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Sample records for qiaamp dna stool

  1. QIAamp及Biomed粪便细菌基因组DNA提取试剂盒提取人肠道细菌基因组DNA质量的差异%Quality differences of bacterial genome DNA extracted from the human intestine by using QIAamp and Biomed DNA Stool Mini Kits

    Institute of Scientific and Technical Information of China (English)

    宋美茹; 姚萍; 张跃新

    2012-01-01

    BACKGROUND: Several studies have showed that there are different qualities of bacterial genome DNA extracted from the humanintestine using different kits. Therefore, it is essential and urgent to select an effective, simple, and excellent DNA stool mini kit.OBJECTIVE: To compare the quality differences of bacterial genome DNA extracted from the human intestinal by using QIAampand Biomed DNA Stool Mini Kits.METHODS: Thirty fresh fecal samples of healthy adult people were selected, and bacterial genome DNA was extracted usingthese two kits. Concentration, absorbance radio of 260 to 280 nm and extraction rate were measured. Species-specific primersfor Lactobacillus group were designed by a set of 16S rDNA-targeted to conduct the general PCR using the genome DNA as thetemplate. The electrophoresis strip's number, brightness and density were compared after gel electrophoresis; subsequently, thenumber of Lactobacillus group was detected by fluorescent real-time PCR.RESULTS AND CONCLUSION: Concentration, extraction rate, expression level, Lactobacillus amount of bacterial genomeDNA extracted from the human intestinal using QIAamp kit were higher than those using Biomed kit (P<0.05 or P<0.01). Itsuggests that the quality of genome DNA extracted with QIAamp kit is more excellent than that with Biomed kit.%背景:有研究表明,不同试剂盒提取的粪便细菌基因组DNA 质量有差别,选择一种操作简便、质量优良的粪便细菌基因组DNA 提取试剂盒成为研究者们关注和急需解决的问题.目的:比较QIAamp 及Biomed 粪便细菌基因组DNA 提取试剂盒提取的人肠道细菌基因组DNA 质量的差异.方法:收集健康成年人新鲜粪便标本30 例,用两种试剂盒分别提取细菌基因组DNA,检测其浓度、吸光度A260/280 nm 值及提取率,设计乳酸菌属16S rDNA 基因特异性引物,以各自所提DNA 为模板,进行常规聚合酶链反应,凝胶电泳后比较条带数量、明暗度及密度,并进行实

  2. [Application of the QIAamp DNA Investigator Kit and Prepfiler Forensic DNA Extraction Kit in genomic DNA extraction from skeletal remains].

    Science.gov (United States)

    Ludwikowska-Pawłowska, Małgorzata; Jacewicz, Renata; Jedrzejczyk, Maciej; Prośniak, Adam; Berent, Jarosław

    2009-01-01

    The report presents an application of the QIAamp DNA Investigator Kit and PrepFiler Forensic DNA Extraction Kit in genomic DNA extraction from post-mortem highly degraded skeletal remains. The analysis included 25 bone samples collected on autopsy. DNA extraction was performed in accordance with the QIAamp DNA Investigator Kit and PrepFiler Forensic DNA Extraction Kit manufacturer's isolation protocols. Amplification was performed on a Biometra termocycler using the AmpFISTR Identifiler PCR Amplification Kit according to the manufacturer's protocol. Typing of PCR products was carried out on an ABI Prism 377 DNA sequencer. The recommended parameters for GeneScan analysis and Genotyper software were followed. The authors demonstrated that the QIAamp DNA Investigator Kit was more effective, convenient and statistically significantly better method which may be employed in DNA extraction from bone specimens.

  3. Improvement in fetal DNA extraction from maternal plasma. Evaluation of the NucliSens Magnetic Extraction system and the QIAamp DSP Virus Kit in comparison with the QIAamp DNA Blood Mini Kit

    DEFF Research Database (Denmark)

    Clausen, Frederik Banch; Krog, Grethe Risum; Rieneck, Klaus

    2007-01-01

    Prenatal diagnostic assays have been developed using free fetal DNA circulating in the maternal blood of pregnant women. Efficient DNA extraction is crucial for a robust analysis. To improve fetal DNA yield, we tested two manual extraction methods--the NucliSens Magnetic Extraction (NMAG) system...... and the QIAamp DSP Virus Kit (QDSP)--against our current standard method, the widely used QIAamp DNA Blood Mini Kit (QDNA)....

  4. An evaluation of the performance of five extraction methods: Chelex® 100, QIAamp® DNA Blood Mini Kit, QIAamp® DNA Investigator Kit, QIAsymphony® DNA Investigator® Kit and DNA IQ™.

    Science.gov (United States)

    Ip, Stephen C Y; Lin, Sze-Wah; Lai, Kam-Ming

    2015-05-01

    DNA left at a crime scene was often limited in amount and far from pristine. To maximize the chance of recovering as much information as possible from such compromised samples, an appropriate extraction method using the available technologies needs to be devised. In this study, we used human blood, buffy coat and a total of 76 simulated touch DNA samples to test the effectiveness of the following five common DNA extraction methods, namely, Chelex® 100, QIAamp® DNA Blood Mini Kit, QIAamp® DNA Investigator Kit, QIAsymphony® DNA Investigator® Kit and DNA IQ™ system, in the recovery of such DNA. We demonstrated that the QIAamp® and QIAsymphony® DNA Investigator® Kits, and the DNA IQ™ system, exhibited a better effectiveness in DNA recovery amongst these methods and yielded extracts with higher success rate in subsequent DNA profiling. These extracts also generated profiles with better intra-colour signal balance. The findings in this work allowed us to propose an extraction approach as follows: 1) casework samples shall be extracted with the QIAamp®/QIAsymphony® DNA Investigator® Kits or the DNA IQ™ system, viz., QIAsymphony® DNA Investigator® Kit and DNA IQ™, due to their higher throughput, are for the touched DNA evidence from the volume crime, while QIAamp® DNA Investigator Kit is preferable for challenging bloodstain samples; and 2) control samples, such as buccal swab, with known identity can be extracted with the Chelex, due to their cheaper cost per sample. Copyright © 2015 Forensic Science Society. Published by Elsevier Ireland Ltd. All rights reserved.

  5. Stool sample storage conditions for the preservation of Giardia intestinalis DNA

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    Salih Kuk

    2012-12-01

    Full Text Available Stool is chemically complex and the extraction of DNA from stool samples is extremely difficult. Haemoglobin breakdown products, such as bilirubin, bile acids and mineral ions, that are present in the stool samples, can inhibit DNA amplification and cause molecular assays to produce false-negative results. Therefore, stool storage conditions are highly important for the diagnosis of intestinal parasites and other microorganisms through molecular approaches. In the current study, stool samples that were positive for Giardia intestinalis were collected from five different patients. Each sample was stored using one out of six different storage conditions [room temperature (RT, +4ºC, -20ºC, 70% alcohol, 10% formaldehyde or 2.5% potassium dichromate] for DNA extraction procedures at one, two, three and four weeks. A modified QIAamp Stool Mini Kit procedure was used to isolate the DNA from stored samples. After DNA isolation, polymerase chain reaction (PCR amplification was performed using primers that target the β-giardin gene. A G. intestinalis-specific 384 bp band was obtained from all of the cyst-containing stool samples that were stored at RT, +4ºC and -20ºC and in 70% alcohol and 2.5% potassium dichromate; however, this band was not produced by samples that had been stored in 10% formaldehyde. Moreover, for the stool samples containing trophozoites, the same G. intestinalis-specific band was only obtained from the samples that were stored in 2.5% potassium dichromate for up to one month. As a result, it appears evident that the most suitable storage condition for stool samples to permit the isolation of G. intestinalis DNA is in 2.5% potassium dichromate; under these conditions, stool samples may be stored for one month.

  6. Compatibility of DNA IQ™, QIAamp(®) DNA Investigator, and QIAsymphony(®) DNA Investigator(®) with various fingerprint treatments.

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    Lin, Sze-Wah; Ip, Stephen C Y; Lam, Tze-Tsun; Tan, Tung-Fai; Yeung, Wai-Lung; Tam, Wai-Ming

    2017-03-01

    Latent fingerprint and touch DNA are the two most important contact evidence for individualization in forensic science which provide complementary information that can lead to direct and unequivocal identification of the culprit. In order to retrieve useful information from both fingerprints and DNA, which are usually mingled together, one strategy is to perform fingerprint examination prior to DNA analysis since common DNA sampling technique such as swabbing could disturb or even destroy fingerprint details. Here, we describe the compatibility of three automatic DNA extraction systems, namely, DNA IQ™, QIAamp(®) DNA Investigator, and QIAsymphony(®) DNA Investigator(®), with respective to the effects of various fingerprint detection techniques. Our results demonstrate that Super Glue fingerprint treatment followed by DNA IQ™ extraction shows better effectiveness in DNA profiling. Aluminum powder dusting offers the least interference to the three DNA extraction systems above. Magnetic powder dusting, on the other hand, strongly impedes DNA recovery. Physical Developer is the most intrusive, which yields profiles with poor quality, including lower peak heights, poor peak height ratios, and poor intra-color balance. In terms of the choice of extraction method, DNA IQ™ system is recommended for sampling after fingerprint treatments, but not the two DNA Investigator systems.

  7. Optimization of Toxoplasma gondii DNA extraction from amniotic fluid using NucliSENS easyMAG and comparison with QIAamp DNA minikit.

    Science.gov (United States)

    Faucher, B; Miermont, F; Ranque, S; Franck, J; Piarroux, R

    2012-06-01

    Antenatal diagnosis of congenital toxoplasmosis relies on PCR in amniotic fluid. Because parasitic load is often low, DNA extraction must be optimized. Manual methods remain widespread although automated methods appear more effective. This study aimed at optimizing an automated method and at comparing it with a widespread manual method: QIAamp DNA minikit. To optimize NucliSens easyMAG, we evaluated the addition of proteinase K pre-treatment and the increase of the amount of silica particles used for the extraction. The optimized method was then compared to QIAamp DNA minikit on samples containing less than 25 tachyzoites/ml. NucliSens easyMAG DNA yield was improved after proteinase K pre-treatment (p DNA amount in samples found positive by PCR was higher after optimized automated extraction than after manual extraction (p Proteinase K pre-treatment should be added to extract DNA from amniotic fluid using NucliSens easyMAG. Using this optimized automated method rather than manual methods would improve the sensitivity of Toxoplasma PCR and simplify the daily workflow.

  8. Optimized microbial DNA extraction from diarrheic stools

    Directory of Open Access Journals (Sweden)

    Donatin Emilie

    2012-12-01

    Full Text Available Abstract Background The detection of enteropathogens in stool specimens increasingly relies on the detection of specific nucleic acid sequences. We observed that such detection was hampered in diarrheic stool specimens and we set-up an improved protocol combining lyophilization of stools prior to a semi-automated DNA extraction. Findings A total of 41 human diarrheic stool specimens comprising of 35 specimens negative for enteropathogens and six specimens positive for Salmonella enterica in culture, were prospectively studied. One 1-mL aliquot of each specimen was lyophilised and total DNA was extracted from lyophilised and non-lyophilised aliquots by combining automatic and phenol-chloroform DNA extraction. DNA was incorporated into real-time PCRs targeting the 16S rRNA gene of Bacteria and the archaea Methanobrevibacter smithii and the chorismate synthase gene of S. enterica. Whereas negative controls consisting in DNA-free water remained negative, M. smithii was detected in 26/41 (63.4% non-lyophilised (Ct value 28.78 ± 9.1 versus 39/41 (95.1% lyophilised aliquots (Ct value 22.04 ± 5.5; bacterial 16S rRNA was detected in 33/41 (80.5% non-lyophilised (Ct value 28.11 ± 5.9 versus 40/41 (97.6% lyophilised aliquots (Ct value 24.94 ± 6.6; and S. enterica was detected in 6/6 (100% non-lyophilized and lyophilized aliquots (Ct value 26.98 ± 4.55 and 26.16 ± 4.97, respectively. S. enterica was not detected in the 35 remaining diarrheal-stool specimens. The proportion of positive specimens was significantly higher after lyophilization for the detection of M. smithii (p = 0.00043 and Bacteria (p = 0.015. Conclusion Lyophilization of diarrheic stool specimens significantly increases the PCR-based detection of microorganisms. The semi-automated protocol described here could be routinely used for the molecular diagnosis of infectious diarrhea.

  9. Accurate, noninvasive detection of Helicobacter pylori DNA from stool samples: potential usefulness for monitoring treatment.

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    Shuber, Anthony P; Ascaño, Jennifer J; Boynton, Kevin A; Mitchell, Anastasia; Frierson, Henry F; El-Rifai, Wa'el; Powell, Steven M

    2002-01-01

    A novel DNA assay demonstrating sensitive and accurate detection of Helicobacter pylori from stool samples is reported. Moreover, in three individuals tested for therapeutic response, the assay showed the disappearance of H. pylori DNA during treatment. Thus, this noninvasive molecular biology-based assay has the potential to be a powerful diagnostic tool given its ability to specifically identify H. pylori DNA.

  10. Accurate, Noninvasive Detection of Helicobacter pylori DNA from Stool Samples: Potential Usefulness for Monitoring Treatment

    OpenAIRE

    Shuber, Anthony P; Ascaño, Jennifer J.; Boynton, Kevin A.; Mitchell, Anastasia; Frierson, Henry F.; El-Rifai, Wa’el; Powell, Steven M

    2002-01-01

    A novel DNA assay demonstrating sensitive and accurate detection of Helicobacter pylori from stool samples is reported. Moreover, in three individuals tested for therapeutic response, the assay showed the disappearance of H. pylori DNA during treatment. Thus, this noninvasive molecular biology-based assay has the potential to be a powerful diagnostic tool given its ability to specifically identify H. pylori DNA.

  11. Microbial diversity in fecal samples depends on DNA extraction method

    DEFF Research Database (Denmark)

    Mirsepasi, Hengameh; Persson, Søren; Struve, Carsten

    2014-01-01

    BACKGROUND: There are challenges, when extracting bacterial DNA from specimens for molecular diagnostics, since fecal samples also contain DNA from human cells and many different substances derived from food, cell residues and medication that can inhibit downstream PCR. The purpose of the study...... was to evaluate two different DNA extraction methods in order to choose the most efficient method for studying intestinal bacterial diversity using Denaturing Gradient Gel Electrophoresis (DGGE). FINDINGS: In this study, a semi-automatic DNA extraction system (easyMag®, BioMérieux, Marcy I'Etoile, France......) and a manual one (QIAamp DNA Stool Mini Kit, Qiagen, Hilden, Germany) were tested on stool samples collected from 3 patients with Inflammatory Bowel disease (IBD) and 5 healthy individuals. DNA extracts obtained by the QIAamp DNA Stool Mini Kit yield a higher amount of DNA compared to DNA extracts obtained...

  12. HUMAN DNA QUANTIFICATION IN THE STOOLS OF PATIENTS WITH COLORECTAL CANCER

    Directory of Open Access Journals (Sweden)

    Yolanda TEIXEIRA

    2015-12-01

    Full Text Available Background - Colorectal cancer is one of the main cause of cancer in the world. Colonoscopy is the best screen method, however the compliance is less than 50%. Quantification of human DNA (hDNA in the feces may be a possible screen non-invasive method that is a consequence of the high proliferation and exfoliation of cancer cells. Objective - To quantify the human DNA in the stools of patients with colorectal cancer or polyps. Methods - Fifty patients with CRC, 26 polyps and 53 with normal colonoscopy were included. Total and human DNA were analyzed from the frozen stools. Results - An increased concentration of hDNA in the stools was observed in colorectal cancer patients compared to controls and polyps. Tumors localized in the left side of the colon had higher concentrations of hDNA. There were no difference between polyps and controls. A cut off of 0.87 ng/mL of human DNA was determined for colorectal cancer patients by the ROC curve, with a sensitivity of 66% and a specificity of 86.8%. For polyps the cut off was 0.41, the sensitivity was 41% and the specificity 77.4%. Conclusion - A higher concentration of hDNA had been found in colorectal cancer patients The quantification of hDNA from the stools can be a trial method for the diagnosis of colorectal cancer.

  13. Molecular Diagnosis of Strongyloides Stercoralis Infection by PCR Detection of Specific DNA in Human Stool Samples

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    Eb Kia

    2011-06-01

    Full Text Available Background: Strongyloidiasis is mostly an asymptomatic infection and diagnosis of latent infec­tions is difficult due to limitations of current parasitological and serological methods. This study was conducted to set up a PCR-based method for molecular diagnosis of Strongyloides stercor­alis infection by detection of copro-DNA in stool samples.Methods: A total of 782 fresh stool samples were collected and examined by agar plate culture. Among those sixteen stool samples, which confirmed to be infected with S. stercoralis were exam­ined as positive control to set up each single and nested PCR, using two primer sets design­ing to amplify partial ribosomal DNA of S. stercoralis genome. Since, single PCR method yielded higher efficacy in detecting positive samples, in the second step, 30 stool samples, which found negative for S. stercoralis by agar plate culture of single stool sample, were examined by sin­gle PCR. Data analysis was performed using McNemar's χ2 test, with consideration of a P-value of <0.05 as indication of significant difference.Results: In amplification of DNA extracted from stool samples, single PCR detected S. stercor­alis DNA target in all 16 positive samples, while nested PCR amplified DNA in only 75% of sam­ples. In the second step, single PCR amplified S. stercoralis extracted DNA in 5 out of 30 sam­ples which were negative by coproculture.Conclusion: Single PCR method amplifying a short (100bp target represented more efficacies for detection of S. stercoralis in faecal examination compared to agar plate culture and nested PCR, which amplified longer target.

  14. An improved DNA isolation technique for PCR detection of Strongyloides stercoralis in stool samples.

    Science.gov (United States)

    Repetto, S A; Alba Soto, C D; Cazorla, S I; Tayeldin, M L; Cuello, S; Lasala, M B; Tekiel, V S; González Cappa, S M

    2013-05-01

    Strongyloides stercoralis is a nematode that causes severe infections in immunocompromised patients. The low parasitic burden of chronically infected patients makes diagnosis difficult to achieve by conventional methods. Here, an in-house (IH) method for the isolation of parasite DNA from stools and a PCR assay for the molecular diagnosis of S. stercoralis were optimized. DNA yield and purity improved with the IH method which included a step of incubation of stool samples with a glycine-SDS buffer and mechanical disruption prior to DNA extraction. For the PCR assay, the addition of bovine serum albumin was required to neutralize inhibitors present in stool. The analytical sensitivity of the PCR using DNA as template, isolated with the IH method, was superior to the commercial one. This study demonstrates that a combined method that adds the step of glycine-SDS buffer incubation plus mechanical disruption prior to DNA isolation with the commercial kit increased PCR sensitivity to levels of the IH method. Finally, our assay was tested on 17 clinical samples. With the IH method for DNA isolation, a S. stercoralis specific band was detected by PCR in the first stool sample in all patients (17/17), while with the commercial kit, our S. stercoralis-specific band was only observed in 7 samples. The superior efficiency of the IH and combined methods over the commercial kit was demonstrated when applied to clinical samples with low parasitic burden. These results show that the DNA extraction procedure is a key to increase sensitivity of the S. stercoralis PCR assay in stool samples. The method developed here could help to improve the molecular diagnosis of S. stercoralis.

  15. Novel circular DNA viruses in stool samples of wild-living chimpanzees.

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    Blinkova, Olga; Victoria, Joseph; Li, Yingying; Keele, Brandon F; Sanz, Crickette; Ndjango, Jean-Bosco N; Peeters, Martine; Travis, Dominic; Lonsdorf, Elizabeth V; Wilson, Michael L; Pusey, Anne E; Hahn, Beatrice H; Delwart, Eric L

    2010-01-01

    Viral particles in stool samples from wild-living chimpanzees were analysed using random PCR amplification and sequencing. Sequences encoding proteins distantly related to the replicase protein of single-stranded circular DNA viruses were identified. Inverse PCR was used to amplify and sequence multiple small circular DNA viral genomes. The viral genomes were related in size and genome organization to vertebrate circoviruses and plant geminiviruses but with a different location for the stem-loop structure involved in rolling circle DNA replication. The replicase genes of these viruses were most closely related to those of the much smaller (approximately 1 kb) plant nanovirus circular DNA chromosomes. Because the viruses have characteristics of both animal and plant viruses, we named them chimpanzee stool-associated circular viruses (ChiSCV). Further metagenomic studies of animal samples will greatly increase our knowledge of viral diversity and evolution.

  16. [Adaptation of a sensitive DNA extraction method for detection of Entamoeba histolytica by real-time polymerase chain reaction].

    Science.gov (United States)

    Pınar, Ahmet; Akyön, Yakut; Alp, Alpaslan; Ergüven, Sibel

    2010-07-01

    This study was aimed to adapt a sensitive DNA extraction protocol in stool samples for real-time polymerase chain reaction (PCR) detection of Entamoeba histolytica which causes important morbidity and mortality worldwide. Stool extraction is a problematic step and has direct effects on PCR sensitivity. In order to improve the sensitivity of E.histolytica detection by real-time PCR, "QIAamp DNA stool minikit (Qiagen, Germany)" was modified by adding an overnight incubation step with proteinase K and sodium dodecyl sulfate (SDS) in this study. Three different extraction methods [(1) original method, (2) cetyltrimethyl-ammonium bromide (CTAB) method, (3) modified method] were evaluated for effects on sensitivity in real-time quantitative PCR (Artus RealArt TM E.histolytica RG PCR Kit, Qiagen Diagnostics, Germany). For this purpose, several concentrations of standard E.histolytica DNA were spiked in parasite-free stool samples and three different extraction protocols were performed. Detection sensitivities of "QIAamp DNA stool minikit" was found 5000 copies/ml and of CTAB method was found 500 copies/ml. Detection sensitivity of the extraction was improved to 5 copies/mL by modified "QIAamp DNA stool minikit" protocol. Since detection sensitivities of nucleic acid extraction protocols from stool samples directly affect the sensitivity of PCR amplification, different extraction protocols for different microorganisms should be evaluated.

  17. Analysis of microsatellite instability in stool DNA of patients with colorectal cancer using denaturing high performance liquid chromatography

    Institute of Scientific and Technical Information of China (English)

    Seok-Byung Lim; Yong Shin; Sang-Geun Jang; Jae-Hyun Park; Jae-Gahb Park; Seung-Yong Jeong; Il-Jin Kim; Dae Yong Kim; Kyung Hae Jung; Hee Jin Chang; Hyo Seong Choi; Dae Kyung Sohn; Hio Chung Kang

    2006-01-01

    AIM: To evaluate the usefulness of denaturing high performance liquid chromatography (DHPLC) for analyzing microsatellite instability (MSI) status in stool DNA of patients with colorectal cancer.METHODS: A total of 80 cancer tissues from patients with primary sporadic colorectal tumor (proximal cancer:27, distal cancer: 53) and matched stool (which were employed for comparison with the tissues) were analyzed for MSI status in BAT 26. DNA samples extracted from stool were evaluated by nested polymerase chain reaction (PCR) and DHPLC for MSI analysis.RESULTS: Six cases (7.5%) of MSI were identified in BAT 26 from 80 cancer tissues. All the stool DNA samples from patients whose cancer tissue showed MSI also displayed MSI in BAT 26.CONCLUSION: As MSI is one of the established fecal DNA markers to screen colorectal cancer, we propose to use DHPLC for the MSI analysis in fecal DNA.

  18. Stool-based DNA testing, a new noninvasive method for colorectal cancer screening, the first report from Iran

    Institute of Scientific and Technical Information of China (English)

    Mohammad Reza Abbaszadegan; Azadeh Aarabi; Alireza Tavasoli; Arash Velayati; Hamid Reza Sima; Hassan Vosooghinia; Mehdi Farzadnia; Hamid Asadzedeh; Mehran Gholamin; Ezzat Dadkhah

    2007-01-01

    AIM: To detect tumor-associated DNA changes in stool samples among Iranian patients with colorectal cancer (CRC) compared to healthy individuals using BAT-26, 16 hypermethylation and long DNA markers.METHODS: Stool DNA was isolated from 45 subjects including 25 CRC patients and 20 healthy individuals using a new, fast and easy extraction method. Long DNA associated with tumor was detected using polymerase chain reaction method. Microsatellite studies were performed utilizing denaturating polyacrylamide gel to determine the instability of BAT-26. Methylation status of p16 promoter was analyzed using methylation-specific PCR (MSP).RESULTS: The results showed a significant difference in existence of long DNA (16 in patients vs 1 in controls, P < 0.001) and/716 (5 in patients vs none in controls, P = 0.043) in the stool samples of two groups. Long DNA was detected in 64% of CRC patients; whereas just one of the healthy individuals was positive for Long DNA. P16 methylation was found in 20% of patients and in none of healthy individuals. Instability of BAT-26 was not detected in any of stool samples.CONCLUSION: We could detect colorectal cancer related genetic alterations by analyzing stool DNA with a sensitivity of 64% and 20% and a specificity of 95% and 100% for Long DNA and p16 respectively.A noninvasive molecular stool-based DNA testing can provide a screening strategy in high-risk individuals.However,additional testing on more samples is necessary from Iranian subjects to determine the exact specificity and sensitivity of these markers.

  19. A New Lab Developed Real Time PCR Assay for Direct Detection of C. Difficle from Stool Sample without DNA Extraction.

    Science.gov (United States)

    Li, Brandon

    2016-09-01

    Clostridium difficile is a major cause of nosocomial antibiotic-associated infectious diarrhea and pseudomembranous colitis. Detection of C. difficile by anaerobic bacterial culture and/or cytotoxicity assays has been largely replaced by rapid enzyme immunoassays (EIA). However, due to the lack of sensitivity of stool EIA, we developed a multiplex real-time PCR assay targeting the C. difficile toxin genes tcdB. stool samples from hospitalized pediatric patients suspected of having C. difficile-associated disease were prospectively collected. Three testing modalities were evaluated, including enriched culture, cepheid Xpert and real-time Pcr (tcdB) on stool samples performed with tcdB gene-specific primers and hydrolysis probes. A total of 150 de-identified clinical specimen were analyzed. The sensitivities of stool real-time Pcr were 95% against cepheid Xpert C. difficile and 93% against enriched culture respectively, with a specificity of 97% and 94%. The lower limit of detection of the stool real-time PCR was 0.5 cFU/ml of per reaction for tcdB. Direct detection of C. difficile toxin genes in stool samples by real-time Pcr showed performance comparable to enriched culture. Real-time PCR of DNA from stool samples is a rapid and cost-effective diagnostic modality for patients that should facilitate appropriate patient management.

  20. A New Lab Developed Real Time PCR Assay for Direct Detection of C. Difficle from Stool Sample without DNA Extraction

    Science.gov (United States)

    Li, Brandon

    2016-01-01

    Clostridium difficile is a major cause of nosocomial antibiotic-associated infectious diarrhea and pseudomembranous colitis. Detection of C. difficile by anaerobic bacterial culture and/or cytotoxicity assays has been largely replaced by rapid enzyme immunoassays (EIA). However, due to the lack of sensitivity of stool EIA, we developed a multiplex real-time PCR assay targeting the C. difficile toxin genes tcdB. stool samples from hospitalized pediatric patients suspected of having C. difficile-associated disease were prospectively collected. Three testing modalities were evaluated, including enriched culture, cepheid Xpert and real-time Pcr (tcdB) on stool samples performed with tcdB gene-specific primers and hydrolysis probes. A total of 150 de-identified clinical specimen were analyzed. The sensitivities of stool real-time Pcr were 95% against cepheid Xpert C. difficile and 93% against enriched culture respectively, with a specificity of 97% and 94%. The lower limit of detection of the stool real-time PCR was 0.5 cFU/ml of per reaction for tcdB. Direct detection of C. difficile toxin genes in stool samples by real-time Pcr showed performance comparable to enriched culture. Real-time PCR of DNA from stool samples is a rapid and cost-effective diagnostic modality for patients that should facilitate appropriate patient management. PMID:27829823

  1. DNA from fecal immunochemical test can replace stool for detection of colonic lesions using a microbiota-based model.

    Science.gov (United States)

    Baxter, Nielson T; Koumpouras, Charles C; Rogers, Mary A M; Ruffin, Mack T; Schloss, Patrick D

    2016-11-14

    There is a significant demand for colorectal cancer (CRC) screening methods that are noninvasive, inexpensive, and capable of accurately detecting early stage tumors. It has been shown that models based on the gut microbiota can complement the fecal occult blood test and fecal immunochemical test (FIT). However, a barrier to microbiota-based screening is the need to collect and store a patient's stool sample. Using stool samples collected from 404 patients, we tested whether the residual buffer containing resuspended feces in FIT cartridges could be used in place of intact stool samples. We found that the bacterial DNA isolated from FIT cartridges largely recapitulated the community structure and membership of patients' stool microbiota and that the abundance of bacteria associated with CRC were conserved. We also found that models for detecting CRC that were generated using bacterial abundances from FIT cartridges were equally predictive as models generated using bacterial abundances from stool. These findings demonstrate the potential for using residual buffer from FIT cartridges in place of stool for microbiota-based screening for CRC. This may reduce the need to collect and process separate stool samples and may facilitate combining FIT and microbiota-based biomarkers into a single test. Additionally, FIT cartridges could constitute a novel data source for studying the role of the microbiome in cancer and other diseases.

  2. Early Adoption of a Multitarget Stool DNA Test for Colorectal Cancer Screening.

    Science.gov (United States)

    Finney Rutten, Lila J; Jacobson, Robert M; Wilson, Patrick M; Jacobson, Debra J; Fan, Chun; Kisiel, John B; Sweetser, Seth; Tulledge-Scheitel, Sidna M; St Sauver, Jennifer L

    2017-05-01

    To characterize early adoption of a novel multitarget stool DNA (MT-sDNA) screening test for colorectal cancer (CRC) screening and to test the hypothesis that adoption differs by demographic characteristics and prior CRC screening behavior and proceeds predictably over time. We used the Rochester Epidemiology Project research infrastructure to assess the use of the MT-sDNA screening test in adults aged 50 to 75 years living in Olmsted County, Minnesota, in 2014 and identified 27,147 individuals eligible or due for screening colonoscopy from November 1, 2014, through November 30, 2015. We used electronic Current Procedure Terminology and Health Care Common Procedure codes to evaluate early adoption of the MT-sDNA screening test in this population and to test whether early adoption varies by age, sex, race, and prior CRC screening behavior. Overall, 2193 (8.1%) and 974 (3.6%) individuals were screened by colonoscopy and MT-sDNA, respectively. Age, sex, race, and prior CRC screening behavior were significantly and independently associated with MT-sDNA screening use compared with colonoscopy use after adjustment for all other variables (Pscreening increased over time and were highest in those aged 50 to 54 years, women, whites, and those who had a history of screening. The use of the MT-sDNA screening test varied predictably by insurance coverage. The rates of colonoscopy decreased over time, whereas overall CRC screening rates remained steady. The results of the present study are generally consistent with predictions derived from prior research and the diffusion of innovation framework, pointing to increasing use of the new screening test over time and early adoption by younger patients, women, whites, and those with prior CRC screening. Copyright © 2017 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc. All rights reserved.

  3. Stool DNA Analysis is Cost-Effective for Colorectal Cancer Surveillance in Patients With Ulcerative Colitis.

    Science.gov (United States)

    Kisiel, John B; Konijeti, Gauree G; Piscitello, Andrew J; Chandra, Tarun; Goss, Thomas F; Ahlquist, David A; Farraye, Francis A; Ananthakrishnan, Ashwin N

    2016-12-01

    Patients with chronic ulcerative colitis are at increased risk for colorectal neoplasia (CRN). Surveillance by white-light endoscopy (WLE) or chromoendoscopy may reduce risk of CRN, but these strategies are underused. Analysis of DNA from stool samples (sDNA) can detect CRN with high levels of sensitivity, but it is not clear if this approach is cost-effective. We simulated these strategies for CRN detection to determine which approach is most cost-effective. We adapted a previously published Markov model to simulate the clinical course of chronic ulcerative colitis, the incidence of cancer or dysplasia, and costs and benefits of care with 4 surveillance strategies: (1) analysis of sDNA and diagnostic chromoendoscopy for patients with positive results, (2) analysis of sDNA with diagnostic WLE for patients with positive results, (3) chromoendoscopy with targeted collection of biopsies, or (4) WLE with random collection of biopsies. Costs were based on 2014 Medicare reimbursement. The primary outcome was the incremental cost-effectiveness ratio (incremental cost/incremental difference in quality-adjusted life-years) compared with no surveillance and a willingness-to-pay threshold of $50,000. All strategies fell below the willingness-to-pay threshold at 2-year intervals. Incremental cost-effectiveness ratios were $16,362 per quality-adjusted life-year for sDNA analysis with diagnostic chromoendoscopy; $18,643 per quality-adjusted life-year for sDNA analysis with diagnostic WLE; $23,830 per quality-adjusted life-year for chromoendoscopy alone; and $27,907 per quality-adjusted life-year for WLE alone. In sensitivity analyses, sDNA analysis with diagnostic chromoendoscopy was more cost-effective than chromoendoscopy alone, up to a cost of $1135 per sDNA test. sDNA analysis remained cost-effective at all rates of compliance; when combined with diagnostic chromoendoscopy, this approach was preferred over chromoendoscopy alone, when the specificity of the sDNA test for CRN

  4. Multitarget stool DNA tests increases colorectal cancer screening among previously noncompliant Medicare patients.

    Science.gov (United States)

    Prince, Mark; Lester, Lynn; Chiniwala, Rupal; Berger, Barry

    2017-01-21

    To determine the uptake of noninvasive multitarget stool DNA (mt-sDNA) in a cohort of colorectal cancer (CRC) screening non-compliant average-risk Medicare patients. This cross sectional primary care office-based study examined mt-sDNA uptake in routine clinical practice among 393 colorectal cancer screening non-compliant Medicare patients ages 50-85 ordered by 77 physicians in a multispecialty group practice (USMD Physician Services, Dallas, TX) from October, 2014-September, 2015. Investigators performed a Health Insurance Portability and Accountability Act compliant retrospective review of electronic health records to identify mt-sDNA use in patients who were either > 10 years since last colonoscopy and/or > 1 year since last fecal occult blood test. Test positive patients were advised to get diagnostic colonoscopy and thereafter patients were characterized by the most clinically significant lesion documented on histopathology of biopsies or excisional tissue. Descriptive statistics were employed. Key outcome measures included mt-sDNA compliance and diagnostic colonoscopy compliance on positive cases. Over 12 mo, 77 providers ordered 393 mt-sDNA studies with 347 completed (88.3% compliance). Patient mean age was 69.8 (50-85) and patients were 64% female. Mt-sDNA was negative in 85.3% (296/347) and positive in 14.7% (51/347). Follow-up colonoscopy was performed in 49 positive patients (96.1% colonoscopy compliance) with two patients lost to follow up. Index findings included: colon cancer (4/49, 8.2%), advanced adenomas (21/49, 42.9%), non-advanced adenomas (15/49, 30.6%), and negative results (9/49, 18.4%). The positive predictive value for advanced colorectal lesions was 51.0% and for any colorectal neoplasia was 81.6%. The mean age of patients with colorectal cancer was 70.3 and all CRC's were localized Stage I (2) and Stage II (2), three were located in the proximal colon and one was located in the distal colon. Mt-sDNA provided medical benefit to screening

  5. Molecular evidence of Orthopoxvirus DNA in capybara (Hydrochoerus hydrochaeris) stool samples.

    Science.gov (United States)

    Dutra, Lara Ambrosio Leal; de Freitas Almeida, Gabriel Magno; Oliveira, Graziele Pereira; Abrahão, Jônatas Santos; Kroon, Erna Geessien; Trindade, Giliane de Souza

    2017-02-01

    Vaccinia virus (VACV) is responsible for outbreaks in Brazil and has immense potential as an emerging virus. VACV can be found naturally circulating in India, Pakistan and South America, where it causes infections characterised by exanthematic lesions in buffaloes, cattle and humans. The transmission cycle of Brazilian VACV has still not been fully characterised; one of the most important gaps in knowledge being the role of wild animals. Capybaras, which are restricted to the Americas, are the world's largest rodents and have peculiar characteristics that make them possible candidates for being part of a natural VACV reservoir. Here, we developed a method for detecting orthopoxvirus DNA in capybara stool samples, and have described for the first time the detection of orthopoxvirus DNA in capybaras samples from three different regions in Brazil. These findings strongly suggest that capybaras might be involved in the natural transmission cycle of VACV and furthermore represent a public health problem, when associated with Brazilian bovine vaccinia outbreaks. This makes infected animals an important factor to be considered when predicting and managing Brazilian VACV outbreaks.

  6. A novel nested multiplex polymerase chain reaction (PCR assay for differential detection of Entamoeba histolytica, E. moshkovskii and E. dispar DNA in stool samples

    Directory of Open Access Journals (Sweden)

    Parija Subhash C

    2007-05-01

    Full Text Available Abstract Background E. histolytica, a pathogenic amoeba, is indistinguishable in its cyst and trophozoite stages from those of non-pathogenic E. moshkovskii and E. dispar by light microscopy. We have developed a nested multiplex PCR targeting a 16S-like rRNA gene for differential detection of all the three morphologically similar forms of E. histolytica, E. moshkovskii and E. dispar simultaneously in stool samples. Results The species specific product size for E. histolytica, E. moshkovskii and E. dispar was 439, 553 and 174 bp respectively, which was clearly different for all the three Entamoeba species. The nested multiplex PCR showed a sensitivity of 94% and specificity of 100% for the demonstration of E. histolytica, E. moshkovskii and E. dispar DNA in stool samples. The PCR was positive for E. histolytica, E. moshkovskii and E. dispar in a total of 190 out of 202 stool specimens (94% sensitive that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture. All the 35 negative control stool samples that were negative for E. histolytica/E. dispar/E. moshkovskii by microscopy and culture were also found negative by the nested multiplex PCR (100% specific. The result from the study shows that only 34.6% of the patient stool samples that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture, were actually positive for pathogenic E. histolytica and the remaining majority of the stool samples were positive for non-pathogenic E. dispar or E. moshkovskii as demonstrated by the use of nested multiplex PCR. Conclusion The present study reports a new nested multiplex PCR strategy for species specific detection and differentiation of E. histolytica, E. dispar and E. moshkovskii DNA in stool specimens. The test is highly specific, sensitive and also rapid, providing the results within 12 hours of receiving stool specimens.

  7. Multitarget stool DNA for colorectal cancer screening:A review and commentary on the United States Preventive Services Draft Guidelines

    Institute of Scientific and Technical Information of China (English)

    Barry M Berger; Bernard Levin; Robert J Hilsden

    2016-01-01

    Multitarget stool DNA(mt-sDNA) testing was approved for average risk colorectal cancer(CRC) screening by the United States Food and Drug Administration and thereafter reimbursed for use by the Medicare program(2014).The United States Preventive Services Task Force(USPSTF) October 2015 draft recommendation for CRC screening included mt-s DNA as an "alternative" screening test that "may be useful in select clinical circumstances",despite its very high sensitivity for early stage CRC.The evidence supporting mt-s DNA for routine screening use is robust.The clinical efficacy of mt-s DNA as measured by sensitivity,specificity,life-years gained(LYG),and CRC deaths averted is similar to or exceeds that of the other more specifically recommended screening options included in the draft document,especially those requiring annual testing adherence.In a population with primarily irregular screening participation,tests with the highest point sensitivity and reasonable specificity are more likely to favorably impact CRC related morbidity and mortality than those depending on annual adherence.This paper reviews the evidence supporting mt-s DNA for routine screening and demonstrates,using USPSTF’s modeling data,that mt-s DNA at three-year intervals provides significant clinical net benefits and fewer complications per LYG than annual fecal immunochemical testing,high sensitivity guaiac based fecal occult blood testing and 10-year colonoscopy screening.

  8. Stool Tests

    Science.gov (United States)

    ... the stomach, intestines, or another part of the gastrointestinal system . A doctor may order a stool collection to ... of bacteria , viruses, or parasites that invade the gastrointestinal system digestive problems, such as the malabsorption of certain ...

  9. A New Lab Developed Real Time PCR Assay for Direct Detection of C. Difficle from Stool Sample without DNA Extraction

    OpenAIRE

    2016-01-01

    Clostridium difficile is a major cause of nosocomial antibiotic-associated infectious diarrhea and pseudomembranous colitis. Detection of C. difficile by anaerobic bacterial culture and/or cytotoxicity assays has been largely replaced by rapid enzyme immunoassays (EIA). However, due to the lack of sensitivity of stool EIA, we developed a multiplex real-time PCR assay targeting the C. difficile toxin genes tcdB. stool samples from hospitalized pediatric patients suspected of having C. difficil...

  10. Detection of Giardia lamblia, Cryptosporidium spp. and Entamoeba histolytica in clinical stool samples by using multiplex real-time PCR after automated DNA isolation.

    Science.gov (United States)

    Van Lint, P; Rossen, J W; Vermeiren, S; Ver Elst, K; Weekx, S; Van Schaeren, J; Jeurissen, A

    2013-01-01

    Diagnosis of intestinal parasites in stool samples is generally still carried out by microscopy; however, this technique is known to suffer from a low sensitivity and is unable to discriminate between certain protozoa. In order to overcome these limitations, a real-time multiplex PCR was evaluated as an alternative approach for diagnosing Giardia lamblia, Cryptosporidium spp. and Entamoeba histolytica in stool samples.Therefore, a total of 631 faecal samples were analysed both by microscopy as well as by real-time PCR following automated DNA extraction. Results showed that real-time PCR exhibited sensitivity and specificity of both 100%, whereas traditional microscopy exhibited sensitivity and specificity of 37.5% and 99.8% respectively. As real-time PCR provides simple, sensitive and specific detection of these three important pathogenic protozoan parasites, this technique, rather than microscopy, has become our diagnostic method of choice for the detection of enteric protozoan parasites for the majority of patients.

  11. Bloody or tarry stools

    Science.gov (United States)

    ... blueberries can also cause black stools. Beets and tomatoes can sometimes make stools appear reddish. In these ... if you notice blood or changes in the color of your stool. You should see your provider ...

  12. Stool Testing for Colorectal Cancer Screening.

    Science.gov (United States)

    Robertson, Douglas J; Imperiale, Thomas F

    2015-10-01

    Colorectal cancer (CRC) screening has been shown to reduce CRC incidence and mortality and is widely recommended. However, despite the demonstrated benefits of screening and ongoing efforts to improve screening rates, a large percentage of the population remains unscreened. Noninvasive stool based tests offer great opportunity to enhance screening uptake. The evidence supporting the use of both fecal immunochemical testing (FIT) and stool DNA (sDNA) has been growing rapidly and both tests are now commercially available for use. Other stool biomarkers (eg, RNA and protein based) are also actively under study both for use independently and as adjuncts to the currently available tests. This mini review provides current, state of the art knowledge about noninvasive stool based screening. It includes a more detailed examination of those tests currently in use (ie, FIT and sDNA) but also provides an overview of stool testing options under development (ie, protein and RNA).

  13. Further evaluation of an updated PCR assay for the detection of Schistosoma mansoni DNA in human stool samples

    Directory of Open Access Journals (Sweden)

    Luciana I Gomes

    2009-12-01

    Full Text Available A previously reported sensitive PCR assay for the detection of Schistosoma mansoni DNA was updated and evaluated. Changes in the DNA extraction method, including the use of a worldwide available commercial kit and the inclusion of additional quality control measures, increased the robustness of the test, as confirmed by the analysis of 67 faecal samples from an endemic area in Brazil. The PCR assay is at hand as a proven, reliable diagnostic test for the control of schistosomiasis in specific settings.

  14. 沼气池污泥微生物总DNA提取方法的比较%Comparison of Three Protocols for Total DNA Extraction of Microorganism in Biogas Digesters

    Institute of Scientific and Technical Information of China (English)

    张琳; 熊格生; 吴莎莎; 卢向阳; 吴俊文; 许凤; 颜亨梅; 刘如石

    2011-01-01

    Fermentation system of biogas production is a complicated ecology system, and more than 99% of the microorganisms in biogas digesters are unculturable. To optimize conditions for cellulose conversion and biogas production in biogas digesters,and also to investigate the microbial diversity in biogas digesters,Chemical lysis method, lysozyme lysis method and QIAamp DNA Stool Mini Kit were used to extract total microbe DNA of biogas digesters, and then the DNA quality, the DNA yield, the size of DNA fragments,and existence of PCR inhibitor were examined,finally PCR-DGGE profiles were carried out to investigate the diversity of microbe in biogas digesters. The results of pulsed-field gel electrophoresis ( PFGE) showed that the DNA obtained from lysozyme lysis method had larger size fragments, better total DNAyield, more widely distributed range of fragments, and more effective PCR amplification efficiency than that of chemical cleavage method and the QIAamp DNA Stool Mini Kit method. The result of PCR and PCR-denaturant gradient gel electrophoresis ( PCR-DGGE) indicated that the DNA templates obtained from lysozyme lysis method had better efficiency of PCR amplification, and revealed an more abundant microbe diversity than that of chemical lysis method and QIAamp DNA Stool Mini Kit. This studies lay a basis to improve the conditions of biogas fermentation and to investigate the quality and quantity of dominant microorganisms.%沼气发酵系统是一个复杂的生态系统,其污泥微生物超过99%是不可培养的.为了优化沼气池纤维素的转化效率、沼气的产率和开展污泥微生物多样性研究,本研究采用化学裂解法、溶菌酶裂解法和QIAamp DNA Stool Mini Kit提取了沼气池污泥样品中微生物的总DNA,对三种方法的DNA得率、纯度、大片段提取效果以及是否含有PCR反应抑制剂进行了研究,最后对16S rRNA基因V3区的扩增产物进行了PCR变性梯度凝胶电泳(PCR-DGGE)分析.与化学裂解法和QIAamp

  15. Comparison of DNA extraction kits and modification of DNA elution procedure for the quantitation of subdominant bacteria from piggery effluents with real-time PCR.

    Science.gov (United States)

    Desneux, Jérémy; Pourcher, Anne-Marie

    2014-08-01

    Four commercial DNA extraction kits and a minor modification in the DNA elution procedure were evaluated for the quantitation of bacteria in pig manure samples. The PowerSoil(®), PowerFecal(®), NucleoSpin(®) Soil kits and QIAamp(®) DNA Stool Mini kit were tested on raw manure samples and on lagoon effluents for their ability to quantify total bacteria and a subdominant bacteria specific of pig manure contamination: Lactobacillus amylovorus. The NucleoSpin(®) Soil kit (NS kit), and to a lesser extent the PowerFecal(®) kit were the most efficient methods. Regardless of the kit utilized, the modified elution procedure increased DNA yield in the lagoon effluent by a factor of 1.4 to 1.8. When tested on 10 piggery effluent samples, compared to the QIAamp kit, the NS kit combined with the modified elution step, increased by a factor up to 1.7 log10 the values of the concentration of L. amylovorus. Regardless of the type of manure, the best DNA quality and the highest concentrations of bacteria were obtained using the NS kit combined with the modification of the elution procedure. The method recommended here significantly improved quantitation of subdominant bacteria in manure. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  16. Stool C difficile toxin

    Science.gov (United States)

    ... toxin; Colitis - toxin; Pseudomembranous - toxin; Necrotizing colitis - toxin; C difficile - toxin ... be analyzed. There are several ways to detect C difficile toxin in the stool sample. Enzyme immunoassay ( ...

  17. Fast and efficient DNA-based method for winter diet analysis from stools of three cervids: moose, red deer, and roe deer.

    Science.gov (United States)

    Czernik, Marta; Taberlet, Pierre; Swisłocka, Magdalena; Czajkowska, Magdalena; Duda, Norbert; Ratkiewicz, Mirosław

    2013-01-01

    Effects of cervid browsing on timber production, especially during winter, lead to economic losses in forest management. The aim of this study was to present an efficient DNA-based method which allows qualitative assessment of the winter diet from stools of moose (Alces alces), roe deer (Capreolus capreolus), and red deer (Cervus elaphus). The preliminary results of the diet composition of the three cervids from Poland were also presented with a special emphasis on moose. The electropherograms of the chloroplast intron trnL (UAA) P6 loop amplification products using g (fluorescence-labeled) and h primers revealed differences in the length of PCR products among various plant species eaten by these herbivores. In addition, the usage of species-specific primers allowed unambiguous identification of different gymnosperms and angiosperms. The preliminary moose diet analysis, based on winter fecal samples from the entire range of moose occurrence in Poland, revealed the presence of 15 to 24 tree, shrub, and herbaceous species. This fast, cost-efficient, and simple method proved also to be reliable for the diet analysis of red deer and roe deer. It may be a valuable tool in forest and conservation management, as well as a way of enhancing ecological studies focusing on the impact of herbivores on the ecosystems and their possible food niche overlap.

  18. Stool Color: When to Worry

    Science.gov (United States)

    Stool color: When to worry Yesterday, my stool color was bright green. Should I be concerned? Answers from Michael ... M.D. Stool comes in a range of colors. All shades of brown and even green are ...

  19. Cytotoxicity of Ru(II) piano-stool complexes with chloroquine and chelating ligands against breast and lung tumor cells: Interactions with DNA and BSA.

    Science.gov (United States)

    Colina-Vegas, Legna; Villarreal, Wilmer; Navarro, Maribel; de Oliveira, Clayton Rodrigues; Graminha, Angélica E; Maia, Pedro Ivo da S; Deflon, Victor M; Ferreira, Antonio G; Cominetti, Marcia Regina; Batista, Alzir A

    2015-12-01

    The synthesis and spectroscopic characterization of nine π-arene piano-stool ruthenium (II) complexes with aromatic dinitrogen chelating ligands or containing chloroquine (CQ), are described in this study: [Ru(η(6)-C10H14)(phen)Cl]PF6 (1), [Ru(η(6)-C10H14)(dphphen)Cl]PF6 (2), [Ru(η(6)-C10H14)(bipy)Cl]PF6 (3), [Ru(η(6)-C10H14)(dmebipy)Cl]PF6 (4) and [Ru(η(6)-C10H14)(bdutbipy)Cl]PF6 (5), [Ru(η(6)-C10H14)(phen)CQ](PF6)2 (6), [Ru(η(6)-C10H14)(dphphen)CQ](PF6)2 (7), [Ru(η(6)-C10H14)(bipy)CQ](PF6)2 (8), [Ru(η(6)-C10H14)(dmebipy)CQ](PF6)2 (9): [1,10-phenanthroline (phen), 4,7-diphenyl-1,10-phenanthroline (dphphen), 2,2'-bipyridine (bipy), 5,5'-dimethyl-2,2'-bipyridine (dmebipy), and 4,4'-di-t-butyl-2,2'-bipyridine (dbutbipy)]. The solid state structures of five ruthenium complexes (1-5) were determined by X-ray crystallography. Electrochemical experiments were performed by cyclic voltammetry to estimate the redox potential of the Ru(II)/Ru(III) couple in each case. Their interactions with DNA and BSA, and activity against four cell lines (L929, A549, MDA-MB-231 and MCF-7) were evaluated. Compounds 2, 6 through 9, interact with DNA which was comparable to the one observed for free chloroquine. The results of fluorescence titration revealed that these complexes strongly quenched the intrinsic fluorescence of BSA following a static quenching procedure. Binding constants (Kb) and the number of binding sites (n~1) were calculated using modified Stern-Volmer equations. The thermodynamic parameters ΔG at different temperatures were calculated and subsequently the values of ΔH and ΔS were also calculated, which revealed that hydrophobic and electrostatic interactions play a major role in the BSA-complex association. The MTT assay results indicated that complexes 2, 5 and 7 showed cytostatic effects at appreciably lower concentrations than those needed for cisplatin, chloroquine and doxorubicin.

  20. Detection of Giardia lamblia, Cryptosporidium spp. and Entamoeba histolytica in clinical stool samples by using multiplex real-time PCR after automated DNA isolation

    NARCIS (Netherlands)

    Van Lint, P; Rossen, J W; Vermeiren, S; Ver Elst, K; Weekx, S; Van Schaeren, J; Jeurissen, A

    2013-01-01

    Diagnosis of intestinal parasites in stool samples is generally still carried out by microscopy; however, this technique is known to suffer from a low sensitivity and is unable to discriminate between certain protozoa. In order to overcome these limitations, a real-time multiplex PCR was evaluated a

  1. Detection of Giardia lamblia, Cryptosporidium spp. and Entamoeba histolytica in clinical stool samples by using multiplex real-time PCR after automated DNA isolation

    NARCIS (Netherlands)

    Van Lint, P; Rossen, J W; Vermeiren, S; Ver Elst, K; Weekx, S; Van Schaeren, J; Jeurissen, A

    2013-01-01

    Diagnosis of intestinal parasites in stool samples is generally still carried out by microscopy; however, this technique is known to suffer from a low sensitivity and is unable to discriminate between certain protozoa. In order to overcome these limitations, a real-time multiplex PCR was evaluated

  2. Screening for enterotoxigenic Bacteroides fragilis in stool samples.

    Science.gov (United States)

    Keenan, Jacqueline I; Aitchison, Alan; Purcell, Rachel V; Greenlees, Rosie; Pearson, John F; Frizelle, Frank A

    2016-08-01

    Bacteroides fragilis is a commensal bacterium found in the gut of most humans, however enterotoxigenic B. fragilis strains (ETBF) have been associated with diarrhoea and colorectal cancer (CRC). The purpose of this study was to establish a method of screening for the Bacteroides fragilis toxin (bft) gene in stool samples, as a means of determining if carriage of ETBF is detected more often in CRC patients than in age-matched healthy controls. Stool samples from 71 patients recently diagnosed with CRC, and 71 age-matched controls, were screened by standard and quantitative PCR using primers specific for the detection of the bft gene. Bacterial template DNA from stool samples was prepared by two methods: a sweep, where all colonies growing on Bacteroides Bile Esculin agar following stool culture for 48 h at 37 °C in an anaerobic environment were swept into sterile water and heat treated; and a direct DNA extraction from each stool sample. The bft gene was detected more frequently from DNA isolated from bacterial sweeps than from matched direct DNA extractions. qPCR was found to be more sensitive than standard PCR in detecting bft. The cumulative total of positive qPCR assays from both sample types revealed that 19 of the CRC patients had evidence of the toxin gene in their stool sample (27%), compared to seven of the age-matched controls (10%). This difference was significant (P = 0.016). Overall, ETBF carriage was detected more often in CRC patient stool samples compared to controls, but disparate findings from the different DNA preparations and testing methods suggests that poor sensitivity may limit molecular detection of ETBF in stool samples. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Isolation of Campylobacter from human stool samples

    Directory of Open Access Journals (Sweden)

    S M Salim

    2014-01-01

    Full Text Available Context: Campylobacter is an undetected cause of diarrhoea especially under 5 years of age in most of the countries. Isolation of this organism is difficult, expensive and cumbersome. Aims: Our objective of this study was to isolate this pathogen from the stool specimens on routinely available blood containing laboratory media using the candle jar for creating the microaerophilic atmosphere in our setup. Settings and Designs: A descriptive study. Materials and Methods: A total of 50 stool samples were inoculated onto selective and non-selective media with and without filtration using a 0.45 μm membrane. The inoculated media were simultaneously incubated in microaerophilic conditions using the Anoxomat as well as in candle jars at temperatures 37°C and 42°C. The culture isolates were confirmed by standard phenotypic tests. A simplex polymerase chain reaction (PCR targeting the 16S ribosomal deoxyribonucleic acid of Campylobacter was performed on the deoxyribonucleic acid (DNA of the culture isolates as well as on the DNA extracted from the stool filtrates. Statistical Analysis: Data was expressed as a proportion. Results: Campylobacter could be isolated in 5 out of 50 stool samples using both the Anoxomat as well as the candle jar. Furthermore, we did not find any difference between the isolation using the selective and blood containing media as well as the different incubation temperatures. All the five were confirmed phenotypically and genotypically to be Campylobacter jejuni. The PCR results corroborated with that of the culture. Conclusions: Isolation by culture was as sensitive as that of the PCR.

  4. Comparison of seven methods for extraction of bacterial DNA from fecal and cecal samples of mice.

    Science.gov (United States)

    Ferrand, Janina; Patron, Kevin; Legrand-Frossi, Christine; Frippiat, Jean-Pol; Merlin, Christophe; Alauzet, Corentine; Lozniewski, Alain

    2014-10-01

    Analysis of bacterial DNA from fecal samples of mice is commonly performed in experimental studies. Although DNA extraction is a critical step in various molecular approaches, the efficiency of methods that may be used for DNA extraction from mice fecal samples has never been evaluated. We compared the efficiencies of six widely used commercial kits (MasterPure™ Gram Positive DNA Purification Kit, QIAamp® DNA Stool Mini Kit; NucliSENS® easyMAG®, ZR Fecal DNA MiniPrep™, FastDNA® SPIN Kit for Feces and FastDNA® SPIN Kit for Soil) and a non-commercial method for DNA isolation from mice feces and cecal contents. DNA quantity and quality were assessed by fluorometry, spectrophotometry, gel electrophoresis and qPCR. Cell lysis efficiencies were evaluated by qPCR targeting three relevant bacteria in spiked specimens. For both feces and intestinal contents, the most efficient extraction method was the FastDNA® SPIN Kit for Soil. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Microbial food safety: Potential of DNA extraction methods for use in diagnostic metagenomics.

    Science.gov (United States)

    Josefsen, Mathilde H; Andersen, Sandra C; Christensen, Julia; Hoorfar, Jeffrey

    2015-07-01

    The efficiency of ten widely applied DNA extraction protocols was evaluated for suitability for diagnostic metagenomics. The protocols were selected based on a thorough literature study. Chicken fecal samples inoculated with about 1×10(3) and 1×10(6) CFU/g Campylobacter jejuni were used as a model. The evaluation was performed based on total DNA yield measured by fluorometry, and quality and quantity of C. jejuni DNA measured by real-time PCR. There was up to a 25-fold variance between the lowest (NucliSens miniMAG, BIOMÉRIEUX) and highest (PowerLyzer PowerSoil DNA Isolation Kit, MO BIO Laboratories) yielding protocols. The PowerLyzer PowerSoil DNA Isolation Kit performed significantly better than all other protocols tested. Selected protocols were modified, i.e., extended heating and homogenization, resulting in increased yields of total DNA. For QIAamp Fast DNA Stool Mini Kit (Qiagen) a 7-fold increase in total DNA was observed following the protocol for human DNA analysis and including a 5 min heating step at 70°C. For the PowerLyzer PowerSoil and the PowerFecal DNA Isolation Kit (MO BIO Laboratories) the total DNA fold increase was 1.6 to 1.8 when including an extra 10 min of bead-vortexing. There was no correlation between the yield of total DNA and the amount of PCR-amplifiable DNA from C. jejuni. The protocols resulting in the highest yield of total DNA did not show correspondingly increased levels of C. jejuni DNA as determined by PCR. In conclusion, substantial variation in the efficiency of the protocols to extract DNA was observed. The highest DNA yield was obtained with the PowerLyzer PowerSoil DNA Isolation Kit, whereas the FastDNA SPIN Kit for Feces (MP Biomedicals) resulted in the highest amount of PCR-amplifiable C. jejuni DNA.

  6. Flushable reagent stool blood test

    Science.gov (United States)

    Stool occult blood test - flushable home test; Fecal occult blood test - flushable home test ... This test is performed at home with disposable pads. You can buy the pads at the drug store without ...

  7. Application of Stool-PCR test for diagnosis of Helicobacter pylori infection in children

    Institute of Scientific and Technical Information of China (English)

    Tahereh Falsafi; Raha Favaedi; Fatemeh Mahjoub; Mehri Najafi

    2009-01-01

    AIM: To evaluate the usefulness of stool-PCR test for diagnosis of Helicobacter pylori ( H pylori) infection in pediatric populations.METHODS: Based on endoscopic features (including nodular gastritis, erosive duodenitis and ulcer) and/or a positive rapid urease test (RUT) obtained during endoscopy, 28 children from a group of children admitted to the Children's Medical Center of Tehran for persistent upper gastrointestinal problems were selected to compare biopsy-based tests with stool-PCR. Their gastric activity and bacterial density were graded by the updated Sydney system, and their first stool after endoscopy was stored at -70℃. Biopsies were cultured on modified campy-blood agar plates and identified by gram-staining, biochemical tests, and PCR. Two methods of phenol-chloroform and boiling were used for DNA extraction from H pylori isolates.Isolation of DNA from stool was performed using a stool DNA extraction kit (Bioneer Inc, Korea). PCR was performed using primers for detection of vacA, cagA,and 16srRNA genes in both isolates and stool.RESULTS: Sixteen out of 28 child patients (57%) were classified as H pylori positive by biopsy-based tests, of which 11 (39%) were also positive by stool-PCR. Sensitivity and specificity of stool-PCR was 62.5% and 92.3% respectively. H pylori was observed in histological sections for 10 out of 11 stool-positive patients. Association was observed between higher score of H pylori in histology and positivity of stool-PCR. Also association was observed between the more severe form of gastritis and a positive stool-PCR.CONCLUSION: Association between higher score of H pylori in histology and a positive stool-PCR make it a very useful test for detection of H pylori active infection in children. We also suggest that a simple stool-PCR method can be a useful test for detection of H pylori virulence genes in stool.

  8. Evaluation of five commercial methods for the extraction and purification of DNA from human faecal samples for downstream molecular detection of the enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis, and Entamoeba spp.

    Science.gov (United States)

    Paulos, Silvia; Mateo, Marta; de Lucio, Aida; Hernández-de Mingo, Marta; Bailo, Begoña; Saugar, José M; Cardona, Guillermo A; Fuentes, Isabel; Mateo, María; Carmena, David

    2016-08-01

    High quality, pure DNA is required for ensuring reliable and reproducible results in molecular diagnosis applications. A number of in-house and commercial methods are available for the extraction and purification of genomic DNA from faecal material, each one offering a specific combination of performance, cost-effectiveness, and easiness of use that should be conveniently evaluated in function of the pathogen of interest. In this comparative study the marketed kits QIAamp DNA stool mini (Qiagen), SpeedTools DNA extraction (Biotools), DNAExtract-VK (Vacunek), PowerFecal DNA isolation (MoBio), and Wizard magnetic DNA purification system (Promega Corporation) were assessed for their efficacy in obtaining DNA of the most relevant enteric protozoan parasites associated to gastrointestinal disease globally. A panel of 113 stool specimens of clinically confirmed patients with cryptosporidiosis (n=29), giardiasis (n=47) and amoebiasis by Entamoeba histolytica (n=3) or E. dispar (n=10) and apparently healthy subjects (n=24) were used for this purpose. Stool samples were aliquoted in five sub-samples and individually processed by each extraction method evaluated. Purified DNA samples were subsequently tested in PCR-based assays routinely used in our laboratory. The five compared methods yielded amplifiable amounts of DNA of the pathogens tested, although performance differences were observed among them depending on the parasite and the infection burden. Methods combining chemical, enzymatic and/or mechanical lysis procedures at temperatures of at least 56°C were proven more efficient for the release of DNA from Cryptosporidium oocysts. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Comparison of two methods of bacterial DNA extraction from human fecal samples contaminated with Clostridium perfringens, Staphylococcus aureus, Salmonella Typhimurium, and Campylobacter jejuni.

    Science.gov (United States)

    Kawase, Jun; Kurosaki, Morito; Kawakami, Yuta; Kashimoto, Takashi; Tsunomori, Yoshie; Sato, Koji; Ikeda, Tetsuya; Yamaguchi, Keiji; Watahiki, Masanori; Shima, Tomoko; Kameyama, Mitsuhiro; Etoh, Yoshiki; Horikawa, Kazumi; Fukushima, Hiroshi; Goto, Ryoichi; Shirabe, Komei

    2014-01-01

    In this study, 2 methods of DNA extraction were evaluated for use in conjunction with the screening system Rapid Foodborne Bacterial Screening 24 (RFBS24), which employs multiplex real-time SYBR Green polymerase chain reaction (SG-PCR) and can simultaneously detect 24 target genes of foodborne pathogens in fecal DNA samples. The QIAamp DNA Stool mini kit (Qkit) and Ultra Clean Fecal DNA Isolation Kit (Ukit) were used for bacterial DNA extraction from fecal samples artificially inoculated with Clostridium perfringens, Staphylococcus aureus, Salmonella Typhimurium, and Campylobacter jejuni. SG-PCR and simplex real-time quantitative PCR (S-qPCR) analyses revealed higher copy numbers (8-234 times) of DNA in samples obtained using Ukit compared with those obtained using Qkit, resulting in lower cycle threshold values for the Ukit samples of the 4 bacteria on SG-PCR analysis. Fecal DNA samples from patients infected during foodborne outbreaks of Salmonella and Campylobacter were also prepared by Qkit and Ukit methods and subjected to RFBS24 analyses. Higher numbers of RFBS24 bacterial target genes were detected in DNA samples obtained using Ukit compared with those obtained using Qkit. Thus, the higher DNA extraction efficiency of the Ukit method compared with Qkit renders the former more useful in achieving improved detection rates of these 4 bacteria in fecal samples using SG-PCR.

  10. A comparison of the efficiency of five different commercial DNA extraction kits for extraction of DNA from faecal samples.

    Science.gov (United States)

    Claassen, Shantelle; du Toit, Elloise; Kaba, Mamadou; Moodley, Clinton; Zar, Heather J; Nicol, Mark P

    2013-08-01

    Differences in the composition of the gut microbiota have been associated with a range of diseases using culture-independent methods. Reliable extraction of nucleic acid is a key step in identifying the composition of the faecal microbiota. Five widely used commercial deoxyribonucleic acid (DNA) extraction kits (QIAsymphony® Virus/Bacteria Midi Kit (kit QS), ZR Fecal DNA MiniPrep™ (kit Z), QIAamp® DNA Stool Mini Kit (kit QA), Ultraclean® Fecal DNA Isolation Kit (kit U) and PowerSoil® DNA Isolation Kit (kit P)) were evaluated, using human faecal samples. Yield, purity and integrity of total genomic DNA were compared spectrophotometrically and using gel electrophoresis. Three bacteria, commonly found in human faeces were quantified using real time polymerase chain reaction (qPCR) and total bacterial diversity was studied using denaturing gradient gel electrophoresis (DGGE) as well as terminal restriction fragment length polymorphism (T-RFLP). The measurements of DNA yield and purity exhibited variations between the five kits tested in this study. Automated kit QS exhibited the best quality and highest quantity of DNA. All kits were shown to be reproducible with CV values≤0.46 for DNA extraction. qPCR results showed that all kits were uniformly efficient for extracting DNA from the selected target bacteria. DGGE and T-RFLP produced the highest diversity scores for DNA extracted using kit Z (H'=2.30 and 1.27) and kit QS (H'=2.16 and 0.94), which also extracted the highest DNA yields compared to the other kits assessed. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Evaluating variation in human gut microbiota profiles due to DNA extraction method and inter-subject differences

    Directory of Open Access Journals (Sweden)

    Brett eWagner Mackenzie

    2015-02-01

    Full Text Available The human gut contains dense and diverse microbial communities which have profound influences on human health. Gaining meaningful insights into these communities requires provision of high quality microbial nucleic acids from human fecal samples, as well as an understanding of the sources of variation and their impacts on the experimental model. We present here a systematic analysis of commonly used microbial DNA extraction methods, and identify significant sources of variation. Five extraction methods (Human Microbiome Project protocol, MoBio PowerSoil DNA Isolation Kit, QIAamp DNA Stool Mini Kit, ZR Fecal DNA MiniPrep, phenol:chloroform-based DNA isolation were evaluated based on the following criteria: DNA yield, quality and integrity, and microbial community structure based on Illumina amplicon sequencing of the V4 region of bacterial and archaeal 16S rRNA genes. Our results indicate that the largest portion of variation within the model was attributed to differences between subjects (biological variation, with a smaller proportion of variation associated with DNA extraction method (technical variation and intra-subject variation. A comprehensive understanding of the potential impact of technical variation on the human gut microbiota will help limit preventable bias, enabling more accurate diversity estimates.

  12. Comparison of DNA extraction kits for detection of Burkholderia pseudomallei in spiked human whole blood using real-time PCR.

