data were collected from a pilot hospital and reforms were carried out and Final questionnaire was prepared. Data were analyzed by descriptive statistics and SPSS 16 software. Results: With the scenario derived from questionnaires, UML diagrams are presented by using Rational Rose 7 software. The results showed that 32.14 percent Indicators of the hospitals were calculated. Database was not designed and 100 percent of the hospital’s clinical governance was required to create a database. Conclusion: Clinical governance unit of hospitals to perform its mission, do not have access to all the needed indicators. Defining of Processes and drawing of models and creating of database are essential for designing of information systems. PMID:27147804

  14. Detection of Rare Drug Resistance Mutations by Digital PCR in a Human Influenza A Virus Model System and Clinical Samples.

    Science.gov (United States)

    Whale, Alexandra S; Bushell, Claire A; Grant, Paul R; Cowen, Simon; Gutierrez-Aguirre, Ion; O'Sullivan, Denise M; Žel, Jana; Milavec, Mojca; Foy, Carole A; Nastouli, Eleni; Garson, Jeremy A; Huggett, Jim F

    2016-02-01

    Digital PCR (dPCR) is being increasingly used for the quantification of sequence variations, including single nucleotide polymorphisms (SNPs), due to its high accuracy and precision in comparison with techniques such as quantitative PCR (qPCR) and melt curve analysis. To develop and evaluate dPCR for SNP detection using DNA, RNA, and clinical samples, an influenza virus model of resistance to oseltamivir (Tamiflu) was used. First, this study was able to recognize and reduce off-target amplification in dPCR quantification, thereby enabling technical sensitivities down to 0.1% SNP abundance at a range of template concentrations, a 50-fold improvement on the qPCR assay used routinely in the clinic. Second, a method was developed for determining the false-positive rate (background) signal. Finally, comparison of dPCR with qPCR results on clinical samples demonstrated the potential impact dPCR could have on clinical research and patient management by earlier (trace) detection of rare drug-resistant sequence variants. Ultimately this could reduce the quantity of ineffective drugs taken and facilitate early switching to alternative medication when available. In the short term such methods could advance our understanding of microbial dynamics and therapeutic responses in a range of infectious diseases such as HIV, viral hepatitis, and tuberculosis. Furthermore, the findings presented here are directly relevant to other diagnostic areas, such as the detection of rare SNPs in malignancy, monitoring of graft rejection, and fetal screening. PMID:26659206

  15. Survey and visual detection of Zaire ebolavirus in clinical samples targeting the nucleoprotein gene in Sierra Leone

    Directory of Open Access Journals (Sweden)

    Jing eYuan

    2015-12-01

    Full Text Available Ebola virus (EBOV can lead to severe hemorrhagic fever with a high risk of death in humans and other primates. To guide treatment and prevent spread of the viral infection, a rapid and sensitive detection method is required for clinical samples. Here, we described and evaluated a reverse transcription loop-mediated isothermal amplification (RT-LAMP method to detect Zaire ebolavirus using the nucleoprotein gene (NP as a target sequence. Two different techniques were used, a calcein/Mn2+ complex chromogenic method and real-time turbidity monitoring. The RT-LAMP assay detected the NP target sequence with a limit of 4.56 copies/μL within 45 min under 61°C, a similar even or increase in sensitivity than that of real-time reverse transcription-polymerase chain reaction (RT-PCR. Additionally, all pseudoviral particles or non- Zaire EBOV genomes were negative for LAMP detection, indicating that the assay was highly specific for EBOV. To appraise the availability of the RT-LAMP method for use in clinical diagnosis of EBOV, of 417 blood or swab samples collected from patients with clinically suspected infections in Sierra Leone, 307 were identified for RT-LAMP-based surveillance of EBOV. Therefore, the highly specific and sensitive RT-LAMP method allows the rapid detection of EBOV, and is a suitable tool for clinical screening, diagnosis, and primary quarantine purposes.

  16. Inflammatory cytokine concentrations in uterine flush and serum samples from dairy cows with clinical or subclinical endometritis.

    Science.gov (United States)

    Kim, Ill-Hwa; Kang, Hyun-Gu; Jeong, Jae-Kwan; Hur, Tai-Young; Jung, Young-Hun

    2014-08-01

    The objective of this study was to compare the concentrations of inflammatory cytokines in uterine flush and serum from healthy postpartum dairy cows and cows with clinical or subclinical endometritis. Clinical endometritis was diagnosed by observation of vaginal discharges (>50% pus) and subclinical endometritis was diagnosed by evaluation of uterine cytology (neutrophils >18%) at 4 weeks postpartum. Uterine flush was obtained from 48 cows at 4, 6, and 8 weeks postpartum for evaluation of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, IL-8, and IL-10 concentrations. Serum samples were obtained from 34 cows just after calving and at 1, 2, 4, 6, and 8 weeks postpartum for evaluation of TNF-α, IL-1β, and IL-6 concentrations. Concentrations of TNF-α, IL-6, and IL-10 were greater (P < 0.05) in cows with clinical endometritis than in cows with subclinical endometritis and healthy controls, whereas concentrations of IL-8 in both cows with clinical and subclinical endometritis were greater (P < 0.005) than in controls. Overall, IL-6 and IL-10 concentrations decreased during the postpartum period. IL-1β concentrations in cows with clinical endometritis decreased (P < 0.0005) during the postpartum, whereas concentrations in cows with subclinical endometritis and controls did not change significantly with time; at 4 weeks postpartum, concentrations were greater (P < 0.0001) in cows with clinical endometritis. There were no significant effects of group, sampling time, or interaction on serum cytokine concentrations. In conclusion, cows with endometritis have greater inflammatory cytokine concentrations in uterine flush than healthy cows, but no differences were observed in serum. PMID:24933095

  17. Comorbidity of psychiatric disorders with Internet addiction in a clinical sample: the effect of personality, defense style and psychopathology.

    Science.gov (United States)

    Floros, Georgios; Siomos, Konstantinos; Stogiannidou, Ariadni; Giouzepas, Ioannis; Garyfallos, Georgios

    2014-12-01

    This study aims to contribute to the understanding of underlying causes for the development of Internet Addiction Disorder (IAD) and assess comorbidity with other mental disorders through the analysis of data from a clinical sample of college students who presented for treatment of IAD. The clinical sample of our study has demonstrated a high percentage of comorbidity with Axis I and II disorders, while the temporal precedence of the establishment of those disorders cannot lead to specific conclusions. Half of the sample (25/50) presented with comorbidity of another Axis I disorder and 38% (19/50) with a concurrent Axis II personality disorder. The majority of Axis I disorders (51.85%) were reported before the onset of IAD, 33.3% after the onset while it was unclear in 14.81% of cases. The examination of a path model demonstrated that important contributions to the understanding of this disorder can be made through concepts from the neurobiological, trait personality paradigm, as well as from the psychodynamic defense style paradigm. Comorbid psychopathology can further exacerbate the presentation of IAD through a direct link, regardless of the underlying personality structure. The clinician treating IAD patients should complete a clinical evaluation for comorbid Axis I and II diagnoses since their presence may signify a more serious presentation.

  18. Molecular method for the characterization of Coxiella burnetii from clinical and environmental samples: variability of genotypes in Spain

    Directory of Open Access Journals (Sweden)

    Jado Isabel

    2012-06-01

    Full Text Available Abstract Background Coxiella burnetii is a highly clonal microorganism which is difficult to culture, requiring BSL3 conditions for its propagation. This leads to a scarce availability of isolates worldwide. On the other hand, published methods of characterization have delineated up to 8 different genomic groups and 36 genotypes. However, all these methodologies, with the exception of one that exhibited limited discriminatory power (3 genotypes, rely on performing between 10 and 20 PCR amplifications or sequencing long fragments of DNA, which make their direct application to clinical samples impracticable and leads to a scarce accessibility of data on the circulation of C. burnetii genotypes. Results To assess the variability of this organism in Spain, we have developed a novel method that consists of a multiplex (8 targets PCR and hybridization with specific probes that reproduce the previous classification of this organism into 8 genomic groups, and up to 16 genotypes. It allows for a direct characterization from clinical and environmental samples in a single run, which will help in the study of the different genotypes circulating in wild and domestic cycles as well as from sporadic human cases and outbreaks. The method has been validated with reference isolates. A high variability of C. burnetii has been found in Spain among 90 samples tested, detecting 10 different genotypes, being those adaA negative associated with acute Q fever cases presenting as fever of intermediate duration with liver involvement and with chronic cases. Genotypes infecting humans are also found in sheep, goats, rats, wild boar and ticks, and the only genotype found in cattle has never been found among our clinical samples. Conclusions This newly developed methodology has permitted to demonstrate that C. burnetii is highly variable in Spain. With the data presented here, cattle seem not to participate in the transmission of C. burnetii to humans in the samples studied

  19. Glycemic Control in a Clinic-Based Sample of Diabetics in M'Bour Senegal

    Science.gov (United States)

    BeLue, Rhonda; Ndiaye, Khadidiatou; NDao, Fatou; Ba, Fatou Niass Niang; Diaw, Mor

    2016-01-01

    Background: Sub-Saharan Africa (SSA) including Senegal is faced with a significant and increasing burden of type 2 diabetes. However, little information is available about diabetes management among Senegalese diabetics. Purpose: The current study aims to describe the level of glycemic control among a convenience sample of diabetics who receive…

  20. A prospective survey of Aspergillus spp. in respiratory tract samples: prevalence, clinical impact and antifungal susceptibility

    DEFF Research Database (Denmark)

    Mortensen, K L; Johansen, H K; Fuursted, K;

    2011-01-01

    ) was evaluated using modified EORTC/MSG criteria. A total of 11,368 airway samples were received. Growth of Aspergillus spp. was found in 129 and 151 patients using routine and extended incubation, respectively. Three patients had proven IA (2%), 11 probable (7%), four had allergic bronchopulmonary aspergillosis...

  1. Usefulness of chorionic villus sampling for prenatal diagnosis of thalassaemia: a clinical study in eastern India

    Directory of Open Access Journals (Sweden)

    Sujoy Dasgupta

    2015-06-01

    Conclusions: Thalassaemia is a prevalent condition in Eastern India. Chorionic villus sampling is an effective and safe method for early diagnosis of fetal thalassaemia that helps to prevent birth of thalassaemic babies. [Int J Reprod Contracept Obstet Gynecol 2015; 4(3.000: 790-794

  2. Reliability and Validity of the Spanish Adaptation of EOSS, Comparing Normal and Clinical Samples

    Science.gov (United States)

    Valero-Aguayo, Luis; Ferro-Garcia, Rafael; Lopez-Bermudez, Miguel Angel; de Huralde, Ma. Angeles Selva-Lopez

    2012-01-01

    The Experiencing of Self Scale (EOSS) was created for the evaluation of Functional Analytic Psychotherapy (Kohlenberg & Tsai, 1991, 2001, 2008) in relation to the concept of the experience of personal self as socially and verbally constructed. This paper presents a reliability and validity study of the EOSS with a Spanish sample (582 participants,…

  3. Genotypic characterization, invasion index and antimicrobial resistance pattern in Listeria monocytogenes strains isolated from clinical samples

    Directory of Open Access Journals (Sweden)

    Behrooz Sadeghi Kalani

    2015-06-01

    Conclusions: Since MLVA is a high-throughput screening method that is fairly inexpensive, easy to accomplish, rapid, and trustworthy, it is well suited to interlaboratory comparisons during epidemiological investigations. Also further studies of larger samples from a variety of sources such as food and animal specimens recommended comparing by MLVA method.

  4. Survey and rapid detection of Klebsiella pneumoniae in clinical samples targeting the rcsA gene in Beijing, China

    Directory of Open Access Journals (Sweden)

    Derong eDong

    2015-05-01

    Full Text Available Klebsiella pneumoniae is a wide-spread nosocomial pathogen. A rapid and sensitive molecular method for the detection of K. pneumoniae in clinical samples is needed to guide therapeutic treatment. In this study, we first described a loop-mediated isothermal amplification (LAMP method for the rapid detection of capsular polysaccharide synthesis regulating gene rcsA from K. pneumoniae in clinical samples by using two methods including real-time turbidity monitoring and fluorescence detection to assess the reaction. Then dissemination of K. pneumoniae strains was investigated from ICU patients in three top hospitals in Beijing, China. The results showed that the detection limit of the LAMP method was 0.115 pg/µl DNA within 60 min under isothermal conditions (61°C, a 100-fold increase in sensitivity compared with conventional PCR. All 30 non- K. pneumoniae strains tested were negative for LAMP detection, indicating the high specificity of the LAMP reaction. To evaluate the application of the LAMP assay to clinical diagnosis, of 110 clinical sputum samples collected from ICU patients with clinically suspected multi-resistant infections in China, a total of 32 K. pneumoniae isolates were identified for LAMP-based surveillance of rcsA. All isolates belonged to nine different K. pneumoniae multilocus sequence typing (MLST groups. Strikingly, of the 32 K. pneumoniae strains, 18 contained the Klebsiella pneumoniae Carbapenemase (KPC-encoding gene blaKPC-2 and had high resistance to β-lactam antibiotics. Moreover, K. pneumoniae WJ-64 was discovered to contain blaKPC-2 and blaNDM-1 genes simultaneously in the isolate. Our data showed the high prevalence of blaKPC-2 among K. pneumoniae and co-occurrence of many resistant genes in the clinical strains signal a rapid and continuing evolution of K. pneumoniae. In conclusion, we have developed a rapid and sensitive visual K. pneumoniae detection LAMP assay, which could be a useful tool for clinical screening

  5. Survey and rapid detection of Klebsiella pneumoniae in clinical samples targeting the rcsA gene in Beijing, China.

    Science.gov (United States)

    Dong, Derong; Liu, Wei; Li, Huan; Wang, Yufei; Li, Xinran; Zou, Dayang; Yang, Zhan; Huang, Simo; Zhou, Dongsheng; Huang, Liuyu; Yuan, Jing

    2015-01-01

    Klebsiella pneumoniae is a wide-spread nosocomial pathogen. A rapid and sensitive molecular method for the detection of K. pneumoniae in clinical samples is needed to guide therapeutic treatment. In this study, we first described a loop-mediated isothermal amplification (LAMP) method for the rapid detection of capsular polysaccharide synthesis regulating gene rcsA from K. pneumoniaein clinical samples by using two methods including real-time turbidity monitoring and fluorescence detection to assess the reaction. Then dissemination of K. pneumoniae strains was investigated from ICU patients in three top hospitals in Beijing, China. The results showed that the detection limit of the LAMP method was 0.115 pg/μl DNA within 60 min under isothermal conditions (61°C), a 100-fold increase in sensitivity compared with conventional PCR. All 30 non- K. pneumoniae strains tested were negative for LAMP detection, indicating the high specificity of the LAMP reaction. To evaluate the application of the LAMP assay to clinical diagnosis, of 110 clinical sputum samples collected from ICU patients with clinically suspected multi-resistant infections in China, a total of 32 K. pneumoniae isolates were identified for LAMP-based surveillance of rcsA. All isolates belonged to nine different K. pneumoniae multilocus sequence typing (MLST) groups. Strikingly, of the 32 K. pneumoniae strains, 18 contained the Klebsiella pneumoniae Carbapenemase (KPC)-encoding gene bla KPC-2 and had high resistance to β-lactam antibiotics. Moreover, K. pneumoniae WJ-64 was discovered to contain bla KPC-2 and bla NDM-1genes simultaneously in the isolate. Our data showed the high prevalence of bla KPC-2 among K. pneumoniae and co-occurrence of many resistant genes in the clinical strains signal a rapid and continuing evolution of K. pneumoniae. In conclusion, we have developed a rapid and sensitive visual K. pneumoniae detection LAMP assay, which could be a useful tool for clinical screening, on

  6. Rapid 16S rRNA next-generation sequencing of polymicrobial clinical samples for diagnosis of complex bacterial infections.

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    Stephen J Salipante

    Full Text Available Classifying individual bacterial species comprising complex, polymicrobial patient specimens remains a challenge for culture-based and molecular microbiology techniques in common clinical use. We therefore adapted practices from metagenomics research to rapidly catalog the bacterial composition of clinical specimens directly from patients, without need for prior culture. We have combined a semiconductor deep sequencing protocol that produces reads spanning 16S ribosomal RNA gene variable regions 1 and 2 (∼360 bp with a de-noising pipeline that significantly improves the fraction of error-free sequences. The resulting sequences can be used to perform accurate genus- or species-level taxonomic assignment. We explore the microbial composition of challenging, heterogeneous clinical specimens by deep sequencing, culture-based strain typing, and Sanger sequencing of bulk PCR product. We report that deep sequencing can catalog bacterial species in mixed specimens from which usable data cannot be obtained by conventional clinical methods. Deep sequencing a collection of sputum samples from cystic fibrosis (CF patients reveals well-described CF pathogens in specimens where they were not detected by standard clinical culture methods, especially for low-prevalence or fastidious bacteria. We also found that sputa submitted for CF diagnostic workup can be divided into a limited number of groups based on the phylogenetic composition of the airway microbiota, suggesting that metagenomic profiling may prove useful as a clinical diagnostic strategy in the future. The described method is sufficiently rapid (theoretically compatible with same-day turnaround times and inexpensive for routine clinical use.

  7. The psychometric properties of the Finnish Young Schema Questionnaire in chronic pain patients and a non-clinical sample.

    Science.gov (United States)

    Saariaho, Tom; Saariaho, Anita; Karila, Irma; Joukamaa, Matti

    2009-03-01

    We investigated the latent factor structure of the Finnish Young Schema Questionnaire (YSQ-S2-extended; short form) in samples of chronic pain patients (n=271) and controls (n=331) with confirmatory factor analysis (CFA). The data in the total sample supported the 18-factor structure as hypothesized by Young, J. E., Klosko, J., & Weishaar, M. E. (2003). Schema therapy: A practitioner's guide. New York: Guilford Press. The diagonally weighted least squares estimation method gave repeatable parameter estimates in successive confirmatory factor analyses (CFA). The internal consistency of the YSQ-S2-extended was adequate to high in both samples and the groups showed equal goodness-of-fit statistics in CFA. This study consisted of the oldest population so far (mean age 47 years) and supported the use of the Finnish version of the YSQ-S2-extended in clinical practice.

  8. Simplified sampling methods for estimating levels of lactobacilli in saliva in dental clinical practice.

    Science.gov (United States)

    Gabre, P; Martinsson, T; Gahnberg, L

    1999-08-01

    The aim of the present study was to evaluate whether estimation of lactobacilli was possible with simplified saliva sampling methods. Dentocult LB (Orion Diagnostica AB, Trosa, Sweden) was used to estimate the number of lactobacilli in saliva sampled by 3 different methods from 96 individuals: (i) Collecting and pouring stimulated saliva over a Dentocult dip-slide; (ii) direct licking of the Dentocult LB dip-slide; (iii) contaminating a wooden spatula with saliva and pressing against the Dentocult dip-slide. The first method was in accordance with the manufacturer's instructions and selected as the 'gold standard'; the other 2 methods were compared with this result. The 2 simplified methods for estimating levels of lactobacilli in saliva showed good reliability and specificity. Sensitivity, defined as the ability to detect individuals with a high number of lactabacilli in saliva, was sufficient for the licking method (85%), but significantly reduced for the wooden spatula method (52%).

  9. Comparison of microRNA expression using different preservation methods of matched psoriatic skin samples

    DEFF Research Database (Denmark)

    Løvendorf, Marianne B; Zibert, John R; Hagedorn, Peter H;

    2012-01-01

    MicroRNAs are non-coding RNA molecules modulating gene expression post-transcriptionally. Formalin-fixed, paraffin-embedding (FFPE) is a standard preservation method often used in clinical practices, but induces RNA degradation. Extracting high-quality RNA from human skin can be challenging as skin...... that microRNA detection in human skin is robust irrespective of preservation method; thus, microRNAs offer an appropriate and flexible approach in clinical practices and for diagnostic purposes in skin disorders....

  10. Application of Atomic Dielectric Resonance Spectroscopy for the screening of blood samples from patients with clinical variant and sporadic CJD

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    Ironside James W

    2007-08-01

    Full Text Available Abstract Background Sub-clinical variant Creutzfeldt-Jakob disease (vCJD infection and reports of vCJD transmission through blood transfusion emphasise the need for blood screening assays to ensure the safety of blood and transplanted tissues. Most assays aim to detect abnormal prion protein (PrPSc, although achieving required sensitivity is a challenge. Methods We have used innovative Atomic Dielectric Resonance Spectroscopy (ADRS, which determines dielectric properties of materials which are established by reflectivity and penetration of radio/micro waves, to analyse blood samples from patients and controls to identify characteristic ADR signatures unique to blood from vCJD and to sCJD patients. Initial sets of blood samples from vCJD, sCJD, non-CJD neurological diseases and normal healthy adults (blood donors were screened as training samples to determine group-specific ADR characteristics, and provided a basis for classification of blinded sets of samples. Results Blood sample groups from vCJD, sCJD, non-CJD neurological diseases and normal healthy adults (blood donors screened by ADRS were classified with 100% specificity and sensitivity, discriminating these by a co-variance expert analysis system. Conclusion ADRS appears capable of recognising and discriminating serum samples from vCJD, sCJD, non-CJD neurological diseases, and normal healthy adults, and might be developed to provide a system for primary screening or confirmatory assay complementary to other screening systems.

  11. Slime production and antibiotic susceptibility in staphylococci isolated from clinical samples

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    Seza Arslan

    2007-02-01

    Full Text Available A total of 187 isolates from several clinical specimens were identified to species level as 129 Staphylococcus aureus strains and 58 coagulase-negative staphylococci (CNS strains by the API Staph System (Biomerieux. Slime production was detected both by the conventional Christensen's method as well as by the Congo red agar method. Seventy-two strains of staphylococci isolates (38.5% were found to be slime producers by Christensen's test tube method whereas 58 strains (31% were slime positive with Congo red agar method. There was no statistically significant difference between the two methods for the detection of slime production (P > 0.05. Susceptibility of isolates against antimicrobial agents was tested by the disk diffusion method. Staphylococcal species had resistance to one or more antibiotics. Among the various antimicrobial agents, oxacillin (71.1% and erythromycin (47.1% showed higher resistance than most of the agents used against all isolates. Oxacillin resistant S. aureus (ORSA and oxacillin resistant coagulase-negative staphylococci (ORCNS, 97 (75.2% and 36 (62.1% respectively were frequently observed in strains isolated from clinical materials. Among the ORSA strains, two strains were resistant to vancomycin. Moreover, 96 (74.4% of 129 S. aureus strains were positive for blactamase enzyme. However, 78 (81.25% of 96 b-lactamase positive S. aureus strains were b-lactamase positive ORSA isolates, but none of them had vancomycin resistance.

  12. TruSeq Stranded mRNA and Total RNA Sample Preparation Kits

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    Total RNA-Seq enabled by ribosomal RNA (rRNA) reduction is compatible with formalin-fixed paraffin embedded (FFPE) samples, which contain potentially critical biological information. The family of TruSeq Stranded Total RNA sample preparation kits provides a unique combination of unmatched data quality for both mRNA and whole-transcriptome analyses, robust interrogation of both standard and low-quality samples and workflows compatible with a wide range of study designs.

  13. Attachment, Obsessive Beliefs and Emotion Dysregulation in Obsessive Compulsive Disorder: A comparison with Normal and Clinical Sample

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    Sevginar Vatan

    2016-06-01

    Full Text Available Objective: The present study aimed at investigating the differences between nonclinical sample and clinical sample (OCD patients in attachment, obsessive beliefs and emotion regulation in Turkish sample. Methods: For these purposes 224 non clinical participants and 101 clinical OCD participants completed questionnaires related to research variables. Experience in Close Relationship Scale (Fraley, Waller & Brennan, 2000 to evaluate attachment, Obsessive Belief Questionnaire to evaluate obsessive beliefs and Difficulties of Emotion Regulation Scale (Gratz & Roemer, 2004 to evaluate emotion regulation abilities were used in this study. Results: A series of t-test for independent samples analyses were done. Findings of the analyses revealed that there was significant differences between two groups in anxiety level of attachment, but not in avoidance level. Also there were significant differences between two groups in all obsessive beliefs subscales such as responsibility and threat perception, perfectionism and need of certainty, the importance of thoughts and control. From emotional regulation perspective there were significant differences in non-acceptance of emotional response, difficulties in engaging goal-directed behavior, impulse control difficulties during emotional distress, limited access to emotional regulation strategies. However there was not significant differences between two groups in clarity about emotions and awareness of emotional arousal. Conclusion: To sum up according to results of this study attachment and obsessive beliefs follow the same pattern with the results in the literature. Moreover the difficulties of emotion regulation abilities thought to improve the knowledge about OCD. Because this part believed not to have enough findings in the OCD literature. The results were discussed in the light of the related literature and dependent recommendations to the area were given.

  14. The association between candidate migraine susceptibility loci and severe migraine phenotype in a clinical sample

    DEFF Research Database (Denmark)

    Esserlind, Ann-Louise; Christensen, Anne Francke; Steinberg, Stacy;

    2016-01-01

    INTRODUCTION: The objective of the study was to follow up and to test whether 12 previously identified migraine-associated single nucleotide polymorphisms were associated as risk factors and/or modifying factors for severe migraine traits in a Danish clinic-based population. METHODS: Semi...... in both a case-control and case-only logistic regression model. RESULTS: We successfully replicated five out of the 12 previously reported loci and confirmed the same direction of effects for all the 12 single nucleotide polymorphisms. In line with the recently published genome-wide association meta...... polymorphisms showed nominal association with many lifetime attacks and prolonged migraine attacks. CONCLUSION: Our study supports previously reported findings on the association of several single nucleotide polymorphisms with migraine. It also suggests that the migraine susceptibility loci may be risk factors...

  15. The frequency of Listeria monocytogenes strains recovered from clinical and non-clinical samples using phenotypic methods and confirmed by PCR

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    abazar pournajaf

    2013-09-01

    Full Text Available Background: Listeria monocytogenes is a facultative intracellular pathogen that causes listeriosis which has extensive clinical manifestations. Infections with L. monocytogenes are a serious threat to immunocompromised persons. The aim of this study was to determine the frequency of L. monocytogenes strains recovered from clinical and non-clinical samples using phenotypic methods and confirmed by PCR. Materials and Methods: In this study, 617 specimens were analyzed. All specimens were cultured in the specific PALCAM agar. Colonies were initially identified by routine biochemical tests. Finally, PCR assays using primers specific for inlA gene were performed. Results: In all, 46 (8.2% L. monocytogenes isolates were recovered from 617 specimens. Fourteen (8.2% strains, including 4 (7.5%, 2 (5.7%, 5 (14.2% and 3 (8.5% isolates were obtained from placental tissue, urine, vaginal and rectal swabs, respectively. In addition, 9 (7.4% strains of L. monocytogenes which were isolated from 107 different dairy products originated from cheese 5 (7.1%, cream 2 (10% and kashk 2 (11.7%, respectively. Among 11 (5.2% strains isolated from 210 different meat products, 5 (5.5%, 4 (7.2% and 2 (3% strains belonged to sausage, meat and poultry extracts, respectively. Finally, 12 (9.2% Listeria strains were recovered from 130 animal specimens that included 6 (10%, 4 (8% and 2 (10% strains from goat, sheep and cattle, respectively. Furthermore, all Listeria isolates (100% were found to be carriers of  inlA gene in PCR assay. Conclusion: The present study showed that the clinical and non-clinical specimens were contaminated with L. monocytogenes. So, it seems necessary to use a simple and standard technique such as PCR for rapid detection of this organism from various sources.

  16. Phylogenetic analysis of Austrian canine distemper virus strains from clinical samples from dogs and wild carnivores.

    Science.gov (United States)

    Benetka, V; Leschnik, M; Affenzeller, N; Möstl, K

    2011-04-01

    Austrian field cases of canine distemper (14 dogs, one badger [Meles meles] and one stone marten [Martes foina]) from 2002 to 2007 were investigated and the case histories were summarised briefly. Phylogenetic analysis of fusion (F) and haemagglutinin (H) gene sequences revealed different canine distemper virus (CDV) lineages circulating in Austria. The majority of CDV strains detected from 2002 to 2004 were well embedded in the European lineage. One Austrian canine sample detected in 2003, with a high similarity to Hungarian sequences from 2005 to 2006, could be assigned to the Arctic group (phocine distemper virus type 2-like). The two canine sequences from 2007 formed a clearly distinct group flanked by sequences detected previously in China and the USA on an intermediate position between the European wildlife and the Asia-1 cluster. The Austrian wildlife strains (2006 and 2007) could be assigned to the European wildlife group and were most closely related to, yet clearly different from, the 2007 canine samples. To elucidate the epidemiological role of Austrian wildlife in the transmission of the disease to dogs and vice versa, H protein residues related to receptor and host specificity (residues 530 and 549) were analysed. All samples showed the amino acids expected for their host of origin, with the exception of a canine sequence from 2007, which had an intermediate position between wildlife and canine viral strains. In the period investigated, canine strains circulating in Austria could be assigned to four different lineages reflecting both a high diversity and probably different origins of virus introduction to Austria in different years.

  17. Concordance of HOMIM and HOMINGS technologies in the microbiome analysis of clinical samples

    Science.gov (United States)

    Mougeot, Jean-Luc C.; Stevens, Craig B.; Cotton, Sean L.; Morton, Darla S.; Krishnan, Keerthana; Brennan, Michael T.; Lockhart, Peter B.; Paster, Bruce J.; Bahrani Mougeot, Farah K.

    2016-01-01

    Background Over 700 bacterial species reside in human oral cavity, many of which are associated with local or distant site infections. Extensive characterization of the oral microbiome depends on the technologies used to determine the presence and proportions of specific bacterial species in various oral sites. Objective The objective of this study was to compare the microbial composition of dental plaque at baseline using Human Oral Microbe Identification Microarray (HOMIM) and Human Oral Microbe Identification using Next Generation Sequencing (HOMINGS) technologies, which are based on 16S rRNA. Methods Dental plaque samples were collected from 96 patients at baseline prior to a dental procedure involving manipulation of gingival tissues. The samples were surveyed for 293 and 597 oral bacterial species via HOMIM and HOMINGS, respectively, based on 16S rRNA gene sequences. We determined the concordance between the two technologies for common species. Genus level analysis was performed using HOMINGS-specific genus identification capabilities. Results HOMINGS detected twice the number of species in the same dental plaque samples compared to HOMIM. For the species detected by both HOMIM and HOMINGS, there was no difference in relative proportions of overall bacterial composition at the species, genus or phylum levels. Additionally, there was no difference in relative proportion for total species per patient between the two technologies. Conclusion HOMINGS significantly expanded oral bacterial species identification compared to HOMIM. The genus and species probes, combined in HOMINGS, provided a more comprehensive representation of oral bacterial community, critical for future characterization of oral microbes in distant site infections. PMID:27065347

  18. Comparison of culture and acid-fast bacilli stain to PCR for detection of Mycobacterium tuberculosis in clinical samples.

    Science.gov (United States)

    Aslanzadeh, J; de la Viuda, M; Fille, M; Smith, W B; Namdari, H

    1998-08-01

    The major drawback in effective use of polymerase chain reaction (PCR) for detecting Mycobacterium tuberculosis (MTB) in clinical samples is the presence of PCR inhibitors and unique cell components of the organism that complicate DNA extraction and subsequent PCR amplification. A PCR assay with a unique multistep DNA extraction method that minimizes these problems was compared in a prospective study to acid-fast bacilli stain (AFBS) and culture for detecting MTB in clinical samples. A total of 254 clinical specimens in two separate studies were processed for MTB by these techniques. While PCR and culture were 100% sensitive and specific, culture required up to 8 weeks of incubation and additional time to perform biochemical testing to identify the isolated micro-organism. Acid-fast bacilli stain had a specificity of about 87% and did not differentiate among Mycobacterial species. In contrast, the results from PCR were available within 48 h and did not require additional testing to attain a final result. Polymerase chain reaction was highly reliable for detection and confirmation and interpretation of positive AFBS results. The assay was easy to perform with a turn around time of about 2 days.

  19. Role of real-time PCR (RT-PCR) in rapid diagnosis of tuberculous mycobacteria in different clinical samples.

    Science.gov (United States)

    2014-02-01

    The study was aimed for molecular detection of mycobacterial DNA in different clinical samples using real-time polymerase chain reaction (RT-PCR) system and rapid diagnosis of tuberculosis. A total of 508 clinical specimens (blood 343, menstrual fluid 53, endometrial tissue 43, body fluid 36, pus from lymph nodes 18, sputum 8, urine 5 and semen 2) were included in this study. We extracted DNA using QIAamp DNA Mini Kit (QIAGEN, Germany) and performed real-time assay using Rotor-Gene Q machine from Corbett Research, Australia for specific amplification of IS6110 sequence of mycobacterial genome. The RT-PCR result was also compared with bacterial culture and acid-fast bacillus staining. RT-PCR assay showed positivity in 52 cases and negative in 456 cases. Corresponding positive results in culture and acid-fast bacillus staining methods were 49 cases and 24 cases respectively. The sensitivity and specificity of detecting Mycobacterium tuberculosis by RT-PCR were 93.87% and 98.69% respectively taking positive culture results as reference standards. The overall positive and negative predictive values were 88.46% and 99.34% respectively. RT-PCR is a useful diagnostic tool for rapid and sensitive detection of mycobacteria in different clinical samples. The easy processing, fast reporting and relative lack of contamination issues make it worthy as a possible replacement to time consuming culture techniques. Moreover, it has added advantage of quantification of mycobacterial DNA, hence bacterial load.

  20. A comparison of new and revised Rorschach measures of schizophrenic functioning in a Serbian clinical sample.

    Science.gov (United States)

    Dzamonja-Ignjatovic, Tamara; Smith, Bruce L; Djuric Jocic, Dragana; Milanovic, Marko

    2013-01-01

    We empirically evaluated indexes derived from the Rorschach Comprehensive System (CS) and the Rorschach Performance Assessment System (R-PAS) that are used for the assessment of psychotic functioning in schizophrenia. We compared the Perceptual Thinking Index (PTI) and the Ego Impairment Index (EII-2) with their revised versions: Thought and Perception Composite (TP-Comp) and EII-3. We evaluated their predictive validity for differentiating schizophrenic from nonschizophrenic patients in a Serbian sample. The sample consisted of 211 (109 men and 102 women, 18-50 years old) inpatients in Serbia who were divided into 2 groups: schizophrenic (100) and nonschizophrenic (111). Test administration, coding, and form quality classification followed CS guidelines. Logistic regression analysis indicated that the new indexes TP-Comp and EII-3 have slightly better predictive power than their counterparts, PTI and EII-2, in identification of schizophrenia, and that TP-Comp performed better than other indexes, although all 4 indexes were successful in differentiating these groups. The results supported the use of TP-Comp in diagnosis of schizophrenia and generally provided evidence for the utility of the Rorschach in evaluating psychosis and for its use in a cross-national context. PMID:23844937

  1. Clinical syndromes, personality and recovery from stress: A study in occupational samples

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    José Miguel Rodríguez Molina

    2012-12-01

    Full Text Available Introduction. Stress manifests itself with different intensity and effects on different people. In many cases it leads to serious health problems or may worsen the prognosis of certain diseases. Stress has been linked to many conditions such as cancer, cardiovascular diseases and infectious diseases. The workplace can be a source of chronic stress. Many variables have been described that allegedly modulate stress response. Aim. To rank the relationship between some of these variables. A model is presented in this study whereby psychopathological personality traits should be related to one of those modulating variables and thus, with the subject's ability to recover from stress. Design. A cross-sectional descriptive design was used. Participants. The sample consisted of 108 volunteers: 15 drivers of Madrid city buses, 44 Iberia flight attendants and 49 waiters in bars in the Community of Madrid. Only 4 bus drivers refused to participate. All flight attendants and waiters consented to be included in the study. Intervention. Tests RESTQ-WORK of Kallus and Jiménez and MCMI of Millon were applied to a sample of 108 workers (bus drivers, bar tender and flight attendants. Outcomes. The hypothesis was verified through Hierarchical Multiple Regression Analysis for each dependant variable.

  2. Development and validation of a clinical trial patient stratification assay that interrogates 27 mutation sites in MAPK pathway genes.