    Directory of Open Access Journals (Sweden)

    Nicole L Podnecky

    Full Text Available Burkholderia pseudomallei, the etiologic agent of melioidosis, is endemic in northern Australia and Southeast Asia and can cause severe septicemia that may lead to death in 20% to 50% of cases. Rapid detection of B. pseudomallei infection is crucial for timely treatment of septic patients. This study evaluated seven commercially available DNA extraction kits to determine the relative recovery of B. pseudomallei DNA from spiked EDTA-containing human whole blood. The evaluation included three manual kits: the QIAamp DNA Mini kit, the QIAamp DNA Blood Mini kit, and the High Pure PCR Template Preparation kit; and four automated systems: the MagNAPure LC using the DNA Isolation Kit I, the MagNAPure Compact using the Nucleic Acid Isolation Kit I, and the QIAcube using the QIAamp DNA Mini kit and the QIAamp DNA Blood Mini kit. Detection of B. pseudomallei DNA extracted by each kit was performed using the B. pseudomallei specific type III secretion real-time PCR (TTS1 assay. Crossing threshold (C T values were used to compare the limit of detection and reproducibility of each kit. This study also compared the DNA concentrations and DNA purity yielded for each kit. The following kits consistently yielded DNA that produced a detectable signal from blood spiked with 5.5×10(4 colony forming units per mL: the High Pure PCR Template Preparation, QIAamp DNA Mini, MagNA Pure Compact, and the QIAcube running the QIAamp DNA Mini and QIAamp DNA Blood Mini kits. The High Pure PCR Template Preparation kit yielded the lowest limit of detection with spiked blood, but when this kit was used with blood from patients with confirmed cases of melioidosis, the bacteria was not reliably detected indicating blood may not be an optimal specimen.

  13. The impact of different DNA extraction methods on the analysis of microbial diversity of oral saliva from healthy youths by polymerase chain reaction-denaturing gradient gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Ming Chen

    2016-03-01

    Conclusion: PCR-DGGE was more accurate in assessing oral microbial diversity by QIAamp DNA Micro Kit. Different individuals had large differences in oral microbial diversity but also had some common microbial dominant communities.

  14. Amplifiability of mitochondrial, microsatellite and amelogenin DNA loci from fecal samples of red brocket deer Mazama americana (Cetartiodactyla, Cervidae).

    Science.gov (United States)

    Oliveira, M L; Duarte, J M B

    2013-01-16

    We tried to amplify mitochondrial, microsatellite and amelogenin loci in DNA from fecal samples of a wild Mazama americana population. Fifty-two deer fecal samples were collected from a 600-ha seasonal semideciduous forest fragment in a subtropical region of Brazil (21°20'S, 47°17'W), with the help of a detection dog; then, stored in ethanol and georeferenced. Among these samples 16 were classified as "fresh" and 36 as "non-fresh". DNA was extracted using the QIAamp(®) DNA Stool Mini Kit. Mitochondrial loci were amplified in 49 of the 52 samples. Five microsatellite loci were amplified by PCR; success in amplification varied according to locus size and sample age. Successful amplifications were achieved in 10/16 of the fresh and in 13/36 of the non-fresh samples; a negative correlation (R = -0.82) was found between successful amplification and locus size. Amplification of the amelogenin locus was successful in 22 of the 52 samples. The difficulty of amplifying nuclear loci in DNA samples extracted from feces collected in the field was evident. Some methodological improvements, including collecting fresh samples, selecting primers for shorter loci and quantifying the extracted DNA by real-time PCR, are suggested to increase amplification success in future studies.

  15. A new method to improve the yield of fetal cell free DNA%一种提高孕妇外周血中游离胎儿DNA产量的新方法

    Institute of Scientific and Technical Information of China (English)

    王谦; 白宁; 郭珊珊; 李凡

    2014-01-01

    目的:探讨提高孕妇外周血中游离胎儿 DNA产量的新方法。方法用QIAamp DNA Blood Mini Kit和QIAamp Circulating Nucleic Acid Kit提取56名孕妇外周血血浆DNA。应用荧光定量聚合酶链反应(PCR)分析样本中游离胎儿DNA的含量。结果与QIAamp DNA Blood Mini Kit相比,经QIAamp Circulating Nucleic Acid Kit提取试剂盒提取的总DNA平均浓度是前者的1.5倍。29名早期妊娠孕妇外周血游离胎儿DNA平均浓度为812拷贝/mL ,27例晚期妊娠为4876拷贝/mL。29名男性胎儿SRY基因平均浓度是前者1.2倍,妊娠早期平均浓度为28.6拷贝/mL ,晚期平均浓度为264拷贝/mL。结论经改良后,采用QIAamp Circulating Nucleic Acid Kit提取试剂盒可以获得更高浓度的胎儿游离 DNA ,可以应用于孕早期和孕中期的无创性产前基因诊断。%Objective To investigate a new method that can improve the yield of fetal cell free DNA .Meth-ods Fetal cell free DNA was extracted from 56 maternal plasma by QIAamp DNA Blood Mini Kit and QIAamp Circulating Nucleic Acid Kit .Results Compared with QIAamp DNA Blood Mini Kit ,the average concentration of total DNA extracted by QIAamp Circulating Nucleic Acid Kit were 1.5 times .Twenty-nine cases of early preg-nancy were 812 copies/mL ,27 cases of late pregnancy is 4 876 copies/mL .Average concentration of 29 cases of male fetal SRY gene were 1.2 times of QIAamp DNA Blood Mini Kit .Average concentration in early pregnancy is 28.6 copies/mL ,that of late is 264 copies/mL .Conclusion We can get a higher concentration of fetal cell free DNA using modified QIAamp Circulating Nucleic Acid Kit .It can be applied in non-invasive prenatal diagnosis of early and mid-term pregnancy .

  16. Evaluation of four different systems for extraction of RNA from stool suspensions using MS-2 coliphage as an exogenous control for RT-PCR inhibition.

    Directory of Open Access Journals (Sweden)

    Lester M Shulman

    Full Text Available Knowing when, and to what extent co-extracted inhibitors interfere with molecular RNA diagnostic assays is of utmost importance. The QIAamp Viral RNA Mini Kit (A; MagNA Pure LC2.0 Automatic extractor (B; KingFisher (C; and NucliSENS EasyMag (D RNA extraction systems were evaluated for extraction efficiency and co-purification of inhibitors from stool suspensions. Real-Time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR of MS-2 coliphage spiked into each system's lysis buffer served as an external control for both. Cycle thresholds (Cts of the MS2 were determined for RNA extracted from stool suspensions containing unknown (n = 93 or varying amounts of inhibitors (n = 92. Stool suspensions from the latter group were also used to determine whether MS-2 and enterovirus rRT-PCR inhibitions were correlated. Specifically 23 RNA extracts from stool suspensions were spiked with enterovirus RNA after extraction and 13 of these stool suspension were spiked with intact enterovirus before extraction. MS2 rRT-PCR inhibition varied for RNAs extracted by the different systems. Inhibition was noted in 12 (13.0%, 26 (28.3%, 7 (7.6%, and 7 (7.6% of the first 93 RNA extracts, and 58 (63.0%, 55 (59.8%, 37 (40.2% and 30 (32.6% of the second 92 extracts for A, B, C, and D, respectively. Furthermore, enterovirus rRT-PCR inhibition correlated with MS2 rRT-PCR inhibition for added enterovirus RNA or virus particles. In conclusion, rRT-PCR for MS-2 RNA is a good predictor of inhibition of enterovirus RNA extracted from stool suspensions. EasyMag performed the best, however all four extraction methods were suitable provided that external controls identified problematic samples.

  17. Evaluation of proper height for squatting stool.

    Science.gov (United States)

    Jung, Hwa S; Jung, Hyung-Shik

    2008-05-01

    Many jobs and activities in people's daily lives have them in squatting postures. Jobs such as housekeeping, farming and welding require various squatting activities. It is speculated that prolonged squatting without any type of supporting stool would gradually and eventually impose musculoskeletal injuries on workers. This study aims to examine the proper height of the stool according to the position of working materials for the squatting worker. A total of 40 male and female college students and 10 female farmers participated in the experiment to find the proper stool height. Student participants were asked to sit and work in three different positions: floor level of 50 mm; ankle level of 200 mm; and knee level of 400 mm. They were then provided with stools of various heights and asked to maintain a squatting work posture. For each working position, they were asked to write down their thoughts on a preferred stool height. A Likert summated rating method as well as pairwise ranking test was applied to evaluate user preference for provided stools under conditions of different working positions. Under a similar experimental procedure, female farmers were asked to indicate their body part discomfort (BPD) on a body chart before and after performing the work. Statistical analysis showed that comparable results were found from both evaluation measures. When working position is below 50 mm, the proper stool height is 100 or should not be higher than 150 mm. When working position is 200 mm, the proper stool height is 150 mm. When working position is 400 mm, the proper stool height is 200 mm. Thus, it is strongly recommended to use proper height of stools with corresponding working position. Moreover, a wearable chair prototype was designed so that workers in a squatting posture do not have to carry and move the stool from one place to another. This stool should ultimately help to relieve physical stress and hence promote the health of squatting workers. This study sought

  18. DNA extraction from protozoan oocysts/cysts in feces for diagnostic PCR.

    Science.gov (United States)

    Hawash, Yousry

    2014-06-01

    PCR detection of intestinal protozoa is often restrained by a poor DNA recovery or by inhibitors present in feces. The need for an extraction protocol that can overcome these obstacles is therefore clear. QIAamp® DNA Stool Mini Kit (Qiagen) was evaluated for its ability to recover DNA from oocysts/cysts directly from feces. Twenty-five Giardia-positive, 15 Cryptosporidium-positive, 15 Entamoeba histolytica-positive, and 45 protozoa-free samples were processed as control by microscopy and immunoassay tests. DNA extracts were amplified using 3 sets of published primers. Following the manufacturer's protocol, the kit showed sensitivity and specificity of 100% towards Giardia and Entamoeba. However, for Cryptosporidium, the sensitivity and specificity were 60% (9/15) and 100%, respectively. A series of optimization experiments involving various steps of the kit's protocol were conducted using Cryptosporidium-positive samples. The best DNA recoveries were gained by raising the lysis temperature to the boiling point for 10 min and the incubation time of the InhibitEX tablet to 5 min. Also, using a pre-cooled ethanol for nucleic acid precipitation and small elution volume (50-100 µl) were valuable. The sensitivity of the amended protocol to Cryptosporidium was raised to 100%. Cryptosporidium DNA was successfully amplified by either the first or the second primer set. When applied on parasite-free feces spiked with variable oocysts/cysts counts, ≈ 2 oocysts/cysts were theoretically enough for detection by PCR. To conclude, the Qiagen kit with the amended protocol was proved to be suitable for protozoan DNA extraction directly from feces and support PCR diagnosis.

  19. Pregnancy Constipation: Are Stool Softeners Safe?

    Science.gov (United States)

    Healthy Lifestyle Pregnancy week by week Is it safe to take stool softeners to treat pregnancy constipation? Answers from Roger ... 2014 Original article: http://www.mayoclinic.org/healthy-lifestyle/pregnancy-week-by-week/expert-answers/pregnancy-constipation/faq- ...

  20. A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle.

    Science.gov (United States)

    Majumdar, Gipsy; Vera, Santiago; Elam, Marshall B; Raghow, Rajendra

    2015-04-01

    We combined the TRIzol method of nucleic acid extraction with QIAamp columns to achieve coextraction of RNA and genomic DNA from peripheral blood mononuclear cells (PBMCs) and biopsied skeletal muscle, both stored at -80 °C for many months. Total RNA was recovered from the upper aqueous phase of TRIzol. The interphase and organic phases were precipitated with ethanol, digested with proteinase K, and filtered through QIAamp MinElute columns to recover DNA. The combined protocol yielded excellent quality and quantity of nucleic acids from archived human PBMCs and muscle and may be easily adapted for other tissues.

  1. Microscale sample preparation for PCR of C. difficile infected stool

    Science.gov (United States)

    Gillers, Sara; Atkinson, Christopher D.; Bartoo, Aaron C.; Mahalanabis, Madhumita; Boylan, Michael O.; Schwartz, John H.; Klapperich, Catherine; Singh, Satish K.

    2015-01-01

    In this paper, we describe the design of a microfluidic sample preparation chip for human stool samples infected with Clostridium difficile. We established a polymerase chain reaction able to distinguish C. difficile in the presence of several other organisms found in the normal intestinal flora. A protocol for on-chip extraction of nucleic acids from clinical samples is described that can detect target DNA down to 5.0×10−3 ng of template. The assay and sample preparation chip were then validated using known positive and known negative clinical samples. The work presented has potential applications in both the developed and developing world. PMID:19505511

  2. 三种粪便总DNA提取方法的比较%A comparison of three protocols for the extraction of total fecal DNA

    Institute of Scientific and Technical Information of China (English)

    申剑; 张宝让; 魏华; 庞小燕; 赵立平

    2008-01-01

    目的 比较不同粪便总DNA提取方法对肠道菌群多样性研究的影响.方法 采用Bead beating法、化学裂解法和QIAamp DNA Stool Mini Kit提取同一份人粪便样品的总DNA,对比3种方法的DNA得率和16SrRNA基因V3区的变性梯度凝胶电泳(DGGE)图谱.结果 Bead beating法的DNA得率约是其他2种方法的2倍;3种方法得到的DGGE图谱的Dice相似性为60%~70%,2条优势条带只出现在Bead beating法图谱中.在2~5min的Bead beating法击打时间里,DNA得率随击打时间的延长有一定的增加,但DGGE图谱无显著变化.结论 不同的DNA提取方法会影响菌群的多样性分析.比较其他2种方法,Bead beating的裂解效率更高,能够检测到更多种类的细菌,更合适肠道菌群组成的分子研究.

  3. Evaluation of ergonomic dental stools through clinical simulation.

    Science.gov (United States)

    Parsell, D E; Weber, M D; Anderson, B C; Cobb, G W

    2000-01-01

    Work-related musculoskeletal pain occurs commonly within the dental community. Three stool designs were utilized in this study: a standard dental stool, a stool with dual arm supports, and a stool with dual arm supports and chest support. Electromyographic data from four muscle groups were collected on 13 clinicians during a simulated crown preparation procedure. Clinical simulation suggests that a potential musculoskeletal benefit to the clinician exists through utilization of dental stool designs which incorporate static arm supports.

  4. Physical Pre-Treatment Improves Efficient DNA Extraction and qPCR Sensitivity from Clostridium Difficile Spores in Faecal Swine Specimens.

    Science.gov (United States)

    Grześkowiak, Łukasz; Zentek, Jürgen; Vahjen, Wilfried

    2016-11-01

    A considerable fraction of the faecal microbiota is spore-forming. Molecular quantification of bacteria may be underestimated if preceded with nucleic acid extraction without special treatment to extract recalcitrant bacterial spores. The objective of this study was to improve the DNA extraction regarding the presence of Clostridium difficile spores in faecal swine specimens. Sow faeces were inoculated with spores of C. difficile (10(6) CFU), frozen at - 30 °C overnight and subjected to DNA extraction. As a preceding step to a standard DNA extraction method (QIAamp DNA stool Mini kit), different physical treatments such as microwave oven heating and repeated bead-beating techniques and a combination of both were applied and compared with each other by means of qPCR. Using a standard DNA extraction method only, C. difficile spores were quantified at 4.96 log copy number/200 mg of faeces. A repeated bead-beating at 6 m/s for 10 min followed by a standard DNA extraction resulted in 5.77 log copy number of spores in inoculated faeces. Heating in a microwave oven at 800 W for 1, 3, 5 and 10 min followed by a standard DNA extraction resulted in a gene quantification of up to 4.89 log copy number. A combination of both methods resulted in the bacterial gene quantity of 5.37 log copy number. Pre-treatment with repeated bead-beating led to the highest quantification of bacteria, and therefore it can be applied for more efficient DNA extraction from spores of C. difficile in faecal specimens.

  5. Stool consistency is strongly associated with gut microbiota richness and composition, enterotypes and bacterial growth rates.

    Science.gov (United States)

    Vandeputte, Doris; Falony, Gwen; Vieira-Silva, Sara; Tito, Raul Y; Joossens, Marie; Raes, Jeroen

    2016-01-01

    The assessment of potentially confounding factors affecting colon microbiota composition is essential to the identification of robust microbiome based disease markers. Here, we investigate the link between gut microbiota variation and stool consistency using Bristol Stool Scale classification, which reflects faecal water content and activity, and is considered a proxy for intestinal colon transit time. Through 16S rDNA Illumina profiling of faecal samples of 53 healthy women, we evaluated associations between microbiome richness, Bacteroidetes:Firmicutes ratio, enterotypes, and genus abundance with self-reported, Bristol Stool Scale-based stool consistency. Each sample's microbiota growth potential was calculated to test whether transit time acts as a selective force on gut bacterial growth rates. Stool consistency strongly correlates with all known major microbiome markers. It is negatively correlated with species richness, positively associated to the Bacteroidetes:Firmicutes ratio, and linked to Akkermansia and Methanobrevibacter abundance. Enterotypes are distinctly distributed over the BSS-scores. Based on the correlations between microbiota growth potential and stool consistency scores within both enterotypes, we hypothesise that accelerated transit contributes to colon ecosystem differentiation. While shorter transit times can be linked to increased abundance of fast growing species in Ruminococcaceae-Bacteroides samples, hinting to a washout avoidance strategy of faster replication, this trend is absent in Prevotella-enterotyped individuals. Within this enterotype adherence to host tissue therefore appears to be a more likely bacterial strategy to cope with washout. The strength of the associations between stool consistency and species richness, enterotypes and community composition emphasises the crucial importance of stool consistency assessment in gut metagenome-wide association studies. Published by the BMJ Publishing Group Limited. For permission to

  6. Optimized Protocol for Simple Extraction of High-Quality Genomic DNA from Clostridium difficile for Whole-Genome Sequencing.

    Science.gov (United States)

    Sim, James Heng Chiak; Anikst, Victoria; Lohith, Akshar; Pourmand, Nader; Banaei, Niaz

    2015-07-01

    Successful sequencing of the Clostridium difficile genome requires high-quality genomic DNA (gDNA) as the starting material. gDNA extraction using conventional methods is laborious. We describe here an optimized method for the simple extraction of C. difficile gDNA using the QIAamp DNA minikit, which yielded high-quality sequence reads on the Illumina MiSeq platform. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  7. BRAF, K-ras and BAT26 mutations in colorectal polyps and stool

    Institute of Scientific and Technical Information of China (English)

    Ying-Min Jin; Bao-Jie Li; Bo Qu; Ya-Ju Du

    2006-01-01

    AIM: To assess the feasibility of using BRAF, K-ras and BAT26 genes as stool-based molecular markers for detection of colorectal adenomas and hyperplastic polyps (HPs).METHODS: We applied PCR-SSCP and direct sequencing to detect BRAF mutations of polyps and paired stool samples. Primer-mediated restriction fragment lengthpolymorphism (RFLP) analysis and mutant-enriched PCR were used in detection of K-ras mutations of polyp tissues and paired stool samples respectively. BAT26, a microsatellite instability marker was examined by detection of small unstable alleles in a poly (A) repeat. RESULTS: No genetic alterations were detected in the 36 colonoscopically normal patients in either tissues or stools. BRAF, K-ras and BAT26 mutations were found in 4 (16%), 10 (40%) and 3 (12%) of 25 adenoma tissues and among them, 75%, 80% and 100% of patients were observed to contain the same mutations in their corresponding stool samples. In HPs, mutations of BRAF and K-fas were detected in the tumor DNA of 2 (11.1%) and 8 (33.3%) of 18 patients respectively, all of whom had identical alterations in their stools. Taken together, the three genetic markers detected 15 (60%) of 25 adenomas and 8 (44.4%) of 18 HPs. The sensitivity of stool detection was 80% for adenomas and 100% for HPs with an overall specificity of 92% for adenomas and 100% for HPs. CONCLUSION: BRAF, K-ras and BAT26 genes have the potential to be molecular markers for colorectal adenomas and HPs, and can be used as non-invasive screening markers for colorectal polyps.

  8. An efficient genomic DNA extraction from whole blood using Nextractor.

    Science.gov (United States)

    Jeong, Tae-Dong; Cho, Young-Uk; Lee, Woochang; Chun, Sail; Min, Won-Ki

    2014-08-05

    We evaluated the performance of the Nextractor NX-48 nucleic acid extractor system for the extraction of genomic DNA from whole blood samples. We compared the performance of the Nextractor to that of the QIAamp DNA Blood Mini Kit and the Maxwell system, using five whole blood samples. Extraction efficiencies were compared based on the total amount of extracted DNA adjusted by input blood volume, and the purity was compared. Polymerase chain reaction analyses were performed using ACTB as a target. The real-time PCR assay was carried out for housekeeping gene GAPDH. Total elapsed time for DNA extraction was compared. Extraction efficiencies for the QIAamp, Maxwell, and Nextractor were 25.4±3.8ng/μL, 9.2±0.6ng/μL, and 31.0±5.6ng/μL, respectively. No significant differences in purity were observed among three methods. DNA extracted using the ACTB was successfully amplified in all three methods. There were no obvious differences in Ct values for GAPDH real-time PCR. Total elapsed time for DNA extraction was about 50min for the QIAamp, 40min for the Maxwell, and 20min for the Nextractor. As the Nextractor is faster and requires less hands-on time than manual procedures, it may be useful for molecular diagnostic testing in clinical laboratories. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Comparison of direct fecal smear microscopy, culture, and polymerase chain reaction for the detection of Blastocystis sp. in human stool samples.

    Science.gov (United States)

    Santos, Herbert J; Rivera, Windell L

    2013-10-01

    To compare the sensitivity and specificity of direct fecal smear microscopy, culture, and polymerase chain reaction in the detection of Blastocystis sp. in human stool. Human stool samples were collected from a community in San Isidro, Rodriguez, Rizal, Philippines. These samples were subjected to direct fecal smear microscopy, culture and polymerase chain reaction to detect the presence of Blastocystis sp. Of the 110 stool samples collected, 28 (25%) were detected positive for the presence of Blastocystis sp. by two or more tests. Culture method detected the highest number of Blastocystis-positive stool samples (n=36), followed by PCR of DNA extracted from culture (n=26), PCR of DNA extracted from stool (n=10), and direct fecal smear (n=9). Compared to culture, the sensitivity of the other detection methods were 66.7% for PCR from culture and 19.4% for both PCR from stool and direct fecal smear. Specificity of the methods was high, with PCR from culture and direct fecal smear having 97.3%, while PCR from stool at 95.9%. In this study, in vitro culture is the best method for detecting Blastocystis sp. in human stool samples. Copyright © 2013 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

  10. Comparison of direct fecal smear microscopy, culture, and polymerase chain reaction for the detection ofBlastocystis sp. in human stool samples

    Institute of Scientific and Technical Information of China (English)

    Herbert J Santos; Windell L Rivera

    2013-01-01

    Objective:To compare the sensitivity and specificity of direct fecal smear microscopy, culture, and polymerase chain reaction in the detection ofBlastocystis sp. in human stool. Methods:Human stool samples were collected from a community inSanIsidro,Rodriguez, Rizal,Philippines.These samples were subjected to direct fecal smear microscopy, culture and polymerase chain reaction to detect the presence of Blastocystissp.Results:Of the110 stool samples collected,28(25%) were detected positive for the presence ofBlastocystis sp. by two or more tests.Culture method detected the highest number ofBlastocystis-positive stool samples (n=36), followed byPCR ofDNA extracted from culture(n=26),PCR ofDNA extracted from stool (n=10), and direct fecal smear(n=9).Compared to culture, the sensitivity of the other detection methods were66.7% forPCR from culture and19.4% for bothPCR from stool and direct fecal smear.Specificity of the methods was high, withPCR from culture and direct fecal smear having 97.3%, whilePCR from stool at95.9%.Conclusions:In this study,in vitroculture is the best method for detectingBlastocystis sp. in human stool samples.

  11. 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.

  12. [Bristol Stool Chart: Prospective and monocentric study of "stools introspection" in healthy subjects].

    Science.gov (United States)

    Amarenco, G

    2014-09-01

    The Bristol Stool Chart (BSC) allows patients to identify their stool form using seven different images with accompanying written descriptors. Stool form was found to correlate better than stool frequency with whole-gut transit as measured by a radio-opaque marker study. This score is widely used in order to verify the presence of a constipation and to evaluate the therapeutic impact of various treatments. In our clinical practice, we was strongly surprised by the facility and the great precision of the patients to report their stool form, meaning that they usually and daily verify these stools. We wanted to precise the goals of a such attitude. Two questionnaires were proposed to healthy and voluntary subjects. Q1 was supposedly presented in order to verify the sensibility of a French version of BSC in a healthy population. Thus, Q1 precised the difficulties or not to understand pictures and written descriptors, asked about exhaustive analysis by means of BSC of stool form and bowel condition. All subjects with history of ano-rectal disorders or specific treatment for bowel dysfunction were excluded. After Q1 fulfilled, Q2 was proposed to the subjects. Q2 was designed to precise the goals of the patient when he look at his stool and the frequency of such an investigation. Finally a specific question concerning the subject opinion about this behavior in terms of bothersome, shame, or metaphysic interrogation. Eighty-five healthy subjects were recruited (42 female and 43 male). Mean age was 37.2 (sd = 15.7). Mean score of BCS was 2.07 (sd =1.05) (2.07 for female and 1.81 for male, P = 0.22). Number of categories of stool form was only 1 in 40%, 2 categories in 31%, 3 in 19%, 4 in 10%. Presence of a constipation defined by category 1 or 2 was found in 17% (23% in F, 12% in M, P = 0.075). Precision of BSC was noted as excellent in 68%, moderated in 18% and poor in 14%. BSC was considered as easy to use in 75%. Frequency of inspection of feces was systematic for 37%, 1

  13. Real-time polymerase chain reaction for detection of Strongyloides stercoralis in stool.

    Science.gov (United States)

    Sultana, Yasmin; Jeoffreys, Neisha; Watts, Matthew R; Gilbert, Gwendolyn L; Lee, Rogan

    2013-06-01

    The use of real-time polymerase chain reaction (PCR) for detection of Strongyloides stercoralis in stool has recently been described. We compared five DNA extraction methods by using normal human stool spiked with Strongyloides ratti and tested by using a real-time PCR. The PowerSoil kit was found to be the best technique in terms of sensitivity and ease of use. The PCR detected DNA extracted from one spiked S. ratti larva diluted 10⁻². The PowerSoil kit was then used to extract DNA from 160 human survey samples. All culture positive specimens with a high and moderate larval load were identified by real-time PCR, but only 15% of specimens with low larval load were positive. Specificity was greater than 99%. The combination of the PowerSoil kit and real-time PCR reliably detected high to moderate larval numbers of S. stercoralis in stools but was less sensitive when the larval load was low.

  14. Leukocyte telomere length variation due to DNA extraction method.

    Science.gov (United States)

    Denham, Joshua; Marques, Francine Z; Charchar, Fadi J

    2014-12-04

    Telomere length is indicative of biological age. Shorter telomeres have been associated with several disease and health states. There are inconsistencies throughout the literature amongst relative telomere length measured by quantitative PCR (qPCR) and different extraction methods or kits used. We quantified whole-blood leukocyte telomere length using the telomere to single copy gene (T/S) ratio by qPCR in 20 young (18-25 yrs) men after extracting DNA using three common extraction methods: Lahiri and Nurnberger (high salt) method, PureLink Genomic DNA Mini kit (Life Technologies) and QiaAmp DNA Mini kit (Qiagen). Telomere length differences of DNA extracted from the three extraction methods was assessed by one-way analysis of variance (ANOVA). DNA purity differed between extraction methods used (P=0.01). Telomere length was impacted by the DNA extraction method used (P=0.01). Telomeres extracted using the Lahiri and Nurnberger method (mean T/S ratio: 2.43, range: 1.57-3.02) and PureLink Genomic DNA Mini Kit (mean T/S ratio: 2.57, range: 2.24-2.80) did not differ (P=0.13). Likewise, QiaAmp and Purelink-extracted telomeres were not statistically different (P=0.14). The Lahiri-extracted telomeres, however, were significantly shorter than those extracted using the QiaAmp DNA Mini Kit (mean T/S ratio: 2.71, range: 2.32-3.02; P=0.003). DNA purity was associated with telomere length. There are discrepancies between the length of leukocyte telomeres extracted from the same individuals according to the DNA extraction method used. DNA purity could be responsible for the discrepancy in telomere length but this will require validation studies. We recommend using the same DNA extraction kit when quantifying leukocyte telomere length by qPCR or when comparing different cohorts to avoid erroneous associations between telomere length and traits of interest.

  15. First identification of eggs of the Asian fish tapeworm Bothriocephalus acheilognathi (Cestoda: Bothriocephalidea) in human stool.

    Science.gov (United States)

    Yera, Hélène; Kuchta, Roman; Brabec, Jan; Peyron, François; Dupouy-Camet, Jean

    2013-06-01

    We report the first case of egg isolation of the Asian fish tapeworm Bothriocephalus acheilognathi (Bothriocephalidea) from human stool. A male patient from Saint Laurent du Maroni (French Guiana) presenting abdominal pain was examined in France for the diagnosis of intestinal parasites. Diphyllobothrium-like eggs were observed in his stool. However, molecular phylogenetic analyses based on sequences of rDNA and COI genes showed that the eggs observed belong to a bothriocephalidean cestode B. acheilognathi. The adult life stages of B. acheilognathi cestodes are known as invasive parasites of a wide spectrum of fish; however, they have not been described to parasitize any mammals. This human infection seems to be accidental and represents a parasite passage through human intestine after the consumption of an infected fish host. Copyright © 2013. Published by Elsevier Ireland Ltd.

  16. Comparison of four commercial DNA extraction kits for the recovery of Bacillus spp. spore DNA from spiked powder samples.

    Science.gov (United States)

    Mölsä, Markos; Kalin-Mänttäri, Laura; Tonteri, Elina; Hemmilä, Heidi; Nikkari, Simo

    2016-09-01

    Bacillus spp. include human pathogens such as Bacillus anthracis, the causative agent of anthrax and a biothreat agent. Bacillus spp. form spores that are physically highly resistant and may remain active over sample handling. We tested four commercial DNA extraction kits (QIAamp DNA Mini Kit, RTP Pathogen Kit, ZR Fungal/Bacterial DNA MiniPrep, and genesig Easy DNA/RNA Extraction kit) for sample inactivation and DNA recovery from two powders (icing sugar and potato flour) spiked with Bacillus thuringiensis spores. The DNA was analysed using a B. thuringiensis-specific real-time PCR assay. The detection limit was 3×10(1)CFU of spiked B. thuringiensis spores with the QIAamp DNA Mini, RTP Pathogen, and genesig Easy DNA/RNA Extraction kits, and 3×10(3)CFU with the ZR Fungal/Bacterial DNA MiniPrep kit. The results showed that manual extraction kits are effective and safe for fast and easy DNA extraction from powder samples even in field conditions. Adding a DNA filtration step to the extraction protocol ensures the removal of Bacillus spp. spores from DNA samples without affecting sensitivity. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Strong-LAMP: A LAMP Assay for Strongyloides spp. Detection in Stool and Urine Samples. Towards the Diagnosis of Human Strongyloidiasis Starting from a Rodent Model

    Science.gov (United States)

    Gandasegui, Javier; Bajo Santos, Cristina; López-Abán, Julio; Saugar, José María; Rodríguez, Esperanza; Vicente, Belén; Muro, Antonio

    2016-01-01

    Background Strongyloides stercoralis, the chief causative agent of human strongyloidiasis, is a nematode globally distributed but mainly endemic in tropical and subtropical regions. Chronic infection is often clinically asymptomatic but it can result in severe hyperinfection syndrome or disseminated strongyloidiasis in immunocompromised patients. There is a great diversity of techniques used in diagnosing the disease, but definitive diagnosis is accomplished by parasitological examination of stool samples for morphological identification of parasite. Until now, no molecular method has been tested in urine samples as an alternative to stool samples for diagnosing strongyloidiasis. This study aimed to evaluate the use of a new molecular LAMP assay in a well-established Wistar rat experimental infection model using both stool and, for the first time, urine samples. The LAMP assay was also clinically evaluated in patients´ stool samples. Methodology/Principal Findings Stool and urine samples were obtained daily during a 28-day period from rats infected subcutaneously with different infective third-stage larvae doses of S. venezuelensis. The dynamics of parasite infection was determined by daily counting the number of eggs per gram of feces from day 1 to 28 post-infection. A set of primers for LAMP assay based on a DNA partial sequence in the 18S rRNA gene from S. venezuelensis was designed. The set up LAMP assay (namely, Strong-LAMP) allowed the sensitive detection of S. venezuelensis DNA in both stool and urine samples obtained from each infection group of rats and was also effective in S. stercoralis DNA amplification in patients´ stool samples with previously confirmed strongyloidiasis by parasitological and real-time PCR tests. Conclusions/Significance Our Strong-LAMP assay is an useful molecular tool in research of a strongyloidiasis experimental infection model in both stool and urine samples. After further validation, the Strong-LAMP could also be potentially

  18. Comparison of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens

    Directory of Open Access Journals (Sweden)

    Nakatsu Cindy H

    2010-05-01

    Full Text Available Abstract Background The influence of diet on intestinal microflora has been investigated mainly using conventional microbiological approaches. Although these studies have advanced knowledge on human intestinal microflora, it is imperative that new methods are applied to facilitate scientific progress. Culture-independent molecular fingerprinting method of Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis (PCR-DGGE has been used to study microbial communities in a variety of environmental samples. However, these protocols must be optimized prior to their application in order to enhance the quality and accuracy of downstream analyses. In this study, the relative efficacy of four commercial DNA extraction kits (Mobio Ultra Clean® Fecal DNA Isolation Kit, M; QIAamp® DNA Stool Mini Kit, Q; FastDNA® SPIN Kit, FSp; FastDNA® SPIN Kit for Soil, FSo were evaluated. Further, PCR-DGGE technique was also assessed for its feasibility in detecting differences in human intestinal bacterial fingerprint profiles. Method Total DNA was extracted from varying weights of human fecal specimens using four different kits, followed by PCR amplification of bacterial 16S rRNA genes, and DGGE separation of the amplicons. Results Regardless of kit, maximum DNA yield was obtained using 10 to 50 mg (wet wt of fecal specimens and similar DGGE profiles were obtained. However, kits FSp and FSo extracted significantly larger amounts of DNA per g dry fecal specimens and produced more bands on their DGGE profiles than kits M and Q due to their use of bead-containing lysing matrix and vigorous shaking step. DGGE of 16S rRNA gene PCR products was suitable for capturing the profiles of human intestinal microbial community and enabled rapid comparative assessment of inter- and intra-subject differences. Conclusion We conclude that extraction kits that incorporated bead-containing lysing matrix and vigorous shaking produced high quality DNA from human fecal

  19. [Procedure and indications of stool examination in parasitology].

    Science.gov (United States)

    Trabelsi, Sonia; Aouinet, Amira; Khaled, Samira

    2012-06-01

    Intestinal parasites are a public health problem in the world especially in tropical and subtropical countries. Despite the improvement in living standards and healthy conditions, these parasitoses remain relatively frequent in Tunisia. Stool specimen examination keeps the fundamental test for screening and diagnosis. It is to directly search the parasite. Respect for the right procedure of collection of stool is an essential step for the reliability and proper interpretation of results of this examination.

  20. Viral load of human bocavirus-1 in stools from children with viral diarrhoea in Paraguay.

    Science.gov (United States)

    Proenca-Modena, J L; Martinez, M; Amarilla, A A; Espínola, E E; Galeano, M E; Fariña, N; Russomando, G; Aquino, V H; Parra, G I; Arruda, E

    2013-12-01

    Since their discovery, four species of human bocavirus (HBoV) have been described in patients with respiratory and gastrointestinal diseases. However, a clear causal association between HBoV-1 and gastroenteritis has not been demonstrated. In this study, we describe the detection and quantification of HBoV-1 in stools from children with acute non-bacterial gastroenteritis using quantitative polymerase chain reaction. HBoV-1 genome was detected in 10.6% of stools with frequent association with rotavirus and norovirus. The median of HBoV-1 viral load was 1.88 × 104 genome/ml, lower than previously shown in secretions of patients with respiratory infections, without any obvious association between high viral load and presence of HBoV as single agent. Thus, although HBoV-1 was frequently detected in these patients, there is no clear causal association of this agent with diarrhoea. Indeed, HBoV-1 DNA in stools of patients with gastroenteritis without respiratory symptoms may be a remnant of previous infections or associated with prolonged shedding of virus in the respiratory or digestive tracts.

  1. Comparison of proteases in DNA extraction via quantitative polymerase chain reaction.

    Science.gov (United States)

    Eychner, Alison M; Lebo, Roberta J; Elkins, Kelly M

    2015-06-01

    We compared four proteases in the QIAamp DNA Investigator Kit (Qiagen) to extract DNA for use in multiplex polymerase chain reaction (PCR) assays. The aim was to evaluate alternate proteases for improved DNA recovery as compared with proteinase K for forensic, biochemical research, genetic paternity and immigration, and molecular diagnostic purposes. The Quantifiler Kit TaqMan quantitative PCR assay was used to measure the recovery of DNA from human blood, semen, buccal cells, breastmilk, and earwax in addition to low-template samples, including diluted samples, computer keyboard swabs, chewing gum, and cigarette butts. All methods yielded amplifiable DNA from all samples.

  2. Rapid diagnosis of diarrhea caused by Shigella sonnei using dipsticks; comparison of rectal swabs, direct stool and stool culture.

    Directory of Open Access Journals (Sweden)

    Claudia Duran

    Full Text Available BACKGROUND: We evaluated a dipstick test for rapid detection of Shigella sonnei on bacterial colonies, directly on stools and from rectal swabs because in actual field situations, most pathologic specimens for diagnosis correspond to stool samples or rectal swabs. METHODOLOGY/PRINCIPAL FINDINGS: The test is based on the detection of S. sonnei lipopolysaccharide (LPS O-side chains using phase I-specific monoclonal antibodies coupled to gold particles, and displayed on a one-step immunochromatographic dipstick. A concentration as low as 5 ng/ml of LPS was detected in distilled water and in reconstituted stools in 6 minutes. This is the optimal time for lecture to avoid errors of interpretation. In distilled water and in reconstituted stools, an unequivocal positive reaction was obtained with 4 x 10(6 CFU/ml of S. sonnei. The specificity was 100% when tested with a battery of Shigella and different unrelated strains. When tested on 342 rectal swabs in Chile, specificity (281/295 was 95.3% (95% CI: 92.9% - 97.7% and sensitivity (47/47 was 100%. Stool cultures and the immunochromatographic test showed concordant results in 95.5 % of cases (328/342 in comparative studies. Positive and negative predictive values were 77% (95% CI: 65% - 86.5% and 100% respectively. When tested on 219 stools in Chile, Vietnam, India and France, specificity (190/198 was 96% (95% CI 92%-98% and sensitivity (21/21 was 100%. Stool cultures and the immunochromatographic test showed concordant results in 96.3 % of cases (211/219 in comparative studies. Positive and negative predictive values were 72.4% (95% CI 56.1%-88.6% and 100 %, respectively. CONCLUSION: This one-step dipstick test performed well for diagnosis of S. sonnei both on stools and on rectal swabs. These data confirm a preliminary study done in Chile.

  3. Comparative analysis of four methods to extract DNA from paraffin-embedded tissues: effect on downstream molecular applications

    Directory of Open Access Journals (Sweden)

    Savelkoul Paul HM

    2010-09-01

    Full Text Available Abstract Background A large portion of tissues stored worldwide for diagnostic purposes is formalin-fixed and paraffin-embedded (FFPE. These FFPE-archived tissues are an extremely valuable source for retrospective (genetic studies. These include mutation screening in cancer-critical genes as well as pathogen detection. In this study we evaluated the impact of several widely used DNA extraction methods on the quality of molecular diagnostics on FFPE tissues. Findings We compared 4 DNA extraction methods from 4 identically processed FFPE mammary-, prostate-, colon- and lung tissues with regard to PCR inhibition, real time SNP detection and amplifiable fragment size. The extraction methods, with and without proteinase K pre-treatment, tested were: 1 heat-treatment, 2 QIAamp DNA-blood-mini-kit, 3 EasyMAG NucliSens and 4 Gentra Capture-Column-kit. Amplifiable DNA fragment size was assessed by multiplexed 200-400-600 bp PCR and appeared highly influenced by the extraction method used. Proteinase K pre-treatment was a prerequisite for proper purification of DNA from FFPE. Extractions with QIAamp, EasyMAG and heat-treatment were found suitable for amplification of fragments up to 400 bp from all tissues, 600 bp amplification was marginally successful (best was QIAamp. QIAamp and EasyMAG extracts were found suitable for downstream real time SNP detection. Gentra extraction was unsuitable. Hands-on time was lowest for heat-treatment, followed by EasyMAG. Conclusions We conclude that the extraction method plays an important role with regard to performance in downstream molecular applications.

  4. Yield of Stool Culture with Isolate Toxin Testing versus a Two-Step Algorithm Including Stool Toxin Testing for Detection of Toxigenic Clostridium difficile▿

    OpenAIRE

    Reller, Megan E.; Lema, Clara A.; Perl, Trish M.; Cai, Mian; Ross, Tracy L.; Speck, Kathleen A.; Carroll, Karen C.

    2007-01-01

    We examined the incremental yield of stool culture (with toxin testing on isolates) versus our two-step algorithm for optimal detection of toxigenic Clostridium difficile. Per the two-step algorithm, stools were screened for C. difficile-associated glutamate dehydrogenase (GDH) antigen and, if positive, tested for toxin by a direct (stool) cell culture cytotoxicity neutralization assay (CCNA). In parallel, stools were cultured for C. difficile and tested for toxin by both indirect (isolate) C...

  5. Comparative Detection and Quantification of Arcobacter butzleri in Stools from Diarrheic and Nondiarrheic People in Southwestern Alberta, Canada.

    Science.gov (United States)

    Webb, Andrew L; Boras, Valerie F; Kruczkiewicz, Peter; Selinger, L Brent; Taboada, Eduardo N; Inglis, G Douglas

    2016-04-01

    Arcobacter butzleri has been linked to enteric disease in humans, but its pathogenicity and epidemiology remain poorly understood. The lack of suitable detection methods is a major limitation. Using comparative genome analysis, we developed PCR primers for direct detection and quantification ofA. butzleri DNA in microbiologically complex matrices. These primers, along with existing molecular and culture-based methods, were used to detectA. butzleri and enteric pathogens in stools of diarrheic and nondiarrheic people (n= 1,596) living in southwestern Alberta, Canada, from May to November 2008. In addition, quantitative PCR was used to compare A. butzleridensities in diarrheic and nondiarrheic stools.Arcobacter butzleriwas detected more often by PCR (59.6%) than by isolation methods (0.8%). Comparison by PCR-based detection found no difference in the prevalence ofA. butzleri between diarrheic (56.7%) and nondiarrheic (45.5%) individuals. Rates of detection in diarrheic stools peaked in June (71.1%) and October (68.7%), but there was no statistically significant correlation between the presence ofA. butzleri and patient age, sex, or place of habitation. Densities ofA. butzleriDNA in diarrheic stools (1.6 ± 0.59 log10 copies mg(-1)) were higher (P= 0.007) than in nondiarrheic stools (1.3 ± 0.63 log10copies mg(-1)). Of the 892 diarrheic samples that were positive for A. butzleri, 74.1% were not positive for other bacterial and/or viral pathogens. The current study supports previous work suggesting that A. butzleri pathogenicity is strain specific and/or dependent on other factors, such as the level of host resistance.

  6. Improved amplification efficiency on stool samples by addition of spermidine and its use for non-invasive detection of colorectal cancer

    KAUST Repository

    Roperch, Jean-Pierre

    2015-05-29

    Background Using quantitative methylation-specific PCR (QM-MSP) is a promising method for colorectal cancer (CRC) diagnosis from stool samples. Difficulty in eliminating PCR inhibitors of this body fluid has been extensively reported. Here, spermidine is presented as PCR facilitator for the detection of stool DNA methylation biomarkers using QM-MSP. We examined its effectiveness with NPY, PENK and WIF1, three biomarkers which we have previously shown to be of relevance to CRC. Results We determined an optimal window for the amplification of the albumin (Alb) gene (100 ng of bisulfite-treated stool DNA added of 1 mM spermidine) at which we report that spermidine acts as a PCR facilitator (AE = 1680%) for SG RT-PCR. We show that the amplification of methylated PENK, NPY and WIF1 is considerably facilitated by QM-MSP as measured by an increase of CMI (Cumulative Methylation Index, i.e. the sum of the three methylation values) by a factor of 1.5 to 23 fold in individual samples, and of 10 fold in a pool of five samples. Conclusions We contend that spermidine greatly reduces the problems of PCR inhibition in stool samples. This observed feature, after validation on a larger sampling, could be used in the development of stool-based CRC diagnosis tests.

  7. Detection of Cryptosporidium sp infection by PCR and modified acid fast staining from potassium dichromate preserved stool

    Directory of Open Access Journals (Sweden)

    Agnes Kurniawan

    2009-09-01

    Full Text Available Aim To identify the frequency of Cryptosporidium infection in children below 3 years old by examining concentrated long term preserved stool using PCR detection of 18S rRNA gene and compared with modified acid fast staining technique.Methods Hundred eighty eight stools from children ≤ 3 years old were stored for 13 months in 2.5% K2Cr2O7 solution at 40C. Cryptosporidium oocysts were isolated by water-ether concentration technique. The concentrates were smeared onto object glass and stained with modified acid fast staining, and the rest of the concentrates were DNA extracted by freezing and thawing cycles and proteinase K digestion, then direct PCR was done to detect 18S rRNA gene.Result The proportion of positive stools for Cryptosporidium sp by acid fast staining from concentrated stools and 18S rRNA PCR were 4.8% and 34.6% respectively, which showed statistically significant difference.Conclusion The frequency of Cryptosporidium infection among children ≤ 3 years old was very high and stool storage in K2Cr2O7 for 13 months did not affect the PCR result. High prevalence of Cryptosporidium infection indicated high transmission in that area and the potential to be transmitted to other individuals such as the immunocompromised. (Med J Indones 2009;18:147-52Key words: 18S rRNA, cryptosporidiosis

  8. Effect of dilution of stool soluble component on growth and development of Strongyloides stercoralis.

    Science.gov (United States)

    Anamnart, Witthaya; Intapan, Pewpan Maleewong; Pattanawongsa, Attarat; Chamavit, Pennapa; Kaewsawat, Supreecha; Maleewong, Wanchai

    2015-06-02

    Dispersion or dilution of stool by water from heavy rainfall may affect Strongyloides stercoralis free-living development producing infective filariform larvae (FL). This study examined effect of water dilution of stool on survival of S. stercoralis free-living development. One g of stool was prepared in water so that its soluble component was diluted sequentially from 1:2 to 1:480. Three dishes were used to compare FL production in three culture conditions: stool suspension, stool sediment deposited in soil, and isolated rhabditiform larvae (RhL) deposited in soil. The fourth dish was for developmental observation of RhL into free-living stages. Numerous FL were generated from undiluted or 1:2 diluted stool and stool sediment placed on soil. However, starting from dilution 1:5, FL production continuously decreased in both stool suspensions and stool sediments placed on soil. RhL isolated from stool dilutions placed on soil gave rise to few FL. Worm mating were seen at 24-30 hours in dilutions 1:20-1:120 only. Highest numbers of FL from indirect free-living cycle were 1/3 of those from control. FL production decreased as stool dilution increased, and reached zero production at 1:160 dilution. Rainfall may disperse or dilute stool so that nutritional supplement for S. stercoralis free-living development is insufficient.

  9. Evaluation of automated and manual DNA purification methods for detecting Ricinus communis DNA during ricin investigations.

    Science.gov (United States)

    Hutchins, Anne S; Astwood, Michael J; Saah, J Royden; Michel, Pierre A; Newton, Bruce R; Dauphin, Leslie A

    2014-03-01

    In April of 2013, letters addressed to the President of United States and other government officials were intercepted and found to be contaminated with ricin, heightening awareness about the need to evaluate laboratory methods for detecting ricin. This study evaluated commercial DNA purification methods for isolating Ricinus communis DNA as measured by real-time polymerase chain reaction (PCR). Four commercially available DNA purification methods (two automated, MagNA Pure compact and MagNA Pure LC, and two manual, MasterPure complete DNA and RNA purification kit and QIAamp DNA blood mini kit) were evaluated. We compared their ability to purify detectable levels of R. communis DNA from four different sample types, including crude preparations of ricin that could be used for biological crimes or acts of bioterrorism. Castor beans, spiked swabs, and spiked powders were included to simulate sample types typically tested during criminal and public health investigations. Real-time PCR analysis indicated that the QIAamp kit resulted in the greatest sensitivity for ricin preparations; the MasterPure kit performed best with spiked powders. The four methods detected equivalent levels by real-time PCR when castor beans and spiked swabs were used. All four methods yielded DNA free of PCR inhibitors as determined by the use of a PCR inhibition control assay. This study demonstrated that DNA purification methods differ in their ability to purify R. communis DNA; therefore, the purification method used for a given sample type can influence the sensitivity of real-time PCR assays for R. communis. Published by Elsevier Ireland Ltd.

  10. Comparison of DNA isolation methods and detection of Salmonella spp. from animal faeces and dust using invA real-time PCR.

    Science.gov (United States)

    Braun, Sascha D; Methner, Ulrich

    2011-01-01

    There is a strong interest to reduce the expenditure for the detection of Salmonella spp. from animal faeces and environmental samples from primary production according to ISO 6579:2002 Annex D by including a rapid and effective method to detect Salmonella spp. already after pre-enrichment in BPW. It has been shown that real-time PCR methods are very effective to detect Salmonella organisms after pre-enrichment of foods. However, materials from primary animal production compose of much higher amounts of substances which might inhibit the sensitivity of real-time PCR. Different techniques of DNA isolation after pre-enrichment of artificially inoculated bovine faecal material were used to compare their detection limit and detection probability using an invA 5' nuclease real-time PCR approach. A detection probability of 100% was shown at 10(5) cfu/ml using the QIAamp DNA Stool Mini Kit (Qiagen, Germany), at 10(4) cfu/ml using the High Pure PCR Template Preparation Kit (Roche, Germany) and at 10(3) cfu/ml using thermal cell lysis or an in-house lab protocol, respectively. In comparison DNA isolation by thermal cell lysis revealed a very good detection limit, low costs and almost no risks of contamination. Furthermore, caecal contents from pigs were analysed by ISO 6579:2002 Annex D and the invA real-time PCR using thermal cell lysis for DNA extraction. As a result neither false positive nor false negative findings were obtained. Inclusion of the real-time PCR after pre-enrichment of samples in BPW followed by bacterial detection of Salmonella only with samples positive with real-time PCR might be a valuable tool to fulfil the international standard of ISO 6579:2002 Annex D but also to diminish the expenditures. However, it must be stated that the modification of an international standard method and its use in routine diagnostic requires the validation and registration of national and/or international competent authorities.

  11. Disposal of children's stools and its association with childhood diarrhea in India.

    Science.gov (United States)

    Bawankule, Rahul; Singh, Abhishek; Kumar, Kaushalendra; Pedgaonkar, Sarang

    2017-01-05

    Children's stool disposal is often overlooked in sanitation programs of any country. Unsafe disposal of children's stool makes children susceptible to many diseases that transmit through faecal-oral route. Therefore, the study aims to examine the magnitude of unsafe disposal of children's stools in India, the factors associated with it and finally its association with childhood diarrhea. Data from the third round of the National Family Health Survey (NFHS-3) conducted in 2005-06 is used to carry out the analysis. The binary logistic regression model is used to examine the factors associated with unsafe disposal of children's stool. Binary logistic regression is also used to examine the association between unsafe disposal of children's stool and childhood diarrhea. Overall, stools of 79% of children in India were disposed of unsafely. The urban-rural gap in the unsafe disposal of children's stool was wide. Mother's illiteracy and lack of exposure to media, the age of the child, religion and caste/tribe of the household head, wealth index, access to toilet facility and urban-rural residence were statistically associated with unsafe disposal of stool. The odds of diarrhea in children whose stools were disposed of unsafely was estimated to be 11% higher (95% CI: 1.01-1.21) than that of children whose stools were disposed of safely. An increase in the unsafe disposal of children's stool in the community also increased the risk of diarrhea in children. We found significant statistical association between children's stool disposal and diarrhea. Therefore, gains in reduction of childhood diarrhea can be achieved in India through the complete elimination of unsafe disposal of children's stools. The sanitation programmes currently being run in India must also focus on safe disposal of children's stool.

  12. Dust mites in a routine clinical stool sample

    Institute of Scientific and Technical Information of China (English)

    Bushra Zia; Hassaan Bin Aftab; Mohammad Faizan Zahid; Joveria Farooqi; Feroze Uddin; Mohammad Asim Beg

    2014-01-01

    We report a case of dust mite carriage in a 56-year-old gentleman. Dust mites eggs and larvae were found in a stool sample which was taken for a routine clinical examination. He was completely asymptomatic with no history of rash, airway disease or other allergic manifestations associated with dust mites. We noticed that the oval structure of mite eggs resembled helminth eggs and therefore may be misidentified during routine clinical analysis. As the patient was otherwise healthy, it was concluded that no rigorous antiparasitic therapy was necessary to eliminate dust mites from his system.

  13. Pathogen quantitation in complex matrices: a multi-operator comparison of DNA extraction methods with a novel assessment of PCR inhibition.

    Directory of Open Access Journals (Sweden)

    Alessandra Pontiroli

    Full Text Available BACKGROUND: Mycobacterium bovis is the aetiological agent of bovine tuberculosis (bTB, an important recrudescent zoonosis, significantly increasing in British herds in recent years. Wildlife reservoirs have been identified for this disease but the mode of transmission to cattle remains unclear. There is evidence that viable M. bovis cells can survive in soil and faeces for over a year. METHODOLOGY/PRINCIPAL FINDINGS: We report a multi-operator blinded trial for a rigorous comparison of five DNA extraction methods from a variety of soil and faecal samples to assess recovery of M. bovis via real-time PCR detection. The methods included four commercial kits: the QIAamp Stool Mini kit with a pre-treatment step, the FastDNA® Spin kit, the UltraClean™ and PowerSoil™ soil kits and a published manual method based on phenol:chloroform purification, termed Griffiths. M. bovis BCG Pasteur spiked samples were extracted by four operators and evaluated using a specific real-time PCR assay. A novel inhibition control assay was used alongside spectrophotometric ratios to monitor the level of inhibitory compounds affecting PCR, DNA yield, and purity. There were statistically significant differences in M. bovis detection between methods of extraction and types of environmental samples; no significant differences were observed between operators. Processing times and costs were also evaluated. To improve M. bovis detection further, the two best performing methods, FastDNA® Spin kit and Griffiths, were optimised and the ABI TaqMan environmental PCR Master mix was adopted, leading to improved sensitivities. CONCLUSIONS: M. bovis was successfully detected in all environmental samples; DNA extraction using FastDNA® Spin kit was the most sensitive method with highest recoveries from all soil types tested. For troublesome faecal samples, we have used and recommend an improved assay based on a reduced volume, resulting in detection limits of 4.25×10(5 cells g(-1

  14. Evaluation of Four Automated Protocols for Extraction of DNA from FTA Cards

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Børsting, Claus; Ferrero-Miliani, Laura

    2013-01-01

    Extraction of DNA using magnetic bead-based techniques on automated DNA extraction instruments provides a fast, reliable, and reproducible method for DNA extraction from various matrices. Here, we have compared the yield and quality of DNA extracted from FTA cards using four automated extraction...... protocols on three different instruments. The extraction processes were repeated up to six times with the same pieces of FTA cards. The sample material on the FTA cards was either blood or buccal cells. With the QIAamp DNA Investigator and QIAsymphony DNA Investigator kits, it was possible to extract DNA...... from the FTA cards in all six rounds of extractions in sufficient amount and quality to obtain complete short tandem repeat (STR) profiles on a QIAcube and a QIAsymphony SP. With the PrepFiler Express kit, almost all the extractable DNA was extracted in the first two rounds of extractions. Furthermore...

  15. DNA extraction and barcode identification of development stages of forensically important flies in the Czech Republic.

    Science.gov (United States)

    Olekšáková, Tereza; Žurovcová, Martina; Klimešová, Vanda; Barták, Miroslav; Šuláková, Hana

    2017-03-21

    Several methods of DNA extraction, coupled with 'DNA barcoding' species identification, were compared using specimens from early developmental stages of forensically important flies from the Calliphoridae and Sarcophagidae families. DNA was extracted at three immature stages - eggs, the first instar larvae, and empty pupal cases (puparia) - using four different extraction methods, namely, one simple 'homemade' extraction buffer protocol and three commercial kits. The extraction conditions, including the amount of proteinase K and incubation times, were optimized. The simple extraction buffer method was successful for half of the eggs and for the first instar larval samples. The DNA Lego Kit and DEP-25 DNA Extraction Kit were useful for DNA extractions from the first instar larvae samples, and the DNA Lego Kit was also successful regarding the extraction from eggs. The QIAamp DNA mini kit was the most effective; the extraction was successful with regard to all sample types - eggs, larvae, and pupari.

  16. Evaluation of Four Automated Protocols for Extraction of DNA from FTA Cards

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Børsting, Claus; Ferrero-Miliani, Laura;

    2013-01-01

    protocols on three different instruments. The extraction processes were repeated up to six times with the same pieces of FTA cards. The sample material on the FTA cards was either blood or buccal cells. With the QIAamp DNA Investigator and QIAsymphony DNA Investigator kits, it was possible to extract DNA......Extraction of DNA using magnetic bead-based techniques on automated DNA extraction instruments provides a fast, reliable, and reproducible method for DNA extraction from various matrices. Here, we have compared the yield and quality of DNA extracted from FTA cards using four automated extraction...... from the FTA cards in all six rounds of extractions in sufficient amount and quality to obtain complete short tandem repeat (STR) profiles on a QIAcube and a QIAsymphony SP. With the PrepFiler Express kit, almost all the extractable DNA was extracted in the first two rounds of extractions. Furthermore...

  17. Ultrasensitive Detection of Shigella Species in Blood and Stool.

    Science.gov (United States)

    Luo, Jieling; Wang, Jiapeng; Mathew, Anup S; Yau, Siu-Tung

    2016-02-16

    A modified immunosensing system with voltage-controlled signal amplification was used to detect Shigella in stool and blood matrixes at the single-digit CFU level. Inactivated Shigella was spiked in these matrixes and detected directly. The detection was completed in 78 min. Detection limits of 21 CFU/mL and 18 CFU/mL were achieved in stool and blood, respectively, corresponding to 2-7 CFUs immobilized on the detecting electrode. The outcome of the detection of extremely low bacterium concentration, i.e., below 100 CFU/mL, blood samples show a random nature. An analysis of the detection probabilities indicates the correlation between the sample volume and the success of detection and suggests that sample volume is critical for ultrasensitive detection of bacteria. The calculated detection limit is qualitatively in agreement with the empirically determined detection limit. The demonstrated ultrasensitive detection of Shigella on the single-digit CFU level suggests the feasibility of the direct detection of the bacterium in the samples without performing a culture.

  18. A loop-mediated isothermal amplification (LAMP assay for early detection of Schistosoma mansoni in stool samples: a diagnostic approach in a murine model.

    Directory of Open Access Journals (Sweden)

    Pedro Fernández-Soto

    2014-09-01

    Full Text Available Human schistosomiasis, mainly due to Schistosoma mansoni species, is one of the most prevalent parasitic diseases worldwide. To overcome the drawbacks of classical parasitological and serological methods in detecting S. mansoni infections, especially in acute stage of the disease, development of cost-effective, simple and rapid molecular methods is still needed for the diagnosis of schistosomiasis. A promising approach is the loop-mediated isothermal amplification (LAMP technology. Compared to PCR-based assays, LAMP has the advantages of reaction simplicity, rapidity, specificity, cost-effectiveness and higher amplification efficiency. Additionally, as results can be inspected by the naked eye, the technique has great potential for use in low-income countries.A sequence corresponding to a mitochondrial S. mansoni minisatellite DNA region was selected as a target for designing a LAMP-based method to detect S. mansoni DNA in stool samples. We used a S. mansoni murine model to obtain well defined stool and sera samples from infected mice with S. mansoni cercariae. Samples were taken weekly from week 0 to 8 post-infection and the Kato-Katz and ELISA techniques were used for monitoring the infection. Primer set designed were tested using a commercial reaction mixture for LAMP assay and an in house mixture to compare results. Specificity of LAMP was tested using 16 DNA samples from different parasites, including several Schistosoma species, and no cross-reactions were found. The detection limit of our LAMP assay (SmMIT-LAMP was 1 fg of S. mansoni DNA. When testing stool samples from infected mice the SmMIT-LAMP detected S. mansoni DNA as soon as 1 week post-infection.We have developed, for the first time, a cost-effective, easy to perform, specific and sensitive LAMP assay for early detection of S. mansoni in stool samples. The method is potentially and readily adaptable for field diagnosis and disease surveillance in schistosomiasis-endemic areas.

  19. Evaluation and In-House Validation of Five DNA Extraction Methods for PCR-based STR Analysis of Bloodstained Denims

    Directory of Open Access Journals (Sweden)

    Henry Perdigon

    2004-06-01

    Full Text Available One type of crime scene evidence commonly submitted for analysis is bloodstain on denim. However, chemicals (e.g., indigo used to produce denim materials may co-purify with DNA and hence, affect subsequent DNA analysis. The present study compared five methods (e.g., standard organic, organic with hydrogen peroxide (H2O2, modified FTA™, organic/Chelex®-Centricon®, and QIAamp® DNA Mini Kit-based procedures for the isolation of blood DNA from denim. A Short Tandem Repeat (STR-based analysis across two to nine STR markers, namely, HUMvWA, HUMTH01, D8S306, HUMFES/FPS, HUMDHFRP2, HUMF13A01, HUMFGA, HUMTPOX, and HUMCSF1PO, was used to evaluate successful amplification of blood DNA extracted from light indigo, dark indigo, indigo-sulfur, pure indigo, sulfur-top, and sulfur-bottom denim materials. The results of the present study support the utility of organic/Chelex®-Centricon® and QIAamp® Kit procedures in extracting PCR-amplifiable DNA from five different types of denim materials for STR analysis. Furthermore, a solid-based method using FTA™ classic cards was modified to provide a simple, rapid, safe, and cost-effective procedure for extracting blood DNA from light, dark indigo and pure indigo denim materials. However, DNA eluted from bloodstained sulfur-dyed denims (e.g., sulfur-top and sulfur-bottom using FTA™ procedure was not readily amplifiable.

  20. Investigation of Entamoeba histolytica in stool specimens by ELISA during a year

    Directory of Open Access Journals (Sweden)

    Mehmet Sinan Dal

    2011-03-01

    Full Text Available The aim of this study was to investigate Entamoebahistolytica in the stool samples that have beensent to microbiology laboratory by ELISA method.Materials and methods: This study was performed on800 stool specimens belonging to different age groupsand different patients sent to Microbiology Laboratory ofa State Hospital between January 2009 and December2009.Results: In this study Entamoeba histolytica/dispar cystsand/or trophozoites were observed in 31 (3.9% out of atotal of 800 stool specimens which were examined bynative-lugol. Entamoeba histolytica specific antigen wasinvestigated by ELISA in all stool specimens. Entamoebahistolytica specific antigen was detected in 12 (1.5% ofthese stool specimens. The diagnosis of amoebiasis forthe patients whose ELISA tests were “positive” and appropriatetherapy was begun.Conclusion: Entamoeba histolytica in the stool samplesshould be investigated and unnecessary treatmentsshould not given to patients. J Clin Exp Invest 2011; 2(1:50-54

  1. Can they or can they not? Nurses' ability to quantify stool in superabsorbent diapers.

    Science.gov (United States)

    Ledbetter, Laura

    2006-08-01

    Estimates of stool output in diapers is not an appropriate guideline to use in determining fluid loss through stool. This study was conducted to determine the accuracy of the ability of the nurses (RNs) and patient care technicians (PCTs) to quantify stool volume in diapers. Size 3 diapers, baby food (green peas), and water were used to simulate combinations of stool and urine and differing degrees of water loss in stool. The results indicated that RNs' and PCTs' assessments of stool volume became less accurate as water loss increased. There were no differences in estimation accuracy between RN and PCTs, and years of experience for RNs or PCTs did not influence accuracy of estimation. It is important to use a holistic approach for determining hydration status in patients, particularly knowledge of signs and symptoms of dehydration.

  2. Value of Tropheryma whipplei quantitative polymerase chain reaction assay for the diagnosis of Whipple disease: usefulness of saliva and stool specimens for first-line screening.

    Science.gov (United States)

    Fenollar, Florence; Laouira, Sonia; Lepidi, Hubert; Rolain, Jean-Marc; Raoult, Didier

    2008-09-01

    Whipple disease (WD) is a chronic infectious disease caused by Tropheryma whipplei. WD DNA has been found in stool and saliva specimens from patients and asymptomatic carriers. A total of 4418 samples that were sent to our center for determination of WD were tested by a T. whipplei-specific quantitative polymerase chain reaction (PCR) based on repetitive sequences. Definite WD was diagnosed in 71 patients, including 55 patients with classic WD (defined by positive results of periodic acid-Schiff staining and/or specific immunohistochemistry of small-bowel biopsy specimens) and 16 patients with localized WD (including patients with endocarditis, neurologic infection, and uveitis). Of the persons without WD, 2.3% had stool specimens positive for T. whipplei by PCR and 0.2% had saliva specimens positive for T. whipplei by PCR. Diagnosis of WD was likely in patients with positive results of both PCR of saliva specimens and PCR of stool specimens (positive predictive value, 95.2%). When the bacterial load was >10(4) colony-forming units per g of stool, the positive predictive value was 100%. A negative result of PCR of a saliva or stool specimen had a negative predictive value of 99.2% for classic WD. For localized WD, positive results of both PCR of saliva specimens and PCR of stool specimens had a sensitivity of 58% (compared with 94% for classic WD). The positive predictive value of testing of blood, cerebrospinal fluid, and urine specimens was 100% for each, and the positive predictive value for testing of duodenal biopsy specimens was 97.5%. T. whipplei-specific quantitative PCR of saliva and stool specimens should be performed as first-line noninvasive screening for WD. When the results for both types of specimens are positive, diagnosis of classic WD should be highly suspected, especially if a high bacterial load is detected. Because PCR of saliva and stool specimens lacks sensitivity for determination of localized WD, invasive samples should be tested on the

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

    OpenAIRE

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

    1992-01-01

    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 sto...

  4. Multicenter Evaluation of Clinical Diagnostic Methods for Detection and Isolation of Campylobacter spp. from Stool.

    Science.gov (United States)

    Fitzgerald, Collette; Patrick, Mary; Gonzalez, Anthony; Akin, Joshua; Polage, Christopher R; Wymore, Kate; Gillim-Ross, Laura; Xavier, Karen; Sadlowski, Jennifer; Monahan, Jan; Hurd, Sharon; Dahlberg, Suzanne; Jerris, Robert; Watson, Renee; Santovenia, Monica; Mitchell, David; Harrison, Cassandra; Tobin-D'Angelo, Melissa; DeMartino, Mary; Pentella, Michael; Razeq, Jafar; Leonard, Celere; Jung, Carrianne; Achong-Bowe, Ria; Evans, Yaaqobah; Jain, Damini; Juni, Billie; Leano, Fe; Robinson, Trisha; Smith, Kirk; Gittelman, Rachel M; Garrigan, Charles; Nachamkin, Irving

    2016-05-01

    The use of culture-independent diagnostic tests (CIDTs), such as stool antigen tests, as standalone tests for the detection of Campylobacter in stool is increasing. We conducted a prospective, multicenter study to evaluate the performance of stool antigen CIDTs compared to culture and PCR for Campylobacter detection. Between July and October 2010, we tested 2,767 stool specimens from patients with gastrointestinal illness with the following methods: four types of Campylobacter selective media, four commercial stool antigen assays, and a commercial PCR assay. Illnesses from which specimens were positive by one or more culture media or at least one CIDT and PCR were designated "cases." A total of 95 specimens (3.4%) met the case definition. The stool antigen CIDTs ranged from 79.6% to 87.6% in sensitivity, 95.9 to 99.5% in specificity, and 41.3 to 84.3% in positive predictive value. Culture alone detected 80/89 (89.9% sensitivity) Campylobacter jejuni/Campylobacter coli-positive cases. Of the 209 noncases that were positive by at least one CIDT, only one (0.48%) was positive by all four stool antigen tests, and 73% were positive by just one stool antigen test. The questionable relevance of unconfirmed positive stool antigen CIDT results was supported by the finding that noncases were less likely than cases to have gastrointestinal symptoms. Thus, while the tests were convenient to use, the sensitivity, specificity, and positive predictive value of Campylobacter stool antigen tests were highly variable. Given the relatively low incidence of Campylobacter disease and the generally poor diagnostic test characteristics, this study calls into question the use of commercially available stool antigen CIDTs as standalone tests for direct detection of Campylobacter in stool. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  5. Patients’ perspectives on providing a stool sample to their GP: a qualitative study

    Science.gov (United States)

    Lecky, Donna M; Hawking, Meredith KD; McNulty, Cliodna AM

    2014-01-01

    Background Stool specimen collection is challenging and informal feedback has indicated that participants find the process difficult. Increasing stool specimen returns would improve the investigation of outbreaks of diarrhoeal and food-borne disease. Aim To explore the barriers to stool sample collection and specimen return to ascertain which factors may help to improve the process. Design and setting Qualitative patient interview study in Gloucester, UK. Method A two-stage purposive sampling process was used to identify patients who had either previous experience or no experience of collecting a stool sample. The interview schedule, based on the theory of planned behaviour, was used to facilitate interviews with 26 patients. Interview transcripts were analysed using a modified framework analysis. Results Barriers to collection included embarrassment, fear of results, concerns around hygiene and contamination, discretion and privacy, and lack of information. Personal gain was identified as the main incentive to collecting and returning a stool sample. The need for an information leaflet on stool collection was emphasised by most patients. Conclusions GPs could make a number of small changes that could make a big difference for patients and potentially increase stool sample return. If they, rather than receptionists, distributed collection kits it may be easier for patients to ask any questions they had regarding collection. In addition, the provision of a stool-collection information leaflet could increase patients’ confidence regarding collecting the sample, and providing drop-off boxes for specimens could help prevent patients’ embarrassment regarding handing their stool over to a receptionist. PMID:25348992

  6. Patients' perspectives on providing a stool sample to their GP: a qualitative study.

    Science.gov (United States)

    Lecky, Donna M; Hawking, Meredith K D; McNulty, Cliodna A M

    2014-11-01

    Stool specimen collection is challenging and informal feedback has indicated that participants find the process difficult. Increasing stool specimen returns would improve the investigation of outbreaks of diarrhoeal and food-borne disease. To explore the barriers to stool sample collection and specimen return to ascertain which factors may help to improve the process. Qualitative patient interview study in Gloucester, UK. A two-stage purposive sampling process was used to identify patients who had either previous experience or no experience of collecting a stool sample. The interview schedule, based on the theory of planned behaviour, was used to facilitate interviews with 26 patients. Interview transcripts were analysed using a modified framework analysis. Barriers to collection included embarrassment, fear of results, concerns around hygiene and contamination, discretion and privacy, and lack of information. Personal gain was identified as the main incentive to collecting and returning a stool sample. The need for an information leaflet on stool collection was emphasised by most patients. GPs could make a number of small changes that could make a big difference for patients and potentially increase stool sample return. If they, rather than receptionists, distributed collection kits it may be easier for patients to ask any questions they had regarding collection. In addition, the provision of a stool-collection information leaflet could increase patients' confidence regarding collecting the sample, and providing drop-off boxes for specimens could help prevent patients' embarrassment regarding handing their stool over to a receptionist. © British Journal of General Practice 2014.

  7. Munchausen Syndrome By Proxy Admitting with Bloody Urine and Stool

    Directory of Open Access Journals (Sweden)

    Tugba Koca

    2014-02-01

    Full Text Available Munchausen syndrome by Proxy is a severe form of child abuse. Disease symptoms and signs are fabricated or imitated by parents or caregivers The child is usually presented to doctors, persistently. A delay in diagnosis may cause severe negative impact on spiritual, physical, mental and social development of the cases and even death. Symptoms usually disappear in the absence of the perpetrators. The diagnosis is extremely difficult. A 21-month-old boy who had applied to many centers due to bleeding from various parts of the body for last six months, and whose symptoms could not be explained with any physical reason after tests were conducted. Finally he was admitted to our center with bloody urine and stools, and diagnosed Munchausen syndrome by proxy. In cases with recurrent hospital admission in whom no apparent disease is diagnosed, Munchausen syndrome by Proxy should be among the differential diagnosis.

  8. Quality control of parasitology stool examination in Tabriz clinical laboratories

    Directory of Open Access Journals (Sweden)

    shahram Khademvatan

    2011-06-01

    Full Text Available The purpose of quality control program was to make doctors and laboratory personnel trust in laboratory results and consequently increasing confidence in laboratory achievements. The quality assurance means raising the level of quality in all tests that lead to raising the level of work efficiency and laboratories including minimum expense for society and minimum time for lab personnel. This study aimed to assess and determine the accuracy and precision of results in Tabriz medical diagnostic laboratories. Materials and Methods: In this retrospective study, 790 stool samples were selected randomly and tested by standard methods.Student t- test, SPSS software and sensitivity and accuracy formulas were used for data analysis. Results: The sensitivity was 62%, 22% and 8% with 95% confidence intervals for worm's eggs, protozoan cysts and trophozoite detection respectively. Conclusion: To elevate quality assurance in clinical diagnostic laboratory, monitoring and check of the laboratories by standard methods continually should be done.

  9. From stool transplants to next-generation microbiota therapeutics.

    Science.gov (United States)

    Petrof, Elaine O; Khoruts, Alexander

    2014-05-01

    The epidemic of Clostridium difficile infection fueled by new virulent strains of the organism has led to increased use of fecal microbiota transplantation (FMT). The procedure is effective for even the most desperate cases after failure of multiple courses of antibiotics. The approach recognizes microbiota to be integral to normal human physiology, and microbiota being used in FMT represents a new class of therapeutics. Imbalance in the composition and altered activity of the microbiota are associated with many diseases. Consequently, there is growing interest in applying FMT to non-C difficile indications. However, this may succeed only if microbiota therapeutics are developed systematically, based on mechanistic understanding, and applying up-to-date principles of microbial ecology. We discuss 2 pathways in the development of this new therapeutic class: whole microbial communities separated from donor stool and an assembly of specific fecal microorganisms grown in vitro. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

  10. DNA

    Science.gov (United States)

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  11. 大熊猫和小熊猫粪便DNA提取的简易方法%A simple protocol for DNA extraction from faeces of the giant panda and lesser panda

    Institute of Scientific and Technical Information of China (English)

    张保卫; 魏辅文; 李明; 吕晓平

    2004-01-01

    采集了大熊猫和小熊猫的新鲜粪便样品,使用100%乙醇保存.通过重复离心富集研究动物的肠道脱落细胞,并使用乙醇和双蒸水洗涤以除去抑制物.用1%的SDS快速裂解细胞,离心除去残渣后,向裂解液中加入蛋白酶进行消化.消化结束后使用等体积的酚/氯仿抽提,乙醇沉淀DNA.用双蒸水溶解粪便DNA后,使用PCR产物纯化试剂盒对粪便DNA进行纯化.电泳检测结果显示,从乙醇保存的大、小熊猫粪便样品中抽提到高质量的粪便DNA.对线粒体控制区、细胞色素b基因、12 S rRNA基因的PCR扩增反应以及测序结果也证实了样品保存方法和DNA抽提方法可靠而高效.此方法使用实验室内常用的分子生物学试剂,不仅克服了分子粪便学研究中常见的抑制物粪便DNA微量降解严重等障碍,与商业化的粪便抽提试剂盒(QIAamp DNA Stool MiniKit,Qiagen)相比还是一种经济的试验方法(抽提反应成本为试剂盒的1/5).文中还对粪便DNA内细菌基因组等背景DNA可能对分子粪便学试验结果的影响进行了探讨.在基于PCR技术的遗传学研究中,对于植食性动物而言,粪便内的背景DNA对目标动物DNA片断的扩增和序列测定未见影响;但对于肉食性动物,则必须考虑被捕食者基因组对试验可能产生的影响,应谨慎对待[动物学报50(3):452-458,2004].

  12. Comparison of eight commercially available kits for DNA extraction from formalin-fixed paraffin-embedded tissues.

    Science.gov (United States)

    Janecka, Anna; Adamczyk, Agnieszka; Gasińska, Anna

    2015-05-01

    A proper extraction method from formalin-fixed paraffin-embedded (FFPE) blocks is essential to obtain DNA of satisfactory quality/quantity. We compared the effectiveness of eight commercially available kits for DNA extraction based on 10 FFPE tissues. Kits differed significantly in terms of DNA yield, purity, and quality. Using the QIAamp DNA FFPE Tissue Kit (Qiagen) and the ReliaPrep FFPE gDNA Miniprep System (Promega), we obtained DNA of the highest quality and acceptable quantity. We also demonstrated that overnight digestion of samples usually improved DNA yield and/or purity. For precious or limited material, double elution is recommended for obtaining up to 42% higher amount of DNA.

  13. Selection of the most suitable method for the extraction of DNA from foods and feeds for species identification

    Directory of Open Access Journals (Sweden)

    Michaela Nesvadbová

    2010-01-01

    Full Text Available High quality and purity of DNA isolated from food and feed is essential for species identification and has unpredictable influences an effect of analysis. In this study, the efficiency of eight different methods for DNA isolation was investigated. For DNA extraction, the raw chicken meet, ham, sausages, tinned lunch meat, pate, tinned feeds for dogs, complete granulated feeds for dogs and chicken flour were used. Kits of several different producers, i.e.: NucleoSpin Food (Marchery-Nagel, Wizard Genomic DNA Purification Kit (Promega, Invisorb Spin Food Kit I (Invitek, Wizard SV Genomic DNA Purification System (Promega, JetQuick Tissue DNA Spin Kit (Genomed, RNA Blue (Top-Bio, JetQuick Blood & Cell Culture Kit (Genomed, QIAamp DNA Mini Kit and QIAamp DNA Blood Mini Kit (Qiagen were employed in the study. Gel agarose electrophoresis for primary verification of DNA quality was performed. The isolates were subsequently assessed for quantity and quality using by spectrophotometer Nanodrop 2000 (Thermo Scientific. To verify of template usability and quality of isolated DNA, the polymerase chain reaction (PCR was used.Differences between isolated DNA from tinned products and meat, ham, sausage, granulated dog feed and chicken flour were found. In tinned food and feed, the DNA was more degraded, DNA content and DNA purity was lower and also PCR amplification was the most difficult. Overall DNA yield and quality have important influence on PCR products amplification. The best results were obtained with NucleoSpin Food and JetQuick Tissue DNA Spin Kit. DNA extracted by these methods proved highest yields, purity and template quality in all foods and feeds and the results of PCR analysis are excellent reproducible. Analyses showed that results depended on different food or feed using and dif­fe­rent isolation system.The results of this work will be utilized to choose the suitable isolating kit for educational course, which is designed for students and

  14. Role of real-time fluorescence PCR in Mycobacterium tuberculosis complex DNA detection from stool samples of children with pulmonary tuberculosis%实时荧光PCR检测儿童肺结核粪便中Mtb-DNA的效果评价

    Institute of Scientific and Technical Information of China (English)

    黄慧谦; 吴驰; 陈建波; 杨燕; 侯志平; 李小勇

    2012-01-01

    目的 应用实时荧光PCR快速检测肺结核患儿粪便中Mtb-DNA,并初步评估其临床效果.方法 收集肺结核住院儿童粪便标本76份,应用实时荧光PCR检测Mtb-DNA,检测结果与20例其他呼吸系统疾病患儿的炎便实时荧光PCR检测Mtb-DNA、76例炎便抗酸杆菌涂片检查以及41例患儿痰标本的涂片和(或)培养、实时荧光PCR检测结果进行比较.结果 粪便实时荧光PCR检测敏感度达到23.68%(18/76),特异度为100.00%(20/20),肺结核患儿粪便PCR阳性率[23.68%(18/76)]明显高于涂片阳性率[6.58%(5/76)],41例患儿中痰涂片和(或)培养阳性例数(15例)和痰PCR检测阳性例数(18例)高于粪便PCR检测阳性例数(11例).结论 实时荧光PCR检测儿童粪便Mtb-DNA,是一种特异度高、比较敏感、非侵入性且相对安全的儿童肺结核快速诊断方法.

  15. Evaluation of five commercial nucleic acid extraction kits for their ability to inactivate Bacillus anthracis spores and comparison of DNA yields from spores and spiked environmental samples.

    Science.gov (United States)

    Dauphin, Leslie A; Moser, Benjamin D; Bowen, Michael D

    2009-01-01

    This study evaluated five commercial extraction kits for their ability to recover DNA from Bacillus anthracis spores and spiked environmental samples. The kits evaluated represent the major types of methodologies which are commercially available for DNA or total nucleic acid extraction, and included the ChargeSwitch gDNA Mini Bacteria Kit, NucliSens Isolation Kit, Puregene Genomic DNA Purification Kit, QIAamp DNA Blood Mini Kit, and the UltraClean Microbial DNA Isolation Kit. Extraction methods were performed using the spores of eight virulent strains of B. anthracis. Viability testing of nucleic acid extracts showed that the UltraClean kit was the most efficient at depleting samples of live B. anthracis spores. TaqMan real-time PCR analysis revealed that the NucliSens, QIAamp and UltraClean kits yielded the best level of detection from spore suspensions. Comparisons of processed samples from spiked swabs and three powder types indicated that DNA extraction using the UltraClean kit resulted in the most consistently positive results and the lowest limit of detection. This study demonstrated that different nucleic extraction methodologies, represented here by various commercial extraction kits, differ in their ability to inactivate live B. anthracis spores as well as DNA yield and purity. In addition, the extraction method used can influence the sensitivity of real-time PCR assays for B. anthracis.

  16. 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.

  17. Reliability and validity of a modified Bristol Stool Form Scale for children

    Science.gov (United States)

    This study sought to: evaluate the ability of children to reliably use a modified Bristol Stool Form Scale for Children (mBSFS-C), evaluate criterion-related validity of the mBSFS-C, and identify the lower age limit for mBSFS-C use. The mBSFS-C comprises 5 stool form types described and depicted in ...

  18. Novel method for digital subtraction of tagged stool in virtual colonoscopy

    Science.gov (United States)

    Guendel, Lutz; Suehling, Michael; Eckert, Helmut

    2008-03-01

    Colon cancer is one of the most frequent causes of death. CT colonography is a novel method for the detection of polyps and early cancer. The general principle of CT colonography includes a cathartic bowel preparation. The resulting discomfort for patients leads to limited patient acceptance and therefore to limited cancer detection rates. Reduced bowel preparation, techniques for stool tagging, and electronic cleansing, however, improve the acceptance rates. Hereby, the high density of oral contrast material highlights residual stool and can be digitally removed. Known subtraction methods cause artifacts: additional 3D objects are introduced and small bowel folds are perforated. We propose a new algorithm that is based on the 2 nd derivative of the image data using the Hessian matrix and the following principal axis transform to detect tiny folds which shall not be subtracted together with tagged stool found by a thresholding method. Since the stool is usually not homogenously tagged with contrast media a detection algorithm for island-like structures is incorporated. The interfaces of air-stool level and colon wall are detected by a 3-dimensional difference of Gaussian module. A 3-dimensional filter smoothes the transitions between removed stool and colon tissue. We evaluated the efficacy of the new algorithm with 10 patient data sets. The results showed no introduced artificial objects and no perforated folds. The artifacts at the air-stool and colon tissue-stool transitions are considerably reduced compared to those known from the literature.

  19. Prevalence of clostridium difficile toxin in diarrhoeal stool samples of patients from a general hospital in Eastern province, Saudi Arabia

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    Sue Elizabeth Shajan, Mohammed Faisal Hashim, Michael A

    2014-04-01

    Full Text Available Introduction: Clostridium difficile is anaerobic spore- forming bacillus, produces two major toxins (Tcd A and Tcd B. Disease caused by toxigenic C.difficile (Tcd varies from mild diarrhea to fulminant disease and death. Aims and Objectives: – This study describes the prevalence of C.difficile toxins (CDT in stool samples from in patients and outpatients of all age groups. Materials and Methods:- A total of 146 samples were examined from 2011 to 2012 were analyzed for the presence of CDT tests, DNA amplification test, and the stool samples were cultured anaerobically on CCFA selective medium for growth- Morphology, identification and other tests. The patient’s details are collected from the medical records. Results: - Out of 146 specimens, only 20 (13.7% were positive for C.difficile toxins. Male and female were 12 (60% and 8(40% respectively, with the majority of them aged between 16 to 71 years. Majority of them were from out patient units (n = 5, 25% with rest from intensive care units (n = 3, 15%, male medical ward (n =3, 15% and surgical wards (n = 1, 5%. All the CDT positive patients had history of prior antibiotic usage before the detection of toxin. Mean duration of antibiotic usage was a 16.75 (±12.75 days, and the mean duration of diarrhea was 4.21 (±4.85 days, 16 patients had underlying medical illness, like hypertension, diabetic mellitus etc; Stool with pus cells and occult blood test was positive among that 18 patients were positive for CDT. The hospitalized patient duration was 20.96 (±16.25 days. Conclusion: – The detection of CDT in the diagnosis of CDI requires vigilance by both clinician and microbiologist to look out for possible infected patients. Antibiotic usage is a known risk factor; thus restricted use of antibiotics may results the reduction of CDI.

  20. Using the Bristol Stool Scale and Parental Report of Stool Consistency as Part of the Rome III Criteria for Functional Constipation in Infants and Toddlers.

    Science.gov (United States)

    Koppen, Ilan J N; Velasco-Benitez, Carlos A; Benninga, Marc A; Di Lorenzo, Carlo; Saps, Miguel

    2016-10-01

    To evaluate among parents of infants and toddlers the agreement between parental report and the Bristol Stool Scale (BSS) in assessing stool consistency and the effect of both methods on determining the prevalence of functional constipation (FC) according to the Rome III criteria. Parents of children ≤48 months of age who were seen for a well-child visit completed a questionnaire about their child's bowel habits during the previous month. Cohen kappa coefficient (κ) was used to measure intrarater agreement between parental report of stool consistency ("hard," "normal," "soft/mucous/liquid") and the BSS (types 1-2, hard; types 3-5, normal; types 6-7, loose/liquid). The prevalence of FC was assessed based on the questionnaire according to the Rome III criteria, comparing both methods of stool consistency assessment. Parents of 1095 children (median age, 15 months; range, 1-48) were included. Only fair agreement existed between the 2 methods of stool consistency assessment (κ = 0.335; P Rome III criteria, using parental report the prevalence of FC was 20.5% and using the BSS the prevalence was 20.9% (P = .87). The agreement between these 2 methods for assessing the prevalence of FC was excellent (κ = 0.95; P < .001). Only fair agreement exists between the BSS and parental report of stool consistency among parents of infants and toddlers. Different methods of stool consistency assessment did not result in a difference in the prevalence of FC. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Detection of pathogenic bacteria in diarrheal stool collected from children in North Lebanon by using conventional stool culture and microarray technique « CLART® Enterobac »

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    Ranin Bechara

    2016-12-01

    Full Text Available Bechara, R., Hosny, M., AL-Kassaa, I., Dabboussi, F., Mallat, H. and Hamze, M. 2016. Detection of pathogenic bacteria in diarrheal stool collected from children in north lebanon by using conventional stool culture and microarray technique « clart® enterobac ». Lebanese Science Journal, 17(2: 233-239. Illness caused by enteropathogens represents an important economic and health burden worldwide. The majority of enteropathogens causes gastrointestinal infections which have a great impact on public health both in developing and developed countries. The aim of this study is to detect and identify the main enteropathogens in Lebanese diarrheal stool from children under 15 years old. The detection was performed by using both conventional method and microarray technique CLART® EnteroBac (Genomica-Spain. Five enteric pathogens, Salmonella spp, Shigella spp, Clostridium difficile B, Enteropathogenic E. coli, Campylobacter jejuni were detected in 80 diarrheal stools, from children under 15 years old. The results showed that CLART® EnteroBac technique have detected enteropathogens in 19% of samples, whereas 1% returned positive using stool culture methods.

  2. Consistency of direct microscopic examination and ELISA in detection of Giardia in stool specimen among children

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    Zohreh Torabi

    2014-09-01

    Full Text Available Objective: To investigate the consistency of direct microscopic examination and ELISA for determination of Giadia in stool specimen. Method: Study population consisted of children with any clinical symptoms of Giardia infestation since last two weeks. Fresh stool specimen was collected from each child. The stools specimens were assessed by two methods of direct microscopic examination and ELISA.The degree of agreement between direct stool exam and ELISA was calculated by Cohen's kappa coefficient. Results: In this study, 124 children with age range 2-12 years were investigated. A total of 64 (61.7% and 79 (65.7% of children had Giardia by direct stool exam and ELISA test respectively. There was association between frequency of constipation and Giardia infection (P=0.036. The Cohen's kappa coefficient calculated for degree of agreement between direct stool exam and ELISA showed κ=0.756 (P<0.001. Conclusions: The frequency of Giardia infection in symptomatic children was high and there was high agreement rate between ELISA and direct stool smear.

  3. A SYBR(®) Green-based real-time PCR method for improved detection of mcr-1-mediated colistin resistance in human stool samples.

    Science.gov (United States)

    Donà, Valentina; Bernasconi, Odette J; Kasraian, Sara; Tinguely, Regula; Endimiani, Andrea

    2017-06-01

    The aim of this study was to design a rapid and sensitive real-time PCR (rt-PCR) method for colistin resistance mcr-1 gene detection in human faecal samples. Stools (n=88) from 36 volunteers were analysed. To isolate mcr-1-producing Enterobacteriaceae, samples were enriched overnight in Luria-Bertani (LB) broth containing 2mg/L colistin and were then plated on selective agar plates with 4mg/L colistin. A SYBR(®) Green-based rt-PCR targeting mcr-1 was then designed. For method validation and to establish the limit of detection (LOD), total DNA was extracted from mcr-1-negative and mcr-1-positive Escherichia coli. rt-PCR was also performed with DNA extracted from 88 native stools and after enriching them in LB broth containing colistin. Based on the culture approach, three unique volunteers resulted colonised with mcr-1-harboring E. coli strains. For culture isolates, rt-PCR exhibited a LOD of 10 genomic copies/reaction, with both sensitivity and specificity of 100%. Nevertheless, when testing native stools, only two of the three mcr-1-positive specimens were detected. However, after enrichment in LB broth containing colistin, the rt-PCR was strongly positive for all culture-positive samples. The average cycle threshold was 22, granting rapid and confident detection of positive specimens within 30 cycles. No false positives were observed for the remaining 85 culture-negative specimens. A rapid rt-PCR for detection of mcr-1 from stool specimens was developed. The detection rate was increased by testing selective broth enrichments. This approach also has the advantage of concomitant isolation of mcr-1-harboring strains for further antimicrobial susceptibility and genetic testing. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

  4. Diagnostic yield of stool culture and predictive factors for positive culture in patients with diarrheal illness.

    Science.gov (United States)

    Lee, Jae Young; Cho, Sun Young; Hwang, Hannah Sun Hae; Ryu, Ja Young; Lee, Jongjin; Song, In Do; Kim, Beom Jin; Kim, Jeong Wook; Chang, Sae Kyung; Choi, Chang Hwan

    2017-07-01

    We aimed to investigate the diagnostic yield of stool cultures and identify predictive factors for positive cultures in patients with diarrheal illness.A total of 13,327 patients who underwent stool cultures due to diarrheal illness were reviewed. Stool cultures were performed for enteric pathogens, including Salmonella, Shigella, Vibrio, Klebsiella oxytoca, and Yersinia. The culture-positive group was compared with the culture-negative group who were randomly selected from culture negative patients.A total of 196 patients (1.47%) were diagnosed with positive stool culture. In 196 culture positive patients, Salmonella spp. (75.0%) was detected most commonly, followed by Vibrio (19.4%). Univariate analyses showed fever (>37.8°C), vomiting, duration and frequency of diarrhea, and high C-reactive protein (CRP) were significantly associated with positive stool culture. Multivariate analysis showed fever (odds ratio [OR], 2.33; 95% confidence interval [CI], 1.25-4.35; P = .008), ≥5/day of diarrhea (OR, 3.52; 95% CI, 1.93-6.44; P 50 mg/L of CRP (OR, 2.27; 95% CI, 1.18-4.36; P = .014) were independent predictors for positive stool culture. OR in patients with all 3 factors was 6.55 (95% CI, 2.56-16.75; P factor.Diagnostic yield of stool culture in patients with diarrheal illness is very low. Fever, frequency of diarrhea, and high CRP are predictors for positive stool cultures. These findings may lead to more discerning and cost-effective utilization of stool culture by clinicians.

  5. Metabolomic study of a diagnostic model for the metabolites of stool fat.

    Science.gov (United States)

    Lee, Choong Hwan; Kim, Jiyoung; Ahn, Su Young; Lee, Sun Young

    2013-01-25

    Metabolomics is a powerful tool for measuring low-molecular-weight metabolites in an organism at a specified time under specific environmental conditions. The aim of this study was to determine the usefulness of metabolomics in identifying the metabolites in stool-fat-positive specimens, and to establish whether the results could be used to predict the long-term prognosis. Fecal specimens were collected from 52 subjects with bowel habit change. The subjects were accessed using Rome III questionnaires and Bristol stool scale form, and followed after three years. The feces samples were centrifuged and the resulting extracts reconstituted for liquid chromatography/mass spectrometry analysis. The datasets were autoscaled, log-transformed, and mean-centered in a column-wise fashion prior to principal-components analysis and partial least-squares-discrimination analysis modeling. Fecal samples from 10 of the 52 patients gave a positive stool-fat result of 30-100 mm; those of the remaining 42 contained neither fatty acids nor neutral fats. The peak intensities of lithocholic acid (p=0.001), lysophosphatidyl ethanolamine (lysoPE) 16:0 (p=0.015), and lysoPE 18:1/0:0 (p=0.014) were correlated with the size of the fatty acid. Subjects with positive stool-fat result showed higher score in Bristol stool scale form than those with negative stool-fat result at initial (p=0.040) and after three years (p=0.012). The metabolomic assay of stool fatty acid revealed mainly lysoPEs and lithocholic acid. The size of the fatty acid was correlated with higher concentrations of lysoPEs and lithocholic acid in stool-fat-test-positive specimens and related to loose stool even after three years of follow-up period.

  6. PURE CULTURE METHOD: GIARDIA LAMBLIA FROM DIFFERENT STOOL SAMPLES

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    H.A YOUSEFI

    2000-03-01

    Full Text Available Introduction. Giardiasis is one of the health problems in the world including Iran. To determine the biochemical and biological problems and also identification of various strains, it is essential to obtain pure culture and then mass production of Giardia lamblia. The goal of this study was to isolate this protozoa purely.
    Methods. Giardia lamblia cysts were isolated from 50 stool samples by use of floating of a four - layer of sucrose method. The cysts were transfered to an inducing solution. Subsequently, they were cultured in a modified culture medium (TYIS-33. Following excystation of trophozoite and its multiplication, the parasite was caltured and purified.
    Findings. Excitation of trophozoite was observed in 40 samples (80 percent from which 22 samples (55 percent yielded pure culture. The doubling time was approximately 13hr and the peak of parasite was observed between third and fourth days.
    Conclusion. The proliferation and growth rate of Giardia lamblia have enabled us to use this method widely. Cystein and ascorbic acid which are present in the induction solution, have a key role in excystation of trophozoite. Purification and passage of samples has facilitated the culture of this parasite in vitro. Therefore this method has yielded better results in comparison with other studies. This is probably due to a decrease in the amount of bovine bile or using different strains of Giardia lamblia in the present study.

  7. Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification.

    Science.gov (United States)

    Devonshire, Alison S; Whale, Alexandra S; Gutteridge, Alice; Jones, Gerwyn; Cowen, Simon; Foy, Carole A; Huggett, Jim F

    2014-10-01

    Circulating cell-free DNA (cfDNA) is becoming an important clinical analyte for prenatal testing, cancer diagnosis and cancer monitoring. The extraction stage is critical in ensuring clinical sensitivity of analytical methods measuring minority nucleic acid fractions, such as foetal-derived sequences in predominantly maternal cfDNA. Consequently, quality controls are required for measurement of extraction efficiency, fragment size bias and yield for validation of cfDNA methods. We evaluated the utility of an external DNA spike for monitoring these parameters in a study comparing three specific cfDNA extraction methods [QIAamp circulating nucleic acid (CNA) kit, NucleoSpin Plasma XS (NS) kit and FitAmp plasma/serum DNA isolation (FA) kit] with the commonly used QIAamp DNA blood mini (DBM) kit. We found that the extraction efficiencies of the kits ranked in the order CNA kit > DBM kit > NS kit > FA kit, and the CNA and NS kits gave a better representation of smaller DNA fragments in the extract than the DBM kit. We investigated means of improved reporting of cfDNA yield by comparing quantitative PCR measurements of seven different reference gene assays in plasma samples and validating these with digital PCR. We noted that the cfDNA quantities based on measurement of some target genes (e.g. TERT) were, on average, more than twofold higher than those of other assays (e.g. ERV3). We conclude that analysis and averaging of multiple reference genes using a GeNorm approach gives a more reliable estimate of total cfDNA quantity.

  8. Antimicrobial resistance of bacterial enteropathogens isolated from stools in Madagascar.

    Science.gov (United States)

    Randrianirina, Frederique; Ratsima, Elisoa Hariniana; Ramparany, Lova; Randremanana, Rindra; Rakotonirina, Hanitra Clara; Andriamanantena, Tahiry; Rakotomanana, Fanjasoa; Rajatonirina, Soatiana; Richard, Vincent; Talarmin, Antoine

    2014-02-25

    Diarrheal diseases are a major public health problem in developing countries, and are one of the main causes of hospital admissions in Madagascar. The Pasteur Institute of Madagascar undertook a study to determine the prevalence and the pathogenicity of bacterial, viral and protozoal enteropathogens in diarrheal and non-diarrheal stools of children aged less than 5 years in Madagascar. We present here the results of the analysis of antimicrobial susceptibility of the bacteria isolated during this study. The study was conducted in the community setting in 14 districts of Madagascar from October 2008 to May 2009. Conventional methods and PCR were used to identify the bacteria; antimicrobial susceptibility was determined using an agar diffusion method for enterobacteriaceae and MICs were measured by an agar dilution method for Campylobacter sp. In addition to the strains isolated during this study, Salmonella sp and Shigella sp isolated at the Pasteur Institute of Madagascar from 2005 to 2009 were included in the analysis to increase the power of the study. Twenty-nine strains of Salmonella sp, 35 strains of Shigella sp, 195 strains of diarrheagenic E. coli, 203 strains of C. jejuni and 71 strains of C. coli isolated in the community setting were tested for antibiotic resistance. Fifty-five strains of Salmonella sp and 129 strains of Shigella sp isolated from patients referred to the Pasteur Institute of Madagascar were also included in the study. Many E. coli and Shigella isolates (around 80%) but fewer Salmonella isolates were resistant to ampicillin and trimethoprim/sulfamethoxazole. A small proportion of strains of each species were resistant to ciprofloxacin and only 3% of E. coli strains presented a resistance to third generation cephalosporins due to the production of extended-spectrum beta-lactamases. The resistance of Campylobacter sp to ampicillin was the most prevalent, whereas less than 5% of isolates were resistant to each of the other antibiotics. The

  9. Investigation of Entamoeba histolytica in stool specimens by direct microscopic examination and ELISA in a hospital

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    Keramettin Yanık

    2011-09-01

    Full Text Available Objectives: Stool antigen assay has been shown to be as sensitive and specific as culture with isoenzyme analysis and to outperform microscopy for the detection of E.histolytica in endemic area. The aim of the present study is to investigate the presence of E.histolytica by direct microscopic examination and ELISA in stool samples, comparatively.Materials and methods: Between September 2010 and May 2011, a total of 975 stool samples of patients in different age groups were sent to microbiology laboratory of Kızıltepe General Hospital. Native-Lugol method and E.histolytica-specific antigen test (Adhesin Ag, Entamoeba CELISA Path was applied to all stool samples.Results: E.histolytica/dispar cysts and/or trophozoites were observed in 21 out of 975 (2.2% stool samples examined by native-Lugol method. In addition, E.histolytica-specific antigen in 975 stool specimens was investigated by ELISA. E.histolytica-specific antigen was determined in 4 patients which had E.histolytica/dispar cysts and/or trophozoites at direct microscopic examination. Although at direct microscopy of 3 patients E.histolytica/dispar cysts and/or trophozoites not observed, E.histolytica-specific antigen was found favorable. A total of 7 (0.7% E.histolytica specific antigen was found in the patient’s stool samples. Patients with E.histolytica-specific antigen were treated.Conclusion: E.histolytica specific antigen in stool samples should be investigated to avoid unnecessary treatment.

  10. Investigation of Entamoeba histolytica in stool specimens by ELISA during a year

    OpenAIRE

    Dal, Tuba

    2015-01-01

    Objectives: The aim of this study was to investigate Entamoeba histolytica in the stool samples that have been sent to microbiology laboratory by ELISA method. Materials and methods: This study was performed on 800 stool specimens belonging to different age groups and different patients sent to Microbiology Laboratory of a State Hospital between January 2009 and December 2009. Results: In this study Entamoeba histolytica/dispar cysts and/or trophozoites were observed in 31 (3.9%) out ...

  11. Blastocystis sp. in Irritable Bowel Syndrome (IBS)--Detection in Stool Aspirates during Colonoscopy.

    Science.gov (United States)

    Ragavan, Nanthiney Devi; Kumar, Suresh; Chye, Tan Tian; Mahadeva, Sanjiv; Shiaw-Hooi, Ho

    2015-01-01

    Blastocystis is one of the most common gut parasites found in the intestinal tract of humans and animals. Its' association with IBS is controversial, possibly as a result of irregular shedding of parasites in stool and variation in stool detection. We aimed to screen for Blastocystis in colonic stool aspirate samples in adult patients with and without IBS undergoing colonoscopy for various indications and measure the interleukin levels (IL-8, IL-3 and IL-5). In addition to standard stool culture techniques, polymerase chain reaction (PCR) techniques were employed to detect and subtype Blastocystis. All the serum samples collected were subjected for ELISA studies to measure the interleukin levels (IL-8, IL-3 and IL-5). Among 109 (IBS n = 35 and non-IBS n = 74) adults, direct stool examination and culture of colonic aspirates were initially negative for Blastocystis. However, PCR analysis detected Blastocystis in 6 (17%) IBS and 4 (5.5%) non-IBS patients. In the six positive IBS patients by PCR method, subtype 3 was shown to be the most predominant (3/6: 50%) followed by subtype 4 (2/6; 33.3%) and subtype 5 (1/6; 16.6%). IL-8 levels were significantly elevated in the IBS Blasto group and IBS group (pBlastocystis-infected stools. Patients with IBS infected with parasite showed an increase in the interleukin levels demonstrate that Blastocystis does have an effect in the immune system.

  12. EFFECT OF INFANT FORMULA SUPPLEMENTED WITH OLIGOSACCHARIDES ON STOOL CHARACTERISTICS OF INFANTS

    Institute of Scientific and Technical Information of China (English)

    TAO Ye-xuan; TANG Qing-ya; FENG Yi; CAI Wei

    2007-01-01

    Objective To determine whether addition of oligosaccharides to a regular infant formula can lead to changes in the colonic function in vivo, particularly the fecal characteristics. Methods One hundred and two health full term infants were randomly assigned to one of two experimental formula groups: oligosaccharide formula (OF) group or regular formula (RF) group. Fifty breast-fed infants served as a control group during the same period. During the 3 weeks' study, stool characteristics, including stooling frequency, stool consistency, pH and color, were recorded daily by parents. Results The mean fecal frequency of the infants in the OF group was significantly more than those of the RF group ( P < 0. 05 ). The stools of the RF group were significantly harder than those in the OF group( P < 0. 001 ). Although the mean stool pH score and stool color score of infants in the OF group were not significantly different from that of infants in the RF group, it was much closer to that of breast-fed infants. Conclusion The addition of oligosaccharides to a normal infant formula could lead to improvements in fecal characteristics.

  13. A conventional polymerase chain reaction-based method for the diagnosis of human schistosomiasis in stool samples from individuals in a low-endemicity area

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    Teiliane Rodrigues Carneiro

    2013-12-01

    Full Text Available The aim of this study was to evaluate the efficacy of a polymerase chain reaction (PCR-based method to detect Schistosoma mansoni DNA in stool samples from individuals living in a low-endemicity area in Brazil. Of the 125 initial stool samples, 80 were ELISA reactive and eggs were identified in 19 of the samples by parasitological examination. For the PCR evaluations, 56 stool samples were selected and divided into five groups. Groups I-IV were scored negative for S. mansoni eggs by parasitological examination. Groups I and II were ELISA reactive, whereas Groups III and IV were ELISA nonreactive. Groups II and III were positive for other intestinal parasites. PCR testing scored eight samples as positive from these four groups. Group V represented the S. mansoni -positive group and it included ELISA-reactive samples that were scored positive for S. mansoni by one or more parasitological examinations (6/19 were positive by Kato-Katz method, 9/17 by saline gradient and 10/13 by Helmintex®. PCR scored 13 of these 19 samples as positive for S. mansoni . We conclude that while none of these methods yielded 100% sensitivity, a combination of techniques should be effective for improving the detection of S. mansoni infection in low-endemicity areas.

  14. A conventional polymerase chain reaction-based method for the diagnosis of human schistosomiasis in stool samples from individuals in a low-endemicity area

    Science.gov (United States)

    Carneiro, Teiliane Rodrigues; Peralta, Regina Helena Saramago; Pinheiro, Marta Cristhiany Cunha; de Oliveira, Sara Menezes; Peralta, José Mauro; Bezerra, Fernando Schemelzer Moraes

    2013-01-01

    The aim of this study was to evaluate the efficacy of a polymerase chain reaction (PCR)-based method to detect Schistosoma mansoni DNA in stool samples from individuals living in a low-endemicity area in Brazil. Of the 125 initial stool samples, 80 were ELISA reactive and eggs were identified in 19 of the samples by parasitological examination. For the PCR evaluations, 56 stool samples were selected and divided into five groups. Groups I-IV were scored negative for S. mansoni eggs by parasitological examination. Groups I and II were ELISA reactive, whereas Groups III and IV were ELISA nonreactive. Groups II and III were positive for other intestinal parasites. PCR testing scored eight samples as positive from these four groups. Group V represented the S. mansoni -positive group and it included ELISA-reactive samples that were scored positive for S. mansoni by one or more parasitological examinations (6/19 were positive by Kato-Katz method, 9/17 by saline gradient and 10/13 by Helmintex®). PCR scored 13 of these 19 samples as positive for S. mansoni . We conclude that while none of these methods yielded 100% sensitivity, a combination of techniques should be effective for improving the detection of S. mansoni infection in low-endemicity areas. PMID:24402156

  15. The impact of different methods of DNA extraction on microbial community measures of BALF samples based on metagenomic data.

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    Wen, Yan; Xiao, Fei; Wang, Chen; Wang, Zhen

    2016-01-01

    It is a challenge to find a better microorganisms DNA extraction method for samples taken from the lower airways for metagenomic sequencing, as the concentrations of bacteria in the alveoli and small airways are likely considerably less than that of the mouth or lower digestive tract. Background DNA from the host, and extraction biases can significantly interfere with microbiota assessment and increase the cost of sequencing. This study aimed to develop an optimized DNA extraction method, which would enable a higher concentration of microbial DNA to be extracted from the samples. We compared the microbiota profiles of the lower airway communities in twelve individuals with IIP. DNA was extracted using three different extraction methods: QIAamp UCP PurePathogen Blood Kit named kit3 in this study, QIAamp UCP Pathogen Mini Kit named kit2, and QIAamp DNA Microbiome Kit named kit1. DNA libraries were constructed according to the manufacturer's instructions (Illumina). The same workflows from Illumina were used to perform cluster generation, template hybridization, isothermal amplification, linearization, blocking, denaturing, and hybridization of the sequencing primers. Raw data was uploaded to MG-RAST v3 and analyzed. A great number of bacterium inhabits the lower airways of patients with IIP, though there is no airway infection. More bacterium was found in mouth or upper airway. DNA concentrations of DNA samples isolated with kit1 with Benzonase were significantly lower than those isolated with the other two kits for BALF and mouthwash samples. Moreover, the ratio of human genome in clean reads of samples isolated with kit1 with Benzonase was remarkably smaller than those isolated with kit2 and kit3. The relative abundance of total bacteria, the total number of taxa, and the relative abundance of taxa in BALF samples as opposed to mouthwash samples with kit1 were significantly higher than for those extracted the other kits. A microbial DNA extraction method with

  16. Microbiome analysis of stool samples from African Americans with colon polyps.

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    Hassan Brim

    Full Text Available BACKGROUND: Colonic polyps are common tumors occurring in ~50% of Western populations with ~10% risk of malignant progression. Dietary agents have been considered the primary environmental exposure to promote colorectal cancer (CRC development. However, the colonic mucosa is permanently in contact with the microbiota and its metabolic products including toxins that also have the potential to trigger oncogenic transformation. AIM: To analyze fecal DNA for microbiota composition and functional potential in African Americans with pre-neoplastic lesions. MATERIALS & METHODS: We analyzed the bacterial composition of stool samples from 6 healthy individuals and 6 patients with colon polyps using 16S ribosomal RNA-based phylogenetic microarray; the Human intestinal Tract Chip (HITChip and 16S rRNA gene barcoded 454 pyrosequencing. The functional potential was determined by sequence-based metagenomics using 454 pyrosequencing. RESULTS: Fecal microbiota profiling of samples from the healthy and polyp patients using both a phylogenetic microarraying (HITChip and barcoded 454 pyrosequencing generated similar results. A distinction between both sets of samples was only obtained when the analysis was performed at the sub-genus level. Most of the species leading to the dissociation were from the Bacteroides group. The metagenomic analysis did not reveal major differences in bacterial gene prevalence/abundances between the two groups even when the analysis and comparisons were restricted to available Bacteroides genomes. CONCLUSION: This study reveals that at the pre-neoplastic stages, there is a trend showing microbiota changes between healthy and colon polyp patients at the sub-genus level. These differences were not reflected at the genome/functions levels. Bacteria and associated functions within the Bacteroides group need to be further analyzed and dissected to pinpoint potential actors in the early colon oncogenic transformation in a large sample size.

  17. 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.

  18. Is "dried stool spots on filter paper method (DSSFP)" more sensitive and effective for detecting Blastocystis spp. and their subtypes by PCR and sequencing?

    Science.gov (United States)

    Seyer, Ayse; Karasartova, Djursun; Ruh, Emrah; Güreser, Ayse Semra; Imir, Turgut; Taylan-Ozkan, Aysegul

    2016-12-01

    PCR and DNA sequencing are currently the diagnostic methods of choice for detection of Blastocystis spp. and their suptypes. Fresh or frozen stool samples have disadvantages in terms of several aspects such as transportation, storage, and existence of PCR inhibitors. Filter paper technology may provide a solution to these issues. The aim of the present study was to detect Blastocystis spp. and their subtypes by employing two different preservation methods: conventional frozen stool (FS) and dried stool spots on filter paper (DSSFP). Concentration and purity of DNA, sensitivity of PCR, and DNA sequencing results obtained from the two methods were also compared. A total of 230 fecal samples were included and separated into two parts: one part of the fecal samples were directly frozen and stored at -20 °C. The remaining portion of the specimens were homogenized with saline and spread onto the filter papers as thin layer with a diameter of approximately 3 cm. After air-dried, the filter papers were stored at room temperature. DSSFP samples were collected by scraping from the filter papers. DNA were extracted by EURx Stool DNA Extraction Kit from both samples. Concentration and purity were measured with Nano-Drop, then PCR and sequencing were conducted for detection of Blastocystis spp. and its genotypes. Pure DNA was obtained with a A260/A280 ratio of 1.7-2.2 in both methods. DNA yield from FS was 25-405 ng/μl and average DNA concentration was 151 ng/μl, while these were 7-339 and 122 ng/μl for DSSFP, respectively. No PCR inhibition was observed in two methods. DNA from DSSFP were found to be stable and PCR were reproducible for at least 1 year. FS-PCR- and DSSFP-PCR-positive samples were 49 (21.3 %) and 58 (25.3 %), respectively (p = 0.078). The 43 specimens were concordantly positive by both FS-PCR and DSSFP-PCR. When the microscopy was taken as the gold standard, sensitivity of DSSFP-PCR and FS-PCR was 95.5 and 86.4 %, while specificity of both

  19. A comparison of DNA collection and retrieval from two swab types (cotton and nylon flocked swab) when processed using three QIAGEN extraction methods.

    Science.gov (United States)

    Brownlow, Robert J; Dagnall, Kathryn E; Ames, Carole E

    2012-05-01

    The Metropolitan Police Service currently uses cotton swabs to retrieve DNA for forensic profiling. Recently, a new nylon flocked swab type has become available from Copan (MicroRheologics, Brescia, Italy) that it is claimed, offers increased sample recovery and release yields. If true, the flocked swab may have important applications in DNA evidence retrieval. This study examines the DNA retrieval capability of cotton and nylon flocked swabs when extracted using three common extraction platforms (QIAcube, BioRobot EZ1 and manually processed QIAamp DNA investigator kit). Results indicate that both swab types are capable of recovering high percentages of DNA (>50%); however, the extraction platform selected was shown to have a significant effect upon DNA retrieval. Across all experiments, the cotton swab combined with the spin-column extractions was shown to be most effective, with the nylon swab and BioRobot EZ1 combination being the least effective. These findings illustrate the importance of extraction method selection.

  20. Helicobacter pylori Stool Antigen Feco-prevalence in Food Workers in Van, Turkey

    Directory of Open Access Journals (Sweden)

    Hanifi Körkoca

    2015-03-01

    Full Text Available Objectives: Helicobacter pylori contributes to the pathogenesis of peptic ulcers, cancer, and may also cause extra gastric infections. These bacteria can be transmitted by means of fecal-oral, oral-oral, and gastro-oral via an infected person. The present study aims to investigate the existence of H.pylori antigens in the stools of workers employed in the food industry. Methods: The existence of the H.pylori stool antigen (HpSA in the stool of food industry workers was researched via the stool antigen test. Results: The H.pylori stool antigen was detected in 74 out of 154 people taking part in this study (48.05%. No statistical differences were found between the HpSA positivity and the branches of their works. Conclusions: The fact that 48.05% HpSA was detected in the workers employed in the food industry reveals the potential significance of these people in terms of the H.pylori infections and the need for further studies on this subject. J Microbiol Infect Dis 2015;5(1: 10-14

  1. Modification of stool's water content in constipated infants: management with an adapted infant formula

    Directory of Open Access Journals (Sweden)

    Alvarez Marina M

    2011-05-01

    Full Text Available Abstract Background Constipation is a common occurrence in formula-fed infants. The aim of this preliminary study was to evaluate the impact of a formula with high levels of lactose and magnesium, in compliance with the official regulations, on stool water content, as well as a parental assessment of constipation. Materials and methods Thirty healthy term-born, formula-fed infants, aged 4-10 weeks, with functional constipation were included. All infants were full-term and fed standard formula. Exclusion criteria were preterm and/or low birth weight, organic constipation, being breast fed or fed a formula specially designed to treat constipation. Stool composition was measured by near-infrared reflectance analysis (NIRA and parents answered questions about crying associated with defecation and stool consistency at baseline and after two weeks of the adapted formula. Results After 2 weeks of the adapted formula, stool water content increased from 71 +/- 8.1% to 84 +/- 5.9%, (p Conclusions This preliminary study suggests that an adapted formula with high levels of lactose and magnesium increases stool water content and improves symptoms of constipation in term-born, formula-fed infants. A larger randomized placebo-controlled trial is indicated.

  2. Detection of Giardia lamblia and Cryptosporidium parvum by direct immunofluorescence assay in stool specimen.

    Science.gov (United States)

    Rahman, M M; Hossain, M A; Paul, S K; Ahmed, S; Islam, A; Ehsan, M A; Alam, M M; Kabir, M R; Sarkar, S R

    2014-07-01

    Giardia and Cryptosporidium are the pathogens which transmitted through contaminated soil and contaminated water are significant causes of diarrhea and nutritional disorders in institutional and community peoples. Children and immune compromise persons are more vulnerable for these infections. Both Giardiasis and Cryptosporidiosis were included in 2004 as WHO Neglected Disease. So this is a major public health problem in developing countries. The present study was carried out to detect the Giardia and Cryptosporidium from diarrheic or patient having loose stool by Direct Immunofluorescence assay. The study was conducted during July 20012 to February 2013 and the work was done in Mymensingh Medical College in the department of Microbiology and in Bangladesh Agricultural University in the department of Veterinary Medicine. A total of 100 loose stools were collected from school children of different area and hospital under sadar upazilla, Mymensingh. The detection of Giardia lamblia and Cryptosporidium parvum showed the individual prevalence 8% and 4% respectively. The highest cyst/oocyst count was 85,000 and 1,000/gm of stool and the lowest being 100 and 50/gm of stool for Giardiasis and Cryptosporidiosis respectively. The detection rate of Giardia and Cryptosporidium by Immunofluorescence assay was relatively higher than the previous study done in Bangladesh and this was the first report from Bangladesh over human stool specimen using Immunofluorescence assay. So, Immunofluorescence assay could be adapted for rapid and accurate detection of Giardia and Cryptosporidium.

  3. Detection of Gastrointestinal Pathogens from Stool Samples on Hemoccult Cards by Multiplex PCR

    Directory of Open Access Journals (Sweden)

    Martin Alberer

    2017-01-01

    Full Text Available Purpose. Up to 30% of international travelers are affected by travelers’ diarrhea (TD. Reliable data on the etiology of TD is lacking. Sufficient laboratory capacity at travel destinations is often unavailable and transporting conventional stool samples to the home country is inconvenient. We evaluated the use of Hemoccult cards for stool sampling combined with a multiplex PCR for the detection of model viral, bacterial, and protozoal TD pathogens. Methods. Following the creation of serial dilutions for each model pathogen, last positive dilution steps (LPDs and thereof calculated last positive sample concentrations (LPCs were compared between conventional stool samples and card samples. Furthermore, card samples were tested after a prolonged time interval simulating storage during a travel duration of up to 6 weeks. Results. The LPDs/LPCs were comparable to testing of conventional stool samples. After storage on Hemoccult cards, the recovery rate was 97.6% for C. jejuni, 100% for E. histolytica, 97.6% for norovirus GI, and 100% for GII. Detection of expected pathogens was possible at weekly intervals up to 42 days. Conclusion. Stool samples on Hemoccult cards stored at room temperature can be used in combination with a multiplex PCR as a reliable tool for testing of TD pathogens.

  4. Yield of stool culture with isolate toxin testing versus a two-step algorithm including stool toxin testing for detection of toxigenic Clostridium difficile.

    Science.gov (United States)

    Reller, Megan E; Lema, Clara A; Perl, Trish M; Cai, Mian; Ross, Tracy L; Speck, Kathleen A; Carroll, Karen C

    2007-11-01

    We examined the incremental yield of stool culture (with toxin testing on isolates) versus our two-step algorithm for optimal detection of toxigenic Clostridium difficile. Per the two-step algorithm, stools were screened for C. difficile-associated glutamate dehydrogenase (GDH) antigen and, if positive, tested for toxin by a direct (stool) cell culture cytotoxicity neutralization assay (CCNA). In parallel, stools were cultured for C. difficile and tested for toxin by both indirect (isolate) CCNA and conventional PCR if the direct CCNA was negative. The "gold standard" for toxigenic C. difficile was detection of C. difficile by the GDH screen or by culture and toxin production by direct or indirect CCNA. We tested 439 specimens from 439 patients. GDH screening detected all culture-positive specimens. The sensitivity of the two-step algorithm was 77% (95% confidence interval [CI], 70 to 84%), and that of culture was 87% (95% CI, 80 to 92%). PCR results correlated completely with those of CCNA testing on isolates (29/29 positive and 32/32 negative, respectively). We conclude that GDH is an excellent screening test and that culture with isolate CCNA testing detects an additional 23% of toxigenic C. difficile missed by direct CCNA. Since culture is tedious and also detects nontoxigenic C. difficile, we conclude that culture is most useful (i) when the direct CCNA is negative but a high clinical suspicion of toxigenic C. difficile remains, (ii) in the evaluation of new diagnostic tests for toxigenic C. difficile (where the best reference standard is essential), and (iii) in epidemiologic studies (where the availability of an isolate allows for strain typing and antimicrobial susceptibility testing).

  5. Enteropathogenic and enteroaggregative E. coli in stools of children with acute gastroenteritis in Davidson County, Tennessee

    Science.gov (United States)

    Foster, Monique A.; Iqbal, Junaid; Zhang, Chengxian; McHenry, Rendie; Cleveland, Brent E.; Romero-Herazo, Yesenia; Fonnesbeck, Chris; Payne, Daniel C.; Chappell, James D.; Halasa, Natasha; Gómez-Duarte, Oscar G.

    2015-01-01

    This prospective acute gastroenteritis (AGE) surveillance was conducted in the inpatient and emergency room settings at a referral pediatric hospital to determine the prevalence of diarrheagenic Escherichia coli (DEC) in children<12 years of age with AGE in Davidson County, Tennessee. Subjects 15 days to 11 years of age, who presented with diarrhea and/or vomiting, were enrolled. Stool specimens were processed for detection of DEC using multiplex polymerase chain reaction. From December 1, 2011, to June 30, 2012, a total of 79 (38%) out of 206 stool specimens from children with AGE tested positive for E. coli. A total of 12 (5.8%) out of 206 stool specimens from children with AGE were positive for a DEC. Eight (67%) out of these 12 were positive for enteropathogenic E. coli, and the remaining 4 were positive for enteroaggregative E. coli. DEC clinical isolates clustered with known E. coli enteropathogens according to multilocus sequencing typing. PMID:26298817

  6. Picky eating in preschool children: Associations with dietary fibre intakes and stool hardness.

    Science.gov (United States)

    Taylor, Caroline M; Northstone, Kate; Wernimont, Susan M; Emmett, Pauline M

    2016-05-01

    It has been suggested that constipation may be associated with picky eating. Constipation is a common condition in childhood and a low intake of dietary fibre may be a risk factor. Differences in fibre intake between picky and non-picky children and its relation to stool consistency is currently not well-understood. Children enrolled in the Avon Longitudinal Study of Parents and Children identified as picky eaters (PE) were compared with non-picky eaters (NPE): (1) to determine dietary fibre intake at 38 months; (2) to investigate whether any difference in dietary fibre intake was predictive of usual stool hardness at 42 months. PE was identified from questionnaires at 24 and 38 months. Usual stool hardness was identified from a questionnaire at 42 months. Dietary intake was assessed at 38 months with a food frequency questionnaire. Dietary fibre intake was lower in PE than NPE (mean difference -1.4 (95% CI -1.6, -1.2) g/day, p hard stools (adjusted multinomial model; OR 1.31, 95% CI 1.07, 1.61; p = 0.010). This was attenuated when dietary fibre was included in the model, suggesting that fibre intake mediated the association (OR 1.16, 95% CI 0.94, 1.43, p = 0.180). Picky eating in 3-year-old children was associated with an increased prevalence of usually having hard stools. This association was mediated by low dietary fibre intake, particularly from vegetables, in PE. For children with PE, dietary advice aimed at increasing fibre intake may help avoid hard stools.

  7. Background levels of carbon-13 reduced in breath and stool by new infant formula.

    Science.gov (United States)

    Boutton, T W; Hopkinson, J M; Benton, D A; Klein, P D

    1988-01-01

    Studies of the absorption and bioavailability of nutrients naturally enriched with 13C require accurate measurements of small increases of 13C in respiratory CO2 and stool carbon. The sensitivity of these measurements would be increased if the natural background of 13C in these excreta were reduced. We have developed a 13C-depleted infant formula based on lactose, whey, and casein from New Zealand cows that consume only C3 vegetation naturally low in 13C. This formula, designated CNRC3, was produced by a commercial infant formula manufacturer and was comparable with a 60:40 whey/casein product. To test the ability of the formula to reduce baseline levels of 13C in infant excreta, 10 formula-fed infants 28-60 days old and free of metabolic disorders were enrolled in the 9-day study. Two stool samples were collected daily. Infants received their usual formula on days 1 and 2 and were switched to CNRC3 formula for days 3-9. On days 2 and 9, seven breath samples were collected at 30-min intervals with a face mask. Breath and stool samples were analyzed for 13C content by gas isotope ratio mass spectrometry. Infants consuming their commercial formula had breath delta 13C values of -21.1 +/- 0.6% over the 3-h collection period; stool values were -22.9 +/- 0.4%. After 7 days on the CNRC3 formula, delta 13C values of breath declined by 5.6% to -26.7 +/- 0.7%; stool values declined by 3.0% to -25.6 +/- 0.5%. The reduced background of 13C achieved by the CNRC3 formula can improve resolution of excess 13C from naturally enriched substrates in infant breath by approximately 50% and in stool by approximately 30%.

  8. PoopMD, a Mobile Health Application, Accurately Identifies Infant Acholic Stools.

    Directory of Open Access Journals (Sweden)

    Amy Franciscovich

    Full Text Available Biliary atresia (BA is the leading cause of pediatric end-stage liver disease in the United States. Education of parents in the perinatal period with stool cards depicting acholic and normal stools has been associated with improved time-to-diagnosis and survival in BA. PoopMD is a mobile application that utilizes a smartphone's camera and color recognition software to analyze an infant's stool and determine if additional follow-up is indicated. PoopMD was developed using custom HTML5/CSS3 and wrapped to work on iOS and Android platforms. In order to define the gold standard regarding stool color, seven pediatricians were asked to review 45 photographs of infant stool and rate them as acholic, normal, or indeterminate. Samples for which 6+ pediatricians demonstrated agreement defined the gold standard, and only these samples were included in the analysis. Accuracy of PoopMD was assessed using an iPhone 5s with incandescent lighting. Variability in analysis of stool photographs as acholic versus normal with intermediate rating weighted as 50% agreement (kappa was compared between three laypeople and one expert user. Variability in output was also assessed between an iPhone 5s and a Samsung Galaxy S4, as well as between incandescent lighting and compact fluorescent lighting. Six-plus pediatricians agreed on 27 normal and 7 acholic photographs; no photographs were defined as indeterminate. The sensitivity was 7/7 (100%. The specificity was 24/27 (89% with 3/27 labeled as indeterminate; no photos of normal stool were labeled as acholic. The Laplace-smoothed positive likelihood ratio was 6.44 (95% CI 2.52 to 16.48 and the negative likelihood ratio was 0.13 (95% CI 0.02 to 0.83. kappauser was 0.68, kappaphone was 0.88, and kappalight was 0.81. Therefore, in this pilot study, PoopMD accurately differentiates acholic from normal color with substantial agreement across users, and almost perfect agreement across two popular smartphones and ambient light

  9. Reaction mechanism of Ru(II) piano-stool complexes: umbrella sampling QM/MM MD study.

    Science.gov (United States)

    Futera, Zdeněk; Burda, Jaroslav V

    2014-07-15

    Biologically relevant interactions of piano-stool ruthenium(II) complexes with ds-DNA are studied in this article by hybrid quantum mechanics-molecular mechanics (QM/MM) computational technique. The whole reaction mechanism is divided into three phases: (i) hydration of the [Ru(II) (η(6) -benzene)(en)Cl](+) complex, (ii) monoadduct formation between the resulting aqua-Ru(II) complex and N7 position of one of the guanines in the ds-DNA oligomer, and (iii) formation of the intrastrand Ru(II) bridge (cross-link) between two adjacent guanines. Free energy profiles of all the reactions are explored by QM/MM MD umbrella sampling approach where the Ru(II) complex and two guanines represent a quantum core, which is described by density functional theory methods. The combined QM/MM scheme is realized by our own software, which was developed to couple several quantum chemical programs (in this study Gaussian 09) and Amber 11 package. Calculated free energy barriers of the both ruthenium hydration and Ru(II)-N7(G) DNA binding process are in good agreement with experimentally measured rate constants. Then, this method was used to study the possibility of cross-link formation. One feasible pathway leading to Ru(II) guanine-guanine cross-link with synchronous releasing of the benzene ligand is predicted. The cross-linking is an exergonic process with the energy barrier lower than for the monoadduct reaction of Ru(II) complex with ds-DNA. Copyright © 2014 Wiley Periodicals, Inc.

  10. Rapid quantification of semen hepatitis B virus DNA by real-time polymerase chain reaction

    Institute of Scientific and Technical Information of China (English)

    Wei-Ping Qian; Li-Ka Shing; Yue-Qiu Tan; Ying Chen; Ying Peng; Zhi Li; Guang-Xiu Lu; Marie C. Lin; Hsiang-Fu Kung; Ming-Ling He

    2005-01-01

    AIM: To examine the sensitivity and accuracy of real-time polymerase chain reaction (PCR) for the quantification of hepatitis B virus (HBV) DNA in semen.METHODS: Hepatitis B viral DNA was isolated from HBV carriers' semen and sera using phenol extraction method and QTAamp DNA blood mini kit (Qiagen, Germany). HBV DNA was detected by conventional PCR and quantified by TaqMan technology-based real-time PCR (quantitative polymerase chain reaction (qPCR)). The detection threshold was 200 copies of HBV DNA for conventional PCR and 10 copies of HBV DNA for real time PCR per reaction.RESULTS: Both methods of phenol extraction and QIAamp DNA blood mini kit were suitable for isolating HBV DNA from semen. The value of the detection thresholds was 500 copies of HBV DNA per mL in the semen. The viral loads were 7.5×107 and 1.67×107 copies of HBV DNA per mL in two HBV infected patients' sera, while 2.L4×105 and 3.02×105 copies of HBV DNA per mL in the semen.CONCLUSION: Real-time PCR is a more sensitive and accurate method to detect and quantify HBV DNA in the semen.

  11. Comparison of polycarbonate and cellulose acetate membrane filters for isolation of Campylobacter concisus from stool samples

    DEFF Research Database (Denmark)

    Linde Nielsen, Hans; Engberg, Jørgen; Ejlertsen, Tove

    2013-01-01

    One thousand seven hundred ninety-one diarrheic stool samples were cultivated for Campylobacter spp. We found a high prevalence of Campylobacter concisus with use of a polycarbonate filter (n = 114) compared to a cellulose acetate filter (n = 79) (P < .0001). The polycarbonate filter is superior ...

  12. Evaluation of the positive predictive value of a rapid Immunochromatographic test to detect Campylobacter in stools

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    Floch Pauline

    2012-12-01

    Full Text Available Abstract The recently developed rapid immunochromatographic tests (ICT have the potential to provide a quick and easy diagnosis of Campylobacter enteritis in comparison to culture. In a previous study we found them sensitive but lacking in specificity. The aim of the present study was to focus on the problem of specificity and determine the positive predictive value (PPV of a positive result of the ImmunoCard Stat! Campy (Meridian Bioscience, Cincinnati, OH, USA. For this purpose, the stools positive by ICT were cultured according to 3 different protocols: Karmali agar, Preston enrichment broth subcultured on Karmali agar, and a filtration method on a blood agar without antibiotics, all incubated for 7 days at 37°C. Out of 609 stools from adults and children with community acquired enteritis, the reference methods detected 25 positive cases (4.1% (culture: 19, specific PCR and ELISA both positive: 6 and the ICT: 31 including the 25 true positives. The PPV was 80.6%. We conclude that ICT is a good method to screen Campylobacter positive stools but because of its lack of specificity the positive stools must be tested by another method.

  13. Organometallic macromolecules with piano stool coordination repeating units: chain configuration and stimulated solution behaviour.

    Science.gov (United States)

    Cao, Kai; Ward, Jonathan; Amos, Ryan C; Jeong, Moon Gon; Kim, Kyoung Taek; Gauthier, Mario; Foucher, Daniel; Wang, Xiaosong

    2014-09-11

    Theoretical calculations illustrate that organometallic macromolecules with piano stool coordination repeating units (Fe-acyl complex) adopt linear chain configuration with a P-Fe-C backbone surrounded by aromatic groups. The macromolecules show molecular weight-dependent and temperature stimulated solution behaviour in DMSO.

  14. Acid fast cysts in diarrheal stool samples of HIV positive patients

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    Anuradha Mokkapati

    2015-02-01

    Full Text Available Background: Gastrointestinal infections are very common in patients with HIV infection or AIDS, and diarrhea is a common clinical presentation of these infections. Acid fast protozoans are very commonly responsible for diarrhea in HIV positive patients leading to death in many cases. Methods: The study group included 50 HIV seropositive patients suffering from diarrhea and the control group included 50 HIV seronegative patients suffering from diarrhea. The stool samples collected were concentrated using formol-ether concentration technique and stained using modified Ziehl-Neelsen's staining procedure. Results: Among the diarrheal stool samples of HIV positive patients (n=50, 17 (34% were positive for acid fast cysts, and among the HIV negative stool samples (n=50, 2 (4% were positive for acid fast cysts. Cryptosporidium oocysts were detected in 15 (30% and Isospora oocysts in 2 (4% of the samples in the study group. Cryptosporidium oocysts were detected in 2 (4% of the samples in the control group. There existed a significant difference between the positivity of HIV-positive and HIV-negative diarrheal stool samples. Conclusion: Timely and effective diagnosis could help in delivering appropriate treatment in an already immunocompromised patient. [Int J Res Med Sci 2015; 3(2.000: 425-428

  15. Not Your Run-of-the-Mill Art-Room Stools

    Science.gov (United States)

    Chrzanowski, Rose-Ann C.

    2010-01-01

    An art room should be a garden of visual stimulation, born of creativity, inquiry, critical thinking and intellectual conversation--and a little collaboration is not a bad thing either! When the author unpacked the new stools for her art room at the high school, she envisioned something more beautiful than the brown masonite circles that…

  16. Active surveillance for carbapenem-resistant Enterobacteriaceae using stool specimens submitted for testing for Clostridium difficile.

    Science.gov (United States)

    Banach, David B; Francois, Jeannette; Blash, Stephanie; Patel, Gopi; Jenkins, Stephen G; LaBombardi, Vincent; Kreiswirth, Barry N; Srinivasan, Arjun; Calfee, David P

    2014-01-01

    Active surveillance to identify asymptomatic carriers of carbapenem-resistant Enterobacteriaceae (CRE) is a recommended strategy for CRE control in healthcare facilities. Active surveillance using stool specimens tested for Clostridium difficile is a relatively low-cost strategy to detect CRE carriers. Further evaluation of this and other risk factor-based active surveillance strategies is warranted.

  17. Development of Poliovirus Extraction Method from Stool Extracts by Using Magnetic Nanoparticles Sensitized with Soluble Poliovirus Receptor

    OpenAIRE

    Arita, Minetaro

    2013-01-01

    A method for extracting poliovirus (PV) from stool extracts was developed. Magnetic nanoparticles sensitized with soluble PV receptor efficiently extracted PV pseudovirus (>99% extraction) or endogenous infectious PVs (>90% extraction) from stool extracts. This method would be useful for extraction of PV from crude biological samples.

  18. 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.

  19. Isolation and characterization of Yersinia-specific bacteriophages from pig stools in Finland.

    Science.gov (United States)

    Salem, M; Virtanen, S; Korkeala, H; Skurnik, M

    2015-03-01

    Bacteriophages infect bacteria, and they are present everywhere in the world including the intestinal tracts of animals. Yersiniosis is a common foodborne infection caused by Yersinia enterocolitica and Yersinia pseudotuberculosis. As these bacteria are frequently isolated from pigs, we wanted to know whether Yersinia-specific bacteriophages are also present in the pig stools and, if so, whether there is a positive or negative association between the prevalence of the Yersinia phages and the pathogenic Yersinia in the stool samples. Altogether 793 pig stool samples collected between November 2010 and March 2012 from 14 Finnish pig farms were screened for the presence of bacteriophages able to infect Y. enterocolitica serotype O:3, O:5,27 or O:9 strains, or Y. pseudotuberculosis serotype O:1a, O:1b or O:3 strains. Yersinia phages were isolated from 90 samples from eight farms. Yersinia enterocolitica O:3 was infected by 59 phages, 28 phages infected serotypes O:3 and O:5,27, and eight phages infected serotypes O:3, O:5,27 and O:9, and Y. pseudotuberculosis O:1a by eight phages. Many phages originating from pigs in the same farm were identical based on their restriction enzyme digestion patterns; 20 clearly different phages were selected for further characterization. Host ranges of these phages were tested with 94 Yersinia strains. Six of the phages infected eight strains, 13 phages infected three strains, and one phage infected only one strain, indicating that the phages had a relatively narrow host range. There was a clear association between the presence of the host bacteria and specific phages in the stools. The isolated bacteriophages may have potential as biocontrol agents for yersiniosis in both humans and pigs in future, and as alternatives or in addition to antibiotics. To our knowledge, this is the first reported isolation of Yersinia-specific phages from pig stool samples. © 2014 The Society for Applied Microbiology.

  20. Validation of a simple stool diary used by caregivers to document diarrhea among young children in a low-income country

    DEFF Research Database (Denmark)

    Grenov, Benedikte; Namusoke, Hanifa; Nabukeera-Barungi, Nicolette;

    2016-01-01

    OBJECTIVES: Development and validation of a simple stool diary for caretakers collecting data on stool frequency and consistency among young children in a low-income country. METHODS: Focus group studies evaluated how diarrhea was understood by caregivers (content validity). The sensitivity......% of caregivers had low scoring abilities after three days. The degree of dehydration (4-point score) was correlated with both increasing stool frequency and liquid stool consistency (+0.2 points (0.07-0.3), p = 0.0018 for six or more diarrheal stools, compared to three or more diarrheal stools per day, and +0...