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    Ken C N Chang

    Full Text Available Somatic mutations identified on genes related to the cancer-developing signaling pathways have drawn attention in the field of personalized medicine in recent years. Treatments developed to target a specific signaling pathway may not be effective when tumor activating mutations occur downstream of the target and bypass the targeted mechanism. For instance, mutations detected in KRAS/BRAF/NRAS genes can lead to EGFR-independent intracellular signaling pathway activation. Most patients with these mutations do not respond well to anti-EGFR treatment. In an effort to detect various mutations in FFPE tissue samples among multiple solid tumor types for patient stratification many mutation assays were evaluated. Since there were more than 30 specific mutations among three targeted RAS/RAF oncogenes that could activate MAPK pathway genes, a custom designed Single Nucleotide Primer Extension (SNPE multiplexing mutation assay was developed and analytically validated as a clinical trial assay. Throughout the process of developing and validating the assay we overcame many technical challenges which include: the designing of PCR primers for FFPE tumor tissue samples versus normal blood samples, designing of probes for detecting consecutive nucleotide double mutations, the kinetics and thermodynamics aspects of probes competition among themselves and against target PCR templates, as well as validating an assay when positive control tumor tissue or cell lines with specific mutations are not available. We used Next Generation sequencing to resolve discordant calls between the SNPE mutation assay and Sanger sequencing. We also applied a triplicate rule to reduce potential false positives and false negatives, and proposed special considerations including pre-define a cut-off percentage for detecting very low mutant copies in the wild-type DNA background.

  3. Association between CLN3 (Neuronal Ceroid Lipofuscinosis, CLN3 type gene expression and clinical characteristics of breast cancer patients

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    Rose-Mary eBoustany

    2015-10-01

    Full Text Available Breast cancer is the most common cancer in women worldwide. Elucidation of underlying biology and molecular pathways is necessary for improving therapeutic options and clinical outcomes. CLN3 protein (CLN3p, deficient in neurodegenerative CLN3 disease is anti-apoptotic, and defects in the CLN3 gene cause accelerated apoptosis of neurons in CLN3 disease and upregulation of ceramide. Dysregulated apoptotic pathways are often implicated in the development of the oncogenic phenotype. Predictably, CLN3 mRNA expression and CLN3 protein were upregulated in a number of human and murine breast cancer cell lines. Here, we determine CLN3 expression in non-tumor vs. tumor samples from fresh and formalin-fixed/paraffin-embedded (FFPE breast tissue and analyze the association between CLN3 overexpression and different clinicopathological characteristics of breast cancer patients. Additionally, gene expression of 28 enzymes involved in sphingolipid metabolism was determined. CLN3 mRNA is overexpressed in tumor vs. non-tumor breast tissue from FFPE and fresh samples, as well as in mouse MCF7 breast cancer compared to MCF10A normal cells. Of the clinicopathological characteristics of tumor grade, age, menopause status, estrogen receptor (ER, progesterone receptor (PR, and human epidermal growth factor receptor 2 (HER2, only absence of HER2 expression correlated with CLN3 overexpression. Sphingolipid genes for ceramide synthases 2 and 6 (CerS2; CerS6, delta(4-desaturase sphingolipid 2 (DEGS2 and acidic sphingomyelinase (SMPD1 displayed higher expression levels in breast cancer vs. control tissue, whereas, ceramide galactosyltransferase (UGT8 was underexpressed in breast cancer samples. CLN3 may be a novel molecular target for cancer drug discovery with the goal of modulation of ceramide pathways.

  4. A STUDY OF METALLO-BETA-LACTATAMASE PRODUCING PSEUDOMONAS AERUGINOSA IN CLINICAL SAMPLES OF S.S.G. HOSPITAL

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    Mehul S Chaudhari Tanuja B Javadekar Govind Ninama Neelam Pandya Jivraj Damor

    2011-04-01

    Full Text Available Introduction: Pseudomonas spp. is common pathogen causing nosocomial infection. Acquired drug resistance is frequent in nosocomial isolates of Pseudomonas spp. Acquired metallo-β-lactamases (MBL in pseudomonas spp. have recently emerged as one of the most worrisome resistance mechanism because of their capacity to hydrolyze all beta-lactam antibiotics including penicillins, cephalosporins and carbapenems, with the exception of aztreonam. Aim: To detect metallo-β-lactamase producing isolates of Pseudomonas aerugenosa from various clinical samples from patients admitted in our hospital. Material and methods : In this studyt we studied the prevalence, following standard methods of isolation and identification techniques of these bacteria from clinical materials Source : Samples of patients from different wards of S.S.G.Hosital are proceeded in Microbiology department , Medical College and S.S.G.Hospital Baroda. Results: Of total study of 150 isolates of Pseudomonas aeruginosa, 8 isolates are resistance to Imipenem . Of 8 samples , all are producing Metallo-Beta-Lactamase enzyme. Conclusion :Infection cause by MBL (metallo-β-lactamase positive isolates of Pseudomonas aerugenosa is important to identify because it poses not only therapeutic problem, but also a serious concern for infection control management. [National J of Med Res 2011; 1(2.000: 60-63

  5. Bipolar disorder in late life: clinical characteristics in a sample of older adults admitted for manic episode

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    Musetti Laura

    2008-07-01

    Full Text Available Abstract Background Although manic episodes in older adults are not rare, little published data exist on late-life manic episodes. Resistance to treatment and concomitant neurological lesions are frequent correlates of elderly mania. The aim of this study was to investigate the prevalence of hospitalizations due to mania in patients older than 64 years through a period of 5 years in an Italian public psychiatric ward. Moreover, we aimed at describing clinical presentation of elderly manic episodes. Methods A retrospective chart review was conducted in order to describe clinical presentation of 20 elderly patients hospitalized for manic episode; moreover, we compared age at onset, the presence of family history for mood disorders, psychosis and irritability between the elderly group and a matched group of 20 younger manic inpatients. Results Seven percent of the whole inpatient elderly people suffered from mania. Half of those patients had a mood disorder age at onset after 50 years and 5 patients were at their first manic episode. Geriatric- and adulthood mania showed similar clinical presentation but younger people had more frequently a mood disorders family history. Conclusion Half of our older manic inpatients consisted of "classic" bipolar patients with an extension of clinical manifestations into later life; the other half of our sample was heterogeneous, even though it was not possible to identify clearly which patients may have had vascular lesions related to the onset of mania.

  6. Human papillomavirus testing by self-sampling: assessment of accuracy in an unsupervised clinical setting

    Science.gov (United States)

    Szarewski, Anne; Cadman, Louise; Mallett, Susan; Austin, Janet; Londesborough, Philip; Waller, Jo; Wardle, Jane; Altman, Douglas G; Cuzick, Jack

    2007-01-01

    Objectives: To compare the performance and acceptability of unsupervised self-sampling with clinician sampling for high-risk human papillomavirus (HPV) types for the first time in a UK screening setting. Setting: Nine hundred and twenty women, from two demographically different centres, attending for routine cervical smear testing Methods: Women performed an unsupervised HPV self-test. Immediately afterwards, a doctor or nurse took an HPV test and cervical smear. Women with an abnormality on any test were offered colposcopy. Results: Twenty-one high-grade and 39 low-grade cervical intraepithelial neoplasias (CINs) were detected. The sensitivity for high-grade disease (CIN2+) for the self HPV test was 81% (95% confidence interval [CI] 60–92), clinician HPV test 100% (95% CI 85–100), cytology 81% (95% CI 60–92). The sensitivity of both HPV tests to detect high- and low-grade cervical neoplasia was much higher than that of cytology (self-test 77% [95%CI 65–86], clinician test 80% [95% CI 68–88], cytology 48% [95% CI 36–61]). For both high-grade alone, and high and low grades together, the specificity was significantly higher for cytology (greater than 95%) than either HPV test (between 82% and 87%). The self-test proved highly acceptable to women and they reported that the instructions were easy to understand irrespective of educational level. Conclusions: Our results suggest that it would be reasonable to offer HPV self-testing to women who are reluctant to attend for cervical smears. This approach should now be directly evaluated among women who have been non-attenders in a cervical screening programme. PMID:17362570

  7. Species and Strain-specific Typing of Cryptosporidium Parasites in Clinical and Environmental Samples

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    Lihua Xiao

    1998-09-01

    Full Text Available Cryptosporidiosis has recently attracted attention as an emerging waterborne and foodborne disease as well as an opportunistic infection in HIV infected individuals. The lack of genetic information, however, has resulted in confusion in the taxonomy of Cryptosporidium parasites and in the development of molecular tools for the identification and typing of oocysts in environmental samples. Phylogenetic analysis of the small subunit ribosomal RNA (SSU rRNA gene has shown that the genus Cryptosporidium is comprised of several distinct species. Our data show the presence of at least four species: C. parvum, C. muris, C. baileyi and C. serpentis (C. meleagridis, C. nasorum and C. felis were not studied. Within each species, there is some sequence variation. Thus, various genotypes (genotype 1, genotype 2, guinea pig genotype, monkey genotype and koala genotype, etc. of C. parvum differ from each other in six regions of the SSU rRNA gene. Information on polymorphism in Cryptosporidium parasites has been used in the development of species and strain-specific diagnostic tools. Use of these tools in the characterization of oocysts various samples indicates that C. parvum genotype 1 is the strain responsible for most human Cryptosporidium infections. In contrast, genotype 2 is probably the major source for environmental contamination of environment, and has been found in most oysters examined from Chesapeake Bay that serve as biologic monitors of surface water. Parasites of Cryptosporidium species other than C. parvum have not been detected in HIV+ individuals, indicating that the disease in humans is caused only by C. parvum.

  8. Clinical utility of reverse phase protein array for molecular classification of breast cancer.

    Science.gov (United States)

    Negm, Ola H; Muftah, Abir A; Aleskandarany, Mohammed A; Hamed, Mohamed R; Ahmad, Dena A J; Nolan, Christopher C; Diez-Rodriguez, Maria; Tighe, Patrick J; Ellis, Ian O; Rakha, Emad A; Green, Andrew R

    2016-01-01

    Reverse Phase Protein Array (RPPA) represents a sensitive and high-throughput technique allowing simultaneous quantitation of protein expression levels in biological samples. This study aimed to confirm the ability of RPPA to classify archival formalin-fixed paraffin-embedded (FFPE) breast cancer tissues into molecular classes used in the Nottingham prognostic index plus (NPI+) determined by immunohistochemistry (IHC). Proteins were extracted from FFPE breast cancer tissues using three extraction protocols: the Q-proteome FFPE Tissue Kit (Qiagen, Hilden, Germany) and two in-house methods using Laemmli buffer with either incubation for 20 min or 2 h at 105 °C. Two preparation methods, full-face sections and macrodissection, were used to assess the yield and quality of protein extracts. Ten biomarkers used for the NPI+ (ER, PgR, HER2, Cytokeratins 5/6 and 7/8, EGFR, HER3, HER4, p53 and Mucin 1) were quantified using RPPA and compared to results determined by IHC. The Q-proteome FFPE Tissue Kit produced significantly higher protein concentration and signal intensities. The intra- and inter-array reproducibility assessment indicated that RPPA using FFPE lysates was a highly reproducible and robust technique. Expression of the biomarkers individually and in combination using RPPA was highly consistent with IHC results. Macrodissection of the invasive tumour component gave more reliable results with the majority of biomarkers determined by IHC, (80 % concordance) compared with full-face sections (60 % concordance). Our results provide evidence for the technical feasibility of RPPA for high-throughput protein expression profiling of FFPE breast cancer tissues. The sensitivity of the technique is related to the quality of extracted protein and purity of tumour tissue. RPPA could provide a quantitative technique alternative to IHC for the biomarkers used in the NPI+.

  9. German Screen for Child Anxiety Related Emotional Disorders (SCARED: Reliability, Validity, and Cross-Informant Agreement in a Clinical Sample

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    Wiegand-Grefe Silke

    2010-06-01

    Full Text Available Abstract Background The psychometric properties and cross-informant agreement of a German translation of the Screen for Child Anxiety Related Emotional Disorders (SCARED were assessed in a clinical sample Methods 102 children and adolescents in outpatient psychotherapy and their parents filled out the SCARED and Youth Self Report/Child Behaviour Checklist (YSR/CBCL. Results The German SCARED showed good internal consistency for both parent and self-report version, and proved to be convergently and discriminantly valid when compared with YSR/CBCL scales. Cross-informant agreement was moderate with children reporting both a larger number as well as higher severity of anxiety symptoms than their parents. Conclusion In conclusion, the German SCARED is a valid and reliable anxiety scale and may be used in a clinical setting

  10. Brain structural correlates of schizotypy and psychosis proneness in a non-clinical healthy volunteer sample.

    Science.gov (United States)

    Nenadic, Igor; Lorenz, Carsten; Langbein, Kerstin; Dietzek, Maren; Smesny, Stefan; Schönfeld, Nils; Fañanás, Lourdes; Sauer, Heinrich; Gaser, Christian

    2015-10-01

    Schizotypal traits are phenotypic risk factors for schizophrenia, associated with biological changes across a putative schizophrenia spectrum. In this study, we tested the hypothesis that brain structural changes in key brain areas relevant to this spectrum (esp. medial and lateral prefrontal cortex) would vary across different degrees of schizotypal trait expression and/or phenotypic markers of psychosis proneness in healthy non-clinical volunteers. We analysed high-resolution 3Tesla magnetic resonance images (MRI) of 59 healthy volunteers using voxel-based morphometry (VBM), correlating grey matter values to the positive and negative symptom factors of the schizotypal personality questionnaire (SPQ, German version) and a measure of psychosis proneness (community assessment of psychic experiences, CAPE). We found positive correlations between positive SPQ dimension and bilateral inferior and right superior frontal cortices, and positive CAPE dimension and left inferior frontal cortex, as well as CAPE negative dimension and right supplementary motor area (SMA) and left inferior parietal cortex. However, only the positive correlation of the right precuneus with negative schizotypy scores was significant after FWE correction for multiple comparisons. Our findings confirm an effect of schizotypal traits and psychosis proneness on brain structure in healthy subjects, providing further support to a biological continuum model. PMID:26164819

  11. Interpreting change on the WAIS-III/WMS-III in clinical samples.

    Science.gov (United States)

    Iverson, G L

    2001-02-01

    Clinicians should note that there is considerable variability in the reliabilities of the index and subtest scores derived from the third editions of the Wechsler Adult Intelligence Scale (WAIS-III) and the Wechsler Memory Scale (WMS-III). The purpose of this article is to review these reliabilities and to illustrate how they can be used to interpret change in patients' performances from test to retest. The WAIS-III IQ and Index scores are consistently the most reliable scores, in terms of both internal consistency and test-retest reliability. The most internally consistent WAIS-III subtests are Vocabulary, Information, Digit Span, Matrix Reasoning, and Arithmetic. Information and Vocabulary have the highest test-retest reliability. On the WMS-III, the Auditory Immediate Index, Immediate Memory Index, Auditory Delayed Index, and General Memory Index are the most reliable, in terms of both internal consistency and test-retest reliability. The Logical Memory I and Verbal Paired Associates I subtests are the most reliable. Data from three clinical groups (i.e., Alzheimer's disease, chronic alcohol abuse, and schizophrenia) were extracted from the Technical Manual [Psychological Corporation (1997). WAIS-III/WMS-III Technical Manual. San Antonio: Harcourt Brace] for the purpose of calculating reliable change estimates. A table of confidence intervals for test-retest measurement error is provided to help the clinician determine if patients have reliably improved or deteriorated on follow-up testing.

  12. HIV-Associated Oral Mucosal Melanin Hyperpigmentation: A Clinical Study in a South African Population Sample

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    R. Chandran

    2016-01-01

    Full Text Available Objective. The aim of the study was to determine the prevalence of HIV-associated oral mucosal melanin hyperpigmentation (HIV-OMH in a specific population of HIV-seropositive South Africans and to analyse the associations between HIV-OMH clinical features and the demographic and immunological characteristics of the study cohort. Material and Methods. This cross-sectional study included 200 HIV-seropositive Black subjects. The collected data comprised age, gender, CD4+ T cell count, viral load, systemic disease, medications, oral site affected by HIV-OMH, extent (localized or generalized, intensity of the pigmentation (dark or light, and smoking and snuff use. Results. Overall, 18.5% of the study cohort had HIV-OMH. Twenty-two and a half percent had OMH that could not with confidence be attributed to HIV infection, and 59% did not have any OMH. There was a significant but weak association between smoking and the presence of HIV-OMH. Conclusions. The prevalence of HIV-OMH in the study population was 18.5%, the gingiva being the most commonly affected site. It appears that the CD4+ T cell count does not play any role in the biopathology of HIV-OMH.

  13. A factor analytic investigation of the Tripartite model of affect in a clinical sample of young Australians

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    Cosgrave Elizabeth M

    2008-09-01

    Full Text Available Abstract Background The Mood and Anxiety Symptom Questionnaire (MASQ was designed to specifically measure the Tripartite model of affect and is proposed to offer a delineation between the core components of anxiety and depression. Factor analytic data from adult clinical samples has shown mixed results; however no studies employing confirmatory factor analysis (CFA have supported the predicted structure of distinct Depression, Anxiety and General Distress factors. The Tripartite model has not been validated in a clinical sample of older adolescents and young adults. The aim of the present study was to examine the validity of the Tripartite model using scale-level data from the MASQ and correlational and confirmatory factor analysis techniques. Methods 137 young people (M = 17.78, SD = 2.63 referred to a specialist mental health service for adolescents and young adults completed the MASQ and diagnostic interview. Results All MASQ scales were highly inter-correlated, with the lowest correlation between the depression- and anxiety-specific scales (r = .59. This pattern of correlations was observed for all participants rating for an Axis-I disorder but not for participants without a current disorder (r = .18. Confirmatory factor analyses were conducted to evaluate the model fit of a number of solutions. The predicted Tripartite structure was not supported. A 2-factor model demonstrated superior model fit and parsimony compared to 1- or 3-factor models. These broad factors represented Depression and Anxiety and were highly correlated (r = .88. Conclusion The present data lend support to the notion that the Tripartite model does not adequately explain the relationship between anxiety and depression in all clinical populations. Indeed, in the present study this model was found to be inappropriate for a help-seeking community sample of older adolescents and young adults.

  14. Evaluation of the WMS-III Rarely Missed Index in a naive clinical sample.

    Science.gov (United States)

    Axelrod, Bradley N; Barlow, Alycia; Paradee, Christine

    2010-01-01

    We examined the WMS-III Rarely Missed Index as a reliable predictor of fabrication of memory difficulties. A total of 31 outpatients referred for neuropsychological evaluation completed the WMS-III Logical Memory Delayed Recognition Test (LMDR) before having heard the stories and again after hearing the stories. Of the 30 items from the LMDR completed by participants who had not heard the stories, 5 were found to significantly differ from chance; only 1 of those items was found to do so in the original RMI studies. Conversely, of the six items in the original RMI study, only one was found to differ from chance in the present study. A Monte Carlo randomization of the original six RMI items found that 69% of random responders fell below cutoffs for incomplete effort, suggesting that those with memory impairment severe enough to result in random responding are likely to be classified as demonstrating less than optimal effort. An examination of response bias found no differences among negative, positive, or no-bias responders in terms of overall memory performance. However, RMI was indicative of incomplete effort more often with individuals presenting with a negative response bias. The present study provides evidence of the limitations of using the RMI to detect incomplete effort using the LMDR. The relative values of specificity versus sensitivity are particularly important in clinical evaluations, with the prevention of false accusations of malingering an important goal. The use of multiple indicators of effort is reinforced by this study, and the conservative interpretation of the RMI in particular is recommended.

  15. The isolation of Candida rugosa and Candida mesorugosa from clinical samples in Ghana.

    Science.gov (United States)

    Adjapong, Gloria; Bartlett, Michael; Hale, Marie; Garrill, Ashley

    2016-03-01

    Members of the Candida rugosa species complex have been described as emerging fungal pathogens and are responsible for a growing number of Candida infections. In this communication we report the isolation of Candida rugosa and Candida mesorugosa in Ghana. To the best of our knowledge this is the first description of this species complex from a clinical setting in Africa.The isolates were identified on the basis of their rRNA gene internal transcribed spacer (ITS) sequences. For one isolate, obtained from sputum, the sequence grouped well with that of C. rugosa. Two other isolates from urine had sequences that grouped with Candida mesorugosa. Morphologically, C. rugosa formed white, wrinkled, and flat colonies on Sabouraud Dextrose Agar (SDA), whereas C. mesorugosa formed white, smooth colonies. On chromogenic medium, the isolates formed small, dry greenish-blue colonies with a pale or white border, similar to C. albicans. The C. rugosa isolate produced pseudohyphae in human serum and on CMA-Tween 80 agar. In contrast, the C. mesorugosa isolates did not generate pseudohyphae in human serum, but generated a few pseudohyphae with abundant blastoconidia on CMA-Tween 80 agar. Growth was observed at 37 °C and 42 °C but not at 45 °C.The two C. mesorugosa isolates had Minimum Inhibitory Concentrations (MICs) of 6 and 48 μg ml(-1) for fluconazole and are thus resistant. The C. rugosa isolate had an MIC of 24 μg ml(-1), indicative of resistance. All three isolates were susceptible to itraconazole and voriconazole (with respective MICs of < 0.125 μg ml(-1)).

  16. Relationship between acne vulgaris and attention-deficit/hyperactivity disorder symptoms in a clinical sample of women*

    Science.gov (United States)

    Bilgic, Ayhan; Bilgic, Özlem; Çolak, Rukiye Sivri; Altınyazar, Hilmi Cevdet

    2016-01-01

    Acne vulgaris has recently been reported to be associated with elevated rates of attention deficit/hyperactivity disorder in epidemiological studies. This report examines childhood and current attention-deficit/hyperactivity disorder symptoms in a clinical sample of female adults. Ninety-one women with acne vulgaris and 53 controls were included in this study. The aforementioned symptoms were measured in participants. No significant differences were found between patients and controls in any of the measurements. Contrary to the findings of epidemiological studies, this study did not uncover a link between acne vulgaris and attention-deficit/hyperactivity disorder PMID:27192533

  17. 慢性乙肝患者石蜡包埋肝组织中HBV cccDNA定量检测方法的建立%Quantification of covalently closed circular DNA of hepatitis B virus in FFPE liver tissues of chronic hepatitis B patients

    Institute of Scientific and Technical Information of China (English)

    韩佳琪; 钟彦伟; 任晓强; 邹正升; 刘树红; 刘学恩; 赵景民; 徐东平

    2011-01-01

    目的 建立慢性乙肝患者微量石蜡包埋肝组织共价闭合环状DNA(HBV cccDNA)定量检测方法.方法 以37例慢性乙肝患者甲醛同定石蜡包埋肝组织为研究对象,提取肝组织HBV DNA,经不降解质粒的ATP依赖的DNA酶(PSAD)消化后,利用滚环扩增加跨缺口实时荧光PCR扩增技术检测肝组织中HBV cccDNA含量,以β-actin为内参照.通过已知浓度的模板DNA进行梯度稀释鉴定该方法的灵敏度,并对该方法进行批内和批间重复性检测.应用该方法分析37例慢性乙肝患者肝组织HBV cccDNA与血清总HBVDNA、肝组织总HBVDNA的关系,以及肝组织HBV cccDNA和HBVDNA与血清HBeAg表达之间的关系.结果 成功建立了微量石蜡包埋肝组织HBV cccDNA的定量检测方法,该方法具有较好的特异性、灵敏度和稳定性.HBeAg阳性者肝组织中cccDNA含量明显高于HBeAg阴性者,HBV cccDNA水平与血清总HBV DNA水平(R2=0.48,P=0.042)及肝组织总HBV DNA水平(R2=0.63,P=0.001)呈正相关.结论 该方法可特异灵敏地定量检测微量石蜡包埋组织切片中的HBV cccDNA.%Objective To establish a method of detecting HBV covalently closed circular DNA (cccDNA) in micro-formalin fixed paraffin imbedding (FFPE) liver biopsy samples.Methods FFPE liver biopsies from 37 patients with chronic hepatitis B were studied.The intrahepatic HBV DNA was extracted and pre-treated with plasmid-safe ATP-dependent DNAse (PSAD), and then amplified by rolling circular amplification (RCA).The HBV cccDNA was quantitatively detected by Taqman real-time PCR with primers located on both sides of the gap of HBV DNA.The human β-actin gene served as the internal control.The sensitivity was tested by serially diluting the DNA templates with known concentrations.The repeatability and stability were evaluated with inter-assay and intra-assay.The level of intrahepatic HBV cccDNA, HBV total DNA, serum HBV DNA and ALT were also compared to find the relations between then

  18. A confirmatory factor analysis of the cognitive capacity screening examination in a clinical sample.

    Science.gov (United States)

    Anderson, D A; Burton, D B; Parker, J D; Godding, P R

    2001-01-01

    Structured mental status examinations offer several advantages over unstructured mental status examinations; however, few have been subjected to advanced psychometric analysis. Confirmatory factor analysis empirically tests the predictive validity of individual test indices within an a priori methodological framework. With such analysis, one can test hypotheses about the structure of latent variability within a given data set. The purpose of this study was to perform a confirmatory factor analysis of the Cognitive Capacity Screening Examination, the most comprehensive of the brief structured mental status examinations. A confirmatory factor analysis of the Cognitive Capacity Screening Examination (CCSE) was performed by applying LISREL 7 to a sample of 924 male veterans, 409 patients from a chemical dependence treatment program, and 515 individuals from a psychology consultation service. Constructs were derived from previous exploratory analysis of the scale. The results of the confirmatory factor analysis and three indices of model fit support an 11 factor model more complex than that originally formulated for the CCSE. However, three of these factors (digit span with interference, complex mental mathematics, and verbal memory) were more sensitive to impairment than other factors, accounting for over 90% of the CCSE total score variance. Although the CCSE is a more complex test than originally envisioned by its designers, it may not be necessary to give all items on the test. Either a subset of the CCSE items (the CCSE-A) or a relatively brief, informal mental status exam may be adequate for many patients. PMID:11912677

  19. Using co-occurrence to evaluate belief coherence in a large non clinical sample.

    Directory of Open Access Journals (Sweden)

    Rachel Pechey

    Full Text Available Much of the recent neuropsychological literature on false beliefs (delusions has tended to focus on individual or single beliefs, with few studies actually investigating the relationship or co-occurrence between different types of co-existing beliefs. Quine and Ullian proposed the hypothesis that our beliefs form an interconnected web in which the beliefs that make up that system must somehow "cohere" with one another and avoid cognitive dissonance. As such beliefs are unlikely to be encapsulated (i.e., exist in isolation from other beliefs. The aim of this preliminary study was to empirically evaluate the probability of belief co-occurrence as one indicator of coherence in a large sample of subjects involving three different thematic sets of beliefs (delusion-like, paranormal & religious, and societal/cultural. Results showed that the degree of belief co-endorsement between beliefs within thematic groupings was greater than random occurrence, lending support to Quine and Ullian's coherentist account. Some associations, however, were relatively weak, providing for well-established examples of cognitive dissonance.

  20. The relationship between temperament and autistic traits in a non-clinical students sample.

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    Ewa Pisula

    Full Text Available Since temperament affects the development of social behaviours and interpersonal relations, the possible links between autistic traits and temperament are of particular interest. The purpose of the study was to explore the relationships between autistic traits and temperamental characteristics in the framework of the Regulative Temperament Theory by Strelau, and the Emotionality, Activity and Sociability theory by Buss and Plomin, with particular emphasis on gender differences. The Autism Spectrum Quotient (AQ, Formal Characteristics of Behaviour--Temperament Inventory and Temperament Survey for Adults were administered. The participants were 593 university students, including 364 females and 229 males. Results showed positive correlations between autistic traits and Emotional Reactivity, Perseveration, Distress, Fear and Anger, and negative correlations with Activity, Briskness, Endurance and Sociability. The results of multiple regression analyses involving the Autism Spectrum Quotient score as a dependent measure were different for females and males. Results of exploratory PCA analysis showed that AQ score, Sociability and Activity loaded one factor (with AQ loading being opposite to two others. High AQ scorers demonstrated higher Emotional Reactivity, Perseveration, Distress and Anger, and lower Briskness, Endurance, Activity and Sociability as compared to norms for the general population. In this study we showed that temperament measures were able to identify items that correlated in parts with autistic traits, while other items were obverse. The relationships between temperament and autistic traits differ slightly between genders. We assume that with regard to the broader autism phenotype, temperaments might be helpful in characterizing healthy control samples.

  1. The relationship between temperament and autistic traits in a non-clinical students sample.

    Science.gov (United States)

    Pisula, Ewa; Kawa, Rafał; Danielewicz, Dorota; Pisula, Wojciech

    2015-01-01

    Since temperament affects the development of social behaviours and interpersonal relations, the possible links between autistic traits and temperament are of particular interest. The purpose of the study was to explore the relationships between autistic traits and temperamental characteristics in the framework of the Regulative Temperament Theory by Strelau, and the Emotionality, Activity and Sociability theory by Buss and Plomin, with particular emphasis on gender differences. The Autism Spectrum Quotient (AQ), Formal Characteristics of Behaviour--Temperament Inventory and Temperament Survey for Adults were administered. The participants were 593 university students, including 364 females and 229 males. Results showed positive correlations between autistic traits and Emotional Reactivity, Perseveration, Distress, Fear and Anger, and negative correlations with Activity, Briskness, Endurance and Sociability. The results of multiple regression analyses involving the Autism Spectrum Quotient score as a dependent measure were different for females and males. Results of exploratory PCA analysis showed that AQ score, Sociability and Activity loaded one factor (with AQ loading being opposite to two others). High AQ scorers demonstrated higher Emotional Reactivity, Perseveration, Distress and Anger, and lower Briskness, Endurance, Activity and Sociability as compared to norms for the general population. In this study we showed that temperament measures were able to identify items that correlated in parts with autistic traits, while other items were obverse. The relationships between temperament and autistic traits differ slightly between genders. We assume that with regard to the broader autism phenotype, temperaments might be helpful in characterizing healthy control samples.

  2. DETECTION & PREVALENCE OF EXTENDED SPECTRUM ΒETA - LACTAMASES AMONG ENTEROBACTERIACEAE SPECIES FROM VARIOUS CLINICAL SAMPLES AT KIMS, AMALAPURAM.

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    Padmaja

    2012-10-01

    Full Text Available ABSTRACT: The present study was conducted in the Department of Microbiology, KIMS, Amalapuram, East Godavari from January 2012 to July 2012. Out of 100 different clinical samples, 50 were culture positive. Of the 100 samples collected, more were from post operative wound sepsis - 44 (44%, followed by cellulites - 20 (20%, Ulcers - 17 (17%, Injuries 15 (15%. Least number of cases are from burns - 4 (4% . Among 50 culture positive cases, 38 (76% isolates belonged to Enterobacteriaceae famil y, followed by Pseudomonas aeruginosa - 8 (16%, followed by Staphylococcus aureus - 4 (8%. Among 38 of Enterobacteriaceae family isolates, 15 were ESBL producers. Among ESBL positiv e strains, more drug resistance was seen to Ceftazidime and Ampicillin (93.33%, followed by Ceftriaxone (86.66%, Aztreonam & Cefotaxime (80%.

  3. Rapid Detection of Methicillin-Resistant Staphylococcus aureus Directly from Sterile or Nonsterile Clinical Samples by a New Molecular Assay

    Science.gov (United States)

    Francois, Patrice; Pittet, Didier; Bento, Manuela; Pepey, Béatrice; Vaudaux, Pierre; Lew, Daniel; Schrenzel, Jacques

    2003-01-01

    A rapid procedure was developed for detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) directly from sterile sites or mixed flora samples (e.g., nose or inguinal swabs). After a rapid conditioning of samples, the method consists of two main steps: (i) immunomagnetic enrichment in S. aureus and (ii) amplification-detection profile on DNA extracts using multiplex quantitative PCR (5′-exonuclease qPCR, TaqMan). The triplex qPCR assay measures simultaneously the following targets: (i) mecA gene, conferring methicillin resistance, common to both S. aureus and Staphylococcus epidermidis; (ii) femA gene from S. aureus; and (iii) femA gene from S. epidermidis. This quantitative approach allows discrimination of the origin of the measured mecA signal. qPCR data were calibrated using two reference strains (MRSA and methicillin-resistant S. epidermidis) processed in parallel to clinical samples. This 96-well format assay allowed analysis of 30 swab samples per run and detection of the presence of MRSA with exquisite sensitivity compared to optimal culture-based techniques. The complete protocol may provide results in less than 6 h (while standard procedure needs 2 to 3 days), thus allowing prompt and cost-effective implementation of contact precautions. PMID:12517857

  4. Associations of Age, Gender, and Subtypes With ADHD Symptoms and Related Comorbidity in a Danish Sample of Clinically Referred Adults

    DEFF Research Database (Denmark)

    Soendergaard, Helle Moeller; Thomsen, Per Hove; Pedersen, Erik;

    2015-01-01

    Objective: The aim was to examine associations of age and gender with ADHD subtypes and subsequently to examine associations of age, gender, and subtypes with comorbid psychiatric disorders. Method: Odds ratios were calculated and logistic regression performed using information from a clinical sa....... Conclusion: When assessing adult ADHD patients' age, gender, subtype, and related comorbid symptom profiles should be taken into account. (J. of Att. Dis. XXXX; XX(X) XX-XX).......Objective: The aim was to examine associations of age and gender with ADHD subtypes and subsequently to examine associations of age, gender, and subtypes with comorbid psychiatric disorders. Method: Odds ratios were calculated and logistic regression performed using information from a clinical...... sample of 155 ADHD adults referred to a Danish specialized ADHD unit from 2010 to 2011. Results: A majority of men (65%) was found in the sample. Most patients were subtyped ADHD combined (78%), followed by ADHD inattentive (18%), and ADHD hyperactive-impulsive (4%). No significant differences were found...

  5. Isolation, Identification, and In Vitro Antifungal Susceptibility Testing of Dermatophytes from Clinical Samples at Sohag University Hospital in Egypt

    Science.gov (United States)

    Shalaby, Mona Fattouh Mohamed; El-din, Asmaa Nasr; El-Hamd, Mohammed Abu

    2016-01-01

    Aim The objective of this study was to isolate, identify, and explore the in-vitro antifungal susceptibility pattern of dermatophytes isolated from clinically suspected cases of dermatophytosis (tinea infections) attending the Dermatology Outpatient Clinic. Methods This study was conducted at Sohag University Hospital from December 2014 to December 2015. Clinical samples (e.g., skin scrapings and hair stumps) were collected under aseptic precautions. The identification of dermatophytes was performed through microscopic examination using 10% potassium hydroxide (KOH) with 40% dimethyl sulphoxide (DMSO) mounts and culture on Sabouraud dextrose agar (SDA) and on Dermasel agar base media, both supplemented with chloramphenicol and cycloheximide. All dermatophytes isolates were subjected to antifungal susceptibility testing using the agar-based disk diffusion (ABDD) method against Clotrimazole, Miconazole, Fluconazole, and Griseofulvin. Data were analyzed via SPSS 16, using Chi square and a screening test (cross-tabulation method). Results A total of 110 patients of dermatophytosis were studied. The patients were clinically diagnosed and mycologically confirmed as having tinea capitis (49), tinea corporis (30), tinea pedis (16), tinea cruris (9), or tinea barbae (6). The dermatophytes isolates belonged to 4 species: Microsporum canis 58 (52.7%), Microsporum gypseum 23 (20.9%), Trichophyton mentagrophytes 18 (16.4%), and Microsporum audouinii 11 (10%). The most effective antifungal drugs tested were Clotrimazole, followed by Miconazole (95.5% and 84.5% of isolates were susceptible, respectively). Conclusion Every patient with a tinea infection should be properly studied for a mycological examination and should be treated accordingly. Dermasel agar is more useful as an identification medium in the isolation of dermatophytes. The ABDD method appears to be a simple, cost-effective, and promising method for the evaluation of antifungal susceptibility of dermatophytes. PMID

  6. Determination of the prevalence of extended spectrumβ-lactamase in clinical samples collected from Dehradun City Hospital

    Institute of Scientific and Technical Information of China (English)

    Narayan Sharma; Ripan Mujumdar; Rajeev Kumar Gautam

    2016-01-01

    Objective:To detect extended spectrumβ-lactamase (ESBL) and determine its prevalence in various clinical samples collected from Dehradun City Hospital. Methods:The samples were first cultured in MacConkey’s agar plates by streak plate method, then identified by Gram staining and biochemical tests. The isolated bacterial strains were then tested for antibiotic susceptibility by Kirby-Bauer method. TheESBL detection is then carried out by double disc diffusion method. Results: Off the 56 samples cultured, 21 strains were identified which were sixEscherichia coli(E. coli), sixKlebsiella, fourProteus, fourPseudomonas aeruginosa (P. aeruginosa) and only oneAcinetobacter. Eight out of 21 (38.1%) strains including three ofE. coli, three ofKlebsiella and two ofP. aeruginosa, were found to be resistance to all five antibiotics (piperacillin, amikacin, ampicillin, gentamicin, and ciprofloxacin). Initial screening using four antibiotics (cefotaxime, ceftazidime, aztreonam and ceftriaxone) and the final confirmatory test using ceftazidime/clavulanic acid and ceftazidime alone showed that 19.05% of all strains isolated wereESBL producers. Individually, 16.67%E. coli, 16.67%Klebsiella pneumoniae, 25%P. aeruginosa and 100%Acinetobacter were found to beESBL producers. Conclusions:Antibiotic resistance byESBL has become a major risk factor worldwide, therefore routine checkup and accordingly prescription are suggested.