  1. An in-depth analysis of a piece of shit: distribution of Schistosoma mansoni and hookworm eggs in human stool.

    Directory of Open Access Journals (Sweden)

    Stefanie J Krauth

    Full Text Available BACKGROUND: An accurate diagnosis of helminth infection is important to improve patient management. However, there is considerable intra- and inter-specimen variation of helminth egg counts in human feces. Homogenization of stool samples has been suggested to improve diagnostic accuracy, but there are no detailed investigations. Rapid disintegration of hookworm eggs constitutes another problem in epidemiological surveys. We studied the spatial distribution of Schistosoma mansoni and hookworm eggs in stool samples, the effect of homogenization, and determined egg counts over time in stool samples stored under different conditions. METHODOLOGY: Whole-stool samples were collected from 222 individuals in a rural part of south Côte d'Ivoire. Samples were cut into four pieces and helminth egg locations from the front to the back and from the center to the surface were analyzed. Some samples were homogenized and fecal egg counts (FECs compared before and after homogenization. The effect of stool storing methods on FECs was investigated over time, comparing stool storage on ice, covering stool samples with a water-soaked tissue, or keeping stool samples in the shade. PRINCIPAL FINDINGS: We found no clear spatial pattern of S. mansoni and hookworm eggs in fecal samples. Homogenization decreased S. mansoni FECs (p = 0.026, while no effect was observed for hookworm and other soil-transmitted helminths. Hookworm FECs decreased over time. Storing stool samples on ice or covered with a moist tissue slowed down hookworm egg decay (p<0.005. CONCLUSIONS/SIGNIFICANCE: Our findings have important implications for helminth diagnosis at the individual patient level and for epidemiological surveys, anthelmintic drug efficacy studies and monitoring of control programs. Specifically, homogenization of fecal samples is recommended for an accurate detection of S. mansoni eggs, while keeping collected stool samples cool and moist delayed the disintegration of

  2. Evaluation of four automated protocols for extraction of DNA from FTA cards.

    Science.gov (United States)

    Stangegaard, Michael; Børsting, Claus; Ferrero-Miliani, Laura; Frank-Hansen, Rune; Poulsen, Lena; Hansen, Anders J; Morling, Niels

    2013-10-01

    Extraction of DNA using magnetic bead-based techniques on automated DNA extraction instruments provides a fast, reliable, and reproducible method for DNA extraction from various matrices. Here, we have compared the yield and quality of DNA extracted from FTA cards using four automated extraction protocols on three different instruments. The extraction processes were repeated up to six times with the same pieces of FTA cards. The sample material on the FTA cards was either blood or buccal cells. With the QIAamp DNA Investigator and QIAsymphony DNA Investigator kits, it was possible to extract DNA from the FTA cards in all six rounds of extractions in sufficient amount and quality to obtain complete short tandem repeat (STR) profiles on a QIAcube and a QIAsymphony SP. With the PrepFiler Express kit, almost all the extractable DNA was extracted in the first two rounds of extractions. Furthermore, we demonstrated that it was possible to successfully extract sufficient DNA for STR profiling from previously processed FTA card pieces that had been stored at 4 °C for up to 1 year. This showed that rare or precious FTA card samples may be saved for future analyses even though some DNA was already extracted from the FTA cards.

  3. Ruthenium(II) piano stool coordination compounds with aminomethylphosphanes: Synthesis, characterisation and preliminary biological study in vitro.

    Science.gov (United States)

    Płotek, Michał; Starosta, Radosław; Komarnicka, Urszula K; Skórska-Stania, Agnieszka; Kołoczek, Przemysław; Kyzioł, Agnieszka

    2017-05-01

    Reaction of {[Ru(η(6)-p-cymene)Cl]2(μ-Cl)2} (1) with aminomethylphosphane derived from morpholine (P{CH2N(CH2CH2)2O}3 (A), PPh2{CH2N(CH2CH2)2O} (B)) or piperazine (P{CH2N(CH2CH2)2NCH2CH3}3 (C), PPh2{CH2N(CH2CH2)2NCH2CH3} (D)) results in four new piano stool ruthenium(II) coordination compounds: [Ru(η(6)-p-cymene)Cl2(A)] (2A), [Ru(η(6)-p-cymene)Cl2(B)] (2B), [Ru(η(6)-p-cymene)Cl2(C)] (2C) and [Ru(η(6)-p-cymene)Cl2(D)] (2D). Every complex was fully characterized using spectroscopic methods ((1)H, (13)C{(1)H}, (31)P{(1)H} NMR and ESI-MS), elemental analysis, X-ray single crystal diffraction and DFT calculations. Preliminary studies of in vitro cytotoxicity on the A549 (human lung adenocarcinoma) and MCF7 (human breast adenocarcinoma) cell lines revealed 2A-2D activity in the same order of magnitude as in the case of cisplatin. Additionally, the study confirmed the ability of 2A-2D to interact with DNA helix and transferrin. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Bryophyte DNA sequences from faeces of an arctic herbivore, barnacle goose (Branta leucopsis)

    NARCIS (Netherlands)

    Stech, M.; Kolvoort, E.; Loonen, M. J. J. E.; Vrieling, K.; Kruijer, J. D.

    2011-01-01

    We tested DNA extraction methods and PCR conditions for the amplification of bryophyte DNA from barnacle goose (Branta leucopsis) faeces collected from Spitsbergen (Svalbard). Both the Qiagen stool kit and a silica-based extraction method received sufficient DNA from fresh and older droppings, as in

  5. Evaluating the Impact of DNA Extraction Method on the Representation of Human Oral Bacterial and Fungal Communities.

    Science.gov (United States)

    Vesty, Anna; Biswas, Kristi; Taylor, Michael W; Gear, Kim; Douglas, Richard G

    2017-01-01

    The application of high-throughput, next-generation sequencing technologies has greatly improved our understanding of the human oral microbiome. While deciphering this diverse microbial community using such approaches is more accurate than traditional culture-based methods, experimental bias introduced during critical steps such as DNA extraction may compromise the results obtained. Here, we systematically evaluate four commonly used microbial DNA extraction methods (MoBio PowerSoil® DNA Isolation Kit, QIAamp® DNA Mini Kit, Zymo Bacterial/Fungal DNA Mini PrepTM, phenol:chloroform-based DNA isolation) based on the following criteria: DNA quality and yield, and microbial community structure based on Illumina amplicon sequencing of the V3-V4 region of the 16S rRNA gene of bacteria and the internal transcribed spacer (ITS) 1 region of fungi. Our results indicate that DNA quality and yield varied significantly with DNA extraction method. Representation of bacterial genera in plaque and saliva samples did not significantly differ across DNA extraction methods and DNA extraction method showed no effect on the recovery of fungal genera from plaque. By contrast, fungal diversity from saliva was affected by DNA extraction method, suggesting that not all protocols are suitable to study the salivary mycobiome.

  6. Quantitative Detection of Shiga Toxins Directly from Stool Specimens of Patients Associated with an Outbreak of Enterohemorrhagic Escherichia coli in Japan—Quantitative Shiga toxin detection from stool during EHEC outbreak

    Directory of Open Access Journals (Sweden)

    Eiki Yamasaki

    2015-10-01

    Full Text Available Detection of Shiga toxins (Stx is important for accurate diagnosis of Enterohemorrhagic Escherichia coli infection. In this study, we quantitatively analyzed Stx protein in nine patients’ stool during an outbreak that occurred in Japan. Highly sensitive immunoassay (bead enzyme-linked immunosorbent assay (bead-ELISA revealed that the concentrations of toxins in stool of patients ranged from 0.71 to 10.44 ng/mL for Stx1 and 2.75 to 51.61 ng/mL for Stx2. To our knowledge, this is the first report that reveals the range of Stx protein concentrations in human stools.

  7. Quantitative Detection of Shiga Toxins Directly from Stool Specimens of Patients Associated with an Outbreak of Enterohemorrhagic Escherichia coli in Japan—Quantitative Shiga toxin detection from stool during EHEC outbreak

    Science.gov (United States)

    Yamasaki, Eiki; Watahiki, Masanori; Isobe, Junko; Sata, Tetsutaro; Nair, G. Balakrish; Kurazono, Hisao

    2015-01-01

    Detection of Shiga toxins (Stx) is important for accurate diagnosis of Enterohemorrhagic Escherichia coli infection. In this study, we quantitatively analyzed Stx protein in nine patients’ stool during an outbreak that occurred in Japan. Highly sensitive immunoassay (bead enzyme-linked immunosorbent assay (bead-ELISA)) revealed that the concentrations of toxins in stool of patients ranged from 0.71 to 10.44 ng/mL for Stx1 and 2.75 to 51.61 ng/mL for Stx2. To our knowledge, this is the first report that reveals the range of Stx protein concentrations in human stools. PMID:26516915

  8. Multiplex PCR method to detect Cyclospora, Cystoisospora, and Microsporidia in stool samples

    Science.gov (United States)

    Taniuchi, Mami; Verweij, Jaco J.; Sethabutr, Orntipa; Bodhidatta, Ladaporn; Garcia, Lynne; Maro, Athanasia; Kumburu, Happiness; Gratz, Jean; Kibiki, Gibson; Houpt, Eric R.

    2011-01-01

    Cyclospora, Cystoisospora, and Microsporidia are eukaryotic enteropathogens that are difficult to detect in stool samples because they require special stains and microscopy. We developed a multiplex PCR reaction with 4 primer sets to amplify Cyclospora cayetanensis, Cystoisospora belli, Enterocytozoon bieneusi, and Encephalitozoon intestinalis. Detection of the amplicon is through specific probes coupled to Luminex beads. Sensitivity of the assay was evaluated using Encephalitozoon intestinalis spores and revealed detection of 101 spores spiked into stool. No cross reactivity was observed. We evaluated the assay on diarrheal specimens from Thailand, Tanzania, Indonesia, and the Netherlands that had been previously tested by microscopy and the assay yielded 87–100% sensitivity and 88–100% specificity. Microscopy negative/PCR positive samples had lower Luminex values suggesting they were true but lower burden infections. In summary this is a convenient single PCR reaction that can detect Cyclospora, Cystoisospora, and Microsporidia without the need for cumbersome microscopic analysis. PMID:21982218

  9. A human gut metaproteomic dataset from stool samples pretreated or not by differential centrifugation

    Directory of Open Access Journals (Sweden)

    Alessandro Tanca

    2015-09-01

    Full Text Available We present a human gut metaproteomic dataset deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD001573. Ten aliquots of a single stool sample collected from a healthy human volunteer were either pretreated by differential centrifugation (DC; N=5 or not centrifuged (NC; N=5. Protein extracts were then processed by filter-aided sample preparation, single-run liquid chromatography and high-resolution mass spectrometry, and peptide identification was carried out using Sequest-HT as search engine within the Proteome Discoverer informatic platform. The dataset described here is also related to the research article entitled “Enrichment or depletion? The impact of stool pretreatment on metaproteomic characterization of the human gut microbiota” published in Proteomics (Tanca et al., 2015, [1].

  10. Morganella morganii causing fatal sepsis in a platelet recipient and also isolated from a donor's stool.

    Science.gov (United States)

    Golubić-Cepulić, B; Budimir, A; Plecko, V; Plenković, F; Mrsić, M; Sarlija, D; Vuk, T; Skrlin, J; Kalenić, S; Labar, B

    2004-06-01

    Bacterial contamination of blood products causes significant patient morbidity and mortality. Contaminated platelet transfusion is a frequent cause of bacteraemia and sepsis because of the storage conditions of platelets. A fatal case of Morganella morganii platelet transfusion associated with sepsis is described, along with procedures traced back to the isolation of M. morganii from a donor's stool. Molecular typing was performed, and the same M. morganii strain was found in blood and post-mortem organ cultures of platelet recipient and platelet bag and in the donor's stool. The route of contamination is unknown. The contamination could be due to either insufficient venipuncture site disinfection or the donor's transient bacteraemia. Patient died 5 days after the transfusion.

  11. DNA extraction from fresh-frozen and formalin-fixed, paraffin-embedded human brain tissue.

    Science.gov (United States)

    Wang, Jian-Hua; Gouda-Vossos, Amany; Dzamko, Nicolas; Halliday, Glenda; Huang, Yue

    2013-10-01

    Both fresh-frozen and formalin-fixed, paraffin-embedded (FFPE) human brain tissues are invaluable resources for molecular genetic studies of central nervous system diseases, especially neurodegenerative disorders. To identify the optimal method for DNA extraction from human brain tissue, we compared methods on differently-processed tissues. Fragments of LRRK2 and MAPT (257 bp and 483 bp/245 bp) were amplified for evaluation. We found that for FFPE samples, the success rate of DNA extraction was greater when using a commercial kit than a laboratory-based method (successful DNA extraction from 76% versus 33% of samples). PCR amplicon size and storage period were key factors influencing the success rate of DNA extraction from FFPE samples. In the fresh-frozen samples, the DNA extraction success rate was 100% using either a commercial kit (QIAamp DNA Micro) or a laboratory-based method (sample boiling in 0.1 mol/L NaOH, followed by proteinase K digestion, and then DNA extraction using Chelex-100) regardless of PCR amplicon length or tissue storage time. Although the present results demonstrate that PCR-amplifiable genomic DNA can be extracted from both fresh-frozen and FFPE samples, fresh brain tissue is recommended for DNA extraction in future neuropathological studies.

  12. Dipstick for rapid diagnosis of Shigella flexneri 2a in stool.

    Directory of Open Access Journals (Sweden)

    Faridabano Nato

    Full Text Available BACKGROUND: Shigellosis or bacillary dysentery, an acute bloody diarrhoea, is a major public health burden in developing countries. In the absence of prompt and appropriate treatment, the infection is often fatal, particularly in young malnourished children. Here, we describe a new diagnostic test for rapid detection, in stool, at the bedside of patients, of Shigella flexneri 2a, the most predominant agent of the endemic form of the disease. METHODOLOGY/PRINCIPAL FINDINGS: The test is based on the detection of S.flexneri 2a lipopolysaccharide (LPS using serotype 2a-specific monoclonal antibodies coupled to gold particles and displayed on one-step immunochromatographic dipstick. A concentration as low as 20 ng/ml of LPS is detected in distilled water and in reconstituted stools in under 15 minutes. The threshold of detection corresponds to a concentration of 5x10(7 CFU/ml of S. flexneri 2a, which provides an unequivocal positive reaction in three minutes in distilled water and reconstituted stools. The specificity is 100% when tested with a battery of Shigella and unrelated strains, in culture. When tested in Vietnam, on clinical samples, the specificity and sensitivity were 99.2 and 91.5%, respectively. A decrease of the sensitivity during the evaluation on stool samples was observed after five weeks at room temperature and was due to moistening of the dipsticks caused by the humidity of the air during the fifth week of the evaluation. This drawback is now overcome by improving the packaging and providing dipsticks individually wrapped in waterproof bags. CONCLUSION: This simple dipstick-bases test represents a powerful tool for case management and epidemiological surveys.

  13. Maintaining Breast Cancer Specimen Integrity and Individual or Simultaneous Extraction of Quality DNA, RNA, and Proteins from Allprotect-Stabilized and Nonstabilized Tissue Samples

    LENUS (Irish Health Repository)

    Mee, Blanaid C.

    2011-12-29

    The Saint James\\'s Hospital Biobank was established in 2008, to develop a high-quality breast tissue BioResource, as a part of the breast cancer clinical care pathway. The aims of this work were: (1) to ascertain the quality of RNA, DNA, and protein in biobanked carcinomas and normal breast tissues, (2) to assess the efficacy of AllPrep® (Qiagen) in isolating RNA, DNA, and protein simultaneously, (3) to compare AllPrep with RNEasy® and QIAamp® (both Qiagen), and (4) to examine the effectiveness of Allprotect® (Qiagen), a new tissue stabilization medium in preserving DNA, RNA, and proteins. One hundred eleven frozen samples of carcinoma and normal breast tissue were analyzed. Tumor and normal tissue morphology were confirmed by frozen sections. Tissue type, tissue treatment (Allprotect vs. no Allprotect), extraction kit, and nucleic acid quantification were analyzed by utilizing a 4 factorial design (SPSS PASW 18 Statistics Software®). QIAamp (DNA isolation), AllPrep (DNA, RNA, and Protein isolation), and RNeasy (RNA isolation) kits were assessed and compared. Mean DNA yield and A260\\/280 values using QIAamp were 33.2 ng\\/μL and 1.86, respectively, and using AllPrep were 23.2 ng\\/μL and 1.94. Mean RNA yield and RNA Integrity Number (RIN) values with RNeasy were 73.4 ng\\/μL and 8.16, respectively, and with AllPrep were 74.8 ng\\/μL and 7.92. Allprotect-treated tissues produced higher RIN values of borderline significance (P=0.055). No discernible loss of RNA stability was detected after 6 h incubation of stabilized or nonstabilized tissues at room temperature or 4°C or in 9 freeze-thaw cycles. Allprotect requires further detailed evaluation, but we consider AllPrep to be an excellent option for the simultaneous extraction of RNA, DNA, and protein from tumor and normal breast tissues. The essential presampling procedures that maintain the diagnostic integrity of pathology specimens do not appear to compromise the quality of molecular isolates.

  14. Stool Tests

    Science.gov (United States)

    ... to determine whether a type of bacteria or parasite may be infecting the intestines. Many microscopic organisms living in the intestines are ... parasites and ova (the egg stage of a parasite) if a child has prolonged diarrhea or other intestinal symptoms. Sometimes, the doctor will collect two or ...

  15. Stool Culture

    Science.gov (United States)

    ... disinfected eggs), raw poultry, uncooked vegetables, and in reptiles; pets such as lizards and turtles may carry ... infections among travelers to Africa, Asia, and Latin America. These strains of E. coli , however, are different ...

  16. Pathogenic Vibrio Strains Isolated from Human Stool and Water Samples from Western Kenya

    Directory of Open Access Journals (Sweden)

    Roselida Achieng Owuor

    2016-03-01

    Full Text Available Objective: Investigate the type of pathogenic Vibrio strains from water and stool samples collected from Migori, SonduMiriu, Nyando and Yala regions in Western Kenya. Methods: A total of 811 samples (596 water and 215 stool samples were collected during the study periods of May to December 2013 and August to September 2014. Pathogenic Vibrio strains were identified through culturing in TCBS Agar, followed by oxidation, string and serological (polyvalent tests, respectively. The PCR analysis was done using combined primers targeting Vibrionaceae 16SrRNA and species specific primers for V. vulnificus and V. cholerae. Results: The results showed the presence of V. vulnificus and V. cholerae. However, V. parahaemolyticus was not found in any of the samples. The PCR results for 16SrRNA, Vib 1, and Vib 2 showed polymorphism in the genes, this was an indication of cross combination of genes from more than one strain in one isolate. Conclusion: The study showed the presence of V. cholerae (Ogawa and Inaba in water and human stool samples. Type B V. vulnificus was detected in the water sample collected from River Migori. This information is of essence in controlling and managing cholera in the western part of Kenya. J Microbiol Infect Dis 2016;6(1: 1-7

  17. Exploring the Efficacy of Pooled Stools in Fecal Microbiota Transplantation for Microbiota-Associated Chronic Diseases.

    Science.gov (United States)

    Kazerouni, Abbas; Wein, Lawrence M

    2017-01-01

    Fecal microbiota transplantation is being assessed as a treatment for chronic microbiota-related diseases such as ulcerative colitis. Results from an initial randomized trial suggest that remission rates depend on unobservable features of the fecal donors and observable features of the patients. We use mathematical modeling to assess the efficacy of pooling stools from different donors during multiple rounds of treatment. In the model, there are two types of patients and two types of donors, where the patient type is observable and the donor type (effective or not effective) is not observable. In the model, clinical outcomes from earlier rounds of treatment are used to estimate the current likelihood that each donor is effective, and then each patient in each round is treated by a pool of donors that are currently deemed to be the most effective. Relative to the no-pooling case, pools of size two or three significantly increase the proportion of patients in remission during the first several rounds of treatment. Although based on data from a single randomized trial, our modeling suggests that pooling of stools - via daily cycling of encapsulated stool from several different donors - may be beneficial in fecal microbiota transplantation for chronic microbiota-related diseases.

  18. The complete genome of klassevirus – a novel picornavirus in pediatric stool

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    Ganem Donald

    2009-06-01

    Full Text Available Abstract Background Diarrhea kills 2 million children worldwide each year, yet an etiological agent is not found in approximately 30–50% of cases. Picornaviral genera such as enterovirus, kobuvirus, cosavirus, parechovirus, hepatovirus, teschovirus, and cardiovirus have all been found in human and animal diarrhea. Modern technologies, especially deep sequencing, allow rapid, high-throughput screening of clinical samples such as stool for new infectious agents associated with human disease. Results A pool of 141 pediatric gastroenteritis samples that were previously found to be negative for known diarrheal viruses was subjected to pyrosequencing. From a total of 937,935 sequence reads, a collection of 849 reads distantly related to Aichi virus were assembled and found to comprise 75% of a novel picornavirus genome. The complete genome was subsequently cloned and found to share 52.3% nucleotide pairwise identity and 38.9% amino acid identity to Aichi virus. The low level of sequence identity suggests a novel picornavirus genus which we have designated klassevirus. Blinded screening of 751 stool specimens from both symptomatic and asymptomatic individuals revealed a second positive case of klassevirus infection, which was subsequently found to be from the index case's 11-month old twin. Conclusion We report the discovery of human klassevirus 1, a member of a novel picornavirus genus, in stool from two infants from Northern California. Further characterization and epidemiological studies will be required to establish whether klasseviruses are significant causes of human infection.

  19. Clinical importance and representation of toxigenic and non-toxigenic Clostridium difficile cultivated from stool samples of hospitalized patients

    Directory of Open Access Journals (Sweden)

    Predrag Stojanovic

    2012-03-01

    Full Text Available The aim of this study was to fortify the clinical importance and representation of toxigenic and non-toxigenic Clostridium difficile isolated from stool samples of hospitalized patients. This survey included 80 hospitalized patients with diarrhea and positive findings of Clostridium difficile in stool samples, and 100 hospitalized patients with formed stool as a control group. Bacteriological examination of a stool samples was conducted using standard microbiological methods. Stool sample were inoculated directly on nutrient media for bacterial cultivation (blood agar using 5% sheep blood, Endo agar, selective Salmonella Shigella agar, Selenite-F broth, CIN agar and Skirrow's medium, and to selective cycloserine-cefoxitin-fructose agar (CCFA (Biomedics, Parg qe tehnicologico, Madrid, Spain for isolation of Clostridium difficile. Clostridium difficile toxin was detected by ELISA-ridascreen Clostridium difficile Toxin A/B (R-Biopharm AG, Germany and ColorPAC ToxinA test (Becton Dickinson, USA. Examination of stool specimens for the presence of parasites (causing diarrhea was done using standard methods (conventional microscopy, commercial concentration test Paraprep S Gold kit (Dia Mondial, France and RIDA®QUICK Cryptosporidium/Giardia Combi test (R-Biopharm AG, Germany. Examination of stool specimens for the presence of fungi (causing diarrhea was performed by standard methods. All stool samples positive for Clostridium difficile were tested for Rota, Noro, Astro and Adeno viruses by ELISA - ridascreen (R-Biopharm AG, Germany. In this research we isolated 99 Clostridium difficile strains from 116 stool samples of 80 hospitalized patients with diarrhea. The 53 (66.25% of patients with diarrhea were positive for toxins A and B, one (1.25% were positive for only toxin B. Non-toxigenic Clostridium difficile isolated from samples of 26 (32.5% patients. However, other pathogenic microorganisms of intestinal tract cultivated from samples of 16 patients

  20. Detection of nosocomial Clostridium difficile infections with toxigenic strains despite negative toxin A and B testing on stool samples.

    Science.gov (United States)

    Stahlmann, J; Schönberg, M; Herrmann, M; von Müller, L

    2014-09-01

    A two-step diagnostic algorithm is recommended to detect Clostridium difficile infections; however, samples are regularly found that are glutamate dehydrogenase (GDH) positive but stool toxin negative. In the present single-centre prospective study we focused on these 'difficult-to-interpret' samples and characterized them by anaerobic culture, toxigenic culture, slpA sequence typing and multiplex PCR (GenoType CDiff). The majority of stool toxin A and B-negative samples have been caused by toxigenic strains including ribotype 027. The multiplex PCR was faster and more sensitive compared with culture and allowed preliminary identification of hypervirulent strains in stool samples on the same day.

  1. Comparative evaluation of eleven commercial DNA extraction kits for real-time PCR detection of Bacillus anthracis spores in spiked dairy samples.

    Science.gov (United States)

    Mertens, Katja; Freund, Lisa; Schmoock, Gernot; Hänsel, Christoph; Melzer, Falk; Elschner, Mandy C

    2014-01-17

    Spores of Bacillus anthracis are highly resistant and can survive conditions used for food preservation. Sample size and complexity represent the major hurdles for pathogen detection in food-related settings. Eleven commercial DNA extraction kits were evaluated for detection of B. anthracis spores by quantitative real-time PCR (qPCR) in dairy products. DNA was extracted from serial dilutions of B. anthracis spores in milk powder, cream cheese, whole milk and buttermilk. Three kits (QIAamp DNA mini kit, Invisorb Food kit I and II) were determined to produce the lowest limit of detections (LODs) with equally good performance. These kits employed lysozyme and proteinase K treatments or proteinase K in combination with cethyltrimethylamonium bromide-mediated (CTAB) precipitation of cell debris for cell disruption and DNA release. The LODs for these three kits were determined as 10(2) spores/ml of distilled water, 10(3)s pores/20 mg of powdered milk and 10(4) spores/100 mg of cream cheese, respectively. Performance testing of the QIAamp DNA mini kit demonstrated a good reproducibility and appropriate detection limits from 10(3)/ml for butter milk, 10(4)/ml for whole milk and 10(4)/100 mg for low fat cream cheese. However, DNA extraction efficiency was strongly inhibited by cream cheese with higher fat contents with an increased LOD of 10(6)/100 mg spores. This study demonstrated that qPCR detection depends directly on the appropriate DNA extraction method for an individual food matrix and bacterial agent.

  2. The Most Favourable Procedure for the Isolation of Cell-Free DNA from the Plasma of Iso-Immunized RHD-Negative Pregnant Women

    Directory of Open Access Journals (Sweden)

    Riyaz Ahmad Rather

    2015-12-01

    Full Text Available Background: The ability to achieve quality recovery of cell-free foetal DNA is important for making non-invasive prenatal diagnoses. In this study, we performed quantitative and qualitative analyses of isolated DNA from maternal plasma, using different DNA-isolation methods. Method: DNA was isolated from 30 iso-immunized women via the QIAamp column-based method, using four different elution volumes and two conventionally based methods. Real-time polymerase chain-reaction quantification of RHD and β-globin genes was performed in order to determine foetal-specific sequences and total genome equivalents, respectively. Results: The column-based method at a 3 μl elution volume yielded the highest quality and quantity of total DNA (67.0±0.6 ng/μL. At a 3 μl elution volume, the β-globin and RHD ‐gene sequences were estimated to be the highest among all isolation procedures, with 2778.13±1.5 and 66.9±0.6 GEq/mL, respectively, and a 100% sensitivity for RHD ‐gene sequence detection. Among the two conventional manual methods, the boiling lysis method yielded a higher DNA concentration (53.8±0.8 ng/μL and purity (1.73±0.05. In addition, the method's sensitivity for foetal-detection sequences was only 80%, whereas the salting-out method's sensitivity was just 70%. Conclusions: This study confirms the theory that the QIAamp method is a specific and sensitive approach for purifying and quantifying plasma DNA, when used in the minimum elution volume.

  3. Glycans affect DNA extraction and induce substantial differences in gut metagenomic studies

    Science.gov (United States)

    Angelakis, Emmanouil; Bachar, Dipankar; Henrissat, Bernard; Armougom, Fabrice; Audoly, Gilles; Lagier, Jean-Christophe; Robert, Catherine; Raoult, Didier

    2016-01-01

    Exopolysaccharides produced by bacterial species and present in feces are extremely inhibitory to DNA restriction and can cause discrepancies in metagenomic studies. We determined the effects of different DNA extraction methods on the apparent composition of the gut microbiota using Illumina MiSeq deep sequencing technology. DNA was extracted from the stool from an obese female using 10 different methods and the choice of DNA extraction method affected the proportional abundance at the phylum level, species richness (Chao index, 227 to 2,714) and diversity (non parametric Shannon, 1.37 to 4.4). Moreover DNA was extracted from stools obtained from 83 different individuals by the fastest extraction assay and by an extraction assay that degradated exopolysaccharides. The fastest extraction method was able to detect 68% to 100% genera and 42% to 95% species whereas the glycan degradation extraction method was able to detect 56% to 93% genera and 25% to 87% species. To allow a good liberation of DNA from exopolysaccharides commonly presented in stools, we recommend the mechanical lysis of stools plus glycan degradation, used here for the first time. Caution must be taken in the interpretation of current metagenomic studies, as the efficiency of DNA extraction varies widely among stool samples. PMID:27188959

  4. Study of Helicobacter pylori genotype status in saliva,dental plaques, stool and gastric biopsy samples

    Institute of Scientific and Technical Information of China (English)

    Hassan Momtaz; Negar Souod; Hossein Dabiri; Meysam Sarshar

    2012-01-01

    AIM:To compare genotype of Helicobacter pylori (H.pylori) isolated from saliva,dental plaques,gastric biopsy,and stool of each patient in order to evaluate the mode of transmission ofH.pylori infection.METHODS:This cross-sectional descriptive study was performed on 300 antral gastric biopsy,saliva,dental plaque and stool samples which were obtained from patients undergoing upper gastrointestinal tract endoscopy referred to endoscopy centre of Hajar hospital of Shahrekord,Iran from March 2010 to February 2011.Initially,H.pylori strains were identified by rapid urease test (RUT) and polymerase chain reaction (PCR)were applied to determine the presence of H.pylori (ureC) and for genotyping of voculating cytotoxin gene A (vacA) and cytotoxin associated gene A (cagA) genes in each specimen.Finally the data were analyzed by using statistical formulas such as Chi-square and Fisher's exact tests to find any significant relationship between these genes and patient's diseases.P < 0.05 was considered statistically significant.RESULTS:Of 300 gastric biopsy samples,77.66%were confirmed to be H.pylori positive by PCR assay while this bacterium were detected in 10.72% of saliva,71.67% of stool samples.We were not able to find it in dental plaque specimens.The prevalence of H.pylori was 90.47% among patients with peptic ulcer disease (PUD),80% among patients with gastric cancer,and 74.13% among patients with none ulcer dyspepsia (NUD) by PCR assay.The evaluation of vacA and cagA genes showed 6 differences between gastric biopsy and saliva specimens and 11 differences between gastric and stool specimens.94.42% ofH.pylori positive specimens were cagA positive and all samples had amplified band both for vacA s and m regions.There was significant relationship between vacA s1a/m1a and PUD diseases (P =0.04),s2/m2 genotype and NUD diseases (P =0.05).No statically significant relationship was found between cagA status with clinical outcomes and vacA genotypes (P =0

  5. Acute diarrhea in HIV infected patient receiving antiretroviral therapy:is there any role of microscopic stool examination at present?

    Institute of Scientific and Technical Information of China (English)

    Beuy Joob; Viroj Wiwanitkit

    2014-01-01

    Objective:To reassess the usefulness of microscopic stool examination for theHIV infected patients with acute diarrhea.Methods:Overall100HIV-infected patients receiving standard antiretroviral therapy who visited to a primary care center(for privacy reason, the name is hereby blinded) with compliant of acute diarrhea were reviewed.In all patients, the standard microscopic stool examination was performed.Results:Of interest, from overall100 indexed cases, there is no case with determined parasite in stool samples.Conclusions:Based on our setting, it seems that there is diagnostic role of using microscopic stool examination for determining possible parasitic infestation inHIV infected patients receiving standard antiretroviral therapy who present with acute diarrhea.

  6. Preponderance of toxigenic Escherichia coli in stool pathogens correlates with toxin detection in accessible drinking-water sources.

    Science.gov (United States)

    Igbokwe, H; Bhattacharyya, S; Gradus, S; Khubbar, M; Griswold, D; Navidad, J; Igwilo, C; Masson-Meyers, D; Azenabor, A A

    2015-02-01

    Since early detection of pathogens and their virulence factors contribute to intervention and control strategies, we assessed the enteropathogens in diarrhoea disease and investigated the link between toxigenic strains of Escherichia coli from stool and drinking-water sources; and determined the expression of toxin genes by antibiotic-resistant E. coli in Lagos, Nigeria. This was compared with isolates from diarrhoeal stool and water from Wisconsin, USA. The new Luminex xTAG GPP (Gastroplex) technique and conventional real-time PCR were used to profile enteric pathogens and E. coli toxin gene isolates, respectively. Results showed the pathogen profile of stool and indicated a relationship between E. coli toxin genes in water and stool from Lagos which was absent in Wisconsin isolates. The Gastroplex technique was efficient for multiple enteric pathogens and toxin gene detection. The co-existence of antibiotic resistance with enteroinvasive E. coli toxin genes suggests an additional prognostic burden on patients.

  7. Human DNA extraction from whole saliva that was fresh or stored for 3, 6 or 12 months using five different protocols.

    Science.gov (United States)

    Garbieri, Thais Francini; Brozoski, Daniel Thomas; Dionísio, Thiago José; Santos, Carlos Ferreira; Neves, Lucimara Teixeira das

    2017-01-01

    This study aimed to compare the quantity and quality of human DNA extracted from saliva that was fresh or frozen for three, six and twelve months using five different DNA extraction protocols: protocol 1 - Oragene™ commercial kit, protocol 2 - QIAamp DNA mini kit, protocol 3 - DNA extraction using ammonium acetate, protocol 4 - Instagene™ Matrix and protocol 5 - Instagene™ Matrix diluted 1:1 using proteinase K and 1% SDS. Briefly, DNA was analyzed using spectrophotometry, electrophoresis and PCR. Results indicated that time spent in storage typically decreased the DNA quantity with the exception of protocol 1. The purity of DNA was generally not affected by storage times for the commercial based protocols, while the purity of the DNA samples extracted by the noncommercial protocols typically decreased when the saliva was stored longer. Only protocol 1 consistently extracted unfragmented DNA samples. In general, DNA samples extracted through protocols 1, 2, 3 and 4, regardless of storage time, were amplified by human specific primers whereas protocol 5 produced almost no samples that were able to be amplified by human specific primers. Depending on the protocol used, it was possible to extract DNA in high quantities and of good quality using whole saliva, and furthermore, for the purposes of DNA extraction, saliva can be reliably stored for relatively long time periods. In summary, a complicated picture emerges when taking into account the extracted DNA's quantity, purity and quality; depending on a given researchers needs, one protocol's particular strengths and costs might be the deciding factor for its employment.

  8. Magnetic resonance colonography without bowel cleansing using oral and rectal stool softeners (fecal cracking) - a feasibility study

    Energy Technology Data Exchange (ETDEWEB)

    Ajaj, Waleed; Lauenstein, Thomas C.; Kuehle, Christiane; Herborn, Christoph U.; Goehde, Susanne C. [University Hospital of Essen, Department of Diagnostic and Interventional Radiology and Neuroradiology, Essen (Germany); Schneemann, Hubert [University Hospital of Essen, Institute of Pharmacy and Pharmaceutical Sciences, Essen (Germany); Ruehm, Stefan G. [David Geffen School of Medicine at UCLA, Department of Radiology, Los Angeles, CA (United States); Goyen, Mathias [University Hospital of Hamburg-Eppendorf, Medical Center, Hamburg (Germany)

    2005-10-01

    The aim of our study was to assess the effect of oral and rectal stool softeners on dark-lumen magnetic resonance (MR) colonography without bowel cleansing. Ten volunteers underwent MR colonography without colonic cleansing. A baseline examination was performed without oral or rectal administration of stool softeners. In a second set, volunteers ingested 60 ml of lactulose 24 h prior to MR examination. In a third examination, water as a rectal enema was replaced by a solution of 0.5%-docusate sodium (DS). A fourth MR examination was performed, in conjunction with both oral administration of lactulose and rectal application of DS. A T1-weighted data set was acquired at scanning times of 0, 5 and 10 min after colonic filling. A fourth data set was acquired 75 s after i.v. injection of contrast agent. Signal intensity of stool was calculated for all colonic segments. Without oral ingestion of lactulose or rectal enema with DS stool signal intensity was high and did not decrease over time. However, lactulose and DS caused a decrease in stool signal intensity. Both substances together led to a decreasing signal intensity of feces. Combination of lactulose and DS provided the lowest signal intensity of stool. Thus, feces could hardly be distinguished from dark rectal enema allowing for the assessment of the colonic wall. (orig.)

  9. Influence of different stool types on muscle activity and lumbar posture among dentists during a simulated dental screening task.

    Science.gov (United States)

    De Bruyne, Mieke A A; Van Renterghem, Benedikt; Baird, Andrew; Palmans, Tanneke; Danneels, Lieven; Dolphens, Mieke

    2016-09-01

    Whereas in the past dental stools typically facilitated a 90° hip angle, a number of currently available alternative designs allow for a more extended hip posture. The present study investigated the influence of different stool types on muscle activity and lumbar posture. Twenty five participants completed a simulated dental procedure on a standard stool, a saddle and the Ghopec. The latter stool comprises a seat pan consisting of a horizontal rear part for the pelvis and an inclinable sloping down front part for the upper legs, with a vertically and horizontally adjustable back rest. Lumbar posture was most close to neutral on the Ghopec, whereas sitting on a standard/saddle stool resulted in more flexed/extended postures respectively. Sitting with a 90° angle (standard stool) resulted in higher activation of back muscles while sitting with a 125° angle (saddle and Ghopec) activated abdominal muscles more, although less in the presence of a backrest (Ghopec). To maintain neutral posture during dental screening, the Ghopec is considered the most suitable design for the tasks undertaken. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. A one-step immune-chromatographic Helicobacter pylori stool antigen test for children was quick, consistent, reliable and specific.

    Science.gov (United States)

    Kalach, Nicolas; Gosset, Pierre; Dehecq, Eric; Decoster, Anne; Georgel, Anne-France; Spyckerelle, Claire; Papadopoulos, Stephanos; Dupont, Christophe; Raymond, Josette

    2017-07-01

    This French study assessed a quick, noninvasive, immuno-chromatographic, Helicobacter pylori (H. pylori) stool antigen test for detecting infections in children. We enrolled 158 children, with a median age of 8.5 years (range eight months to 17 years), with digestive symptoms suggesting upper gastrointestinal tract disease. Upper digestive endoscopy was performed with gastric biopsy specimens for histology, a rapid urease test, culture test and quantitative real-time polymerase chain reaction. The H. pylori stool antigen test was performed twice for each child and the results were compared to the reference method. The reference methods showed that 23 (14.6%) of the 158 children tested were H. pylori positive. The H. pylori stool antigen test showed 91.3% sensitivity, with a 95% confidence interval (95% CI) of 86.9-95.6 and 97% specificity (95% CI 94.3-99.6), 30.84 positive likelihood ratio and 0.09 negative likelihood ratio. The test accuracy was 96.2% (95% CI 93.2-99.1). The two blinded independent observers produced identical H. pylori stool antigen test results and the Kappa coefficient for the H. pylori stool antigen test was one. The H. pylori stool antigen test was found to be a consistent, reliable, quick and specific test for detecting the H. pylori infection in children. ©2017 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.

  11. Lactulose administration for correction of stool disturbances in patients with rheumatic diseases

    Directory of Open Access Journals (Sweden)

    A E Karateev

    2008-01-01

    Full Text Available Objective. To study efficacy and tolerability of Normase® (lactulose, Doctor Reddy’s laboratories for correction of stool disturbances in pts with rheumatic diseases (RD. Material and methods. 50 pts with RD (female with mean age 64,2+19,6 years suffering from constipation (stool frequency less than 3 times a week at least for 3 months were included. Lactulose 30 ml was administered once a day in the evening. In the absence of effect dose was increased to 60 ml, in case of prominent effect — decreased to 15 ml. Duration of treatment was 2 weeks. Dejection frequency from 8 lh to 14 ,h day and changes of general health connected with bowels function (on VAS were used as outcome measures. Results. Results of treatment were assessed in 48 pts. Two pts were lost to follow up, in two the drug was withdrawn due to adverse events (haemorrhoidal bleeding increase in one and prominent wind in another. Stool normalization was achieved in 12, significant improvement (increase of dejection frequency at least by 50% — in 19 (39,6%, insignificant improvement (increase of dejection frequency less than by 50% — in 9 (18,7% pts. In 6 pts (12,5% effect was absent. Mean dejection frequency significantly increased from 1,8+0,4 to 4,1 ±1,7 times a week (p<0,001 Conclusion. Lactulose is effective and relatively safe drug for treatment of chronic constipation in pts with RD and can be recommended for wide administration in rheumatological practice.

  12. Designing fecal microbiota transplant trials that account for differences in donor stool efficacy.

    Science.gov (United States)

    Olesen, Scott W; Gurry, Thomas; Alm, Eric J

    2017-01-01

    Fecal microbiota transplantation is a highly effective intervention for patients suffering from recurrent Clostridium difficile, a common hospital-acquired infection. Fecal microbiota transplantation's success as a therapy for C. difficile has inspired interest in performing clinical trials that experiment with fecal microbiota transplantation as a therapy for other conditions like inflammatory bowel disease, obesity, diabetes, and Parkinson's disease. Results from clinical trials that use fecal microbiota transplantation to treat inflammatory bowel disease suggest that, for at least one condition beyond C. difficile, most fecal microbiota transplantation donors produce stool that is not efficacious. The optimal strategies for identifying and using efficacious donors have not been investigated. We therefore examined the optimal Bayesian response-adaptive strategy for allocating patients to donors and formulated a computationally tractable myopic heuristic. This heuristic computes the probability that a donor is efficacious by updating prior expectations about the efficacy of fecal microbiota transplantation, the placebo rate, and the fraction of donors that produce efficacious stool. In simulations designed to mimic a recent fecal microbiota transplantation clinical trial, for which traditional power calculations predict 100% statistical power, we found that accounting for differences in donor stool efficacy reduced the predicted statistical power to 9%. For these simulations, using the heuristic Bayesian allocation strategy more than quadrupled the statistical power to 39%. We use the results of similar simulations to make recommendations about the number of patients, the number of donors, and the choice of clinical endpoint that clinical trials should use to optimize their ability to detect if fecal microbiota transplantation is effective for treating a condition.

  13. DETECTION OF HELICOBACTER PYLORI STOOL ANTIGEN BY NON-INVASIVE ENZYME IMMUNOASSAY

    Institute of Scientific and Technical Information of China (English)

    鄢盛恺; 林其燧; 宋耀虹; 王树琴

    2003-01-01

    Objective. To evaluate the clinical utility of a new non-invasive enzyme immunoassay(EIA) for the diagnosis of Helicobacter pylori (H.pylori) infection. Methods. Stool specimens of 63 patients were collected and tested by using a commercial kit for detecting Helicobacter pylori stool antigen (HpSA), of which 61 patients also underwent 13C-Urea breath test (13C-UBT). The tissue samples of 31 patients were obtained endoscopically and were examined with histologic technique (Warthin-Starry silver stain).Regarded 13C-UBT as a golden standard, HpSA test and histologic techniques were evaluated. Using this method,we also investigated the positive rate of H.Pylori infection in children in Beijing.Results.The sensitivity and specificity of HpSA test were 94.7% and 95.1% respectively; the positive and negative predictive values were 97.3% and 91.7% respectively; and the accuracy was 95.1%.The results showed the prevalence of H.pylori infection was 26.0% in children (3~18 years) of district of Xicheng in Beijing. After treatment, HpSA seems to disappear rapidly(3~5 days) from the feces. Conclusion. The detection of HpSA in stool samples by HpSA test is a rapid noninvasive test for detecting H.pylori infection, and has both high sensitivity and high specificity. It is suitable for screening and diagnosis of H.pylori infection, monitoring the treatment efficacy in routine in all hospitals.

  14. Diagnosis of Strongyloides stercoralis: Detection of parasite-derived DNA in urine.

    Science.gov (United States)

    Lodh, Nilanjan; Caro, Reynaldo; Sofer, Shterna; Scott, Alan; Krolewiecki, Alejandro; Shiff, Clive

    2016-11-01

    Detecting infections of Strongyloides stercoralis is arduous and has low sensitivity. Clinically this is a major problem because chronic infections may disseminate in the host and lead to a life threatening condition. Epidemiologically, S. stercoralis is often missed in surveys as it is difficult to identify by standard stool examination procedures. We present, for the first time, evidence that the infection can be detected in filtered urine samples collected and processed in the field and subsequently assayed for the presence of parasite DNA. Urine specimens (∼40mL) were collected from 125 test and control individuals living in rural and peri-urban regions of Northern Argentina. From the same individuals, fresh stool specimens were processed using three different copropological methods. Urine specimens were filtered in the field through a 12.5cm Whatman No. 3 filter. The filters were dried and packed individually in sealable plastic bags with desiccant and shipped to a laboratory where DNA was recovered from the filter and PCR-amplified with primers specific to a dispersed repetitive sequence. Prevalence of S. stercoralis infection by stool culture and direct examination was 35/125 (28%), In contrast, PCR-based detection of parasite-specific trans-renal DNA in urine indicated that 56/125 (44.8%) carried the parasite. Of the patients that tested positive for urine-based parasite DNA, approximately half also tested positive in their stool specimens. There were 6.4% of cases where parasite larvae were seen in the stool but no DNA was amplified from the urine. As proof of principle, DNA amplification from urine residue reveals significantly more cases of S. stercoralis infection than the current standard stool examination techniques. Additional work is required to establish the relative utility, sensitivity and specificity of urine-based analysis compared to parasitological and nucleic acid detection from stool for clinical and epidemiological detection for S

  15. The rapid detection of Cryptosporidium and Giardia species in clinical stools using the Quik Chek immunoassay.

    Science.gov (United States)

    Alexander, Claire L; Niebel, Marc; Jones, Brian

    2013-12-01

    Diagnostic testing in the United Kingdom for Cryptosporidium and Giardia species is routinely performed by microscopy. In this study, two hundred stool samples from human clinical cases were examined for the presence of these two parasites comparing microscopy with an antigen immunoassay, Quik Chek (Techlab, Inc.). The Quik Chek assay was shown to have a sensitivity and specificity for Cryptosporidium detection of 87.6% and 98.9% respectively and for Giardia detection, 93.3% and 99.4% respectively. The high correlation with microscopy data provides evidence to support implementation of this rapid test within diagnostic microbiology laboratories.

  16. Enteropathogenic and enteroaggregative E. coli in stools of children with acute gastroenteritis in Davidson County, Tennessee.

    Science.gov (United States)

    Foster, Monique A; Iqbal, Junaid; Zhang, Chengxian; McHenry, Rendie; Cleveland, Brent E; Romero-Herazo, Yesenia; Fonnesbeck, Chris; Payne, Daniel C; Chappell, James D; Halasa, Natasha; Gómez-Duarte, Oscar G

    2015-11-01

    This prospective acute gastroenteritis (AGE) surveillance was conducted in the inpatient and emergency room settings at a referral pediatric hospital to determine the prevalence of diarrheagenic Escherichia coli (DEC) in children E. coli. A total of 12 (5.8%) out of 206 stool specimens from children with AGE were positive for a DEC. Eight (67%) out of these 12 were positive for enteropathogenic E. coli, and the remaining 4 were positive for enteroaggregative E. coli. DEC clinical isolates clustered with known E. coli enteropathogens according to multilocus sequencing typing.

  17. First detection and genotyping of Giardia intestinalis in stool samples collected from children in Ghazni Province, eastern Afghanistan and evaluation of the PCR assay in formalin-fixed specimens.

    Science.gov (United States)

    Lass, Anna; Karanis, Panagiotis; Korzeniewski, Krzysztof

    2017-08-01

    It is estimated that faecal-orally transmitted diseases are common in Afghanistan, as a consequence of poor hygienic standards of life and widespread contamination of water and food with both human and animal faeces. However, there is little information in the literature concerning infections caused by intestinal parasites in the Afghan population. In this study, we report the occurrence of Giardia intestinalis assemblages (A and B) in formalin-fixed stool samples collected from 245 Afghan schoolchildren living in Ghazni Province in eastern Afghanistan. Detection of the parasite's DNA and genotyping was performed using real-time PCR, specific to the β-giardin gene of G. intestinalis. Positive results were recorded in 52 (21.2%) samples. Genotyping was successful in 39 faecal samples and showed the predominance of assemblage B of G. intestinalis in this population (15 assemblage A and 24 assemblage B). Co-infection with both genotypes A and B was detected in four samples. Additionally, we evaluated the effect of 10% buffered formalin fixative on the detection of G. intestinalis DNA using real-time PCR and nested PCR characterised by different lengths of PCR products (74 and 479 bp, respectively). The human faeces containing the Giardia cysts were tested for 16 weeks. Amplification of G. intestinalis DNA with real-time PCR was possible up to 6 weeks of preservation of stool sample in formalin, compared to only 2 weeks with nested PCR. This suggests that real-time PCR is a more suitable tool in cases where stool samples have to be kept in formalin for longer periods of time.

  18. Prevalence and Multiple Drug Resistance of Shigella sonnei Isolated from Diarrheal Stool of Children

    Directory of Open Access Journals (Sweden)

    MM Dallal

    2015-11-01

    Full Text Available Background : Science the discovery of antibiotic, the incidence of antibiotic resistance has been inevitable. Although there has been many study in these area but problem still exists. The aim of this research was to study the serotyping and multiple antibiotic resistance patterns of Shigell sonnei isolated from diarrheal stool of children.Methods : The stool samples of children from zero to fourteen years of age admitted at Children Medical Center in Tehran were tested over period of twelvemonth. Identification of isolates was carried out according to standard methods and the antibiotic susceptibility test was performed using Kirby Bauer disk method.Results : Of the 200 samples analyzed 6 (3% were tested positive for Shigella sonnei The antibiotic resistance patterns showed that all were résistance to tetracycline, streptomycin, clindamycin and cortimoxazol, and 66.7% of the samples had multiples resistance to above antibiotics.Conclusion:The results showed that multiple antibiotics resistance of Shigella sonnei is increasing, therefore awareness about the prevention by improved hygiene and proper medication are needed to reduce the burden of the preventable infectious diseases among young children.  

  19. Multisite clinical evaluation of a rapid test for Entamoeba histolytica in stool.

    Science.gov (United States)

    Verkerke, Hans P; Hanbury, Blake; Siddique, Abdullah; Samie, Amidou; Haque, Rashidul; Herbein, Joel; Petri, William A

    2015-02-01

    Rapid point-of-care detection of enteric protozoa in diarrheal stool is desirable in clinical and research settings to efficiently determine the etiology of diarrhea. We analyzed the ability of the third-generation E. histolytica Quik Chek assay developed by Techlab to detect amebic antigens in fecal samples collected from independent study populations in South Africa and Bangladesh. We compared the performance of this recently released rapid test to that of the commercially available ProSpecT Entamoeba histolytica microplate assay from Remel and the E. histolytica II enzyme-linked immunosorbent assay (ELISA) from Techlab, using real-time and nested-PCR for Entamoeba species to resolve any discrepant results. After discrepant resolution, The E. histolytica Quik Chek assay exhibited sensitivity and specificity compared to the E. histolytica II ELISA of 98.0% (95% confidence interval [CI], 92.9% to 99.8%) and 100% (95% CI, 99.0% to 100%), respectively. Compared to the ProSpecT microplate assay, the E. histolytica Quik Chek (Quik Chek) assay exhibited 97.0% sensitivity (95% CI, 91.5% to 99.4%) and 100% specificity (95% CI, 99.0% to 100%). Our results indicate that the Quik Chek is a robust assay for the specific detection of E. histolytica trophozoites in unfixed frozen clinical stool samples.

  20. Oral Supplementation with Bovine Colostrum Decreases Intestinal Permeability and Stool Concentrations of Zonulin in Athletes

    Directory of Open Access Journals (Sweden)

    Maciej Hałasa

    2017-04-01

    Full Text Available Increased intestinal permeability has been implicated in various pathologies, has various causes, and can develop during vigorous athletic training. Colostrum bovinum is a natural supplement with a wide range of supposed positive health effects, including reduction of intestine permeability. We assessed influence of colostrum supplementation on intestinal permeability related parameters in a group of 16 athletes during peak training for competition. This double-blind placebo-controlled study compared supplementation for 20 days with 500 mg of colostrum bovinum or placebo (whey. Gut permeability status was assayed by differential absorption of lactulose and mannitol (L/M test and stool zonulin concentration. Baseline L/M tests found that six of the participants (75% in the colostrum group had increased intestinal permeability. After supplementation, the test values were within the normal range and were significantly lower than at baseline. The colostrum group Δ values produced by comparing the post-intervention and baseline results were also significantly lower than the placebo group Δ values. The differences in stool zonulin concentration were smaller than those in the L/M test, but were significant when the Δ values due to intervention were compared between the colostrum group and the placebo group. Colostrum bovinum supplementation was safe and effective in decreasing of intestinal permeability in this series of athletes at increased risk of its elevation.

  1. A novel ELISA test for laboratory diagnosis of Blastocystis spp. in human stool specimens.

    Science.gov (United States)

    Dogruman-Al, Funda; Turk, Songul; Adiyaman-Korkmaz, Gulcan; Hananel, Amit; Levi, Lital; Kopelowitz, June; Babai, Oded; Gross, Shimon; Greenberg, Zvi; Herschkovitz, Yoav; Mumcuoglu, Ipek

    2015-02-01

    Detection of Blastocystis is routinely performed by microscopy, culture, and formyl-ether (ethyl acetate) concentration technique (FECT). Yet, these methods require special skilled personnel, are time consuming, and often involve processing that may cause misdiagnosis. The aim of this work is to demonstrate the usefulness of a newly introduced ELISA test for the detection of Blastocystis antigens in stool samples (CoproELISA(TM) Blastocystis, Savyon Diagnostics) as a proper alternative to currently used methods, especially microscopy. A cohort of 179 fresh/frozen clinical stool samples was tested by the ELISA test, and results were compared to consensus methods comprised of microscopic examination of Lugol's iodine staining, culture, and immunofluorescence assay (IFA). The new ELISA test was able to detect fewer than 10(3) cells, recognized subtypes 1, 2, 3, and 5 (comprising >95 % of human Blastocystis infections), and exhibited similar reactivity when comparing formalin-preserved samples to fresh/frozen samples. The test demonstrated 92 % sensitivity, 87 % specificity, and 89 % accuracy when culture, and IFA or microscopy consensus results were taken as reference. When the consensus was comprised of culture and IFA, the test demonstrated sensitivity, specificity, and accuracy of 82, 86, and 84 %, respectively. In contrast, the sensitivity of Lugol staining microscopy was only 18 %. This work presents a unique ELISA test that provides an alternative to the use of microscopy, currently most widely used method. The test enables high-throughput screening and diagnosis of Blastocystis, adaptation to automatic procedures.

  2. Toward a Broadly Applicable Force Field for d(6)-Piano Stool Complexes.

    Science.gov (United States)

    Schmid, Maurus H; Ward, Thomas R; Meuwly, Markus

    2013-05-14

    Three-legged piano stool complexes are prototypical organometallic complexes relevant to a wide range of chemically relevant questions. Force field parametrization of transition-metal complexes is difficult and underdeveloped, and metal-specific force fields and software are required. Here we report our efforts to derive parameters for the conventional CHARMM and the Valbond-CHARMM force fields for d(6)-piano stool complexes. In Valbond-CHARMM, the usual angular term is replaced with hybrid orbital strength functions. These functions describe the energy not only of distorted bond angles around the minimum but also at very large distortions. Structure optimizations led to a good agreement between the calculated force field and the X-ray structures. They were comparable to RMSDs obtained between X-ray and DFT structures. In addition, and contrary to treating the systems with DFT, molecular dynamics simulations on the multiple nanosecond time scale are possible and allow to compute meaningful structural and energetic observables. Explicit solvent simulations of the complexes in methanol and water allow to determine the solvent distribution around the complexes. The parametrization presented here will be a useful starting point for dynamics investigations of catalysts in structurally more demanding environments.

  3. Multisite Clinical Evaluation of a Rapid Test for Entamoeba histolytica in Stool

    Science.gov (United States)

    Verkerke, Hans P.; Hanbury, Blake; Siddique, Abdullah; Samie, Amidou; Haque, Rashidul; Herbein, Joel

    2014-01-01

    Rapid point-of-care detection of enteric protozoa in diarrheal stool is desirable in clinical and research settings to efficiently determine the etiology of diarrhea. We analyzed the ability of the third-generation E. histolytica Quik Chek assay developed by Techlab to detect amebic antigens in fecal samples collected from independent study populations in South Africa and Bangladesh. We compared the performance of this recently released rapid test to that of the commercially available ProSpecT Entamoeba histolytica microplate assay from Remel and the E. histolytica II enzyme-linked immunosorbent assay (ELISA) from Techlab, using real-time and nested-PCR for Entamoeba species to resolve any discrepant results. After discrepant resolution, The E. histolytica Quik Chek assay exhibited sensitivity and specificity compared to the E. histolytica II ELISA of 98.0% (95% confidence interval [CI], 92.9% to 99.8%) and 100% (95% CI, 99.0% to 100%), respectively. Compared to the ProSpecT microplate assay, the E. histolytica Quik Chek (Quik Chek) assay exhibited 97.0% sensitivity (95% CI, 91.5% to 99.4%) and 100% specificity (95% CI, 99.0% to 100%). Our results indicate that the Quik Chek is a robust assay for the specific detection of E. histolytica trophozoites in unfixed frozen clinical stool samples. PMID:25428152

  4. Diagnosis of human fascioliasis by stool and blood techniques: update for the present global scenario.

    Science.gov (United States)

    Mas-Coma, S; Bargues, M D; Valero, M A

    2014-12-01

    Before the 1990s, human fascioliasis diagnosis focused on individual patients in hospitals or health centres. Case reports were mainly from developed countries and usually concerned isolated human infection in animal endemic areas. From the mid-1990s onwards, due to the progressive description of human endemic areas and human infection reports in developing countries, but also new knowledge on clinical manifestations and pathology, new situations, hitherto neglected, entered in the global scenario. Human fascioliasis has proved to be pronouncedly more heterogeneous than previously thought, including different transmission patterns and epidemiological situations. Stool and blood techniques, the main tools for diagnosis in humans, have been improved for both patient and survey diagnosis. Present availabilities for human diagnosis are reviewed focusing on advantages and weaknesses, sample management, egg differentiation, qualitative and quantitative diagnosis, antibody and antigen detection, post-treatment monitoring and post-control surveillance. Main conclusions refer to the pronounced difficulties of diagnosing fascioliasis in humans given the different infection phases and parasite migration capacities, clinical heterogeneity, immunological complexity, different epidemiological situations and transmission patterns, the lack of a diagnostic technique covering all needs and situations, and the advisability for a combined use of different techniques, at least including a stool technique and a blood technique.

  5. Rapid microfluidic solid-phase extraction system for hyper-methylated DNA enrichment and epigenetic analysis

    NARCIS (Netherlands)

    De, Arpita; Sparreboom, Wouter; van den Berg, Albert; Carlen, Edwin

    Genetic sequence and hyper-methylation profile information from the promoter regions of tumor suppressor genes are important for cancer disease investigation. Since hyper-methylated DNA (hm-DNA) is typically present in ultra-low concentrations in biological samples, such as stool, urine, and saliva,

  6. Detection of Helicobacter pylori by Real-Time PCR for 16s rRNA in Stools of NonInfected Healthy Children, Using ELISA Antigen Stool Test as the Gold Standard.

    Science.gov (United States)

    George, Sergio; Mamani, Nora; Lucero, Yalda; Torres, Juan Pablo; Farfán, Mauricio; Lagomarcino, Anne J; Orellana, Andrea; O'Ryan, Miguel

    2016-12-01

    We previously detected Helicobacter pylori infection by stool antigen ELISA assay in 33-41% of asymptomatic Chilean children between 2-3 years of age, of which 11-20% had a transient infection and 21-22% a persistent infection. A total of 88% of ELISA-positive samples were also rtPCR positive, while 37/133 (33%) of ELISA-negative stool samples were rtPCR positive. The significance of a ELISA-negative/rtPCR-positive sample requires clarification. We aimed to determine whether rtPCR is able to detect persistent infections not detected by ELISA. We selected 36 children with an ELISA-negative/rtPCR-positive stool sample, of which 25 were never H. pylori infected according to ELISA, and 11 had a transient infection with an ELISA-positive sample before or after the discordant sample. At least two additional consecutive ELISA-negative samples per child were tested in duplicate by rtPCR for the 16s rRNA gene. A total of 14 of 78 (17.9%) rtPCR reactions were positive, but only 4/78 (5.1%) were positive in both duplicates, representing a total of 3/36 (8.3%) children with an additional rtPCR-positive sample, only one of whom was persistently negative by ELISA. One child with a transient infection had two positive rtPCR reactions despite negative ELISA samples. In H. pylori noninfected or transiently infected children, as determined by stool ELISA, additional ELISA-negative/rtPCR-positive stool samples were found in 8.3% of children, but a possible persistent infection was only identified in 2.7% of children. Thus, the characterization of infection dynamics in children is not being misrepresented by application of stool ELISA. Furthermore, rtPCR does not significantly improve dynamic characterization. © 2016 John Wiley & Sons Ltd.

  7. Parasites in stool samples in the environment of Ilha da Marambaia, Rio de Janeiro, Brazil: an approach in public health

    Directory of Open Access Journals (Sweden)

    Beatriz Coronato

    2012-04-01

    Full Text Available This research aimed to describe the frequency of parasites in stool samples in the environment of Ilha da Marambaia, Rio de Janeiro, Brazil. One hundred and five stool samples were collected and processed by the coproparasitological techniques ethyl acetate sedimentation and centrifuge-flotation using saturated sugar solution. Parasites were detected in 81.9% of the samples, hookworm being the most prevalent, followed by Trichuris vulpis. Ascaris sp. eggs were also found. A high level of evolutive forms of parasites with public health risk was found in stool samples of the environment studied. We propose that health education programs, allied to an improvement of human and animal health care, must be employed to reduce the environmental contamination.

  8. Prevalence of amoebiasis in a model research community and its confirmation using stool antigen elisa for Entamoeba histolytica.

    Science.gov (United States)

    Akhtar, Tasleem; Khan, Aamir Ghafoor; Ahmed, Israr; Nazli, Rubina; Haider, Jamila

    2016-09-01

    Entamoeba histolytica (E. histolytica) produces an invasive disease called amoebiasis, which commonly produces diarrhea with or without blood in both children and adults, leading to high morbidity and mortality. Entamoeba dispar (E. Dispar) is a non invasive, non pathogenic organism. Both Entamoeba histolytica and Entamoeba Dispar look alike on microscopy and therefore cannot be differentiated unless checked on ELISA, PCR or other specific method. To calculate the actual prevalence of pathogenic amoebiasis in children by comparing the stool microscopy with ELISA stool antigen i.e. gold standard. Across sectional, comparative study. Children under five years in a community village Budhni, District Peshawar. A sample of 288 children aged Entamoeba histolytica. The specificity and sensitivity of microscopic method was calculated against ELISA. Data was analyzed using statistical computer software package SPSS version 10.0. A total of 288 stool specimens were collected and examined for Entamoeba histolytica. Out of these 36(12.5%) stools were positive for E. histolyticaon microscopy while 14(4.9%) were positive on ELISA. Out of 14 ELISA positive samples, 10 samples were also positive on microscopy while 4 were ELISA positive but microscopy negative. About 22 samples, which were positive on microscopy were negative on ELISA indicating that these samples might have been of E. Dispar which is non pathogenic protozoa. The sensitivity and specificity of microscopic method was 71.4% and 90.5% respectively, as against stool antigen test. Actual prevalence of Entamoeba histolytica is low in the area. Stool ELISA was able to differentiate between pathogenic Entamoeba histolytica and the non-pathogenic Entamoeba dispar and thus can minimize unnecessary antiamoebic treatment in these children.

  9. Assessing stool quantities generated by three specific Kato-Katz thick smear templates employed in different settings.

    Science.gov (United States)

    Leuenberger, Andrea; Nassoro, Tatu; Said, Khadija; Fenner, Lukas; Sikalengo, George; Letang, Emilio; Montresor, Antonio; Zhou, Xiao-Nong; Steinmann, Peter; Marti, Hanspeter; Utzinger, Jürg; Knopp, Stefanie

    2016-07-01

    The Kato-Katz technique is recommended for the diagnosis of helminth infections in epidemiological surveys, drug efficacy studies and monitoring of control interventions. We assessed the comparability of the average amount of faeces generated by three Kato-Katz templates included in test kits from two different providers. Nine hundred Kato-Katz thick smear preparations were done; 300 per kit. Empty slides, slides plus Kato-Katz template filled with stool and slides plus stool after careful removal of the template were weighed to the nearest 0.1 mg. The average amount of stool that was generated on the slide was calculated for each template, stratified by standard categories of stool consistency (i.e. mushy, soft, sausage-shaped, hard and clumpy). The average amount of stool generated on slides was 40.7 mg (95 % confidence interval (CI): 40.0-41.4 mg), 40.3 mg (95 % CI: 39.7-40.9 mg) and 42.8 mg (95 % CI: 42.2-43.3 mg) for the standard Vestergaard Frandsen template, and two different templates from the Chinese Center for Disease Control and Prevention (China CDC), respectively. Mushy stool resulted in considerably lower average weights when the Vestergaard Frandsen (37.0 mg; 95 % CI: 34.9-39.0 mg) or new China CDC templates (37.4 mg; 95 % CI: 35.9-38.9 mg) were used, compared to the old China CDC template (42.2 mg; 95 % CI: 40.7-43.7 mg) and compared to other stool consistency categories. The average amount of stool generated by three specific Kato-Katz templates was similar (40.3-42.8 mg). Since the multiplication factor is somewhat arbitrary and small changes only have little effect on infection intensity categories, it is suggested that the standard multiplication factor of 24 should be kept for the calculation of eggs per gram of faeces for all investigated templates.

  10. Comparison of DNA yield and STR success rates from different tissues in embalmed bodies.

    Science.gov (United States)

    Wheeler, Amanda; Czado, Natalia; Gangitano, David; Turnbough, Meredith; Hughes-Stamm, Sheree

    2017-01-01

    Formalin fixation is commonly used to preserve tissue sections for pathological testing and embalming cadavers for medical dissection or burial. DNA extracted from formalin-fixed tissues may also provide an alternative source of genetic material for medical diagnosis and forensic casework, such as identifying unknown embalmed human remains. Formaldehyde causes DNA damage, chemical modifications, and degradation, thereby reducing the quantity and quality of DNA available for downstream genetic analyses. By comparing the DNA yield, level of DNA degradation, and short tandem repeat (STR) success of various tissue types, this study is the first of its kind to provide some guidance on which samples from embalmed bodies are likely to generate more complete STR profiles. Tissue samples were dissected from three male embalmed cadavers and included bone, cartilage, hair, muscle, internal organs, skin, teeth, and nail clippings. DNA was purified from all samples using the QIAamp® FFPE Tissue Kit (Qiagen), quantified using the QuantiFiler® Trio DNA Quantification kit (Life Technologies), and genotyped using the GlobalFiler® PCR Amplification Kit (Life Technologies). Results of this study showed variation in DNA quantity and STR success between different types of tissues and some variation between cadavers. Overall, bone marrow samples resulted in the highest DNA yields, the least DNA degradation, and greatest STR success. However, several muscle, hair, and nail samples generated higher STR success rates than traditionally harvested bone and tooth samples. A key advantage to preferentially using these tissue samples over bone (and marrow) and teeth is their comparative ease and speed of collection from the cadaver and processing during DNA extraction. Results also indicate that soft tissues affected by lividity (blood pooling) may experience greater exposure to formalin, resulting in more DNA damage and reduced downstream STR success than tissues under compression. Overall

  11. Dietary shift and dysbiosis may trigger mucous stools in giant pandas (Ailuropoda melanoleuca

    Directory of Open Access Journals (Sweden)

    Candace L Williams

    2016-05-01

    Full Text Available Dietary shifts can result in dysbiosis between the host and its gastrointestinal tract (GIT microbiota, leading to negative outcomes including inflammation. Giant pandas (Ailuropoda melanoleuca are physiologically classified as carnivores; however, they consume a herbivorous diet with dramatic seasonal feeding shifts and episodes of chronic GIT distress with symptoms including abdominal pain, loss of appetite and the excretion of mucous stools (mucoids. These episodes adversely affect the overall nutritional and health status of giant pandas. Here, we examined the fecal microbiota of two giant pandas’ normal and mucoid stools and compared these microbiota to baseline samples from a season with historically few episodes. To identify the microbiota present, we isolated and sequenced 16S rRNA using next-generation sequencing. Mucoids occurred following a seasonal feeding switch from predominately bamboo culm (stalk to leaves. All fecal samples displayed low diversity and were dominated by bacterial in the phyla Firmicutes and to a lesser extent, the Proteobacteria. Fecal samples immediately prior to mucoid episodes had lower microbial diversity compared to baseline samples, followed by increased diversity in mucoids. Mucoids were mostly comprised of common mucosal-associated taxa including Streptococcus and Leuconostoc species, and exhibited increased abundance for bacteria in the family Pasteurellaceae. Taken together, these findings indicate that diet-induced intestinal dysbiosis in giant pandas likely results in an expulsion of the mucosal lining in the form of mucoids. We suggest that these occurrences serve to reset their GIT microbiota, as giant pandas have retained a carnivorous GIT anatomy while shifting to an herbivorous diet.