  7. WITHDRAWN: Clinical fitting of CAD/CAM zirconia single crowns generated from digital intraoral impressions based on active wavefront sampling.

    Science.gov (United States)

    Scotti, Roberto; Cardelli, Paolo; Baldissara, Paolo; Monaco, Carlo

    2011-10-17

    OBJECTIVES: The aim of this clinical trial was to test the accuracy of single all-ceramic zirconia crowns resulting from digital intraoral impressions with active wavefront sampling technology by measuring the marginal and internal fits of the crowns. MATERIALS AND METHODS: Thirty-seven teeth (24 anterior and 13 posterior) in fifteen patients were restored with single zirconia-ceramic crowns (Lava/Lava Ceram; 3M ESPE) generated from a digital intraoral scanner (Lava Chairside Oral Scanner; 3M ESPE). Before definitive insertion, silicone replicas were produced for all 37 crowns. The sample was cut in four sections; each section was evaluated in four points: marginal gap, mid-axial wall, axio-occlusal edge and centro-occlusal. A total of 592 measurements (148 for each evaluation point) was examined using stereomicroscopy with a magnification of 50×. The Mann-Whitney U test was used to evaluate whether there were differences between anterior and posterior values (alpha=0.05). RESULTS: The mean values for each point were: 48.65μm (SD 29.45μm) for the marginal gap, 112.25μm (SD 55.54μm) at the mid-axial wall, 137.81μm (SD 71.31μm) at the axio-occlusal edge of the abutments, and 157.25μm (SD 75.51μm) at the centro-occlusal location. No statistical differences were found between the anterior and posterior group for each point (p-values: P1=0.39; P2=0.38; P3=0.07; P4=0.30). CONCLUSIONS: The marginal and internal fitting values obtained were within literature agreed as clinically acceptable for both anterior and posterior teeth. CLINICAL RELEVANCE: Single crown restorations obtained using digital intraoral impressions based on active wavefront sampling technology presented enough accuracy to be used as an alternative to the conventional impression techniques. PMID:22027653

  8. Genotyping of human parvovirus B19 in clinical samples from Brazil and Paraguay using heteroduplex mobility assay, single-stranded conformation polymorphism and nucleotide sequencing

    Directory of Open Access Journals (Sweden)

    Marcos César Lima de Mendonça

    2011-06-01

    Full Text Available Heteroduplex mobility assay, single-stranded conformation polymorphism and nucleotide sequencing were utilised to genotype human parvovirus B19 samples from Brazil and Paraguay. Ninety-seven serum samples were collected from individuals presenting with abortion or erythema infectiosum, arthropathies, severe anaemia and transient aplastic crisis; two additional skin samples were collected by biopsy. After the procedure, all clinical samples were classified as genotype 1.

  9. Genotyping of human parvovirus B19 in clinical samples from Brazil and Paraguay using heteroduplex mobility assay, single-stranded conformation polymorphism and nucleotide sequencing.

    Science.gov (United States)

    Mendonça, Marcos César Lima de; Ferreira, Ana Maria de Amorim; Santos, Marta Gonçalves Matos dos; Oviedo, Elva Cristina; Bello, Maria Sônia Dal; Siqueira, Marilda Mendonça; Maceira, Juan Manuel Piñeiro; von Hubinger, Maria Genoveva; Couceiro, José Nelson dos Santos Silva

    2011-06-01

    Heteroduplex mobility assay, single-stranded conformation polymorphism and nucleotide sequencing were utilised to genotype human parvovirus B19 samples from Brazil and Paraguay. Ninety-seven serum samples were collected from individuals presenting with abortion or erythema infectiosum, arthropathies, severe anaemia and transient aplastic crisis; two additional skin samples were collected by biopsy. After the procedure, all clinical samples were classified as genotype 1.

  10. A clinical trial alert tool to recruit large patient samples and assess selection bias in general practice research

    Directory of Open Access Journals (Sweden)

    Scheidt-Nave Christa

    2011-02-01

    Full Text Available Abstract Background Many research projects in general practice face problems when recruiting patients, often resulting in low recruitment rates and an unknown selection bias, thus limiting their value for health services research. The objective of the study is to evaluate the recruitment performance of the practice staff in 25 participating general practices when using a clinical trial alert (CTA tool. Methods The CTA tool was developed for an osteoporosis survey of patients at risk for osteoporosis and fractures. The tool used data from electronic patient records (EPRs to automatically identify the population at risk (net sample, to apply eligibility criteria, to contact eligible patients, to enrol and survey at least 200 patients per practice. The effects of the CTA intervention were evaluated on the basis of recruitment efficiency and selection bias. Results The CTA tool identified a net sample of 16,067 patients (range 162 to 1,316 per practice, of which the practice staff reviewed 5,161 (32% cases for eligibility. They excluded 3,248 patients and contacted 1,913 patients. Of these, 1,526 patients (range 4 to 202 per practice were successfully enrolled and surveyed. This made up 9% of the net sample and 80% of the patients contacted. Men and older patients were underrepresented in the study population. Conclusion Although the recruitment target was unreachable for most practices, the practice staff in the participating practices used the CTA tool successfully to identify, document and survey a large patient sample. The tool also helped the research team to precisely determine a slight selection bias.

  11. Detection of Bordetella pertussis from Clinical Samples by Culture and End-Point PCR in Malaysian Patients

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    Tan Xue Ting

    2013-01-01

    Full Text Available Pertussis or whooping cough is a highly infectious respiratory disease caused by Bordetella pertussis. In vaccinating countries, infants, adolescents, and adults are relevant patients groups. A total of 707 clinical specimens were received from major hospitals in Malaysia in year 2011. These specimens were cultured on Regan-Lowe charcoal agar and subjected to end-point PCR, which amplified the repetitive insertion sequence IS481 and pertussis toxin promoter gene. Out of these specimens, 275 were positive: 4 by culture only, 6 by both end-point PCR and culture, and 265 by end-point PCR only. The majority of the positive cases were from ≤3 months old patients (77.1% (. There was no significant association between type of samples collected and end-point PCR results (. Our study showed that the end-point PCR technique was able to pick up more positive cases compared to culture method.

  12. Validation of the Marijuana Effect Expectancies Questionnaire (MEEQ in a Non-Clinical French-Speaking Adolescent Sample

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    Emilie Schmits

    2016-02-01

    Full Text Available Teenagers commonly use cannabis. Expectancies related to the effects of cannabis play an important role in its consumption and are frequently measured with the Marijuana Effect Expectancies Questionnaire (MEEQ. This study aims to assess the psychometric properties (factor structure, internal consistency reliability, criterion validity of the French MEEQ. A sample of 1,343 non-clinical teenagers (14–18 years were recruited to answer a self-report questionnaire; 877 of them responded twice (one-year interval. A four-factor structure was obtained: Cognitive Impairment and Negative, Relaxation and Social Facilitation, Perceptual Enhancement and Craving and Negative Behavioral Effect Expectancies. It is concluded that the French MEEQ constitutes an appropriate tool to measure cannabis effect expectancies among adolescents.

  13. Clinical and neuropsychological assessment of attention and ADHD comorbidity in a sample of children and adolescents with idiopathic epilepsy

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    Celia Regina Carvalho Machado da Costa

    2015-02-01

    Full Text Available Children with epilepsy present significant problems concerning attention and comorbidity with attention deficit hyperactivity disorder (ADHD. Objective To determine the prevalence of attention complaints, ADHD diagnosis and attention profile in a sample of children and adolescents with idiopathic epilepsy. Method 36 children and adolescents with idiopathic epilepsy and 37 genre and age matched healthy controls underwent several procedures to diagnose their neuropsychological profile and comorbidity with ADHD. Results The prevalence of ADHD was higher in patients with epilepsy [χ2= 4.1, p = 0.043, 6 (16.7% vs 1 (2.7%], with worse results in attention related WISC items and factors in patients with epilepsy comparing to the controls, but not between patients with and without ADHD. Clinical characteristics did not influence those results. Conclusion This study found a greater prevalence of problems wih attention in pediatric patients with idiopathic epilepsy, but not a distinct profile between those with or without ADHD.

  14. Data analysis considerations for detecting copy number changes in formalin-fixed, paraffin-embedded tissues.

    Science.gov (United States)

    Jacobs, Sharoni

    2012-11-01

    The Whole Genome Sampling Analysis (WGSA) assay in combination with Affymetrix GeneChip Mapping Arrays is used for copy number analysis of high-quality DNA samples (i.e., samples that have been collected from blood, fresh or frozen tissue, or cell lines). Formalin-fixed, paraffin-embedded (FFPE) samples, however, represent the most prevalent form of archived clinical samples, but they provide additional challenges for molecular assays. FFPE processing usually results in the degradation of FFPE DNA and in the contamination and chemical modification of these DNA samples. In this article, we describe the steps needed to obtain reliable copy number predictions from degraded and contaminated FFPE samples.

  15. Evaluation of the hyplex® TBC PCR test for detection of Mycobacterium tuberculosis complex in clinical samples

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    Hoffmann Harald

    2010-03-01

    Full Text Available Abstract Background Tuberculosis (TB is one of the major public health concerns worldwide. The detection of the pathogen Mycobacterium tuberculosis complex (MTBC as early as possible has a great impact on the effective control of the spread of the disease. In our study, we evaluated the hyplex® TBC PCR test (BAG Health Care GmbH, a novel assay using a nucleic acid amplification technique (NAAT with reverse hybridisation and ELISA read out for the rapid detection of M. tuberculosis directly in clinical samples. Results A total of 581 respiratory and non-respiratory specimens from our pneumological hospital and the National TB Institute of Uzbekistan were used for the evaluation of the PCR assay. Of these, 292 were classified as TB samples and 289 as non-TB samples based on the results of the TB cultures as reference method. The PCR results were initially used to optimise the cut-off value of the hyplex® TBC test system by means of a ROC analysis. The overall sensitivity of the assay was determined to be 83.1%. In smear-positive TB samples, the sensitivity of the hyplex® TBC PCR test was estimated to 93.4% versus 45.1% in smear-negative samples. The specificity of the test was 99.25%. Of the two specimens (0.75% with false-positive PCR results, one yielded a culture positive for non-tuberculous mycobacteria. Based on the assumption of a prevalence of 8% TB positives among the samples in our diagnostic TB laboratory, the positive and negative predictive values were estimated to 90.4% and 98.5%, respectively. Conclusions The hyplex® TBC PCR test is an accurate NAAT assay for a rapid and reliable detection of M. tuberculosis in various respiratory and non-respiratory specimens. Compared to many other conventional NAAT assays, the hyplex® TBC PCR test is in a low price segment which makes it an attractive option for developing and emerging countries with high TB burdens.

  16. Acceptability, effectiveness, and cost-effectiveness of internet-based exposure treatment for irritable bowel syndrome in a clinical sample: a randomized controlled trial

    OpenAIRE

    Andréewitch Sergej; Lindfors Perjohan; Hedman Erik; Andersson Erik; Andersson Gerhard; Ljótsson Brjánn; Rück Christian; Lindefors Nils

    2011-01-01

    Background: Internet-based cognitive behavior therapy (ICBT) has shown promising effects in the treatment of irritable bowel syndrome (IBS). However, to date no study has used a design where participants have been sampled solely from a clinical population. We aimed to investigate the acceptability, effectiveness, and cost-effectiveness of ICBT for IBS using a consecutively recruited sample from a gastroenterological clinic. less thanbrgreater than less thanbrgreater thanMethods: Sixty-one pat...

  17. Clinical expression of bipolar disorder type I as a function of age and polarity at onset: convergent findings in samples from France and the United States.

    OpenAIRE

    Etain, Bruno; Lajnef, Mohamed; Bellivier, Frank; Mathieu, Flavie; Raust, Aurélie; Cochet, Barbara; Gard, Sébastien; M'Bailara, Katia; Kahn, Jean-Pierre; Elgrabli, Orly; Cohen, Renaud,; Jamain, Stéphane; Vieta, Eduard; Leboyer, Marion; Henry, Chantal

    2012-01-01

    BACKGROUND: The clinical presentation, course, and comorbidities of bipolar disorder type I are highly heterogeneous, and this variability remains poorly predictable. Certain onset characteristics (eg, age and polarity at onset) may delineate subgroups differing in clinical expression and outcome. METHOD: We retrospectively investigated the association between both age and polarity at onset and the clinical characteristics of bipolar I disorder (DSM-IV) in 2 independent adult samples: 480 Fre...

  18. Immuno-fluorescence based Vi capsular polysaccharide detection for specific recognition of Salmonella enterica serovar Typhi in clinical samples.

    Science.gov (United States)

    Pandey, Satish K; Vinayaka, Aaydha C; Rishi, Dharam B; Rishi, Praveen; Suri, C Raman

    2014-09-01

    Typhoid fever is a life threatening bacterial infection that remains a major global health concern. This continued high burden associated with significant morbidity and mortality rate demands specific and rapid detection technique. This work reports a new sandwich type fluorescence immunoassay format using polymyxin B, a cationic receptor molecule, as a binder agent while anti-Vi antibody served as the capturing agent for specifically detecting Salmonella enterica serovar Typhi. Anti-Vi IgG antibody raised against Vi-BSA conjugate revealed affinity of 7.779nM(-1) signifying immunodominancy of O-acetyls groups in Vi polysaccharide. The detection limit of the developed assay was around 10(1) cellsmL(-1) of Vi expressing Salmonella enterica serovar Typhi with a correlation coefficient (R(2)) equal to 0.97. Positive response obtained for all the tested serovar Typhi clinical isolates as well as the pathogen spiked blood samples recommended specificity and accuracy of Vi antigen as a biomarker during typhoid fever. The intra- and inter-assay precision with Vi spiked samples were satisfactory revealing coefficient of variance (CV%) with a mean of 4.05% and 5.97% respectively. This may be the novel attempt and constructive report on the fluorescence based detection of Vi antigen of serovar Typhi in the epidemic as well as pandemic outbreaks.

  19. Targeted therapies in cancer - challenges and chances offered by newly developed techniques for protein analysis in clinical tissues.

    Science.gov (United States)

    Malinowsky, K; Wolff, C; Gündisch, S; Berg, D; Becker, Kf

    2010-12-19

    In recent years, new anticancer therapies have accompanied the classical approaches of surgery and radio- and chemotherapy. These new forms of treatment aim to inhibit specific molecular targets namely altered or deregulated proteins, which offer the possibility of individualized therapies.The specificity and efficiency of these new approaches, however, bring about a number of challenges. First of all, it is essential to specifically identify and quantify protein targets in tumor tissues for the reasonable use of such targeted therapies. Additionally, it has become even more obvious in recent years that the presence of a target protein is not always sufficient to predict the outcome of targeted therapies. The deregulation of downstream signaling molecules might also play an important role in the success of such therapeutic approaches. For these reasons, the analysis of tumor-specific protein expression profiles prior to therapy has been suggested as the most effective way to predict possible therapeutic results. To further elucidate signaling networks underlying cancer development and to identify new targets, it is necessary to implement tools that allow the rapid, precise, inexpensive and simultaneous analysis of many network components while requiring only a small amount of clinical material.Reverse phase protein microarray (RPPA) is a promising technology that meets these requirements while enabling the quantitative measurement of proteins. Together with recently developed protocols for the extraction of proteins from formalin-fixed, paraffin-embedded (FFPE) tissues, RPPA may provide the means to quantify therapeutic targets and diagnostic markers in the near future and reliably screen for new protein targets.With the possibility to quantitatively analyze DNA, RNA and protein from a single FFPE tissue sample, the methods are available for integrated patient profiling at all levels of gene expression, thus allowing optimal patient stratification for

  20. Targeted therapies in cancer - challenges and chances offered by newly developed techniques for protein analysis in clinical tissues

    Directory of Open Access Journals (Sweden)

    K Malinowsky, C Wolff, S Gündisch, D Berg, KF Becker

    2011-01-01

    Full Text Available In recent years, new anticancer therapies have accompanied the classical approaches of surgery and radio- and chemotherapy. These new forms of treatment aim to inhibit specific molecular targets namely altered or deregulated proteins, which offer the possibility of individualized therapies.The specificity and efficiency of these new approaches, however, bring about a number of challenges. First of all, it is essential to specifically identify and quantify protein targets in tumor tissues for the reasonable use of such targeted therapies. Additionally, it has become even more obvious in recent years that the presence of a target protein is not always sufficient to predict the outcome of targeted therapies. The deregulation of downstream signaling molecules might also play an important role in the success of such therapeutic approaches. For these reasons, the analysis of tumor-specific protein expression profiles prior to therapy has been suggested as the most effective way to predict possible therapeutic results. To further elucidate signaling networks underlying cancer development and to identify new targets, it is necessary to implement tools that allow the rapid, precise, inexpensive and simultaneous analysis of many network components while requiring only a small amount of clinical material.Reverse phase protein microarray (RPPA is a promising technology that meets these requirements while enabling the quantitative measurement of proteins. Together with recently developed protocols for the extraction of proteins from formalin-fixed, paraffin-embedded (FFPE tissues, RPPA may provide the means to quantify therapeutic targets and diagnostic markers in the near future and reliably screen for new protein targets.With the possibility to quantitatively analyze DNA, RNA and protein from a single FFPE tissue sample, the methods are available for integrated patient profiling at all levels of gene expression, thus allowing optimal patient stratification

  1. Clinical validation of three short forms of the Dutch Wechsler Memory Scale-Fourth Edition (WMS-IV-NL) in a mixed clinical sample.

    Science.gov (United States)

    Bouman, Zita; Hendriks, Marc P H; Van Der Veld, William M; Aldenkamp, Albert P; Kessels, Roy P C

    2016-06-01

    The reliability and validity of three short forms of the Dutch version of the Wechsler Memory Scale-Fourth Edition (WMS-IV-NL) were evaluated in a mixed clinical sample of 235 patients. The short forms were based on the WMS-IV Flexible Approach, that is, a 3-subtest combination (Older Adult Battery for Adults) and two 2-subtest combinations (Logical Memory and Visual Reproduction and Logical Memory and Designs), which can be used to estimate the Immediate, Delayed, Auditory and Visual Memory Indices. All short forms showed good reliability coefficients. As expected, for adults (16-69 years old) the 3-subtest short form was consistently more accurate (predictive accuracy ranged from 73% to 100%) than both 2-subtest short forms (range = 61%-80%). Furthermore, for older adults (65-90 years old), the predictive accuracy of the 2-subtest short form ranged from 75% to 100%. These results suggest that caution is warranted when using the WMS-IV-NL Flexible Approach short forms to estimate all four indices.

  2. Deparaffinization with mineral oil: a simple procedure for extraction of high-quality DNA from archival formalin-fixed paraffin-embedded samples.

    Science.gov (United States)

    Heikal, Nahla; Nussenzveig, Roberto H; Agarwal, Archana M

    2014-09-01

    Extracting DNA from formalin-fixed paraffin-embedded (FFPE) archival samples remains difficult. Successful polymerase chain reactions (PCR) with DNA extracted from FFPE samples is still very low. We extracted DNA from 12 recent and old archival FFPE bone marrow trephine biopsies by use of a simple protocol on the basis of deparaffinization with molecular biology-grade mineral oil followed by DNA extraction with the Qiagen FFPE kit. Comparison of this deparaffinization method with standard protocols, for example, xylene or Hemo-D with subsequent rehydration using graded ethanols, was investigated. The quality and quantity of extracted DNA were tested by a combination of ultraviolet spectroscopy, analysis on a Caliper LabChip GX, and real-time PCR combined with high-resolution melt analysis. Highest quality PCR-amplifiable DNA was obtained by deparaffinization with mineral oil, whereas more variable results were obtained for the other 2 deparaffinization procedures. This result was confirmed by real-time PCR and high-resolution melt analysis. Besides improvements in the quality of extracted DNA, use of mineral oil for deparaffinization has the added benefit of decreased time (20 vs. 75 min) and a significant reduction of hands-on labor (1 step vs. multiple hands-on centrifugation and decanting steps).

  3. Diffuse myocardial fibrosis evaluation using cardiac magnetic resonance T1 mapping: sample size considerations for clinical trials

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    Liu Songtao

    2012-12-01

    Full Text Available Abstract Background Cardiac magnetic resonance (CMR T1 mapping has been used to characterize myocardial diffuse fibrosis. The aim of this study is to determine the reproducibility and sample size of CMR fibrosis measurements that would be applicable in clinical trials. Methods A modified Look-Locker with inversion recovery (MOLLI sequence was used to determine myocardial T1 values pre-, and 12 and 25min post-administration of a gadolinium-based contrast agent at 3 Tesla. For 24 healthy subjects (8 men; 29 ± 6 years, two separate scans were obtained a with a bolus of 0.15mmol/kg of gadopentate dimeglumine and b 0.1mmol/kg of gadobenate dimeglumine, respectively, with averaged of 51 ± 34 days between two scans. Separately, 25 heart failure subjects (12 men; 63 ± 14 years, were evaluated after a bolus of 0.15mmol/kg of gadopentate dimeglumine. Myocardial partition coefficient (λ was calculated according to (ΔR1myocardium/ΔR1blood, and ECV was derived from λ by adjusting (1-hematocrit. Results Mean ECV and λ were both significantly higher in HF subjects than healthy (ECV: 0.287 ± 0.034 vs. 0.267 ± 0.028, p=0.002; λ: 0.481 ± 0.052 vs. 442 ± 0.037, p Conclusion ECV and λ quantification have a low variability across scans, and could be a viable tool for evaluating clinical trial outcome.

  4. Stability of Self-Reported Arousal to Sexual Fantasies Involving Children in a Clinical Sample of Pedophiles and Hebephiles.

    Science.gov (United States)

    Grundmann, Dorit; Krupp, Jurian; Scherner, Gerold; Amelung, Till; Beier, Klaus M

    2016-07-01

    In forensic research, there is a controversial discussion concerning the changeability or stability of pedophilia. Seto (2012) conceptualized pedophilia as a sexual age orientation characterized by an early onset, correlations with sexual and romantic behavior, and stability over time. However, empirical data are sparse and are mostly based on samples of detected offenders. The present study examined self-reported arousal to sexual fantasies involving children in a clinical sample of pedo-/hebephiles. In Study 1, retrospective self-reports on the age of onset and duration of sexual interest in minors were examined. In Study 2, the stability and variability of self-reported arousal to sexual fantasies involving children were evaluated prospectively. Non-prosecuted self-identifying pedo-/hebephilic men seeking professional help were recruited within the Berlin Prevention Project Dunkelfeld. Between 2005 and 2013, 494 participants completed the intake assessment. Self-reported data were collected via questionnaire focusing on sexual arousal to fantasies during masturbation involving prepubescent and/or early pubescent minors. Subsequent assessments of sexual arousal were obtained for 121 of the participants. The average time between the first and last assessment was approximately 29 months. Spearman's correlation coefficients examined the between-group rank-order and Wilcoxon signed-rank tests examined the within-individual mean-level stability. The majority of subjects reported an early onset of their pedo-/hebephilic sexual arousal. The rank-order stability was medium to high. Over the investigated period, the majority of subjects showed no or only minimal decrease or increase of self-reported sexual arousal. These results suggested that sexual arousal to fantasies involving prepubescent and/or early pubescent children is stable. Furthermore, the results support the conceptualization of pedo-/hebephilia as a sexual age orientation in men. PMID:27113471

  5. Stability of Self-Reported Arousal to Sexual Fantasies Involving Children in a Clinical Sample of Pedophiles and Hebephiles.

    Science.gov (United States)

    Grundmann, Dorit; Krupp, Jurian; Scherner, Gerold; Amelung, Till; Beier, Klaus M

    2016-07-01

    In forensic research, there is a controversial discussion concerning the changeability or stability of pedophilia. Seto (2012) conceptualized pedophilia as a sexual age orientation characterized by an early onset, correlations with sexual and romantic behavior, and stability over time. However, empirical data are sparse and are mostly based on samples of detected offenders. The present study examined self-reported arousal to sexual fantasies involving children in a clinical sample of pedo-/hebephiles. In Study 1, retrospective self-reports on the age of onset and duration of sexual interest in minors were examined. In Study 2, the stability and variability of self-reported arousal to sexual fantasies involving children were evaluated prospectively. Non-prosecuted self-identifying pedo-/hebephilic men seeking professional help were recruited within the Berlin Prevention Project Dunkelfeld. Between 2005 and 2013, 494 participants completed the intake assessment. Self-reported data were collected via questionnaire focusing on sexual arousal to fantasies during masturbation involving prepubescent and/or early pubescent minors. Subsequent assessments of sexual arousal were obtained for 121 of the participants. The average time between the first and last assessment was approximately 29 months. Spearman's correlation coefficients examined the between-group rank-order and Wilcoxon signed-rank tests examined the within-individual mean-level stability. The majority of subjects reported an early onset of their pedo-/hebephilic sexual arousal. The rank-order stability was medium to high. Over the investigated period, the majority of subjects showed no or only minimal decrease or increase of self-reported sexual arousal. These results suggested that sexual arousal to fantasies involving prepubescent and/or early pubescent children is stable. Furthermore, the results support the conceptualization of pedo-/hebephilia as a sexual age orientation in men.

  6. Birth Order and Sibling Gender Ratio of a Clinical Sample of Children and Adolescents Diagnosed with Attention Deficit Hyperactivity Disorder

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    Ahmad Ghanizadeh

    2012-09-01

    Full Text Available Objective: It is not clear whether sibling’s gender ratio is associated with attention deficit hyperactivity disorder (ADHD. This study examines whether inattentiveness severity and hyperactivity/impulsivity severity are associated with birth order of children with ADHD.Method: Participants are a clinical sample of 173 children and adolescents with ADHD and 43 ones without ADHD. Diagnoses were made using Diagnostic and Statistical Manual of Mental Disorders forth edition-Text Revision (DSM-IV-TR, diagnostic criteria according to face-to-face interview with the children and their parents. ADHD DSM-IV checklist was used to measure inattentiveness and hyperactivity/impulsivity scores.Results: The association of birth order and diagnosis of ADHD was not statistically significant after adjusting for covariate factors. The gender ratio of siblings is not associated with ADHD.Conclusion: Birth order and siblings gender ratio are independent of ADHD diagnosis. The results of this study support the fact that genetic factors rather than environmental factor of birth order is associated with ADHD. Moreover, contrary to autism, the current results do not suggest the androgen theory for ADHD.

  7. Cryptic and rare Aspergillus species in Brazil: prevalence in clinical samples and in vitro susceptibility to triazoles.

    Science.gov (United States)

    Negri, C E; Gonçalves, S S; Xafranski, H; Bergamasco, M D; Aquino, V R; Castro, P T O; Colombo, A L

    2014-10-01

    Aspergillus spp. are among the most common causes of opportunistic invasive fungal infections in tertiary care hospitals. Little is known about the prevalence and in vitro susceptibility of Aspergillus species in Latin America, because there are few medical centers able to perform accurate identification at the species level. The purpose of this study was to analyze the distribution of cryptic and rare Aspergillus species among clinical samples from 133 patients with suspected aspergillosis admitted in 12 medical centers in Brazil and to analyze the in vitro activity of different antifungal drugs. The identification of Aspergillus species was performed based on a polyphasic approach, as well as sequencing analysis of the internal transcribed spacer (ITS) region, calmodulin, and β-tubulin genes and phylogenetic analysis when necessary. The in vitro susceptibility tests with voriconazole, posaconazole, and itraconazole were performed according to the CLSI M38-A2 document (2008). We demonstrated a high prevalence of cryptic species causing human infection. Only three isolates, representing the species Aspergillus thermomutatus, A. ochraceus, and A. calidoustus, showed less in vitro susceptibility to at least one of the triazoles tested. Accurate identifications of Aspergillus at the species level and with in vitro susceptibility tests are important because some species may present unique resistance patterns against specific antifungal drugs.

  8. Sensitive and direct detection of receptor binding specificity of highly pathogenic avian influenza A virus in clinical samples.

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    Tadanobu Takahashi

    Full Text Available Influenza A virus (IAV recognizes two types of N-acetylneuraminic acid (Neu5Ac by galactose (Gal linkages, Neu5Acα2,3Gal and Neu5Acα2,6Gal. Avian IAV preferentially binds to Neu5Acα2,3Gal linkage, while human IAV preferentially binds to Neu5Acα2,6Gal linkage, as a virus receptor. Shift in receptor binding specificity of avian IAV from Neu5Acα2,3Gal linkage to Neu5Acα2,6Gal linkage is generally believed to be a critical factor for its transmission ability among humans. Surveillance of this shift of highly pathogenic H5N1 avian IAV (HPAI is thought to be a very important for prediction and prevention of a catastrophic pandemic of HPAI among humans. In this study, we demonstrated that receptor binding specificity of IAV bound to sialo-glycoconjugates was sensitively detected by quantifying the HA gene with real-time reverse-transcription-PCR. The new assay enabled direct detection of receptor binding specificity of HPAIs in chicken clinical samples including trachea and cloaca swabs in only less than 4 h.

  9. Clinical Utility of Subtyping Binge Eating Disorder by History of Anorexia or Bulimia Nervosa in a Treatment Sample

    Science.gov (United States)

    Utzinger, Linsey M.; Mitchell, James E.; Cao, Li; Crosby, Ross D.; Crow, Scott J.; Wonderlich, Stephen A.; Peterson, Carol B.

    2016-01-01

    Objective This study examined whether having a history of anorexia nervosa (AN) or bulimia nervosa (BN) is associated with response to treatment in adults with binge eating disorder (BED). Method Data from 189 adults diagnosed with BED who were randomly assigned to one of three group cognitive-behavioral (CBT) treatments were analyzed to compare those with and without a history of AN/BN. Results A total of 16% of the sample had a history of AN/BN. The BED subgroup with a history of AN/BN presented with higher rates of mood disorders and greater eating-related symptom severity at baseline. Participants with a history of AN/BN also had higher global eating disorder (ED) symptoms at end of treatment (EOT), and more frequent objective binge-eating episodes at EOT and 12-month follow-up. Discussion These findings suggest that in adults with BED, a history of AN/BN is predictive of greater eating-related symptom severity following group-based CBT and poorer short- and long-term binge-eating outcomes. These findings suggest that considering ED history in the treatment of adults with BED may be clinically useful. PMID:25959549

  10. Impact of obesity on the clinical profile of a population-based sample with chronic obstructive pulmonary disease.

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    Francisco García-Rio

    Full Text Available AIMS: To characterize the distribution of BMI in a population-based sample of COPD patients and to evaluate the impact of obesity on their health status, exercise tolerance, systemic inflammation and comorbidity. METHODS: A population-based sample of 3,797 subjects aged 40-80 years from the EPI-SCAN study was selected. Subjects were categorized according their body mass index (BMI as underweight (<18.5 kg/m2, normal weight (18.5-24.9 kg/m2, overweight (25.0-29.9 kg/m2 or obese (BMI≥30.0 kg/m2. Subjects were evaluated with post-bronchodilator spirometry and 6-minute walk tests. Smoking habits, respiratory symptoms, generic and specific quality of life, daily physical activities, comorbidities and systemic inflammatory biomarkers were recorded. RESULTS: The prevalence of obesity or being overweight was higher in the 382 COPD patients than in the subjects without airflow limitation (29.4%, 95%CI 24.8-33.9% vs. 24.3, 95%CI 22.9-25.8; and 44.7%, 95%CI 39.7-49.6% vs. 43.0%, 95%CI 41.3-44.6, respectively; p = 0.020. In the COPD subgroup, obese subjects presented more dyspnea and less chronic cough, chronic bronchitis or chronic phlegm than normal-weight patients, as well as a worse health status. Moreover, reduced exercise tolerance and higher plasmatic C-reactive protein levels were found in the obese patients, who also presented a greater prevalence of cardiovascular disease (adjusted odds ratio 4.796, 95%CI 1.806-12.736, p = 0.002. CONCLUSIONS: In a population-based sample, obesity is more prevalent in COPD patients than in subjects without airflow limitation. Furthermore, obesity affects the clinical manifestations, quality of life and exercise tolerance of COPD patients, and it may contribute to a phenotype characterized by increased systemic inflammation and greater frequency of cardiovascular comorbidity.

  11. Incidence of Cyp51 A key mutations in Aspergillus fumigatus-a study on primary clinical samples of immunocompromised patients in the period of 1995-2013.

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    Birgit Spiess

    Full Text Available As the incidence of azole resistance in Aspergillus fumigatus is rising and the diagnosis of invasive aspergillosis (IA in immunocompromised patients is rarely based on positive culture yield, we screened our Aspergillus DNA sample collection for the occurrence of azole resistance mediating cyp51 A key mutations. Using two established, a modified and a novel polymerase chain reaction (PCR assays followed by DNA sequence analysis to detect the most frequent mutations in the A. fumigatus cyp51 A gene conferring azole resistance (TR34 (tandem repeat, L98H and M220 alterations. We analyzed two itraconazole and voriconazole and two multi-azole resistant clinical isolates and screened 181 DNA aliquots derived from clinical samples (blood, bronchoalveolar lavage (BAL, biopsies, cerebrospinal fluid (CSF of 155 immunocompromised patients of our Aspergillus DNA sample collection, previously tested positive for Aspergillus DNA and collected between 1995 and 2013. Using a novel PCR assay for the detection of the cyp51 A 46 bp tandem repeat (TR46 directly from clinical samples, we found the alteration in a TR46/Y121F/T289A positive clinical isolate. Fifty stored DNA aliquots from clinical samples were TR46 negative. DNA sequence analysis revealed a single L98H mutation in 2010, two times the L98H alteration combined with TR34 in 2011 and 2012 and a so far unknown N90K mutation in 1998. In addition, four clinical isolates were tested positive for the TR34/L98H combination in the year 2012. We consider our assay of epidemiological relevance to detect A. fumigatus azole resistance in culture-negative clinical samples of immunocompromised patients; a prospective study is ongoing.

  12. ITS1 PCR-RFLP Diagnosis and Characterization of Leishmania in Clinical Samples and Strains from Cases of Human Cutaneous Leishmaniasis in States of the Mexican Southeast

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    Amalia Monroy-Ostria

    2014-01-01

    Full Text Available American cutaneous leishmaniasis includes a spectrum of clinical forms localized cutaneous, diffuse cutaneous, and mucocutaneous leishmaniasis which can be caused by different strains of Leishmania belonging to the L. mexicana or L. braziliensis complexes which may coexist in the same endemic area. We evaluated the PCR-RFLP assay of the ITS1 genes for direct identification of Leishmania species in 163 clinical samples and 21 Mexican isolates of Leishmania. In relation to the Mexican isolates of Leishmania 52% displayed a pattern similar to the L. (L. mexicana, 5% showed a mixed pattern compatible with L. (L. mexicana and L. (V. braziliensis, eight with L. (L. amazonensis and L. (L. mexicana, and one to L. (V. braziliensis. Most of the clinical samples, 109/116 (94%, gave a pattern similar to that of the L. mexicana, two clinical samples gave similar patterns to that of Leishmania braziliensis, and 5 samples gave patterns that suggest a coinfection of L. (L. mexicana and L. (V. braziliensis or L. (L. mexicana and L. (L. amazonensis. The ITS1 PCR-RFLP assay is a multipurpose tool for diagnosis of Leishmania from clinical samples and enables determination of the infecting species of New World Leishmania in the field in relatively short time and low cost.

  13. Molecular identification of Coccidioides immitis in formalin-fixed, paraffin-embedded (FFPE) tissues from a Colombian patient.

    Science.gov (United States)

    Canteros, Cristina E; Vélez H, Alejandro; Toranzo, Adriana I; Suárez-Alvarez, Roberto; Tobón O, Ángela; Jimenez A, María del Pilar; Restrepo M, Ángela

    2015-06-01

    Coccidioides immitis and C. posadasii are the etiologic agents of coccidioidomycosis, an endemic fungal disease of the Americas. In Colombia, this mycosis is uncommon, and only five cases, two of them imported, have been documented.By means of DNA sequencing, C. immitis was identified in formalin-fixed, paraffin-embedded archival tissues samples from the 5th Colombian patient diagnosed in 1997. The patient was born in Pinto, Department of Magdalena, and had never visited other geographic regions, a reason to consider that the mycosis had been acquired locally.This species is primarily found in California although it has been occasionally reported in other geographic areas such as Mexico and Brazil. This is the first indigenous report of C. immitis-associated coccidioidomycosis in a Colombian patient. PMID:25908652

  14. Lower urinary tract symptoms associated with neurological conditions: Observations on a clinical sample of outpatients neurorehabilitation service

    Directory of Open Access Journals (Sweden)

    Fabrizio Torelli

    2015-07-01

    Full Text Available Objectives: The overall aims of this study were to investigate the lower urinary tract symptoms (LUTS associated with neurological conditions and their prevalence and impact on a clinical sample of outpatients of a neurorehabilitation service. Materials and methods: We reviewed the files of 132 patients treated in our neurorehabilitation service from December 2012 to December 2013. Patients were divided into several subgroups based on the neurological diagnosis: Multiple Sclerosis (MS, other demyelinating diseases, Peripheral Neuropathy, neurovascular disorders (ND, neoplastic disease, traumatic brain injury (TBI, Parkinson and Parkinsonism, spinal cord injuries (SCI. Urinary status was based on medical evaluations of history of LUTS, type, degree, onset and duration of symptoms. We tried to analyze prevalence, kind of disorder, timing of presentation (if before or after the neurological onset and eventual persistence of urological disorders (in the main group and in all subgroups. Results: At the time of admission to our rehabilitation service, LUTS were observed in 14 out of 132 cases (11%. A high proportion of these outpatients (64.2% presented bothersome urinary symptoms such as incontinence, frequency and urgency (storage LUTS. The most frequent symptom was urinary urge incontinence (42.8%. This symptom was found to be prevalent in the multiple sclerosis and neurovascular disorders. In 93% the urinary symptoms arose as a result of neurologic conditions and 78.5% did not present a complete recovery of urological symptoms in spite of improved selfreported functional activity limitations. None of these patients performed urological rehabilitation. Conclusions: Neurological disorders are a significant issue in rehabilitation services and it can lead to lower tract dysfunction, which causes LUTS. Storage symptoms are more common, especially urge incontinence. Current literature reports that a further optimization of the rehabilitation potential

  15. Detection of Mycobacterium tuberculosis by Using Loop-Mediated Isothermal Amplification Combined with a Lateral Flow Dipstick in Clinical Samples

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    Thongchai Kaewphinit

    2013-01-01

    Full Text Available Tuberculosis (TB is a communicable disease caused by the bacterium Mycobacterium tuberculosis (MTB and is a persistent problem in the developing countries. Loop-mediated isothermal amplification (LAMP allows DNA to be amplified rapidly at a constant temperature. Here, a LAMP method was combined with a chromatographic lateral-flow dipstick (LFD to detect IS6110 gene of M. tuberculosis specifically and rapidly. The reaction was optimized at 63°C for 60 min, and the amplified DNA hybridized to an FITC-labeled oligonucleotide probe for 5 min was detected at the LFD test line 5 min after application. Excluding the step of DNA extraction, the test results could be generated approximately within 1 h. In addition to the advantage of short assay time, this technique could avoid the contact of carcinogenic ethidium bromide due to the exclusion of the electrophoresis analysis step. Furthermore, the data indicated that LAMP-LFD could detect M. tuberculosis genomic DNA as little as 5 pg. The technique showed a significant specificity since no cross-hybridization to M. intracellulare (MIC, M. fortuitum (MFT, M. avium (MAV, M. kansasii (MKS, and M. gordonae (MGD genomic DNAs was observed. In the clinical unknown samples test, the sensitivity of LAMP-LFD was 98.92 % and the specificity was 100 % compared to those of the standard culture assay. Based on its sensitivity, specificity, rapidity, low cost, and convenience, LAMP-LFD could be applicable for use in both laboratories and epidemiological surveys of MTB.

  16. Differential survival following trastuzumab treatment based on quantitative HER2 expression and HER2 homodimers in a clinic-based cohort of patients with metastatic breast cancer

    Directory of Open Access Journals (Sweden)

    Masuda Norikazu

    2010-02-01

    Full Text Available Abstract Background We have recently described the correlation between quantitative measures of HER2 expression or HER2 homodimers by the HERmark assay and objective response (RR, time-to progression (TTP, and overall survival (OS in an expanded access cohort of trastuzumab-treated HER2-positive patients with metastatic breast cancer (MBC who were stringently selected by fluorescence in situ hybridization (FISH. Multivariate analyses suggested a continuum of HER2 expression that correlated with outcome following trastuzumab. Here we investigate the relationship between HER2 expression or HER2 homodimers and OS in a clinic-based population of patients with MBC selected primarily by IHC. Methods HERmark, a proximity-based assay designed to detect and quantitate protein expression and dimerization in formalin-fixed paraffin-embedded (FFPE tissues, was used to measure HER2 expression and HER2 homodimers in FFPE samples from patients with MBC. Assay results were correlated with OS using univariate Kaplan-Meier, hazard function plots, and multivariate Cox regression analyses. Results Initial analyses revealed a parabolic relationship between continuous measures of HER2 expression and risk of death, suggesting that the assumption of linearity for the HER2 expression measurements may be inappropriate in subsequent multivariate analyses. Cox regression analyses using the categorized variable of HER2 expression level demonstrated that higher HER2 levels predicted better survival outcomes following trastuzumab treatment in the high HER2-expressing group. Conclusions These data suggest that the quantitative amount of HER2 expression measured by Hermark may be a new useful marker to identify a more relevant target population for trastuzumab treatment in patients with MBC.

  17. Using Rasch Measurement to Create a Quality of Sleep Scale for a Non-Clinical Sample Based on the Pittsburgh Sleep Quality Index (PSQI

    Directory of Open Access Journals (Sweden)

    Panayiotis Panayides

    2013-02-01

    Full Text Available Originally, the aim of the present study was to investigate the psychometric properties and the appropriateness of the Greek version of the PSQI for a non-clinical sample. However, the scale was deemed not to be appropriate and results suggested some major modifications (study 1. The modified scale was administered to a second sample of Cypriots and was shown to be unidimensional and to have a high degree of reliability (study 2. The items define a theoretical linear quality of sleep continuum of increasing difficulty and cover a wide range of that continuum. Furthermore, a 3-point (instead of the original 4-point Likert scale was shown to be optimal and the scale was found to be appropriate for a non-clinical sample. The resulting scale is suitable for research purposes in studies regarding quality of sleep in academia, medicine and marketing. It could be used either for individuals or for large scale samples.

  18. Analysis on drug-resistance and molecular epidemiology of Acinetobacter baumannii isolated from the clinical samples in two Chinese hospitals

    Institute of Scientific and Technical Information of China (English)

    WEI FENG SHI; ZHI MI HUANG; NING XU

    2006-01-01

    In the present study, the drug-resistance genes encoding β-lactamases, aminoglycoside modifying enzymes, DNA topoisomerases and integron as well as their molecular epidemiology were investigated by means of analyzing the drug-resistance and molecular epidemiology of Acinebacter baumannii isolated from the clinical samples in two hospitals in Changzhou and Huzhou city of Jiangsu and Zhejiang province from July 2000 to March 2005. The minimal inhibitory concentrations (MICs) of these 307 isolates were detected by automatic microbiological system, and 35 strains against 5-fluoroquinolones were performed by agar dilution assay. Meanwhile, the resistant genes in 80 isolates were amplified by PCR with identification by DNA sequencer. It was found that most of the 307 isolates of A. baumannii were resistant to multiple antibiotics tested, in which the resistance rates of the isolates against piperacillin, piperacillin/tazobactam, amoxacillin/clavulanic acid, cefotaxime, ceftazidime,cefepime, gentamicin, amikacin, ciprofloxacin, chloramphenicol and sulfamethoxazole/trimethoprim were all above 35%, but those of imipenem and meropenem were quite low, ranged only 2.6% and 3.3 %. In addition, it was also demonstrated that the positive rates of TEM and SHV β-lactamase genes accounted for 93.8% and 22.5% respectively, and those of the aminoglycoside-modifying enzyme genes including aacC1, aacC2, aacC3, aacC4, aacC4A, aphA6, ant(2")-Ⅰ and ant(3")-Ⅰ were 58.8%, 8.8%, 7.5%, 28.8%, 45.0%, 2.5%, 28.8% and 65.0% respectively. The mutations in the quinolone-resistant determining region (QRDR) of gyrA and parC genes indicated that substitution in Ser-83 residue of GyrA protein was most frequently occurred among strains with MIC for ciprofloxacin of more than 4 μg/ml, whereas a double mutation at Ser-83 residue of gyrA and Ser-80 of parC was found in strains with MIC of ciprofloxacin of more than 8 μg/ml. As to the positive rates of class 1 integron (Int Ⅰ -1) and qacE△1-sul

  19. Opening the archives for state of the art tumour genetic research: sample processing for array-CGH using decalcified, formalin-fixed, paraffin-embedded tissue-derived DNA samples

    OpenAIRE

    Bovee Judith VMG; Hogendoorn Pancras CW; Meijer Danielle; Verbeke Sofie LJ; de Jong Danielle; Szuhai Károly

    2011-01-01

    Abstract Background Molecular genetic studies on rare tumour entities, such as bone tumours, often require the use of decalcified, formalin-fixed, paraffin-embedded tissue (dFFPE) samples. Regardless of which decalcification procedure is used, this introduces a vast breakdown of DNA that precludes the possibility of further molecular genetic testing. We set out to establish a robust protocol that would overcome these intrinsic hurdles for bone tumour research. Findings The goal of our study w...

  20. Evaluation of a single-tube fluorogenic RT-PCR assay for detection of bovine respiratory syncytial virus in clinical samples

    DEFF Research Database (Denmark)

    Hakhverdyan, Mikhayil; Hägglund, Sara; Larsen, Lars Erik;

    2005-01-01

    understanding of the virus. In this study, a BRSV fluorogenic reverse transcription PCR (fRT-PCR) assay, based on TaqMan principle, was developed and evaluated on a large number of clinical samples, representing various cases of natural and experimental BRSV infections. By using a single-step closed-tube format...

  1. Mothers' and Fathers' Ratings of Family Relationship Quality: Associations with Preadolescent and Adolescent Anxiety and Depressive Symptoms in a Clinical Sample

    Science.gov (United States)

    Queen, Alexander H.; Stewart, Lindsay M.; Ehrenreich-May, Jill; Pincus, Donna B.

    2013-01-01

    This study examined the independent associations among three family relationship quality factors--cohesion, expressiveness, and conflict--with youth self-reported depressive and anxiety symptoms in a clinical sample of anxious and depressed youth. Ratings of family relationship quality were obtained through both mother and father report. The…

  2. PCR assay based on DNA coding for 16S rRNA for detection and identification of mycobacteria in clinical samples.

    Science.gov (United States)

    Kox, L F; van Leeuwen, J; Knijper, S; Jansen, H M; Kolk, A H

    1995-01-01

    A PCR and a reverse cross blot hybridization assay were developed for the detection and identification of mycobacteria in clinical samples. The PCR amplifies a part of the DNA coding for 16S rRNA with a set of primers that is specific for the genus Mycobacterium and that flanks species-specific sequences within the genes coding for 16S rRNA. The PCR product is analyzed in a reverse cross blot hybridization assay with probes specific for M. tuberculosis complex (pTub1), M. avium (pAvi3), M. intracellulare (pInt5 and pInt7), M. kansasii complex-M. scrofulaceum complex (pKan1), M. xenopi (pXen1), M. fortuitum (pFor1), M. smegmatis (pSme1), and Mycobacterium spp. (pMyc5a). The PCR assay can detect 10 fg of DNA, the equivalent of two mycobacteria. The specificities of the probes were tested with 108 mycobacterial strains (33 species) and 31 nonmycobacterial strains (of 17 genera). The probes pAvi3, pInt5, pInt7, pKan1, pXen1, and pMyc5a were specific. With probes pTub1, pFor1, and pSme1, slight cross hybridization occurred. However, the mycobacterial strains from which the cross-hybridizing PCR products were derived belonged to nonpathogenic or nonopportunistic species which do not occur in clinical samples. The test was used on 31 different clinical specimens obtained from patients suspected of having mycobacterial disease, including a patient with a double mycobacterial infection. The samples included sputum, bronchoalveolar lavage, tissue biopsy samples, cerebrospinal fluid, pus, peritoneal fluid, pleural fluid, and blood. The results of the PCR assay agreed with those of conventional identification methods or with clinical data, showing that the test can be used for the direct and rapid detection and identification of mycobacteria in clinical samples. PMID:8586707

  3. Clinical forensic sample collection techniques following consensual intercourse in volunteers - cervical canal brush compared to conventional swabs.

    Science.gov (United States)

    Joki-Erkkilä, Minna; Tuomisto, Sari; Seppänen, Mervi; Huhtala, Heini; Ahola, Arja; Rainio, Juha; Karhunen, Pekka J

    2014-10-01

    The purpose of the research was to evaluate gynecological evidence collection techniques; the benefit of cervical canal brush sample compared to vaginal fornix and cervical swab samples and the time frame for detecting Y-chromosomal material QiAmp DNA Mini Kit(®) and Quantifiler Y Human Male DNA Quantification Kit(®) in adult volunteers following consensual intercourse. Eighty-four adult female volunteers following consensual intercourse were recruited for the study. By combining all sample collecting techniques, 81.0% of the volunteers were Y-DNA positive. Up to 60 h the conventional swab sampling techniques detected more Y-DNA positive samples when compared to the brush technique. However, after 60 h, the cervical canal brush sample technique showed its benefit by detecting 27.3% (6/22) of Y-DNA positive samples, which were Y-DNA negative in both conventional swab sampling techniques. By combining swab and brush techniques, 75% of the volunteers were still Y-DNA positive in 72-144 post-coital hours. The rate of measurable Y-DNA decreased approximately 3% per hour. Despite reported consensual intercourse, 6.8% (3/44) of volunteers were Y-DNA negative within 48 h. Y-DNA was not detected after 144 post-coital hours (6 days). In conclusion, the brush as a forensic evidence collection method may provide additional biological trace evidence from the cervical canal, although the best biological trace evidence collection can be obtained by combining all three sampling techniques. The time frame for gynecological forensic evidence sample collection should be considered to be at least a week if sexual violence is suspected.

  4. The influence of variations in eating disorder-related symptoms on processing of emotional faces in a non-clinical female sample: An eye-tracking study.

    Science.gov (United States)

    Sharpe, Emma; Wallis, Deborah J; Ridout, Nathan

    2016-06-30

    This study aimed to: (i) determine if the attention bias towards angry faces reported in eating disorders generalises to a non-clinical sample varying in eating disorder-related symptoms; (ii) examine if the bias occurs during initial orientation or later strategic processing; and (iii) confirm previous findings of impaired facial emotion recognition in non-clinical disordered eating. Fifty-two females viewed a series of face-pairs (happy or angry paired with neutral) whilst their attentional deployment was continuously monitored using an eye-tracker. They subsequently identified the emotion portrayed in a separate series of faces. The highest (n=18) and lowest scorers (n=17) on the Eating Disorders Inventory (EDI) were compared on the attention and facial emotion recognition tasks. Those with relatively high scores exhibited impaired facial emotion recognition, confirming previous findings in similar non-clinical samples. They also displayed biased attention away from emotional faces during later strategic processing, which is consistent with previously observed impairments in clinical samples. These differences were related to drive-for-thinness. Although we found no evidence of a bias towards angry faces, it is plausible that the observed impairments in emotion recognition and avoidance of emotional faces could disrupt social functioning and act as a risk factor for the development of eating disorders. PMID:27138825

  5. A study of trait anhedonia in non-clinical Chinese samples: evidence from the Chapman Scales for Physical and Social Anhedonia.

    Directory of Open Access Journals (Sweden)

    Raymond C K Chan

    Full Text Available BACKGROUND: Recent studies suggest that anhedonia, an inability to experience pleasure, can be measured as an enduring trait in non-clinical samples. In order to examine trait anhedonia in a non-clinical sample, we examined the properties of a range of widely used questionnaires capturing anhedonia. METHODS: 887 young adults were recruited from colleges. All of them were administered a set of checklists, including Chapman Scale for Social Anhedonia (CRSAS and the Chapman Scale for Physical Anhedonia Scale (CPAS, The Temporal Experience of Pleasure Scale(TEPS, and The Schizotypal Personality Questionnaire (SPQ. RESULTS: Males showed significantly higher level of physical (F = 5.09, p<0.001 and social (F = 4.38, p<0.005 anhedonia than females. As expected, individuals with schizotypal personality features also demonstrated significantly higher scores of physical (t = 3.81, p<0.001 and social (t = 7.33, p<0.001 trait anhedonia than individuals without SPD features, but no difference on self-report anticipatory and consummatory pleasure experience. CONCLUSIONS: Concerning the comparison on each item of physical and social anhedonia, the results indicated that individuals with SPD feature exhibited higher than individuals without SPD features on more items of social anhedonia than physical anhedonia scale. These preliminary findings suggested that trait anhedonia can be identified a non-clinical sample. Exploring the demographic and clinical correlates of trait anhedonia in the general population may provide clues to the pathogenesis of psychotic disorder.

  6. Effects of state and trait anxiety on selective attention to threatening stimuli in a non-clinical sample of school children

    Directory of Open Access Journals (Sweden)

    Jeniffer Ortega Marín

    2015-01-01

    Full Text Available Attentional biases, consisting of a preferential processing of threatening stimuli, have been found in anxious adults as predicted by several cognitive models. However, studies with non-clinical samples of children have provided mixed results. therefore, the aim of this research was to determine the effects of state and trait anxiety on the selective attention towards threatening stimuli in a non-clinical sample of school children (age: 8 to 13, n = 110 using the dot-probe task. This study did not reveal an effect of trait anxiety on selective attention towards threatening stimuli. However, a significant difference was found between participants with low state anxiety and high state anxiety. Nevertheless, the effect size was small. Specifically, participants with low state anxiety showed a bias towards threatening stimuli. Overall, the findings of this research with a non-clinical sample of school children suggest that attentional biases towards threatening information, which has been repeatedly found in anxious adults, are not necessarily inherent to non-clinical anxiety in children and on the other hand, the relationship between attentional biases and anxiety in this population might be moderated by other cognitive processes.

  7. Complete solubilization of formalin-fixed, paraffin-embedded tissue may improve proteomic studies.

    Science.gov (United States)

    Shi, Shan-Rong; Taylor, Clive R; Fowler, Carol B; Mason, Jeffrey T

    2013-04-01

    Tissue-based proteomic approaches (tissue proteomics) are essential for discovering and evaluating biomarkers for personalized medicine. In any proteomics study, the most critical issue is sample extraction and preparation. This problem is especially difficult when recovering proteins from formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, improving and standardizing protein extraction from FFPE tissue is a critical need because of the millions of archival FFPE tissues available in tissue banks worldwide. Recent progress in the application of heat-induced antigen retrieval principles for protein extraction from FFPE tissue has resulted in a number of published FFPE tissue proteomics studies. However, there is currently no consensus on the optimal protocol for protein extraction from FFPE tissue or accepted standards for quantitative evaluation of the extracts. Standardization is critical to ensure the accurate evaluation of FFPE protein extracts by proteomic methods such as reverse phase protein arrays, which is now in clinical use. In our view, complete solubilization of FFPE tissue samples is the best way to achieve the goal of standardizing the recovery of proteins from FFPE tissues. However, further studies are recommended to develop standardized protein extraction methods to ensure quantitative and qualitative reproducibility in the recovery of proteins from FFPE tissues.

  8. Evaluation of Factors Affecting Continuous Performance Test Identical Pairs Version Score of Schizophrenic Patients in a Japanese Clinical Sample

    Directory of Open Access Journals (Sweden)

    Takayoshi Koide

    2012-01-01

    Full Text Available Aim. Cognitive impairment in schizophrenia strongly relates to social outcome and is a good candidate for endophenotypes. When we accurately measure drug efficacy or effects of genes or variants relevant to schizophrenia on cognitive impairment, clinical factors that can affect scores on cognitive tests, such as age and severity of symptoms, should be considered. To elucidate the effect of clinical factors, we conducted multiple regression analysis using scores of the Continuous Performance Test Identical Pairs Version (CPT-IP, which is often used to measure attention/vigilance in schizophrenia. Methods. We conducted the CPT-IP (4-4 digit and examined clinical information (sex, age, education years, onset age, duration of illness, chlorpromazine-equivalent dose, and Positive and Negative Symptom Scale (PANSS scores in 126 schizophrenia patients in Japanese population. Multiple regression analysis was used to evaluate the effect of clinical factors. Results. Age, chlorpromazine-equivalent dose, and PANSS-negative symptom score were associated with mean d′ score in patients. These three clinical factors explained about 28% of the variance in mean d′ score. Conclusions. As conclusion, CPT-IP score in schizophrenia patients is influenced by age, chlorpromazine-equivalent dose and PANSS negative symptom score.

  9. Clinical and neuropsychological assessment of executive function in a sample of children and adolescents with idiopathic epilepsy

    Directory of Open Access Journals (Sweden)

    Andrea Bandeira de Lima

    2014-12-01

    Full Text Available Objective To compare the executive functions of children and adolescents with idiopathic epilepsy with a control group and to correlate with clinical data, intelligence and academic performance. Method Cross-sectional, descriptive and analytical study. Thirty-one cases and thirty-five controls were evaluated by the WCST (Wisconsin Card Sorting Test.The results were compared with clinical data (seizure type and frequency, disease duration and number of antiepileptic drugs used, IQ (WISC-III and academic performance (APT. Results Patients with epilepsy had poorer executive function scores. There was no positive linear correlation between test scores and epilepsy variables. There was a positive association between academic performance and some executive function results. Conclusion Children with well controlled idiopathic epilepsy may show deficits in executive functions in spite of clinical variables. Those deficits may influence academic performance.

  10. Different EV enrichment methods suitable for clinical settings yield different subpopulations of urinary extracellular vesicles from human samples

    OpenAIRE

    Royo, Felix; Zuñiga-Garcia, Patricia; Sanchez-Mosquera, Pilar; Egia, Ainara; Perez, Amparo; Loizaga, Ana; Arceo, Raquel; Lacasa, Isabel; Rabade, Ainara; Arrieta, Edurne; Bilbao, Roberto; Unda, Miguel; Carracedo, Arkaitz; Falcon-Perez, Juan M.

    2016-01-01

    Urine sample analysis is irreplaceable as a non-invasive method for disease diagnosis and follow-up. However, in urine samples, non-degraded protein and RNA may be only found in urinary extracellular vesicles (uEVs). In recent years, various methods of uEV enrichment using low volumes of urine and unsophisticated equipment have been developed, with variable success. We compared the results of the differential ultracentrifugation procedure with 4 of these methods. The methods tested were a lec...

  11. Epidemiology and clinical outcome of virus-positive respiratory samples in ventilated patients: a prospective cohort study.

    OpenAIRE

    Daubin, Cédric; Parienti, Jean-Jacques; Vincent, Sophie; Vabret, Astrid; du Cheyron, Damien; Ramakers, Michel; Freymuth, François; Charbonneau, Pierre

    2006-01-01

    INTRODUCTION: Respiratory viruses are a major cause of respiratory tract infections. The prevalence of a virus-positive respiratory sample and its significance in patients requiring mechanical ventilation remain unknown. METHODS: We conducted a cohort study in all consecutive adults ventilated for more than 48 hours admitted to a 22-bed medical intensive care unit during a 12-month period. Respiratory samples at the time of intubation were assessed by culture, by indirect immunofluorescence a...

  12. Rapid Detection of Methicillin-Resistant Staphylococcus aureus Directly from Sterile or Nonsterile Clinical Samples by a New Molecular Assay

    OpenAIRE

    Francois, Patrice; Pittet, Didier; Bento, Manuela; Pepey, Béatrice; Vaudaux, Pierre; Lew, Daniel; Schrenzel, Jacques

    2003-01-01

    A rapid procedure was developed for detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) directly from sterile sites or mixed flora samples (e.g., nose or inguinal swabs). After a rapid conditioning of samples, the method consists of two main steps: (i) immunomagnetic enrichment in S. aureus and (ii) amplification-detection profile on DNA extracts using multiplex quantitative PCR (5′-exonuclease qPCR, TaqMan). The triplex qPCR assay measures simultaneously the fo...

  13. Assessing Eating Disorder Risk: The Pivotal Role of Achievement Anxiety, Depression and Female Gender in Non-Clinical Samples

    OpenAIRE

    Christos C. Frangos; Fragkos, Konstantinos C.

    2013-01-01

    The objective of the present study was to assess factors predicting eating disorder risk in a sample of undergraduate students. A structured questionnaire was employed on a random sample (n = 1865) consisting of the following sections: demographics, SCOFF (Sick, Control, One stone, Fat, Food) questionnaire for screening eating disorders and the Achievement Anxiety Test and the Depression, Anxiety and Stress Scale. The students at risk for eating disorders (SCOFF score ≥2) were 39.7%. Eating d...

  14. Posttraumatic stress disorder with and without alcohol use disorders: Diagnostic and clinical correlates in a psychiatric sample

    OpenAIRE

    Ray, Lara A.; Capone, Christy; Sheets, Erin; Young, Diane; Chelminski, Iwona; Zimmerman, Mark

    2009-01-01

    This study compared outpatients (n = 196) with PTSD versus PTSD + alcohol use disorders (AUD) on clinical measures. PTSD + AUD patients were more likely to meet criteria for Borderline and Antisocial Personality Disorders. Emotion dysregulation may help account for the relationship between PTSD and AUD.

  15. Indicators of Anxiety and Depression in Women with the Fragile X Premutation: Assessment of a Clinical Sample

    Science.gov (United States)

    Lachiewicz, A.; Dawson, D.; Spiridigliozzi, G.; Cuccaro, M.; Lachiewicz, M.; McConkie-Rosell, A.

    2010-01-01

    Background: Current research suggests that depression and anxiety may be common problems in women with the fragile X (FMR1) premutation. Methods: To learn more about this in a clinical setting, we asked 33 women with the FMR1 premutation and 20 women without the FMR1 premutation to complete the Brief Carroll Depression Scale (Brief CDS) and the…

  16. Autism Spectrum Disorders as a Qualitatively Distinct Category from Typical Behavior in a Large, Clinically Ascertained Sample

    Science.gov (United States)

    Frazier, Thomas W.; Youngstrom, Eric A.; Sinclair, Leslie; Kubu, Cynthia S.; Law, Paul; Rezai, Ali; Constantino, John N.; Eng, Charis

    2010-01-01

    The present study evaluated the hypothesis that autism spectrum disorders (ASDs) are best represented as a discrete category distinct from typical behavior within autism-affected families. The latent structure, categorical versus dimensional, of ASDs informs future diagnostic revisions, clinical assessment, and the design of future research. Data…

  17. Associations between delusion proneness and personality structure in non-clinical participants : Comparison between young and elderly samples

    NARCIS (Netherlands)

    Laroi, Frank; Van der Linden, Martial; DeFruyt, Filip; van Os, Jim; Aleman, Andre

    2006-01-01

    Background: Few studies have explored the prevalence of delusions in the non-clinical, elderly population. In addition, the association between personality structure and delusions remains poorly investigated. The aims of the present study were, first, to explore the relation between age and the prev

  18. Associations between delusion proneness and personality structure in non-clinical participants: Comparison between young and elderly samples

    NARCIS (Netherlands)

    Laroi, F.; Van der Linden, M.; DeFruyt, F.; Van Os, J.; Aleman, A.

    2006-01-01

    Background: Few studies have explored the prevalence of delusions in the non-clinical, elderly population. In addition, the association between personality structure and delusions remains poorly investigated. The aims of the present study were, first, to explore the relation between age and the prev

  19. Acceptability, effectiveness, and cost-effectiveness of internet-based exposure treatment for irritable bowel syndrome in a clinical sample: a randomized controlled trial

    Directory of Open Access Journals (Sweden)

    Andréewitch Sergej

    2011-10-01

    Full Text Available Abstract Background Internet-based cognitive behavior therapy (ICBT has shown promising effects in the treatment of irritable bowel syndrome (IBS. However, to date no study has used a design where participants have been sampled solely from a clinical population. We aimed to investigate the acceptability, effectiveness, and cost-effectiveness of ICBT for IBS using a consecutively recruited sample from a gastroenterological clinic. Methods Sixty-one patients were randomized to 10 weeks of ICBT (n = 30 or a waiting list control (n = 31. The ICBT was guided by an online therapist and emphasized acceptance of symptoms through exposure and mindfulness training. Severity of IBS symptoms was measured with the Gastrointestinal symptom rating scale - IBS version (GSRS-IBS. Patients in both groups were assessed at pre- and post-treatment while only the ICBT group was assessed 12 months after treatment completion. Health economic data were also gathered at all assessment points and analyzed using bootstrap sampling. Results Fifty of 61 patients (82% completed the post-treatment assessment and 20 of 30 patients (67% in the ICBT group were assessed at 12-month follow-up. The ICBT group demonstrated significantly (p d = 0.77 (95% CI: 0.19-1.34. Similar effects were noted on measures of quality of life and IBS-related fear and avoidance behaviors. Improvements in the ICBT group were maintained at 12-month follow-up. The ICBT condition was found to be more cost-effective than the waiting list, with an 87% chance of leading to reduced societal costs combined with clinical effectiveness. The cost-effectiveness was sustained over the 12-month period. Conclusions ICBT proved to be a cost-effective treatment when delivered to a sample recruited from a gastroenterological clinic. However, many of the included patients dropped out of the study and the overall treatment effects were smaller than previous studies with referred and self-referred samples. ICBT may

  20. Comparative analysis of genetic diversity and incidence of virulence factors and antibiotic resistance among enterococcal populations from raw fruit and vegetable foods, water and soil, and clinical samples.

    Science.gov (United States)

    Abriouel, Hikmate; Omar, Nabil Ben; Molinos, Antonio Cobo; López, Rosario Lucas; Grande, Maria José; Martínez-Viedma, Pilar; Ortega, Elena; Cañamero, Magdalena Martínez; Galvez, Antonio

    2008-03-31

    A comparative study was carried out among enterococci isolated from fruits and vegetable foods, water and soil, and clinical samples. Results indicate strong differences in the numbers of enterococcal species found in different environments as well as their abundance. While Enterococcus faecalis was clearly the predominant species in clinical samples, Enterococcus faecium predominated in vegetables, and it slightly outnumbered E. faecalis in water samples. Other species (Enterococcus hirae, Enterococcus mundtii, Enterococcus durans, Enterococcus gallinarum and Enterococcus casseliflavus) were found more frequently in vegetables, water, and specially in soil. Isolates from vegetable foods showed a lower incidence of antibiotic resistance compared to clinical isolates for most antimicrobials tested, especially erythromycin, tetracycline, chloramphenicol, ciprofloxacin, levofloxacin, gentamicin and streptomycin for E. faecalis, and quinupristin/dalfopristin, ampicillin, penicillin, ciprofloxacin, levofloxacin, rifampicin, choramphenicol, gentamicin and nitrofurantoin for E. faecium. E. faecium isolates from vegetable foods and water showed an average lower number of antibiotic resistance traits (2.95 and 3.09 traits for vegetable and water isolates, respectively) compared to clinical samples (7.5 traits). Multi-resistant strains were also frequent among clinical E. faecalis isolates (5.46 traits on average). None of E. faecalis or E. faecium isolates from vegetable foods, water and soil showed beta-haemolytic activity, while 25.64% of clinical E. faecalis did. A 51.28% of E. faecalis clinical isolates tested positive for the cylA, cylB, cylM set of genes, while some or all of these genes were missing in the rest of isolates. In clinical E. faecalis and E. faecium isolates, the genetic determinants for the enterococcal surface protein gene (esp), the collagen adhesin gene (ace) and the sex pheromone gene ccf (as well as cob in E. faecalis) showed a clearly higher

  1. Validity of Footprint Analysis to Determine Flatfoot Using Clinical Diagnosis as the Gold Standard in a Random Sample Aged 40 Years and Older

    OpenAIRE

    Pita-Fernández, Salvador; González-Martín, Cristina; Seoane-Pillado, Teresa; López-Calviño, Beatriz; Pértega-Díaz, Sonia; Gil-Guillén, Vicente

    2015-01-01

    Background Research is needed to determine the prevalence and variables associated with the diagnosis of flatfoot, and to evaluate the validity of three footprint analysis methods for diagnosing flatfoot, using clinical diagnosis as a benchmark. Methods We conducted a cross-sectional study of a population-based random sample ≥40 years old (n = 1002) in A Coruña, Spain. Anthropometric variables, Charlson’s comorbidity score, and podiatric examination (including measurement of Clarke’s angle, t...

  2. Gender ratio in a clinical population sample, age of diagnosis and duration of assessment in children and adults with autism spectrum disorder.

    OpenAIRE

    Rutherford, Marion; McKenzie, Karen; Johnson, Tess; Catchpole, Ciara; O'Hare, Anne; McClure, Iain; Forsyth, Kirsty; McCartney, Deborah; Murray, Aja Louise

    2016-01-01

    This article reports on gender ratio, age of diagnosis and the duration of assessment procedures in autism spectrum disorder diagnosis in a national study which included all types of clinical services for children and adults. Findings are reported from a retrospective case note analysis undertaken with a representative sample of 150 Scottish children and adults recently diagnosed with autism spectrum disorder. The study reports key findings that the gender ratio in this consecutively referred...

  3. Daytime of Sampling, Tooth-Brushing and Ascorbic Acid Influence Salivary Thiobarbituric Acid Reacting Substances – A Potential Clinical Marker of Gingival Status

    Directory of Open Access Journals (Sweden)

    Július Hodosy

    2005-01-01

    Sialic acid content of saliva is not influenced significantly by any of the investigated factors. Conclusion. TBARS levels in saliva are affected by daytime of sampling, tooth-brushing and ascorbic acid pre-treatment. These results must be considered in clinical research using salivary TBARS levels. Sialic acid seems not to be a major component of TBARS in saliva. Further studies should clarify the molecular compounds of salivary TBARS and uncover the role of oral microbial factors.

  4. Development of Novel PCR Assays To Detect Azole Resistance-Mediating Mutations of the Aspergillus fumigatus cyp51A Gene in Primary Clinical Samples from Neutropenic Patients

    OpenAIRE

    Spiess, Birgit; Seifarth, Wolfgang; Merker, Natalia; Howard, Susan J.; Reinwald, Mark; Dietz, Anne; Hofmann, Wolf-Karsten; Buchheidt, Dieter

    2012-01-01

    The increasing incidence of azole resistance in Aspergillus fumigatus causing invasive aspergillosis (IA) in immunocompromised/hematological patients emphasizes the need to improve the detection of resistance-mediating cyp51A gene mutations from primary clinical samples, particularly as the diagnosis of invasive aspergillosis is rarely based on a positive culture yield in this group of patients. We generated primers from the unique sequence of the Aspergillus fumigatus cyp51A gene to establis...

  5. A randomised clinical trial on cardiotocography plus fetal blood sampling versus cardiotocography plus ST-analysis of the fetal electrocardiogram (STAN®) for intrapartum monitoring

    OpenAIRE

    Rijnders Robbert JP; Porath Martina M; Oei S Guid; Nijhuis Jan G; Mol Ben WJ; van Lith Jan MM; van Geijn Herman P; Drogtrop Addy P; Bijvoet Saskia M; van Beek Erik; Moons Karel GM; Westerhuis Michelle EMH; Schuitemaker Nico WE; van der Tweel Ingeborg; Visser Gerard HA

    2007-01-01

    Abstract Background Cardiotocography (CTG) is worldwide the method for fetal surveillance during labour. However, CTG alone shows many false positive test results and without fetal blood sampling (FBS), it results in an increase in operative deliveries without improvement of fetal outcome. FBS requires additional expertise, is invasive and has often to be repeated during labour. Two clinical trials have shown that a combination of CTG and ST-analysis of the fetal electrocardiogram (ECG) reduc...

  6. A rapid DNA extraction method from culture and clinical samples. Suitable for the detection of human cytomegalovirus by the polymerase chain reaction.

    Science.gov (United States)

    Zandotti, C; De Lamballerie, X; Guignole-Vignoli, C; Bollet, C; De Micco, P

    1993-02-01

    We propose an one-step DNA extraction method suitable for the polymerase chain reaction. This procedure utilizes Chelex 100, a chelating in exchange resin. This technique was compared with a traditional technique (proteinase K lysis, phenol-chloroform extraction and ethanol precipitation) for isolation of human cytomegalovirus DNA from clinical samples. The procedure using Chelex 100 appeared to be a simple and fast extraction method for human cytomegalovirus DNA.

  7. Identification of Anxiety Sensitivity Classes and Clinical Cut-Scores in a Sample of Adult Smokers: Results from a Factor Mixture Model

    OpenAIRE

    Allan, Nicholas P.; Raines, Amanda M.; Capron, Daniel W.; Norr, Aaron M.; Zvolensky, Michael J.; Schmidt, Norman B.

    2014-01-01

    Anxiety sensitivity (AS), a multidimensional construct, has been implicated in the development and maintenance of anxiety and related disorders. Recent evidence suggests that AS is a dimensional-categorical construct within individuals. Factor mixture modeling was conducted in a sample of 579 adult smokers (M age = 36.87 years, SD = 13.47) to examine the underlying structure. Participants completed the Anxiety Sensitivity Index-3 and were also given a Structured Clinical Interview for DSM-IV-...