  12. Comparison of Various Methods in the Diagnosis of Entamoeba histolytica in Stool and Serum Specimens.

    Science.gov (United States)

    Uslu, Hakan; Aktas, Osman; Uyanik, Muhammet Hamidullah

    2016-06-01

    Entamoeba histolytica is indistinguishable from Entamoeba dispar in direct microscopic examination. A definitive diagnosis of E. histolytica is important in terms of the treatment of the patient and to avoid unnecessary costs. This study's aim is to determine the prevalence of E. histolytica and to make a comparison of the different diagnostic tests in the patients specimens defined as E. histolytica/E. dispar infection. Faecal and serum specimens of 90 patients defined as E. histolytica/E. dispar with microscopy (wet mount examination with 0.85% saline and Lugol's iodine) were examined. Stool samples were examined by trichrome staining for trophozoites and cysts and by immunoassay methods for specific adhesin antigens (Wampole (®) E. histolytica II antigen testing) and for specific serine-rich 30 kD membrane protein (Serazym(®) E. histolytica antigen testing). Anti-E. histolytica antibodies were investigated using a latex slide test and indirect hemagglutination methods in serum specimens. Presence of E. histolytica was not confirmed in 31.1% cases with trichrome staining, 62.2% of the Wampole antigen test, 64.4%, of the Serazym antigen test, 73.3% of the indirect hemagglutination test and 75.6%. of the latex agglutination. Considering the common results from Wampole and Serazym antigen testing as a reference standard, the specificity/sensitivity is 100/53.85% for trichrome staining, 75.00/98.11% for the latex agglutination test and 78.57/96.77% for the indirect hemagglutination test. It has been shown that investigation of E. histolytica in stools by direct wet-smear microscopy alone can cause significant false positive results. To obtain a reliable diagnosis for E. histolytica and to avoid unnecessary treatment for this parasite, at least one more specific assay, particularly an antigen testing and microscopy, is required.

  13. Diagnostic microRNA markers to screen for sporadic human colon cancer in stool: I. Proof of principle.

    Science.gov (United States)

    Ahmed, Farid E; Ahmed, Nancy C; Vos, Paul W; Bonnerup, Chris; Atkins, James N; Casey, Michelle; Nuovo, Gerard J; Naziri, Wade; Wiley, John E; Mota, Helvecio; Allison, Ron R

    2013-01-01

    To present proof-of-principle application for employing micro(mi)RNAs as diagnostic markers for colon cancer, we carried out global microarray expression studies on stool samples obtained from fifteen individuals (three controls, and three each with TNM stage 0-1, stage 2, stage 3, and stage 4 colon cancer), using Affymetrix GeneChip miRNA 3.0 Array, to select for a panel of miRNA genes for subsequent focused semi-quantitative polymerase chain reaction (PCR) analysis studies. Microarray results showed 202 preferentially expressed miRNA genes that were either increased (141 miRNAs), or reduced (61 miRNAs) in expression. We then conducted a stem-loop reverse transcriptase (RT)-TaqMan® minor groove binding (MGB) probes, followed by a modified qPCR expression study on 20 selected miRNAs. Twelve of the miRNAs exhibited increased and 8 decreased expression in stool from 60 individuals (20 controls, 20 with tumor-lymph node-metastatic (TNM) stage 0-1, 10 with stage 2, five with stage 3, and 5 with stage 4 colon cancer) to quantitatively monitor miRNA changes at various TNM stages of colon cancer progression. We also used laser-capture microdissection (LCM) of colon mucosal epithelial tissue samples (three control samples, and three samples from each of the four stages of colon cancer, for a total of 15 samples) to find concordance or lack thereof with stool findings. The reference housekeeping pseudogene-free ribosomal gene (18S rRNA), which shows little variation in expression, was employed as a normalization standard for relative PCR quantification. Results of the PCR analyses confirmed that twelve miRNAs (miR-7, miR-17, miR-20a, miR-21, miR-92a, miR-96, miR-106a, miR-134, miR-183, miR-196a, miR-199a-3p and miR214) had an increased expression in the stool of patients with colon cancer, and that later TNM carcinoma stages exhibited a more pronounced expression than did adenomas. On the other hand, eight miRNAs (miR-9, miR-29b, miR-127-5p, miR-138, miR-143, miR-146a, mi

  14. Effect of room temperature transport vials on DNA quality and phylogenetic composition of faecal microbiota of elderly adults and infants.

    LENUS (Irish Health Repository)

    Hill, Cian J

    2016-05-01

    Alterations in intestinal microbiota have been correlated with a growing number of diseases. Investigating the faecal microbiota is widely used as a non-invasive and ethically simple proxy for intestinal biopsies. There is an urgent need for collection and transport media that would allow faecal sampling at distance from the processing laboratory, obviating the need for same-day DNA extraction recommended by previous studies of freezing and processing methods for stool. We compared the faecal bacterial DNA quality and apparent phylogenetic composition derived using a commercial kit for stool storage and transport (DNA Genotek OMNIgene GUT) with that of freshly extracted samples, 22 from infants and 20 from older adults.

  15. Detection of Campylobacter species and Arcobacter butzleri in stool samples by use of real-time multiplex PCR

    NARCIS (Netherlands)

    R.F. de Boer (Richard); A. Ott (Alewijn); P. Güren (Pinar); E. van Zanten; A.F. van Belkum (Alex); A.M.D. Kooistra-Smid

    2013-01-01

    textabstractThe presence of Campylobacter (or Campylobacter-like) species in stools from patients suspected of infectious gastroenteritis (n = 493) was investigated using real-time PCR for detection of Arcobacter butzleri (hsp60 gene), Campylobacter coli (ceuE gene), Campylobacter jejuni (mapA), fiv

  16. Comparison of Routine Method with Antibody and Antigen Ones for Diagnosing Giardia-Entamoeba Histolytica in Stool and Blood

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    Gharavi, MJ. (PhD

    2015-01-01

    Full Text Available Background and Objectives: Giardia lamblia and Entamoeba histolytica are the most prevalent human intestinal pathogenic protozoa, worldwide. The clinical features of Giardia infection are acute diarrhea, a chronic condition with continuous diarrhea and malabsorption. Entamoeba histolytica invade intestinal tract without any typical clinical indications, and it can involve liver and other organs too. Therefore, we aimed to study these protozoa by serological and parasitological methods. Material and Methods: In this comparative study, the stool and blood specimens were collected from 1025 patients selected via simple random sampling in three different laboratories located in Tehran and Karaj, Iran (2012. Formalin Detergent test was performed on all samples. Both serum and stool positive samples of this method were analyzed for antigen and antibodies related to Giardia lamblia and Entamoeba histolytica, respectively. Results: of 1025 stool specimens, 76 (4.7% were positive for Giardia lamblia and 19 (1.8% for Entamoeba histolytica using Formalin-detergent method. In ELISA, 81 (7.9% coproantibodies to Giardia lamblia and 24 (2.3% coproantibodies to Entamoeba histolytica, 78 (7.6% corproantigen for Giardia lamblia, and 5 (0.4% for Entamoeba histolytica were observed. circulatory antibodies to Entamoeba histlytica were detected in 22 cases (2.1% Conclusion: Sensitivity of microscopic method compared to serological methods is higher than 90%; therefore, Formalin-detergent method can be the best method for stool examination.

  17. Detection of Campylobacter species and Arcobacter butzleri in stool samples by use of real-time multiplex PCR

    NARCIS (Netherlands)

    R.F. de Boer (Richard); A. Ott (Alewijn); P. Güren (Pinar); E. van Zanten; A.F. van Belkum (Alex); A.M.D. Kooistra-Smid

    2013-01-01

    textabstractThe presence of Campylobacter (or Campylobacter-like) species in stools from patients suspected of infectious gastroenteritis (n = 493) was investigated using real-time PCR for detection of Arcobacter butzleri (hsp60 gene), Campylobacter coli (ceuE gene), Campylobacter jejuni (mapA),

  18. 75 FR 38124 - In the Matter of Certain Foldable Stools; Notice of a Commission Determination Not To Review an...

    Science.gov (United States)

    2010-07-01

    ... investigation, as amended, named several respondents including the following: Amazon.com , Inc. of Seattle...'') of Denver, Colorado. 74 FR. 65155-6 (Dec. 9, 2009). The complaint, as amended, alleges violations of... foldable stools by reason of infringement of U.S. Patent No. D460,566. 75 FR 6706 (Feb. 10, 2010)....

  19. The accuracy of the Helicobacter pylori stool antigen test in diagnosing H-pylori in treated and untreated patients

    NARCIS (Netherlands)

    Arents, NL; van Zwet, AA; Thijs, JC; de Jong, A; Pool, MO; Kleibeuker, JH

    Objective and design To evaluate the performance of the Helicobacter pylori stool antigen test (HpSA test) in detecting H. pylori infection and monitoring the effect of treatment. This was done in two separate studies using either a biopsy or the C-13-urea breath test based 'gold standard' (in

  20. Clinical value of Helicobacter pylori stool antigen test, ImmunoCard STAT HpSA, for detecting H pylori infection

    Institute of Scientific and Technical Information of China (English)

    Yi-Hui Li; Hong Guo; Peng-Bin Zhang; Xiao-Yan Zhao; Si-Ping Da

    2004-01-01

    AIM: To evaluate the reliability of the Helicobacter pylori stool antigen test, ImmunoCard STAT HpSA, for detecting H pylori infection.METHODS: Stool specimens were collected from 53 patients who received upper endoscopy examination due to gastrointestinal symptoms. ImmunoCard STAT HpSA wasused to detect H pylori stool antigens. H pyloriinfection wasdetected based on three different tests: the urease test, Warthin-Starry staining and culture. H pylori status wasdefined as positive when both the urease test and histology or culture alone was positive.RESULTS: Sensitivity, specificity, positive predictive and negative predictive values and the total accuracy of ImmunoCard STAT HpSA for the diagnosis of H pylorinfection were 92.6% (25/27), 88.5% (23/26), 89.3% (25/28), 92%(23/25) and 90.6% (48/53), respectively.CONCLUSION: The stool antigen test, ImmunoCard STAT HpSA, is a simple noninvasive and accurate test for the diagnosis of H pyloriinfection.

  1. A Cross-Sectional Study on the Perceptions and Practices of Teenagers With Inflammatory Bowel Disease About Repeated Stool Sampling

    NARCIS (Netherlands)

    Heida, Anke; Dijkstra, Alie; Dantuma, Sietske K.; van Rheenen, Patrick F.

    2016-01-01

    Purpose: Repeated stool sampling to monitor disease activity is increasingly used in teenagers with inflammatory bowel disease (IBD). Knowledge about their perceptions and practices regarding collection of feces will increase the success rate of this monitoring strategy. Methods: We sent a survey to

  2. Evaluation of stool microbiota signatures in two cohorts of Asian (Singapore and Indonesia newborns at risk of atopy

    Directory of Open Access Journals (Sweden)

    Chua Kaw Yan

    2011-08-01

    Full Text Available Abstract Background Studies have suggested that demographic and lifestyle factors could shape the composition of fecal microbiota in early life. This study evaluated infant stool microbiota signatures in two Asian populations, Singapore (n = 42 and Indonesia (n = 32 with contrasting socioeconomic development, and examined the putative influences of demographic factors on these human fecal associated bacterial signatures. Results Longitudinal analysis showed associations of geographical origin with Clostridium leptum, Atopobium and Bifidobacterium groups. Mode of delivery had the largest effect on stool microbiota signatures influencing the abundance of four bacterial groups. Significantly higher abundance of bacterial members belonging to the Bacteroides-Prevotella, Bifidobacterium and Atopobium groups, but lower abundance of Lactobacilli-Enterococci group members, were observed in vaginal delivered compared to caesarean delivered infants. Demographic factors influencing the structure of infants stool microbiota during the first year of life included breastfeeding, age of weaning, sibship size and exposure to antibiotics. Conclusions Differences in stool microbiota signatures were observed in relation to various demographic factors. These features may confound studies relating to the association of the structure of fecal microbiota and the predisposition to human modern disease.

  3. Comparison of microscopy, real-time PCR and a rapid immunoassay for the detection of Giardia lamblia in human stool specimens

    NARCIS (Netherlands)

    Schuurman, T.; Lankamp, P.; van Belkum, A.; Kooistra-Smid, M.; van Zwet, A.

    2007-01-01

    Giardia lamblia is one of the most common intestinal parasites worldwide, with microscopy being the diagnostic reference standard for use with human stools. However, microscopy is time-consuming, labour-intensive and lacks sensitivity when single stools are examined. In the present study, microscopy

  4. Comparison of microscopy, real-time PCR and a rapid immunoassay for the detection of Giardia lamblia in human stool specimens

    NARCIS (Netherlands)

    Schuurman, T.; Lankamp, P.; van Belkum, A.; Kooistra-Smid, M.; van Zwet, A.

    2007-01-01

    Giardia lamblia is one of the most common intestinal parasites worldwide, with microscopy being the diagnostic reference standard for use with human stools. However, microscopy is time-consuming, labour-intensive and lacks sensitivity when single stools are examined. In the present study,

  5. Septicemia Caused by Dysgonic Fermenter 3 in a Severely Immunocompromised Patient and Isolation of the Same Microorganism from a Stool Specimen

    Science.gov (United States)

    Grob, René; Zbinden, Reinhard; Ruef, Christian; Hackenthal, Matthias; Diesterweg, Ingrid; Altwegg, Martin; von Graevenitz, Alexander

    1999-01-01

    Dysgonic fermenter 3 (DF-3)-associated bacteremia occurred in a febrile patient with acute myelocytic leukemia during aplasia. Another DF-3 isolate, identical by ribotyping, was grown 10 weeks later from stool collected in the absence of diarrhea. This is the first case in which DF-3 was isolated from blood and stool specimens from the same patient. PMID:10203539

  6. Development and initial evaluation of a lateral flow dipstick test for antigen detection of Entamoeba histolytica in stool sample.

    Science.gov (United States)

    Saidin, Syazwan; Yunus, Muhammad Hafiznur; Othman, Nurulhasanah; Lim, Yvonne Ai-Lian; Mohamed, Zeehaida; Zakaria, Nik Zairi; Noordin, Rahmah

    2017-05-01

    Entamoeba histolytica infection remains a public health concern in developing countries. Early diagnosis of amoebiasis can avoid disease complications, thus this study was aimed at developing a test that can rapidly detect the parasite antigens in stool samples. Rabbits were individually immunized with recombinant pyruvate phosphate dikinase (rPPDK) and E. histolytica excretory-secretory antigens to produce polyclonal antibodies. A rapid dipstick test was produced using anti-rPPDK PAb lined on the dipstick as capture reagent and anti-EhESA PAb conjugated to colloidal gold as the detector reagent. Using E. histolytica-spiked in stool sample of a healthy individual, the detection limit of the dipstick test was found to be 1000 cells ml(-1). Meanwhile when rPPDK was spiked in the stool sample, the minimum concentration detected by the dipstick test was 0.1 μg ml(-1). The performances of the dipstick, commercial Techlab E. histolytica II enzyme-linked immunosorbent assays (ELISA) and real-time PCR were compared using 70 stool samples from patients infected with Entamoeba species (n = 45) and other intestinal pathogens (n = 25). When compared to real-time PCR, the diagnostic sensitivity of the dipstick for detection of E. histolytica was 65.4% (n = 17/26); while the diagnostic specificity when tested with stool samples containing other intestinal pathogens was 92% (23/25). In contrast, Techlab E. histolytica II ELISA detected 19.2% (5/26) of the E. histolytica-positive samples as compared to real-time PCR. The lateral flow dipstick test produced in this study enabled rapid detection of E. histolytica, thus it showed good potential to be further developed into a diagnostic tool for intestinal amoebiasis.

  7. [Investigation of the presence of Blastocystis spp. in stool samples with microscopic, culture and molecular methods].

    Science.gov (United States)

    Adıyaman Korkmaz, Gülcan; Doğruman Al, Funda; Mumcuoğlu, İpek

    2015-01-01

    Blastocystis species are enteric protozoa frequently detected in human and animals. Seventeen subtypes (STs) have now been identified, nine of them isolating from humans. The pleomorphic structure and genetic diversity of Blastocystis spp. and the absence of standardized diagnostic methods complicate the evaluation of current data. Microscopic methods such as native-lugol and trichrome staining are most frequently used methods in routine diagnosis, while culture and molecular methods are preferred for research purposes. The aims of this study were to investigate the presence of Blastocystis spp. in the stool samples of patients with gastrointestinal complaints by microscopic and culture methods, and to detect the subtypes of isolates by polymerase chain reaction (PCR). A total of 350 stool samples collected from patients with diarrhea (n= 157) and without diarrhea (n= 193) were included in the study. Presence of Blastocystis spp. in the samples were investigated by native-lugol examination, trichrome staining and direct fluorescent antibody (DFA) methods. Ringer's solution containing 10% horse serum and 0.05% asparagine was used for cultivation. The cultures were evaluated after 3-4 days of incubation at 37°C by microscopic examination. The subtypes of Blastocystis spp. strains isolated from the cultures have been identified by PCR using sequence-tagged site primers. A total of 66 (19%) stool samples, of them 26 (16.6%) were from diarrheal and 40 (21%) from non-diarrheal cases, yielded Blastocystis sp. growth in culture. Among the evaluated samples, 12% (42/350) were found positive with native-lugol examination, 17% (58/350) with trichrome staining, and 19% (66/350) with DFA method. The agreement of culture and native-lugol method was estimated as strong (κ= 0.752), while it was very strong between culture with trichrome staining and DFA methods (κ= 0.922 and κ= 1.00, respectively). When the culture was accepted as reference method, the sensitivity and

  8. Rice-based oral rehydration solution decreases the stool volume in acute diarrhoea.

    Science.gov (United States)

    Molla, A M; Ahmed, S M; Greenough, W B

    1985-01-01

    A ramdomized trial using oral rehydration solutions (ORS) with rice or glucose was carried out in 342 patients with acute watery diarrhea in the outpatient ward of the International Centre for Diarrheal Disease Research, Bangladesh, during an epidemic of cholera between December 1982-March 1983. On admission, 75% of these patients had severe dehydration and 70% were positive for Vibrio cholerae. There were 185 children aged under 10 years and 157 adults; 169 patients were treated with rice-ORS and 173 with glucose-ORS. Patients in both groups were comparable in age and body weight as well as the duration and severity of illness. Patients with severe dehydration were first rehydrated intravenously, and then treated with ORS. Those with moderate dehydration received ORS from the start. The mean stool output in the first 24 hours in children treated with rice-ORS was less than that in those treated with glucose-ORS (155 vs 204 ml/kg 24h; P0.01). The same was the case for the adult patients, the corresponding values for stool output being 115 versus 159 ml/kg24h (P0.05); the corresponding intakes in adult patients were, respectively, 180.5 and 247 ml/kg24 hours. A gain of about 10% of the body weight on admission was observed in all the groups. 6 cases (4 children and 2 adults) who failed to respond to oral rehydration after intravenous therapy all belonged to the glucose-ORS group. The study shows that, even under epidemic conditions of severe cholera or in cholera-like diarrhea, the glucose or sucrose solutions can be replaced by rice powder with improved results. Glucose and sucrose are manufactured products which are expensive and not always available in countries where diarrheal diseases are a problem. Rice, a staple food in many of these countries, reduces the fluid requirements when used in ORS and also provides increased nutrition even in the acute stage of illness.

  9. gbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental, Stool, and Historical Continuous Plankton Recorder Samples.

    Science.gov (United States)

    Vezzulli, Luigi; Stauder, Monica; Grande, Chiara; Pezzati, Elisabetta; Verheye, Hans M; Owens, Nicholas J P; Pruzzo, Carla

    2015-01-01

    The Vibrio cholerae N-acetyl glucosamine-binding protein A (GbpA) is a chitin-binding protein involved in V. cholerae attachment to environmental chitin surfaces and human intestinal cells. We previously investigated the distribution and genetic variations of gbpA in a large collection of V. cholerae strains and found that the gene is consistently present and highly conserved in this species. Primers and probe were designed from the gbpA sequence of V. cholerae and a new Taq-based qPCR protocol was developed for diagnostic detection and quantification of the bacterium in environmental and stool samples. In addition, the positions of primers targeting the gbpA gene region were selected to obtain a short amplified fragment of 206 bp and the protocol was optimized for the analysis of formalin-fixed samples, such as historical Continuous Plankton Recorder (CPR) samples. Overall, the method is sensitive (50 gene copies), highly specific for V. cholerae and failed to amplify strains of the closely-related species Vibrio mimicus. The sensitivity of the assay applied to environmental and stool samples spiked with V. cholerae ATCC 39315 was comparable to that of pure cultures and was of 102 genomic units/l for drinking and seawater samples, 101 genomic units/g for sediment and 102 genomic units/g for bivalve and stool samples. The method also performs well when tested on artificially formalin-fixed and degraded genomic samples and was able to amplify V. cholerae DNA in historical CPR samples, the earliest of which date back to August 1966. The detection of V. cholerae in CPR samples collected in cholera endemic areas such as the Benguela Current Large Marine Ecosystem (BCLME) is of particular significance and represents a proof of concept for the possible use of the CPR technology and the developed qPCR assay in cholera studies.

  10. gbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental, Stool, and Historical Continuous Plankton Recorder Samples

    Science.gov (United States)

    Vezzulli, Luigi; Stauder, Monica; Grande, Chiara; Pezzati, Elisabetta; Verheye, Hans M.; Owens, Nicholas J. P.; Pruzzo, Carla

    2015-01-01

    The Vibrio cholerae N-acetyl glucosamine-binding protein A (GbpA) is a chitin-binding protein involved in V. cholerae attachment to environmental chitin surfaces and human intestinal cells. We previously investigated the distribution and genetic variations of gbpA in a large collection of V. cholerae strains and found that the gene is consistently present and highly conserved in this species. Primers and probe were designed from the gbpA sequence of V. cholerae and a new Taq-based qPCR protocol was developed for diagnostic detection and quantification of the bacterium in environmental and stool samples. In addition, the positions of primers targeting the gbpA gene region were selected to obtain a short amplified fragment of 206 bp and the protocol was optimized for the analysis of formalin-fixed samples, such as historical Continuous Plankton Recorder (CPR) samples. Overall, the method is sensitive (50 gene copies), highly specific for V. cholerae and failed to amplify strains of the closely-related species Vibrio mimicus. The sensitivity of the assay applied to environmental and stool samples spiked with V. cholerae ATCC 39315 was comparable to that of pure cultures and was of 102 genomic units/l for drinking and seawater samples, 101 genomic units/g for sediment and 102 genomic units/g for bivalve and stool samples. The method also performs well when tested on artificially formalin-fixed and degraded genomic samples and was able to amplify V. cholerae DNA in historical CPR samples, the earliest of which date back to August 1966. The detection of V. cholerae in CPR samples collected in cholera endemic areas such as the Benguela Current Large Marine Ecosystem (BCLME) is of particular significance and represents a proof of concept for the possible use of the CPR technology and the developed qPCR assay in cholera studies. PMID:25915771

  11. gbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental, Stool, and Historical Continuous Plankton Recorder Samples.

    Directory of Open Access Journals (Sweden)

    Luigi Vezzulli

    Full Text Available The Vibrio cholerae N-acetyl glucosamine-binding protein A (GbpA is a chitin-binding protein involved in V. cholerae attachment to environmental chitin surfaces and human intestinal cells. We previously investigated the distribution and genetic variations of gbpA in a large collection of V. cholerae strains and found that the gene is consistently present and highly conserved in this species. Primers and probe were designed from the gbpA sequence of V. cholerae and a new Taq-based qPCR protocol was developed for diagnostic detection and quantification of the bacterium in environmental and stool samples. In addition, the positions of primers targeting the gbpA gene region were selected to obtain a short amplified fragment of 206 bp and the protocol was optimized for the analysis of formalin-fixed samples, such as historical Continuous Plankton Recorder (CPR samples. Overall, the method is sensitive (50 gene copies, highly specific for V. cholerae and failed to amplify strains of the closely-related species Vibrio mimicus. The sensitivity of the assay applied to environmental and stool samples spiked with V. cholerae ATCC 39315 was comparable to that of pure cultures and was of 102 genomic units/l for drinking and seawater samples, 101 genomic units/g for sediment and 102 genomic units/g for bivalve and stool samples. The method also performs well when tested on artificially formalin-fixed and degraded genomic samples and was able to amplify V. cholerae DNA in historical CPR samples, the earliest of which date back to August 1966. The detection of V. cholerae in CPR samples collected in cholera endemic areas such as the Benguela Current Large Marine Ecosystem (BCLME is of particular significance and represents a proof of concept for the possible use of the CPR technology and the developed qPCR assay in cholera studies.

  12. Fecal Fat Analyses in Chronic Pancreatitis Importance of Fat Ingestion before Stool Collection

    Science.gov (United States)

    Engjom, Trond; Jurmy, Palwasha; Tjora, Erling; Gilja, Odd Helge; Dimcevski, Georg

    2017-01-01

    Objective Quantitative determination of fecal fat still is the gold standard for measuring malabsorption. We evaluated the importance of standardized food intake before and under the collection of feces. Material and Methods In a project, evaluating patients with suspected chronic pancreatitis (CP) and healthy volunteers (HC), stools were collected for 72 hours coupled to registration of nutritional intake over five consecutive days. Patient groups were created by a modified Layer score, which includes imaging findings, clinical parameters and pancreas function testing. Results We found 12 patients with CP, 11 patients without CP and 13 healthy individuals in our database. Median fecal fat in CP patients was 12 g/day, in non-CP patients 5 g/day and in healthy controls 5 g/day. Median fat absorption coefficient was 81% in those with chronic pancreatitis, 92% in those without CP and 92% in healthy controls. Corresponding median fat intake was 65 g/day, 68 g/day and 81 g/day in the respective groups. Spearman Rank Order Correlation between fecal fat (g/d) and fat absorption coefficient in all study subjects (n = 36) was good (-0.88 (p<0.001)). When we stratified groups according to fat intake, correlation between fecal fat and fat absorption was also good (-0.86 to -0.95). Conclusion In the diagnoses of fat malabsorption, calculating the ratio of fat absorption did not give additional information compared to fecal fat. PMID:28095460

  13. Comparison of stool containment in cloth and single-use diapers using a simulated infant feces.

    Science.gov (United States)

    Kubiak, M; Kressner, B; Raynor, W; Davis, J; Syverson, R E

    1993-03-01

    Single-use diapers and cloth diapers with vinyl pants were compared for their relative abilities to contain stool within the diaper. Artificial feces with carbon black as an additive allowed a quantitative measure of fecal containment by image analysis in 60 infants. This method showed complete containment of feces in the diaper in 50% of the single-use diapers whereas only 10% of the cloth diapers showed complete containment. In infants where the border of the vinyl pants was used as the boundary of containment with the cloth diapers, complete containment occurred only 33% of the time. Fluorescein dye ratings for containment/leakage in 69 infants showed that 83% of single-use diapers and 30% of the cloth diapers were rated as having no or minor leakage of feces. Cultures were taken of laundered vinyl pants that had previously been used over cloth diapers to determine microbial contamination. Thirty-nine percent of the pants contained Gram-negative, lactose-fermenting bacilli indicating fecal contamination. This study comparing single-use and cloth diapers for containment of artificial feces by use of image analysis and fluorescein dye ratings showed better containment by single-use diapers. The study also raises the question of possible spread of feces-borne pathogens by the vinyl pants used over cloth diapers, particularly in a day-care center.

  14. Systematic detection and association of Entamoeba species in stool samples from selected sites in India.

    Science.gov (United States)

    Nath, J; Banyal, N; Gautam, D S; Ghosh, S K; Singha, B; Paul, J

    2015-01-01

    This study developed a fast and high throughput dot-blot technique to evaluate the presence of Entamoeba in stool samples (n = 643) followed by a PCR-based method to validate and differentiate the two species E. histolytica and E. dispar. The prevalence rate of the parasite has been detected in a cross-sectional study carried out in the population of the Eastern and Northern parts of India. Of the various demographic features, prevalence was highest in the monsoon season (P = 0·017), in the <15 years age group (P = 0·015). In HIV-positive individuals, the prevalence rate was significantly high (P = 0·008) in patients with a CD4 cell count <200 as well as in patients without antiretroviral therapy (ART) (P = 0·011). Our analysis further confirmed that risk factors such as toilet facilities, living conditions, hygienic practices, drinking water source, occupation and level of education are important predictors as they were found to contribute significantly in the prevalence of the parasite.

  15. Safety Assessment of Bacteroides uniformis CECT 7771 Isolated from Stools of Healthy Breast-Fed Infants.

    Directory of Open Access Journals (Sweden)

    M Leonor Fernández-Murga

    Full Text Available Bacteroides uniformis CECT 7771 is a potential probiotic strain, originally isolated from the stools of healthy breast-feed infants. The strain showed pre-clinical efficacy in a mouse obesity model. The objective of this study was to evaluate its potential toxicity and translocation ability after acute oral administration to mice.A safety study was conducted in immunocompetent and immunosuppressed C57BL-6 mice. Both mouse groups (n = 10 per group were fed orally 2 x 10(9 colony forming units (cfu/day of B. uniformis CECT 7771 or placebo by gavage for 6 days. Throughout this time, feed and water intake and body weight were monitored. Afterwards, mice were sacrificed and biological samples were collected to analyze blood and urine biochemistry, inflammatory and immune markers; gut mucosal histology and bacterial translocation to peripheral tissues. The results demonstrated that acute ingestion of this Bacteroides strain had no adverse effects on the animals' general health status or food intake, nor did it affect biochemical indicators of liver, kidney and pancreatic function or gut mucosal histology. Findings also demonstrated that administration did not lead to bacterial translocation to blood, liver or mesenteric lymph nodes. B. uniformis CECT 7771 also downregulated gene and protein expression (iNOS and PPAR-γ and inflammatory cytokines induced by immunosuppression.The findings indicate that the acute oral consumption of B. uniformis CECT 7771 does not raise safety concerns in mice. Further studies in humans should be conducted.

  16. Safety Assessment of Bacteroides uniformis CECT 7771 Isolated from Stools of Healthy Breast-Fed Infants.

    Science.gov (United States)

    Fernández-Murga, M Leonor; Sanz, Yolanda

    2016-01-01

    Bacteroides uniformis CECT 7771 is a potential probiotic strain, originally isolated from the stools of healthy breast-feed infants. The strain showed pre-clinical efficacy in a mouse obesity model. The objective of this study was to evaluate its potential toxicity and translocation ability after acute oral administration to mice. A safety study was conducted in immunocompetent and immunosuppressed C57BL-6 mice. Both mouse groups (n = 10 per group) were fed orally 2 x 10(9) colony forming units (cfu)/day of B. uniformis CECT 7771 or placebo by gavage for 6 days. Throughout this time, feed and water intake and body weight were monitored. Afterwards, mice were sacrificed and biological samples were collected to analyze blood and urine biochemistry, inflammatory and immune markers; gut mucosal histology and bacterial translocation to peripheral tissues. The results demonstrated that acute ingestion of this Bacteroides strain had no adverse effects on the animals' general health status or food intake, nor did it affect biochemical indicators of liver, kidney and pancreatic function or gut mucosal histology. Findings also demonstrated that administration did not lead to bacterial translocation to blood, liver or mesenteric lymph nodes. B. uniformis CECT 7771 also downregulated gene and protein expression (iNOS and PPAR-γ) and inflammatory cytokines induced by immunosuppression. The findings indicate that the acute oral consumption of B. uniformis CECT 7771 does not raise safety concerns in mice. Further studies in humans should be conducted.

  17. [Isolation rate and susceptibilities of candida species from blood, vascular catheter, urine and stool].

    Science.gov (United States)

    Tashiro, Masato; Murakami, Hinako; Yoshizawa, Sadako; Tateda, Kazuhiro; Yamaguchi, Keizo

    2012-03-01

    We evaluated species distribution and antifungal susceptibility of Candida isolates during 2002-2008. Of 177 Candida isolates from blood, species distribution was 90 (51%) Candida albicans, 30 (17%) C. parapsilosis, 22 (12%) C. glabrata, 6 (3%) C. tropicalis and 29 (16%) other Candida spp.. Of 162 Candida isolates from vascular catheter, species distribution was 87 (54%) C. albicans, 14 (9%) C. parapsilosis, 36 (22%) C. glabrata, 5 (3%), C. tropicalis, 2 (1%) C. krusei and 18 (11%) other Candida spp.. Of 1889 Candida isolates from urine, species distribution was 1165 (62%) C. albicans, 22 (1%) C. parapsilosis, 484 (26%) C. glabrata, 83 (4%) C. tropicalis, 26 (1%) C. krusei and 109 (6%) other Candida spp.. Of 782 Candida isolates from stool, species distribution was 425 (54%) C. albicans, 3 (1%) C. parapsilosis, 103 (13%) C. glabrata, 28 (4%) C. tropicalis, 5 (1%), C. krusei and 218 (28%) other Candida spp. Both C. albicans and non-Candida spp. isolated from urine increased slightly over the past 7 years. Flucytosine, fluconazole, itraconazole and micafungin still have strong activity against Candida isolates.

  18. Helicobacter pylori Seropositivity and Stool Antigen in Patients With Hyperemesis Gravidarum

    Directory of Open Access Journals (Sweden)

    2006-01-01

    Full Text Available The objective of this paper is to investigate whether Helicobacter pylori is an etiologic factor in hyperemesis gravidarum. Thirty one patients with hyperemesis gravidarum and twenty nine pregnant controls without hyperemesis gravidarum were included in this prospective study. All pregnant women were examined both for Helicobacter pylori serum immunoglobulin G antibodies (HpIgG Ab, showing chronic infection, and Helicobacter pylori stool antigens (HpSA, showing active gastrointestinal colonization. Chi-square and Student t tests were used accordingly for statistical analysis. Helicobacter pylori seropositivity was 67.7% in the patients with hyperemesis gravidarum and 79.3% in the control group ( χ 2 =1.02,P=.31 . HpSA was detected in 22.6% of patients with hyperemesis gravidarum, whereas 6.9% of patients in the control group. The difference was not statistically significant ( χ 2 =2.89,P=.08 . In this study, no relation was found between Helicobacter pylori and hyperemesis gravidarum. The low social status of women in both groups could be one of the reasons for the high prevalence of Hp infection.

  19. Pomegranate extract induces ellagitannin metabolite formation and changes stool microbiota in healthy volunteers.

    Science.gov (United States)

    Li, Zhaoping; Henning, Susanne M; Lee, Ru-Po; Lu, Qing-Yi; Summanen, Paula H; Thames, Gail; Corbett, Karen; Downes, Julia; Tseng, Chi-Hong; Finegold, Sydney M; Heber, David

    2015-08-01

    The health benefits of pomegranate (POM) consumption are attributed to ellagitannins and their metabolites, formed and absorbed in the intestine by the microbiota. In this study twenty healthy participants consumed 1000 mg of POM extract daily for four weeks. Based on urinary and fecal content of the POM metabolite urolithin A (UA), we observed three distinct groups: (1) individuals with no baseline UA presence but induction of UA formation by POM extract consumption (n = 9); (2) baseline UA formation which was enhanced by POM extract consumption (N = 5) and (3) no baseline UA production, which was not inducible (N = 6). Compared to baseline the phylum Actinobacteria was increased and Firmicutes decreased significantly in individuals forming UA (producers). Verrucomicrobia (Akkermansia muciniphila) was 33 and 47-fold higher in stool samples of UA producers compared to non-producers at baseline and after 4 weeks, respectively. In UA producers, the genera Butyrivibrio, Enterobacter, Escherichia, Lactobacillus, Prevotella, Serratia and Veillonella were increased and Collinsella decreased significantly at week 4 compared to baseline. The consumption of pomegranate resulted in the formation of its metabolites in some but not all participants. POM extract consumption may induce health benefits secondary to changes in the microbiota.

  20. Performances of Four Helicobacter pylori Serological Detection Kits Using Stool Antigen Test as Gold Standard

    Science.gov (United States)

    2016-01-01

    The aim was to determine the performances of four Helicobacter pylori serological detection kits in different target groups, using Amplified IDEIA™ Hp StAR™ as gold standard. Kits studied were Rapid Immunochromatoghraphic Hexagon, Helicoblot 2.1, an EIA IgG kit and EIA IgA kit. Methods: Stool and blood samples were collected from 162 apparently healthy participants (control) and 60 Type 2 diabetes mellitus (T2DM) patients. Results: The performances of the four serological detection kits were found to be affected by gender, age, health status and ethnicity of the participants. In the control group, the Helicoblot 2.1 kit had the best performance (AUC = 0.85; p<0.05, accuracy = 86.4%), followed by EIA IgG (AUC = 0.75; p<0.05, accuracy = 75.2%). The Rapid Hexagon and EIA IgA kits had relatively poor performances. In the T2DM subgroup, the kits H2.1 and EIA IgG had best performances, with accuracies of 96.5% and 93.1% respectively. The performance of EIA IgG improved with adjustment of its cut-off value. Conclusion: The performances of the detection kits were affected by various factors which should be taken into consideration. PMID:27736910

  1. Genetic analysis for brix weight per stool and its component traits in sugarcane (Saccharum officmarum)

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Brix weight per stool (BW) of sugarcane is a complex trait, which is the final product of a combination of many components. Diallel cross experiments were conducted during a period of two years for BW and its five component traits, including stalk diameter (SD), stalk length (SL), stalk number (SN), stalk weight (SW), and brix scale (BS) of sugarcane. Phenotypic data of all the six traits were analyzed by mixed linear model and their phenotype variances were portioned into additive (A),dominance (D), additive×environment interaction (AE) and dominance×environment interaction (DE) effects, and the correlations of A, D, AE and DE effects between BW and its components were estimated. Conditional analysis was employed to investigate the contribution of the components traits to the variances of A, D, AE and DE effects of BW. It was observed that the heritabilities of BW were significantly attributed to A, D and DE by 23.9%, 30.9% and 28.5%, respectively. The variance of A effect for BW was significantly affected by SL, SN and BS by 25.3%, 93.7% and 17.4%, respectively. The variances of D and DE effects for BW were also significantly influenced by all the five components by 5.1%~85.5%. These determinants might be helpful in sugarcane breeding and provide valuable information for multiple-trait improvement of BW.

  2. Detection of Shiga Toxin-Producing Strains of Escherichia coli (O157:H7 isolated from specimens of urinary and stool by Multiplex-PCR method

    Directory of Open Access Journals (Sweden)

    seyed mohammad nayeb Nayeb Aghaee

    2006-06-01

    Full Text Available Background: Infection of shiga toxin-producing Strains of Escherichia coli has some other dangerous and deadly effects such as hemolytic uremic syndrome (HUS in addition to diarrhea. Its diagnosis is difficult and there is no information on It's incidence in Iran. The goal of this study is to evaluate and detect shiga toxinproducing strains of E. coli isolated from urinary and stool specimens by multiplex- PCR method. Materials and Methods: One hundred samples out of 500 collected samples were screened. The selected samples have more chance of heaving different kinds of O157. After DNA extraction from the isolated strains, the multiplex PCR reaction for both stx1 and stx2 genes performed and consequently amplicons were examined by agarose gel electrophoresis. Samples were analyzed after painting by ethidium bromide. Findings: Only 3 cases (3% of the studied sample were positive for the presence of STEC. All three cases were related to stx2. Two of them were related from feces samples and one of them was isolated from the urine samples. Conclusion: Although it seems that the incidence and prevalence of gastrointestinal infections by STEC are low in Iran, But because of the serious complications of the STEC infection such as HUS and HC and failure of detection of these strains by routine methods it is necessary to use molecular and serological diagnostic methods, especially in childhood clinics for recognizing doubtful cases.

  3. Efficiency of EGFR mutation analysis for small microdissected cytological specimens using multitech DNA extraction solution.

    Science.gov (United States)

    Oh, Seo Young; Lee, Hoon Taek

    2015-07-01

    The microdissection method has greatly facilitated the isolation of pure cell populations for accurate analysis of mutations. However, the absence of coverslips in these preparations leads to poor resolution of cellular morphological features. In the current study, the authors developed the MultiTech DNA extraction solution to improve the visualization of cell morphology for microdissection and tested it for the preservation of morphological properties of cells, quality of DNA, and ability to detect mutations. A total of 121 cytological samples, including fine-needle aspirates, sputum, pleural fluid, and bronchial washings, were selected from hospital archives. DNA extracted from microdissected cells was evaluated by epidermal growth factor receptor (EGFR) mutation analysis using pyrosequencing, Sanger sequencing, and peptide nucleic acid (PNA)-mediated real-time polymerase chain reaction clamping. Morphological features of cells as well as DNA quality and quantity were analyzed in several cytological samples to assess the performance of the MultiTech DNA extraction solution. The results were compared with previous EGFR mutation tests. The MultiTech DNA extraction solution improved the morphology of archived stained cells before microdissection and provided a higher DNA yield than the commercial QIAamp DNA Mini Kit in samples containing a minimal number of cells (25-50 cells). The authors were able to detect identical EGFR mutations by using different analysis platforms and consistently identified these mutations in samples comprising as few as 25 microdissected cells. The MultiTech DNA extraction solution is a reliable medium that improves the resolution of cell morphology during microdissection. It was particularly useful in EGFR mutations of samples containing a small number of cells. © 2015 American Cancer Society.

  4. Evaluation of Methods to Improve the Extraction and Recovery of DNA from Cotton Swabs for Forensic Analysis

    Science.gov (United States)

    Adamowicz, Michael S.; Stasulli, Dominique M.; Sobestanovich, Emily M.; Bille, Todd W.

    2014-01-01

    Samples for forensic DNA analysis are often collected from a wide variety of objects using cotton or nylon tipped swabs. Testing has shown that significant quantities of DNA are retained on the swab, however, and subsequently lost. When processing evidentiary samples, the recovery of the maximum amount of available DNA is critical, potentially dictating whether a usable profile can be derived from a piece of evidence or not. The QIAamp DNA Investigator extraction kit was used with its recommended protocol for swabs (one hour incubation at 56°C) as a baseline. Results indicate that over 50% of the recoverable DNA may be retained on the cotton swab tip, or otherwise lost, for both blood and buccal cell samples when using this protocol. The protocol’s incubation time and temperature were altered, as was incubating while shaking or stationary to test for increases in recovery efficiency. An additional step was then tested that included periodic re-suspension of the swab tip in the extraction buffer during incubation. Aliquots of liquid blood or a buccal cell suspension were deposited and dried on cotton swabs and compared with swab-less controls. The concentration of DNA in each extract was quantified and STR analysis was performed to assess the quality of the extracted DNA. Stationary incubations and those performed at 65°C did not result in significant gains in DNA yield. Samples incubated for 24 hours yielded less DNA. Increased yields were observed with three and 18 hour incubation periods. Increases in DNA yields were also observed using a swab re-suspension method for both cell types. The swab re-suspension method yielded an average two-fold increase in recovered DNA yield with buccal cells and an average three-fold increase with blood cells. These findings demonstrate that more of the DNA collected on swabs can be recovered with specific protocol alterations. PMID:25549111

  5. Evaluation of methods to improve the extraction and recovery of DNA from cotton swabs for forensic analysis.

    Science.gov (United States)

    Adamowicz, Michael S; Stasulli, Dominique M; Sobestanovich, Emily M; Bille, Todd W

    2014-01-01

    Samples for forensic DNA analysis are often collected from a wide variety of objects using cotton or nylon tipped swabs. Testing has shown that significant quantities of DNA are retained on the swab, however, and subsequently lost. When processing evidentiary samples, the recovery of the maximum amount of available DNA is critical, potentially dictating whether a usable profile can be derived from a piece of evidence or not. The QIAamp DNA Investigator extraction kit was used with its recommended protocol for swabs (one hour incubation at 56°C) as a baseline. Results indicate that over 50% of the recoverable DNA may be retained on the cotton swab tip, or otherwise lost, for both blood and buccal cell samples when using this protocol. The protocol's incubation time and temperature were altered, as was incubating while shaking or stationary to test for increases in recovery efficiency. An additional step was then tested that included periodic re-suspension of the swab tip in the extraction buffer during incubation. Aliquots of liquid blood or a buccal cell suspension were deposited and dried on cotton swabs and compared with swab-less controls. The concentration of DNA in each extract was quantified and STR analysis was performed to assess the quality of the extracted DNA. Stationary incubations and those performed at 65°C did not result in significant gains in DNA yield. Samples incubated for 24 hours yielded less DNA. Increased yields were observed with three and 18 hour incubation periods. Increases in DNA yields were also observed using a swab re-suspension method for both cell types. The swab re-suspension method yielded an average two-fold increase in recovered DNA yield with buccal cells and an average three-fold increase with blood cells. These findings demonstrate that more of the DNA collected on swabs can be recovered with specific protocol alterations.

  6. Evaluation of methods to improve the extraction and recovery of DNA from cotton swabs for forensic analysis.

    Directory of Open Access Journals (Sweden)

    Michael S Adamowicz

    Full Text Available Samples for forensic DNA analysis are often collected from a wide variety of objects using cotton or nylon tipped swabs. Testing has shown that significant quantities of DNA are retained on the swab, however, and subsequently lost. When processing evidentiary samples, the recovery of the maximum amount of available DNA is critical, potentially dictating whether a usable profile can be derived from a piece of evidence or not. The QIAamp DNA Investigator extraction kit was used with its recommended protocol for swabs (one hour incubation at 56°C as a baseline. Results indicate that over 50% of the recoverable DNA may be retained on the cotton swab tip, or otherwise lost, for both blood and buccal cell samples when using this protocol. The protocol's incubation time and temperature were altered, as was incubating while shaking or stationary to test for increases in recovery efficiency. An additional step was then tested that included periodic re-suspension of the swab tip in the extraction buffer during incubation. Aliquots of liquid blood or a buccal cell suspension were deposited and dried on cotton swabs and compared with swab-less controls. The concentration of DNA in each extract was quantified and STR analysis was performed to assess the quality of the extracted DNA. Stationary incubations and those performed at 65°C did not result in significant gains in DNA yield. Samples incubated for 24 hours yielded less DNA. Increased yields were observed with three and 18 hour incubation periods. Increases in DNA yields were also observed using a swab re-suspension method for both cell types. The swab re-suspension method yielded an average two-fold increase in recovered DNA yield with buccal cells and an average three-fold increase with blood cells. These findings demonstrate that more of the DNA collected on swabs can be recovered with specific protocol alterations.

  7. Ancient DNA

    DEFF Research Database (Denmark)

    Willerslev, Eske; Cooper, Alan

    2004-01-01

    ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair......ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair...

  8. Estimation of age-related DNA degradation from formalin-fixed and paraffin-embedded tissue according to the extraction methods.

    Science.gov (United States)

    Watanabe, Mototsugu; Hashida, Shinsuke; Yamamoto, Hiromasa; Matsubara, Takehiro; Ohtsuka, Tomoaki; Suzawa, Ken; Maki, Yuho; Soh, Junichi; Asano, Hiroaki; Tsukuda, Kazunori; Toyooka, Shinichi; Miyoshi, Shinichiro

    2017-09-01

    Techniques for the extraction and use of nucleic acids from formalin-fixed and paraffin-embedded (FFPE) tissues, preserved over long time periods in libraries, have been developed. However, DNA extracted from FFPE tissues is generally damaged, and long-term storage may affect DNA quality. Therefore, it is important to elucidate the effect of long-term storage on FFPE tissues and evaluate the techniques used to extract DNA from them. In the present study, the yield, purity, and integrity of DNA in FFPE tissue samples was evaluated. Two DNA extraction techniques were used: A silica-binding DNA collection method using QIAamp DNA FFPE Tissue kit (QIA) and a total tissue DNA collection method using a WaxFree DNA extraction kit (WAX). A total of 25 FFPE tissues from lung adenocarcinomas were studied, which had been surgically resected and fixed at Okayama University Hospital prior to examination and subsequent storage at room temperature for 0.5, 3, 6, 9 and 12 years. Extracted DNA was quantified using ultraviolet absorbance, fluorescent dye, and quantitative polymerase chain reaction (qPCR). The quality of the DNA was defined by the absorbance ratio of 260 to 280 nm (A260/280) and Q-score, which is the quantitative value of qPCR product size ratio. The results demonstrated that the yield of total DNA extracted using WAX was significantly greater than when QIA was used (Pextracted using WAX included more contaminants and was significantly more fragmented compared with DNA extracted using QIA (Pextraction (QIA P=0.02, WAX P=0.03; 0.5 years vs. 3 years, QIA Pextraction methods are viable depending on whether high yield or high quality of extracted DNA is required. However, due to the increased degradation with age, storage time limits the available DNA in FFPE tissues regardless of the extraction method.

  9. Electron microscopy identification of microsporidia (enterocytozoon bieneusi and cyclospora cayetanensis from stool samples of HIV infected patients

    Directory of Open Access Journals (Sweden)

    Satheeshkumar S

    2004-01-01

    Full Text Available Microsporidia (Enterocytozoon bieneusi and Cyclospora cayetanensis have been reported worldwide causing diarrhoea in AIDS patients. Stool samples from HIV infected patients were subjected to routine examination for parasites, followed by special staining techniques to detect microsporidia and Cyclospora cayetanensis. Confirmed positive cases of these parasites were further processed for electron microscopy identity of the parasites and characteristic details. Scanning and transmission electron microscopy showed better morphological and structural details of the parasites.

  10. Antimicrobial susceptibility and β-lactamase production in Bacillus cereus isolates from stool of patients, food and environment samples

    Directory of Open Access Journals (Sweden)

    Savić Dejana

    2016-01-01

    Full Text Available Background/Aim. Bacillus cereus (B. cereus usually ingested by food can cause two types of diseases: vomiting due to the presence of emetic toxin and diarrheal syndrome, due to the presence of diarrheal toxins. Systemic manifestations can also occur. The severe forms of disease demand antibiotic treatmant. The aim of this study was to determine the differences in antibiotic susceptibility and β-lactamase activity of B. cereus isolates from stools of humans, food and environment. Methods. Identification of B. cereus was performed with selective medium, classical biochemical test and polymerase chain reaction (PCR with primers specific for bal gene. Thirty isolates from each group were analysed for antibiotic susceptibility using the disk-diffusion assay. Production of β-lactamase was determined by cefinase test, and double-disc method. Results. All strains identified as B. cereus using classical biochemical test, yielded 533 bp fragment with PCR. Isolates from all the three groups were susceptible to imipenem, vancomycin, and erythromycin. All isolates were susceptible to ciprofloxacin but one from the environment. A statistically significant difference between the groups was confirmed to tetracycline and trimethoprim-sulphamethoxazole sensitivity. A total of 28/30 (93.33% samples from the foods and 25/30 (83.33% samples from environment were approved sensitive to tetracycline, while 10/30 (33.33% isolates from stools were sensitive. Opposite to this result, high susceptibility to trimethoprim-sulphamethoxazole was shown in samples from stools (100%, while isolates from foods (63.33% and from environment (70% had low susceptibility. All samples produced β-lactamases. Conclusion. The strains of B. cereus from all the three groups showed high rate of sensitivity to most tested antibiotics, except to tetracycline in samples from human stool and to trimethoprim-sulphamethoxazole in samples from food and environment. The production of

  11. Assessing stool quantities generated by three specific Kato-Katz thick smear templates employed in different settings.

    OpenAIRE

    Leuenberger, Andrea; Nassoro, Tatu; Said, Khadija; Fenner, Lukas; Sikalengo, George; Letang, Emilio; Montresor, Antonio; Zhou, Xiao-Nong; Steinmann, Peter; Marti, Hanspeter; Utzinger, Jürg; Knopp, Stefanie

    2016-01-01

    Background The Kato-Katz technique is recommended for the diagnosis of helminth infections in epidemiological surveys, drug efficacy studies and monitoring of control interventions. We assessed the comparability of the average amount of faeces generated by three Kato-Katz templates included in test kits from two different providers. Methods Nine hundred Kato-Katz thick smear preparations were done; 300 per kit. Empty slides, slides plus Kato-Katz template filled with stool and slides plus sto...

  12. Real-time PCR using SYBR Green for the detection of Shigella spp. in food and stool samples.

    Science.gov (United States)

    Mokhtari, W; Nsaibia, S; Gharbi, A; Aouni, M

    2013-02-01

    Shigella spp are exquisitely fastidious Gram negative organisms that frequently get missed in the detection by traditional culture methods. For this reason, this work has adapted a classical PCR for detection of Shigella in food and stool specimens to real-time PCR using the SYBR Green format. This method follows a melting curve analysis to be more rapid and provide both qualitative and quantitative data about the targeted pathogen. A total of 117 stool samples with diarrhea and 102 food samples were analyzed in Public Health Regional Laboratory of Nabeul by traditional culture methods and real-time PCR. To validate the real-time PCR assay, an experiment was conducted with both spiked and naturally contaminated stool samples. All Shigella strains tested were ipaH positive and all non-Shigella strains yielded no amplification products. The melting temperature (T(m) = 81.5 ± 0.5 °C) was consistently specific for the amplicon. Correlation coefficients of standard curves constructed using the quantification cycle (C(q)) versus copy numbers of Shigella showed good linearity (R² = 0.995; slope = 2.952) and the minimum level of detection was 1.5 × 10³ CFU/g feces. All food samples analyzed were negative for Shigella by standard culture methods, whereas ipaH was detected in 8.8% culture negative food products. Moreover, the ipaH specific PCR system increased the detection rate over that by culture alone from 1.7% to 11.1% among patients with diarrhea. The data presented here shows that the SYBR Green I was suitable for use in the real-time PCR assay, which provided a specific, sensitive and efficient method for the detection and quantification of Shigella spp in food and stool samples.

  13. Simultaneous investigation of influenza and enteric viruses in the stools of adult patients consulting in general practice for acute diarrhea

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    Arena Christophe

    2012-06-01

    Full Text Available Abstract Background Gastrointestinal symptoms are not an uncommon manifestation of an influenza virus infection. In the present study, we aimed to investigate the presence of influenza viruses in the stools of adult patients consulting their general practitioner for uncomplicated acute diarrhea (AD and the proportion of concurrent infections by enteric and influenza viruses. Method A case-control study was conducted from December 2010 to April 2011. Stool specimens were collected and tested for influenza viruses A (seasonal A/H3N2 and pandemic A/H1N1 and B, and for four enteric viruses (astrovirus, group A rotavirus, human enteric adenovirus, norovirus of genogroups I – NoVGI - and genogroup II - NoVGII. Results General practitioners enrolled 138 cases and 93 controls. Of the 138 stool specimens collected, 92 (66.7% were positive for at least one of the four enteric viruses analysed and 10 (7.2% tested positive for one influenza virus. None of these 10 influenza positive patients reported respiratory symptoms. In five influenza-positive patients (3.6%, we also detected one enteric virus, with 4 of them being positive for influenza B (2 had co-detection with NoVGI, 1 with NoVGII, and 1 with astrovirus. None of the 93 controls tested positive for one of the enteric and/or other influenza viruses we investigated. Conclusions In this study we showed that the simultaneous detection of influenza and enteric viruses is not a rare event. We have also reported, for the first time in general practice, the presence of seasonal and pandemic influenza viruses in the stools of adult patients consulting for uncomplicated AD. A simultaneous investigation of enteric and influenza viruses in patients complaining of gastrointestinal symptoms could be useful for future studies to better identify the agents responsible for AD.

  14. Evaluation of rapid commercial enzyme immunoassay for detection of Giardia lamblia in formalin-preserved stool specimens.

    OpenAIRE

    E. H. Scheffler; Van Etta, L L

    1994-01-01

    Two hundred twenty-three formalin-preserved stool specimens were evaluated by using ProSpecT Giardia Rapid Assay (membrane bound) (Alexon, Inc., Sunnyvale, Calif.). Enzyme immunoassay (EIA) results were compared with those by conventional microscopic examination. Two hundred four specimens were negative by both methods, and 13 (6.3%) were positive. Five specimens were negative by initial microscopic exam and positive by EIA; three of these specimens were found to be positive upon extensive mi...

  15. Evaluation of Helicobacter pylori antigen positivity in stool samples of patients with dyspeptic complaints in a tertiary care hospital

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    Mehmet Burak Selek

    2013-12-01

    Full Text Available Objective: Helicobacter pylori is a microorganism associatedwith gastritis, peptic ulcer disease and gastriccancer. We aimed to figure out the positivity rate in stoolsamples of outpatients with dyspeptic complaints visitinggastroenterology department and to evaluate its relationwith age, gender and seasonal changes.Methods: Between January 01, 2012 and December 31,2012, stool samples of 330 adult outpatients admitted togastroenterology department are investigated with an immunochromatographictest kit using monoclonal antibodiesfor detection of H. pylori antigen.Results: Among 330 patients’ stool samples tested, 67(20.3% were positive. 18.6% of men and 22.2% of womenwere detected as positive. According to age groups,17.1% patients were positive for 15-35 age groups,27.1% patients were positive for 36-55 age groups and18.2% patients were positive for above 56. Seasonal differenceof H. pylori antigen positivity in stool samples wasstatistically significant (p=0.001. Highest positivity rate29.7% was detected for winter months (December-January-February. According to logistic regression analysis,winter is found as a risk factor with statistically significant2.295 times greater risk [p=0001, Exp (B = 2.925, 95.0%C.I. for EXP (B = 1.668-5.129].Conclusion: H. pylori antigen positivity rate of our study islower than other previously conducted studies in Turkey.But, positivity rates are higher among women comparedto men, concordant with other studies. Even more, detectionof high positivity rates in winter shows primary infectionand/or relapse can be affected by seasonal changes.Key words: Helicobacter pylori, gastroenterology, stool antigen test

  16. Dipstick test for rapid diagnosis of Shigella dysenteriae 1 in bacterial cultures and its potential use on stool samples.

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    Neelam Taneja

    Full Text Available BACKGROUND: We describe a test for rapid detection of S. dysenteriae 1 in bacterial cultures and in stools, at the bedside of patients. METHODOLOGY/PRINCIPAL FINDINGS: The test is based on the detection of S. dysenteriae 1 lipopolysaccharide (LPS using serotype 1-specific monoclonal antibodies coupled to gold particles and displayed on a one-step immunochromatographic dipstick. A concentration as low as 15 ng/ml of LPS was detected in distilled water and in reconstituted stools in 10 minutes. In distilled water and in reconstituted stools, an unequivocal positive reaction was obtained with 1.6×10⁶ CFU/ml and 4.9×10⁶ CFU/ml of S. dysenteriae 1, respectively. Optimal conditions to read the test have been determined to limit the risk of ambiguous results due to appearance of a faint yellow test band in some negative samples. The specificity was 100% when tested with a battery of Shigella and unrelated strains in culture. When tested on 328 clinical samples in India, Vietnam, Senegal and France by laboratory technicians and in Democratic Republic of Congo by a field technician, the specificity (312/316 was 98.7% (95% CI:96.6-99.6% and the sensitivity (11/12 was 91.7% (95% CI:59.8-99.6%. Stool cultures and the immunochromatographic test showed concordant results in 98.4 % of cases (323/328 in comparative studies. Positive and negative predictive values were 73.3% (95% CI:44.8-91.1% and 99.7% (95% CI:98-100%. CONCLUSION: The initial findings presented here for a simple dipstick-based test to diagnose S. dysenteriae 1 demonstrates its promising potential to become a powerful tool for case management and epidemiological surveys.

  17. Stool screening of Syrian refugees and asylum seekers in Germany, 2013/2014: Identification of Sabin like polioviruses.

    Science.gov (United States)

    Böttcher, Sindy; Neubauer, Katrin; Baillot, Armin; Rieder, Gabriele; Adam, Maja; Diedrich, Sabine

    2015-10-01

    Germany is a partner of the Global Polio Eradication Initiative. Assurance of polio free status is based on enterovirus surveillance, which focuses on patients with signs of acute flaccid paralysis or aseptic meningitis/encephalitis, representing the key symptoms of poliovirus infection. In response to the wild poliovirus outbreak in Syria 2013 and high number of refugees coming from Syria to Germany, stool samples from 629 Syrian refugees/asylum seekers aged refugees and asylum seekers at that time.

  18. Stool antigen tests in the diagnosis of Helicobacter pylori infection before and after eradication therapy

    Institute of Scientific and Technical Information of China (English)

    Lea Veijola; Eveliina Myllyluoma; Riitta Korpela; Hilpi Rautelin

    2005-01-01

    Aim: To evaluate two enzyme immunoassay-based stool antigen tests, Premier Platinum HpSA and Amplified IDEIA HpStAR, and one rapid test, ImmunoCard STAT! HpSA, in the primary diagnosis of Helicobacter pylori (H pylori) infection and after eradication therapy.METHODS: Altogether 1 574 adult subjects were screened with a whole-blood H pylori antibody test and positive results were confirmed with locally validated serology and 13C-urea breath test. All 185 subjects,confirmed to be H pylori positive, and 97 H pylorinegative individuals, randomly selected from the screened study population and with negative results in serology and UBT, were enrolled. After eradication therapy the results of 182 subjects were assessed.RESULTS: At baseline, the sensitivity of HpSA and HpStAR was 91.9% and 96.2%, respectively, and specificity was 95.9% for both tests. ImmunoCard had sensitivity of 93.0% but specificity of only 88.7%. After eradication therapy, HpSA and HpStAR had sernsitivity of 81.3% and 100%, and specificity of 97.0% and 97.6%,respectively. ImmunoCard had sensitivity of 93.8% and specificity of 97.0%. HpSA, HpStAR, and ImmunoCard had PPV 77%, 80%, and 75%, and NPV 98%, 100%,and 99%, respectively.CONCLUSION: In primary diagnosis, the EIA-based tests performed well. After eradication therapy, negative results were highly accurate for all the three tests.HpStAR had the best overall performance.

  19. Long-term effect of wholemeal bread on stool weight, transit time, fecal bile acids, fats, and neutral sterols.

    Science.gov (United States)

    Eastwood, M A; Elton, R A; Smith, J H

    1986-03-01

    Stool weight, fecal constituents, bile acids, fat, neutral sterols, and intestinal transit time were recorded in 28 subjects over 18 mo. During the first 12 mo the subjects ate white bread. They were studied for an initial period of 7 days, and after 6 mo (study period 1). For the first 6 mo they ate their usual intake of bread, they then increased their white bread intake by 62 g/day for 6 mo (study period 2). The subjects ate a self-selected diet throughout the 18 mo study. During the last 6 mo (study period 3) the subjects replaced white bread by the same amount of wholemeal bread as in study period 2. No increase in stool weight occurred until study period 3 when there was an increase of 20%. There developed a linear relationship between stool weight and intestinal transit time which was not found during the initial first and second study periods. A seasonal influence on serum cholesterol was not observed during the wholemeal bread period. Fecal bile acid excretion was unchanged throughout the experiment.

  20. Metagenomic analysis of the shrew enteric virome reveals novel viruses related to human stool-associated viruses.

    Science.gov (United States)

    Sasaki, Michihito; Orba, Yasuko; Ueno, Keisuke; Ishii, Akihiro; Moonga, Ladslav; Hang'ombe, Bernard M; Mweene, Aaron S; Ito, Kimihito; Sawa, Hirofumi

    2015-02-01

    Shrews are small insectivorous mammals that are distributed worldwide. Similar to rodents, shrews live on the ground and are commonly found near human residences. In this study, we investigated the enteric virome of wild shrews in the genus Crocidura using a sequence-independent viral metagenomics approach. A large portion of the shrew enteric virome was composed of insect viruses, whilst novel viruses including cyclovirus, picornavirus and picorna-like virus were also identified. Several cycloviruses, including variants of human cycloviruses detected in cerebrospinal fluid and stools, were detected in wild shrews at a high prevalence rate. The identified picornavirus was distantly related to human parechovirus, inferring the presence of a new genus in this family. The identified picorna-like viruses were characterized as different species of calhevirus 1, which was discovered previously in human stools. Complete or nearly complete genome sequences of these novel viruses were determined in this study and then were subjected to further genetic characterization. Our study provides an initial view of the diversity and distinctiveness of the shrew enteric virome and highlights unique novel viruses related to human stool-associated viruses.

  1. Application of Nuclear Magnetic Resonance to Detect Toxigenic Clostridium difficile from Stool Specimens: A Proof of Concept.

    Science.gov (United States)

    Yang, Paul; Hash, Sara; Park, Katherine; Wong, Charlene; Doraisamy, Loganathan; Petterson, Jonas; Petti, Cathy A; Ward, Pamela M; Lee, Seung Heon; Menon, Suresh; She, Rosemary C

    2017-03-01

    We evaluated the performance of an early prototype core molecular mirroring nuclear magnetic resonance detection platform (Mentor-100) to detect toxigenic Clostridium difficile from stool. This technology uses customized nanoparticles bound to target specific oligonucleotide probes that form binaries in the presence of nucleic acid from the target microorganism. Liquid patient stool specimens were seeded with C. difficile or other Clostridium species to determine the analytical sensitivity and specificity. Samples underwent nucleic acid extraction and target amplification with probes conjugated with iron nanoparticles. Signal from nuclear magnetic resonance spin-spin relaxation time was measured to detect the presence or absence of toxigenic C. difficile. The limit of detection was difficile. No cross-reactivity was observed with nontoxigenic C. difficile, Clostridium sordellii, Clostridium perfringens, Bacillus subtilis, or Paenibacillus polymyxa at 10(8) colony forming units/mL. Correlation studies using frozen stool samples yielded a sensitivity of 88.4% (61 of 69) and a specificity of 87.0% (40 of 46) as compared with a commercial PCR assay for C. difficile. The area under the curve in the receiver operating characteristic curve analysis was 0.922. The prototype molecular mirroring platform showed promising performance for pathogen detection from clinical specimens. The platform design has the potential to offer a novel, low-cost alternative to currently available nucleic acid-based tests. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  2. Diagnostic values of Helicobacter pylori diagnostic tests: stool antigen test, urea breath test, rapid urease test, serology and histology

    Directory of Open Access Journals (Sweden)

    Shadi Kazemi

    2011-01-01

    Full Text Available Background: The purpose of this study is to compare validity of 5 diagnostic tests of helicobacter pylori with each other: stool antigen test, urea breath test (UBT, rapid urease test (RUT, serology and histology. Methods: A total of 94 patients who had indication of endoscopy entered the study. All of the 5 tests were performed for each patient. When the results of at least 2 tests were positive (except serology, Helicobacter pylori infection was considered to be positive. The sensitivity, specificity, positive predictive value, negative predictive value, accuracy and area under receiver operating characteristic (ROC curve of these 5 tests were determined. Results: The sensitivity, specificity, positive predictive value, negative predictive value, accuracy and area under ROC curve of these 5 tests are as below, respectively. Histology: 89%, 78%, 93%, 91%, 85% and 0.881; RUT: 93%, 75%, 95%, 94%, 86% and 0.831; serology: 50%, 54%, 46%, 61%, 52% and 0.563; stool antigen test: 96%, 83%, 98%, 96%, 91% and 0.897; UBT: 89%, 73%, 92%, 90%, 82% and 0.892. Conclusions: Stool antigen test is the most accurate test for Helicobacter pylori diagnosis before eradication of these bacteria.

  3. Prevalence of Entamoeba spp. in Stool Samples of Patients with Amebiasis Suspect by Native-Lugol and ELISA.

    Science.gov (United States)

    Beyhan, Yunus Emre; Yılmaz, Hasan; Taş Cengiz, Zeynep

    2016-06-01

    This study aimed to determine the prevalence of Entamoeba spp. in suspected stool samples submitted to our laboratory. In this retrospective study, stool samples of 998 patients with suspected amebiasis were sent from various clinics and services to our laboratory and were investigated by native-Lugol and enzyme-linked immunosorbent assay (ELISA) [for Entamoeba spp. antigen (Ridascreen® Entamoeba)] between January 2010 and December 2014. By the end of the study, it was shown that 8.5% (85) of 997 patients, 7.45% (39) of males and 9.8% (46) of females whom amoeba antigen inspected in their stool samples, were positive. No parasite was identified by the saline-Lugol method. The highest antigen positivity was detected in the 25-44-year-old group with 11% positivity, and a high positivity of 23.2% was seen in March. These results demonstrate that amebiasis is still a major health concern for our region. Although no parasite was detected during microscopic examinations, the detection of antigen positivity by ELISA reveals that microscopic examinations require experience and utilizing only microscopic examinations may lead to overlooks. To obtain more reliable results in diagnosis, ELISA analyses that use E. histolytica-specific monoclonal antibodies should be applied in addition to microscopic methods.