  8. Clinical Implications of Rabphillin-3A-Like Gene Alterations in Breast Cancer.

    Directory of Open Access Journals (Sweden)

    Balananda-Dhurjati Kumar Putcha

    Full Text Available For the rabphillin-3A-like (RPH3AL gene, a putative tumor suppressor, the clinical significance of genetic alterations in breast cancers was evaluated. DNA and RNA were extracted from formalin-fixed, paraffin-embedded (FFPE cancers and matching normal tissues. DNA samples were assessed for loss of heterozygosity (LOH at the 17p13.3 locus of RPH3AL and the 17p13.1 locus of the tumor suppressor, TP53. RPH3AL was sequenced, and single nucleotide polymorphisms (SNPs were genotyped. RNA samples were evaluated for expression of RPH3AL, and FFPE tissues were profiled for its phenotypic expression. Alterations in RPH3AL were correlated with clinicopathological features, LOH of TP53, and patient survival. Of 121 cancers, 80 had LOH at one of the RPH3AL locus. LOH of RHP3AL was associated with nodal metastasis, advanced stage, large tumor size, and poor survival. Although ~50% were positive for LOH at the RPH3AL and TP53 loci, 19 of 105 exhibited LOH only at the RPH3AL locus. Of these, 12 were non-Hispanic Caucasians (Whites, 15 had large tumors, and 12 were older (>50 years. Patients exhibiting LOH at both loci had shorter survival than those without LOH at these loci (log-rank, P = 0.014. LOH at the TP53 locus alone was not associated with survival. Analyses of RPH3AL identified missense point mutations in 19 of 125 cases, a SNP (C>A in the 5'untranslated region at -25 (5'UTR-25 in 26 of 104, and a SNP (G>T in the intronic region at 43 bp downstream to exon-6 (intron-6-43 in 79 of 118. Genotype C/A or A/A of the SNP at 5'UTR-25 and genotype T/T of a SNP at intron-6-43 were predominantly in Whites. Low levels of RNA and protein expression of RPH3AL were present in cancers relative to normal tissues. Thus, genetic alterations in RPH3AL are associated with aggressive behavior of breast cancers and with short survival of patients.

  9. Schizotypal personality questionnaire - brief revised (updated): An update of norms, factor structure, and item content in a large non-clinical young adult sample.

    Science.gov (United States)

    Davidson, Charlie A; Hoffman, Lesa; Spaulding, William D

    2016-04-30

    This study updates and provides evidence for the dimensionality, reliability, and validity of a standard instrument for detection and measurement of schizotypy in non-clinical young adults. Schizotypy represents a set of traits on which both nonclinical and schizophrenia-spectrum populations vary meaningfully. These traits are linked to biological, cognitive, and social dimensions of serious mental illness (SMI), to clinical and subclinical variation in personal and social functioning, and to risk for SMI. Reliable and valid identification of schizotypal traits has important implications for clinical practice and research. Four consecutive independent samples of undergraduates were administered the SPQ-BR (N=2552). Confirmatory factor analyses suggested a minor item wording change improved reliability, and this Updated questionnaire was implemented for three-quarters of the sample (SPQ-BRU). A, single-order, nine-factor structure had acceptable psychometric properties. The best fitting second-order structure included four higher-order factors that distinguished Social Anxiety and Interpersonal factors. This differentiation was supported by differential relationships with treatment history. The Disorganized factor had the greatest unique relationship with personal and family treatment history. With few exceptions, factor loadings showed stability across samples. Overall, the higher-order and lower-order factors of schizotypy demonstrated reliability and convergent and discriminant validity; detailed psychometric data are presented in a supplement. PMID:27086255

  10. Evaluating 3D printing to solve the sample-to-device interface for LRS and POC diagnostics: example of an interlock meter-mix device for metering and lysing clinical urine samples.

    Science.gov (United States)

    Jue, Erik; Schoepp, Nathan G; Witters, Daan; Ismagilov, Rustem F

    2016-05-21

    This paper evaluates the potential of 3D printing, a semi-automated additive prototyping technology, as a means to design and prototype a sample-to-device interface, amenable to diagnostics in limited-resource settings, where speed, accuracy and user-friendly design are critical components. As a test case, we built and validated an interlock meter-mix device for accurately metering and lysing human urine samples for use in downstream nucleic acid amplification. Two plungers and a multivalve generated and controlled fluid flow through the device and demonstrate the utility of 3D printing to create leak-free seals. Device operation consists of three simple steps that must be performed sequentially, eliminating manual pipetting and vortexing to provide rapid (5 to 10 s) and accurate metering and mixing. Bretherton's prediction was applied, using the bond number to guide a design that prevents potentially biohazardous samples from leaking from the device. We employed multi-material 3D printing technology, which allows composites with rigid and elastomeric properties to be printed as a single part. To validate the meter-mix device with a clinically relevant sample, we used urine spiked with inactivated Chlamydia trachomatis and Neisseria gonorrhoeae. A downstream nucleic acid amplification by quantitative PCR (qPCR) confirmed there was no statistically significant difference between samples metered and mixed using the standard protocol and those prepared with the meter-mix device, showing the 3D-printed device could accurately meter, mix and dispense a human urine sample without loss of nucleic acids. Although there are some limitations to 3D printing capabilities (e.g. dimension limitations related to support material used in the printing process), the advantages of customizability, modularity and rapid prototyping illustrate the utility of 3D printing for developing sample-to-device interfaces for diagnostics. PMID:27122199

  11. Evaluating 3D printing to solve the sample-to-device interface for LRS and POC diagnostics: example of an interlock meter-mix device for metering and lysing clinical urine samples.

    Science.gov (United States)

    Jue, Erik; Schoepp, Nathan G; Witters, Daan; Ismagilov, Rustem F

    2016-05-21

    This paper evaluates the potential of 3D printing, a semi-automated additive prototyping technology, as a means to design and prototype a sample-to-device interface, amenable to diagnostics in limited-resource settings, where speed, accuracy and user-friendly design are critical components. As a test case, we built and validated an interlock meter-mix device for accurately metering and lysing human urine samples for use in downstream nucleic acid amplification. Two plungers and a multivalve generated and controlled fluid flow through the device and demonstrate the utility of 3D printing to create leak-free seals. Device operation consists of three simple steps that must be performed sequentially, eliminating manual pipetting and vortexing to provide rapid (5 to 10 s) and accurate metering and mixing. Bretherton's prediction was applied, using the bond number to guide a design that prevents potentially biohazardous samples from leaking from the device. We employed multi-material 3D printing technology, which allows composites with rigid and elastomeric properties to be printed as a single part. To validate the meter-mix device with a clinically relevant sample, we used urine spiked with inactivated Chlamydia trachomatis and Neisseria gonorrhoeae. A downstream nucleic acid amplification by quantitative PCR (qPCR) confirmed there was no statistically significant difference between samples metered and mixed using the standard protocol and those prepared with the meter-mix device, showing the 3D-printed device could accurately meter, mix and dispense a human urine sample without loss of nucleic acids. Although there are some limitations to 3D printing capabilities (e.g. dimension limitations related to support material used in the printing process), the advantages of customizability, modularity and rapid prototyping illustrate the utility of 3D printing for developing sample-to-device interfaces for diagnostics.

  12. Cluster Analysis of the Yale Global Tic Severity Scale (YGTSS): Symptom Dimensions and Clinical Correlates in an Outpatient Youth Sample

    Science.gov (United States)

    Kircanski, Katharina; Woods, Douglas W.; Chang, Susanna W.; Ricketts, Emily J.; Piacentini, John C.

    2010-01-01

    Tic disorders are heterogeneous, with symptoms varying widely both within and across patients. Exploration of symptom clusters may aid in the identification of symptom dimensions of empirical and treatment import. This article presents the results of two studies investigating tic symptom clusters using a sample of 99 youth (M age = 10.7, 81% male,…

  13. A Psychometric Evaluation of the Behavioral Inhibition Questionnaire in a Non-Clinical Sample of Dutch Children and Adolescents

    Science.gov (United States)

    Broeren, Suzanne; Muris, Peter

    2010-01-01

    The Behavioral Inhibition Questionnaire (BIQ) is a parent-rating scale for measuring temperamental characteristics referring to shyness, fearfulness, and withdrawal in young, preschool children. The present study evaluated the psychometric properties of the BIQ in a Dutch community sample of children with a broad age range. For this purpose, the…

  14. A psychometric evaluation of the behavioral inhibition questionnaire in a non-clinical sample of Dutch children and adolescents.

    NARCIS (Netherlands)

    S.M.L. Broeren (Suzanne); P.E.H.M. Muris (Peter)

    2010-01-01

    textabstractThe Behavioral Inhibition Questionnaire (BIQ) is a parent-rating scale for measuring temperamental characteristics referring to shyness, fearfulness, and withdrawal in young, preschool children. The present study evaluated the psychometric properties of the BIQ in a Dutch community sampl

  15. High-Throughput Amplicon-Based Copy Number Detection of 11 Genes in Formalin-Fixed Paraffin-Embedded Ovarian Tumour Samples by MLPA-Seq.

    Science.gov (United States)

    Kondrashova, Olga; Love, Clare J; Lunke, Sebastian; Hsu, Arthur L; Waring, Paul M; Taylor, Graham R

    2015-01-01

    Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity.

  16. Characterization of Erysipelothrix species isolates from clinically affected pigs, environmental samples, and vaccine strains from six recent swine erysipelas outbreaks in the United States.

    Science.gov (United States)

    Bender, J S; Shen, H G; Irwin, C K; Schwartz, K J; Opriessnig, T

    2010-10-01

    The aim of this study was to characterize Erysipelothrix sp. isolates from clinically affected pigs and their environment and compare them to the Erysipelothrix sp. vaccines used at the sites. Samples were collected during swine erysipelas outbreaks in vaccinated pigs in six Midwest United States swine operations from 2007 to 2009. Pig tissue samples were collected from 1 to 3 pigs from each site. Environmental samples (manure, feed, central-line water, oral fluids, and swabs collected from walls, feed lines, air inlets, exhaust fans, and nipple drinkers) and live vaccine samples were collected following the isolation of Erysipelothrix spp. from clinically affected pigs. All Erysipelothrix sp. isolates obtained were further characterized by serotyping. Selected isolates were further characterized by PCR assays for genotype (E. rhusiopathiae, E. tonsillarum, Erysipelothrix sp. strain 1, and Erysipelothrix sp. strain 2) and surface protective antigen (spa) type (A, B1, B2, and C). All 26 isolates obtained from affected pigs were E. rhusiopathiae, specifically, serotypes 1a, 1b, 2, and 21. From environmental samples, 56 isolates were obtained and 52/56 were E. rhusiopathiae (serotypes 1a, 1b, 2, 6, 9, 12, and 21), 3/56 were Erysipelothrix sp. strain 1 (serotypes 13 and untypeable), and one was a novel species designated Erysipelothrix sp. strain 3 (serotype untypeable). Four of six vaccines used at the sites were commercially available products and contained live E. rhusiopathiae serotype 1a. Of the remaining two vaccines, one was an autogenous live vaccine and contained live E. rhusiopathiae serotype 2 and one was a commercially produced inactivated vaccine and was described by the manufacturer to contain serotype 2 antigen. All E. rhusiopathiae isolates were positive for spaA. All Erysipelothrix sp. strain 1 isolates and the novel Erysipelothrix sp. strain 3 isolate were negative for all currently known spa types (A, B1, B2, and C). These results indicate that

  17. Molecular beacon-based real-time PCR detection of primary isolates of Salmonella Typhimurium and Salmonella Enteritidis in environmental and clinical samples

    Directory of Open Access Journals (Sweden)

    Emmanuel Maria A

    2009-05-01

    Full Text Available Abstract Background A fast and simple two-step multiplex real-time PCR assay has been developed to replace the traditional, laborious Salmonella serotyping procedure. Molecular beacons were incorporated into the assay as probes for target DNA. Target sequences were regions of the invA, prot6E and fliC genes specific for Salmonella spp. Salmonella Enteritidis and Salmonella Typhimurium, respectively, the two most clinically relevant serotypes. An internal amplification positive control was included in the experiment to ensure the optimal functioning of the PCR and detect possible PCR inhibition. Three sets of primers were used for the amplification of the target sequences. The results were compared to those of the Kauffmann-White antigenic classification scheme. Results The assay was 100% sensitive and specific, correctly identifying all 44 Salmonella strains, all 21 samples of S. Enteritidis and all 17 samples of S. Typhimurium tested in this work. Therefore, the entire experiment had specificity and sensitivity of 100%. The detection limit was down to 10 copies of DNA target per 25 μl reaction. Conclusion The assay can amplify and analyse a large number of samples in approximately 8 hours, compared to the 4 to 5 days conventional identification takes, and is thus considered a very promising method for detecting the two major serotypes of Salmonella quickly and accurately from clinical and environmental samples.

  18. Rapid detection of Candida albicans in clinical samples by DNA amplification of common regions from C. albicans-secreted aspartic proteinase genes.

    Science.gov (United States)

    Flahaut, M; Sanglard, D; Monod, M; Bille, J; Rossier, M

    1998-02-01

    Laboratory diagnosis based on genomic amplification methods such as PCR may provide an alternative and more sensitive method than conventional culture for the early detection of deep-seated candidiasis, an increasing cause of morbidity and mortality among immunocompromised patients. A novel method of DNA extraction from clinical samples based on treatment with proteinase K and isolation of DNA on a silica membrane was developed. The targets used for DNA amplification were the Candida albicans-secreted aspartic proteinase (SAP) genes, a multiple-gene family of at least seven members in C. albicans. A single pair of primers was designed in order to detect six of these SAP genes and, subsequently, to increase the sensitivity of the test. Detection of the PCR product by enzyme-linked immunosorbent assay was found to be as sensitive as Southern blotting with an SAP-labeled probe. The sensitivity of the assay was 1 cell/ml from serially diluted Candida cultures and 1 to 4 cells/ml from seeded blood specimens. The sensitivity and specificity of the present assay were tested in a retrospective study performed blindly with 156 clinical samples and were 100 and 98%, respectively, compared with the results of culture. For the subset of blood culture samples (n = 124), the sensitivity and the specificity were 100%. The two false-positive PCR samples came from patients treated with azole antifungal agents, indicating that PCR was probably able to detect damaged organisms that could not be recovered by culture.

  19. Diagnosis of rubella infection by detecting specific immunoglobulin M antibodies in saliva samples: a clinic-based study in Niterói, RJ, Brazil

    Directory of Open Access Journals (Sweden)

    Oliveira Solange Artimos de

    2000-01-01

    Full Text Available This study was designed to investigate whether saliva could be a feasible alternative to serum for the diagnosis of recent rubella infection in a clinic setting. Forty-five paired blood and saliva samples collected 1 to 29 days after onset of illness were tested for specific immunoglobulin (Ig M by antibody-capture radioimmunoassay (MACRIA. Rubella IgM was detected in all serum samples and in 38 (84.4% saliva specimens. Forty-six serum and saliva samples from other patients with rash diseases were tested by MACRIA for control purposes and two saliva specimens were reactive. The saliva test had specificity of 96%. These results indicate that salivary IgM detection may be a convenient non-invasive alternative to serum for investigation of recent rubella cases, especially for disease surveillance and control programmes.

  20. Evaluation of factors affecting real-time PCR performance for diagnosis of Entamoeba histolytica and Entamoeba dispar in clinical stool samples.

    Science.gov (United States)

    Forsell, Joakim; Koskiniemi, Satu; Hedberg, Ida; Edebro, Helén; Evengård, Birgitta; Granlund, Margareta

    2015-09-01

    Although PCR offers the potential for sensitive detection of parasites there are several pitfalls for optimal performance, especially when DNA is extracted from a complex sample material such as stool. With the aid of a sensitive inhibitor control in a duplex real-time PCR (qPCR) for identification of Entamoeba histolytica and Entamoeba dispar we have evaluated factors that influenced the performance of the qPCR and have suggested a rationale to be used in the analysis of clinical samples. Pre-PCR processing was found to be of outmost importance for an optimal amplification since inhibitors caused false-negative results when higher amounts of sample were used. Stool sampling with a flocked swab (ESwab, Copan), yielding on average 173 mg, gave positive qPCR results in samples with cysts of E. dispar that were negative in serially diluted stool samples. The degree of inhibition found varied between samples and was not an on-off phenomenon. Even low-grade inhibition, shown as an increase of two cycles in the qPCR for the inhibitor control, could lead to false negativity in samples with low amounts of parasites. Lack of amplification in the qPCR due to inhibition could be overcome by dilution of the extracted DNA by 1/10-1/20. We also describe the use of guanidinium thiocyanate buffer for transport and storage of samples as well as a time-saving semi-automated DNA extraction method in an Arrow instrument (Nordiag) preceded by bead beating.

  1. The contribution of social rank and attachment theory to depression in a non clinical sample of adolescents

    OpenAIRE

    Puissant, Sylvia Pinna; Gauthier, Jean-Marie; Van Oirbeek, R. KUL

    2011-01-01

    This study explores the relative contribution of the overall quality of attachment to the mother, to the father and to peers (Inventory of Parent and Peer Attachment scales), the style of attachment towards peers (Attachment Questionnaire for Children scale), the social rank variables (submissive behavior and social comparison), and sex and age variables in predicting the depression score (Center of Epidemiological Studies Depression Scale) on a non-psychiatric sample of 13-18 year old adoles...

  2. Is real-time PCR better than conventional PCR for Mycobacterium tuberculosis complex detection in clinical samples?

    Science.gov (United States)

    Tortoli, Enrico; Urbano, Pasquale; Marcelli, Fiorella; Simonetti, Tullia M; Cirillo, Daniela M

    2012-08-01

    Cobas Amplicor MTB and later Cobas TaqMan MTB were used to test a very large series of consecutive specimens received for tuberculosis diagnosis. Performance parameters were estimated and compared overall and for separate specimen categories. Both systems showed excellent specificity, and that of TaqMan was the higher. The sensitivities were similar but satisfactory only with respiratory specimens and smear-positive samples.

  3. FACE Analysis as a Fast and Reliable Methodology to Monitor the Sulfation and Total Amount of Chondroitin Sulfate in Biological Samples of Clinical Importance

    Directory of Open Access Journals (Sweden)

    Evgenia Karousou

    2014-06-01

    Full Text Available Glycosaminoglycans (GAGs due to their hydrophilic character and high anionic charge densities play important roles in various (pathophysiological processes. The identification and quantification of GAGs in biological samples and tissues could be useful prognostic and diagnostic tools in pathological conditions. Despite the noteworthy progress in the development of sensitive and accurate methodologies for the determination of GAGs, there is a significant lack in methodologies regarding sample preparation and reliable fast analysis methods enabling the simultaneous analysis of several biological samples. In this report, developed protocols for the isolation of GAGs in biological samples were applied to analyze various sulfated chondroitin sulfate- and hyaluronan-derived disaccharides using fluorophore-assisted carbohydrate electrophoresis (FACE. Applications to biologic samples of clinical importance include blood serum, lens capsule tissue and urine. The sample preparation protocol followed by FACE analysis allows quantification with an optimal linearity over the concentration range 1.0–220.0 µg/mL, affording a limit of quantitation of 50 ng of disaccharides. Validation of FACE results was performed by capillary electrophoresis and high performance liquid chromatography techniques.

  4. Electrochemical genosensor array for the simultaneous detection of multiple high-risk human papillomavirus sequences in clinical samples

    Energy Technology Data Exchange (ETDEWEB)

    Civit, Laia [Nanobiotechnology and Bioanalysis Group, Departament d' Enginyeria Quimica, Universitat Rovira i Virgili, 43007 Tarragona (Spain); Fragoso, Alex, E-mail: alex.fragoso@urv.cat [Nanobiotechnology and Bioanalysis Group, Departament d' Enginyeria Quimica, Universitat Rovira i Virgili, 43007 Tarragona (Spain); Hoelters, Sebastian; Duerst, Matthias [Department for Gynecology, Jena University Hospital, Friedrich-Schiller-University Jena, D-07743 Jena (Germany); O' Sullivan, Ciara K., E-mail: ciara.osullivan@urv.cat [Nanobiotechnology and Bioanalysis Group, Departament d' Enginyeria Quimica, Universitat Rovira i Virgili, 43007 Tarragona (Spain); Institucio Catalana de Recerca i Estudis Avancats, Passeig Lluis Companys 23, 08010 Barcelona (Spain)

    2012-02-17

    Highlights: Black-Right-Pointing-Pointer High-risk human papillomavirus is detected in virtually all-invasive cervical cancers. Black-Right-Pointing-Pointer Electrochemical genosensor for simultaneous detection of multiple high-risk HPV applied to cervical scrape samples. Black-Right-Pointing-Pointer Excellent correlation with HPV genotyping carried out within a hospital laboratory. - Abstract: An electrochemical genosensor array for the simultaneous detection of three high-risk human papillomavirus (HPV) DNA sequences, HPV16, 18 and 45, exhibiting high sensitivity and selectivity is presented. The electrodes of a 4 Multiplication-Sign 4 array were modified via co-immobilization of a 1:100 (mol/mol) mixture of a thiolated probe and an oligoethyleneglycol-terminated bipodal thiol. Detection of synthetic and PCR products was carried out in a sandwich type format, with the target hybridized between a surface immobilized probe and a horseradish peroxidase-labelled secondary reporter probe. The detection limits obtained in the detection of each individual target were in the pM range, allowing the application of this sensor for the detection of samples obtained from PCR amplification of cervical scrape samples. The results obtained exhibited an excellent correlation with the HPV genotyping carried out within a hospital laboratory. Multiplexing and cross-reactivity studies demonstrated high selectivity over potential interfering sequences, facilitating application of the developed platform for the high-throughput screening of multiple high-risk DNA sequences.

  5. Bifactor analysis and construct validity of the Five Facet Mindfulness Questionnaire (FFMQ in non-clinical Spanish samples

    Directory of Open Access Journals (Sweden)

    Jaume eAguado

    2015-04-01

    Full Text Available The objective of the present study was to examine the dimensionality, reliability, and construct validity of the Five Facet Mindfulness Questionnaire (FFMQ in three Spanish samples using structural equation modelling (SEM. Pooling the FFMQ data from 3 Spanish samples (n= 1191, we estimated the fit of two competing models (correlated five-factor vs. bifactor via confirmatory factor analysis. The factorial invariance of the best fitting model across meditative practice was also addressed. The pattern of relationships between the FFMQ latent dimensions and anxiety, depression, and distress was analysed using SEM. FFMQ reliability was examined by computing the omega and omega hierarchical coefficients. The bifactor model, which accounted for the covariance among FFMQ items with regard to one general factor (mindfulness and five orthogonal factors (observing, describing, acting with awareness, non-judgment, and non-reactivity, fit the FFMQ structure better than the correlated five-factor model. The relationships between the latent variables and their manifest indicators were not invariant across the meditative experience. Observing items had significant loadings on the general mindfulness factor, but only in the meditator sub-sample. The SEM analysis revealed significant links between mindfulness and symptoms of depression and stress. When the general factor was partialled out, the acting with awareness facet did not show adequate reliability. The FFMQ shows a robust bifactor structure among Spanish individuals. Nevertheless, the Observing subscale does not seem to be adequate for assessing mindfulness in individuals without meditative experience.

  6. Bifactor analysis and construct validity of the five facet mindfulness questionnaire (FFMQ) in non-clinical Spanish samples.

    Science.gov (United States)

    Aguado, Jaume; Luciano, Juan V; Cebolla, Ausias; Serrano-Blanco, Antoni; Soler, Joaquim; García-Campayo, Javier

    2015-01-01

    The objective of the present study was to examine the dimensionality, reliability, and construct validity of the Five Facet Mindfulness Questionnaire (FFMQ) in three Spanish samples using structural equation modeling (SEM). Pooling the FFMQ data from 3 Spanish samples (n = 1191), we estimated the fit of two competing models (correlated five-factor vs. bifactor) via confirmatory factor analysis. The factorial invariance of the best fitting model across meditative practice was also addressed. The pattern of relationships between the FFMQ latent dimensions and anxiety, depression, and distress was analyzed using SEM. FFMQ reliability was examined by computing the omega and omega hierarchical coefficients. The bifactor model, which accounted for the covariance among FFMQ items with regard to one general factor (mindfulness) and five orthogonal factors (observing, describing, acting with awareness, non-judgment, and non-reactivity), fit the FFMQ structure better than the correlated five-factor model. The relationships between the latent variables and their manifest indicators were not invariant across the meditative experience. Observing items had significant loadings on the general mindfulness factor, but only in the meditator sub-sample. The SEM analysis revealed significant links between mindfulness and symptoms of depression and stress. When the general factor was partialled out, the acting with awareness facet did not show adequate reliability. The FFMQ shows a robust bifactor structure among Spanish individuals. Nevertheless, the Observing subscale does not seem to be adequate for assessing mindfulness in individuals without meditative experience. PMID:25914664

  7. Molecular detection and characterization of infectious laryngotracheitis virus (Gallid herpesvirus-1) from clinical samples of commercial poultry flocks in India.

    Science.gov (United States)

    Gowthaman, Vasudevan; Singh, Sambhu Dayal; Dhama, Kuldeep; Barathidasan, Rajamani; Mathapati, Basavaraj S; Srinivasan, Palani; Saravanan, Sellappan; Ramakrishnan, Muthannan Andavar

    2014-01-01

    Although the existence of infectious laryngotracheitis virus (ILTV) in India was first reported in 1964, no reports are available regarding its molecular detection and characterization. The present study was aimed to detect and characterize ILTV from recent respiratory disease complex (RDC) outbreaks of commercial poultry flocks in different parts of the country by using envelope glycoprotein G gene (US4 gene) based PCR and sequencing. A total of thirty two flocks with a history of RDC were investigated. Overall, all the strains/breeds of birds and all ages of birds are equally susceptible and depending on the severity, the clinical signs and gross lesions were varied. Out of 32 flocks investigated 10 were found positive for ILTV infection by PCR. The phylogenetic analyses of eight representative sequences in the present study deciphered that Indian ILT viruses are closely related to chicken embryo origin vaccine strains of Italy, USA, China and Brazil.

  8. Changes in disengagement coping mediate changes in affect following mindfulness-based cognitive therapy in a non-clinical sample.

    Science.gov (United States)

    Cousin, Gaëtan; Crane, Catherine

    2016-08-01

    Past research has shown that mindfulness-based interventions increase positive affect in non-clinical populations. However, the mechanisms underlying this increase are poorly understood. On the basis of previous empirical and theoretical accounts, we hypothesized that a decreased use of disengagement coping strategies in daily life would explain the benefits of a mindfulness-based intervention in terms of increased positive affect. We analysed the data of 75 healthy adult participants (58 women; 17 men) of different ages (M = 49 years old; SD = 13; age range 19-81) who had been randomly allocated to 8-week Mindfulness-Based Cognitive Therapy (MBCT) or to a waitlist control group. The results confirmed our hypothesis: Participants in the MBCT group showed significant improvements in positive affect compared to the control group, with decreased use of disengagement coping styles mediating these improvements. The implications of this study are discussed. PMID:26385256

  9. Changes in disengagement coping mediate changes in affect following mindfulness-based cognitive therapy in a non-clinical sample.

    Science.gov (United States)

    Cousin, Gaëtan; Crane, Catherine

    2016-08-01

    Past research has shown that mindfulness-based interventions increase positive affect in non-clinical populations. However, the mechanisms underlying this increase are poorly understood. On the basis of previous empirical and theoretical accounts, we hypothesized that a decreased use of disengagement coping strategies in daily life would explain the benefits of a mindfulness-based intervention in terms of increased positive affect. We analysed the data of 75 healthy adult participants (58 women; 17 men) of different ages (M = 49 years old; SD = 13; age range 19-81) who had been randomly allocated to 8-week Mindfulness-Based Cognitive Therapy (MBCT) or to a waitlist control group. The results confirmed our hypothesis: Participants in the MBCT group showed significant improvements in positive affect compared to the control group, with decreased use of disengagement coping styles mediating these improvements. The implications of this study are discussed.

  10. Rapid detection of methicillin-resistant Staphylococcus aureus directly from clinical samples: methods, effectiveness and cost considerations

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    Stürenburg, Enno

    2009-07-01

    Full Text Available Methicillin-resistant Staphylococcus aureus (MRSA isolates is a serious public health problem whose ever-increasing rate is commensurate with the pressure it is exerting on the healthcare system. At present, more than 20% of clinical S. aureus isolates in German hospitals are methicillin resistant. Strategies from low-prevalence countries show that this development is not necessarily inevitable. In the Scandinavian countries and the Netherlands, thanks to a rigorous prevention programme, MRSA prevalence has been kept at an acceptably low level (<1–3%. Central to these ‘search and destroy’ control strategies is an admission screening using several MRSA swabs taken from mucocutaneous colonisation sites of high-risk patients (‘MRSA surveillance’. It has also been reported that the speed with which MRSA carriage is detected has an important role to play, as it is a key component of any effective strategy to prevent the pathogen from spreading. Since MRSA culturing involves a 2–3 day delay before the final results are available, rapid detection techniques (commonly referred to as ‘MRSA rapid tests’ using PCR methods and, most recently, rapid culturing methods have been developed. The implementation of rapid tests reduces the time of detection of MRSA carriers from 48–72 to 2–5 h. Clinical evaluation data have shown that MRSA can thus be detected with very high sensitivity. Specificity however is sometimes impaired due to false-positive PCR signals occurring in mixed flora specimens. In order to rule out any false-positive PCR results, a culture screen must always be carried out simultaneously.The data provide preliminary evidence that a PCR assay can reduce nosocomial MRSA transmission in high-risk patients or high-risk areas, whereas an approach that screens all patients admitted to the hospital is probably not effective. Information concerning the cost-effectiveness of rapid MRSA tests is still sparse and thus the issue remains

  11. Molecular characterization of Leptospira sp by multilocus variable number tandem repeat analysis (MLVA from clinical samples: a case report

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    Hélène Pailhoriès

    2015-08-01

    Full Text Available Leptospirosis is a zoonotic infection for which diagnosis is difficult. It has appeared as a global emerging infectious disease over recent years. Genotype determination often requires a Leptospira strain obtained by culture, which is a long and fastidious technique. A method based on multilocus variable number tandem repeat analysis (MLVA to determine the genotype of Leptospira interrogans, performed directly on blood or urine samples, is proposed. This method was applied to a fatal case of leptospirosis for which the geographical origin of infection was unknown. This technique will allow a genotype to be obtained for L. interrogans, even when cultures remain negative.

  12. Simple, Rapid Mycobacterium ulcerans Disease Diagnosis from Clinical Samples by Fluorescence of Mycolactone on Thin Layer Chromatography.

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    Anita Wadagni

    2015-11-01

    Full Text Available Mycobacterium ulcerans infection, known as Buruli ulcer, is a disease of the skin and subcutaneous tissues which is an important but neglected tropical disease with its major impact in rural parts of West and Central Africa where facilities for diagnosis and management are poorly developed. We evaluated fluorescent thin layer chromatography (f-TLC for detection of mycolactone in the laboratory using samples from patients with Buruli ulcer and patients with similar lesions that gave a negative result on PCR for the IS2404 repeat sequence of M. ulcerans.Mycolactone and DNA extracts from fine needle aspiration (FNA, swabs and biopsy specimen were used to determine the sensitivity and specificity of f-TLC when compared with PCR for the IS2404. For 71 IS2404 PCR positive and 28 PCR negative samples the sensitivity was 73.2% and specificity of 85.7% for f-TLC. The sensitivity was similar for swabs (73%, FNAs (75% and biopsies (70%.We have shown that mycolactone can be detected from M. ulcerans infected skin tissue by f-TLC technique. The technique is simple, easy to perform and read with minimal costs. In this study it was undertaken by a member of the group from each endemic country. It is a potentially implementable tool at the district level after evaluation in larger field studies.

  13. Differential diagnosis of Entamoeba spp. in clinical stool samples using SYBR green real-time polymerase chain reaction.

    Science.gov (United States)

    Gomes, Thiago Dos Santos; Garcia, Mariana Coimbra; de Souza Cunha, Flavia; Werneck de Macedo, Heloisa; Peralta, José Mauro; Peralta, Regina Helena Saramago

    2014-01-01

    Amoebiasis, a disease caused by Entamoeba histolytica, is usually diagnosed by microscopic examination, which does not differentiate the morphologically identical species of the E. histolytica/E. dispar complex. Furthermore, morphologically similar species such as Entamoeba hartmanni contribute to misidentification. Therefore, there is a need for more sensitive and specific methods. This study standardized a multiplex real-time PCR system for E. histolytica and E. dispar and a single real-time PCR for E. hartmanni. The multiplex protocol detected up to 0.0143 pg of E. histolytica DNA and 0.5156 pg of E. dispar DNA, and the average melting temperature (T(m)) was 73 °C and 70 °C, respectively. For E. hartmanni, the T(m) was 73 °C and the amplification was successful down to 0.03 fg of plasmid DNA. Negative controls and other intestinal parasites presented no amplification. Among the 48 samples tested, E. dispar DNA was detected in 37; none exhibited E. histolytica DNA and 11 were negative in the multiplex protocol. In 4 of these 11 samples, however, E. hartmanni DNA was amplified. SYBR Green is demonstrated to be an interesting option and these combined PCR reactions can improve laboratory diagnosis of amoebiasis in developing countries.

  14. Differential diagnosis of Entamoeba spp. in clinical stool samples using SYBR green real-time polymerase chain reaction.

    Science.gov (United States)

    Gomes, Thiago Dos Santos; Garcia, Mariana Coimbra; de Souza Cunha, Flavia; Werneck de Macedo, Heloisa; Peralta, José Mauro; Peralta, Regina Helena Saramago

    2014-01-01

    Amoebiasis, a disease caused by Entamoeba histolytica, is usually diagnosed by microscopic examination, which does not differentiate the morphologically identical species of the E. histolytica/E. dispar complex. Furthermore, morphologically similar species such as Entamoeba hartmanni contribute to misidentification. Therefore, there is a need for more sensitive and specific methods. This study standardized a multiplex real-time PCR system for E. histolytica and E. dispar and a single real-time PCR for E. hartmanni. The multiplex protocol detected up to 0.0143 pg of E. histolytica DNA and 0.5156 pg of E. dispar DNA, and the average melting temperature (T(m)) was 73 °C and 70 °C, respectively. For E. hartmanni, the T(m) was 73 °C and the amplification was successful down to 0.03 fg of plasmid DNA. Negative controls and other intestinal parasites presented no amplification. Among the 48 samples tested, E. dispar DNA was detected in 37; none exhibited E. histolytica DNA and 11 were negative in the multiplex protocol. In 4 of these 11 samples, however, E. hartmanni DNA was amplified. SYBR Green is demonstrated to be an interesting option and these combined PCR reactions can improve laboratory diagnosis of amoebiasis in developing countries. PMID:24693242

  15. Gene expression data from acetaminophen-induced toxicity in human hepatic in vitro systems and clinical liver samples

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    Robim M. Rodrigues

    2016-06-01

    Full Text Available This data set is composed of transcriptomics analyses of (i liver samples from patients suffering from acetaminophen-induced acute liver failure (ALF and (ii hepatic cell systems exposed to acetaminophen and their respective controls. The in vitro systems include widely employed cell lines i.e. HepaRG and HepG2 cells as well as a novel stem cell-derived model i.e. human skin-precursors-derived hepatocyte-like cells (hSKP-HPC. Data from primary human hepatocytes was also added to the data set “Open TG-GATEs: a large-scale toxicogenomics database” (Igarashi et al., 2015 [1]. Changes in gene expression due to acetaminophen intoxication as well as comparative information between human in vivo and in vitro samples are provided. The microarray data have been deposited in NCBI׳s Gene Expression Omnibus and are accessible through GEO Series accession number GEO: GSE74000. The provided data is used to evaluate the predictive capacity of each hepatic in vitro system and can be directly compared with large-scale publically available toxicogenomics databases. Further interpretation and discussion of these data feature in the corresponding research article “Toxicogenomics-based prediction of acetaminophen-induced liver injury using human hepatic cell systems” (Rodrigues et al., 2016 [2].

  16. Differential Diagnosis of Entamoeba spp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Thiago dos Santos Gomes

    2014-01-01

    Full Text Available Amoebiasis, a disease caused by Entamoeba histolytica, is usually diagnosed by microscopic examination, which does not differentiate the morphologically identical species of the E. histolytica/E. dispar complex. Furthermore, morphologically similar species such as Entamoeba hartmanni contribute to misidentification. Therefore, there is a need for more sensitive and specific methods. This study standardized a multiplex real-time PCR system for E. histolytica and E. dispar and a single real-time PCR for E. hartmanni. The multiplex protocol detected up to 0.0143 pg of E. histolytica DNA and 0.5156 pg of E. dispar DNA, and the average melting temperature (Tm was 73°C and 70°C, respectively. For E. hartmanni, the Tm was 73°C and the amplification was successful down to 0.03 fg of plasmid DNA. Negative controls and other intestinal parasites presented no amplification. Among the 48 samples tested, E. dispar DNA was detected in 37; none exhibited E. histolytica DNA and 11 were negative in the multiplex protocol. In 4 of these 11 samples, however, E. hartmanni DNA was amplified. SYBR Green is demonstrated to be an interesting option and these combined PCR reactions can improve laboratory diagnosis of amoebiasis in developing countries.

  17. Assessing Eating Disorder Risk: The Pivotal Role of Achievement Anxiety, Depression and Female Gender in Non-Clinical Samples

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    Christos C. Frangos

    2013-03-01

    Full Text Available The objective of the present study was to assess factors predicting eating disorder risk in a sample of undergraduate students. A structured questionnaire was employed on a random sample (n = 1865 consisting of the following sections: demographics, SCOFF (Sick, Control, One stone, Fat, Food questionnaire for screening eating disorders and the Achievement Anxiety Test and the Depression, Anxiety and Stress Scale. The students at risk for eating disorders (SCOFF score ≥2 were 39.7%. Eating disorder risk was more frequent in females, students with divorced parents, students who lived alone, students who were seeking a romantic relationship or were married, students who were at a post-secondary vocational institute/college (private-public educational level and who were more likely to have marks under merit level. Also, the mean scores for the psychological factors of depression, stress and anxiety were higher in students with eating disorder risk. A logistic regression model was produced depicting that depression, stress, female gender, being married and searching for a romantic relationship were risk factors of having an eating disorder risk. The suggested psychological model examined with structural equation modelling signified the role of academic anxiety as an immediate precursor of general anxiety. Hence, college populations in Greece need organized infrastructures of nutrition health services and campaigns to assist in reducing the risk of eating disorders.

  18. Evaluation of kDNA PCR hybridization and ITS1 nPCR methods in different clinical samples for visceral leishmaniasis diagnosis in dogs with and without clinical signs

    Energy Technology Data Exchange (ETDEWEB)

    Ferreira, Aline Leandra C.; Carregal, Virginia M.; Leite, Rodrigo S.; Ferreira, Sidney A.; Andrade, Antero Silva R., E-mail: alineleandra@hotmail.com, E-mail: streptos@hotmail.com, E-mail: rleite2005@gmail.com, E-mail: vidasnino@yahoo.com.br, E-mail: antero@cdtn.br [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil). Lab. de Radiobiologia; Melo, Maria N., E-mail: melo@icb.ufmg.br [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Parasitologia

    2013-07-01

    Visceral leishmaniasis (VL) in Brazil is caused by Leishmania infantum and dogs are considered the main domestic reservoirs of this parasite. The VL control program in Brazil emphasizes the use of serological surveys, followed by elimination of seropositive dogs. However, serologic tests have limitations in terms of sensitivity and specificity. Molecular methods such as PCR (Polymerase Chain Reaction) associated with hybridization using {sup 32}P radiolabeled DNA probes (kDNA PCR hybridization) are useful tools in this scenario, since they are more specific and sensitive than conventional methods. A variety of samples can be employed with PCR; however non-invasive procedures are the most adequate. One of main obstacles for implementation of PCR in the canine visceral leishmaniasis (CVL) diagnosis is the lack of standardization. Few studies up to the moment compared the effectiveness of the different PCR methods and clinical samples available. The objective of this study was to compare the kDNA PCR hybridization and the Internal Transcribed Spacer 1 nested PCR (ITS1 nPCR) methods and four types of clinical samples for the diagnosis of CVL in dogs with and without clinical signs of the disease. The methods were compared using samples of conjunctival swab (SC), bone marrow (BM), skin (S) and peripheral blood (PB). A group of 60 mongrel dogs, all positive in serological and parasitological tests, were equally divided in two groups: S (with clinical signs) and A (without clinical signs). The frequencies of positive results for the kDNA PCR hybridization in the S group were: CS 97% (29/30), BM 83 % (25/30), S 63% (19/30) and PB 4 7% (14/30). By the same method the following results were obtained in the A group: CS 70% (21/30), BM 63% (19/30), S 57% (17/30) and PB 17% (5/30). The ITS1 nPCR allowed the following positivities for the S group: CS 83% (25/30), BM 97% (29/30), S 83% (25/30) and PB 70% (21/30). For the A group the following results were obtained: CS and BM 83

  19. Prevalence of virulence factors and antibiotic resistance in vancomycin-resistant Enterococcus faecium isolated from sewage and clinical samples in Iran

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    Jahangiri S

    2010-01-01

    Full Text Available Purpose: The purpose of the present study was to perform a molecular epidemiological survey by investigating the antibiotic resistance and the presence of known virulence factors in Enterococcus faecium isolates in Iran. The data collected from this study would allow us to control the spread and develop strategies for treatment of the enterococcal infections. Materials and Methods: In this study, 156 vancomycin-sensitive E. faecium (VSEF; 58 and vancomycin-resistant E. faecium (VREF; 98 samples were isolated from clinical specimen and sewage treatment plants (STPs. These isolates were screened for the presence of genes encoding for aggregation substance (asa1, cytolysin (cyl, enterococcal surface protein (esp, gelatinase (gelE and hyaluronidase (hyl by polymerase chain reaction (PCR. Results: Although significantly different, the results showed the presence of hyl and esp genes in both clinical (41 and 75%, respectively and sewage (3.2 and 41%, respectively isolates. Sensitivity of all isolates to seven antibiotics was examined. The results of the clinical isolates showed that the majority of esp positive isolates were also resistant to vancomycin, ciprofloxacin and erythromycin. Furthermore, cyl, gelE and asa1 were not found in either clinical or STP isolates. Finally, we determined the distinct types of isolates using Pulse Field Gel Electrophoresis (PFGE, which confirmed that most of the isolates were clonally unrelated. Conclusion: Our results demonstrated that higher number of the clinical E. faecium isolates carried virulence genes than the isolates from STP. Finally, the lack of the genes in clinical and STP isolates confirmed that these genes do not transfer horizontally.

  20. Detection of vancomycin resistance in enterococcus species isolated from clinical samples and feces of colonized patients by phenotypic and genotypic methods

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    Priyanka Paul Biswas

    2016-01-01

    Full Text Available Background: The aim of this study was to find out the clinical correlation between the presence of vancomycin-resistant genes (van A and van B and their expression as detected by phenotypic tests in colonized patients and in clinical isolates. Materials and Methods: Enterococci were isolated from various clinical samples and also from fecal specimens of colonized patients at the time of admission, after 48 h and after 5 days of admission. Identification to species level was done using standard methods. Vancomycin susceptibility in Enterococci was detected by disc diffusion test. Minimum inhibitory concentration was determined by agar dilution method. Multiplex polymerase chain reaction (PCR was used to detect the presence of van genes. Results: Out of all the clinical and fecal samples processed, 12.0% isolates were either vancomycin resistant or vancomycin intermediate. Further, these isolates carried van A or van B genes as confirmed by PCR methods. Expression of van A gene was found to be more in Enterococcus faecalis (28.3% as compared to Enterococcus faecium (25.0% in both clinical and fecal isolates. 16.6% strains of E. faecium and 15.0% strains each of E. faecalis and Enterococcus gallinarum were found to carry van B genes. The overall prevalence of vancomycin resistant Enterococci (VRE in colonized patients was about 9.6%. Prior administration of antibiotics had significant effect (P = 0.001 on VRE carriage. Urinary tract infection was the most common infection caused by vancomycin susceptible Enterococci (VSE, 105/214 (49.0% and VRE, 13/36 (36.1%. There was no significant difference (P = 0.112 in the distribution of VRE and VSE in different infection types. Both clinical and fecal VRE showed maximum resistance to penicillin, ampicillin, and piperacillin. Resistance to linezolid was 2.8% in clinically isolated VRE. Conclusion: VRE in our study were found to be resistant to a number of commonly used antibiotics. The frequency of isolation

  1. Numerical and structural genomic aberrations are reliably detectable in tissue microarrays of formalin-fixed paraffin-embedded tumor samples by fluorescence in-situ hybridization.

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    Heike Horn

    Full Text Available Few data are available regarding the reliability of fluorescence in-situ hybridization (FISH, especially for chromosomal deletions, in high-throughput settings using tissue microarrays (TMAs. We performed a comprehensive FISH study for the detection of chromosomal translocations and deletions in formalin-fixed and paraffin-embedded (FFPE tumor specimens arranged in TMA format. We analyzed 46 B-cell lymphoma (B-NHL specimens with known karyotypes for translocations of IGH-, BCL2-, BCL6- and MYC-genes. Locus-specific DNA probes were used for the detection of deletions in chromosome bands 6q21 and 9p21 in 62 follicular lymphomas (FL and six malignant mesothelioma (MM samples, respectively. To test for aberrant signals generated by truncation of nuclei following sectioning of FFPE tissue samples, cell line dilutions with 9p21-deletions were embedded into paraffin blocks. The overall TMA hybridization efficiency was 94%. FISH results regarding translocations matched karyotyping data in 93%. As for chromosomal deletions, sectioning artefacts occurred in 17% to 25% of cells, suggesting that the proportion of cells showing deletions should exceed 25% to be reliably detectable. In conclusion, FISH represents a robust tool for the detection of structural as well as numerical aberrations in FFPE tissue samples in a TMA-based high-throughput setting, when rigorous cut-off values and appropriate controls are maintained, and, of note, was superior to quantitative PCR approaches.

  2. Numerical and structural genomic aberrations are reliably detectable in tissue microarrays of formalin-fixed paraffin-embedded tumor samples by fluorescence in-situ hybridization.

    Science.gov (United States)

    Horn, Heike; Bausinger, Julia; Staiger, Annette M; Sohn, Maximilian; Schmelter, Christopher; Gruber, Kim; Kalla, Claudia; Ott, M Michaela; Rosenwald, Andreas; Ott, German

    2014-01-01

    Few data are available regarding the reliability of fluorescence in-situ hybridization (FISH), especially for chromosomal deletions, in high-throughput settings using tissue microarrays (TMAs). We performed a comprehensive FISH study for the detection of chromosomal translocations and deletions in formalin-fixed and paraffin-embedded (FFPE) tumor specimens arranged in TMA format. We analyzed 46 B-cell lymphoma (B-NHL) specimens with known karyotypes for translocations of IGH-, BCL2-, BCL6- and MYC-genes. Locus-specific DNA probes were used for the detection of deletions in chromosome bands 6q21 and 9p21 in 62 follicular lymphomas (FL) and six malignant mesothelioma (MM) samples, respectively. To test for aberrant signals generated by truncation of nuclei following sectioning of FFPE tissue samples, cell line dilutions with 9p21-deletions were embedded into paraffin blocks. The overall TMA hybridization efficiency was 94%. FISH results regarding translocations matched karyotyping data in 93%. As for chromosomal deletions, sectioning artefacts occurred in 17% to 25% of cells, suggesting that the proportion of cells showing deletions should exceed 25% to be reliably detectable. In conclusion, FISH represents a robust tool for the detection of structural as well as numerical aberrations in FFPE tissue samples in a TMA-based high-throughput setting, when rigorous cut-off values and appropriate controls are maintained, and, of note, was superior to quantitative PCR approaches.

  3. Optimization and clinical validation of a Real-Time PCR protocol for direct detection of Trichomonas vaginalis in pooled urine samples

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    WHA Zandijk

    2009-12-01

    Full Text Available Background and Objectives: A new Real- Time PCR protocol for the detection of Trichomonas vaginalis in pooled urine"nsamples has been optimized and validated."nMaterials and Methods: The amplification protocol, targeting a 2kb repeated gene in the T. vaginalis genome, was optimized"nby varying PCR parameters. As a reference method, a Real-Time PCR protocol targeting the beta-tubulin gene (Y. Versluis"net al, 2006, Int J STD AIDS 17:642 was used. Clinical validation was performed with pooled urine samples obtained from"npatients of the sexually transmitted diseases clinic of a university hospital (n=963; from February – June 2007."nResults: Positive samples with the new optimized technique is 1.1% (n=10, while the beta-tubulin real-time PCR method"ngenerated four positives (0.3%."nConclusion: The new RT- PCR protocol is a sensitive (1.000 and specific (0.993 procedure to detect and to identify T."nvaginalis in urine samples.

  4. Prediction of treatment outcome in a clinical sample of problem drinkers: self-efficacy, alcohol expectancies, and readiness to change.

    Science.gov (United States)

    Demmel, Ralf; Beck, Beate; Lammers, André

    2003-01-01

    Cognitive processes related to client motivation are important mediators of alcoholism treatment outcome. The present study aimed to expand previous research on client motivation and treatment outcome by establishing the predictive utility of self-efficacy, alcohol expectancies, and readiness to change in a sample of alcohol-dependent inpatients (N = 83). Treatment outcome was assessed three months following discharge. According to self-reported alcohol use, 22 clients were classified as abstainers and 41 clients as relapsers. Twenty participants were lost to follow-up. Readiness to change and anticipated reinforcement from alcohol predicted abstinence at follow-up. Client motivation was unrelated to both frequency and quantity of alcohol use. In accordance with social learning theory, self-efficacy was inversely correlated with alcohol expectancies. The results of the present study suggest that once abstinence has been violated factors other than pretreatment motivation determine drinking behavior.

  5. Prediction of treatment outcome in a clinical sample of problem drinkers: self-efficacy, alcohol expectancies, and readiness to change

    Directory of Open Access Journals (Sweden)

    Demmel, Ralf

    2003-10-01

    Full Text Available Cognitive processes related to client motivation are important mediators of alcoholism treatment outcome. The present study aimed to expand previous research on client motivation and treatment outcome by establishing the predictive utility of self-efficacy, alcohol expectancies, and readiness to change in a sample of alcohol-dependent inpatients (N = 83. Treatment outcome was assessed three months following discharge. According to self-reported alcohol use, 22 clients were classified as abstainers and 41 clients as relapsers. Twenty participants were lost to follow-up. Readiness to change and anticipated reinforcement from alcohol predicted abstinence at follow-up. Client motivation was unrelated to both frequency and quantity of alcohol use. In accordance with social learning theory, self-efficacy was inversely correlated with alcohol expectancies. The results of the present study suggest that once abstinence has been violated factors other than pretreatment motivation determine drinking behavior.

  6. Loneliness mediates the relationship between emotion dysregulation and bulimia nervosa/binge eating disorder psychopathology in a clinical sample.

    Science.gov (United States)

    Southward, Matthew W; Christensen, Kara A; Fettich, Karla C; Weissman, Jessica; Berona, Johnny; Chen, Eunice Y

    2014-12-01

    Emotion dysregulation has been linked to binge eating disorder (BED) and bulimia nervosa (BN) although the mechanisms by which it affects BN/BED psychopathology are unclear. This study tested loneliness as a mediator between emotion dysregulation and BN/BED psychopathology. A treatment-seeking sample of 107 women with BN or BED was assessed for loneliness (UCLA Loneliness Scale), emotion dysregulation (Difficulties in Emotion Regulation Scale), and BN/BED psychopathology (Eating Disorder Examination) before treatment. Hierarchical linear regressions and bootstrapping mediation models were run. Greater overall emotion dysregulation was associated with greater BN/BED psychopathology, mediated by loneliness (95 % CI 0.03, 0.09). Emotion dysregulation, however, did not mediate between loneliness and BN/BED psychopathology (95 % CI −0.01, 0.01). Targeting loneliness may effectively treat emotional aspects of BN/BED in women.

  7. Detection of Bartonella henselae DNA in clinical samples including peripheral blood of immune competent and immune compromised patients by three nested amplifications

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    Karina Hatamoto Kawasato

    2013-02-01

    Full Text Available Bacteria of the genus Bartonella are emerging pathogens detected in lymph node biopsies and aspirates probably caused by increased concentration of bacteria. Twenty-three samples of 18 patients with clinical, laboratory and/or epidemiological data suggesting bartonellosis were subjected to three nested amplifications targeting a fragment of the 60-kDa heat shock protein (HSP, the internal transcribed spacer 16S-23S rRNA (ITS and the cell division (FtsZ of Bartonella henselae, in order to improve detection in clinical samples. In the first amplification 01, 04 and 05 samples, were positive by HSP (4.3%, FtsZ (17.4% and ITS (21.7%, respectively. After the second round six positive samples were identified by nested-HSP (26%, eight by nested-ITS (34.8% and 18 by nested-FtsZ (78.2%, corresponding to 10 peripheral blood samples, five lymph node biopsies, two skin biopsies and one lymph node aspirate. The nested-FtsZ was more sensitive than nested-HSP and nested-ITS (p < 0.0001, enabling the detection of Bartonella henselae DNA in 15 of 18 patients (83.3%. In this study, three nested-PCR that should be specific for Bartonella henselae amplification were developed, but only the nested-FtsZ did not amplify DNA from Bartonella quintana. We conclude that nested amplifications increased detection of B. henselae DNA, and that the nested-FtsZ was the most sensitive and the only specific to B. henselae in different biological samples. As all samples detected by nested-HSP and nested-ITS, were also by nested-FtsZ, we infer that in our series infections were caused by Bartonella henselae. The high number of positive blood samples draws attention to the use of this biological material in the investigation of bartonellosis, regardless of the immune status of patients. This fact is important in the case of critically ill patients and young children to avoid more invasive procedures such as lymph nodes biopsies and aspirates.

  8. Diagnosing dementia and normal aging: clinical relevance of brain ratios and cognitive performance in a Brazilian sample

    Directory of Open Access Journals (Sweden)

    M.L.F. Chaves

    1999-09-01

    Full Text Available The main objective of the present study was to evaluate the diagnostic value (clinical application of brain measures and cognitive function. Alzheimer and multiinfarct patients (N = 30 and normal subjects over the age of 50 (N = 40 were submitted to a medical, neurological and cognitive investigation. The cognitive tests applied were Mini-Mental, word span, digit span, logical memory, spatial recognition span, Boston naming test, praxis, and calculation tests. The brain ratios calculated were the ventricle-brain, bifrontal, bicaudate, third ventricle, and suprasellar cistern measures. These data were obtained from a brain computer tomography scan, and the cutoff values from receiver operating characteristic curves. We analyzed the diagnostic parameters provided by these ratios and compared them to those obtained by cognitive evaluation. The sensitivity and specificity of cognitive tests were higher than brain measures, although dementia patients presented higher ratios, showing poorer cognitive performances than normal individuals. Normal controls over the age of 70 presented higher measures than younger groups, but similar cognitive performance. We found diffuse losses of tissue from the central nervous system related to distribution of cerebrospinal fluid in dementia patients. The likelihood of case identification by functional impairment was higher than when changes of the structure of the central nervous system were used. Cognitive evaluation still seems to be the best method to screen individuals from the community, especially for developing countries, where the cost of brain imaging precludes its use for screening and initial assessment of dementia.

  9. Detection of E. coli O157:H7 and Shigella dysenteriae toxins in clinical samples by PCR-ELISA

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    Jafar Amani

    2015-06-01

    Full Text Available Shiga toxin producing bacteria are potential causes of serious human disease such as hemorrhagic colitis, severe inflammations of ileocolonic regions of gastrointestinal tract, thrombocytopenia, septicemia, malignant disorders in urinary ducts, hemolytic uremic syndrome (HUS Shiga toxin 1 (stx1, shiga toxin 2 (stx2, or a combination of both are responsible for most clinical symptoms of these diseases. A lot of methods have been developed so far to detect shiga toxins such as cell culture, ELISA, and RFPLA, but due to high costs and labor time in addition to low sensitivity, they have not received much attention. In this study, PCR-ELISA method was used to detect genes encoding shiga toxins 1 and 2 (stx1 and stx2. To detect stx1 and stx2 genes, two primer pairs were designed for Multiplex-PCR then PCR-ELISA. PCR products (490 and 275, respectively were subsequently verified by sequencing. Sensitivity and specificity of PCR-ELISA method were determined by using genome serial dilution and Enterobacteriastrains. PCR-ELISA method used in this study proved to be a rapid and precise approach to detect different types of shiga toxins and can be used to detect bacterial genes encoding shiga toxins.

  10. Two to five repeated measurements per patient reduced the required sample size considerably in a randomized clinical trial for patients with inflammatory rheumatic diseases

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    Smedslund Geir

    2013-02-01

    Full Text Available Abstract Background Patient reported outcomes are accepted as important outcome measures in rheumatology. The fluctuating symptoms in patients with rheumatic diseases have serious implications for sample size in clinical trials. We estimated the effects of measuring the outcome 1-5 times on the sample size required in a two-armed trial. Findings In a randomized controlled trial that evaluated the effects of a mindfulness-based group intervention for patients with inflammatory arthritis (n=71, the outcome variables Numerical Rating Scales (NRS (pain, fatigue, disease activity, self-care ability, and emotional wellbeing and General Health Questionnaire (GHQ-20 were measured five times before and after the intervention. For each variable we calculated the necessary sample sizes for obtaining 80% power (α=.05 for one up to five measurements. Two, three, and four measures reduced the required sample sizes by 15%, 21%, and 24%, respectively. With three (and five measures, the required sample size per group was reduced from 56 to 39 (32 for the GHQ-20, from 71 to 60 (55 for pain, 96 to 71 (73 for fatigue, 57 to 51 (48 for disease activity, 59 to 44 (45 for self-care, and 47 to 37 (33 for emotional wellbeing. Conclusions Measuring the outcomes five times rather than once reduced the necessary sample size by an average of 27%. When planning a study, researchers should carefully compare the advantages and disadvantages of increasing sample size versus employing three to five repeated measurements in order to obtain the required statistical power.

  11. Characterization of dengue virus infections in a sample of patients suggests unique clinical, immunological, and virological profiles that impact on the diagnosis of dengue and dengue hemorrhagic fever.

    Science.gov (United States)

    Senaratne, Thamarasi; Wimalaratne, Harith; Alahakoon, D G S; Gunawardane, Nirmali; Carr, Jillian; Noordeen, Faseeha

    2016-10-01

    Dengue virus (DENV) infections are increasing with respect to incidence and severity in the Central Province of Sri Lanka. The objective of this study was to define the clinical, immunological, and virological profiles of patients admitted to the General Hospital, Kandy with clinically apparent dengue. Demographic, clinical, hematological parameters, liver enzymes (ALT and AST), and blood samples were collected from 292 patients with fever dengue fever/dengue hemorrhagic fever (DF/DHF). Samples were analyzed for, anti-DENV IgM, IgG, and DENV nucleic acid. Myalgia was the commonest complaint by 65% of the patients. Packed cell volume was >45% in 27% of the patients while 42.12% had reduced platelets and 62.67% had reduced white blood cell counts. In contrast to other studies, positive tourniquet test (PTT) and petechiae were not major indicators of DENV infection or severity of the disease. Clinical profiles were significantly different between DF and DHF/DSS and showed many similarities to that reported elsewhere. Altogether, 43 patients (14.73%) were viremic as detected by RT-PCR; 181 patients (62%) were positive for anti-DENV IgM, and 245 (84%) patients were positive for anti-DENV IgG. In combination, anti-DENV IgM and RT-PCR assays detected 224 (77.5%) of DENV infected cases, thus improving the DENV diagnosis rate. Hence, the diagnostic utility of PTT, anti-DENV IgM/IgG serology, or RT-PCR used alone in the early phase of illness is low in Sri Lanka but the diagnostic value can be improved by a combination of serology and RT-PCR. J. Med. Virol. 88:1703-1710, 2016. © 2016 Wiley Periodicals, Inc. PMID:26990973

  12. Analytical investigations of toxic p-phenylenediamine (PPD) levels in clinical urine samples with special focus on MALDI-MS/MS.

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    Hooff, Gero P; van Huizen, Nick A; Meesters, Roland J W; Zijlstra, Eduard E; Abdelraheem, Mohamed; Abdelraheem, Waleed; Hamdouk, Mohamed; Lindemans, Jan; Luider, Theo M

    2011-01-01

    Para-phenylenediamine (PPD) is a common chromophoric ingredient in oxidative hair-dyes. In some African countries like Sudan, Egypt and Morocco but also in India this chemical is used alone or in combination with colouring extracts like Henna for dyeing of the hair or the skin. Excessive dermal exposure to PPD mainly leads to the N-mono- and N,N'-diacetylated products (MAPPD, DAPPD) by N-acetyltransferase 1 and 2 (NAT1 and 2) catalyzed reactions. Metabolites and PPD are mainly excreted via renal clearance. Despite a low risk of intoxication when used in due form, there are numerous cases of acute intoxication in those countries every year. At the ENT Hospital - Khartoum (Sudan) alone more than 300 cases are reported every year (~10% fatal), mostly caused by either an accidental or intended (suicidal) high systemic exposure to pure PPD. Intoxication leads to a severe clinical syndrome including laryngeal edema, rhabdomyolysis and subsequent renal failure, neurotoxicity and acute toxic hepatitis. To date, there is no defined clinical treatment or antidote available and treatment is largely supportive. Herein, we show the development of a quick on-site identification assay to facilitate differential diagnosis in the clinic and, more importantly, the implementation of an advanced analytical platform for future in-depth investigations of PPD intoxication and metabolism is described. The current work shows a sensitive (~25 µM) wet chemistry assay, a validated MALDI-MS/MS and HPLC-UV assay for the determination of PPD and its metabolites in human urine. We show the feasibility of the methods for measuring PPD over a range of 50-1000 µM. The validation criteria included linearity, lower limit of quantification (LLOQ), accuracy and precision, recovery and stability. Finally, PPD concentrations were determined in clinical urine samples of cases of acute intoxication and the applied technique was expanded to identify MAPPD and DAPPD in the identical samples.

  13. Analytical investigations of toxic p-phenylenediamine (PPD levels in clinical urine samples with special focus on MALDI-MS/MS.

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    Gero P Hooff

    Full Text Available Para-phenylenediamine (PPD is a common chromophoric ingredient in oxidative hair-dyes. In some African countries like Sudan, Egypt and Morocco but also in India this chemical is used alone or in combination with colouring extracts like Henna for dyeing of the hair or the skin. Excessive dermal exposure to PPD mainly leads to the N-mono- and N,N'-diacetylated products (MAPPD, DAPPD by N-acetyltransferase 1 and 2 (NAT1 and 2 catalyzed reactions. Metabolites and PPD are mainly excreted via renal clearance. Despite a low risk of intoxication when used in due form, there are numerous cases of acute intoxication in those countries every year. At the ENT Hospital - Khartoum (Sudan alone more than 300 cases are reported every year (~10% fatal, mostly caused by either an accidental or intended (suicidal high systemic exposure to pure PPD. Intoxication leads to a severe clinical syndrome including laryngeal edema, rhabdomyolysis and subsequent renal failure, neurotoxicity and acute toxic hepatitis. To date, there is no defined clinical treatment or antidote available and treatment is largely supportive. Herein, we show the development of a quick on-site identification assay to facilitate differential diagnosis in the clinic and, more importantly, the implementation of an advanced analytical platform for future in-depth investigations of PPD intoxication and metabolism is described. The current work shows a sensitive (~25 µM wet chemistry assay, a validated MALDI-MS/MS and HPLC-UV assay for the determination of PPD and its metabolites in human urine. We show the feasibility of the methods for measuring PPD over a range of 50-1000 µM. The validation criteria included linearity, lower limit of quantification (LLOQ, accuracy and precision, recovery and stability. Finally, PPD concentrations were determined in clinical urine samples of cases of acute intoxication and the applied technique was expanded to identify MAPPD and DAPPD in the identical samples.

  14. Hemólise produzida por Candida tropicalis isoladas de amostras clínicas Hemolysis produced by Candida tropicalis isolates from clinical samples

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    Emanuele Júlio Galvão de França

    2010-06-01

    Full Text Available INTRODUÇÃO: Leveduras do gênero Candida são responsáveis pela maioria das infecções fúngicas em humanos. Candida tropicalis tem sido uma das mais comumente isoladas dentre as espécies não-albicans. O objetivo foi analisar a hemólise in vitro promovida por isolados clínicos de C. tropicalis provenientes de sangue e outras amostras clínicas de pacientes internados no Hospital Universitário da UEL, PR-Brasil. MÉTODOS: Foi avaliada a hemólise promovida por 28 isolados clínicos de C. tropicalis, sendo os isolados agrupados em classes de acordo com os níveis de hemólise. RESULTADOS: A maioria dos isolados de sangue apresentou hemólise fraca (+, enquanto as classes de hemólise forte (+++ e muito forte (++++ foram as predominantes nos isolados de outras amostras clínicas como urina, lesão de unha e secreção traqueal, embora não tenham sido detectadas diferenças estatísticas (p>0,05. CONCLUSÕES: Isolados de C. tropicalis, obtidos de diferentes amostras clínicas, apresentam capacidade de promover hemólise in vitro.INTRODUCTION: Yeasts belonging to the genus Candida are responsible for the majority of fungal infections in humans. Candida tropicalis has been one of most commonly isolated non-albicans species. To analyze in vitro hemolysis promoted by clinical isolates of C. tropicalis obtained from blood and other clinical samples from hospitalized patients at the University Hospital of Londrina State University, Paraná, Brazil. METHODS: The hemolysis promoted by 28 clinical isolates of C. tropicalis was evaluated, and the isolates were grouped into classes according to the hemolysis levels. RESULTS: The majority of the blood isolates showed weak hemolysis (+, while the classes of strong hemolysis (+++ and very strong hemolysis (++++ predominated among isolates from other clinical samples such as urine, nail lesions and tracheal secretions. However, no statistical differences were detected (p> 0.05. CONCLUSIONS: Isolates of C

  15. Molecular characterization of vancomycin-resistant Enterococcus faecium strains isolated from carriage and clinical samples in a tertiary hospital, Turkey.

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    Gozalan, Aysegul; Coskun-Ari, Fatma Filiz; Ozdem, Birsen; Unaldi, Ozlem; Celikbilek, Nevreste; Kirca, Fisun; Aydogan, Sibel; Muderris, Tuba; Guven, Tumer; Acikgoz, Ziya Cibali; Durmaz, Riza

    2015-07-01

    This study aimed to determine the presence of vancomycin resistance (vanA and vanB) and virulence genes (esp, asa1, gelE, ace, hyl, cylA, cpd and ebpA) in vancomycin-resistant Enterococcus faecium (VREfm) strains and to analyse the clonal relationships among the strains. E. faecium strains were identified from rectal and clinical specimens by biochemical tests and the API-20 Strep kit. Susceptibility testing was performed using disc-diffusion and broth-dilution methods. PFGE was used for molecular typing of the VREfm strains. The vancomycin resistance and virulence genes were amplified by two-step multiplex PCR. All 55 VREfm isolates were resistant to penicillin G, ampicillin and high-level gentamicin but were susceptible to quinupristin/dalfopristin and linezolid. Multiplex PCR analysis indicated that all isolates harboured vanA and that 41 (75 %) were positive for virulence genes. The esp gene was the most common virulence factor and was detected in nine (41 %) invasive and 32 (96.7 %) non-invasive isolates. Multiple virulence genes were observed only in two non-invasive isolates; one harboured esp and ebpA and the other harboured esp, ebpA, asa1, gelE and cpd. PFGE typing yielded 16 different types, seven of which were clusters with two to 14 strains each. The clustering rates of the rectal swab, blood and urine isolates were 72.7 %, 61.5 % and 87.5 %, respectively. The genetic similarity observed among the VREfm isolates indicated cross-transmission in the hospital. Further studies on the virulence factors present in the strains might provide insight into the acquisition of these traits and their contribution to increased prevalence of VREfm.

  16. Molecular characterization of vancomycin-resistant Enterococcus faecium strains isolated from carriage and clinical samples in a tertiary hospital, Turkey.

    Science.gov (United States)

    Gozalan, Aysegul; Coskun-Ari, Fatma Filiz; Ozdem, Birsen; Unaldi, Ozlem; Celikbilek, Nevreste; Kirca, Fisun; Aydogan, Sibel; Muderris, Tuba; Guven, Tumer; Acikgoz, Ziya Cibali; Durmaz, Riza

    2015-07-01

    This study aimed to determine the presence of vancomycin resistance (vanA and vanB) and virulence genes (esp, asa1, gelE, ace, hyl, cylA, cpd and ebpA) in vancomycin-resistant Enterococcus faecium (VREfm) strains and to analyse the clonal relationships among the strains. E. faecium strains were identified from rectal and clinical specimens by biochemical tests and the API-20 Strep kit. Susceptibility testing was performed using disc-diffusion and broth-dilution methods. PFGE was used for molecular typing of the VREfm strains. The vancomycin resistance and virulence genes were amplified by two-step multiplex PCR. All 55 VREfm isolates were resistant to penicillin G, ampicillin and high-level gentamicin but were susceptible to quinupristin/dalfopristin and linezolid. Multiplex PCR analysis indicated that all isolates harboured vanA and that 41 (75 %) were positive for virulence genes. The esp gene was the most common virulence factor and was detected in nine (41 %) invasive and 32 (96.7 %) non-invasive isolates. Multiple virulence genes were observed only in two non-invasive isolates; one harboured esp and ebpA and the other harboured esp, ebpA, asa1, gelE and cpd. PFGE typing yielded 16 different types, seven of which were clusters with two to 14 strains each. The clustering rates of the rectal swab, blood and urine isolates were 72.7 %, 61.5 % and 87.5 %, respectively. The genetic similarity observed among the VREfm isolates indicated cross-transmission in the hospital. Further studies on the virulence factors present in the strains might provide insight into the acquisition of these traits and their contribution to increased prevalence of VREfm. PMID:25976005

  17. The Social Appearance Anxiety Scale in Italian adolescent populations: Construct validation and group discrimination in community and clinical eating disorders samples

    OpenAIRE

    Dakanalis, Antonios; Carrà, Giuseppe; Calogero, Rachel M.; Zanetti, M. Assunta; Volpato, Chiara; Riva, Giuseppe; Clerici, Massimo; Cipresso, Pietro

    2015-01-01

    Anxiety in situations where one’s overall appearance (including body shape) may be negatively evaluated is hypothesized to play a central role in Eating Disorders (EDs) and in their co-occurrence with Social Anxiety Disorder (SAD). Three studies were conducted among community (N = 1995) and clinical (N = 703) ED samples of 11- to 18-year-old Italian girls and boys to (a) evaluate the psychometric qualities and measurement equivalence/invariance (ME/I) of the Social Appearance Anxiety (SAA) Sc...

  18. The self-report Version of the LSAS-CA: Psychometric Properties of the French Version in a non-clinical adolescent sample

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    Emilie Schmits

    2014-02-01

    Full Text Available The Liebowitz Social Anxiety Scale (LSAS is one of the most popular measures of social anxiety in adults. The LSAS has been adapted for clinical assessment of children and adolescents (LSAS-CA. The psychometric properties of the self-report version of the LSAS-CA (LSAS-CA-SR have been investigated in a Spanish population. However, no study to date has adapted and validated this scale in French. The purpose of this study was to develop a French version of the LSAS-CA-SR and to assess its score reliability and structural validity in a French-speaking community sample. The sample was made up of 1,343 teenagers from secondary schools, aged between 14 and 18 years. Confirmatory factor analyses established the structural validity of the French version of the LSAS-CA-SR and good psychometric properties, including reliable internal consistency, were observed.

  19. Expression profiling of blood samples from an SU5416 Phase III metastatic colorectal cancer clinical trial: a novel strategy for biomarker identification

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    Smolich Beverly D

    2003-02-01

    Full Text Available Abstract Background Microarray-based gene expression profiling is a powerful approach for the identification of molecular biomarkers of disease, particularly in human cancers. Utility of this approach to measure responses to therapy is less well established, in part due to challenges in obtaining serial biopsies. Identification of suitable surrogate tissues will help minimize limitations imposed by those challenges. This study describes an approach used to identify gene expression changes that might serve as surrogate biomarkers of drug activity. Methods Expression profiling using microarrays was applied to peripheral blood mononuclear cell (PBMC samples obtained from patients with advanced colorectal cancer participating in a Phase III clinical trial. The PBMC samples were harvested pre-treatment and at the end of the first 6-week cycle from patients receiving standard of care chemotherapy or standard of care plus SU5416, a vascular endothelial growth factor (VEGF receptor tyrosine kinase (RTK inhibitor. Results from matched pairs of PBMC samples from 23 patients were queried for expression changes that consistently correlated with SU5416 administration. Results Thirteen transcripts met this selection criterion; six were further tested by quantitative RT-PCR analysis of 62 additional samples from this trial and a second SU5416 Phase III trial of similar design. This method confirmed four of these transcripts (CD24, lactoferrin, lipocalin 2, and MMP-9 as potential biomarkers of drug treatment. Discriminant analysis showed that expression profiles of these 4 transcripts could be used to classify patients by treatment arm in a predictive fashion. Conclusions These results establish a foundation for the further exploration of peripheral blood cells as a surrogate system for biomarker analyses in clinical oncology studies.