  4. Tumor M2-pyruvate kinase in stool as a biomarker for diagnosis of colorectal cancer: A meta-analysis

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    Jin-Xi Huang

    2014-01-01

    Full Text Available Objective: The aim of this meta-analysis was to evaluate the diagnosis value of tumor M2-pyruvate kinase (M2-PK in stool as a biomarker for diagnosis of colorectal cancer. Materials and Methods: By searching the databases of Cochrane Library, PubMed, China national knowledge Information and Wanfang, the diagnosis study related to tumor M2-PK in stool as a biomarker for diagnosis of colorectal cancer were screened and included in this study. The pooled sensitivity, specificity, positive likelihood ratio (+LR, negative likelihood ratio (−LR and the receiver operating characteristic curve (ROC were calculated by stata 11.0 software. Results: According to the including criteria, 14 trials including 1990 subjects were finally included in this meta-analysis. The pooled diagnosis sensitivity, specificity, +LR, −LR and area under curve were 0.78 (95% confidence interval [CI]: 0.74-0.81, 0.77 (95% CI: 0.76-0.79, 4.38 (95% CI: 3.27-5.88, 0.28 (95% CI: 0.23-0.34 and 0.86 (95% CI: 0.834-0.89. No statistical publication bias was found in this study. Conclusion: Tumor M2-PK in stool can be a useful biomarker in the diagnosis of colorectal cancer with relative high sensitivity and specificity.

  5. Application of density gradient for the isolation of the fecal microbial stool component and the potential use thereof.

    Science.gov (United States)

    Hevia, Arancha; Delgado, Susana; Margolles, Abelardo; Sánchez, Borja

    2015-11-19

    The idea of considering the gut microbiota as a virtual human organ has led to the concept of fecal microbiota transplantation (FMT), which has recently been extremely successful in the treatment of cases of recurrent Clostridium difficile infection. Administration of safe, viable, and representative fecal microbiota is crucial for FMT. To our knowledge, suitable techniques and systematic conditions for separating the fecal microbiota from stool samples have not been thoroughly investigated. In this work we show the potential to separate stool microorganisms from the rest of fecal material using a procedure with a Nycodenz® density gradient, yielding 10(10) viable bacteria per two grams of feces. This procedure did not affect the original microbiota composition in terms of viability, distribution and proportions, as assessed by a phylogenetic metagenomic approach. Obtaining the fecal microbiota by concentration and separation of the microorganisms from the rest of the stool components would allow the standardization of its recovery and its long-term preservation. FMT or similar microbiota restoration therapies could be used for the treatment of several disorders, or even for aesthetic purposes, so the method described in our work may contribute to the setting of the basis for the development of safe and standardized products.

  6. Comparison of two commercial DNA extraction kits for the analysis of nasopharyngeal bacterial communities

    Directory of Open Access Journals (Sweden)

    Keith A. Crandall

    2016-04-01

    Full Text Available Characterization of microbial communities via next-generation sequencing (NGS requires an extraction ofmicrobial DNA. Methodological differences in DNA extraction protocols may bias results and complicate inter-study comparisons. Here we compare the effect of two commonly used commercial kits (Norgen and Qiagenfor the extraction of total DNA on estimatingnasopharyngeal microbiome diversity. The nasopharynxis a reservoir for pathogens associated with respiratory illnesses and a key player in understandingairway microbial dynamics. Total DNA from nasal washes corresponding to 30 asthmatic children was extracted using theQiagenQIAamp DNA and NorgenRNA/DNA Purification kits and analyzed via IlluminaMiSeq16S rRNA V4 ampliconsequencing. The Norgen samples included more sequence reads and OTUs per sample than the Qiagen samples, but OTU counts per sample varied proportionallybetween groups (r = 0.732.Microbial profiles varied slightly between sample pairs, but alpha- and beta-diversity indices (PCoAand clustering showed highsimilarity between Norgen and Qiagenmicrobiomes. Moreover, no significant differences in community structure (PERMANOVA and adonis tests and taxa proportions (Kruskal-Wallis test were observed betweenkits. Finally, aProcrustes analysis also showed low dissimilarity (M2 = 0.173; P< 0.001 between the PCoAs of the two DNA extraction kits. Contrary to what has been observed in previous studies comparing DNA extraction methods, our 16S NGS analysis of nasopharyngeal washes did not reveal significant differences in community composition or structure between kits. Our findingssuggest congruence between column-based chromatography kits and supportthe comparison of microbiomeprofilesacross nasopharyngeal metataxonomic studies.

  7. Single Lysis-Salting Out Method of Genomic DNA Extraction From Dried Blood Spots.

    Science.gov (United States)

    Shaik, Muntaj; Shivanna, Devaraju Kuramkote; Kamate, Mahesh; Ab, Vedamurthy; Tp, Kruthika-Vinod

    2016-11-01

    Dried blood spots (DBS) are an important form of bio-sampling and valuable approach for storing blood samples for genetic studies. This has necessitated in developing an effective protocol to isolate genomic DNA (gDNA) from DBS samples.In this study, we have elucidated a dependable and non-hazardous "single lysis-salting out" (SLSO) protocol of gDNA extraction from DBS and compared against the available commercial kits. For the purpose of this study, blood spots were collected on S&S 903 filter cards from 10 healthy volunteers and 30 patients with glutaric aciduria type I (GA-I). The gDNA was extracted from theseDBS samples by SLSO, QIAamp® gDNA Micro kit and innuPREP forensic kit methods. The quantity and quality of gDNA obtained from these methods were determined by measuring the absorbance using a Nanodrop spectrophotometer. The SLSO method showed four-fold and eight-fold increased yield of gDNA in healthy volunteers and patient samples, respectively, compared to commercial kits (p<0.0001). The protocol was also found to be cost efficient, reducing the per sample cost to almost half. The suitability of this method for genetic studies was confirmed by performing R402W genotyping by RFLP in GA-I patients. The genotyping results showed the presence of R402W mutation in 20% (6/30) of patients. The SLSO method was found to be inexpensive, non-hazardous and a suitable technique for isolating gDNA from DBS samples for genetic studies. © 2016 Wiley Periodicals, Inc.

  8. Development of TaqMan-based quantitative PCR for sensitive and selective detection of toxigenic Clostridium difficile in human stools.

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    Hiroyuki Kubota

    Full Text Available Clostridium difficile is the main cause of nosocomial diarrhea, but is also found in asymptomatic subjects that are potentially involved in transmission of C. difficile infection. A sensitive and accurate detection method of C. difficile, especially toxigenic strains is indispensable for the epidemiological investigation.TaqMan-based quantitative-PCR (qPCR method for targeting 16S rRNA, tcdB, and tcdA genes of C. difficile was developed. The detection limit and accuracy of qPCR were evaluated by analyzing stool samples spiked with known amounts of C. difficile. A total of 235 stool specimens collected from 82 elderly nursing home residents were examined by qPCR, and the validity was evaluated by comparing the detection result with that by C. difficile selective culture (CDSC.The analysis of C. difficile-spiked stools confirmed that qPCR quantified whole C. difficile (TcdA+TcdB+, TcdA-TcdB+, and TcdA-TcdB- types, TcdB-producing strains (TcdA+TcdB+ and TcdA-TcdB+ types, and TcdA-producing strains (TcdA+TcdB+ type, respectively, with a lower detection limit of 103 cells/g of stool. Of the 235 specimens examined, 12 specimens (5.1% were C. difficile-positive by qPCR: TcdA+TcdB+ strain in six specimens and TcdA-TcdB- strain in the other six. CDSC detected C. difficile in 9 of the 12 specimens, and toxigenic types of the isolates from the 9 specimens were consistent with those identified by qPCR, supporting the validity of our qPCR method. Moreover, the qPCR examination revealed that the carriage rate of whole C. difficile and that of toxigenic strains in the 82 subjects over a 6-month period ranged from 2.4 to 6.8% and 1.2 to 3.8%, respectively. An average qPCR count of C. difficile detected was 104.5 cells/g of stool, suggesting that C. difficile constituted a very small fraction of intestinal microbiota.Our qPCR method should be an effective tool for both clinical diagnosis and epidemiological investigation of C. difficile.

  9. The prevalence of enteric RNA viruses in stools from diarrheic and non-diarrheic people in southwestern Alberta, Canada.

    Science.gov (United States)

    Leblanc, Danielle; Inglis, G Douglas; Boras, Valerie F; Brassard, Julie; Houde, Alain

    2017-01-01

    Southwestern Alberta is a region of Canada that has high rates of enteritis as well as high densities of livestock. The presence of enteric RNA viruses, specifically norovirus (NoV) GI, GII, GIII, GIV; sapovirus (SaV); rotavirus (RV); and astrovirus (AstV), was evaluated in stools from diarrheic (n = 2281) and non-diarrheic (n = 173) people over a 1-year period in 2008 and 2009. Diarrheic individuals lived in rural (46.6 %) and urban (53.4 %) settings and ranged in age from less than 1 month to 102 years, and the highest prevalence of infection in these individuals was in November. In all, viruses were detected in diarrheic stools from 388 individuals (17.0 %). NoV GII was the most frequently detected virus (8.0 %; n = 182) followed by SaV (4.3 %; n = 97), RV (2.0 %; n = 46), AstV (1.8 %; n = 42), NoV GI (0.9 %; n = 20), and NoV GIV (0.1 %; n = 1). Animal NoV GIII was never detected. The prevalence of mixed viral infections in diarrheic individuals was 2.8 % (n = 11). Children from 1 to 5 years of age accounted for the highest prevalence of positive stools, followed by the elderly individuals (≥70 years). Only NoV GII (1.2 %; n = 2) and SaV (1.2 %; n = 2) were detected in stools from non-diarrheic people. Sequence analysis of a subset of stools revealed homology to NoV, SaV and RV sequences from humans but not to strains from non-human animals. The results of this study do not support the hypothesis that viruses of animal origin have a significant impact on the occurrence of acute gastroenteritis caused by RNA enteric viruses in people living in southwestern Alberta.

  10. Ancient DNA analyses of museum specimens from selected Presbytis (primate: Colobinae) based on partial Cyt b sequences

    Science.gov (United States)

    Aifat, N. R.; Yaakop, S.; Md-Zain, B. M.

    2016-11-01

    The IUCN Red List of Threatened Species has categorized Malaysian primates from being data deficient to critically endanger. Thus, ancient DNA analyses hold great potential to understand phylogeny, phylogeography and population history of extinct and extant species. Museum samples are one of the alternatives to provide important sources of biological materials for a large proportion of ancient DNA studies. In this study, a total of six museum skin samples from species Presbytis hosei (4 samples) and Presbytis frontata (2 samples), aged between 43 and 124 years old were extracted to obtain the DNA. Extraction was done by using QIAGEN QIAamp DNA Investigator Kit and the ability of this kit to extract museum skin samples was tested by amplification of partial Cyt b sequence using species-specific designed primer. Two primer pairs were designed specifically for P. hosei and P. frontata, respectively. These primer pairs proved to be efficient in amplifying 200bp of the targeted species in the optimized PCR conditions. The performance of the sequences were tested to determine genetic distance of genus Presbytis in Malaysia. From the analyses, P. hosei is closely related to P. chrysomelas and P. frontata with the value of 0.095 and 0.106, respectively. Cyt b gave a clear data in determining relationships among Bornean species. Thus, with the optimized condition, museum specimens can be used for molecular systematic studies of the Malaysian primates.

  11. Detecting an elusive invasive species: a diagnostic PCR to detect Burmese python in Florida waters and an assessment of persistence of environmental DNA.

    Science.gov (United States)

    Piaggio, Antoinette J; Engeman, Richard M; Hopken, Matthew W; Humphrey, John S; Keacher, Kandy L; Bruce, William E; Avery, Michael L

    2014-03-01

    Recent studies have demonstrated that detection of environmental DNA (eDNA) from aquatic vertebrates in water bodies is possible. The Burmese python, Python bivittatus, is a semi-aquatic, invasive species in Florida where its elusive nature and cryptic coloration make its detection difficult. Our goal was to develop a diagnostic PCR to detect P. bivittatus from water-borne eDNA, which could assist managers in monitoring this invasive species. First, we used captive P. bivittatus to determine whether reptilian DNA could be isolated and amplified from water samples. We also evaluated the efficacy of two DNA isolation methods and two DNA extraction kits commonly used in eDNA preparation. A fragment of the mitochondrial cytochrome b gene from P. bivittatus was detected in all water samples isolated with the sodium acetate precipitate and the QIAamp DNA Micro Kit. Next, we designed P. bivittatus-specific primers and assessed the degradation rate of eDNA in water. Our primers did not amplify DNA from closely related species, and we found that P. bivittatus DNA was consistently detectable up to 96 h. Finally, we sampled water from six field sites in south Florida. Samples from five sites, where P. bivittatus has been observed, tested positive for eDNA. The final site was negative and had no prior documented evidence of P. bivittatus. This study shows P. bivittatus eDNA can be isolated from water samples; thus, this method is a new and promising technique for the management of invasive reptiles.

  12. Bovine coronavirus detection in a collection of diarrheic stool samples positive for group a bovine rotavirus

    Directory of Open Access Journals (Sweden)

    Aline Fernandes Barry

    2009-11-01

    Full Text Available Neonatal diarrhea is an important cause of economic losses for cattle farmers. The main viral etiologies of enteric diseases are group A rotaviruses (GARV and the bovine coronavirus (BCoV. Although both viruses infect calves of the same age, the occurrence of mixed infections is still under studied. The present study describes the co-infection of BCoV and GARV in stool samples. Forty-four diarrheic fecal samples from calves up to 60 days old that had previously tested positive for GARV by SS-PAGE were analyzed using semi-nested PCR for BCoV. A product with 251 bp of the BCoV nucleoprotein gene was amplified in 15.9% (7/44 of the samples, demonstrating that co-infection is not an unusual event. These results reinforce the need for testing for both GARV and BCoV, even in fecal samples that previously tested positive for one virus.A diarreia neonatal é uma importante causa de perdas econômicas para a criação de bovinos. Os principais agentes etiológicos virais das doenças entéricas são o rotavírus bovino grupo A (GARV e o coronavírus bovino (BCoV. Embora ambos os vírus infectem bezerros na mesma faixa etária, infecções mistas ainda são pouco estudadas. O presente trabalho descreve a identificação do BCoV em amostras de fezes positivas para o GARV, caracterizando a ocorrência de infecções mistas. Quarenta e quatro amostras de fezes diarreicas de bezerros com até 60 dias de idade, previamente identificadas como positivas para o GARV bovino por meio da técnica de SS-PAGE, foram avaliadas quanto a presença do BCoV pela técnica de semi-nested PCR. Um produto com 251 pb do gene da nucleoproteína do BCoV foi amplificado em 15,9% (7/44 das amostras de fezes demonstrando que a co-infecção não é um evento raro. Esse resultado enfatizada a importância da realização simultânea do diagnóstico para esses dois importantes vírus entéricos de bezerros em surtos de diarreia neonatal tanto em rebanhos bovinos leiteiros quanto de

  13. Normative values for stool frequency and form using Rome III diagnostic criteria for functional constipation in adults: systematic review with meta-analysis.

    Science.gov (United States)

    Miller, Larry E; Ibarra, Alvin; Ouwehand, Arthur C; Zimmermann, Angela K

    2017-01-01

    When designing clinical trials focused on functional constipation therapies, understanding the normative values of populations selected using the Rome III criteria is important for estimating baseline symptom severity, and for power analysis and sample size calculations. The objective of this review was to determine normative ranges for stool frequency and form in adults with functional constipation (Rome III criteria). Eligible studies reported stool frequency or form; random effects meta-analysis was performed with subgroup analyses to explore sources of heterogeneity. A total of 25 studies (43 groups, 2292 subjects) were included. Pooled estimates were 2.7 (95% CI 2.4-3.0) for weekly stools and 2.4 (95% CI 2.1-2.6) for stool form (Bristol scale). Heterogeneity was high for both outcomes (both I(2)=96%, P<0.001). Subgroup analysis revealed that weekly bowel movement frequency was higher in larger than in smaller studies (3.1 vs. 2.3, P<0.001) and in studies conducted in Europe compared with those in the Americas (3.1 vs. 2.2, P=0.02). For stool form, the use of a daily diary versus subject recall was the sole explanatory variable (2.5 vs. 2.1, P<0.05). We conclude that adults with functional constipation have significant variation in stool frequency and form, explained in part by geography and study design.

  14. Effect of an α-lactalbumin-enriched infant formula supplemented with oligofructose on fecal microbiota, stool characteristics, and hydration status: a randomized, double-blind, controlled trial.

    Science.gov (United States)

    Wernimont, Susan; Northington, Robert; Kullen, Martin J; Yao, Manjiang; Bettler, Jodi

    2015-04-01

    To evaluate the impact of oligofructose (OF)-supplemented infant formula on fecal microbiota, stool characteristics, and hydration. Ninety-five formula-fed infants were randomized to α-lactalbumin-enriched control formula (CF) or identical formula with 3.0 g/L OF (EF) for 8 weeks; 50 infants fed human milk (HM) were included. Eighty-four infants completed the study, 70 met per-protocol criteria. Over 8 weeks, bifidobacteria increased more in EF than CF group (0.70 vs. 0.16 log10 bacterial counts/g dry feces, P = .008); EF was not significantly different from HM group (P = .32). EF group stool consistency was intermediate between CF and HM groups; at week 8, EF group had softer stools than CF (5-point scale: 1 = hard, 5 = watery; consistency score 3.46 vs. 2.82, P = .015) without significant differences in stool frequency. Physician-assessed hydration status was normal for all infants. Infant formula with 3.0 g/L OF promoted bifidobacteria growth and softer stools without adversely affecting stool frequency or hydration. © The Author(s) 2014.

  15. Breast cancer proliferative activity: Is it the source of serum free DNA?

    African Journals Online (AJOL)

    Taha I. Hewala

    2013-04-13

    Apr 13, 2013 ... 165 Horria Avenue, El Hadara, Alexandria 21561, Egypt. Received 30 December ... serum of patients with various types of cancers including breast cancer.1 However, the .... to the QIAamp mini spin column. After washing, the ...

  16. Comparison of DNA extraction protocols for Mycobacterium Tuberculosis in diagnosis of tuberculous meningitis by real-time polymerase chain reaction

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    Rajeev Thakur

    2011-01-01

    Full Text Available Background: Several nucleic acid amplification techniques are available for detection of Mycobacterium tuberculosis (MTB in pulmonary and extrapulmonary samples, but insufficient data are available on the diagnostic utility of these techniques in tubercular meningitis where bacilli load is less. The success of final amplification and detection of nucleic acid depends on successful extraction of DNA from the organism. Aims: We performed this study to compare four methods of extraction of MTB DNA from cerebrospinal fluid (CSF samples so as to select one method of DNA extraction for amplification of nucleic acid from clinical samples. Materials and Methods: Four methods of extracting MTB DNA from CSF samples for testing by real-time polymerase chain reaction (PCR were compared: QIAGEN R protocol for DNA purification using QIAamp spin procedure (manual, AMPLICOR R respiratory specimen preparation kit, MagNA Pure R kit extraction, combined manual DNA extraction with automated extraction by MagNA Pure R . Real-time PCR was performed on COBAS TaqMan 48 Analyzer R with known positive and negative controls. Results: The detection limit for the combined manual and MagNA Pure R extraction protocol was found to be 100 copies of MTB DNA per reaction as against 1,000 copies of MTB DNA per reaction by the QIAGEN R , AMPLICOR R , and the MagNA Pure R extraction protocol. Conclusion: The real-time PCR assay employing the combination of manual extraction steps with MagNA Pure R extraction protocol for extraction of MTB DNA proved to be better than other extraction methods in analytical sensitivity, but could not detect less than 10 2 bacilli /ml.

  17. Comprehensive and Rapid Real-Time PCR Analysis of 21 Foodborne Outbreaks

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    Hiroshi Fukushima

    2009-01-01

    Full Text Available A set of four duplex SYBR Green I PCR (SG-PCR assay combined with DNA extraction using QIAamp DNA Stool Mini kit was evaluated for the detection of foodborne bacteria from 21 foodborne outbreaks. The causative pathogens were detected in almost all cases in 2 hours or less. The first run was for the detection of 8 main foodborne pathogens in 5 stool specimens within 2 hours and the second run was for the detection of other unusual suspect pathogens within a further 45 minutes. After 2 to 4 days, the causative agents were isolated and identified. The results proved that for comprehensive and rapid molecular diagnosis in foodborne outbreaks, Duplex SG-PCR assay is not only very useful, but is also economically viable for one-step differentiation of causative pathogens in fecal specimens obtained from symptomatic patients. This then allows for effective diagnosis and management of foodborne outbreaks.

  18. Detection of Campylobacter in Stool and Determination of Significance by Culture, Enzyme Immunoassay, and PCR in Developing Countries

    Science.gov (United States)

    Platts-Mills, James A.; Liu, Jie; Gratz, Jean; Mduma, Esto; Amour, Caroline; Swai, Ndealilia; Taniuchi, Mami; Begum, Sharmin; Peñataro Yori, Pablo; Tilley, Drake H.; Lee, Gwenyth; Shen, Zeli; Whary, Mark T.; Fox, James G.; McGrath, Monica; Kosek, Margaret; Haque, Rashidul

    2014-01-01

    Campylobacter is a common bacterial enteropathogen that can be detected in stool by culture, enzyme immunoassay (EIA), or PCR. We compared culture for C. jejuni/C. coli, EIA (ProSpecT), and duplex PCR to distinguish Campylobacter jejuni/C. coli and non-jejuni/coli Campylobacter on 432 diarrheal and matched control stool samples from infants in a multisite longitudinal study of enteric infections in Tanzania, Bangladesh, and Peru. The sensitivity and specificity of culture were 8.5% and 97.6%, respectively, compared with the results of EIA and 8.7% and 98.0%, respectively, compared with the results of PCR for C. jejuni/C. coli. Most (71.6%) EIA-positive samples were positive by PCR for C. jejuni/C. coli, but 27.6% were positive for non-jejuni/coli Campylobacter species. Sequencing of 16S rRNA from 53 of these non-jejuni/coli Campylobacter samples showed that it most closely matched the 16S rRNA of C. hyointestinalis subsp. lawsonii (56%), C. troglodytis (33%), C. upsaliensis (7.7%), and C. jejuni/C. coli (2.6%). Campylobacter-negative stool spiked with each of the above-mentioned Campylobacter species revealed reactivity with EIA. PCR detection of Campylobacter species was strongly associated with diarrhea in Peru (odds ratio [OR] = 3.66, P Campylobacter jejuni/C. coli infections represented less than half of all infections with Campylobacter species. In sum, in infants in developing country settings, the ProSpecT EIA and PCR for Campylobacter reveal extremely high rates of positivity. We propose the use of PCR because it retains high sensitivity, can ascertain burden, and can distinguish between Campylobacter infections at the species level. PMID:24452175

  19. Evaluation of a single procedure allowing the isolation of enteropathogenic Yersinia along with other bacterial enteropathogens from human stools.

    Science.gov (United States)

    Savin, Cyril; Leclercq, Alexandre; Carniel, Elisabeth

    2012-01-01

    Enteropathogenic Yersinia are among the most frequent agents of human diarrhea in temperate and cold countries. However, the incidence of yersiniosis is largely underestimated because of the peculiar growth characteristics of pathogenic Yersinia, which make their isolation from poly-contaminated samples difficult. The use of specific procedures for Yersinia isolation is required, but is expensive and time consuming, and therefore is not systematically performed in clinical pathology laboratories. A means to circumvent this problem would be to use a single procedure for the isolation of all bacterial enteropathogens. Since the Statens Serum Institut enteric medium (SSI) has been reported to allow the growth at 37°C of most gram-negative bacteria, including Yersinia, our study aimed at evaluating its performances for Yersinia isolation, as compared to the commonly used Yersinia-specific semi-selective Cefsulodin-Irgasan-Novobiocin medium (CIN) incubated at 28°C. Our results show that Yersinia pseudotuberculosis growth was strongly inhibited on SSI at 37°C, and therefore that this medium is not suitable for the isolation of this species. All Yersinia enterocolitica strains tested grew on SSI, while some non-pathogenic Yersinia species were inhibited. The morphology of Y. enterocolitica colonies on SSI allowed their differentiation from various other gram-negative bacteria commonly isolated from stool samples. However, in artificially contaminated human stools, the recovery of Y. enterocolitica colonies on SSI at 37°C was difficult and was 3 logs less sensitive than on CIN at 28°C. Therefore, despite its limitations, the use of a specific procedure (CIN incubated at 28°C) is still required for an efficient isolation of enteropathogenic Yersinia from stools.

  20. Improved method for extraction and detection of Helicobacter pylori DNA in formalin-fixed paraffin embedded gastric biopsies using laser micro-dissection.

    Science.gov (United States)

    Loayza, María Fernanda; Villavicencio, Fernando Xavier; Santander, Stephanie Carolina; Baldeón, Manuel; Ponce, Lourdes Karina; Salvador, Iván; Vivar Díaz, Nicolás

    2015-01-01

    To assess the molecular events exerted by Helicobacter pylori interacting directly with gastric epithelial cells, an improved procedure for microbial DNA isolation from stained hematoxilin-eosin gastric biopsies was developed based on laser micro-dissection (LM) [1]. Few articles have described the use of LM to select and detect H. pylori genome from formalin-fixed paraffin embedded gastric tissue [2]. To improve the yield and quality of DNA isolated from H. pylori contacting intestinal epithelial cells, the following conditions were established after modification of the QIAamp DNA Micro kit. •Use of at least 25 cut sections of 10-20 μm of diameter and 3 μm thick with more than 10 bacteria in each cut.•Lysis with 30 μL of tissue lysis buffer and 20 μL of proteinase K (PK) with the tube in an upside-down position.•The use of thin purification columns with 35 μL of elution buffer. The mean of DNA concentration obtained from 25 LM cut sections was 1.94± 0 .16 ng/μL, and it was efficiently amplified with qPCR in a Bio Rad iCycler instrument. The LM can improve the sample selection and DNA extraction for molecular analysis of H. pylori associated with human gastric epithelium.

  1. A comparison of different pre-lysis methods and extraction kits for recovery of Streptococcus agalacticae (Lancefield group B Streptococcus) DNA from whole blood.

    Science.gov (United States)

    Burke, Rachael M; McKenna, James P; Cox, Ciara; Coyle, Peter V; Shields, Michael D; Fairley, Derek J

    2016-10-01

    Sub-optimal recovery of bacterial DNA from whole blood samples can limit the sensitivity of molecular assays to detect pathogenic bacteria. We compared 3 different pre-lysis protocols (none, mechanical pre-lysis and achromopeptidase pre-lysis) and 5 commercially available DNA extraction platforms for direct detection of Group B Streptococcus (GBS) in spiked whole blood samples, without enrichment culture. DNA was extracted using the QIAamp Blood Mini kit (Qiagen), UCP Pathogen Mini kit (Qiagen), QuickGene DNA Whole Blood kit S (Fuji), Speed Xtract Nucleic Acid Kit 200 (Qiagen) and MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche Diagnostics Corp). Mechanical pre-lysis increased yields of bacterial genomic DNA by 51.3 fold (95% confidence interval; 31.6-85.1, pextraction methods. Including a pre-lysis step can improve the limits of detection of GBS using PCR or other molecular methods without need for culture. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Improved method for extraction and detection of Helicobacter pylori DNA in formalin-fixed paraffin embedded gastric biopsies using laser micro-dissection

    Science.gov (United States)

    Loayza, María Fernanda; Villavicencio, Fernando Xavier; Santander, Stephanie Carolina; Baldeón, Manuel; Ponce, Lourdes Karina; Salvador, Iván; Vivar Díaz, Nicolás

    2014-01-01

    To assess the molecular events exerted by Helicobacter pylori interacting directly with gastric epithelial cells, an improved procedure for microbial DNA isolation from stained hematoxilin-eosin gastric biopsies was developed based on laser micro-dissection (LM) [1]. Few articles have described the use of LM to select and detect H. pylori genome from formalin-fixed paraffin embedded gastric tissue [2]. To improve the yield and quality of DNA isolated from H. pylori contacting intestinal epithelial cells, the following conditions were established after modification of the QIAamp DNA Micro kit. • Use of at least 25 cut sections of 10–20 μm of diameter and 3 μm thick with more than 10 bacteria in each cut. • Lysis with 30 μL of tissue lysis buffer and 20 μL of proteinase K (PK) with the tube in an upside-down position. • The use of thin purification columns with 35 μL of elution buffer. The mean of DNA concentration obtained from 25 LM cut sections was 1.94± 0 .16 ng/μL, and it was efficiently amplified with qPCR in a Bio Rad iCycler instrument. The LM can improve the sample selection and DNA extraction for molecular analysis of H. pylori associated with human gastric epithelium. PMID:26150965

  3. Discovery of a novel human picornavirus in a stool sample from a pediatric patient presenting with fever of unknown origin.

    Science.gov (United States)

    Jones, Morris S; Lukashov, Vladimir V; Ganac, Robert D; Schnurr, David P

    2007-07-01

    Fever of unknown origin (FUO) is a serious problem in the United States. An unidentified agent was cultured from the stool of an infant who presented with FUO. This virus showed growth in HFDK cells and suckling mice. Using DNase sequence-independent single-primer amplification, we identified several nucleotide sequences with a high homology to Theiler's murine encephalomyelitis virus. Nearly full-length viral genome sequencing and phylogenetic analysis demonstrate that this virus is a member of the Cardiovirus genus of the Picornaviridae family.

  4. DETECTION OF HELICOBACTER PYLORI ANTIGEN IN STOOL BY ENZYME-LINKED IMMUNOSORBENT ASSAY AND COMPARISON WITH CONVENTIONAL METHODS

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    Rajesh Kumar

    2016-06-01

    Full Text Available Helicobacter pylori (H. pylori bacteria are ‘slow’ bacterial pathogens and are associated with gastritis, peptic ulcers, gastric adenocarcinoma and gastric Mucosa-Associated Lymphoid Type (MALT B-cell lymphomas. Several methods, both invasive and noninvasive, are available for detection of H. pylori infection. Invasive methods involve endoscopy and examination of gastric biopsies, e.g. by culture, rapid urease test or histology and are not appropriate for large-scale population studies. Non-invasive methods include the urea breath test, serology and stool antigen test. The latter approach is non-invasive, does not require highly specialized equipment and unlike serology is more likely to provide evidence of active rather than past infection. Furthermore, it may be more appropriate for use in paediatric patients, where techniques such as serology are insensitive and invasive methods are undesirable. Additionally, it may be used for treatment follow-up purposes. Pathogen-specific stool antigen tests are a valid alternative to the Urea Breath Test for non-invasive detection of H. pylori. METHODOLOGY A total of 120 patients who underwent upper gastrointestinal endoscopy for various gastrointestinal disturbances like dyspepsia were included in the study. Stool samples were obtained from the patient on the day of endoscopy and stored at – 20oC. Three biopsy samples were collected, two from the gastric antrum and one from the corpus. One biopsy sample from the antrum was used for performing Rapid urease test at the Endoscopy room and the other two samples were placed in 10% formalin and sent to the laboratory for histopathological examination. RESULTS Sensitivity, specificity, positive and negative predictive values of ELISA was 100%, 77%, 52% and 100% respectively. CONCLUSION H. pylori stool antigen (HpSA is suitable to use particularly in developing countries and for selection of patients for endoscopy. Detection of HpSA shows high sensitivity

  5. Isolation of pathogenic Escherichia coli from stool samples of diarrhoeal patients with history of raw milk consumption

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    M. N. Brahmbhatt

    2013-07-01

    Full Text Available Aim: To detect the occurrence of pathogenic Escherichia coli from stool samples of diarrhoeal patients with history of raw milk consumption and to determine the public health significance of isolates, especially their role in causing human diseases.Materials and Methods: Atotal of 100 stool samples from diarrhoeal patients, with history of raw milk consumption were collected from primary health centres in and around Anand city, under aseptic conditions and a total of 50 raw milk samples were collected from milk vendors, retail shops located in Anand city in sterilized sample bottles. MacConkey broth was used for the enrichment of all the samples and inoculation was done on MacConkey agar and EMB agar was used as the selective media. This was followed by the confirmation of isolates using biochemical tests. For the serotyping,E. coli isolates were sent to the National Salmonella and Escherichia Centre, Central Research Institute (CRI, Kasauli, Himachal Pradesh.Detection of virulence genes was performed using PCR technique.Results: During the present investigation, 26 (52% E. coli isolates from 50 milk samples and 59 (59% E. coli isolates from 100 stool samples were recovered. Out of 85 E. coli isolates sent for serotyping, 74 isolates could be typed which were further distributed into 13 different serogroups O2, O4, O8, O17, O22, O25, O29, O36, O45, O60, O90, O116 and O172, whereas 8 isolates were found untypable and 3 isolates were reported rough isolates. Of the 59 E. coli isolates from stool samples of diarrhoeal patients tested, 15 isolates (25.42% were reported to be positive for stx genes, among that 6 (10.16% were positive for stx1 gene, 9 (15.25% isolates were positive for stx2 gene, while 3 isolates (5.08% were positive for eaeA gene. In this study, 21 E. coliisolates were found to be Shiga toxin producing E. coli (STEC while none of the isolates were positive for the serotype O157. Conclusions: Our present findings indicate that raw

  6. Incidence of Giardia lamblia Subspecies by PCR-RFLP in Stool Specimens of Hospitalized Children at Urmia Mutahhari Hospital, West Azerbaijan Province, Iran.

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    Khosro Hazrati Tappeh

    2014-12-01

    Full Text Available Giardia lamblia is one of the most prevalent intestinal flagellate protozoa that infects a wide range of vertebrate hosts causing severe intestinal disorder in children.This study was performed to determine subspecies of G.lamblia by the PCR-RFLP method, targeting the glutamate dehydrogenase(gdhlocus, in hospitalized children at Urmia Mutahhari Hospital, West Azerbaijan Province,Iran and determining the infection transformational storages in this area.Overall, 720 stool specimens were collected from the hospitalized children, 34 samples were positive and Giardia cysts were detected under the microscope. Cysts were partially purified by the sucrose density gradient method and then washed with sterile distilled water to remove effectively the PCR inhibitors. Genomic DNA of G. lamblia isolates was extracted by freeze-thaw cycles followed by phenol/ chloroform/isoamyl alcohol method. The single step PCR-RFLP assay was used to differentiate the assemblages between A and B, which were found in humans. In this method, 432 bp expected size was amplified, and then for detection of subspecies, specific restriction RsaI and BspLI enzymes were used.Totally 34 samples were positive in terms of Giardia cyst out of 720 examined samples microscopically, so the parasite spread rate is reported 4.72%. Analysis PCR-RFLP on these samples revealed that 28 samples (93.3% have the genotype BIII and 2 samples (6.7% belong to the subgroup BIV.PCR-RFLP is a proper analytical method for determining the genotype among parasite types, using the glutamate dehydrogenizes zone's genes. Based on the results, an animal origin of infection cycle is suggested.

  7. Is PCR the Next Reference Standard for the Diagnosis of Schistosoma in Stool? A Comparison with Microscopy in Senegal and Kenya.

    Directory of Open Access Journals (Sweden)

    Lynn Meurs

    Full Text Available The current reference test for the detection of S. mansoni in endemic areas is stool microscopy based on one or more Kato-Katz stool smears. However, stool microscopy has several shortcomings that greatly affect the efficacy of current schistosomiasis control programs. A highly specific multiplex real-time polymerase chain reaction (PCR targeting the Schistosoma internal transcriber-spacer-2 sequence (ITS2 was developed by our group a few years ago, but so far this PCR has been applied mostly on urine samples. Here, we performed more in-depth evaluation of the ITS2 PCR as an alternative method to standard microscopy for the detection and quantification of Schistosoma spp. in stool samples.Microscopy and PCR were performed in a Senegalese community (n = 197 in an area with high S. mansoni transmission and co-occurrence of S. haematobium, and in Kenyan schoolchildren (n = 760 from an area with comparatively low S. mansoni transmission. Despite the differences in Schistosoma endemicity the PCR performed very similarly in both areas; 13-15% more infections were detected by PCR when comparing to microscopy of a single stool sample. Even when 2-3 stool samples were used for microscopy, PCR on one stool sample detected more infections, especially in people with light-intensity infections and in children from low-risk schools. The low prevalence of soil-transmitted helminthiasis in both populations was confirmed by an additional multiplex PCR.The ITS2-based PCR was more sensitive than standard microscopy in detecting Schistosoma spp. This would be particularly useful for S. mansoni detection in low transmission areas, and post-control settings, and as such improve schistosomiasis control programs, epidemiological research, and quality control of microscopy. Moreover, it can be complemented with other (multiplex real-time PCRs to detect a wider range of helminths and thus enhance effectiveness of current integrated control and elimination strategies for

  8. Heat-stabilised rice bran consumption by colorectal cancer survivors modulates stool metabolite profiles and metabolic networks: a randomised controlled trial.

    Science.gov (United States)

    Brown, Dustin G; Borresen, Erica C; Brown, Regina J; Ryan, Elizabeth P

    2017-05-01

    Rice bran (RB) consumption has been shown to reduce colorectal cancer (CRC) growth in mice and modify the human stool microbiome. Changes in host and microbial metabolism induced by RB consumption was hypothesised to modulate the stool metabolite profile in favour of promoting gut health and inhibiting CRC growth. The objective was to integrate gut microbial metabolite profiles and identify metabolic pathway networks for CRC chemoprevention using non-targeted metabolomics. In all, nineteen CRC survivors participated in a parallel randomised controlled dietary intervention trial that included daily consumption of study-provided foods with heat-stabilised RB (30 g/d) or no additional ingredient (control). Stool samples were collected at baseline and 4 weeks and analysed using GC-MS and ultra-performance liquid chromatography-MS. Stool metabolomics revealed 93 significantly different metabolites in individuals consuming RB. A 264-fold increase in β-hydroxyisovaleroylcarnitine and 18-fold increase in β-hydroxyisovalerate exemplified changes in leucine, isoleucine and valine metabolism in the RB group. A total of thirty-nine stool metabolites were significantly different between RB and control groups, including increased hesperidin (28-fold) and narirutin (14-fold). Metabolic pathways impacted in the RB group over time included advanced glycation end products, steroids and bile acids. Fatty acid, leucine/valine and vitamin B6 metabolic pathways were increased in RB compared with control. There were 453 metabolites identified in the RB food metabolome, thirty-nine of which were identified in stool from RB consumers. RB consumption favourably modulated the stool metabolome of CRC survivors and these findings suggest the need for continued dietary CRC chemoprevention efforts.

  9. Comparison of four DNA extraction methods for the detection of Mycobacterium leprae from Ziehl-Neelsen-stained microscopic slides.

    Science.gov (United States)

    Ruiz-Fuentes, Jenny Laura; Díaz, Alexis; Entenza, Anayma Elena; Frión, Yahima; Suárez, Odelaisy; Torres, Pedro; de Armas, Yaxsier; Acosta, Lucrecia

    2015-12-01

    The diagnosis of leprosy has been a challenge due to the low sensibility of the conventional methods and the impossibility of culturing the causative organism. In this study, four methods for Mycobacterium leprae nucleic-acid extraction from Ziehl-Neelsen-stained slides (ZNS slides) were compared: Phenol/chloroform, Chelex 100 resin, and two commercial kits (Wizard Genomic DNA Purification Kit and QIAamp DNA Mini Kit). DNA was extracted from four groups of slides: a high-codification-slide group (bacteriological index [BI]⩾4), a low-codification-slide group (BI=1), a negative-slide group (BI=0), and a negative-control-slide group (BI=0). Quality DNA was evidenced by the amplification of specific repetitive element present in M. leprae genomic DNA (RLEP) using a nested polymerase chain reaction. This is the first report comparing four different extraction methods for obtaining M. leprae DNA from ZNS slides in Cuban patients, and applied in molecular diagnosis. Good-quality DNA and positive amplification were detected in the high-codification-slide group with the four methods, while from the low-codification-slide group only the QIAGEN and phenol-chloroform methods obtained amplification of M. leprae. In the negative-slide group, only the QIAGEN method was able to obtain DNA with sufficient quality for positive amplification of the RLEP region. No amplification was observed in the negative-control-slide group by any method. Patients with ZNS negative slides can still transmit the infection, and molecular methods can help identify and treat them, interrupting the chain of transmission and preventing the onset of disabilities. The ZNS slides can be sent easily to reference laboratories for later molecular analysis that can be useful not only to improve the diagnosis, but also for the application of other molecular techniques. Copyright © 2015 Asian-African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.

  10. From an Easily Accessible Pentacarbonylcobalt(I) Salt to Piano Stool Cations [(arene)Co(CO)2 ]().

    Science.gov (United States)

    Meier, Stefan C; Holz, Albina; Schmidt, Alexei; Kratzert, Daniel; Himmel, Daniel; Krossing, Ingo

    2017-08-10

    The facile synthesis of a pentacarbonyl cobalt(I) salt without the need for a super acid as solvent is presented. This salt, [Co(CO)5 ](+) [Al(OR(F) )4 ](-) {R(F) =C(CF3 )3 }, readily accessible on a multigram scale, undergoes substitution reactions with arenes yielding the hitherto unknown class of two-legged cobalt piano stool complexes [(arene)Co(CO)2 ](+) with four different arene ligands. Such a substitution chemistry would have been impossible in superacid solution, as the arenes used would have been oxidized and/or protonated. Thus, the general approach described herein may have a wide synthetic use. Additionally, the thermochemistry of the piano-stool complexes is shown to be not easy to describe computationally and most of the established DFT methods overestimate the reaction energies. Only CCSD(T) calculations close to the basis set limit gave energies fully agreeing with the experiment. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Detection and differentiation of norovirus genogroups I and II from clinical stool specimens using real-time PCR.

    Science.gov (United States)

    Ramanan, Poornima; Espy, M J; Khare, Reeti; Binnicker, M J

    2017-04-01

    A real-time RT-PCR assay was designed to detect and differentiate norovirus genogroups I (GI) and II (GII), with primers and probes targeting the nonstructural polyprotein gene. Stool samples (n = 100) submitted for routine testing by the BioFire FilmArray® GI panel were also tested by the norovirus GI/GII real-time PCR assays. When compared to the FilmArray GI panel, the norovirus real-time PCR assay demonstrated a sensitivity of 77.5% (62/80) and specificity of 95% (19/20). Specimens yielding discordant results (n = 19) were tested at two outside laboratories for adjudication. Following discordant resolution, the adjusted sensitivity and specificity of the norovirus real-time PCR assays were 96.9% (63/65) and 100% (35/35), respectively. These results suggest that the real-time PCR assays are able to accurately detect and differentiate norovirus GI/GII from clinical stool specimens. Furthermore, our report highlights a potential issue with the specificity of the BioFire FilmArray® norovirus assay, which warrants additional investigation.

  12. High-throughput multiplex quantitative polymerase chain reaction method for Giardia lamblia and Cryptosporidium species detection in stool samples.

    Science.gov (United States)

    Nurminen, Noora; Juuti, Rosa; Oikarinen, Sami; Fan, Yue-Mei; Lehto, Kirsi-Maarit; Mangani, Charles; Maleta, Kenneth; Ashorn, Per; Hyöty, Heikki

    2015-06-01

    Giardia lamblia and Cryptosporidium species belong to a complex group of pathogens that cause diseases hampering development and socioeconomic improvements in the developing countries. Both pathogens are recognized as significant causes of diarrhea and nutritional disorders. However, further studies are needed to clarify the role of parasitic infections, especially asymptomatic infections in malnutrition and stunting. We developed a high-throughput multiplex quantitative polymerase chain reaction (qPCR) method for G. lamblia and Cryptosporidium spp. detection in stool samples. The sensitivity and specificity of the method were ensured by analyzing confirmed positive samples acquired from diagnostics laboratories and participating in an external quality control round. Its capability to detect asymptomatic G. lamblia and Cryptosporidium spp. infections was confirmed by analyzing stool samples collected from 44 asymptomatic 6-month-old infants living in an endemic region in Malawi. Of these, five samples were found to be positive for G. lamblia and two for Cryptosporidium spp. In conclusion, the developed method is suitable for large-scale studies evaluating the occurrence of G. lamblia and Cryptosporidium spp. in endemic regions and for clinical diagnostics of these infections.

  13. Exploring the concurrent presence of hepatitis A virus genome in serum, stool, saliva, and urine samples of hepatitis A patients.

    Science.gov (United States)

    Joshi, Madhuri S; Bhalla, Shilpa; Kalrao, Vijay R; Dhongade, Ramchandra K; Chitambar, Shobha D

    2014-04-01

    The use of saliva and urine as an alternative to serum samples for detection of anti-hepatitis A virus (HAV) IgM antibodies has been documented. However, these samples remain underreported or unexplored for shedding of HAV. To address this issue, paired serum, stool, saliva, and urine samples collected from hepatitis A patients were screened by reverse transcription polymerase chain reaction for detection of HAV RNA. HAV RNA was detected in 67.6% (44/65), 52.3% (34/65), 8.7% (5/57), and 12.3% (8/65) of the serum, stool, saliva, and urine samples, respectively. Phylogenetic analysis of nucleotide sequences obtained for partial RNA polymerase region grouped HAV strains from all of the clinical samples of the study in subgenotype IIIA. Low frequency of HAV nucleic acid in saliva and urine samples indicates limited utility of these samples in genomic studies on HAV but suggests its potential for transmission and infection of hepatitis A.

  14. Comparação entre dois métodos de detecção de DNA de papilomavírus humano em carcinoma epidermoide de lábio Comparison between two methods for human papilomavírus DNA detection in lip squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Adriana Demathe

    2010-04-01

    Full Text Available Introdução: Recentemente o papilomavírus humano (HPV tem sido associado à carcinogênese oral. A metodologia empregada na detecção do vírus é uma das maiores causas observadas da grande variabilidade nas taxas de detecção do HPV. Objetivo: Este estudo comparou a sensibilidade de detecção do DNA do HPV em casos de carcinoma epidermoide de lábio utilizando a amplificação do DNA viral por reação em cadeia da polimerase (PCR ou nPCR. Material e método: Foram utilizadas 33 amostras provenientes de casos de carcinoma epidermoide de lábio. Para as extrações do DNA utilizou-se o sistema QIAamp DNA Mini Kit. Como controle interno utilizou-se o gene da b-globina. Das 33 amostras iniciais, 30 foram positivas para o gene b-globina, sendo utilizadas para detectar o DNA viral. Comparou-se a amplificação do DNA viral pelos métodos da PCR com os oligonucleotídeos MY09/MY11 e nPCR, empregando-se os pares de oligonucleotídeos iniciadores MY09/MY11 e, na segunda etapa, o par GP5+/GP6+. O controle positivo para a presença do DNA do HPV utilizado foi a linhagem de células HeLa e, como controle negativo, a mistura de amplificação sem DNA. A análise dos produtos de PCR e nPCR para HPV foi realizada por eletroforese em gel de poliacrilamida a 8%. Resultados: Utilizando-se o método da PCR, a amplificação do DNA do HPV foi constatada em dois casos. Com a nPCR foi verificada presença de DNA viral em 13 das 30 amostras. Conclusão: Com a utilização da nPCR, a detecção do HPV nos casos estudados aumentou mais de seis vezes.Introduction: Human papillomavirus (HPV has been currently associated with oral carcinogesis. The methodology applied in virus detection is one of the main reasons for the great variability observed in HPV detection. Objectives: This study compared HPV DNA detection efficiency in lip squamous cell carcinoma samples (SCC using viral DNA amplification by PCR or nPCR. Methods: Thirty three samples of lip squamous cell

  15. Flavobacterium faecale sp. nov., an agarase-producing species isolated from stools of Antarctic penguins.

    Science.gov (United States)

    Kim, Jin Ho; Choi, Bo Hyun; Jo, Minho; Kim, Sun Chang; Lee, Pyung Cheon

    2014-08-01

    Taxonomic studies were performed on an agarase-producing strain, designated WV33(T), isolated from faeces of Antarctic penguins. Cells of strain WV33(T) were Gram-staining-negative, strictly aerobic, orange and rod-shaped. Strain WV33(T) displayed agarase activity and was able to utilize galactose as a sole carbon source. 16S rRNA gene sequence analysis revealed that strain WV33(T) was closely related to Flavobacterium algicola TC2(T) (98.0% similarity), F. frigidarium ATCC 700810(T) (96.9%) and F. frigoris LMG 21922(T) (96.1%). The predominant cellular fatty acids were iso-C(15 : 1) G, iso-C(15 : 0), C(15 : 0), C(16 : 0) and summed feature 3 (comprising iso-C(15 : 0) 2-OH and/or C(16 : 1)ω7c). Menaquinone 6 (MK-6) was the sole quinone identified, and the major pigment was zeaxanthin. The major polar lipid was phosphatidylethanolamine. DNA-DNA relatedness of strain WV33(T) with respect to its closest phylogenetic neighbours was 25% for F. algicola NBRC 102673(T), 23% for F. frigidarium DSM 17623(T) and 21% for F. frigoris DSM 15719(T). The DNA G+C content of strain WV33(T) was 37±0.6 mol%. Based on the phenotypic, chemotaxonomic and phylogenetic data, strain WV33(T) is concluded to represent a novel species of the genus Flavobacterium, for which the name Flavobacterium faecale sp. nov. is proposed. The type strain is WV33(T) ( = KCTC 32457(T) = CECT 8384(T)).

  16. Comportamento fisiológico de goiabeira serrana quando multiplicada por mergulhia de cepa Physiological behavior of feijoa multiplied by stool layring

    Directory of Open Access Journals (Sweden)

    MS. Mielke

    1994-04-01

    Full Text Available Com o objetivo de estudar alguns fatores fisiológicos envolvidos na formação e desenvolvimento de raízes em mudas de goiabeira serrana (Feijoa sellowiana Berg, multiplicadas por mergulhia de cepa, foi realizado um experimento em Pelotas-RS, com mudas decepadas em julho de 91 e sobre as quais foi realizada amontoa em outubro de 91. A avaliação foi feita em agosto de 92, sendo observadas as variáveis: teor de clorofila total, área foliar, numero e diâmetro de brotações, número e percentagem de brotações enraizadas e peso da matéria seca das raízes. As variáveis teor de clorofila, área foliar e diâmetro médio das brotações não apresentaram influência sobre a matéria seca das raízes. O incremento no número de brotações causou um aumento no número de brotações enraizadas, entretanto, não apresentou influência na percentagem de brotações enraizadas. O aumento no número de brotações por cepa ocasionou um decréscimo em seu diâmetro médio. É demonstrado que é possível multiplicar plantas de goiabeira serrana através de mergulhia de cepa.The aim of this research was to study some physiological factors involved in root growth and development in sprouts of feijoa Feijoa settowiana Berg., multiplied by stool layring. The experiment was conducted in Pelotas,RS, Brazil, using sprouts cut in July 91, which were covered with soil in October 91. In August 92 the following variables were evaluated: total chlorophyll content, leaf area, stool number and diameter, number and percentage of rooted stools and root dry-matter weight. The variables chlorophyll content, leaf area and stool average diameter, did not show influence on root dry matter. The increase in number of stools caused an increase in the number but not in the percentage of rooted stools. The increase in stool number per plant caused a decrease in stool average diameter. It is demonstrated that it is possible to multiply feijoa plants through stool layring.

  17. 结肠弯曲菌Real-time PCR检测方法的建立%Real-time PCR development for identification of Campylobacter coli from stool specimen

    Institute of Scientific and Technical Information of China (English)

    乔博; 许学斌; 顾一心; 刘国栋; 赵飞; 何利华; 张建中; 张茂俊

    2012-01-01

    目的 建立结肠弯曲菌实时PCR检测方法并评价该方法的可行性.方法 BLAST与Vector NTI Suite 6.0筛选出结肠弯曲菌的特异基因ceuE作为检测靶基因,Primer 5.0和Oligo6.0设计引物和探针,优化实时 PCR反应体系并评价其最低检测限、特异性、可重复性.结果 与结论 在结肠弯曲菌的ceuE特异基因上设计引物和探针,经优化了的实时PCR反应体系最低检测限为5.62CFU,特异度为100%,变异系数为0.15%~1.30%,本研究为结肠弯曲菌的快速检测提供了一种参考方法.%The aim of this study is to develop the real-time PCR assays to detect and qualify C. coli from stool specimen. The primers and probe were designed based on the specific sequence of ceuE gene in C. coli using Primer 5.0 and Vector NTI Suite 6.0 e. The PCR assay was optimized with the reference C. coli strains. The standard curve based on the dilutions of genomic DNA showed a linear relationship between log CFU/mL and threshold cycles. The detectable limitation was 5.62 CFU/mL by using purified DNA from bacteria culture. The reproducibility of this assay was assessed by calculating the variation of the threshold cycle value and the slope from test repeats for the same strains and different strains. Our results indicate that the developed assay has high sensitivity and specificity for identification of C. coli.

  18. Normative values for stool frequency and form using Rome III diagnostic criteria for functional constipation in adults: systematic review with meta-analysis

    Science.gov (United States)

    Miller, Larry E.; Ibarra, Alvin; Ouwehand, Arthur C.; Zimmermann, Angela K.

    2017-01-01

    When designing clinical trials focused on functional constipation therapies, understanding the normative values of populations selected using the Rome III criteria is important for estimating baseline symptom severity, and for power analysis and sample size calculations. The objective of this review was to determine normative ranges for stool frequency and form in adults with functional constipation (Rome III criteria). Eligible studies reported stool frequency or form; random effects meta-analysis was performed with subgroup analyses to explore sources of heterogeneity. A total of 25 studies (43 groups, 2292 subjects) were included. Pooled estimates were 2.7 (95% CI 2.4-3.0) for weekly stools and 2.4 (95% CI 2.1-2.6) for stool form (Bristol scale). Heterogeneity was high for both outcomes (both I2=96%, Precall was the sole explanatory variable (2.5 vs. 2.1, P<0.05). We conclude that adults with functional constipation have significant variation in stool frequency and form, explained in part by geography and study design.

  19. Two sequential PCR amplifications for detection of Schistosoma mansoni in stool samples with low parasite load

    Directory of Open Access Journals (Sweden)

    Maria Cristina Carvalho do Espírito-Santo

    2012-10-01

    Full Text Available Schistosomiasis constitutes a major public health problem, with an estimated 200 million individuals infected worldwide and 700 million people living in risk areas. In Brazil there are areas of high, medium and low endemicity. Studies have shown that in endemic areas with a low prevalence of Schistosoma infection the sensitivity of parasitological methods is clearly reduced. Consequently diagnosis is often impeded due to the presence of false-negative results. The aim of this study is to present the PCR reamplification (Re-PCR protocol for the detection of Schistosoma mansoni in samples with low parasite load (with less than 100 eggs per gram (epg of feces. Three methods were used for the lysis of the envelopes of the S. mansoni eggs and two techniques of DNA extraction were carried out. Extracted DNA was quantified, and the results suggested that the extraction technique, which mixed glass beads with a guanidine isothiocyanate/phenol/chloroform (GT solution, produced good results. PCR reamplification was conducted and detection sensitivity was found to be five eggs per 500 mg of artificially marked feces. The results achieved using these methods suggest that they are potentially viable for the detection of Schistosoma infection with low parasite load.

  20. Comparison of efficiency of various DNA extraction methods from cysts of Giardia intestinalis measured by PCR and TaqMan real time PCR.

    Science.gov (United States)

    Adamska, M; Leońska-Duniec, A; Maciejewska, A; Sawczuk, M; Skotarczak, B

    2010-12-01

    The aim of the presented study was to work out an effective method of extraction of DNA from Giardia intestinalis cysts as well as a sensitive and specific method for detection of DNA of this protozoan using a polymerase chain reaction (PCR). Twelve protocols for DNA extraction have been compared. Purification and extraction of DNA were preceded by additional actions in order to destroy the cysts' wall. The highest effectiveness of DNA extraction was obtained in case of alternating application of freezing the samples in liquid nitrogen and their incubation in water bath in the temperature of 100 degrees C, and then the extraction with the QIAamp DNA Tissue Mini Kit (QIAGEN)--T kit--with an all night long incubation with proteinase K in 56 degrees C. Effectiveness of DNA extraction with the use of each kit after extraction with each treatment was measured by nested PCR product of beta-giardin gene fragment and C(T) values of real time PCR of the SSU rRNA gene of G. intestinalis. The detection limit, defined as the lowest number detected in 100% cases, was 100 cysts per 200 microl when effectiveness was evaluated with nested PCR and 50 oocysts with real time PCR after extraction DNA with T kit. Results of our comparative studies have shown that all stages preceding the molecular detection of G. intestinalis DNA are equally important, and materially influence on the final effect and this version of method seems to be very useful for the sensitive detection of DNA of G. intestinalis.

  1. Comparison of efficiency of various DNA extraction methods from cysts of Giardia intestinalis measured by PCR and TaqMan real time PCR

    Directory of Open Access Journals (Sweden)

    Adamska M.

    2010-12-01

    Full Text Available The aim of the presented study was to work out an effective method of extraction of DNA from Giardia intestinalis cysts as well as a sensitive and specific method for detection of DNA of this protozoan using a polymerase chain reaction (PCR. Twelve protocols for DNA extraction have been compared. Purification and extraction of DNA were preceded by additional actions in order to destroy the cysts’ wall. The highest effectiveness of DNA extraction was obtained in case of alternating application of freezing the samples in liquid nitrogen and their incubation in water bath in the temperature of 100 ˚C, and then the extraction with the QIAamp DNA Tissue Mini Kit (QIAGEN – T kit – with an all night long incubation with proteinase K in 56 ˚C. Effectiveness of DNA extraction with the use of each kit after extraction with each treatment was measured by nested PCR product of β-giardin gene fragment and CT values of real time PCR of the SSU rRNA gene of G. intestinalis. The detection limit, defined as the lowest number detected in 100 % cases, was 100 cysts per 200 μl when effectiveness was evaluated with nested PCR and 50 oocysts with real time PCR after extraction DNA with T kit. Results of our comparative studies have shown that all stages preceding the molecular detection of G. intestinalis DNA are equally important, and materially influence on the final effect and this version of method seems to be very useful for the sensitive detection of DNA of G. intestinalis.

  2. Short-term effect of prebiotics administration on stool characteristics and serum cytokines dynamics in very young children with acute diarrhea.

    Science.gov (United States)

    Vaisman, Nachum; Press, Josef; Leibovitz, Eugene; Boehm, Güenther; Barak, Vivian

    2010-07-01

    We investigated the effect of a mixture of long-chain fructo-oligosaccharides, galacto-oligosaccharides and acidic oligosaccharides on the number and consistency of stools and on immune system biomarkers in 104 supplemented and non-supplemented subjects (aged 9-24 months) with acute diarrhea. Interleukin-1 (IL-1), IL-1RA, IL-6, IL-8, IL-10, TNF-α and sIL-2R cytokine levels were determined. The significant decrease in number of stools and increase in stool consistency in the supplemented group was of little clinical relevance. The only significant change in pro- and anti-inflammatory cytokines was decreased TNF-α levels in the supplemented group. Prebiotic supplementation during acute diarrhea episodes did not influence the clinical course.

  3. Short-Term Effect of Prebiotics Administration on Stool Characteristics and Serum Cytokines Dynamics in Very Young Children with Acute Diarrhea

    Directory of Open Access Journals (Sweden)

    Nachum Vaisman

    2010-06-01

    Full Text Available We investigated the effect of a mixture of long-chain fructo-oligosaccharides, galacto-oligosaccharides and acidic oligosaccharides on the number and consistency of stools and on immune system biomarkers in 104 supplemented and non-supplemented subjects (aged 9–24 months with acute diarrhea. Interleukin-1 (IL-1, IL-1RA, IL-6, IL-8, IL-10, TNF-α and sIL-2R cytokine levels were determined. The significant decrease in number of stools and increase in stool consistency in the supplemented group was of little clinical relevance. The only significant change in pro- and anti-inflammatory cytokines was decreased TNF-α levels in the supplemented group. Prebiotic supplementation during acute diarrhea episodes did not influence the clinical course.

  4. Diagnosis of soil-transmitted helminths in the era of preventive chemotherapy: effect of multiple stool sampling and use of different diagnostic techniques.

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    Stefanie Knopp

    Full Text Available BACKGROUND: Soil-transmitted helminth infections are common throughout the tropics and subtropics and they disproportionately affect the poorest of the poor. In view of a growing global commitment to control soil-transmitted helminthiasis, there is a need to elucidate the effect of repeated stool sampling and the use of different diagnostic methods in areas targeted for preventive chemotherapy that are characterized by low-infection intensities. In this study, we focused on schoolchildren on Unguja Island, Zanzibar, an area where anthelminthic drugs have been repeatedly administered over the past decade. METHODOLOGY/PRINCIPAL FINDINGS: Three serial stool samples from each of 342 schoolchildren were examined using the Kato-Katz (K-K, Koga agar plate (KAP, and Baermann (BM techniques. These methods were used individually or in combination for the diagnosis of Ascaris lumbricoides (K-K, Trichuris trichiura (K-K, hookworm (K-K and KAP, and Strongyloides stercoralis (KAP and BM. The examination of multiple stool samples instead of a single one resulted in an increase of the observed prevalence; e.g., an increase of 161% for hookworm using the K-K method. The diagnostic sensitivity of single stool sampling ranged between 20.7% for BM to detect S. stercoralis and 84.2% for K-K to diagnose A. lumbricoides. Highest sensitivities were observed when different diagnostic approaches were combined. The observed prevalences for T. trichiura, hookworm, A. lumbricoides, and S. stercoralis were 47.9%, 22.5%, 16.5%, and 10.8% after examining 3 stool samples. These values are close to the 'true' prevalences predicted by a mathematical model. CONCLUSION/SIGNIFICANCE: Rigorous epidemiologic surveillance of soil-transmitted helminthiasis in the era of preventive chemotherapy is facilitated by multiple stool sampling bolstered by different diagnostic techniques.

  5. DETECTION OF HELICOBACTER ANTIGEN IN STOOL SAMPLES AND ITS RELATION TO H. PYLORI POSITIVE CHOLECYSTITIS IN EGYPTIAN PATIENTS WITH CHRONIC CALCULAR CHOLECYSTITIS.

    Science.gov (United States)

    Hassan, Ehsan H; Gerges, Shawkat S; Ahmed, Rehab; Mostafa, Zeinab M; Al-Hamid, Hager Abd; Abd El-Galil, Heba; Thabet, Suzan

    2015-12-01

    Evidences supporting the association between H. pylori infection and chronic cholecystitis could be found by using direct culture or staining of H. pylori in gallbladder tissues as well as indirect techniques. Stool antigen test has been widely used due to its noninvasive nature. Various stool antigen tests were developed to detect H. pylori using an enzyme immunoassay (EIA) based on monoclonal or polyclonal antibodies This study evaluated the frequency of H. pylori antigen in stool samples of patients with chronic calcular cholecystitis as regard gall bladder histopathological changes. Fifty patients were included presented with symptomatic qholecystolithiasis recruited from the outpatient clinic of National Hepatology and Tropical Medicine Research Institute during 2014-2015. Full history and clinical examination and abdominal ultrasonography were performed. Stool samples were collected, prepared and examined for detection of H. pylori antigen. Cholecystectomy was done for all patients; 45 patients (90%) by laparoscopic Cholecystectomy and 5 patients (10%) by open surgery and removed gallbladders were submitted to pathology department for detection of H. pylori in tissue under microscope using Giemsa stain. The results showed that (82%) were females with mean age (42.6 +/- 1 years). The mean BMI was (29 + 7.2) H. pylori-specific antigen in stool samples was detected in 40% of patients and 38% were detected in patients; tissue, with significant correlation between H. pylori-specific antigen in stool and in tissue. Histopathological pictures infection in tissue were 68.4% mucosal erosions, 63.2% mucosal atrophy, 57.9% mucosal hyperplasia, 26.3% metaplasia, 42.1% musculosa hypertrophy, 26.3% fibrosis, but lymphoid aggregates were in 42.1% of cases.

  6. Simple fecal flotation is a superior alternative to guadruple Kato Katz smear examination for the detection of hookworm eggs in human stool

    DEFF Research Database (Denmark)

    Inpankaew, Tawin; Schär, Fabian; Khieu, Virak

    2014-01-01

    BACKGROUND: Microscopy-based identification of eggs in stool offers simple, reliable and economical options for assessing the prevalence and intensity of hookworm infections, and for monitoring the success of helminth control programs. This study was conducted to evaluate and compare the diagnostic...... parameters of the Kato-Katz (KK) and simple sodium nitrate flotation technique (SNF) in terms of detection and quantification of hookworm eggs, with PCR as an additional reference test in stool, collected as part of a baseline cross-sectional study in Cambodia. METHODS/PRINCIPLE FINDINGS: Fecal samples...

  7. Pattern of shedding of small, round-structured virus particles in stools of patients of outbreaks of food-poisoning from raw oysters.

    Science.gov (United States)

    Haruki, K; Seto, Y; Murakami, T; Kimura, T

    1991-01-01

    The pattern of shedding of the small, round-structured virus (SRSV) particles in the stools of patients who suffered from food-poisoning due to raw oysters was investigated. The duration and concentration of fecal shedding of the SRSV particles were studied by electron microscopic examinations of stool specimens obtained during the course of illness to see a relation of viral shedding to day of illness. It was found that the fecal shedding of the SRSV particles occurred within five days of illness; thereafter, the concentration of the SRSV particles in feces rapidly decreased within a few days during the course of illness.

  8. Epidemiology of enteric pathogens found in stool symptomatic patients selected in a northern Bari region population between 2000 and 2009

    Directory of Open Access Journals (Sweden)

    Maria Antonietta Distasi

    2011-06-01

    Full Text Available Enteritis occur primarily in children and elderly. In order to analyse the frequency of single specific causative agents of enteritis, 5072 stool cultures have been evaluated at the Andria Hospital laboratory from 2000 to 2009. Cultures for Salmonella, Shigella, Campylobacter were performed, and, on samples from children under 6 years old, testing for presence of Adenovirus and Rotavirus was carried out. After inoculation on specific media, bacterial identification was performed via the VITEK system (bioMérieux, supplemented by serological identification for Salmonella and Shigella.Virus detection was performed by DIARLEX Dasit system. During the study period, 716 (14.1% samples were found positive for the presence of pathogen microorganisms. In the trimesters from February to May and November to January viral etiology was prevalent, whereas the bacterial one prevailed from June to October. In addition, during 2009, an increase of Campylobacter isolates was observed, with consequent reduction of Salmonella isolates.

  9. Evaluation of a new real-time PCR assay for the direct detection of diarrheagenic Escherichia coli in stool specimens.

    Science.gov (United States)

    Eigner, U; Hiergeist, A; Veldenzer, A; Rohlfs, M; Schwarz, R; Holfelder, M

    2017-02-01

    Diarrheagenic E. coli (DEC) are one of the most common causes for diarrhea worldwide, especially in children. We evaluated the rapid RIDA ® GENE (RG) real-time multiplex PCR assays (R-Biopharm, Darmstadt, Germany) for the detection of the most important diarrheagenic E. coli. Three hundred fifteen liquid or non-formed stool specimens were examined. The results of the RG multiplex assays were compared to specific PCR methods. The sensitivity and specificity of the RG PCRs were as follows, 100%/100% for the detection of EHEC, 96.3% and 99% for EPEC, 100% and 100% for the detection of EAEC, ETEC and EIEC, respectively. Overall, the RG real-time PCR system for the detection of DEC tested in this study provided reliable and rapid results and shows the ability as a useful addendum for the detection of diarrheagenic E. coli in the medical laboratory.

  10. Performance of stool cultures before and after a 3-day hospitalization: fewer cultures, better for patients and for money.

    Science.gov (United States)

    Le Guern, Rémi; Loïez, Caroline; Grandbastien, Bruno; Courcol, René; Wallet, Frédéric

    2013-09-01

    We evaluated retrospectively the yield of stool culture (SC) depending on the length of hospitalization, and we characterized the patients missed by the 3-day rejection rule. SC detects bacterial enteric pathogens (Campylobacter spp., Salmonella enterica, Yersinia spp., Shigella spp.). During this 5-year study period, 13,039 SCs were requested, and 376 were positive (2.9%). The yield of SC dropped from 11.7% before 3 days of hospitalization to 0.7% after 3 days in children and 4.3% to 0.3% in adults. Finally, only 13 clinically relevant cases (0.2% of SC prescribed after 3 days) were undiagnosed by strict application of the 3-day rule. In conclusion, rejection of SC prescribed after 3 days of hospitalization allows to reduce workload by 37.8% for children and 65.7% for adults, representing a cost of €12,500 ($16,250) per year in our hospital. © 2013.