  20. Rapid Drug Susceptibility Testing of Drug-Resistant Mycobacterium tuberculosis Isolates Directly from Clinical Samples by Use of Amplicon Sequencing: a Proof-of-Concept Study

    Science.gov (United States)

    Anderson, Julia; Lemmer, Darrin; Lehmkuhl, Erik; Georghiou, Sophia B.; Heaton, Hannah; Wiggins, Kristin; Gillece, John D.; Schupp, James M.; Catanzaro, Donald G.; Crudu, Valeriu; Cohen, Ted; Rodwell, Timothy C.; Engelthaler, David M.

    2016-01-01

    Increasingly complex drug-resistant tuberculosis (DR-TB) is a major global health concern and one of the primary reasons why TB is now the leading infectious cause of death worldwide. Rapid characterization of a DR-TB patient's complete drug resistance profile would facilitate individualized treatment in place of empirical treatment, improve treatment outcomes, prevent amplification of resistance, and reduce the transmission of DR-TB. The use of targeted next-generation sequencing (NGS) to obtain drug resistance profiles directly from patient sputum samples has the potential to enable comprehensive evidence-based treatment plans to be implemented quickly, rather than in weeks to months, which is currently needed for phenotypic drug susceptibility testing (DST) results. In this pilot study, we evaluated the performance of amplicon sequencing of Mycobacterium tuberculosis DNA from patient sputum samples using a tabletop NGS technology and automated data analysis to provide a rapid DST solution (the Next Gen-RDST assay). One hundred sixty-six out of 176 (94.3%) sputum samples from the Republic of Moldova yielded complete Next Gen-RDST assay profiles for 7 drugs of interest. We found a high level of concordance of our Next Gen-RDST assay results with phenotypic DST (97.0%) and pyrosequencing (97.8%) results from the same clinical samples. Our Next Gen-RDST assay was also able to estimate the proportion of resistant-to-wild-type alleles down to mixtures of ≤1%, which demonstrates the ability to detect very low levels of resistant variants not detected by pyrosequencing and possibly below the threshold for phenotypic growth methods. The assay as described here could be used as a clinical or surveillance tool. PMID:27225403

  1. Rapid Drug Susceptibility Testing of Drug-Resistant Mycobacterium tuberculosis Isolates Directly from Clinical Samples by Use of Amplicon Sequencing: a Proof-of-Concept Study.

    Science.gov (United States)

    Colman, Rebecca E; Anderson, Julia; Lemmer, Darrin; Lehmkuhl, Erik; Georghiou, Sophia B; Heaton, Hannah; Wiggins, Kristin; Gillece, John D; Schupp, James M; Catanzaro, Donald G; Crudu, Valeriu; Cohen, Ted; Rodwell, Timothy C; Engelthaler, David M

    2016-08-01

    Increasingly complex drug-resistant tuberculosis (DR-TB) is a major global health concern and one of the primary reasons why TB is now the leading infectious cause of death worldwide. Rapid characterization of a DR-TB patient's complete drug resistance profile would facilitate individualized treatment in place of empirical treatment, improve treatment outcomes, prevent amplification of resistance, and reduce the transmission of DR-TB. The use of targeted next-generation sequencing (NGS) to obtain drug resistance profiles directly from patient sputum samples has the potential to enable comprehensive evidence-based treatment plans to be implemented quickly, rather than in weeks to months, which is currently needed for phenotypic drug susceptibility testing (DST) results. In this pilot study, we evaluated the performance of amplicon sequencing of Mycobacterium tuberculosis DNA from patient sputum samples using a tabletop NGS technology and automated data analysis to provide a rapid DST solution (the Next Gen-RDST assay). One hundred sixty-six out of 176 (94.3%) sputum samples from the Republic of Moldova yielded complete Next Gen-RDST assay profiles for 7 drugs of interest. We found a high level of concordance of our Next Gen-RDST assay results with phenotypic DST (97.0%) and pyrosequencing (97.8%) results from the same clinical samples. Our Next Gen-RDST assay was also able to estimate the proportion of resistant-to-wild-type alleles down to mixtures of ≤1%, which demonstrates the ability to detect very low levels of resistant variants not detected by pyrosequencing and possibly below the threshold for phenotypic growth methods. The assay as described here could be used as a clinical or surveillance tool. PMID:27225403

  2. Molecular identification and antifungal susceptibility profiles of Candida parapsilosis complex species isolated from culture collection of clinical samples

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    Fábio Silvestre Ataides

    2015-08-01

    Full Text Available AbstractINTRODUCTION:Candida parapsilosis is a common yeast species found in cases of onychomycosis and candidemia associated with infected intravascular devices. In this study, we differentiated Candida parapsilosis sensu stricto, Candida orthopsilosis , and Candida metapsilosis from a culture collection containing blood and subungual scraping samples. Furthermore, we assessed the in vitro antifungal susceptibility of these species to fluconazole, itraconazole, voriconazole, posaconazole, amphotericin B, and caspofungin.METHODS:Differentiation of C. parapsilosis complex species was performed by amplification of the secondary alcohol dehydrogenase (SADH gene and digestion by the restriction enzyme Ban I. All isolates were evaluated for the determination of minimal inhibitory concentrations using Etest, a method for antifungal susceptibility testing.RESULTS:Among the 87 isolates, 78 (89.7% were identified as C. parapsilosis sensu stricto , five (5.7% were identified as C. orthopsilosis , and four (4.6% were identified as C. metapsilosis . Analysis of antifungal susceptibility showed that C. parapsilosis sensu strictoisolates were less susceptible to amphotericin B and itraconazole. One C. parapsilosis sensu stricto isolate was resistant to amphotericin B and itraconazole. Moreover, 10.2% of C. parapsilosis sensu stricto isolates were resistant to caspofungin. Two C. parapsilosis sensu strictoisolates and one C. metapsilosis isolate were susceptible to fluconazole in a dose-dependent manner.CONCLUSIONS:We reported the first molecular identification of C. parapsilosiscomplex species in State of Goiás, Brazil. Additionally, we showed that although the three species exhibited differences in antifungal susceptibility profiles, the primary susceptibility of this species was to caspofungin.

  3. SELDI-TOF-MS determination of hepcidin in clinical samples using stable isotope labelled hepcidin as an internal standard

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    Johnson Philip J

    2008-10-01

    Full Text Available Abstract Background Hepcidin is a 25-residue peptide hormone crucial to iron homeostasis. It is essential to measure the concentration of hepcidin in cells, tissues and body fluids to understand its mechanisms and roles in physiology and pathophysiology. With a mass of 2791 Da hepcidin is readily detectable by mass spectrometry and LC-ESI, MALDI and SELDI have been used to estimate systemic hepcidin concentrations by analysing serum or urine. However, peak heights in mass spectra may not always reflect concentrations in samples due to competition during binding steps and variations in ionisation efficiency. Thus the purpose of this study was to develop a robust assay for measuring hepcidin using a stable isotope labelled hepcidin spiking approach in conjunction with SELDI-TOF-MS. Results We synthesised and re-folded hepcidin labelled with 13C/15N phenylalanine at position 9 to generate an internal standard for mass spectrometry experiments. This labelled hepcidin is 10 Daltons heavier than the endogenous peptides and does not overlap with the isotopic envelope of the endogenous hepcidin or other common peaks in human serum or urine mass spectra and can be distinguished in low resolution mass spectrometers. We report the validation of adding labelled hepcidin into serum followed by SELDI analysis to generate an improved assay for hepcidin. Conclusion We demonstrate that without utilising a spiking approach the hepcidin peak height in SELDI spectra gives a good indication of hepcidin concentration. However, a stable isotope labelled hepcidin spiking approach provides a more robust assay, measures the absolute concentration of hepcidin and should facilitate inter-laboratory hepcidin comparisons.

  4. First report of mecC MRSA in human samples from Austria: molecular characteristics and clinical data

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    H. Kerschner

    2015-01-01

    Full Text Available Reports of mecC methicillin-resistant Staphylococcus aureus (MRSA strains have been published from several European countries. We describe the first six mecC MRSA isolates of human origin from Austria and report the application of a rapid PCR test. Candidate isolates (n = 295 received between 2009 and 2013 were investigated phenotypically by cefoxitin screening and streaking on ChromID MRSA plates. The presence of mecC was confirmed in six isolates from blood cultures, wound swabs and screening samples of four female and two male patients (age range 7–89 years by an in-house PCR method and the new Genspeed MRSA test (Greiner Bio-One, Kremsmünster, Austria. The mecC MRSA were further characterized by whole genome sequencing, multilocus sequence and spa typing. Antimicrobial susceptibility testing was performed by Eucast disk-diffusion method and Vitek 2. The six mecC MRSA isolates were from two clonal lineages (CC130, including a new single-locus variant, and CC599 and four different spa types (t843, t1535, t3256, t5930. Analysis for virulence factor genes yielded lukED, eta, etd2 and edin-B (CC130 isolates and tst, lukED, eta and sel (ST599 isolates. The Genspeed MRSA test identified mecC in all isolates whereas Vitek 2 failed to detect methicillin resistance in one isolate. The strains were susceptible to a wide range of non-β-lactam antibiotics. All patients were successfully treated or decolonized. mecC MRSA are present in Austria as colonizers but may also cause infections. Thus, laboratories must choose appropriate test methods such as cefoxitin screening and confirmation using molecular assays specifically targeting mecC.

  5. Associations between parenting behavior and anxiety in a rodent model and a clinical sample: relationship to peripheral BDNF levels.

    Science.gov (United States)

    Dalle Molle, R; Portella, A K; Goldani, M Z; Kapczinski, F P; Leistner-Segal, S; Leistner-Segala, S; Salum, G A; Manfro, G G; Silveira, P P

    2012-01-01

    Adverse early-life environment is associated with anxiety-like behaviors and disorders. Brain-derived neurotrophic factor (BDNF) is sensitive to this environment and could be a marker of underlying brain changes. We aimed at evaluating the development of anxiety-like behaviors in a rat model of early adversity, as well as the possible association with BDNF levels. Similar associations were investigated in a sample of adolescent humans. For the rat study, Wistar rat litters were divided into: early-life stress (ELS, limited access to nesting material) and control groups. Maternal behavior was observed from days 1 to 9 of life and, as adults, rats were subjected to behavioral testing and BDNF measurements in plasma, hippocampus, amygdala and periaqueductal gray. For the human study, 129 adolescents were evaluated for anxiety symptoms and perceived parental care. Serum BDNF levels and the Val66Met polymorphism of the BDNF gene were investigated. We found that ELS dams showed more pure contact, that is, contact with low care and high control, toward pups, and their adult offspring demonstrated higher anxiety-like behaviors and plasma BDNF. Also the pure contact correlated positively with adult peripheral BDNF. Similarly in humans, there was a positive correlation between maternal overprotection and serum BDNF only in Met carriers. We also found negative correlations between maternal warmth and separation anxiety, social phobia and school phobia. Finally, our translational approach revealed that ELS, mediated through variations in maternal care, is associated with anxiety in both rats and humans and increased peripheral BDNF may be marking these phenomena.

  6. Optimization and Isolation of Dermatophytes from Clinical Samples and In Vitro Antifungal Susceptibility Testing By Disc Diffusion Method

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    Amodkumar Yadav

    2013-06-01

    Full Text Available India is a large subcontinent with remarkably varied topography, situated within the tropical and subtropical belts of the world. In the study patients with Tinea infections were examined clinically by dermatologist. Isolation, confirmatory test were done as per the standard procedure, and Antifungal Susceptibility test was done by Disc diffusion method.Other conditions such as seborrheic dermatitis, psoriasis, alopecia areata, folliculitis and pseudopelade may mimic ringworm of head and other tinea must be identified. A total of sixty six patients of dermatophytosis were studied. Males were predominantly affected 51 (77% cases as compared to females15 (23% cases. Male to female ratio was 3.4:1. Most common age group affected was 21-30 years with 20 cases (23%. Least common age group affected was 61-70 years with 3 cases (4%.Tinea corporis was more common in the age group 21-30 years with 13 cases (37.14% and in males with 29 cases (82.85% than females with 6 cases (17.15%.Tinea unguium was more common in the age group of 31-40 years with 6 cases (37.5% and in males with 10 cases (62.5% than females with 6 cases (37.5%.Tinea cruris was more common in the age group 51-60 years with 2 cases (40% and was more common in males with 5 cases (100%.In tinea pedis, one case was seen in the age group of 11-20 years and the other in the age group of 41-50 and 51-60 years, and was more common in males with 3 cases (100%.Tinea barbae was more common in the age group 21-30 years with 2 cases (66.66% .Tinea capitis was more common in the age group of 31-40 years with 2 cases (66.66% and was more common in females with 3 cases (100%. Tinea manuum was more common in the age group of 31-40 years and in males with 1 case (100%. In males, commonest infection was T. corporis while in female commonest infection was T.corporis.rate of direct microscopy and culture (78.79%. About 89.47% of the dermatophytes grew faster in DTM with compare to SDA, so the growth rate of

  7. Isolation and speciation of Enterococci from various clinical samples and their antimicrobial susceptibility pattern with special reference to high level Aminoglycoside resistance

    Directory of Open Access Journals (Sweden)

    :Saroj Golia, Nirmala AR, Asha S Kamath B

    2014-07-01

    Full Text Available Background and Objectives: Enterococci are important nosocomial agents and strains resistant to penicillin and other antibiotics occur frequently. Enterococci are intrinsically resistant to cephalosporins and offer low level resistance to aminoglycosides. In penicillin sensitive strains, synergism occurs with combination treatment with penicillin and aminoglycoside. Serious infections caused by them are treated with penicillin and aminoglycoside combination. But the synergistic effect is lost, when the strain develops high level aminoglycoside resistance. The choice of drug for infections due to such strains is vancomycin. The present study was carried out to isolate and speciate Enterococci from various clinical samples, to know the susceptibility pattern of the isolates, to determine the High Level Aminoglycoside Resistance (HLAR among Enterococcal isolates. Methods: A total of One hundred Enterococcal species isolated from various clinical samples were identified by various biochemical reactions. Antimicrobial susceptibility testing and HLAR were determined by Kirby- Bauer disc diffusion method. Results: Out of 100 Enterococcal isolates, 59 were E. faecalis, 38 were E. faecium, 3 were other Enterococcal species. Among these 53 isolates showed High Level Aminoglycoside Resistance. Conclusion: Present study shows the presence of drug resistance to most of commonly used antibiotics and HLAR is also more in E.faecium compared to E.fecalis.

  8. The expression of small regulatory RNAs in clinical samples reflects the different life styles of Staphylococcus aureus in colonization vs. infection.

    Directory of Open Access Journals (Sweden)

    Juan Song

    Full Text Available Small RNAs (sRNAs are involved in the post-transcriptional regulation of metabolic pathways and in responses to stress and virulence. We analyzed the expression levels of five sRNAs of Staphylococcus aureus during human colonization or infection. Total RNA was isolated from nasal carriers, abscesses and cystic fibrosis patients (20 subjects per condition. The expression levels of the sRNAs were measured in the clinical samples and compared with those of the corresponding strains grown in vitro. Five sRNAs were encoded and expressed in all clinical strains in vitro. In vivo, the global expression of the five sRNAs was extremely variable in the abscessed patients, more homogeneous in the cystic fibrosis patients, and highly uniform in the nasal carrier samples. The expression levels of the sRNAs in vivo resembled those obtained at exponential phase or late exponential phase of growth in vitro, for three and one sRNA respectively; while for one sRNA, the expression was always higher in vivo as compared to in vitro growth. The in vitro conditions do not uniformly mimic the in vivo conditions for sRNA expression. Nasal colonization is associated with a unique expression pattern of sRNA that might reflect the commensalism of S. aureus in this niche.

  9. Convergent and divergent validity of K-SADS-PL anxiety and attention deficit hyperactivity disorder diagnoses in a clinical sample of school-aged children.

    Science.gov (United States)

    Villabø, Marianne A; Oerbeck, Beate; Skirbekk, Benedicte; Hansen, Berit Hjelde; Kristensen, Hanne

    2016-07-01

    Background The Schedule for Affective Disorders and Schizophrenia for School Age Children, Present and Lifetime Version (K-SADS-PL) is a commonly used diagnostic interview both in research and clinical settings, yet published data on the psychometric properties of the interview generated diagnoses are scarce. Aims To examine the convergent and divergent validity of the Norwegian version of the K-SADS-PL current diagnoses of anxiety disorders and attention deficit hyperactivity disorder (ADHD). Method Participants were 105 children aged 7-13 years referred for treatment at child mental health clinics and 36 controls. Diagnostic status was determined based on K-SADS-PL interviews with the mothers. Child and mother reported child symptoms of anxiety on the Multidimensional Anxiety Scale for Children and teachers reported anxiety symptoms on the Teacher Report Form. Mother and teacher reported on symptoms of ADHD on the Disruptive Behavior Rating Scale. Results Rating scale data from multiple informants in a clinical sample and healthy controls supported the convergent and divergent validity of K-SADS-PL anxiety diagnoses combined, and, specifically, the diagnoses of separation anxiety disorder, social phobia, and specific phobia. Support was also observed for convergent and divergent validity of ADHD diagnoses, including the predominately inattentive subtype. Conclusion The K-SADS-PL generates valid diagnoses of anxiety disorders and ADHD. PMID:26836986

  10. Detection of KPC Carbapenemase in Pseudomonas aeruginosa Isolated From Clinical Samples Using Modified Hodge Test and Boronic Acid Phenotypic Methods and Their Comparison With the Polymerase Chain Reaction

    Science.gov (United States)

    Falahat, Saeed; Shojapour, Mana; Sadeghi, Abdorrahim

    2016-01-01

    Background Bacterial resistance to antibiotics has become a major source of concern for public health. Pseudomonas aeruginosa strains are important opportunistic pathogens. These bacteria have a high resistance to a wide range of existing antimicrobials and antibiotics. Objectives The present study was performed to evaluate the frequency of KPC in P. aeruginosa isolated from clinical samples of educational hospitals of Arak University of Medical Sciences, using the mentioned phenotypic and genotypic methods. Materials and Methods One hundred and eight non-duplicate clinical isolates of P. aeruginosa were collected from hospitals of Arak University of Medical Sciences, Arak, Iran. Antibacterial susceptibility was determined by the disk diffusion method. KPC production was confirmed by the Modified Hodge Test (MHT), which is a phenotypic test, and combined-disk test with boronic acid and the Polymerase Chain Reaction (PCR). Results In the present study, 13 isolates (12%) of P. aeruginosa were positive for KPC, using PCR. Comparison of the two phenotypic methods used in this study showed that boronic acid is more sensitive than MHT in identification of KPC-producing strains (84.6% vs. 77%). Conclusions Utilization of reliable methods for identifying carbapenemase-producing strains and determining their antibiotic resistance pattern could have a very important role in treatment of infections caused by these strains. A substantial amount of P. aeruginosa isolated from clinical samples of hospitals in Arak (Iran) produce KPC carbapenemase. Due to their low specificity, MHT and boronic acid phenotypic methods could not completely identify KPC-producing P. aeruginosa. However, the sensitivity of boronic acid phenotypic method in detection of KPC was higher than MHT.

  11. Infection frequency of Epstein-Barr virus in subgingival samples from patients with different periodontal status and its correlation with clinical parameters

    Institute of Scientific and Technical Information of China (English)

    WU Yan-min; YAN Jie; CHEN Li-li; SUN Wei-lian; GU Zhi-yuan

    2006-01-01

    Objective: To detect the infection frequencies of different genotypes of Epstein-Barr virus (EBV) in subgingival samples from chronic periodontitis (CP) patients, and to discuss the correlation between infection with EBV and clinical parameters. Methods: Nested-PCR assay was used to detect EBV-1 and EBV-2 in subgingival samples from 65 CP patients, 65ivitis patients and 24 periodontally healthy individuals. The amplicons were further identified by restriction fragment length polymorphism analysis (RFLP) with endonucleases Afa I and Stu I. Clinical parameters mainly included bleeding on probing riodontally healthy individuals, the infection frequencies were 47.7% , 24.6% and 16.7% for EBV-1, and 15.4% , 7.7% and 0% for EBV-2,ectively. In 2 out of the 65 CP patients co-infection of EBV-1 and EBV-2 was found. The positive rate of EBV-1 in chronic periodontitis patients was higher than that in gingivitis patients (P=0.01) and periodontally healthy individuals (P=-0.01). But no .05) or in EBV-2frequency among the three groups (P>0.05). In CP patients, higher mean BOP value was found in EBV-1 or EBV-2 positive egative ones (P<0.01), but with no statistical difference in the mean PD or AL value between EBV positive and negative patients (P>0.05). After initial periodontal treatment, 12 out of the 21 EBV-1 positive CP patients did not s a sensitive, specific and stable method to detect EBV-1 and EBV-2 in subgingival samples. Subgingival infection with EBV-1 is closely associated with chronic correlated with BOP.

  12. Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

    Science.gov (United States)

    Cura, Carolina I.; Duffy, Tomas; Lucero, Raúl H.; Bisio, Margarita; Péneau, Julie; Jimenez-Coello, Matilde; Calabuig, Eva; Gimenez, María J.; Valencia Ayala, Edward; Kjos, Sonia A.; Santalla, José; Mahaney, Susan M.; Cayo, Nelly M.; Nagel, Claudia; Barcán, Laura; Málaga Machaca, Edith S.; Acosta Viana, Karla Y.; Brutus, Laurent; Ocampo, Susana B.; Aznar, Christine; Cuba Cuba, Cesar A.; Gürtler, Ricardo E.; Ramsey, Janine M.; Ribeiro, Isabela; VandeBerg, John L.; Yadon, Zaida E.; Osuna, Antonio; Schijman, Alejandro G.

    2015-01-01

    Background Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). Methods/Principal Findings The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. Conclusions/Significance Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production. PMID:25993316

  13. The Trauma of Commercial Sexual Exploitation of Youth: A Comparison of CSE Victims to Sexual Abuse Victims in a Clinical Sample.

    Science.gov (United States)

    Cole, Jennifer; Sprang, Ginny; Lee, Robert; Cohen, Judith

    2016-01-01

    This study examined the demographic features, trauma profiles, clinical severity indicators, problem behaviors, and service utilization characteristics of youth victims of commercial sexual exploitation (CSE) compared with a matched sample of sexually abused/assaulted youth who were not exploited in commercial sex. Secondary data analysis and propensity score matching were used to select a sample of 215 help-seeking youth who were exploited in prostitution (n = 43) or who were sexually abused/assaulted but not exploited in prostitution (n = 172) from the National Child Traumatic Stress Network Core Data Set (NCTSN CDS). Propensity Score Matching was used to select a comparison sample based on age, race, ethnicity, and primary residence. Statistically significant differences were noted between the groups on standardized (e.g., UCLA Posttraumatic Stress Disorder Reaction Index [PTSD-RI], Child Behavior Checklist [CBCL]) and other measures of emotional and behavioral problems (e.g., avoidance and hyperarousal symptoms, dissociation, truancy, running away, conduct disorder, sexualized behaviors, and substance abuse). This study provides useful insight into the symptom and service utilization profiles of youth exploited in commercial sex as compared with youth with other types of sexually exploitive experiences. Targeted screening and event-sensitive measures are recommended to more accurately identify youth exploited in commercial sex. More research is needed to determine if and what modifications to trauma therapies may be required to address the more severe symptomatology and behavior problems associated with youth exploited in commercial sex. PMID:25381275

  14. Development of a set of multiplex standard polymerase chain reaction assays for the identification of infectious agents from aborted bovine clinical samples.

    Science.gov (United States)

    Tramuta, Clara; Lacerenza, Daniela; Zoppi, Simona; Goria, Mariella; Dondo, Alessandro; Ferroglio, Ezio; Nebbia, Patrizia; Rosati, Sergio

    2011-07-01

    The current study describes the development of a set of 5 multiplex polymerase chain reaction (mPCR) assays for the simultaneous detection of abortive infection agents in bovine fetal tissues, including Brucella spp., Leptospira spp., and Campylobacter fetus (mPCR1); Hammondia heydorni, Neospora caninum, and Toxoplasma gondii (mPCR2); Coxiella burnetii and Chlamydophila psittaci (mPCR3); Mycoplasma bovis, Mycoplasma bovigenitalium, and Ureaplasma diversum (mPCR4); and Bovine viral diarrhea virus (BVDV) and Bovine herpesvirus-1 (BoHV-1; mPCR5). The protocol was tested on different tissue samples collected from 50 aborted bovine fetuses, and it showed that out of the 50 fetuses, 7 (14%, mPCR2) were PCR-positive for N. caninum, 4 (8%, mPCR5) were PCR-positive for BVDV, and 2 (4%, mPCR4) were PCR-positive for U. diversum. The results obtained by using each multiplex PCR were 100% concordant with those obtained by using the respective PCR assays targeting single genes on the same specimens. Moreover, all multiplex PCR assays on clinical samples were compared with reference methods, obtaining a perfect accordance in all samples and confirming the validity of the set of multiplex PCR assays. The proposed set of multiplex PCR assays is, therefore, suitable for the simultaneous detection of the main infectious agents responsible for bovine abortion.

  15. Cross-cultural confirmation of bi-factor models of a symptom distress measure: Symptom Checklist-90-Revised in clinical samples.

    Science.gov (United States)

    Urbán, Róbert; Arrindell, Willem A; Demetrovics, Zsolt; Unoka, Zsolt; Timman, Reinier

    2016-05-30

    Four decades have elapsed since the introduction for clinical and research purposes of the Symptom Checklist-90(-R). Yet, its underlying dimensional structure has not been clearly delineated. A shift has been observed in the methods utilized-from predominantly exploratory factor analytic in nature in the first two decades or so to different confirmatory methods in recent years. A need remains to search for a structure that remains invariant across samples and nations. In that context, the present study attempted to replicate and extend recent findings yielded in a Hungarian general population sample (N=2,874) with two psychiatric patient samples from Hungary (N=972) and The Netherlands (N=1,902). In doing so, four models were contrasted: the one-factor model, Derogatis' nine factor model, a second-ordered factor model, and a bi-factor model. The bi-factor model was shown to yield the closest fit to the data in both countries. Further studies are needed to determine the stable number and kind of subscale scores that reflect the specific (primary) symptoms best, that is, those subscales with minimal shared variance with the overall general psychological distress dimension. PMID:27039011

  16. 改良醋酸铵盐析法提取福尔马林固定石蜡包埋组织DNA%The improved ammonium acetate method in the application of DNA extraction from formalin-fixed and paraffin-embedded ( FFPE ) tissues

    Institute of Scientific and Technical Information of China (English)

    周勤虎; 丁梅; 王保捷; 庞灏; 徐振亮; 张晨; 冯春梅; 王巍巍

    2012-01-01

    目的 采用改良醋酸铵盐析法提取福尔马林固定石蜡包埋组织DNA,并评价其应用价值.方法 取死后24h内人体肾组织用10%中性福尔马林溶液固定7d,石蜡包埋.采用酚/氯仿法、Chelex- 100法及改良醋酸铵盐析法提取DNA.经用紫外分光光度计法、AGE及PCR-PAGE检测比较不同方法提取DNA的质量.结果 改良醋酸铵盐析法、酚/氯仿法、Chelex-100法OD260/OD280比值分别为1.962±0.195、2.110±0.470、1.018±0.124,两两比较均有显著性差异(P<0.05);3种方法提取DNA含量(μg)分别为0.515±0.447、0.328±0.345、5.346±1.994; AGE电泳图谱可印证上述结果;PCR-PAGE检测显示改良醋酸铵盐析法提取DNA的谱带清晰度好于其它两种方法.结论 改良醋酸铵盐析法更适合微量福尔马林固定石蜡包埋组织样本DNA的提取.%Objective The modified ammonium acetate method was used to extract DNA from the formalin-fixed and paraffin-embedded ( FFPE) tissues and its application was evaluated. Methods The human kidney tissues were obtained by autopsy within 24 hours post-death and then were embedded in paraffin after fixing with 10% neutral formalin solution for 7 days. The DNA from FFPE tissues were extracted by the methods of modified ammonium acetate, phenol/chloroform, and Chelex-100. The quality and the quantity of the DNA extracted by three different methods were evaluated by UV spectrophotometer, AGE and PCR-PAGE. Results The ratio of OD260/OD280 in modified ammonium acetate method was 1. 962 ±0. 195. There was a significant difference (p <0. 05), in comparison with the ratio in phenol/chloroform (2. 110 ±0. 470) and Chelex-100 (1. 018 ±0. 124). The yields (μg) of DNA extracted by three different methods were 0. 515 ± 0. 447, 0. 328 ± 0. 345 and 5. 346 ± 1.994, separately. Furthermore, the above results were confirmed by AGE and the PCR-PAGE. DNA extracted by the modified ammonium acetate method showed more clear bands than that by

  17. Correlates of perceptual distortions in clinical and non-clinical populations using the Cardiff Anomalous Perceptions Scale (CAPS): associations with anxiety and depression and a re-validation using a representative population sample.

    Science.gov (United States)

    Bell, Vaughan; Halligan, Peter W; Pugh, Katherine; Freeman, Daniel

    2011-10-30

    Although the literature on hallucinations in psychiatric patients shows clear links with anxiety and depression, associations of affect with a wider array of anomalous perceptual experiences have been much less studied. This study investigated patients with psychosis (N=29) and a non-clinical population (N=193) using the Cardiff Anomalous Perceptions Scale (CAPS), a measure of perceptual distortion and associated distress, intrusiveness and frequency; along with measures of depression, anxiety and worry. The study also allowed a re-validation of the CAPS in a more representative sample of the UK population. Moderate, reliable correlations with depression, anxiety and worry were found in the non-clinical population with the association being stronger in psychotic patients. The study re-confirmed that anomalous perceptual experiences are common in the general population and that a significant minority (11.9%) have higher levels than the mean of psychotic patients. Scale reliability and validity were also re-confirmed, and the CAPS score was found to be unrelated to age or gender in either sample. As in the original study, factor analysis produced a three-factor solution, although factor theme was not fully replicated: as before, a cluster of first-rank symptoms emerged, but with equivocal evidence for a temporal lobe factor and no replication of a 'chemosensation' component. PMID:21703692

  18. A Robust Protocol for Using Multiplexed Droplet Digital PCR to Quantify Somatic Copy Number Alterations in Clinical Tissue Specimens

    Science.gov (United States)

    Hughesman, Curtis B.; Lu, X. J. David; Liu, Kelly Y. P.; Zhu, Yuqi; Poh, Catherine F.; Haynes, Charles

    2016-01-01

    The ability of droplet digital PCR (ddPCR) to accurately determine the concentrations of amplifiable targets makes it a promising platform for measuring copy number alterations (CNAs) in genomic biomarkers. However, its application to clinical samples, particularly formalin-fixed paraffin-embedded specimens, will require strategies to reliably determine CNAs in DNA of limited quantity and quality. When applied to cancerous tissue, those methods must also account for global genetic instability and the associated probability that the abundance(s) of one or more chosen reference loci do not represent the average ploidy of cells comprising the specimen. Here we present an experimental design strategy and associated data analysis tool that enables accurate determination of CNAs in a panel of biomarkers using multiplexed ddPCR. The method includes strategies to optimize primer and probes design to cleanly segregate droplets in the data output from reaction wells amplifying multiple independent templates, and to correct for bias from artifacts such as DNA fragmentation. We demonstrate how a panel of reference loci can be used to determine a stable CNA-neutral benchmark. These innovations, when taken together, provide a comprehensive strategy that can be used to reliably detect biomarker CNAs in DNA extracted from either frozen or FFPE tissue biopsies. PMID:27537682

  19. The influence of psychoeducation on regulating biological rhythm in a sample of patients with bipolar II disorder: a randomized clinical trial

    Directory of Open Access Journals (Sweden)

    Faria AD

    2014-06-01

    Full Text Available Augusto Duarte Faria,1 Luciano Dias de Mattos Souza,2 Taiane de Azevedo Cardoso,2 Karen Amaral Tavares Pinheiro,2 Ricardo Tavares Pinheiro,2 Ricardo Azevedo da Silva,2 Karen Jansen21Department of Clinical and Health Psychology, Universidade Federal do Rio Grande – FURG, Rio Grande, RS, Brazil; 2Health and Behavior Postgraduate Program, Universidade Católica de Pelotas – UCPEL, Pelotas, RS, BrazilIntroduction: Changes in biological rhythm are among the various characteristics of bipolar disorder, and have long been associated with the functional impairment of the disease. There are only a few viable options of psychosocial interventions that deal with this specific topic; one of them is psychoeducation, a model that, although it has been used by practitioners for some time, only recently have studies shown its efficacy in clinical practice.Aim: To assess if patients undergoing psychosocial intervention in addition to a pharmacological treatment have better regulation of their biological rhythm than those only using medication.Method: This study is a randomized clinical trial that compares a standard medication intervention to an intervention combined with drugs and psychoeducation. The evaluation of the biological rhythm was made using the Biological Rhythm Interview of Assessment in Neuropsychiatry, an 18-item scale divided in four areas (sleep, activity, social rhythm, and eating pattern. The combined intervention consisted of medication and a short-term psychoeducation model summarized in a protocol of six individual sessions of 1 hour each.Results: The sample consisted of 61 patients with bipolar II disorder, but during the study, there were 14 losses to follow-up. Therefore, the final sample consisted of 45 individuals (26 for standard intervention and 19 for combined. The results showed that, in this sample and time period evaluated, the combined treatment of medication and psychoeducation had no statistically significant impact on the

  20. COMPARATIVE EFFECTIVENESS OF DISINFECTANTS WITH PHE NOL ON MULTIDRUG RESISTANT BACTERIA AND FUNGI ISOLATED FRO M THE CLINICAL SAMPLE – AN IN VITRO PRELIMINARY STUDY

    Directory of Open Access Journals (Sweden)

    Mannur

    2013-05-01

    Full Text Available ABSTRACT: BACKGROUND: The emergence of multidrug resistant (MDR microor ganism infections has generated considerable attention in the recent past . The primary goal of infection control procedures is to prevent cross contamination betwee n patients as well as between patients and health care providers. This can be achieved by the safe disposal of body fluids and cultures having such microorganisms from the hospital ward and labo ratory. This study aimed at comparing the efficacy of comme rcially available disinfectants on microorganisms isolated from clinical samples. MATERIAL AND METHOD: Four commonly used disinfectants namely Savlon, 70% Ethanol, Dettol an d Lysol were tested for their Antibacterial and Anti fungal effects against multidrug resistant bac teria and fungi isolated from clinical samples from inpatients admitted to Sri Siddhartha Medical Colle ge Hospital, Tumkur. Multi- drug resistant (MDR microorganisms E. coli, S. aureus, K. pneumoniae, P . aeruginosa, C. albicans, A. niger and A flavus isolated from clinical samples were used for testin g the effectiveness of disinfectants. Agar Well- diffusion method using Mueller Hinton Agar and Modi fied Muller Hinton agar was used to assess the effectiveness of disinfectants. Phenol was consider ed to be the standard disinfectant to which other disinfectants were compared. The zone of inhibitio n (ZOI for each isolate was measured. These were compared to the ZOI of Phenol (40mm. The resu lts were statistically analyzed by descriptive statistical methods. RESULTS: The ZOI against S. aureus and E. coli of Savlon was 33mm & 34mm and for Dettol 38mm & 37mm, respectively. But for K . pneumoniae the ZOI was 29mm & 30mm and for P. aeruginosa was 24mm and 30mm for Savlon and Dettol, respectively. The ZOI for 70% ethanol ranged from 32mm to 34 mm on all the isolates. The ZOI for Lysol ranged from 22mm to 24mm on isolates. CONCLUSION: The disinfectants -- Savlon and Dettol can be used as

  1. Diagnosis of canine visceral leishmaniasis with radiolabelled probes: comparison of the kDNA PCR-hybridization with three molecular methods in different clinical samples

    International Nuclear Information System (INIS)

    Leishmania (Leishmania) chagasi is responsible for visceral leishmaniasis (VL) in Brazil and the dog is the main domestic reservoir. Disease control is based on the elimination of infected animals and the use of a sensitive and specific diagnostic test is necessary. The Brazilian VL control program emphasizes serologic surveys, mainly using the enzyme-linked immunosorbent assay (ELISA) and the immunofluorescence antibody test (IFAT), followed by the elimination of the seropositive dogs. However, these techniques present limitations in terms of sensitivity and specificity. The Polymerase Chain Reaction (PCR) associated to hybridization with DNA probes labeled with 32P has been recognized as a valuable tool for Leishmania identification. In this study, the sensitivity of kDNA PCR hybridization method was compared with three other molecular methods: Internal Transcribed Spacer 1 Nested PCR (ITS-1nPCR), Leishmania nested PCR (LnPCR) and Seminested kDNA PCR (kDNA snPCR). The comparison was performed in different clinical specimens: conjunctival swab, skin, blood and bone marrow. A group of thirty symptomatic dogs, positive in the parasitological and serological tests, was used. When. The techniques targeting kDNA mini-circles (kDNA snPCR and KDNA PCR-hybridization) showed the worst result for blood samples. The KDNA-PCR hybridization showed the best sensitivity for conjunctival swab. By comparing the samples on the basis of positivity obtained by the sum of all methods, the blood showed the worst outcome (71/120).The bone marrow showed the highest positivity (106/120), followed by conjunctival swab (100/120) and skin (89/120). Since the bone marrow samples are unsuitable for routine epidemiological surveys, the conjunctival swab was recommended because it allows high sensitivity, especially when associated with kDNA PCR hybridization method, and is a noninvasive sampling method. (author)

  2. Diagnosis of canine visceral leishmaniasis with radiolabelled probes: comparison of the kDNA PCR-hybridization with three molecular methods in different clinical samples

    Energy Technology Data Exchange (ETDEWEB)

    Ferreira, Aline Leandra C.; Ferreira, Sidney A.; Carregal, Virginia M.; Andrade, Antero Silva R., E-mail: antero@cdtn.br [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil). Lab. de Radiobiologia; Melo, Maria N., E-mail: melo@icb.ufmg.br [Departamento de Parasitologia. Instituto de Ciencias Biologicas. Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil)

    2011-07-01

    Leishmania (Leishmania) chagasi is responsible for visceral leishmaniasis (VL) in Brazil and the dog is the main domestic reservoir. Disease control is based on the elimination of infected animals and the use of a sensitive and specific diagnostic test is necessary. The Brazilian VL control program emphasizes serologic surveys, mainly using the enzyme-linked immunosorbent assay (ELISA) and the immunofluorescence antibody test (IFAT), followed by the elimination of the seropositive dogs. However, these techniques present limitations in terms of sensitivity and specificity. The Polymerase Chain Reaction (PCR) associated to hybridization with DNA probes labeled with {sup 32}P has been recognized as a valuable tool for Leishmania identification. In this study, the sensitivity of kDNA PCR hybridization method was compared with three other molecular methods: Internal Transcribed Spacer 1 Nested PCR (ITS-1nPCR), Leishmania nested PCR (LnPCR) and Seminested kDNA PCR (kDNA snPCR). The comparison was performed in different clinical specimens: conjunctival swab, skin, blood and bone marrow. A group of thirty symptomatic dogs, positive in the parasitological and serological tests, was used. When. The techniques targeting kDNA mini-circles (kDNA snPCR and KDNA PCR-hybridization) showed the worst result for blood samples. The KDNA-PCR hybridization showed the best sensitivity for conjunctival swab. By comparing the samples on the basis of positivity obtained by the sum of all methods, the blood showed the worst outcome (71/120).The bone marrow showed the highest positivity (106/120), followed by conjunctival swab (100/120) and skin (89/120). Since the bone marrow samples are unsuitable for routine epidemiological surveys, the conjunctival swab was recommended because it allows high sensitivity, especially when associated with kDNA PCR hybridization method, and is a noninvasive sampling method. (author)

  3. Evidence for different mechanisms of ‘unhooking’ for melphalan and cisplatin-induced DNA interstrand cross-links in vitro and in clinical acquired resistant tumour samples

    International Nuclear Information System (INIS)

    DNA interstrand cross-links (ICLs) are critical lesions produced by several cancer chemotherapy agents including platinum drugs and nitrogen mustards. We have previously shown in haematological (multiple myeloma) and solid tumours (ovarian cancer) that clinical sensitivity to such agents can result from a defect in DNA ICL processing leading to their persistence. Conversely, enhanced repair can result in clinical acquired resistance following chemotherapy. The repair of ICLs is complex but it is assumed that the ‘unhooking’ step is common to all ICLs. Using a modification of the single cell gel electrophoresis (Comet) assay we measured the formation and unhooking of melphalan and cisplatin-induced ICLs in cell lines and clinical samples. DNA damage response in the form of γ-H2AX foci formation and the formation of RAD51 foci as a marker of homologous recombination were also determined. Real-time PCR of 84 genes involved in DNA damage signalling pathways was also examined pre- and post-treatment. Plasma cells from multiple myeloma patients known to be clinically resistant to melphalan showed significant unhooking of melphalan-induced ICLs at 48 hours, but did not unhook cisplatin-induced ICLs. In ovarian cancer cells obtained from patients following platinum-based chemotherapy, unhooking of cisplatin-induced ICLs was observed at 48 hours, but no unhooking of melphalan-induced ICLs. In vitro, A549 cells were proficient at unhooking both melphalan and cisplatin-induced ICLs. γ-H2AX foci formation closely followed the formation of ICLs for both drugs, and rapidly declined following the peak of formation. RPMI8226 cells unhooked melphalan, but not cisplatin-induced ICLs. In these cells, although cross-links form with cisplatin, the γ-H2AX response is weak. In A549 cells, addition of 3nM gemcitabine resulted in complete inhibition of cisplatin-induced ICL unhooking but no effect on repair of melphalan ICLs. The RAD51 foci response was both drug and cell line

  4. Gender ratio in a clinical population sample, age of diagnosis and duration of assessment in children and adults with autism spectrum disorder.

    Science.gov (United States)

    Rutherford, Marion; McKenzie, Karen; Johnson, Tess; Catchpole, Ciara; O'Hare, Anne; McClure, Iain; Forsyth, Kirsty; McCartney, Deborah; Murray, Aja

    2016-07-01

    This article reports on gender ratio, age of diagnosis and the duration of assessment procedures in autism spectrum disorder diagnosis in a national study which included all types of clinical services for children and adults. Findings are reported from a retrospective case note analysis undertaken with a representative sample of 150 Scottish children and adults recently diagnosed with autism spectrum disorder. The study reports key findings that the gender ratio in this consecutively referred cohort is lower than anticipated in some age groups and reduces with increasing age. The gender ratio in children, together with the significant difference in the mean age of referral and diagnosis for girls compared to boys, adds evidence of delayed recognition of autism spectrum disorder in younger girls. There was no significant difference in duration of assessment for males and females suggesting that delays in diagnosis of females occur prior to referral for assessment. Implications for practice and research are considered.

  5. Adult attention deficit hyperactivity disorder symptoms and five-factor model traits in a clinical sample: a structural equation modeling approach.

    Science.gov (United States)

    Knouse, Laura E; Traeger, Lara; O'Cleirigh, Conall; Safren, Steven A

    2013-10-01

    Relationships among attention deficit hyperactivity disorder (ADHD) symptoms and adult personality traits have not been examined in larger clinically diagnosed samples. We collected multisource ADHD symptom and self-report NEO Five-Factor Inventory (Costa and McCrae [Odessa, FL: Psychological Assessment Resources, Inc, 1992) data from 117 adults with ADHD and tested symptom-trait associations using structural equation modeling. The final model fit the data. Inattention was positively associated with neuroticism and negatively associated with conscientiousness. On the basis of ADHD expression in adulthood, hyperactivity and impulsivity were estimated as separate constructs and showed differential relationships to extraversion and agreeableness. A significant positive relationship between hyperactivity and conscientiousness arose in the context of other pathways. ADHD symptoms are reliably associated with personality traits, suggesting a complex interplay across development that warrants prospective study into adulthood. PMID:24080671

  6. Occurrence and characteristics of extended-spectrum β-lactamases producing Escherichia coli in foods of animal origin and human clinical samples in Chhattisgarh, India

    Directory of Open Access Journals (Sweden)

    Bhoomika

    2016-09-01

    Full Text Available Aim: To assess the prevalence of antimicrobial resistance producing extended-spectrum β-lactamases (ESBL (blaTEM, blaSHV, and blaCTX-M genes in Escherichia coli isolated from chicken meat, chevon meat, raw milk, and human urine and stool samples collected from tribal districts of Chhattisgarh, viz., Jagdalpur, Dantewada, Kondagaon, and Kanker. Materials and Methods: A total of 330 samples, comprising 98 chicken meat, 82 chevon meat, 90 raw milk, and 60 human urine and stool samples, were processed for isolation of E. coli. Isolates were confirmed biochemically and further tested against commonly used antibiotics to know their resistant pattern. The resistant isolates were tested for ESBL production by phenotypic method followed by characterization with molecular method using multiplex-polymerase chain reaction technique. Results: Overall 57.87% (191/330 samples were found positive for E. coli, which include 66.32% (65/98 chicken meat, 46.34% (38/82 chevon meat, 81.11% (73/90 raw milk, and 25% (15/60 human urine and stool samples. Isolates showed the highest resistance against cefotaxime (41.36% followed by oxytetracycline (34.03%, ampicillin (29.31%, cephalexin (24.60%, cefixime (16.75%, and ceftazidime (13.08%. Phenotypic method detected 10.99% (21/191 isolates as presumptive ESBL producers, however, molecular method detected 3.66% (7/191, 2.09% (4/191, and 0.00% (0/191 prevalence of blaTEM, blaCTX-M, and blaSHV, respectively. Conclusion: The present study indicates a high prevalence of E. coli in raw chicken meat, chevon meat, and milk due to poor hygienic practices. The antibiotic susceptibility test detected the presence of the resistance pattern against ESBL in E. coli isolated from raw chicken meat, chevon meat, milk, and also in human clinical samples is of great concern. The appearance of E. coli in the human food chain is alarming and requires adaptation of hygienic practices and stipulate use of antibiotics.

  7. Performance of the digene LQ, RH and PS HPVs genotyping systems on clinical samples and comparison with HC2 and PCR-based Linear Array

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    Godínez Jose M

    2011-11-01

    Full Text Available Abstract Background Certain Human Papillomaviruses (HPVs are the infectious agents involved in cervical cancer development. Detection of HPVs DNA is part of the cervical cancer screening protocols and HPVs genotyping has been proposed for its inclusion in these preventive programs. The aim of this study was to evaluate three novel genotyping tests, namely Qiagen LQ, RH and PS, in clinical samples with and without abnormalities. For this, 305 cervical samples were processed and the results of the evaluated techniques were compared with those obtained in the HPVs diagnostic process in our lab, by using HC2 and Linear Array (LA technologies. Results The concordances and kappa statistics (k for each technique compared with HC2 were 98.69% (k = 0.94 for LQ, 98.03% (k = 0.91 for RH and 91.80% (k = 0.82 for PS. There was a very good agreement in HPVs type-specific concordance for the most prevalent types HPV16 (kappa range = 0.83-0.90, HPV18 (k.r.= 0.74-0.80 and HPV45 (k.r.= 0.82-0.90. Conclusions The three tests showed an overall good concordance for HPVs detection when compared with HR-HC2 system. LQ and RH rendered lower detection rate for multiple infections than LA genotyping. However, our understanding of the clinical significance of multiple HPVs infections is still incomplete and therefore the relevance of the lower ability to detect multiple infections needs to be evaluated.

  8. Molecular structure and transferability of Tn1546-like elements in Enterococcus faecium isolates from clinical, sewage, and surface water samples in Iran.

    Science.gov (United States)

    Talebi, M; Pourshafie, M R; Katouli, M; Möllby, R

    2008-03-01

    The molecular structure and transferability of Tn1546 in 143 vancomycin-resistant Enterococcus faecium (VREF) isolates obtained from patients (n = 49), surface water (n = 28), and urban and hospital sewage (n = 66) in Tehran, Iran, were investigated. Molecular characterization of Tn1546 elements in vanA VREF was performed using a combination of restriction fragment length polymorphism analysis and DNA sequencing of the internal PCR fragments of vanA transposons. Long-PCR amplification showed that the molecular size of Tn1546 elements varied from 10.8 to 12.8 kb. The molecular analysis of Tn1546 showed that 45 isolates (31.5%) harbored a deletion/mutation upstream from nucleotide 170. No horizontal transfer of Tn1546 was observed following filter-mating conjugation with these isolates. Nevertheless, the rates of transferability for other isolates were 10(-5) to 10(-6) per donor. Insertion sequences IS1216V and IS1542 were present in 103 (72%) and 138 (96.5%) of the isolates, respectively. The molecular analysis of Tn1546 elements resulted in three genomic organizations. The genomic organization lineage 1 was dominated by the isolates from clinical samples (3.4%), lineage 2 was dominated mostly by sewage isolates (24.5%), and lineage 3 contained isolates obtained from all sources (72.1%). The genetic diversity determined using pulsed-field gel electrophoresis (PFGE) revealed a single E. faecium clone, designated 44, which was common to the samples obtained from clinical specimens and hospital and municipal sewage. Furthermore, the results suggest that lineage 3 Tn1546 was highly disseminated among our enterococcal isolates in different PFGE patterns. PMID:18192406

  9. Human papillomavirus 16 E2 interacts with neuregulin receptor degradation protein 1 affecting ErbB-3 expression in vitro and in clinical samples of cervical lesions.

    Science.gov (United States)

    Paolini, Francesca; Curzio, Gianfranca; Melucci, Elisa; Terrenato, Irene; Antoniani, Barbara; Carosi, Mariantonia; Mottolese, Marcella; Vici, Patrizia; Mariani, Luciano; Venuti, Aldo

    2016-05-01

    The ErbB tyrosine kinase receptors play a key role in regulating many cellular functions and human papillomaviruses (HPVs) may interact with transductional pathway of different growth factor receptors. Here, these interactions were analysed in W12 cell line carrying HPV 16 genome and in clinical samples. W12 cells, in which HPV16 becomes integrated during passages, were utilised to detect viral and ErbB family expression at early (W12E) and late passages (W12G). Interestingly, a strong reduction of ErbB-3 expression was observed in W12G. Loss of the E2 and E5 viral genes occurs in W12G and this may affect ErbB-3 receptor expression. E2 and E5 rescue experiments demonstrated that only E2 gene was able to restore ErbB-3 expression. E2 is a transcriptional factor but the expression levels of ErbB3 were unaffected and ErbB-3 promoter did not show any consensus sequence for E2, thus E2 may interact in another way with ErbB3. Indeed, HPV 16 E2 can modulate ErbB-3 by interacting with the ubiquitin ligase neuregulin receptor degradation protein 1 (Nrdp-1) that is involved in the regulation of this receptor, via ubiquitination and degradation. E2 co-immunoprecipitated in a complex with Nrdp-1 leading to hypothesise an involvement of this interaction in ErbB-3 regulation. In addition, 90% of the clinical samples with integrated virus and E2 loss showed no or low ErbB-3 positivity, confirming in vitro results. In conclusion, the new discovered interaction of HPV-16 E2 with Nrdp-1 may affect ErbB-3 expression. PMID:26963794

  10. Bisulfite-Based DNA Methylation Analysis from Recent and Archived Formalin-Fixed, Paraffin Embedded Colorectal Tissue Samples.

    Science.gov (United States)

    Kalmár, Alexandra; Péterfia, Bálint; Hollósi, Péter; Wichmann, Barnabás; Bodor, András; Patai, Árpád V; Schöller, Andrea; Krenács, Tibor; Tulassay, Zsolt; Molnár, Béla

    2015-09-01

    We aimed to test the applicability of formalin-fixed and paraffin-embedded (FFPE) tissue samples for gene specific DNA methylation analysis after using two commercially available DNA isolation kits. Genomic DNA was isolated from 5 colorectal adenocarcinomas and 5 normal adjacent tissues from "recent", collected within 6 months, and "archived", collected more than 5 years ago, FFPE tissues using either High Pure FFPET DNA Isolation kit or QIAamp DNA FFPE Tissue kit. DNA methylation analysis of MAL, SFRP1 and SFRP2 genes, known to be hypermethylated in CRC, was performed using methylation-sensitive high resolution melting (MS-HRM) analysis and sequencing. QIAamp (Q) method resulted in slightly higher recovery in archived (HP: 1.22 ± 3.18 μg DNA; Q: 3.00 ± 4.04 μg DNA) and significantly (p < 0.05) higher recovery in recent samples compared to High Pure method (HP) (HP: 4.10 ± 2.91 μg DNA; Q: 11.51 ± 7.50 μg DNA). Both OD260/280 and OD260/230 ratios were lower, but still high in the High Pure isolated archived and recent samples compared to those isolated with QIAamp. Identical DNA methylation patterns were detected for all 3 genes tested by MS-HRM with both isolation kits in the recent group. However, despite of higher DNA recovery in QIAamp slightly more reproducible methylation results were obtained from High Pure isolated archived samples. Sequencing confirmed DNA hypermethylation in CRCs. In conclusion, reproducible DNA methylation patterns were obtained from recent samples using both isolation kits. However, long term storage may affect the reliability of the results leading to moderate differences between the efficiency of isolation kits.

  11. Occurrence of Ambler Class B Metallo-β-Lactamase Gene in Imipenem-Resistant Pseudomonas Aeruginosa Strains Isolated from Clinical Samples

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    Zeynab Golshani

    2014-02-01

    Full Text Available Background: 5TMetallo-β-lactamase (MBLs can hydrolyze a broad spectrum of beta-lactams, including penicillins, cephalosporins, and carbapenems. Genes encoding these enzymes are located on the plasmid that can easily be transferred to other bacteria. The aim of this study was to isolate and identify the Pseudomonas aeruginosa strains encoding VIM1 gene, in clinical samples, using the PCR technique. Materials and Methods: During a 4 month period, 100 strains of Pseudomonas aeruginosa from clinical specimens were collected. Standard tests were performed to identify strains of Pseudomonas aeruginosa. Resistance to antibiotics was examined and then the PCR was used to detect VIM1gene. Results:In this study, the highest rates of resistance to antibiotics, amikacin and cefotaxime was observed (65% and 62%, the lowest resistance to antibiotics piperacillin (48% and imipenem and cefepime with 55% resistance was reported. DDST method was performed for 37 strains for the MBl detection. Among the 37 isolate, 30 strains were MBL-producing with imipenem-EDTA method. Twelve strains (18% were carriers of VIM1 gene using the PCR method. Conclusion: In the present study, the prevalence of strains producing MBL genes in strains of hospitals is a growing trend; correct prescription of medications can prevent the spread of resistant pathogens. It is suggested that molecular methods for rapid detection of resistance genes can be used to prevent the spread of this genes.

  12. Mapping the Personality Psychopathology Five domains onto DSM-IV personality disorders in Dutch clinical and forensic samples: implications for DSM-5.

    Science.gov (United States)

    Sellbom, Martin; Smid, Wineke; de Saeger, Hilde; Smit, Naomi; Kamphuis, Jan H

    2014-01-01

    The Personality Psychopathology Five (PSY-5) model represents 5 broadband dimensional personality domains that align with the originally proposed DSM-5 personality trait system, which was eventually placed in Section III for further study. The main objective of this study was to examine the associations between the PSY-5 model and personality disorder criteria. More specifically, we aimed to determine if the PSY-5 domain scales converged with the alternative DSM-5 Section III model for personality disorders, with a particular emphasis on the personality trait profiles proposed for each of the specific personality disorder types. Two samples from The Netherlands consisting of clinical patients from a personality disorder treatment program (n = 190) and forensic psychiatric hospital (n = 162) were used. All patients had been administered the MMPI-2 (from which MMPI-2-RF PSY-5 scales were scored) and structured clinical interviews to assess personality disorder criteria. Results based on Poisson or negative binomial regression models showed statistically significant and meaningful associations for the hypothesized PSY-5 domains for each of the 6 personality disorders, with a few minor exceptions that are discussed in detail. Implications for these findings are also discussed.

  13. Codetection of Trichomonas vaginalis and Candida albicans by PCR in urine samples in a low-risk population attended in a clinic first level in central Veracruz, Mexico.

    Science.gov (United States)

    López-Monteon, A; Gómez-Figueroa, F S; Ramos-Poceros, G; Guzmán-Gómez, D; Ramos-Ligonio, A

    2013-01-01

    The aim of this study is to estimate the prevalence of Trichomonas vaginalis and Candida albicans in low-risk patients treated at a first level clinic (primary health care represents the first level of contact of individuals, families, and the community with the system national health). Using a cross-sectional study in patients treated in clinical laboratory of the Sanitary District no. 7 of the city of Orizaba during the months June-July, 252 urine samples were collected for the identification of T. vaginalis and C. albicans by PCR. Furthermore, we analyzed the sociodemographic characteristics of the studied population. We observed an overall prevalence of 23.41% (95% CI 22.10-24.72) for T. vaginalis and 38.88% (95% CI 37.73-40.03) for C. albicans. There was also presence of coinfection in 14.28% (95% CI 13.10-15.46), which was associated with the presence of pain. Most of the positive cases were observed in women house-maker (80%, 95% CI 50.36-48.98). The results of this study provide evidence that the majority of positive cases observed in the studied population are presented in an asymptomatic form and usually are not associated with any risk factor.

  14. Codetection of Trichomonas vaginalis and Candida albicans by PCR in Urine Samples in a Low-Risk Population Attended in a Clinic First Level in Central Veracruz, Mexico

    Directory of Open Access Journals (Sweden)

    A. López-Monteon

    2013-01-01

    Full Text Available The aim of this study is to estimate the prevalence of Trichomonas vaginalis and Candida albicans in low-risk patients treated at a first level clinic (primary health care represents the first level of contact of individuals, families, and the community with the system national health. Using a cross-sectional study in patients treated in clinical laboratory of the Sanitary District no. 7 of the city of Orizaba during the months June-July, 252 urine samples were collected for the identification of T. vaginalis and C. albicans by PCR. Furthermore, we analyzed the sociodemographic characteristics of the studied population. We observed an overall prevalence of 23.41% (95% CI 22.10–24.72 for T. vaginalis and 38.88% (95% CI 37.73–40.03 for C. albicans. There was also presence of coinfection in 14.28% (95% CI 13.10–15.46, which was associated with the presence of pain. Most of the positive cases were observed in women house-maker (80%, 95% CI 50.36–48.98. The results of this study provide evidence that the majority of positive cases observed in the studied population are presented in an asymptomatic form and usually are not associated with any risk factor.

  15. Distribution of Candida Species in different clinical samples and their virulence: Biofilm formation, proteinase and phospholipase production: A study on hospitalized patients in Southern India

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    Vinitha Mohandas

    2011-01-01

    Full Text Available Introduction: Candida species are normal inhabitants of the skin and mucosa. The importance of epidemiological monitoring of yeasts involved in pathogenic processes is unquestionable due to the increase of these infections over the last decade; Materials and Methods: The clinical samples from the respiratory tract (sputum, bronchial wash, tracheal secretions, saliva, blood, urine, middle ear discharge, vitreous fluid, corneal ulcer, and plastic devices (endotracheal tube, catheter tip, suction tip were collected and cultured. The species of Candida isolated were identified. Results: A total of 111 isolates of Candida species were recovered from 250 diverse clinical sources. C. albicans (39.64% was the most isolated species, although the Candida non albicans species with 60.36% showed the major prevalence. In blood cultures, C. krusei (38.23% and C. albicans (20.58% were isolated frequently. C. albicans (63.27% was the predominant species in mucosal surface. Urinary tract infections caused by yeasts were more frequent in hospitalized patients, C. krusei (50.0% being commonly isolated, followed by C. albicans (25.0%. Discussion: Several virulence factors like, biofilm, proteinase, phospholipase, etc. contribute to the pathogenecity. Early detection of virulence factors by Candida is useful in clinical decision making. We therefore have aimed at demonstrating the formation of biofilm using the method proposed by Branchini et al, (1994. The proteinase produced by Candida was estimated as per the method of Staib et al, (1965. Phospholipase assay was carried out as per the method of Samaranayake et al, (2005. Conclusions : The data suggests that the capacity of Candida species to produce biofilm may be a reflection of the pathogenic potential of the isolates. C. krusei and C. tropicalis showed strong slime production. The non-Candida albicans produced more proteinase than C. albicans. C. albicans produced higher levels of phospholipase than non

  16. Validation of a Multiplex Real-Time PCR Assay for Detection of Mycobacterium spp., Mycobacterium tuberculosis Complex, and Mycobacterium avium Complex Directly from Clinical Samples by Use of the BD Max Open System.

    Science.gov (United States)

    Rocchetti, Talita T; Silbert, Suzane; Gostnell, Alicia; Kubasek, Carly; Widen, Raymond

    2016-06-01

    A multiplex real-time PCR was validated on the BD Max open system to detect different Mycobacterium tuberculosis complex, Mycobacterium avium complex, and Mycobacterium spp. directly from clinical samples. The PCR results were compared to those with traditional cultures. The multiplex PCR assay was found to be a specific and sensitive method for the rapid detection of mycobacteria directly from clinical specimens.

  17. Dose-response effect between cannabis use and psychosis liability in a non-clinical population: evidence from a snowball sample.

    Science.gov (United States)

    Ruiz-Veguilla, Miguel; Barrigón, María Luisa; Hernández, Laureano; Rubio, José Luis; Gurpegui, Manuel; Sarramea, Fernando; Cervilla, Jorge; Gutiérrez, Blanca; James, Anthony; Ferrin, Maite

    2013-08-01

    This study aimed to explore the associations between daily cannabis use and the specific profiles of subclinical symptoms in a non-clinical population obtained through snowball sampling, taking into account alcohol use, other drug use, social exclusion and age at onset of cannabis use. We included 85 daily cannabis users and 100 non-daily cannabis users. Both the case and the control populations were identified by snowball sampling. Daily cannabis use was associated with more alcohol intake and other drug use, as well as with early onset in the use of cannabis. Daily cannabis use appeared to exert a dose-response effect on first-rank symptoms, mania symptoms and auditory hallucinations, even after adjusting for sex, age, other drug use, social exclusion and age at onset of cannabis use. The paranoid dimension was only associated with the heaviest consumption of cannabis. Initial age of cannabis use modified the effects of daily cannabis use on the first-rank and voices experiences. Daily cannabis use was associated with significantly more first-rank and voices experiences among those subjects who started to use cannabis before 17 years of age. Our study supports the association of psychotic experiences with cannabis use even among non-psychotic subjects.

  18. The Social Appearance Anxiety Scale in Italian Adolescent Populations: Construct Validation and Group Discrimination in Community and Clinical Eating Disorders Samples.

    Science.gov (United States)

    Dakanalis, Antonios; Carrà, Giuseppe; Calogero, Rachel; Zanetti, M Assunta; Volpato, Chiara; Riva, Giuseppe; Clerici, Massimo; Cipresso, Pietro

    2016-02-01

    Anxiety in situations where one's overall appearance (including body shape) may be negatively evaluated is hypothesized to play a central role in Eating Disorders (EDs) and in their co-occurrence with Social Anxiety Disorder (SAD). Three studies were conducted among community (N = 1995) and clinical (N = 703) ED samples of 11- to 18-year-old Italian girls and boys to (a) evaluate the psychometric qualities and measurement equivalence/invariance (ME/I) of the Social Appearance Anxiety (SAA) Scale (SAAS) and (b) determine to what extent SAA or other situational domains of social anxiety related to EDs distinguish adolescents with an ED only from those with SAD. Results upheld the one-factor structure and ME/I of the SAAS across samples, gender, age categories, and diagnostic status (i.e., ED participants with and without comorbid SAD). The SAAS demonstrated high internal consistency and 3-week test-retest reliability. The strength of the inter-relationships between SAAS and measures of body image, teasing about appearance, ED symptoms, depression, social anxiety, avoidance, and distress, as well as the ability of SAAS to discriminate community adolescents with high and low levels of ED symptoms and community participants from ED participants provided construct validity evidence. Only SAA strongly differentiated adolescents with any ED from those with comorbid SAD (23.2 %). Latent mean comparisons across all study groups were performed and discussed.

  19. Development of a diagnostic real-time polymerase chain reaction assay for the detection of invasive Haemophilus influenzae in clinical samples.

    Science.gov (United States)

    Meyler, Kenneth L; Meehan, Mary; Bennett, Desiree; Cunney, Robert; Cafferkey, Mary

    2012-12-01

    Since the introduction of the Haemophilus influenzae serotype b vaccine, invasive H. influenzae disease has become dominated by nontypeable (NT) strains. Several widely used molecular diagnostic methods have been shown to lack sensitivity or specificity in the detection of some of these strains. Novel real-time assays targeting the fucK, licA, and ompP2 genes were developed and evaluated. The fucK assay detected all strains of H. influenzae tested (n = 116) and had an analytical sensitivity of 10 genome copies/polymerase chain reaction (PCR). This assay detected both serotype b and NT H. influenzae in 12 previously positive specimens (culture and/or bexA PCR) and also detected H. influenzae in a further 5 of 883 culture-negative blood and cerebrospinal fluid (CSF) samples. The fucK assay has excellent potential as a diagnostic test for detection of typeable and nontypeable strains of invasive H. influenzae in clinical samples of blood and CSF. PMID:23017260

  20. Distribution of contagious and environmental mastitis agents isolated from milk samples collected from clinically health buffalo cows between brazilian dry and rainy seasons of the year

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    R.P. Maia

    2010-02-01

    Full Text Available The present study was performed to evaluate the microbiological characteristics of clinically health quarters submitted to milking and also to observe the distribution of contagious and environmental agents between brazilian dry and rainy seasons of the year. During nine months 734 quarters from 37 buffalo cows were submitted monthly to udder inspection, palpation and strip cup test before milking. 734 asseptic milk samples were inoculated in 10% ovine blood agar and in MacConkey agar media, then incubated for 72 hours at 37oC. Among the 580 isolated microrganisms, 182 (31,38% were recovered from samples collected during the rainy season and 398 (68,62% from the dry season. In the rainy period the most prevalent agents were: bacteria from the genus Corynebacterium sp (53,30%, Staphylococcus sp (19,78% and Rhodococcus equi (13,74%. In the dry period, the commonest ones were: Corynebacterium sp (44,97%, Staphylococcus sp (18,84% and Micrococcus sp (9,55%. The results demonstrated that the methods used to select health quarters in brazilian dairy buffalo farms allow the transmission of contagious bacteria during both seasons of the year, maintaining Ital.J.Anim.Sci. vol. 6, (Suppl. 2, 896-899, 2007 897 VIII World Buffalo Congress agents known to cause mainly subclinical inflammatory reactions that compromise cronically the physiology and production of the mammary gland.

  1. Investigating the Prevalence of Pervasive Developmental Disorders According to Sex in a Sample of Iranian Children Referred to Medical-Rehabilitation Centers and Psychiatrics Clinics

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    K. Khushabi

    2006-04-01

    Full Text Available Introduction & Objective: According to significance of pervasive developmental disorders (PDD in children and the increasing rate of its prevalence in referred patients to clinic in recent years and due to absence of any report about the rate of PPD in our country, this study was carried out. The aim of this study was to determine the prevalence of PPD in a sample of Iranian children who referred to medical and rehabilitation centers.Materials & Methods: 248 children who referred to three medical-rehabilitation centers were participated in the research. Accessible sampling with diagnosis of PDD based on DSM-IV criteria was chosen. The obtained data were analyzed using descriptive statistics methods such as percent and frequency distribution. Results: Autistics disorder was most prevalent among pervasive developmental disorders. In this research Autistic disorder (proportion 4/1 to 1, Asperger disorder (proportion 3 to 1 and childhood disintegrative disease were more prevalent in boys than girls. Ret disorders was observed only in girls and pervasive developmental disease (NOS was seen in both sexes. Conclusion: The results showed that pervasive developmental disorders are 4 times more prevalent in boys than girls and the findings of this research were consistent with those of previous studies.

  2. The Social Appearance Anxiety Scale in Italian Adolescent Populations: Construct Validation and Group Discrimination in Community and Clinical Eating Disorders Samples.

    Science.gov (United States)

    Dakanalis, Antonios; Carrà, Giuseppe; Calogero, Rachel; Zanetti, M Assunta; Volpato, Chiara; Riva, Giuseppe; Clerici, Massimo; Cipresso, Pietro

    2016-02-01

    Anxiety in situations where one's overall appearance (including body shape) may be negatively evaluated is hypothesized to play a central role in Eating Disorders (EDs) and in their co-occurrence with Social Anxiety Disorder (SAD). Three studies were conducted among community (N = 1995) and clinical (N = 703) ED samples of 11- to 18-year-old Italian girls and boys to (a) evaluate the psychometric qualities and measurement equivalence/invariance (ME/I) of the Social Appearance Anxiety (SAA) Scale (SAAS) and (b) determine to what extent SAA or other situational domains of social anxiety related to EDs distinguish adolescents with an ED only from those with SAD. Results upheld the one-factor structure and ME/I of the SAAS across samples, gender, age categories, and diagnostic status (i.e., ED participants with and without comorbid SAD). The SAAS demonstrated high internal consistency and 3-week test-retest reliability. The strength of the inter-relationships between SAAS and measures of body image, teasing about appearance, ED symptoms, depression, social anxiety, avoidance, and distress, as well as the ability of SAAS to discriminate community adolescents with high and low levels of ED symptoms and community participants from ED participants provided construct validity evidence. Only SAA strongly differentiated adolescents with any ED from those with comorbid SAD (23.2 %). Latent mean comparisons across all study groups were performed and discussed. PMID:25976291

  3. Development of a diagnostic real-time polymerase chain reaction assay for the detection of invasive Haemophilus influenzae in clinical samples.

    LENUS (Irish Health Repository)

    Meyler, Kenneth L

    2012-12-01

    Since the introduction of the Haemophilus influenzae serotype b vaccine, invasive H. influenzae disease has become dominated by nontypeable (NT) strains. Several widely used molecular diagnostic methods have been shown to lack sensitivity or specificity in the detection of some of these strains. Novel real-time assays targeting the fucK, licA, and ompP2 genes were developed and evaluated. The fucK assay detected all strains of H. influenzae tested (n = 116) and had an analytical sensitivity of 10 genome copies\\/polymerase chain reaction (PCR). This assay detected both serotype b and NT H. influenzae in 12 previously positive specimens (culture and\\/or bexA PCR) and also detected H. influenzae in a further 5 of 883 culture-negative blood and cerebrospinal fluid (CSF) samples. The fucK assay has excellent potential as a diagnostic test for detection of typeable and nontypeable strains of invasive H. influenzae in clinical samples of blood and CSF.

  4. Rapid and accurate detection of Mycobacterium tuberculosis in sputum samples by Cepheid Xpert MTB/RIF assay--a clinical validation study.

    Directory of Open Access Journals (Sweden)

    Andrea Rachow

    Full Text Available BACKGROUND: A crucial impediment to global tuberculosis control is the lack of an accurate, rapid diagnostic test for detection of patients with active TB. A new, rapid diagnostic method, (Cepheid Xpert MTB/RIF Assay, is an automated sample preparation and real-time PCR instrument, which was shown to have good potential as an alternative to current reference standard sputum microscopy and culture. METHODS: We performed a clinical validation study on diagnostic accuracy of the Xpert MTB/RIF Assay in a TB and HIV endemic setting. Sputum samples from 292 consecutively enrolled adults from Mbeya, Tanzania, with suspected TB were subject to analysis by the Xpert MTB/RIF Assay. The diagnostic performance of Xpert MTB/RIF Assay was compared to standard sputum smear microscopy and culture. Confirmed Mycobacterium tuberculosis in a positive culture was used as a reference standard for TB diagnosis. RESULTS: Xpert MTB/RIF Assay achieved 88.4% (95%CI = 78.4% to 94.9% sensitivity among patients with a positive culture and 99% (95%CI = 94.7% to 100.0% specificity in patients who had no TB. HIV status did not affect test performance in 172 HIV-infected patients (58.9% of all participants. Seven additional cases (9.1% of 77 were detected by Xpert MTB/RIF Assay among the group of patients with clinical TB who were culture negative. Within 45 sputum samples which grew non-tuberculous mycobacteria the assay's specificity was 97.8% (95%CI = 88.2% to 99.9%. CONCLUSIONS: The Xpert MTB/RIF Assay is a highly sensitive, specific and rapid method for diagnosing TB which has potential to complement the current reference standard of TB diagnostics and increase its overall sensitivity. Its usefulness in detecting sputum smear and culture negative patients needs further study. Further evaluation in high burden TB and HIV areas under programmatic health care settings to ascertain applicability, cost-effectiveness, robustness and local acceptance are required.

  5. Clinical significance of quantitative detection of Brucella DNA in serum samples%布鲁菌病血清DNA定量检测的临床意义

    Institute of Scientific and Technical Information of China (English)

    郑文艳; 张鞠玲; 张专才; 闫飞; 曲芬

    2013-01-01

    目的:分析布鲁菌病血清DNA定量检测与布鲁菌病临床表现的相关性,从而探讨其对布鲁菌病的诊断价值。方法选择2010年5月-2011年6月内蒙古自治区100例布鲁菌血清凝集试验阳性(玻片凝集试验+~++++)的患者。采用荧光定量PCR法测定血清布鲁菌DNA含量,分析血清布鲁菌DNA检测结果与临床表现的相关性。结果100例布鲁菌血清凝集试验阳性患者中,23例有明显的临床症状,包括发热(91.3%)、全身关节疼痛(78.3%)、乏力(87.0%)、多汗(65.2%)等全身不适症状,其中的20例血清布鲁菌DNA定量值明显升高[范围(1.30~9.27)×106 copies/ml,平均(2.00±7.87)×106 copies/ml],检测时间仅3 h,灵敏度、特异度和准确度分别达到86.96%、97.40%和95.00%。结论血清布鲁菌DNA定量检测可以为布鲁菌病的诊断提供准确、快