  11. Planococcus faecalis sp. nov., a carotenoid-producing species isolated from stools of Antarctic penguins.

    Science.gov (United States)

    Kim, Jin Ho; Kang, Hyung Jun; Yu, Byung Jo; Kim, Sun Chang; Lee, Pyung Cheon

    2015-10-01

    Taxonomic studies were performed on a novel carotenoid-producing strain, designated AJ003T, isolated from faeces of Antarctic penguins. Cells of strain AJ003T were aerobic, Gram-stain-positive, cocci-shaped and orange. Strain AJ003T was capable of growing in a broad temperature range, including sub-zero growth (below − 20 to 30 °C). 16S rRNA gene sequence analysis revealed that strain AJ003T was closely related to Planococcus halocryophilus Or1T (97.4 % similarity), Planococcus antarcticus DSM 14505T (97.3 %), Planococcus kocurii NCIMB 629T (97.3 %), and Planococcus donghaensis JH1T (97.1 %). The predominant cellular fatty acids were anteiso-C15 : 0, and iso-C16 : 0.MK-7 and MK-8 were the quinones identified, and the major pigment was glycosyl-4,4′-diaponeurosporen-4′-ol-4-oic acid. The major polar lipid was phosphatidylglycerol. DNA–DNA relatedness of strain AJ003T with respect to its closest phylogenetic neighbours was 38.2 ± 0.5 % for Planococcus halocryophilus DSM 24743T, 32.2 ± 0.2 % for Planococcus antarcticus DSM 14505T, 21.0 ± 0.3 % for Planococcus kocurii DSM 20747T and 18.6 ± 1.4 % for Planococcus donghaensis KCTC 13050T. The DNA G+C content of strain AJ003T was 40.0 ± 0.6 mol%. Based on the phenotypic, chemotaxonomic and phylogenetic data, strain AJ003T is concluded to represent a novel species of the genus Planococcus, for which the name Planococcus faecalis sp. nov. is proposed. The type strain is AJ003T ( = KCTC 33580T = CECT 8759T).

  12. Geographical Variation in Antibiotic-Resistant Escherichia coli Isolates from Stool, Cow-Dung and Drinking Water

    Science.gov (United States)

    Sahoo, Krushna Chandra; Tamhankar, Ashok J.; Sahoo, Soumyakanta; Sahu, Priyadarshi Soumyaranjan; Klintz, Senia Rosales; Lundborg, Cecilia Stålsby

    2012-01-01

    Little information is available on relationships between the biophysical environment and antibiotic resistance. This study was conducted to investigate the antibiotic resistance pattern of Escherichia coli isolated from child stool samples, cow-dung and drinking water from the non-coastal (230 households) and coastal (187 households) regions of Odisha, India. Susceptibility testing of E. coli isolates (n = 696) to the following antibiotics: tetracycline, ampicillin/sulbactam, cefuroxime, cefotaxime, cefixime, cotrimoxazole, amikacin, ciprofloxacin, norfloxacin and nalidixic acid was performed by the disk diffusion method. Ciprofloxacin minimum inhibitory concentration (MIC) values were determined for ciprofloxacin-resistant isolates (n = 83). Resistance to at least one antibiotic was detected in 90% or more of the E. coli isolates. Ciprofloxacin MIC values ranged from 8 to 32 µg/mL. The odds ratio (OR) of resistance in E. coli isolates from children’s stool (OR = 3.1, 95% CI 1.18–8.01), cow-dung (OR = 3.6, 95% CI 1.59–8.03, P = 0.002) and drinking water (OR = 3.8, 95% CI 1.00–14.44, P = 0.049) were higher in non-coastal compared to coastal region. Similarly, the co-resistance in cow-dung (OR = 2.5, 95% CI 1.39–4.37, P = 0.002) and drinking water (OR = 3.2, 95% CI 1.36–7.41, P = 0.008) as well as the multi-resistance in cow-dung (OR = 2.2, 95% CI 1.12–4.34, P = 0.022) and drinking water (OR = 2.7, 95% CI 1.06–7.07, P = 0.036) were also higher in the non-coastal compared to the coastal region. PMID:22690160

  13. Geographical variation in antibiotic-resistant Escherichia coli isolates from stool, cow-dung and drinking water.

    Science.gov (United States)

    Sahoo, Krushna Chandra; Tamhankar, Ashok J; Sahoo, Soumyakanta; Sahu, Priyadarshi Soumyaranjan; Klintz, Senia Rosales; Lundborg, Cecilia Stålsby

    2012-03-01

    Little information is available on relationships between the biophysical environment and antibiotic resistance. This study was conducted to investigate the antibiotic resistance pattern of Escherichia coli isolated from child stool samples, cow-dung and drinking water from the non-coastal (230 households) and coastal (187 households) regions of Odisha, India. Susceptibility testing of E. coli isolates (n = 696) to the following antibiotics: tetracycline, ampicillin/sulbactam, cefuroxime, cefotaxime, cefixime, cotrimoxazole, amikacin, ciprofloxacin, norfloxacin and nalidixic acid was performed by the disk diffusion method. Ciprofloxacin minimum inhibitory concentration (MIC) values were determined for ciprofloxacin-resistant isolates (n = 83). Resistance to at least one antibiotic was detected in 90% or more of the E. coli isolates. Ciprofloxacin MIC values ranged from 8 to 32 µg/mL. The odds ratio (OR) of resistance in E. coli isolates from children's stool (OR = 3.1, 95% CI 1.18-8.01), cow-dung (OR = 3.6, 95% CI 1.59-8.03, P = 0.002) and drinking water (OR = 3.8, 95% CI 1.00-14.44, P = 0.049) were higher in non-coastal compared to coastal region. Similarly, the co-resistance in cow-dung (OR = 2.5, 95% CI 1.39-4.37, P = 0.002) and drinking water (OR = 3.2, 95% CI 1.36-7.41, P = 0.008) as well as the multi-resistance in cow-dung (OR = 2.2, 95% CI 1.12-4.34, P = 0.022) and drinking water (OR = 2.7, 95% CI 1.06-7.07, P = 0.036) were also higher in the non-coastal compared to the coastal region.

  14. Geographical Variation in Antibiotic-Resistant Escherichia coli Isolates from Stool, Cow-Dung and Drinking Water

    Directory of Open Access Journals (Sweden)

    Cecilia Stålsby Lundborg

    2012-03-01

    Full Text Available Little information is available on relationships between the biophysical environment and antibiotic resistance. This study was conducted to investigate the antibiotic resistance pattern of Escherichia coli isolated from child stool samples, cow-dung and drinking water from the non-coastal (230 households and coastal (187 households regions of Odisha, India. Susceptibility testing of E. coli isolates (n = 696 to the following antibiotics: tetracycline, ampicillin/sulbactam, cefuroxime, cefotaxime, cefixime, cotrimoxazole, amikacin, ciprofloxacin, norfloxacin and nalidixic acid was performed by the disk diffusion method. Ciprofloxacin minimum inhibitory concentration (MIC values were determined for ciprofloxacin-resistant isolates (n = 83. Resistance to at least one antibiotic was detected in 90% or more of the E. coli isolates. Ciprofloxacin MIC values ranged from 8 to 32 µg/mL. The odds ratio (OR of resistance in E. coli isolates from children’s stool (OR = 3.1, 95% CI 1.18–8.01, cow-dung (OR = 3.6, 95% CI 1.59–8.03, P = 0.002 and drinking water (OR = 3.8, 95% CI 1.00–14.44, P = 0.049 were higher in non-coastal compared to coastal region. Similarly, the co-resistance in cow-dung (OR = 2.5, 95% CI 1.39–4.37, P = 0.002 and drinking water (OR = 3.2, 95% CI 1.36–7.41, P = 0.008 as well as the multi-resistance in cow-dung (OR = 2.2, 95% CI 1.12–4.34, P = 0.022 and drinking water (OR = 2.7, 95% CI 1.06–7.07, P = 0.036 were also higher in the non-coastal compared to the coastal region.

  15. [Polio vaccination and stool screening in German reception centers for asylum seekers, November 2013-January 2014 : What was implemented?].

    Science.gov (United States)

    Zeitlmann, Nadine; George, Maja; Falkenhorst, Gerhard

    2016-05-01

    Following the polio outbreak in Syria and the rising number of Syrian asylum seekers in Germany in 2013, the Robert Koch Institute recommended - within the context of existing vaccination recommendations for asylum seekers - on 01/11/2013 to prioritize polio vaccination of Syrian asylum seekers and stool screening in a target group of Syrian asylum seekers aged less than three years. The article evaluates the implementation of this recommendation in German asylum seeker reception centres (RC) to gain further knowledge on the vaccination practices in RCs and to identify opportunities for improving future recommendations. The electronic questionnaire was sent by email to all German RCs, asking for general information on the RC, existing vaccination efforts, the main obstacles for implementation of the recommendations, the number of incoming and vaccinated asylum seekers, and asylum seekers screened for poliovirus in the period from 01/11/2013 to 31/01/2014. The RCs rated the feasibility of the recommendation and the provided multilingual information material. All of the 20 identified RCs responded. During the study period, 33.874 asylum seekers arrived in the RCs. Of those with available information about possession of a vaccination record, on average 1.6 % did have one. All RCs offered timely vaccination to Syrian asylum seekers younger than three years. In this target group, eight RC achieved vaccination coverages of ≥ 80 %. Stool screening coverage was ≥ 80 % in five of 19 RCs. Eleven RCs rated the recommendation as very well/well implementable. Staff shortages and language barriers were mentioned as the main implementation obstacles. Similar future recommendations for asylum seekers in RCs should be accompanied by informational material in additional languages. Staff shortages hampering implementation could be overcome through collaborations with non-governmental organizations.

  16. Comparative evaluation of different DNA extraction methods for HPV genotyping by linear array and INNO-LiPA.

    Science.gov (United States)

    Donà, Maria Gabriella; Benevolo, Maria; Pimpinelli, Fulvia; Battista, Mara; Rollo, Francesca; Stivali, Francesca; Moscarelli, Antonella; Giuliani, Massimo; Di Carlo, Aldo; Vocaturo, Amina

    2011-06-01

    In order to investigate the influence of DNA extraction on two PCR-based HPV genotyping tests (Linear Array, Roche and INNO-LiPA Extra, Innogenetics), three different procedures were used to purify DNA from 28 cervico-vaginal samples tested previously by the Hybrid Capture 2: the AmpliLute Liquid Media Extraction kit (Roche), the QIAamp DNA Blood mini kit (QIAGEN), and the NucliSENS EasyMAG automated platform (bioMérieux). All HC2-positive samples were found positive by both assays, independently of the extract used. Type-specific concordance (i.e., identical HPV type-specific profile in all the extracts of the same sample) was observed in 55% and 75% of the cases testing samples by the Linear Array and the INNO-LiPA, respectively. Using the DNA extracted with the two manual methods the results were concordant in 75% of the cases both for the Linear Array and the INNO-LiPA. When comparing the Linear Array results obtained on either of the two manual extracts with those obtained following automated extraction, 65% of the samples showed type-specific concordance in both cases. The INNO-LiPA results were concordant in 80% of the cases comparing the AmpliLute versus the automated extract, while concordant results were observed in 90% of the cases when comparing the QIAGEN versus the automated extract. In conclusion, the Linear Array and INNO-LiPA results are affected by the method of DNA extraction. Consequently, different HPV type-specific profiles may be observed using different extracts of the same sample. The use of consistent protocols for DNA purification is a priority to guarantee intra-assay reproducibility over time.

  17. High prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae detected in the human gut using an improved DNA detection protocol.

    Directory of Open Access Journals (Sweden)

    Bédis Dridi

    Full Text Available BACKGROUND: The low and variable prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae DNA in human stool contrasts with the paramount role of these methanogenic Archaea in digestion processes. We hypothesized that this contrast is a consequence of the inefficiencies of current protocols for archaeon DNA extraction. We developed a new protocol for the extraction and PCR-based detection of M. smithii and M. stadtmanae DNA in human stool. METHODOLOGY/PRINCIPAL FINDINGS: Stool specimens collected from 700 individuals were filtered, mechanically lysed twice, and incubated overnight with proteinase K prior to DNA extraction using a commercial DNA extraction kit. Total DNA was used as a template for quantitative real-time PCR targeting M. smithii and M. stadtmanae 16S rRNA and rpoB genes. Amplification of 16S rRNA and rpoB yielded positive detection of M. smithii in 95.7% and M. stadtmanae in 29.4% of specimens. Sequencing of 16S rRNA gene PCR products from 30 randomly selected specimens (15 for M. smithii and 15 for M. stadtmanae yielded a sequence similarity of 99-100% using the reference M. smithii ATCC 35061 and M. stadtmanae DSM 3091 sequences. CONCLUSIONS/SIGNIFICANCE: In contrast to previous reports, these data indicate a high prevalence of the methanogens M. smithii and M. stadtmanae in the human gut, with the former being an almost ubiquitous inhabitant of the intestinal microbiome.

  18. Antibiotic resistance among Escherichia coli isolates from stool samples of children aged 3 to 14 years from Ujjain, India

    Science.gov (United States)

    2013-01-01

    Background Antibiotic resistance is a major global public health concern, particularly in settings where few treatment options are available. Limited research has been done on antibiotic resistance in Escherichia coli of Indian children at community level. Therefore we studied antibiotic resistance patterns in E. coli isolates from stool samples of children aged 3-14 years from Ujjain, Central India, to investigate associations of resistance with demographic variables. Methods Children, 3-14 years of age, were included from 30 randomly selected villages of Palwa demographic surveillance site, Ujjain, India. Parents were interviewed using a questionnaire, and stool samples were collected from participating children. E. coli were isolated from stool samples (n = 529), and susceptibility testing to 18 different antibiotics was done using standard methods. Results The proportions of isolates resistant to various antibiotics were, nalidixic acid, (45%), tetracycline (37%), ampicillin (37%), sulfamethoxazole/trimethoprim (29%) and amoxicillin/clavulanic acid (29%). No isolates were resistant to imipenem. Overall, 72% of isolates were resistant to at least one antibiotic and 33% were multi-drug resistant. High rates of cross-resistance were seen for 15 (83%) of the antibiotics studied. E. coli isolates from children with literate mothers were more resistant to penicillins and fluoroquinolones. ESBL-producers comprised 9% of the isolates. Conclusion Antibiotic resistance and cross-resistance were common in E. coli from stools of children. Resistance rates were associated with maternal literacy. PMID:24124728

  19. Diagnostic accuracy of a rapid fecal test to confirm H pylori eradication after therapy: Prospective comparison with a laboratory stool test

    Institute of Scientific and Technical Information of China (English)

    Lucio Trevisani; Viviana Cifalà; Nadia Fusetti; Giuseppe Gilli; Paola Tombesi; Marco Torchiaro; Sergio Boccia; Vincenzo Abbasciano

    2007-01-01

    AIM: To investigate the clinical performances of rapid stool test (ImmunoCard STAT HpSA, Meridian Diagnostic Inc.) in the evaluation of eradication therapy of H pylori and to compare it with a well-known and validated laboratory stool test (Amplified IDEA Hp StAR, Dako).METHODS: Stool samples of 122 patients were evaluated after eradication therapy of H pylori. H pyloristatus was assessed by 13C-urea breath test (UBT).Stool specimens were tested using either the rapid immunoassay kit or the laboratory immunoassay kit.RESULTS: Forty-three patients were infected and 79 non-infected. Sensitivity and specificity of ImmunoCard STAT and Hp StAR were 58.14% and 76.4%, and 97.47% and 98.73%, respectively (P > 0.05). Overall agreement between the two tests was 92.6% (113 of 122 cases).CONCLUSION: ImmunoCard STAT seems to have rather low performances, and it cannot be regarded as a reliable tool in the post-treatment setting. Also Hp StAR cannot be recommended to confirm H pylori eradication after treatment.

  20. Short-term effect of prebiotics administration on stool characteristics and serum cytokines dynamics in very young children with acute diarrhea

    NARCIS (Netherlands)

    N. Vaisman (Nachum); J. Press (Josef); E. Leibovitz (Eugene); G. Boehm (Günther); V. Barak (Vivian)

    2010-01-01

    textabstractWe investigated the effect of a mixture of long-chain fructo-oligosaccharides, galacto-oligosaccharides and acidic oligosaccharides on the number and consistency of stools and on immune system biomarkers in 104 supplemented and non-supplemented subjects (aged 9-24 months) with acute diar

  1. Comparison of invasive methods and two different stool antigen tests for diagnosis of H pylori infection in patients with gastric bleeding

    Institute of Scientific and Technical Information of China (English)

    Ebru Demiray; (O)zlem Yilmaz; Cihat (S)arki(s); Müjde Soytürk; (I)lkay (S)im(s)ek

    2006-01-01

    AIM: To compare two different H pylori stool antigen tests as noninvasive diagnostic methods.METHODS: The study population consisted of 22 upper gastrointestinal system bleeding patients. Urea breath test (UBT), rapid urease test (RUT) and histopathological examination were applied to all patients. Stool specimens from these patients were examined by rapid STRIP!HpSA and one step simple H pylori antigen cassette test for the detection of H pylori antigens.RESULTS: For these 22 patients, 15 (68.2%) were diagnosed as positive and seven (31.8%) were diagnosed negative for H pylori infection by the gold standard methods. Whereas 10 (45.5%) were positive and 12 (54.5%) were diagnosed negative by the rapid STRIP!HpSA test. The sensitivity, specificity, positive and negative predictive values were 60%, 86%, 90% and 50%, respectively. When compared to the gold standard methods, these differences were not significant.However, six patients (27.3%) were positive, and 16(72.7%) were negative by the simple H pylori stool antigen cassette test. The sensitivity, specificity, positive and negative predictive values were 33%, 86%, 83% and 38%, respectively. Compared to the gold standard methods, the simple H pylori stool antigen cassette test results were significantly different (P = 0.012).CONCLUSION: Rapid STRIP!HpSA test could be used as a routine diagnostic tool in the microbiology laboratory for assessing clinical significance and eradication control of H pylori in upper gastrointestinal system bleeding patients.

  2. Differentiation of Entamoeba histolytica/Entamoeba dispar by the polymerase chain reaction in stool samples of patients with gastrointestinal symptoms in the Sanliurfa Province.

    Science.gov (United States)

    Zeyrek, Fadile Yıldız; Turgay, Nevin; Unver, Aysegül; Ustün, Sebnem; Akarca, Ulus; Töz, Seray

    2013-01-01

    We aimed to diagnose amebiasis and also identify Entamoeba histolytica (E. histolytica) and Entamoeba dispar (E. dispar) in patients with gastrointestinal symptoms in an endemic region in Turkey. Stool samples obtained from 181 patients with gastrointestinal symptoms from the Harran University Hospital of Sanliurfa were examined for the diagnosis of amebiasis by the three methods which are as follows:- In house polymerase chain reaction (PCR) targeting the 135 base pair region located on the small-subunit ribosomal RNA (SSU rRNA) gene to differentiate E. histolytica from E. dispar; and the commercial kit, RIDASCREEN® stool ELISA, that identifies Entamoeba sensu lato antigen and microscopical examination of Trichrome stained smears of stool samples. Positivity for E. histolytica/E. dispar complex was found to be 79 (43.6%) by microscopy versus 83 (45.9%) by PCR out of 181 stool samples. A total of 45 patients were found to be positive by the antigen detection method. PCR and microscopy were both positive in 59 samples. The number of patients infected with E. dispar (39.8%) was found to be higher than E. histolytica (3.3%) while 5 patients (2.8%) had mixed E. histolytica+E. dispar infections according to PCR results. Routine diagnosis of amebiasis by a combination of microscopy and antigen detection technique should be complemented with a PCR assay as a reference test for sensitive differentiation of both species.

  3. Normal tissue complication probability (NTCP) models for late rectal bleeding, stool frequency and fecal incontinence after radiotherapy in prostate cancer patients

    NARCIS (Netherlands)

    Schaake, Wouter; van der Schaaf, Arjen; van Dijk, Lisanne V.; Bongaerts, Alfons H. H.; van den Bergh, Alfons C. M.; Langendijk, Johannes A.

    2016-01-01

    Background and purpose: Curative radiotherapy for prostate cancer may lead to anorectal side effects, including rectal bleeding, fecal incontinence, increased stool frequency and rectal pain. The main objective of this study was to develop multivariable NTCP models for these side effects. Material a

  4. Diagnostic accuracy of a rapid fecal test to confirm H pylori eradication after therapy: Prospective comparison with a laboratory stool test

    Science.gov (United States)

    Trevisani, Lucio; Cifalà, Viviana; Fusetti, Nadia; Gilli, Giuseppe; Tombesi, Paola; Torchiaro, Marco; Boccia, Sergio; Abbasciano, Vincenzo

    2007-01-01

    AIM: To investigate the clinical performances of rapid stool test (ImmunoCard STAT HpSA, Meridian Diagnostic Inc.) in the evaluation of eradication therapy of H pylori and to compare it with a well-known and validated laboratory stool test (Amplified IDEA Hp StAR, Dako). METHODS: Stool samples of 122 patients were evaluated after eradication therapy of H pylori. H pylori status was assessed by 13C-urea breath test (UBT). Stool specimens were tested using either the rapid immunoassay kit or the laboratory immunoassay kit. RESULTS: Forty-three patients were infected and 79 non-infected. Sensitivity and specificity of ImmunoCard STAT and Hp StAR were 58.14% and 76.4%, and 97.47% and 98.73%, respectively (P > 0.05). Overall agreement between the two tests was 92.6% (113 of 122 cases). CONCLUSION: ImmunoCard STAT seems to have rather low performances, and it cannot be regarded as a reliable tool in the post-treatment setting. Also Hp StAR cannot be recommended to confirm H pylori eradication after treatment. PMID:17724805

  5. An uncooked vegan diet shifts the profile of human fecal microflora: computerized analysis of direct stool sample gas-liquid chromatography profiles of bacterial cellular fatty acids.

    Science.gov (United States)

    Peltonen, R; Ling, W H; Hänninen, O; Eerola, E

    1992-01-01

    The effect of an uncooked extreme vegan diet on fecal microflora was studied by direct stool sample gas-liquid chromatography (GLC) of bacterial cellular fatty acids and by quantitative bacterial culture by using classical microbiological techniques of isolation, identification, and enumeration of different bacterial species. Eighteen volunteers were divided randomly into two groups. The test group received an uncooked vegan diet for 1 month and a conventional diet of mixed Western type for the other month of the study. The control group consumed a conventional diet throughout the study period. Stool samples were collected. Bacterial cellular fatty acids were extracted directly from the stool samples and measured by GLC. Computerized analysis of the resulting fatty acid profiles was performed. Such a profile represents all bacterial cellular fatty acids in a sample and thus reflects its microflora and can be used to detect changes, differences, or similarities of bacterial flora between individual samples or sample groups. GLC profiles changed significantly in the test group after the induction and discontinuation of the vegan diet but not in the control group at any time, whereas quantitative bacterial culture did not detect any significant change in fecal bacteriology in either of the groups. The results suggest that an uncooked extreme vegan diet alters the fecal bacterial flora significantly when it is measured by direct stool sample GLC of bacterial fatty acids. PMID:1482187

  6. The Diagnostic Accuracy of the M2 Pyruvate Kinase Quick Stool Test--A Rapid Office Based Assay Test for the Detection of Colorectal Cancer.

    Directory of Open Access Journals (Sweden)

    Suresh Sithambaram

    Full Text Available M2 pyruvate kinase (M2PK is an oncoprotein secreted by colorectal cancers in stools. This the first report on the accuracy of a rapid stool test in the detection of colorectal cancer (CRC.To determine the sensitivity, specificity and positive and negative predictive value of a rapid, point of care stool test M2 PK- the M2PK Quick.Consecutive cases of endoscopically diagnosed and histological proven CRC were recruited. Stools were collected by patients and tested with the immunochromatographic M2PK Quick Test (Schebo Biotech AC, Giessen, Germany. Controls were consecutively chosen from patients without any significant colorectal or gastrointestinal disease undergoing colonoscopy. CRC was staged according to the AJCC staging manual (7th Edition and location of tumor defined as proximal or distal.The sensitivity, specificity, positive predictive value, negative predictive value and overall accuracy were: 93%, 97.5%, 94.9%, 96.5% and 96.0% respectively. The positive predictive value for proximal tumors was significantly lower compared to distal tumors. No differences were seen between the different stages of the tumor.The M2-PK Quick, rapid, point-of-care test is a highly accurate test in the detection of CRC. It is easy and convenient to perform and a useful diagnostic test for the detection of CRC in a clinical practice setting.

  7. Comparison of 2 chromogenic media for the detection of extended-spectrum β-lactamase producing Enterobacteriaceae stool carriage in nursing home residents.

    Science.gov (United States)

    Blane, Beth; Brodrick, Hayley J; Gouliouris, Theodore; Ambridge, Kirsty E; Kidney, Angela D; Ludden, Catherine M; Limmathurotsakul, Direk; Török, M Estée; Peacock, Sharon J

    2016-03-01

    ChromID ESBL agar and Brilliance ESBL agar were compared for the isolation of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae from 298 stools. These had comparable sensitivity and selectivity for the 116 positive samples. Pre-enrichment with cefpodoxime and extending incubation to 48 hours after direct plating both significantly increased sensitivity but reduced selectivity of both agars.

  8. Simple fecal flotation is a superior alternative to guadruple Kato Katz smear examination for the detection of hookworm eggs in human stool

    DEFF Research Database (Denmark)

    Inpankaew, Tawin; Schär, Fabian; Khieu, Virak;

    2014-01-01

    BACKGROUND: Microscopy-based identification of eggs in stool offers simple, reliable and economical options for assessing the prevalence and intensity of hookworm infections, and for monitoring the success of helminth control programs. This study was conducted to evaluate and compare the diagnost...

  9. Effect of Mass Stool Examination and Mass Treatment For Decreasing Intestinal Helminth and Protozoan Infection Rates in Bolivian Children: A Cross-Sectional Study

    Science.gov (United States)

    Còrdova Vidal, Claudia; Strauss, Wilma; Ikoma, Toshikazu; Endoh, Kazuo; Yamamoto, Masaharu

    2016-01-01

    Bolivia is one of the countries with a high intestinal helminth and protozoan infection rate. Despite the high prevalence of the parasitic infection, nationwide preventive measures for Bolivian children have not yet been implemented. We evaluated the effect of mass stool examination and treatment as a strategy for decreasing the infection rate. This study was conducted between 2013 and 2015 in children aged 2–18 years. A total of 2,033 stool samples (575 in 2013, 815 in 2014 and 642 in 2015) were collected and examined using the formalin-ether medical sedimentation method. As an anthelminthic medicine, nitazoxanide was given to all infected children within 2 months post-examination, each year. The effect of mass stool examination and treatment was evaluated based on the changes in the overall or individual parasitic infection rates during the study period. The overall parasitic infection rate decreased significantly from 65.2% in 2013 to 43.0% in 2015; a 22.2 percentage point decrease (PHymenolepis nana, decreased significantly from 9.0% in 2013 to 6.4% in 2014 to 3.4% in 2015 (P<0.001). Prevalence of the most common pathogenic protozoan infection, Entamoeba histolytica, decreased significantly from 19.0% in 2013 to 3.0% in 2015 (P<0.001). Conversely, the rate of Giardia intestinalis increased significantly from 16.5% in 2013 to 21.2% in 2015 (P<0.01). Mass stool examination and treatment for intestinal helminth and protozoan infections was effective for decreasing the overall parasitic infection rate in the study population, excluding Giardia intestinalis. Further studies on the long-term effect of mass stool examination and treatment for decreasing all intestinal parasitic infection rates in Bolivian children are needed. PMID:27923058

  10. Simple fecal flotation is a superior alternative to guadruple Kato Katz smear examination for the detection of hookworm eggs in human stool.

    Directory of Open Access Journals (Sweden)

    Tawin Inpankaew

    2014-12-01

    Full Text Available Microscopy-based identification of eggs in stool offers simple, reliable and economical options for assessing the prevalence and intensity of hookworm infections, and for monitoring the success of helminth control programs. This study was conducted to evaluate and compare the diagnostic parameters of the Kato-Katz (KK and simple sodium nitrate flotation technique (SNF in terms of detection and quantification of hookworm eggs, with PCR as an additional reference test in stool, collected as part of a baseline cross-sectional study in Cambodia.Fecal samples collected from 205 people in Dong village, Rovieng district, Preah Vihear province, Cambodia were subjected to KK, SNF and PCR for the detection (and in case of microscopy-based methods, quantification of hookworm eggs in stool. The prevalence of hookworm detected using a combination of three techniques (gold standard was 61.0%. PCR displayed a highest sensitivity for hookworm detection (92.0% followed by SNF (44.0% and quadruple KK smears (36.0% compared to the gold standard. The overall eggs per gram feces from SNF tended to be higher than for quadruple KK and the SNF proved superior for detecting low egg burdens.As a reference, PCR demonstrated the higher sensitivity compared to SNF and the quadruple KK method for detection of hookworm in human stool. For microscopic-based quantification, a single SNF proved superior to the quadruple KK for the detection of hookworm eggs in stool, in particular for low egg burdens. In addition, the SNF is cost-effective and easily accessible in resource poor countries.

  11. Simple fecal flotation is a superior alternative to guadruple Kato Katz smear examination for the detection of hookworm eggs in human stool.

    Science.gov (United States)

    Inpankaew, Tawin; Schär, Fabian; Khieu, Virak; Muth, Sinuon; Dalsgaard, Anders; Marti, Hanspeter; Traub, Rebecca J; Odermatt, Peter

    2014-12-01

    Microscopy-based identification of eggs in stool offers simple, reliable and economical options for assessing the prevalence and intensity of hookworm infections, and for monitoring the success of helminth control programs. This study was conducted to evaluate and compare the diagnostic parameters of the Kato-Katz (KK) and simple sodium nitrate flotation technique (SNF) in terms of detection and quantification of hookworm eggs, with PCR as an additional reference test in stool, collected as part of a baseline cross-sectional study in Cambodia. Fecal samples collected from 205 people in Dong village, Rovieng district, Preah Vihear province, Cambodia were subjected to KK, SNF and PCR for the detection (and in case of microscopy-based methods, quantification) of hookworm eggs in stool. The prevalence of hookworm detected using a combination of three techniques (gold standard) was 61.0%. PCR displayed a highest sensitivity for hookworm detection (92.0%) followed by SNF (44.0%) and quadruple KK smears (36.0%) compared to the gold standard. The overall eggs per gram feces from SNF tended to be higher than for quadruple KK and the SNF proved superior for detecting low egg burdens. As a reference, PCR demonstrated the higher sensitivity compared to SNF and the quadruple KK method for detection of hookworm in human stool. For microscopic-based quantification, a single SNF proved superior to the quadruple KK for the detection of hookworm eggs in stool, in particular for low egg burdens. In addition, the SNF is cost-effective and easily accessible in resource poor countries.

  12. Diagnostic efficacy of monoclonal antibody based sandwich enzyme linked immunosorbent assay (ELISA for detection of Fasciola gigantica excretory/secretory antigens in both serum and stool

    Directory of Open Access Journals (Sweden)

    Zoheiry Mona K

    2011-09-01

    Full Text Available Abstract Background This research was carried out to develop a reliable monoclonal antibody (MoAb-based sandwich enzyme linked immunosorbent assay (ELISA for the diagnosis of active Fasciola gigantica infection in both serum and stool for comparative purposes. Methods From a panel of MoAbs raised against F. gigantica excretory/secretory antigens (ES Ags, a pair (12B/11D/3F and 10A/9D/10G was chosen due to its high reactivity and strict specificity to F. gigantica antigen by indirect ELISA. Results The two MoAbs were of the IgG1 and IgG2a subclasses, respectively. Using SDS-PAGE and EITB, the selected MoAbs recognized 83, 64, 45 and 26 kDa bands of ES Ags. The lower detection limit of ELISA assay was 3 ng/ml. In stool, the sensitivity, specificity and diagnostic efficacy of ELISA was 96%, 98.2 and 97.1%; while in serum they were 94%, 94.6% and 94.3%, respectively. Moreover, a positive correlation was found between ova count in stool of F. gigantica infected patients and the OD readings of ELISA in both stool and serum samples (r = 0.730, p Conclusions These data showed that the use of MoAb-based sandwich ELISA for the detection of F. gigantica coproantigens in stool specimens was superior to serum samples; it provides a highly efficient, non-invasive technique for the diagnosis of active F. gigantica infection.

  13. Repeated stool sampling and use of multiple techniques enhance the sensitivity of helminth diagnosis: a cross-sectional survey in southern Lao People's Democratic Republic.

    Science.gov (United States)

    Sayasone, Somphou; Utzinger, Jürg; Akkhavong, Kongsap; Odermatt, Peter

    2015-01-01

    Intestinal parasitic infections are common in Lao People's Democratic Republic (Lao PDR). We investigated the accuracy of the Kato-Katz (KK) technique in relation to varying stool sampling efforts, and determined the effect of the concurrent use of a quantitative formalin-ethyl acetate concentration technique (FECT) for helminth diagnosis and appraisal of concomitant infections. The study was carried out between March and May 2006 in Champasack province, southern Lao PDR. Overall, 485 individuals aged ≥6 months who provided three stool samples were included in the final analysis. All stool samples were subjected to the KK technique. Additionally, one stool sample per individual was processed by FECT. Diagnosis was done under a light microscope by experienced laboratory technicians. Analysis of three stool samples with KK plus a single FECT was considered as diagnostic 'gold' standard and resulted in prevalence estimates of hookworm, Opisthorchis viverrini, Ascaris lumbricoides, Trichuris trichiura and Schistosoma mekongi infection of 77.9%, 65.0%, 33.4%, 26.2% and 24.3%, respectively. As expected, a single KK and a single FECT missed a considerable number of infections. While our diagnostic 'gold' standard produced similar results than those obtained by a mathematical model for most helminth infections, the 'true' prevalence predicted by the model for S. mekongi (28.1%) was somewhat higher than after multiple KK plus a single FECT (24.3%). In the current setting, triplicate KK plus a single FECT diagnosed helminth infections with high sensitivity. Hence, such a diagnostic approach might be utilised for generating high-quality baseline data, assessing anthelminthic drug efficacy and rigorous monitoring of community interventions.

  14. Comparison of three stool antigen assays with the 13C- urea breath test for the primary diagnosis of Helicobacter pylori infection and monitoring treatment outcome.

    LENUS (Irish Health Repository)

    Hooton, Carmel

    2012-02-03

    BACKGROUND: The urea breath test (UBT) is the gold-standard non-invasive test for the detection of Helicobacter pylori infection, however, the lack of availability of the UBT due to the high cost of the test, and in particular the need for expensive analytical instrumentation, limits the usefulness of this method. Stool antigen assays may offer an alternative non-invasive method for the diagnosis of infection. OBJECTIVE: To compare the accuracy of three stool antigen assays (HpSA, IDEIA HpStAR, and ImmunoCard STAT) against the UBT for the primary diagnosis of H. pylori infection and for monitoring treatment outcome. METHODS: A total of 102 patients attending two gastroenterology day-case clinics for the investigation of dyspepsia were included. Each patient provided breath and stool samples for analysis. Patients who tested positive for H. pylori by the validated UBT were prescribed triple therapy and invited to return for repeat breath and stool sample analysis 6 weeks post-treatment. RESULTS: Of the 102 patients tested, 48 were diagnosed with H. pylori infection by the UBT. The HpSA assay interpreted 38 of these as positive (79% sensitive). Of the 54 UBT-negative patients the HpSA assay interpreted all 54 as negative (100% specific). The IDEIA HpStAR assay correctly identified 44 patients as positive (92% sensitive) and 50 as negative (92.5% specific). The ImmunoCard STAT assay interpreted 38 patients as positive (79% sensitive) and 52 as negative (96.3% specific). CONCLUSION: The findings indicate that the IDEIA HpStAR stool antigen kit is the most accurate assay of the three assays evaluated, and possibly represents a viable alternative to the UBT for the primary diagnosis of H. pylori infection and for monitoring treatment outcome.

  15. Blood Group Determination using DNA extracted from Exfoliated Primary Teeth at Various Time Durations and Temperatures: A PCR Study

    Science.gov (United States)

    Bhat, Sham S; Salman, Afreen; Hegde, Sundeep

    2016-01-01

    Aim To determine polymerase chain reaction (PCR)-based blood group on tooth pulp obtained from teeth stored for 1 month, 6 months, and 1 year following extraction and to evaluate the stability of deoxyribonucleic acid (DNA) in primary tooth subjected to a temperature of 200°C ± 5°C for 15 minutes. Materials and methods Dental pulp tissue was collected from 40 exfoliated primary teeth stored for various time durations and temperature and preserved at 4°C till DNA extraction was carried out. Deoxyribonucleic acid was extracted using silica membrane-based spin-column procedure of QIAamp DNA minikit from BioRad. Deoxyribonucleic acid was subjected to PCR amplification and monoplex allele-specific PCR primers for ABO genotyping. Statistical analysis used The data were analyzed by comparison (based on percentage). Results In our study, overall, 85% samples showed a DNA yield. Cent percent results were obtained for samples studied at the end of 1 month followed by 90 and 80% for samples studied for 6 months and 1 year respectively. Heated samples showed 70% result. Conclusion Polymerase chain reaction was found to be an effective method for blood group determination for teeth stored at various time durations and temperatures. However, as the time interval increased, the number of positive results obtained decreased. How to cite this article Pai RK, Bhat SS, Salman A, Hegde S. Blood Group Determination using DNA extracted from Exfoliated Primary Teeth at Various Time Durations and Temperatures: A PCR Study. Int J Clin Pediatr Dent 2016;9(4):308-312. PMID:28127161

  16. Evaluation of diagnostic accuracy of two rapid stool antigen tests using an immunochromatographic assay to detect Helicobacter pylori.

    Science.gov (United States)

    da Silva-Etto, Joyce Matie Kinoshita; Mattar, Rejane; Villares-Lopes, Cibele Aparecida; Marques, Sergio Barbosa; Carrilho, Flair José

    2017-05-05

    The stool antigen assay for H. pylori infection diagnosis with monoclonal antibodies is a simple and recommended technique by the Maastricht V/Florence consensus report. Recently, Pylori K-Set K-1219 (Coris Bioconcept Sprl, Belgium) and HP-F23 (Symbiosys, Brazil) have been made commercially available in Brazil. Thus, the aim of this study was to evaluate the diagnostic accuracies of these two rapid stool antigen tests by immunochromatographic assays (index tests) for the clinical practice. A total of 98 patients who underwent upper gastrointestinal endoscopy and (13)C-urea breath test entered the study. H. pylori infection status was defined by the combination of the rapid urease test and the (13)C-urea breath test (reference standard). Two observers who were aware of H. pylori status performed the reading of index tests. Diagnostic accuracy (sensitivity, specificity, positive predictive value, negative predictive value with 95% confidence intervals, positive likelihood ratio, negative likelihood ratio and kappa index measure of agreement) were determined. The index tests where in perfect agreement with the H. pylori status with kappa values of 0.87 for Pylori K-Set K-1219 and 0.92 for HP-F23. The sensitivity of HP-F23 was 97.9% (IC95%: 87.5-100) and specificity was 93.8% (IC95%; 84-97.2).The positive likelihood ratio was 15.8, and the negative likelihood ratio was 0.02. The Pylori K-Set K-1219 had a sensitivity of 87.7% (IC95%: 74.5-94.9) and a specificity of 100% (IC95%: 91.6-100); the positive likelihood ratio was ∞, and the negative likelihood ratio was 0.1. The test line on the cassette device of HP-F23 was stronger than of the Pylori K-Set K-1219. The HP-F23 test performed better in clinical practice. Nonetheless, the (13)C-urea breath test is more reliable technique. Moreover, caution must be paid to the trace or clear pale test line readings that were observed in false positive and false negative results, leading to incorrect management of the patient

  17. Efficiency of Direct Microscopy of Stool Samples Using an Antigen-Specific Adhesin Test for Entamoeba Histolytica

    Science.gov (United States)

    İrvem, Arzu; Özdil, Kamil; Çalışkan, Zuhal; Yücel, Muhterem

    2016-01-01

    Background: E. histolytica is among the common causes of acute gastroenteritis. The pathogenic species E. histolytica and the nonpathogenic species E. dispar cannot be morphologically differentiated, although correct identification of these protozoans is important for treatment and public health. In many laboratories, the screening of leukocytes, erythrocytes, amoebic cysts, trophozoites and parasite eggs is performed using Native-Lugol’s iodine for pre-diagnosis. Aims: In this study, we aimed to investigate the frequency of E. histolytica in stool samples collected from 788 patients residing in the Anatolian region of İstanbul who presented with gastrointestinal complaints. We used the information obtained to evaluate the effectiveness of microscopic examinations when used in combination with the E. histolytica adhesin antigen test. Study Design: Retrospective cross-sectional study Methods: Preparations of stool samples stained with Native-Lugol’s iodine were evaluated using the E. histolytica adhesin test and examined using standard light microscopy at ×40 magnification. Pearson’s Chi-square and Fisher’s exact tests were used for statistical analysis. Logistic regression analysis was used for multivariate analysis. Results: Of 788 samples, 38 (4.8%) were positive for E. histolytica adhesin antigens. When evaluated together with the presences of erythrocytes, leukocytes, cysts, and trophozoites, respectively, using logistic regression analysis, leukocyte positivity was significantly higher. The odds ratio of leukocyte positivity increased adhesin test-positivity by 2,530-fold (95% CI=1.01–6.330). Adhesin test-positivity was significant (p=0.047). Conclusion: In line with these findings, the consistency between the presence of cysts and erythrocytes and adhesin test-positivity was found to be highly significant, but that of higher levels of leukocytes was found to be discordant. It was concluded that leukocytes and trophozoites were easily misjudged

  18. Comparison of DNA extraction methods for polymerase chain reaction amplification of guanaco (Lama guanicoe) fecal DNA samples.

    Science.gov (United States)

    Espinosa, M I; Bertin, A; Squeo, F A; Cortés, A; Gouin, N

    2015-01-23

    Feces-based population genetic studies have become increasingly popular. However, polymerase chain reaction (PCR) amplification rates from fecal material vary depending on the species, populations, loci, and extraction protocols. Here, we assessed the PCR amplification success of three microsatellite markers and a segment of the mitochondrial control region of DNA extracted from field-collected feces of guanaco (Lama guanicoe) using two protocols - Qiagen DNA Stool Kit and 2 cetyltrimethylammonium bromide/phenol:chloroform:isoamyl alcohol (2CTAB/PCI) method. Chelex resin treatment to remove inhibitors was also tested. Our results show that the mitochondrial locus was the most difficult to amplify. PCR success rates improved for all markers after Chelex treatment of extracted DNA, and 2CTAB/PCI method (95.83%) appeared to perform slightly better than stool kit (91.67%) for the nuclear markers. Amplification success was significantly influenced by the extraction method, Chelex treatment, and locus (P 0.89), but they decreased slightly after treatment for amplification of nuclear markers and markedly after treatment for amplification of the mitochondrial control region. Thus, we showed that Chelex treatment gives high PCR success, especially for nuclear markers, and adequate DNA extraction rates can be achieved from L. guanicoe feces even from non-fresh fecal material. Although not significant, 2CTAB/PCI method tended to provide higher successful amplification rates on a whole set of samples, suggesting that the method could be particularly useful when using small sample sizes.

  19. Heterogeneity of Campylobacter species isolated from serial stool specimens of Egyptian children using pulsed field gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Atef M. El-Gendy

    2013-03-01

    Full Text Available Background: The genus Campylobacter spp. is a common cause of human acute bacteria lenteritis and travellers’ diarrhoea worldwide.Objective: To determine whether multiple serial isolations of Campylobacter spp., when obtained from a single child, represented the same or a different organism.Methods: In a birth cohort study conducted in Egypt, numerous children showed serial isolations of Campylobacter spp. Of these, 13 children were selected from different households based on the successive isolation of six or more Campylobacter isolates from stool samples.Results: Eighty isolates were recovered and identified as either Campylobacter coli (n = 25 or Campylobacter jejuni (n = 55. Pulsed-field gel electrophoresis (PFGE revealed the presence of 38 unique C. jejuni and 24 C. coli profiles at a similarity level of ≥ 90%. Although no seriallyidentical isolates were detected in six children, others demonstrated at least one identical couple of isolates; all identified serially between one to six weeks. Two children demonstrated > 80% similar couples of isolates that appeared seven months apart. PFGE could be a useful tool for differentiating reinfection, relapse and convalescent excretion phases.Conclusion: Our data suggest that Campylobacter infection in children is a complex process; children are exposed to multiple species in endemic environments and strains of the same bacterium appear to be shed serially between one to six weeks after the first exposure. Isolates that persisted for longer periods were relatively less similar, as shown from the results of this study.

  20. Pancreatic exocrine insufficiency: Comparing fecal elastase 1 with 72-h stool for fecal fat estimation.

    Science.gov (United States)

    Chowdhury, Sudipta Dhar; Kurien, Reuben Thomas; Ramachandran, Anup; Joseph, Anjilivelil Joseph; Simon, Ebby George; Dutta, Amit Kumar; David, Deepu; Kumar C, Bharath; Samuel, Prassana; Balasubramaniam, K A

    2016-11-01

    Identification of pancreatic exocrine insufficiency (PEI) is important in the management of chronic pancreatitis. The 72-h stool for fecal fat estimation (FFE) has long been considered a gold standard indirect test for the diagnosis of PEI. However, the test is cumbersome for both patients and laboratory personnel alike. In this study, we aimed to assess fecal elastase 1 (FE1) as an alternate to FFE for the diagnosis of PEI. In all, 87 consecutive patients diagnosed with chronic pancreatitis were included in this study. FFE and FE1 estimation was done for all the patients. For FE1, two cutoffs (analysis. The sensitivity, specificity, and positive and negative predictive value and PABAK (prevalence and bias adjusted kappa) for FE1 <100 μg was 84.9, 47.6, 83.6, 50, and 0.52, respectively. For FE1 <200 μg, it was 90.9, 9.5, 75.95, 25, and 0.43, respectively. FE1 is a sensitive test; however, it does not have a good agreement with FFE. FE1 may be used as screening test for PEI in patients with chronic pancreatitis.

  1. Automatic Recognition of Human Parasite Cysts on Microscopic Stools Images using Principal Component Analysis and Probabilistic Neural Network

    Directory of Open Access Journals (Sweden)

    Beaudelaire Saha Tchinda

    2015-09-01

    Full Text Available Parasites live in a host and get its food from or at the expensive of that host. Cysts represent a form of resistance and spread of parasites. The manual diagnosis of microscopic stools images is time-consuming and depends on the human expert. In this paper, we propose an automatic recognition system that can be used to identify various intestinal parasite cysts from their microscopic digital images. We employ image pixel feature to train the probabilistic neural networks (PNN. Probabilistic neural networks are suitable for classification problems. The main novelty is the use of features vectors extracted directly from the image pixel. For this goal, microscopic images are previously segmented to separate the parasite image from the background. The extracted parasite is then resized to 12x12 image features vector. For dimensionality reduction, the principal component analysis basis projection has been used. 12x12 extracted features were orthogonalized into two principal components variables that consist the input vector of the PNN. The PNN is trained using 540 microscopic images of the parasite. The proposed approach was tested successfully on 540 samples of protozoan cysts obtained from 9 kinds of intestinal parasites.

  2. Glycerol Jelly (GJ) mount: a new and simple method for routine stool examination using a modified glycerol jelly reagent.

    Science.gov (United States)

    Abdel-Hamid, M Y

    2001-08-01

    Wet mount is the basic primary technique for stool examination in laboratories, allowing only the use of X10 and X40 objectives which do not sometimes reveal relevant details to make an exact identification of certain protozoa. In a modification of the Glycerol Jelly (GJ) reagent, which is used in permanent preparation of helminths, five concentrations were prepared and tested for fixing the cover glass instantly while maintaining the high translucency of the fecal films. GJ reagent (7 gm gelatine dissolved in 50 ml boiling water added to 10 ml glycerol) gave satisfactory results especially with iodine and alkaline Methylene blue mounts which stained the cytological structures of protozoa while the GJ reagent enabled the examiner to use X100 oil immersion objective immediately and consequently identify protozoa with certainty and make an accurate identification. Identification of Cryptosporidium parvum oocysts by GJ wet mount, inspite of its small size, was the most impressive. GJ fecal films were examined up to 8 weeks of preparation and they were valid and reliable. GJ mount is an easy, fast and cheap technique for examining the fecal direct smear with the oil-immersion lense.

  3. Toxin Profile, Biofilm Formation, and Molecular Characterization of Emetic Toxin-Producing Bacillus cereus Group Isolates from Human Stools.

    Science.gov (United States)

    Oh, Su Kyung; Chang, Hyun-Joo; Choi, Sung-Wook; Ok, Gyeongsik; Lee, Nari

    2015-11-01

    Emetic toxin-producing Bacillus cereus group species are an important problem, because the staple food for Korean is grains such as rice. In this study, we determined the prevalence (24 of 129 isolates) of emetic B. cereus in 36,745 stool samples from sporadic food-poisoning cases in Korea between 2007 and 2008. The toxin gene profile, toxin production, and biofilm-forming ability of the emetic B. cereus isolates were investigated. Repetitive element sequence polymorphism polymerase chain reaction fingerprints (rep-PCR) were also used to assess the intraspecific biodiversity of these isolates. Emetic B. cereus was present in 0.07% of the sporadic food-poisoning cases. The 24 emetic isolates identified all carried the nheABC and entFM genes and produced NHE enterotoxin. However, they did not have hemolysin BL toxin or related genes. A relationship between biofilm formation and toxin production was not observed in this study. The rep-PCR fingerprints of the B. cereus isolates were not influenced by the presence of toxin genes, or biofilm-forming ability. The rep-PCR assay discriminated emetic B. cereus isolates from nonemetic isolates, even if this assay did not perfectly discriminate these isolates. Further study on emetic isolates possessing a high degree of diversity may be necessary to evaluate the performance of the subtyping assay to discriminate emetic and nonemetic B. cereus isolates and could provide a more accurate indication of the risk from B. cereus strains.

  4. The stool microbiota of insulin resistant women with recent gestational diabetes, a high risk group for type 2 diabetes.

    Science.gov (United States)

    Fugmann, Marina; Breier, Michaela; Rottenkolber, Marietta; Banning, Friederike; Ferrari, Uta; Sacco, Vanessa; Grallert, Harald; Parhofer, Klaus G; Seissler, Jochen; Clavel, Thomas; Lechner, Andreas

    2015-08-17

    The gut microbiota has been linked to metabolic diseases. However, information on the microbiome of young adults at risk for type 2 diabetes (T2D) is lacking. The aim of this cross-sectional analysis was to investigate whether insulin resistant women with previous gestational diabetes (pGDM), a high risk group for T2D, differ in their stool microbiota from women after a normoglycemic pregnancy (controls). Bacterial communities were analyzed by high-throughput 16S rRNA gene sequencing using fecal samples from 42 pGDM and 35 control subjects 3-16 months after delivery. Clinical characterization included a 5-point OGTT, anthropometrics, clinical chemistry markers and a food frequency questionnaire. Women with a Prevotellaceae-dominated intestinal microbiome were overrepresented in the pGDM group (p microbiota are already present in young adults at risk for T2D and that further investigations of a potential pathophysiological role of gut bacteria in early T2D development are warranted.

  5. Pilot Dietary Intervention with Heat-Stabilized Rice Bran Modulates Stool Microbiota and Metabolites in Healthy Adults

    Directory of Open Access Journals (Sweden)

    Amy M. Sheflin

    2015-02-01

    Full Text Available Heat-stabilized rice bran (SRB has been shown to regulate blood lipids and glucose, modulate gut mucosal immunity and inhibit colorectal cancer in animal and human studies. However, SRB’s effects on gut microbial composition and metabolism and the resulting implications for health remain largely unknown. A pilot, randomized-controlled trial was developed to investigate the effects of eating 30 g/day SRB on the stool microbiome and metabolome. Seven healthy participants consumed a study meal and snack daily for 28 days. The microbiome and metabolome were characterized using 454 pyrosequencing and gas chromatography-mass spectrometry (GC-MS at baseline, two and four weeks post-intervention. Increases in eight operational taxonomic units (OTUs, including three from Bifidobacterium and Ruminococcus genera, were observed after two and four weeks of SRB consumption (p < 0.01. Branched chain fatty acids, secondary bile acids and eleven other putative microbial metabolites were significantly elevated in the SRB group after four weeks. The largest metabolite change was a rice bran component, indole-2-carboxylic acid, which showed a mean 12% increase with SRB consumption. These data support the feasibility of dietary SRB intervention in adults and support that SRB consumption can affect gut microbial metabolism. These findings warrant future investigations of larger cohorts evaluating SRB’s effects on intestinal health.

  6. DNA Nanotechnology

    Science.gov (United States)

    Taniguchi, Masateru; Kawai, Tomoji

    2002-11-01

    DNA is one candidate of promising molecules for molecular electronic devices, since it has the double helix structure with pi-electron bases for electron transport, the address at 0.4 nm intervals, and the self-assembly. Electrical conductivity and nanostructure of DNA and modified DNA molecules are investigated in order to research the application of DNA in nanoelectronic devices. It has been revealed that DNA is a wide-gap semiconductor in the absence of doping. The conductivity of DNA has been controlled by chemical doping, electric field doping, and photo-doping. It has found that Poly(dG)[middle dot]Poly(dC) has the best conductivity and can function as a conducting nanowire. The pattern of DNA network is controlled by changing the concentration of the DNA solution.

  7. DNA Methylation

    OpenAIRE

    Alokail, Majed S.; Alenad, Amal M.

    2015-01-01

    The DNA of E. coli contains 19,120 6-methyladenines and 12,045 5-methylcytosines in addition to the four regular bases and these are formed by the postreplicative action of three DNA methyltransferases. The majority of the methylated bases are formed by the Dam and Dcm methyltransferases encoded by the dam (DNA adenine methyltransferase) and dcm (DNA cytosine methyltransferase) genes. Although not essential, Dam methylation is important for strand discrimination during repair of replication e...

  8. Real-Time Polymerase Chain Reaction in Stool Detects Transmission of Strongyloides stercoralis from an Infected Donor to Solid Organ Transplant Recipients.

    Science.gov (United States)

    Gómez-Junyent, Joan; Paredes-Zapata, David; de las Parras, Esperanza Rodríguez; González-Costello, José; Ruiz-Arranz, Ángel; Cañizares, Rosario; Saugar, José María; Muñoz, José

    2016-04-01

    Solid organ transplant recipients can acquire Strongyloides stercoralis from an infected donor. The diagnosis of S. stercoralis in immunocompromised individuals may be challenging due to a lower sensitivity of available parasitological and serological methods, compared with immunocompetent individuals. Recently, a real-time polymerase chain reaction (RT-PCR) in stool has been developed for S. stercoralis diagnosis. We report two cases of S. stercoralis infection transmitted by a donor to two solid organ transplant recipients, who were diagnosed with RT-PCR in stool. This test could play an important role inS. stercoralis diagnosis in immunosuppressed patients, facilitating rapid treatment initiation and reducing the risk of severe strongyloidiasis. Adherence to current recommendations of screening among donors and recipients from endemic areas is also urgently needed.

  9. Fecal Collection and Stabilization Methods for Improved Fecal DNA Test for Colorectal Cancer in a Screening Setting

    Directory of Open Access Journals (Sweden)

    Francesca Maria Carozzi

    2013-01-01

    Full Text Available Early detection of CRC and adenomas reduces CRC-related mortality. The optimal screening test for CRC is still a subject of debate, and molecular stool sample analysis could provide a valid alternative to conventional methods in terms of compliance and practicability. Seven fecal DNA storage systems were evaluated in two successive phases. In the first phase of the study was selected the preservative buffer able to ensure the best human DNA recovery. In the second phase was evaluated human DNA stability, amplificability and integrity in DNA extracted from selected buffer. Results showed that the best performance was obtained in samples stored in 100 mM EDTA buffer and Genefec buffer. Likewise buffer addition yielded a significant increase in DNA stability and integrity without PCR inhibition, compared to the matched aliquots with no buffer added. Our study shows that samples collected in stabilization solution stabilize DNA so that intact nucleic acids, are more effectively detectable in the molecular assay. DNA buffer preservation and storage conditions could be useful to guarantee the most consistent yield in human DNA. Stabilization buffer addition to stool samples prior to transport presents an easily implemented solution that appears to be highly effective. Overall DNA extracted from faeces preserved in preservative buffer can feasibility been used for molecular analysis leading to an increase of assay sensitivity.

  10. Molecular prevalence of Entamoeba histolytica/dispar infection among patients attending four health centres in north-west Ethiopia.

    Science.gov (United States)

    Yimer, Mulat; Zenebe, Yohannes; Mulu, Wondemagegn; Abera, Bayeh; Saugar, José M

    2017-01-01

    The prevalence of amoebiasis is often overestimated owing to its epidemiological overlap with the non-pathogenic Entamoeba dispar To provide evidence for this conjecture, a cross-sectional study was conducted from November 2013 to January 2015. A range of 180-200 µg of semi-solid and formed stools and 200 µL of diarrhoeic stool samples were used for DNA extraction from microscopically E. histolytica/dispar positive samples using the QIAamp® DNA Stool Mini Kit according to manufacturers' instructions. Nested PCR targeting 18S ribosomal RNA gene was used. In 422 microscopically positive E. histolytica/dispar stools, molecular prevalence revealed that E. histolytica infestation was present in only 1.7% (95% confidence interval [CI], 0.47-2.93) and E. dispar was found in 42.2% (95% CI, 37.49-46.91), while 56.2% (95% CI, 51.47-60.93) had neither E. histolytica nor E. dispar (P histolytica is rarer in our study areas than was previously believed. Hence, accurate differentiation of E. histolytica and E. dispar is crucial.

  11. Comparison of methods for detection of Blastocystis infection in routinely submitted stool samples, and also in IBS/IBD Patients in Ankara, Turkey.

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    Funda Dogruman-Al

    Full Text Available BACKGROUND: This study compared diagnostic methods for identifying Blastocystis in stool samples, and evaluated the frequency of detection of Blastocystis in patients with irritable bowel syndrome (IBS and inflammatory bowel disease (IBD. RESULTS AND DISCUSSION: From a set of 105 stool specimens submitted for routine parasitological analysis, 30 were identified as positive for Blastocystis by the culture method. From that group of 30 positives, Lugol's stain, trichrome staining, and an immunofluorescence assay identified 11, 15, and 26 samples as positive respectively. Using culture as a standard, the sensitivity of Lugol's stain was 36.7%, trichrome staining was 50%, and the IFA stain was 86.7%. The specificity of Lugol's stain was 91%, trichrome staining was 100%, and the IFA stain was 97.3%. In the group of 27 IBS and IBD patients, using all methods combined, we detected Blastocystis in 67% (18/27 of the patients. Blastocystis was detected in 33% (2/6 of IBD patients and 76% (16/21 of IBS patients. For comparison, trichrome staining alone, the method most frequently used in many countries, would have only identified Blastocystis infection in 29% (6/21 of the IBS patients. No parasitic co-infections were identified in the IBS/IBD patients. Most Blastocystis-positive IBS/IBD patients were over 36 with an average length of illness of 4.9 years. CONCLUSIONS: Most IBS patients in this study were infected with Blastocystis. IFA staining may be a useful alternative to stool culture, especially if stool specimens have been chemically preserved.

  12. Isolation and Characterization of Escherichia coli Sequence Type 131 and Other Antimicrobial-Resistant Gram-Negative Bacilli from Clinical Stool Samples from Veterans.

    Science.gov (United States)

    Mohamed, Muhanad; Clabots, Connie; Porter, Stephen B; Thuras, Paul; Johnson, James R

    2016-08-01

    Emerging multidrug-resistant (MDR) Gram-negative bacilli (GNB), including Escherichia coli sequence type 131 (ST131) and its resistance-associated H30 subclone, constitute an ever-growing public health threat. Their reservoirs and transmission pathways are incompletely defined. To assess diarrheal stools as a potential reservoir for ST131-H30 and other MDR GNB, we cultured 100 clinical stool samples from a Veterans Affairs Medical Center clinical laboratory (October to December 2011) for fluoroquinolone- and extended-spectrum cephalosporin (ESC)-resistant E. coli and other GNB, plus total E. coli We then characterized selected resistant and susceptible E. coli isolates by clonal group, phylogenetic group, virulence genotype, and pulsotype and screened all isolates for antimicrobial resistance. Overall, 79 of 100 stool samples yielded GNB (52 E. coli; 48 other GNB). Fifteen samples yielded fluoroquinolone-resistant E. coli (10 were ST131, of which 9 were H30), 6 yielded ESC-resistant E. coli (2 were ST131, both non-H30), and 31 yielded susceptible E. coli (1 was ST131, non-H30), for 13 total ST131-positive samples. Fourteen non-E. coli GNB were ESC resistant, and three were fluoroquinolone resistant. Regardless of species, almost half (46%) of the fluoroquinolone-resistant and/or ESC-resistant non-E. coli GNB were resistant to at least three drug classes. Fecal ST131 isolates closely resembled reference clinical ST131 isolates according to virulence genotypes and pulsed-field gel electrophoresis (PFGE) profiles. Thus, a substantial minority (30%) of veterans with diarrhea who undergo stool testing excrete antibiotic-resistant GNB, including E. coli ST131. Consequently, diarrhea may pose transmission risks for more than just diarrheal pathogens and may help disseminate clinically relevant ST131 strains and other MDR GNB within hospitals and the community. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  13. Clinical and Analytical Evaluation of a Single-Vial Stool Collection Device with Formalin-Free Fixative for Improved Processing and Comprehensive Detection of Gastrointestinal Parasites.

    Science.gov (United States)

    Couturier, Brianne A; Jensen, Ryan; Arias, Nora; Heffron, Michael; Gubler, Elyse; Case, Kristin; Gowans, Jason; Couturier, Marc Roger

    2015-08-01

    Microscopic examination of feces is a standard laboratory method for diagnosing gastrointestinal parasite infections. In North America, the ovum and parasite (O&P) examination is typically performed using stool that is chemically fixed in polyvinyl alcohol (PVA) and formalin, after which the stool is concentrated by filtration to enhance sensitivity. Mini Parasep solvent-free (SF) tubes allow collection and concentration within a single collection vial. The goal of the study was to determine whether consolidated processing and concentration with the Parasep tubes using an alcohol-based fixative (Alcorfix) provide O&P examinations equivalent to or better than those done by processing of PVA-formalin-fixed stool using a SpinCon concentration device. Parasep tubes revealed filtration performance equivalent to that of the SpinCon concentration device using PVA-formalin-fixed stool containing protozoa. Specimens cocollected in Parasep tubes containing PVA-formalin and Alcorfix revealed comparable morphology and staining for various protozoa. Alcorfix effectively fixed live Cryptosporidium and microsporidia such that morphology and staining were conserved for modified acid-fast and modified trichrome stains. A work flow analysis revealed significant time savings for batches of 10 or 30 O&P specimens in tubes with Alcorfix compared to the amount of time that it took to analyze the same number of specimens in tubes with PVA-formalin. The direct hands-on time savings with Mini Parasep tubes were 17 min and 41 s and 32 min and 1 s for batches of 10 and 30 specimens, respectively. Parasep tubes containing Alcorfix provide significant work flow advantages to laboratories that process medium to high volumes of O&P specimens by streamlining processing and converting to a single tube. These improvements in work flow, reduction of the amount of formalin used in the laboratory, and equivalent microscopy results are attractive advancements in O&P testing for North American

  14. Detection of enteropathogens associated with travelers’ diarrhea using a multiplex Luminex-based assay performed on stool samples smeared on Whatman FTA Elute cards

    Science.gov (United States)

    Lalani, Tahaniyat; Tisdale, Michele D; Maguire, Jason D; Wongsrichanalai, Chansuda; Riddle, Mark S; Tribble, David R

    2015-01-01

    We evaluated the limits of detection (LoD) for an 11-plex PCR-Luminex assay performed on Whatman FTA Elute cards smeared with stool containing pathogens associated with travelers’ diarrhea. LoDs ranged between 102-105 CFU, PFU or cysts/g for most pathogens except Cryptosporidium. Campylobacter and norovirus LoD increased with prolonged storage of cards. PMID:26072151

  15. Comparison of 2 chromogenic media for the detection of extended-spectrum β-lactamase producing Enterobacteriaceae stool carriage in nursing home residents

    Science.gov (United States)

    Blane, Beth; Brodrick, Hayley J.; Gouliouris, Theodore; Ambridge, Kirsty E.; Kidney, Angela D.; Ludden, Catherine M.; Limmathurotsakul, Direk; Török, M. Estée; Peacock, Sharon J.

    2016-01-01

    ChromID ESBL agar and Brilliance ESBL agar were compared for the isolation of extended-spectrum β-lactamase (ESBL)–producing Enterobacteriaceae from 298 stools. These had comparable sensitivity and selectivity for the 116 positive samples. Pre-enrichment with cefpodoxime and extending incubation to 48 hours after direct plating both significantly increased sensitivity but reduced selectivity of both agars. PMID:26712266

  16. Evaluation of the First Stool Pass Time Induced by Marketed Sorbitol in Benzodiazepine Intoxicated Patients: A Randomized Clinical Trial

    Directory of Open Access Journals (Sweden)

    Farzad Gheshlaghi

    2012-01-01

    Full Text Available Background: Poisoning, a common worldwide problem, seeks its own treatments toimprove, especially by the forthcoming evidence based medicine (EBM.Charcoal/sorbitol slurry (CSS administration is one of these methods with greatdebates around it which needs to be investigated more.Methods: In this clinical trial, 105 cases of benzodiazepine toxicity with at least 3symptoms and no contraindication for sorbitol prescription were divided into 3 groups.Patient grouping was based on the sorbitol manufacturing factory. Sorbitol wasprescribed for the patients, with one kind of sorbitol for the patients of each group. Themeasured variable was the time passed up to the presence of charcoal-mixed stool andthe gathered data were analyzed by ANOVA test using SPSS software.Results: The average age was 25.8±8.4 in females and 24±8.5 years for males(P=0.641. The average follow up time was 8.3±3.1 hours for females and 8.1±2.7hours for males (P=0.30. The average pass time from drug ingestion, was 46±23.15min in females and 40±23.15 min in males (P=0.132. Interestingly, no sorbitol was ableto increase in transit time in the study population despite a follow-up interval about 3times more than expected (based on the reference.Conclusion: No increase in transit time was seen by sorbitol prescription in our casesdespite the appropriate follow-up interval. This ineffectiveness may be somehow due tothe absence of standard sorbitol amounts in those products, but we assume that it ismainly due to the population-based bowel habits (i.e. constipated ones with essentialprolonged transit time.

  17. Synthesis, crystal structure and cytotoxic activity of ruthenium(II) piano-stool complex with N,N-chelating ligand

    Science.gov (United States)

    Rogala, Patrycja; Jabłońska-Wawrzycka, Agnieszka; Kazimierczuk, Katarzyna; Borek, Agnieszka; Błażejczyk, Agnieszka; Wietrzyk, Joanna; Barszcz, Barbara

    2016-12-01

    A mononuclear compound of the general formula [(η6-p-cymene)RuIICl(2,2‧-PyBIm)]PF6 has been synthesized from a bidentate N,N-donor ligand, viz. 2,-(2‧-pyridyl)benzimidazole (2,2‧-PyBIm) and the corresponding chloro-complex [(η6-p-cymene)Ru(μ-Cl)Cl]2 (precursor). The isolated coordination compound was characterized by IR, UV-vis and 1H, 13C NMR spectroscopies. The single crystal X-ray analysis of the complex reveals that the asymmetric part of the unit cell consists of two symmetrically independent, [(η6-p-cymene)RuCl(2,2‧-PyBIm)]+ cationic complexes. Each cation exhibits a pseudo-octahedral three-legged piano-stool geometry, in which three "legs" are occupied by one chloride ion and two nitrogen donor atoms of the chelating ligand 2,2‧-PyBIm. The Hirshfeld surface analysis of obtained complex was determined, too. The ionic nature of the compound is identified by a strong band at around 830 cm-1 due to the νP-F stretching mode of the PF6- counter ion. The electronic spectrum of this monomeric complex displays high intensity bands in the ultraviolet region assignable to π→π*/n→π* transitions, as well as a band attributable to the metal-to-ligand charge transfer (MLCT) dπ(Ru)→π*(L) transition. Additionally, the complex has been screened for its cytotoxicity against three human cancer lines: non-small cell lung carcinoma (A549), colon adenocarcinoma (HT29) and breast adenocarcinoma (MCF-7) as well as normal mice fibroblast cells (BALB/3T3). The complex demonstrated a moderate antiproliferative activity against the cell lines tested.

  18. Rapid isolation of gluten-digesting bacteria from human stool and saliva by using gliadin-containing plates

    Science.gov (United States)

    Sarantopoulos, Christos; Ongchangco, Deryn; Sry, Jeremy; Cesario, Thomas

    2014-01-01

    The number of individuals with gluten intolerance has increased dramatically over the last years. To date, the only therapy for gluten intolerance is the complete avoidance of dietary gluten. To sustain a strictly gluten-free diet, however, is very challenging. Therefore, there is need for a non-dietary therapy. Any such treatment must appreciate that the immunogenic part of gluten are gliadin peptides which are poorly degraded by the enzymes of the gastrointestinal tract. Probiotic therapy and oral enzyme therapy containing gluten-degrading bacteria (GDB) and their gliadin-digesting enzymes are possible new approaches for the treatment of gluten intolerance, however effectively isolating GDB for these treatments is problematic. The goal of this study was to develop an easy technique to isolate GDB rapidly and efficiently with the hope it might lead to newer ways of developing either probiotics or traditional medicines to treat gluten intolerance. Several researchers have already isolated successfully GDB by using gluten minimal or limited agar plates. Although these plates can be used to isolate bacteria which can tolerate gluten, further assays are needed to investigate if the same bacteria can also digest gluten. The agar plates we developed can detect bacteria which cannot only tolerate gluten but are able to digest it as well. Therefore, we were able to combine two steps into one step. Using such technologies, we were able to isolate five GDB from saliva and stool, and identified three bacterial reference strains with gluten-degrading activity. The technique we developed to isolate bacteria with gluten-degrading activity is fast, effective, and easy to use. The GDB isolated by our technology could have potential as part of a probiotic or enzymatic therapy for people with gluten intolerance. PMID:25519429

  19. Rapid isolation of gluten-digesting bacteria from human stool and saliva by using gliadin-containing plates.

    Science.gov (United States)

    Berger, Martina; Sarantopoulos, Christos; Ongchangco, Deryn; Sry, Jeremy; Cesario, Thomas

    2015-07-01

    The number of individuals with gluten intolerance has increased dramatically over the last years. To date, the only therapy for gluten intolerance is the complete avoidance of dietary gluten. To sustain a strictly gluten-free diet, however, is very challenging. Therefore, there is need for a non-dietary therapy. Any such treatment must appreciate that the immunogenic part of gluten are gliadin peptides which are poorly degraded by the enzymes of the gastrointestinal tract. Probiotic therapy and oral enzyme therapy containing gluten-degrading bacteria (GDB) and their gliadin-digesting enzymes are possible new approaches for the treatment of gluten intolerance, however effectively isolating GDB for these treatments is problematic. The goal of this study was to develop an easy technique to isolate GDB rapidly and efficiently with the hope it might lead to newer ways of developing either probiotics or traditional medicines to treat gluten intolerance. Several researchers have already isolated successfully GDB by using gluten minimal or limited agar plates. Although these plates can be used to isolate bacteria which can tolerate gluten, further assays are needed to investigate if the same bacteria can also digest gluten. The agar plates we developed can detect bacteria which cannot only tolerate gluten but are able to digest it as well. Therefore, we were able to combine two steps into one step. Using such technologies, we were able to isolate five GDB from saliva and stool, and identified three bacterial reference strains with gluten-degrading activity. The technique we developed to isolate bacteria with gluten-degrading activity is fast, effective, and easy to use. The GDB isolated by our technology could have potential as part of a probiotic or enzymatic therapy for people with gluten intolerance.

  20. Dna Sequencing

    Science.gov (United States)

    Tabor, Stanley; Richardson, Charles C.

    1995-04-25

    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  1. Stool guaiac test

    Science.gov (United States)

    ... of the esophagus ( esophagitis ) Inflammation of the stomach ( gastritis ) from GI infections Hemorrhoids Crohn disease or ulcerative ... varices Colon cancer Colorectal polyps Crohn disease Esophagitis Gastritis Hemorrhoids Peptic ulcer Tumor Review Date 1/28/ ...

  2. [Evaluation of Two Methods (Nativ-Lugol Preparation and Enzyme-Linked Immunosorbent Assay) for Detection of Entamoeba histolytica in Stool Samples].

    Science.gov (United States)

    Alver, Oktay; Topaç, Tuncay; Töre, Okan

    2015-09-01

    This study aims to compare the performance of Native-Lugol examination and EIA Antigen Detection Test using stool samples obtained from patients diagnosed as clinical gastroenteritis and submitted to the Parasitology Laboratory in Uludağ University between January 2010 and February 2011. The stool samples taken from 116 patients and sent to the laboratory of parasitology from various clinics including outpatient services have been investigated using Native-Lugol examination and EIA Antigen Detection Kit (Wampole® E. histolytica II Techlab®, Inc., Blacksburg, Virginia) methods on all the samples. In one of 116 stool samples (%0,86), E. histolytica/E. dispar cysts and/or trophozoites were detected by using direct microscobic (nativ-lugol) method. E. histolytica specific antigen was detected in 34 (29.3%) out of the sample set, and the patients were given adequate treatment. The highest rate of E. histolytica specific antigen positivity were observed in 11-19 age group. On account of the fact that the sensitivity of direct microscopy is quite low, it is concluded that, from the viewpoint of preventing the amebiasis suspected patients from false diagnosis and hence from receiving inadequate treatment, the use of the ELISA method is more appropriate and advantageous, as it is cost effective and does not require highly qualified staff.

  3. Teaching retirement financial literacy in an undergraduate gerontology classroom: broadening the concept of the tripod or three-legged stool of retirement income utilizing active learning.

    Science.gov (United States)

    Baker, Hallie E; Brown, Pamela Pitman

    2015-01-01

    The three-legged stool concept is widely used in gerontological and geriatric education as an explanation on how one should fiscally approach his or her retirement. Financial managers, planners, retirees, business owners, even the Social Security Administration uses this metaphor of fiscal soundness in retirement planning. Gerontologists are moving away from the "tripod of retirement income" and "three-legged stool" term, as more often market work is needed for financial security. This activity focuses on the tripod or three-legged stool concepts of retirement planning using active learning, allowing the students to work collaboratively in a group, reflect upon the activity, and most importantly have fun. The game also allows for an expansion of the tripod concepts into the four pillars of economic security, broaching the use of personal assets and the possible need for longer employment. Game scenarios also emphasize macro- and microlevel forces, such as race, gender, health status, education, or marital status, which can influence timing of retirement or the level of retirement income available. The authors include instructions on how to set up the learning experience including worksheets, as well as reflection questions posed throughout the process.

  4. Detection and differentiation of Cryptosporidium by real-time polymerase chain reaction in stool samples from patients in Rio de Janeiro, Brazil

    Directory of Open Access Journals (Sweden)

    Roberta Flávia Ribeiro Rolando

    2012-06-01

    Full Text Available This study reports the first genetic characterisation of Cryptosporidium isolates in Brazil using real-time polymerase chain reaction (RT-PCR. A total of 1,197 faecal specimens from children and 10 specimens from human immunodeficiency virus-infected patients were collected between 1999-2010 and screened using microscopy. Forty-eight Cryptosporidium oocyst-positive isolates were identified and analysed using a generic TaqMan assay targeting the 18S rRNA to detect Cryptosporidium species and two other TaqMan assays to identify Cryptosporidium hominis and Cryptosporidium parvum. The 18S rRNA assay detected Cryptosporidium species in all 48 of the stool specimens. The C. parvum TaqMan assay correctly identified five/48 stool samples, while 37/48 stool specimens were correctly amplified in the C. hominis TaqMan assay. The results obtained in this study support previous findings showing that C. hominis infections are more prevalent than C. parvum infections in Brazil and they demonstrate that the TaqMan RT-PCR procedure is a simple, fast and valuable tool for the detection and differentiation of Cryptosporidium species.

  5. 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.

  6. 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.

  7. 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; 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 (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. PMID:24693242

  8. A filter paper-based microdevice for low-cost, rapid, and automated DNA extraction and amplification from diverse sample types.

    Science.gov (United States)

    Gan, Wupeng; Zhuang, Bin; Zhang, Pengfei; Han, Junping; Li, Cai-Xia; Liu, Peng

    2014-10-07

    A plastic microfluidic device that integrates a filter disc as a DNA capture phase was successfully developed for low-cost, rapid and automated DNA extraction and PCR amplification from various raw samples. The microdevice was constructed by sandwiching a piece of Fusion 5 filter, as well as a PDMS (polydimethylsiloxane) membrane, between two PMMA (poly(methyl methacrylate)) layers. An automated DNA extraction from 1 μL of human whole blood can be finished on the chip in 7 minutes by sequentially aspirating NaOH, HCl, and water through the filter. The filter disc containing extracted DNA was then taken out directly for PCR. On-chip DNA purification from 0.25-1 μL of human whole blood yielded 8.1-21.8 ng of DNA, higher than those obtained using QIAamp® DNA Micro kits. To realize DNA extraction from raw samples, an additional sample loading chamber containing a filter net with an 80 μm mesh size was designed in front of the extraction chamber to accommodate sample materials. Real-world samples, including whole blood, dried blood stains on Whatman® 903 paper, dried blood stains on FTA™ cards, buccal swabs, saliva, and cigarette butts, can all be processed in the system in 8 minutes. In addition, multiplex amplification of 15 STR (short tandem repeat) loci and Sanger-based DNA sequencing of the 520 bp GJB2 gene were accomplished from the filters that contained extracted DNA from blood. To further prove the feasibility of integrating this extraction method with downstream analyses, "in situ" PCR amplifications were successfully performed in the DNA extraction chamber following DNA purification from blood and blood stains without DNA elution. Using a modified protocol to bond the PDMS and PMMA, our plastic PDMS devices withstood the PCR process without any leakage. This study represents a significant step towards the practical application of on-chip DNA extraction methods, as well as the development of fully integrated genetic analytical systems.

  9. The Bristol Stool Form Scale: its translation to Portuguese, cultural adaptation and validation Traducción, adaptación cultural y validación de la "Bristol Stool Form Scale" Tradução, adaptação cultural e validação da Bristol Stool Form Scale para a população brasileira

    Directory of Open Access Journals (Sweden)

    Anna Paula Martinez

    2012-06-01

    Full Text Available The Bristol Stool Form Scale is used for describing feces. The objective of this research was its translation, cultural adaptation and validation for Brazil. The methodology was translation, back-translation and discussion. Validation involved 85 nurses, 80 doctors, and 80 patients, who correlated images of seven types of feces with the descriptions. Results: there was a difference in sex distribution, with males predominating among the doctors and females among nurses and patients. In relation to concordance between definitions and pictures, the highest percentage was in type 5 in all three groups and the lowest was in types 6 and 7 for the doctors, in type 3 for the nurses, and type 6 for the patients. The general Kappa index was 0.826. Conclusion: the scale demonstrated high reliability for all the groups studied.La "Bristol Stool Form Scale" es usada para describir las heces. Objetivo: traducción, adaptación cultural y la validación para ser utilizada en Brasil. Metodología: Fue realizada la traducción, la traducción inversa y la discusión final. Para validar, se incluyeron 85 enfermeros y 80 médicos y pacientes que correlacionaron diseños de siete tipos de heces con descripciones. Resultados - Hubo diferencia en cuanto a la distribución del sexo con predominio masculino entre los médicos y femenino para los enfermeros y pacientes. Con respecto a la concordancia entre los conceptos y las imágenes, la mayor concordancia fue del tipo 5 en cuanto que el de menor correspondencia para los médicos fueron los tipos 6 y 7, para los enfermeros el 3 y el 6 para los pacientes. El índice de Kappa general fue de 0,826. Conclusión: Los valores obtenidos demuestran la alta confiabilidad de este cuestionario con respecto a los grupos estudiados.A Escala de Bristol para Consistência de Fezes é usada na descrição de fezes. O objetivo deste estudo foi realizar a tradução, adaptação cultural e validação para o Brasil, dessa escala. Como

  10. HPV16 detection by qPCR method in relation to quantity and quality of DNA extracted from archival formalin fixed and paraffin embedded head and neck cancer tissues by three commercially available kits.

    Science.gov (United States)

    Biesaga, Beata; Janecka, Anna; Mucha-Małecka, Anna; Adamczyk, Agnieszka; Szostek, Sława; Słonina, Dorota; Halaszka, Krzysztof; Przewoźnik, Marcin

    2016-10-01

    The aim of the present study was to compare HPV16 detection by quantitative polymerase chain reaction (qPCR) in relation to the quantity and quality of DNA isolated from 21 formalin fixed and paraffin embedded (FFPE) head and neck cancer tissues by three commercially available kits: EX-WAX™ DNA Extraction Kit (M) (Merck Millipore, Darmstadt, Germany), QIAamp(®) DNA FFPE Tissue (Q) (Qiagen, Hilden, Germany) and ReliaPrep™ FFPE gDNA Miniprep System (P) (Promega, Madison, USA). Quantity of extracted DNA was assessed spectrophometrically and fluorometrically. Its quality was analyzed using A260/280 and A260/230 ratios and the β-actin fragment amplifiability in qPCR. HPV16 presence was detected by qPCR, using specific primers and TaqMan probe. HPV infection was found in 8 DNA samples extracted with M kit (38.1%) and in 7 (33.3%) isolated with Q and P kits. Three samples from M and Q kits were characterized by HPV16 positivity and lack of β-actin amplifiability. They had significantly lower A260/280 ratio (M: 1.6±0.0, p=0.044 and Q: 1.7±0.0, p=0.016) compared to samples with both fragments amplification (M: 1.7±0.0 and Q: 1.9±0.0). Therefore, for HPV detection by qPCR in FFPE tissues we recommend ReliaPrep™ FFPE gDNA Miniprep System.

  11. Scientific Opinion on the substantiation of a health claim related to beta-palmitate and contribution to softening of stools pursuant to Article 14 of Regulation (EC No 1924/2006

    Directory of Open Access Journals (Sweden)

    EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA

    2014-02-01

    Full Text Available Following an application from Specialised Nutrition Europe (formerly IDACE, submitted for authorisation of a health claim pursuant to Article 14 of Regulation (EC No 1924/2006 via the Competent Authority of France, the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA was asked to deliver an opinion on the scientific substantiation of a health claim related to beta-palmitate and contribution to softening of stools. The food constituent, beta-palmitate, that is the subject of the health claim, is sufficiently characterised. Contribution to softening of stools is a beneficial physiological effect for infants. In weighing the evidence the Panel took into account that, out of two human intervention studies with important methodological limitations, one suggested a stool-softening effect of beta-palmitate whereas the second did not, that one animal study did not support a stool-softening effect of beta-palmitate, and that the evidence provided for a mechanism by which beta-palmitate could contribute to the softening of stools is weak. The Panel concludes that a cause and effect relationship has not been established between the consumption of beta-palmitate and softening of stools.

  12. DNA glue

    DEFF Research Database (Denmark)

    Filichev, Vyacheslav V; Astakhova, Irina V.; Malakhov, Andrei D.

    2008-01-01

    Significant alterations in thermal stability of parallel DNA triplexes and antiparallel duplexes were observed upon changing the attachment of ethynylpyrenes from para to ortho in the structure of phenylmethylglycerol inserted as a bulge into DNA (TINA). Insertions of two ortho-TINAs as a pseudo...

  13. Variation in the limit-of-detection of the ProSpecT Campylobacter microplate enzyme immunoassay in stools spiked with emerging Campylobacter species.

    Science.gov (United States)

    Bojanić, Krunoslav; Midwinter, Anne Camilla; Marshall, Jonathan Craig; Rogers, Lynn Elizabeth; Biggs, Patrick Jon; Acke, Els

    2016-08-01

    Campylobacter enteritis in humans is primarily associated with C. jejuni/coli infection. The impact of other Campylobacter spp. is likely to be underestimated due to the bias of culture methods towards Campylobacter jejuni/coli diagnosis. Stool antigen tests are becoming increasingly popular and appear generally less species-specific. A review of independent studies of the ProSpecT® Campylobacter Microplate enzyme immunoassay (EIA) developed for C. jejuni/coli showed comparable diagnostic results to culture methods but the examination of non-jejuni/coli Campylobacter spp. was limited and the limit-of-detection (LOD), where reported, varied between studies. This study investigated LOD of EIA for Campylobacter upsaliensis, Campylobacter hyointestinalis and Campylobacter helveticus spiked in human stools. Multiple stools and Campylobacter isolates were used in three different concentrations (10(4)-10(9)CFU/ml) to reflect sample heterogeneity. All Campylobacter species evaluated were detectable by EIA. Multivariate analysis showed LOD varied between Campylobacter spp. and faecal consistency as fixed effects and individual faecal samples as random effects. EIA showed excellent performance in replicate testing for both within and between batches of reagents, in agreement between visual and spectrophotometric reading of results, and returned no discordance between the bacterial concentrations within independent dilution test runs (positive results with lower but not higher concentrations). This study shows how limitations in experimental procedures lead to an overestimation of consistency and uniformity of LOD for EIA that may not hold under routine use in diagnostic laboratories. Benefits and limitations for clinical practice and the influence on estimates of performance characteristics from detection of multiple Campylobacter spp. by EIA are discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Evaluation of CHROMagar STEC and STEC O104 chromogenic agar media for detection of Shiga Toxin-producing Escherichia coli in stool specimens.

    Science.gov (United States)

    Gouali, Malika; Ruckly, Corinne; Carle, Isabelle; Lejay-Collin, Monique; Weill, François-Xavier

    2013-03-01

    The performance of CHROMagar STEC and CHROMagar STEC O104 (CHROMagar Microbiology, Paris, France) media for the detection of Shiga toxin-producing Escherichia coli (STEC) was assessed with 329 stool specimens collected over 14 months from patients with suspected STEC infections (June 2011 to August 2012). The CHROMagar STEC medium, after an enrichment broth step, allowed the recovery of the STEC strain from 32 of the 39 (82.1%) Shiga toxin-positive stool specimens, whereas the standard procedure involving Drigalski agar allowed the recovery of only three additional STEC strains. The isolates that grew on CHROMagar STEC medium belonged to 15 serotypes, including the prevalent non-sorbitol-fermenting (NSF) O157:H7, O26:H11, and O104:H4 serotypes. The sensitivity, specificity, and positive and negative predictive values for the CHROMagar STEC medium were between 89.1% and 91.4%, 83.7% and 86.7%, 40% and 51.3%, and 98% and 98.8%, respectively, depending on whether or not stx-negative eae-positive E. coli was considered atypical enteropathogenic E. coli (EPEC) or STEC that had lost Shiga toxin genes during infection. In conclusion, the good performance of CHROMagar STEC agar medium, in particular, the high negative predictive value, and its capacity to identify NSF O157:H7 as well as common non-O157 STEC may be useful for clinical bacteriology, public health, and reference laboratories; it could be used in addition to a method targeting Shiga toxins (detection of stx genes by PCR, immunodetection of Shiga toxins in stool specimens, or Vero cell cytotoxicity assay) as an alternative to O157 culture medium. This combined approach should allow rapid visualization of both putative O157 and non-O157 STEC colonies for subsequent characterization, essential for real-time surveillance of STEC infections and investigations of outbreaks.

  15. Presence of Tablet Remnants of Nevirapine Extended-Release in Stools and Its Impact on Virological Outcome in HIV-1-Infected Patients: A Prospective Cohort Study.

    Directory of Open Access Journals (Sweden)

    Yi-Chieh Lee

    Full Text Available Nevirapine extended-release (NVP-XR taken once daily remains an effective antiretroviral agent for patients infected with HIV-1 strains that do not harbor resistance mutations. Presence of tablet remnants of NVP XR in stools was reported in 1.19% and 3.05% of subjects in two clinical trials. However, the prevalence may have been underestimated because the information was retrospectively collected in the studies.Between April and December 2014, we prospectively inquired about the frequency of noticing tablet remnants of NVP XR in stools in HIV-1-infected patients who switched to antiretroviral regimens containing NVP XR plus 2 nucleos(tide reverse-transcriptase inhibitors. Patients were invited to participate in therapeutic drug monitoring of plasma concentrations of NVP 12 or 24 hours after taking the previous dose (C12 and C24, respectively of NVP XR using high-performance liquid chromatography. The information on clinical characteristics, including plasma HIV RNA load and CD4 lymphocyte count, at baseline and during follow-up was recorded.During the 9-month study period, 272 patients switched to NVP XR-based regimens and 60 (22.1% noticed tablet remnants of NVP XR in stools, in whom 54.2% reported noticing the tablet remnants at least once weekly. Compared with patients who did not notice tablet remnants, those who noticed tablet remnants had a higher mean CD4 lymphocyte count (629 vs 495 cells/mm3, P = 0.0002 and a similar mean plasma HIV RNA load (1.57 vs 1.61 log10 copies/mL, P = 0.76 on switch. At about 12 and 24 weeks after switch, patients who noticed tablet remnants continued to have a similar mean plasma HIV RNA load (1.39 vs 1.43 log10 copies/mL, P = 0.43; and 1.30 vs 1.37 log10 copies/mL, P = 0.26, respectively, but had a lower median NVP C12 (3640 vs 4730 ng/mL, P = 0.06, and a similar median NVP C24 (3220 vs 3330 ng/ml, P = 0.95 when compared with those who did not notice tablet remnants.The presence of tablet remnants of NVP XR in

  16. Comparison of the Compositions of the Stool Microbiotas of Infants Fed Goat Milk Formula, Cow Milk-Based Formula, or Breast Milk

    OpenAIRE

    Tannock, Gerald W.; Lawley, Blair; Munro, Karen; Gowri Pathmanathan, Siva; Zhou, Shao J.; Makrides, Maria; Robert A Gibson; Sullivan, Thomas; Prosser, Colin G.; Lowry, Dianne; Alison J. Hodgkinson

    2013-01-01

    The aim of the study was to compare the compositions of the fecal microbiotas of infants fed goat milk formula to those of infants fed cow milk formula or breast milk as the gold standard. Pyrosequencing of 16S rRNA gene sequences was used in the analysis of the microbiotas in stool samples collected from 90 Australian babies (30 in each group) at 2 months of age. Beta-diversity analysis of total microbiota sequences and Lachnospiraceae sequences revealed that they were more similar in breast...

  17. DNA methylation

    DEFF Research Database (Denmark)

    Williams, Kristine; Christensen, Jesper; Helin, Kristian

    2012-01-01

    DNA methylation is involved in key cellular processes, including X-chromosome inactivation, imprinting and transcriptional silencing of specific genes and repetitive elements. DNA methylation patterns are frequently perturbed in human diseases such as imprinting disorders and cancer. The recent...... discovery that the three members of the TET protein family can convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) has provided a potential mechanism leading to DNA demethylation. Moreover, the demonstration that TET2 is frequently mutated in haematopoietic tumours suggests that the TET...... proteins are important regulators of cellular identity. Here, we review the current knowledge regarding the function of the TET proteins, and discuss various mechanisms by which they contribute to transcriptional control. We propose that the TET proteins have an important role in regulating DNA methylation...

  18. DNA data

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Raw DNA chromatogram data produced by the ABI 373, 377, 3130 and 3730 automated sequencing machines in ABI format. These are from fish (primarily Sebastes spp.,...

  19. DNA adductomics.

    Science.gov (United States)

    Balbo, Silvia; Turesky, Robert J; Villalta, Peter W

    2014-03-17

    Systems toxicology is a broad-based approach to describe many of the toxicological features that occur within a living system under stress or subjected to exogenous or endogenous exposures. The ultimate goal is to capture an overview of all exposures and the ensuing biological responses of the body. The term exposome has been employed to refer to the totality of all exposures, and systems toxicology investigates how the exposome influences health effects and consequences of exposures over a lifetime. The tools to advance systems toxicology include high-throughput transcriptomics, proteomics, metabolomics, and adductomics, which is still in its infancy. A well-established methodology for the comprehensive measurement of DNA damage resulting from every day exposures is not fully developed. During the past several decades, the (32)P-postlabeling technique has been employed to screen the damage to DNA induced by multiple classes of genotoxicants; however, more robust, specific, and quantitative methods have been sought to identify and quantify DNA adducts. Although triple quadrupole and ion trap mass spectrometry, particularly when using multistage scanning (LC-MS(n)), have shown promise in the field of DNA adductomics, it is anticipated that high-resolution and accurate-mass LC-MS(n) instrumentation will play a major role in assessing global DNA damage. Targeted adductomics should also benefit greatly from improved triple quadrupole technology. Once the analytical MS methods are fully mature, DNA adductomics along with other -omics tools will contribute greatly to the field of systems toxicology.

  20. Microbiota-Gut-Brain Axis: Yeast Species Isolated from Stool Samples of Children with Suspected or Diagnosed Autism Spectrum Disorders and In Vitro Susceptibility Against Nystatin and Fluconazole.

    Science.gov (United States)

    Kantarcioglu, A Serda; Kiraz, Nuri; Aydin, Ahmet

    2016-02-01

    Autism spectrum disorder (ASD) is a general term for a group of complex neurodevelopmental disorders of brain development that limits a person's ability to function normally. Etiology has not been clearly defined up to date. However, gut microbiota and the bidirectional communication between the gastrointestinal tract and brain, the so-called microbiota-gut-brain axis, are hypothesized, which may be involved in the etiology of several mental disorders. Recent reports suggest that Candida, particularly Candida albicans, growth in intestines may cause lower absorption of carbohydrates and minerals and higher toxin levels which are thought to contribute autistic behaviors. The aim of this study was to identify the 3-year deposited yeasts isolated from stool samples of children with diagnosed or suspected ASD and to determine in vitro activity of nystatin and fluconazole against these isolates using Clinical Laboratory Standards Institute M27-A3 guidelines. A 17-year retrospective assessment was also done using our laboratory records. Among the species identified, intrinsically fluconazole-resistant Candida krusei (19.8 %) and Candida glabrata (14.8 %) with elevated MICs were remarkable. Overall, C. albicans (57.4 %) was the most commonly isolated species in 17 years. The species identification and/or antifungal susceptibility tests have to be performed using the strain isolated from stool sample, to select the appropriate antifungal agent, if antimycotic therapy is needed.

  1. Recombinase Polymerase Amplification Compared to Real-Time Polymerase Chain Reaction Test for the Detection of Fasciola hepatica in Human Stool.

    Science.gov (United States)

    Cabada, Miguel M; Malaga, Jose L; Castellanos-Gonzalez, Alejandro; Bagwell, Kelli A; Naeger, Patrick A; Rogers, Hayley K; Maharsi, Safa; Mbaka, Maryann; White, A Clinton

    2017-02-08

    Fasciola hepatica is the most widely distributed trematode infection in the world. Control efforts may be hindered by the lack of diagnostic capacity especially in remote endemic areas. Polymerase chain reaction (PCR)-based methods offer high sensitivity and specificity but require expensive technology. However, the recombinase polymerase amplification (RPA) is an efficient isothermal method that eliminates the need for a thermal cycler and has a high deployment potential to resource-limited settings. We report on the characterization of RPA and PCR tests to detect Fasciola infection in clinical stool samples with low egg burdens. The sensitivity of the RPA and PCR were 87% and 66%, respectively. Both tests were 100% specific showing no cross-reactivity with trematode, cestode, or nematode parasites. In addition, RPA and PCR were able to detect 47% and 26% of infections not detected by microscopy, respectively. The RPA adapted to a lateral flow platform was more sensitive than gel-based detection of the reaction products. In conclusion, the Fasciola RPA is a highly sensitive and specific test to diagnose chronic infection using stool samples. The Fasciola RPA lateral flow has the potential for deployment to endemic areas after further characterization. © The American Society of Tropical Medicine and Hygiene.

  2. Performance of chromID Clostridium difficile agar compared with BBL C. difficile selective agar for detection of C. difficile in stool specimens.

    Science.gov (United States)

    Han, Sang Bong; Chang, Jiyoung; Shin, Sang Hyun; Park, Kang Gyun; Lee, Gun Dong; Park, Yong Gyu; Park, Yeon-Joon

    2014-09-01

    We evaluated the performance of a new chromogenic medium for detection of Clostridium difficile, chromID C. difficile agar (CDIF; bioMérieux, France), by comparison with BBL C. difficile Selective Agar (CDSA; Becton Dickinson and Company, USA). After heat pre-treatment (80℃, 5 min), 185 diarrheal stool samples were inoculated onto the two media types and incubated anaerobically for 24 hr and 48 hr for CDIF and for 48 hr and 72 hr for CDSA. All typical colonies on each medium were examined by Gram staining, and the gram-positive rods confirmed to contain the tpi gene by PCR were identified as C. difficile. C. difficile was recovered from 36 samples by using a combination of the two media. The sensitivity with CDIF 48 hr was highest (100%) and was significantly higher than that with CDIF 24 hr (58.3%; P<0.001), because samples with a low burden of C. difficile tended to require prolonged incubation up to 48 hr (P<0.001). The specificity of CDIF 24 hr and CDIF 48 hr (99.3% and 90.6%, respectively) was significantly higher than that of CDSA 48 hr and CDSA 72 hr (72.5% and 67.1%, respectively; P<0.001). CDIF was effective for detecting C. difficile in heat-pretreated stool specimens, thus reducing unnecessary testing for toxin production in non-C. difficile isolates and turnaround time.

  3. Performance of the Fecal Immunochemical Test for Colorectal Cancer Screening Using Different Stool-Collection Devices: Preliminary Results from a Randomized Controlled Trial.

    Science.gov (United States)

    Shin, Hye Young; Suh, Mina; Baik, Hyung Won; Choi, Kui Son; Park, Boyoung; Jun, Jae Kwan; Hwang, Sang-Hyun; Kim, Byung Chang; Lee, Chan Wha; Oh, Jae Hwan; Lee, You Kyoung; Han, Dong Soo; Lee, Do-Hoon

    2016-11-15

    We are in the process of conducting a randomized trial to determine whether compliance with the fecal immunochemical test (FIT) for colorectal cancer screening differs according to the stool-collection method. This study was an interim analysis of the performance of two stool-collection devices (sampling bottle vs conventional container). In total, 1,701 individuals (age range, 50 to 74 years) were randomized into the sampling bottle group (intervention arm) or the conventional container group (control arm). In both groups, we evaluated the FIT positivity rate, the positive predictive value for advanced neoplasia, and the detection rate for advanced neoplasia. The FIT positivity rates were 4.1% for the sampling bottles and 2.0% for the conventional containers; these values were significantly different. The positive predictive values for advanced neoplasia in the sampling bottles and conventional containers were 11.1% (95% confidence interval [CI], -3.4 to 25.6) and 12.0% (95% CI, -0.7 to 24.7), respectively. The detection rates for advanced neoplasia in the sampling bottles and conventional containers were 4.5 per 1,000 persons (95% CI, 2.0 to 11.0) and 2.4 per 1,000 persons (95% CI, 0.0 to 5.0), respectively. The impact of these findings on FIT screening performance was unclear in this interim analysis. This impact should therefore be evaluated in the final analysis following the final enrollment period.

  4. Multicenter clinical evaluation of the portrait toxigenic C. difficile assay for detection of toxigenic Clostridium difficile strains in clinical stool specimens.

    Science.gov (United States)

    Buchan, Blake W; Mackey, Tami-Lea A; Daly, Judy A; Alger, Garrison; Denys, Gerald A; Peterson, Lance R; Kehl, Sue C; Ledeboer, Nathan A

    2012-12-01

    We compared the Portrait Toxigenic C. difficile Assay, a new semiautomated sample-to-result molecular test, to a toxigenic bacterial culture/cell cytotoxin neutralization assay (TBC/CCNA) for the detection of toxigenic Clostridium difficile in 549 stool specimens. Stool specimens were also tested by one of three alternative FDA-cleared molecular tests for toxigenic C. difficile (Xpert C. difficile, Illumigene C. difficile, or GeneOhm Cdiff). The sensitivities and specificities of the molecular tests compared to TBC/CCNA were as follows: 98.2% and 92.8% for the Portrait assay, 100% and 91.7% for the Xpert assay, 93.3% and 95.1% for the Illumigene assay, and 97.4% and 98.5% for the GeneOhm assay, respectively. The majority of Portrait false-positive results (20/31; 64.5%) were also positive for C. difficile by an alternative molecular test, suggesting an increased sensitivity compared to the culture-based "gold standard" method. The Portrait test detected an assay input of 30 CFU in 100% of spiked samples and detected an input of 10 CFU in 96.7% of samples tested.

  5. DNA expressions - A formal notation for DNA

    NARCIS (Netherlands)

    Vliet, Rudy van

    2015-01-01

    We describe a formal notation for DNA molecules that may contain nicks and gaps. The resulting DNA expressions denote formal DNA molecules. Different DNA expressions may denote the same molecule. Such DNA expressions are called equivalent. We examine which DNA expressions are minimal, which

  6. DNA expressions - A formal notation for DNA

    NARCIS (Netherlands)

    Vliet, Rudy van

    2015-01-01

    We describe a formal notation for DNA molecules that may contain nicks and gaps. The resulting DNA expressions denote formal DNA molecules. Different DNA expressions may denote the same molecule. Such DNA expressions are called equivalent. We examine which DNA expressions are minimal, which

  7. DNA and RNA sensor

    Institute of Scientific and Technical Information of China (English)

    LIU; Tao; LIN; Lin; ZHAO; Hong; JIANG; Long

    2005-01-01

    This review summarizes recent advances in DNA sensor. Major areas of DNA sensor covered in this review include immobilization methods of DNA, general techniques of DNA detection and application of nanoparticles in DNA sensor.

  8. What Is Mitochondrial DNA?

    Science.gov (United States)

    ... DNA What is mitochondrial DNA? What is mitochondrial DNA? Although most DNA is packaged in chromosomes within ... proteins. For more information about mitochondria and mitochondrial DNA: Molecular Expressions, a web site from the Florida ...

  9. Genotyping of Giardia duodenalis isolates from human subjects in Zabul, using PCR-RFLP

    Directory of Open Access Journals (Sweden)

    Maryam Abedi

    2016-09-01

    Full Text Available Objective: To uncover the molecular prevalence of Giardia duodenalis by PCR-restriction fragment length polymorphism (RFLP in Zabul city, Iran. Methods: Twenty-four stool samples were collected from 215 patients with suspected giardiasis by microscopic examination. To increase the sensitivity of the PCR, the total genomic DNA from isolates was extracted by applying glass beads and the QIAamp Kit. A one-step PCRRFLP method, targeting the glutamate dehydrogenase gene, was utilized to differentiate the assemblages A and B among isolates. Results: The PCR fragment was determined from 30 isolates, RFLP assay of 24 isolates showed 24 (100 isolates as Genotype B group BIII. Conclusions: The results with the glutamate dehydrogenase gene assay demonstrated that the predominant subtype of Giardia duodenalis in the area is BIII, which showed animals are the main reservoir of the isolates in this area.

  10. DNA vaccines

    Science.gov (United States)

    Gregersen, Jens-Peter

    2001-12-01

    Immunization by genes encoding immunogens, rather than with the immunogen itself, has opened up new possibilities for vaccine research and development and offers chances for new applications and indications for future vaccines. The underlying mechanisms of antigen processing, immune presentation and regulation of immune responses raise high expectations for new and more effective prophylactic or therapeutic vaccines, particularly for vaccines against chronic or persistent infectious diseases and tumors. Our current knowledge and experience of DNA vaccination is summarized and critically reviewed with particular attention to basic immunological mechanisms, the construction of plasmids, screening for protective immunogens to be encoded by these plasmids, modes of application, pharmacokinetics, safety and immunotoxicological aspects. DNA vaccines have the potential to accelerate the research phase of new vaccines and to improve the chances of success, since finding new immunogens with the desired properties is at least technically less demanding than for conventional vaccines. However, on the way to innovative vaccine products, several hurdles have to be overcome. The efficacy of DNA vaccines in humans appears to be much less than indicated by early studies in mice. Open questions remain concerning the persistence and distribution of inoculated plasmid DNA in vivo, its potential to express antigens inappropriately, or the potentially deleterious ability to insert genes into the host cell's genome. Furthermore, the possibility of inducing immunotolerance or autoimmune diseases also needs to be investigated more thoroughly, in order to arrive at a well-founded consensus, which justifies the widespread application of DNA vaccines in a healthy population.

  11. DNA nanotechnology

    Directory of Open Access Journals (Sweden)

    Nadrian C Seeman

    2003-01-01

    We are all aware that the DNA found in cells is a double helix consisting of two antiparallel strands held together by specific hydrogen-bonded base pairs; adenine (A always pairs with thymine (T, and guanine (G always pairs with cytosine (C. The specificity of this base pairing and the ability to ensure that it occurs in this fashion (and not some other1 is key to the use of DNA in materials applications. The double helical arrangement of the two molecules leads to a linear helix axis, linear not in the geometrical sense of being a straight line, but in the topological sense of being unbranched. Genetic engineers discovered in the 1970s how to splice together pieces of DNA to add new genes to DNA molecules2, and synthetic chemists worked out convenient syntheses for short pieces of DNA (up to ∼100–150 units in the 1980s3. Regardless of the impact of these technologies on biological systems, hooking together linear molecules leads only to longer linear molecules, with circles, knots, and catenanes perhaps resulting from time to time.

  12. Fecal collection, ambient preservation, and DNA extraction for PCR amplification of bacterial and human markers from human feces.

    Science.gov (United States)

    Nechvatal, Jordan M; Ram, Jeffrey L; Basson, Marc D; Namprachan, Phanramphoei; Niec, Stephanie R; Badsha, Kawsar Z; Matherly, Larry H; Majumdar, Adhip P N; Kato, Ikuko

    2008-02-01

    Feces contain intestinal bacteria and exfoliated epithelial cells that may provide useful information concerning gastrointestinal tract health. Intestinal bacteria that synthesize or metabolize potential carcinogens and produce anti-tumorigenic products may have relevance to colorectal cancer, the second most common cause of cancer deaths in the USA. To facilitate epidemiological studies relating bacterial and epithelial cell DNA and RNA markers, preservative/extraction methods suitable for self-collection and shipping of fecal samples at room temperature were tested. Purification and PCR amplification of fecal DNA were compared after preservation of stool samples in RNAlater (R) or Paxgene (P), or after drying over silica gel (S) or on Whatman FTA cards (W). Comparisons were made to samples frozen in liquid nitrogen (N2). DNA purification methods included Whatman (accompanying FTA cards), Mo-Bio Fecal (MB), Qiagen Stool (QS), and others. Extraction methods were compared for amount of DNA extracted, DNA amplifiable in a real-time SYBR-Green quantitative PCR format, and the presence of PCR inhibitors. DNA can be extracted after room temperature storage for five days from W, R, S and P, and from N2 frozen samples. High amounts of total DNA and PCR-amplifiable Bacteroides spp. DNA (34%+/-9% of total DNA) with relatively little PCR inhibition were especially obtained with QS extraction applied to R preserved samples (method QS-R). DNA for human reduced folate carrier (SLC19A1) genomic sequence was also detected in 90% of the QS-R extracts. Thus, fecal DNA is well preserved by methods suitable for self-collection that may be useful in future molecular epidemiological studies of intestinal bacteria and human cancer markers.

  13. 《矮凳桥风情》的民间文化意蕴%Folk Culture Connotation of"The Low Stool Bridge Customs"

    Institute of Scientific and Technical Information of China (English)

    康馨

    2016-01-01

    Lin Jinlan has devoted all his life to the creation of short stories whose series of novels-"The Low Stool Bridge Cus-toms"was published in the 80's.Known as the"strange stories","The Low Stool Bridge Customs"is different from his previ-ous styles."The Low Stool Bridge Customs"is rooted in the folklife and expresses the fate and experience of the common people through his folk value standpoint.Lin Jinlan combined the archetype image of Wenzhou culture with characters'survival envi-ronment and character,related the popular and elegant dialect to mandarin,which shows his efforts of returning to mother tongue.Regional color dense image and original and unique novel language contributed to the works full of cultural implication of the aesthetic characteristics.%林斤澜毕生致力于短篇小说创作,发表于新时期的《矮凳桥风情》系列小说与他之前的作品风格迥异,被称为“怪味小说”。《矮凳桥风情》的创作扎根于民间,以民间价值立场叙写普通人的命运遭际。林斤澜将温州文化中保留的原型形象与人物的生存环境和性格特征相结合,以通俗典雅的方言土语与普通话相结合,做出了小说语言回归母语的努力。地域色彩浓厚的形象与原始而别致的小说语言,促成了作品充满文化意蕴的审美特质。

  14. DNA origami nanopores for controlling DNA translocation.

    Science.gov (United States)

    Hernández-Ainsa, Silvia; Bell, Nicholas A W; Thacker, Vivek V; Göpfrich, Kerstin; Misiunas, Karolis; Fuentes-Perez, Maria Eugenia; Moreno-Herrero, Fernando; Keyser, Ulrich F

    2013-07-23

    We combine DNA origami structures with glass nanocapillaries to reversibly form hybrid DNA origami nanopores. Trapping of the DNA origami onto the nanocapillary is proven by imaging fluorescently labeled DNA origami structures and simultaneous ionic current measurements of the trapping events. We then show two applications highlighting the versatility of these DNA origami nanopores. First, by tuning the pore size we can control the folding of dsDNA molecules ("physical control"). Second, we show that the specific introduction of binding sites in the DNA origami nanopore allows selective detection of ssDNA as a function of the DNA sequence ("chemical control").

  15. Comparison of sensitivity and faecal egg counts of Mini-FLOTAC using fixed stool samples and Kato-Katz technique for the diagnosis of Schistosoma mansoni and soil-transmitted helminths.

    Science.gov (United States)

    Coulibaly, Jean T; Ouattara, Mamadou; Becker, Sören L; Lo, Nathan C; Keiser, Jennifer; N'Goran, Eliézer K; Ianniello, Davide; Rinaldi, Laura; Cringoli, Giuseppe; Utzinger, Jürg

    2016-12-01

    Accurate diagnostic tools for human helminthiasis are crucial for epidemiological surveys, improved patient management, and evaluation of community-based intervention studies. However, the diagnosis of intestinal schistosomiasis and soil-transmitted helminthiasis heavily relies on stool microscopy using the Kato-Katz technique, which has a low sensitivity. The Mini-FLOTAC method is an alternative microscopy-based technique, but its diagnostic performance using sodium acetate-acetic acid-formalin-(SAF)-fixed stool specimens has not been validated. The fixation of stool samples for later examination in a laboratory may reduce logistical and financial barriers of prevalence surveys by not requiring field laboratories. We compared the diagnostic accuracy of the Kato-Katz technique using fresh stool samples with the Mini-FLOTAC technique, employing matched stool samples that were fixed in SAF. Three consecutive stool samples from 149 school-aged children in Côte d'Ivoire were subjected to quintuplicate Kato-Katz thick smears examined on the same day. From the remaining stool, approximately 2g was fixed in 10ml of SAF for about 3 months, and then subjected to the Mini-FLOTAC method, using two flotation solutions (FS2 and FS7). The combined results of multiple Kato-Katz and Mini-FLOTAC readings revealed prevalences of Schistosoma mansoni, Trichuris trichiura and hookworm of 99.3%, 72.5% and 7.4%, respectively. Employing a Bayesian latent class analysis to estimate the true sensitivity of the diagnostic approaches, the sensitivity of Mini-FLOTAC using FS2 was 50.1% (95% Bayesian credible interval (BCI): 30.9-70.2%) for hookworm and 68.0% (95% BCI: 34.9-93.5%) for T. trichiura. When applying Mini-FLOTAC using FS7, the sensitivity was 89.9% (95% BCI: 86.9-97.4%) for S. mansoni, 37.2% (95% BCI: 17.2-60.6%) for hookworm and 67.7% (95% BCI: 33.0-93.0%) for T. trichiura. The specificity ranged from 80.1-95.0% in all Mini-FLOTAC tests. Mini-FLOTAC revealed higher arithmetic mean

  16. Unusual mode of protein binding by a cytotoxic π-arene ruthenium(ii) piano-stool compound containing an O,S-chelating ligand.

    Science.gov (United States)

    Hildebrandt, Jana; Görls, Helmar; Häfner, Norman; Ferraro, Giarita; Dürst, Matthias; Runnebaum, Ingo B; Weigand, Wolfgang; Merlino, Antonello

    2016-08-02

    A new pseudo-octahedral π-arene ruthenium(ii) piano-stool compound, containing an O,S-bidentate ligand (compound 1) and showing significant cytotoxic activity in vitro, was synthesized and characterized. In solution stability and interaction with the model protein bovine pancreatic ribonuclease (RNase A) were investigated by using UV-Vis absorption spectroscopy. Its crystal structure and that of the adduct formed upon reaction with RNase A were obtained by X-ray crystallography. The comparison between the structure of purified compound 1 and that of the fragment bound to RNase A reveals an unusual mode of protein binding that includes ligand exchange and alteration of coordination sphere geometry.

  17. Epidemiological trends and patterns of antimicrobial resistance of Shigella spp. isolated from stool cultures in two different populations in Southern Israel.

    Science.gov (United States)

    Peleg, Itai; Givon-Lavi, Noga; Leibovitz, Eugene; Broides, Arnon

    2014-03-01

    Southern Israel is inhabited by Bedouins, living in conditions similar to developing countries and Jews, living in conditions similar to developed countries. We determined the epidemiology of Shigella spp. in these populations. We retrospectively reviewed Shigella spp. stool isolations between 2005-2009. Overall, 3295 isolates were analyzed. S. sonnei was isolated in 2057/3295 (62.4%) and S. flexneri in 1058 (32.1%). S. sonnei was isolated in 1567/1707 (91.8%) from Jewish patients and S. flexneri in 931/1542 (60.4%) from Bedouin patients. Ampicillin resistance increased linearly from 217/373 (58.2%) in 2005 to 186/256 (72.7%) in 2009, (P Shigella spp. to ampicilin and trimethoprim-sulfamethoxazole were found in Jewish patients: 1527/1706 (89.5%) versus 977/1542 (63.4%) (P Shigella spp. infections can differ in populations residing in the same geographical area.

  18. Epidemiology of extended-spectrum β-lactamase producing Escherichia coli in the stools of returning Japanese travelers, and the risk factors for colonization.

    Directory of Open Access Journals (Sweden)

    Kenichiro Yaita

    Full Text Available OBJECTIVE: Travel overseas has recently been considered a risk factor for colonization with drug-resistant bacteria. The purpose of this study was to establish the epidemiology and risk factors associated with the acquisition of drug-resistant bacteria by Japanese travelers. METHODS: Between October 2011 and September 2012, we screened the stools of 68 Japanese returning travelers for extended-spectrum β-lactamase (ESBL producing Escherichia coli. All specimens were sampled for clinical reasons. Based on the results, the participants were divided into an ESBL-producing E. coli positive group (18 cases; 26% and an ESBL-producing E. coli negative group (50 cases; 74%, and a case-control study was performed. Microbiological analyses of ESBL-producing strains, including susceptibility tests, screening tests for metallo-β-lactamase, polymerase chain reaction amplification and sequencing of blaCTX-M genes, multilocus sequence typing, and whole genome sequencing, were also conducted. RESULTS: In a univariate comparison, travel to India was a risk factor (Odds Ratio 13.6, 95% Confidence Interval 3.0-75.0, p<0.0001. There were no statistical differences in the characteristics of the travel, such as backpacking, purpose of travel, interval between travel return and sampling stool, and duration of travel. Although 10 of 13 analyzed strains (77% produced CTX-M-15, no ST131 clone was detected. CONCLUSION: We must be aware of the possibilities of acquiring ESBL-producing E. coli during travel in order to prevent the spread of these bacteria not only in Japan but globally.

  19. Development and Evaluation of a Real-Time PCR Assay for Detection and Quantification of Blastocystis Parasites in Human Stool Samples: Prospective Study of Patients with Hematological Malignancies▿

    Science.gov (United States)

    Poirier, Philippe; Wawrzyniak, Ivan; Albert, Aurélie; El Alaoui, Hicham; Delbac, Frédéric; Livrelli, Valérie

    2011-01-01

    Blastocystis anaerobic parasites are widespread worldwide in the digestive tract of many animal species, including humans. Epidemiological Blastocystis studies are often limited by the poor sensitivity of standard parasitological assays for its detection. This report presents a highly sensitive real-time quantitative PCR (qPCR) assay developed to detect Blastocystis parasites in stool samples. The assay targets a partial sequence of the Blastocystis small ribosomal subunit (SSU) rRNA gene, allowing subtyping (ST) of Blastocystis isolates by direct sequencing of qPCR products. This qPCR method was assessed in a prospective study of 186 patients belonging to two cohorts—a group of 94 immunocompromised patients presenting hematological malignancies and a control group of 92 nonimmunocompromised patients. Direct-light microscopy and xenic in vitro stool culture analysis showed only 29% and 52% sensitivity, respectively, compared to our qPCR assay. Of the 27 (14.5%) Blastocystis-positive patients, 8 (4%) experienced digestive symptoms. No correlation was found between symptomatic patients and immune status, parasite load, or parasite subtypes, although subtyping of all isolates revealed a high (63.0%) prevalence of ST4. Two unexpected avian subtypes were found, i.e., ST6 and ST7, which are frequently isolated in Asia but rarely present in Western countries. In conclusion, this qPCR proved by far the most sensitive of the tested methods and allowed subtype determination by direct sequencing of qPCR products. New diagnostic tools such as the qPCR are essential for evaluating the clinical relevance of Blastocystis subtypes and their role in acute or chronic digestive disorders. PMID:21177897

  20. Schistosoma mansoni Infections in young children: when are schistosome antigens in urine, eggs in stool and antibodies to eggs first detectable?

    Directory of Open Access Journals (Sweden)

    J Russell Stothard

    Full Text Available BACKGROUND: in uganda, control of intestinal schistosomiasis with preventive chemotherapy is typically focused towards treatment of school-aged children; the needs of younger children are presently being investigated as in lakeshore communities very young children can be infected. In the context of future epidemiological monitoring, we sought to compare the detection thresholds of available diagnostic tools for Schistosoma mansoni and estimate a likely age of first infection for these children. METHODS AND FINDINGS: a total of 242 infants and preschool children (134 boys and 108 girls, mean age 2.9 years, minimum 5 months and maximum 5 years were examined from Bugoigo, a well-known disease endemic village on Lake Albert. Schistosome antigens in urine, eggs in stool and host antibodies to eggs were inspected to reveal a general prevalence of 47.5% (CI(95 41.1-54.0%, as ascertained by a positive criterion from at least one diagnostic method. Although children as young as 6 months old could be found infected, the average age of infected children was between 3¼-3¾ years, when diagnostic techniques became broadly congruent. CONCLUSION: whilst different assays have particular (disadvantages, direct detection of eggs in stool was least sensitive having a temporal lag behind antigen and antibody methods. Setting precisely a general age of first infection is problematic but if present Ugandan policies continue, a large proportion of infected children could wait up to 3-4 years before receiving first medication. To better tailor treatment needs for this younger ageclass, we suggest that the circulating cathodic antigen urine dipstick method to be used as an epidemiological indicator.

  1. Occurrence of Listeria species in meat, chicken products and human stools in Assiut city, Egypt with PCR use for rapid identification of Listeria monocytogenes

    Directory of Open Access Journals (Sweden)

    Ashraf Mohamed Abd El-Malek

    Full Text Available The present research was conducted to check the presence of Listeria spp. in some meat and chicken products purchased from retail supermarkets in Assiut (Egypt. A total of 100 samples including 25 samples each of minced frozen beef, luncheon, frozen chicken legs and frozen chicken breast fillets were collected over a 7-month period between January and July 2009 and analyzed for the presence of Listeria spp. In addition, 28 stool cultures examined for Listeria spp. from hospitalized children resident in Assiut Pediatric University Hospital with diarrhea or fever. Out of the total 100 meat samples examined, Listeria spp. were detected in 8 (32% of minced frozen beef, 8 (32% of luncheon, 13 (52% of frozen chicken leg and 14 (56% of frozen chicken fillet samples analyzed, respectively. Regarding the examined 28 stool cultures from hospitalized children with underlying disease in Assiut Univ. hospital, 2 (7.14% were found positive for Listeria spp. For identification of L. monocytogenes using polymerase chain reaction (PCR, two primers were selected to detect 217-pb fragment ofthe prfA (transcriptional activator of the virulence factor gene for L. monocytogenes. 13 selected Listeria isolates displayed beta-haemolysis on sheep blood agar and positive CAMP test were further identified using PCR. PCR results showed that L. monocytogenes were confirmed in one of minced imported frozen meat examined, two of luncheon samples and two of frozen chicken legs with the total incidence of 5 isolates (5% from the total 100 examined food samples. This suggests the presence of a significant public health hazard linked to the consumption of these meat and chicken products sold in Assiut city contaminated with L. monocytogenes. The public health significance of these pathogens as well as recommended sanitary measures was discussed. [Veterinary World 2010; 3(8.000: 353-359

  2. Diffuse and enteroaggregative patterns of adherence of Escherichia coli isolated from stools of children in northeastern Brazil

    Directory of Open Access Journals (Sweden)

    Scaletsky Isabel Cristina Affonso

    2001-01-01

    Full Text Available Childhood diarrheal diseases remain highly endemic in northeastern Brazil. The attributable fraction of all diarrheal diseases among children less than 2 years of age due to Escherichia coli was examined in a 2-year prospective study in two large urban centers of Brazil. Between May 1997 and June 1999, fecal E. coli isolates from 237 children with diarrhea (217 acute and 20 persistent cases and 231 children without diarrhea (controls attending two hospitals in Northeast Brazil were tested for their pattern of adherence to HEp-2 cells and for colony hybridization with DNA probes specific for the six pathotypes of diarrheagenic E. coli. Enteroinvasive E. coli, enterotoxigenic E. coli and enterohemorrhagic E. coli were not isolated from any children. Diffusely adherent E. coli (DAEC and enteroaggregative E. coli (EAEC were the most frequent isolates with similar frequencies from children with or without diarrhea. Atypical EPEC (EAF-negative strains were isolated with similiar frequency from both cases (5.5% and controls (5.6%. Enteropathogenic E. coli (typical EPEC strains, characterized by localized adherence pattern of adherence, hybridization with the EAF probe, and belonging to the classical O serogroups, were significantly associated with diarrhea (P = 0.03. These E. coli strains associated with diarrhea accounted for 9% of all children with diarrhea. Collectively, in Northeast Brazil, E. coli strains comprise a small proportion of severe diarrhea prevalence in children.

  3. Improved methods for isolating DNA from Ostertagia ostertagi eggs in cattle feces.

    Science.gov (United States)

    Harmon, Aaron F; Zarlenga, Dante S; Hildreth, Michael B

    2006-02-18

    A multiplex PCR assay for differentiating strongyle eggs from cattle has recently been described; however, the egg disruption and DNA extraction procedures, though effective, are inadequate for large studies or clinical application. The purpose of this research was to evaluate methods for disrupting trichostrongyle eggs, then assess commercial kits for extracting egg DNA using Ostertagia ostertagi as a model species. Egg disruption procedures tested included probe sonication, bath sonication, bead beating, boiling, microwaving, proteinase K/SDS digestion, freezing, and various combinations of the above with the incorporation of sodium dodecyl sulfate. These procedures were evaluated in conjunction with four commercial DNA extraction kits: DNA Stool mini kit and DNeasy Plant kit (Qiagen), Fast DNA kit (QBiogene), and the MAP extraction kit (Tetracore). Results showed that egg disruption was best accomplished with the bead beater and ceramic beads, resulting in 100% disruption within 1min. When DNA extraction was preceded by the isolation of eggs from feces, all procedures except the Fast DNA kit produced PCR-ready DNA from at least two eggs. The DNeasy Plant kit allowed consistent detection of DNA released from one egg. Due to the morphological similarities among trichostrongyle eggs in ruminants, strongyle eggs in equids, and hookworm eggs, the methods described herein may have broad application to other nematodes.

  4. MDA-MSP技术检测粪便miR34b/c甲基化及其在结直肠癌早期诊断中的意义%Clinical significance of detection of stool miR34b/c methylation by MDA-MSP in patients with colorectal cancer

    Institute of Scientific and Technical Information of China (English)

    张丰云; 管静芝; 赵慧霞; 李秋文; 董伟伟; 段昕妤; 朱建华; 王如良; 郝怡鑫

    2013-01-01

    AIM: To investigate the value of detection of stool miR34b/c methylation in the diagnosis of colorectal cancer. METHODS: Multiple displacement amplifica- tion (MDA) was used to amplify bisulfite modified genomic DNA, and methylation-specific PCR (MSP) was used to analyze methylation of miR34b/c in colorectal cancer tissue and stool DNA from 126 patients with colorectal cancer and stool DNA in 64 patients with benign diseases. RESULTS: In 126 cancer specimens and matched tumor-adjacent tissue specimens, 95.2% (120/126) and 11.9% (15/126) showed methylation of miR34b/c, and there is a significant difference in the rate of methylation between them (P 0.05). The rate of methylation of miR34b/c in stool DNA was significantly higher in cancer patients than in patients with benign disease (90.2% vs 7.8%, P < 0.01). The sensitivity and specificity of detection of miR34b/c methylation in diagnosis of colorectal cancer were 90.2% and 92.2%, respectively. CONCLUSION: The hypermethylation of miR34b/c is frequent in colorectal cancer and may be used as a novel diagnostic biomarker for colorectal cancer. MDA and MSP techniques provide ideal tools for analysis of methylation in trace DNA samples.%目的:探讨粪便miR34b/c甲基化状态的检测在结直肠癌早期诊断中的意义.方法:从126例结直肠癌患者癌组织、癌旁组织、粪便和64例正常对照者的粪便中分别提取DNA,采用多重置换扩增(multiple displacement amplification,MDA)技术对经过亚硫酸氢盐修饰样本进行全基因组扩增,结合甲基化特异性PCR(methylation-specific PCR,MSP)检测组织和粪便中miR34b/c基因甲基化状态.结果:结直肠癌癌组织miR34b/c基因的甲基化阳性率为95.2%(120/126),对应的癌旁正常组织为11.9%(15/126),两者比较有显著差异(P<0.01); miR34b/c甲基化状态与各临床病理参数无显著相关(P>0.05).结直肠癌粪便miR34b/c甲基化阳性率为90.2%(111/123),显著高于正常对照7.8%(5/64),

  5. DNA nanostructure immobilization to lithographic DNA arrays

    Science.gov (United States)

    Negrete, Omar D.

    Although DNA is well known for its genetic role in biology, DNA has also been sought-after as a material for the self-assembly of biological and electronic devices. Examples of DNA nanostructure construction include DNA tiled self-assembly and DNA Origami, where by controlling the sequence and concentration of DNA molecules, the rational design of geometric DNA nanostructures is possible. The assembly of DNA nanostructures takes place in solution and thus they are in disorder and require further organization to construct circuitry or devices. Hence, it is essential for future applications of this technology to develop methods to direct the placement of DNA nanostructures on a surface. To address this challenge my research examines the use of DNA microarrays to capture DNA nanostructures via DNA hybridization. Modern DNA arrays offer a high-density of sequence-specific molecular recognition sites where the addressable placement of DNA nanostructures can be achieved. Using Maskless Array Synthesizer (MAS) technology, I have characterized photolithographic DNA arrays for the hybridization of DNA complexes like large DNA molecules (> 1 kb), DNA-gold nanoparticle conjugates, and DNA Origami. Although modern photolithographic DNA arrays can possess a high-density of sequence (106/cm2), the printed DNA areas are on the order of tens of microns. Thus, I have also developed a method to reduce the DNA array spot size to nanoscale dimensions through the combined use of electron beam lithography with photolithographic DNA synthesis. This work addresses the key elements towards developing a surface patterning technology that takes advantage of DNA base-pairing for both molecular sub-assembly and surface patterning.

  6. Semi - quantitative detection of hepatitis E virus RNA from stools of patients with hepatitis E%戊型肝炎患者粪便中病毒RNA的半定量检测

    Institute of Scientific and Technical Information of China (English)

    吕海芹; 张建琼; 赵宇; 林陵; 董莉

    2000-01-01

    目的 探讨戊型肝炎病毒(HEV)RNA半定量检测的意义和可行性,为HEV RNA的定量PCR(多聚酶链反应)检测提供科学依据。方法 在逆转录-套式PCR基础上,采用样品倍比稀释法对戊型肝炎患者粪便中病毒RNA作半定量检测。结果 20份粪便标本中有14份检测结果阳性,其中病毒RNA拷贝水平为8×105、8×107、8×108、8×109PCR单位·ml-1的分别为2、10、1、1份。对同一标本而言,在不同稀释度时,其PCR产物在量上无显著差异。结论定量PCR技术检测戊型肝炎病毒RNA是可行的,但应以起始模板来定量而不是以终产物定量。%Objective To investigate the significance and feasibility of quantification of hepatitis E virus RNA from stools of pa-tients with hepatitis E and give scientific proof for quantitative detection of it by RT- nested PCR and detection of RNA of viruses. Meth-ods A semi - quantitative detection of HEV RNA was made by serial dilution of the specimens on the basis of RT - nested PCR. Results There were 14 specimens positive anong the 20 clinical specimens and the copies of RNA were about 8×105 PCR unit·ml- 1 for 2 spec-imens,8×107pCR unit·ml- 1 for 10 specimens,8×108 PCR unit·ml- 1 and 8×109PCR unit·ml- 1 each for 1 specimen. But there was lit-tle difference among the amount of final products for HEV RNA RT- nested PCR. Conclusion The quantification of HEV RNA by RT-nested PCR is feasible but the quantity of HEV RNA should be determined by the initial cDNA not PCR products.

  7. Lactobacillus plantarum PADA FESES INDIVIDU DEWASA SEHAT YANG MENGONSUMSI Lactobacillus plantarum IS-10506 DARI DADIH [Lactobacillus plantarum in Stool of Apparently Healthy Adults Consuming Lactobacillus plantarum IS-10506 from Dadih

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    Azmier Adib*

    2013-12-01

    Full Text Available A placebo double blind pre-post human study was conducted in apparently healthy adults. There were two treatment groups consisting of Group A and B representing probiotic and placebo group, respectively. Twenty four participants were randomly assigned, each supplemented with either placebo or probiotic Lactobacillus plantarum IS-10506. The micro encapsulated powder was given at a dose of 2.6x1010 CFU/day for 21 consecutive days. Stool samples were collected before and after the supplementation. The fresh stool samples were analyzed for the viability of Lactobacillus sp. by conventional plate count method in MRS agar. Some stool samples were kept frozen to be analyzed by using real time PCR to trace back the availability of Lactobacillus plantarum with species specific primer. The Lactobacillus sp. in stools of healthy adults given microencapsulated probiotic Lactobacillus plantarum IS-10506 powder was significantly more than those who consumed microencapsulated placebo powder. Molecular detection by qPCR confirmed the availability of Lactobacillus plantarum in fecal samples of the probiotic group after given the supplementation for 21 days. The molecular detection validation confirmed that probiotic Lactobacillus plantarum was available in the fecal samples of the probiotic group of healthy adults. However, the availability and viability of Lactobacillus plantarum were not consistently found in the intestinal tract.

  8. Performance of the chromID Salmonella Elite chromogenic agar in comparison with CHROMagar™ Salmonella, Oxoid™ Brilliance™ Salmonella and Hektoen agars for the isolation of Salmonella from stool specimens.

    Science.gov (United States)

    Martiny, Delphine; Dediste, Anne; Anglade, Claire; Vlaes, Linda; Moens, Catherine; Mohamed, Souad; Vandenberg, Olivier

    2016-10-01

    chromID™ Salmonella Elite is compared with 3 culture media commonly used for Salmonella isolation from stool specimens. As results were equivalent to other chromogenic media (100% sensitivity, 98% specificity), only financial arguments should guide the choice for a medium with respect to another.

  9. Estimating the sensitivity and specificity of Kato-Katz stool examination technique for detection of hookworms, Ascaris lumbricoides and Trichuris trichiura infections in humans in the absence of a 'gold standard'.

    Science.gov (United States)

    Tarafder, M R; Carabin, H; Joseph, L; Balolong, E; Olveda, R; McGarvey, S T

    2010-03-15

    The accuracy of the Kato-Katz technique in identifying individuals with soil-transmitted helminth (STH) infections is limited by day-to-day variation in helminth egg excretion, confusion with other parasites and the laboratory technicians' experience. We aimed to estimate the sensitivity and specificity of the Kato-Katz technique to detect infection with Ascaris lumbricoides, hookworm and Trichuris trichiura using a Bayesian approach in the absence of a 'gold standard'. Data were obtained from a longitudinal study conducted between January 2004 and December 2005 in Samar Province, the Philippines. Each participant provided between one and three stool samples over consecutive days. Stool samples were examined using the Kato-Katz technique and reported as positive or negative for STHs. In the presence of measurement error, the true status of each individual is considered as latent data. Using a Bayesian method, we calculated marginal posterior densities of sensitivity and specificity parameters from the product of the likelihood function of observed and latent data. A uniform prior distribution was used (beta distribution: alpha=1, beta=1). A total of 5624 individuals provided at least one stool sample. One, two and three stool samples were provided by 1582, 1893 and 2149 individuals, respectively. All STHs showed variation in test results from day to day. Sensitivity estimates of the Kato-Katz technique for one stool sample were 96.9% (95% Bayesian Credible Interval [BCI]: 96.1%, 97.6%), 65.2% (60.0%, 69.8%) and 91.4% (90.5%, 92.3%), for A. lumbricoides, hookworm and T. trichiura, respectively. Specificity estimates for one stool sample were 96.1% (95.5%, 96.7%), 93.8% (92.4%, 95.4%) and 94.4% (93.2%, 95.5%), for A. lumbricoides, hookworm and T. trichiura, respectively. Our results show that the Kato-Katz technique can perform with reasonable accuracy with one day's stool collection for A. lumbricoides and T. trichiura. Low sensitivity of the Kato-Katz for detection

  10. Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP system for a highly sensitive detection of enterovirus in the stool samples of acute flaccid paralysis cases

    Directory of Open Access Journals (Sweden)

    Wakita Takaji

    2009-12-01

    Full Text Available Abstract Background In the global eradication program for poliomyelitis, the laboratory diagnosis plays a critical role by isolating poliovirus (PV from the stool samples of acute flaccid paralysis (AFP cases. In this study, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP system for a rapid and highly sensitive detection of enterovirus including PV to identify stool samples positive for enterovirus including PV. Methods A primer set was designed for RT-LAMP to detect enterovirus preferably those with PV-like 5'NTRs of the viral genome. The sensitivity of RT-LAMP system was evaluated with prototype strains of enterovirus. Detection of enterovirus from stool extracts was examined by using RT-LAMP system. Results We detected at least 400 copies of the viral genomes of PV(Sabin strains within 90 min by RT-LAMP with the primer set. This RT-LAMP system showed a preference for Human enterovirus species C (HEV-C strains including PV, but exhibited less sensitivity to the prototype strains of HEV-A and HEV-B (detection limits of 7,400 to 28,000 copies. Stool extracts, from which PV, HEV-C, or HEV-A was isolated in the cell culture system, were mostly positive by RT-LAMP method (positive rates of 15/16 (= 94%, 13/14 (= 93%, and 4/4 (= 100%, respectively. The positive rate of this RT-LAMP system for stool extracts from which HEV-B was isolated was lower than that of HEV-C (positive rate of 11/21 (= 52%. In the stool samples, which were negative for enterovirus isolation by the cell culture system, we found that two samples were positive for RT-LAMP (positive rates of 2/38 (= 5.3%. In these samples, enterovirus 96 was identified by sequence analysis utilizing a seminested PCR system. Conclusions RT-LAMP system developed in this study showed a high sensitivity comparable to that of the cell culture system for the detection of PV, HEV-A, and HEV-C, but less sensitivity to HEV-B. This RT-LAMP system would be useful for the

  11. Real-time PCR for detection of Strongyloides stercoralis in human stool samples from Côte d'Ivoire: diagnostic accuracy, inter-laboratory comparison and patterns of hookworm co-infection.

    Science.gov (United States)

    Becker, Sören L; Piraisoody, Nivetha; Kramme, Stefanie; Marti, Hanspeter; Silué, Kigbafori D; Panning, Marcus; Nickel, Beatrice; Kern, Winfried V; Herrmann, Mathias; Hatz, Christoph F; N'Goran, Eliézer K; Utzinger, Jürg; von Müller, Lutz

    2015-10-01

    Human infections with the helminth species Strongyloides stercoralis encompass a wide clinical spectrum, ranging from asymptomatic carriage to life-threatening disease. The diagnosis of S. stercoralis is cumbersome and the sensitivity of conventional stool microscopy is low. New molecular tools have been developed to increase sensitivity. We compared the diagnostic accuracy of real-time PCR with microscopy for the detection of S. stercoralis and hookworm in human stool samples, and investigated the inter-laboratory agreement of S. stercoralis-specific real-time PCR in two European laboratories. Stool specimens from 256 randomly selected individuals in rural Côte d'Ivoire were examined using three microscopic techniques (i.e. Kato-Katz, Koga agar plate (KAP) and Baermann (BM)). Additionally, ethanol-fixed stool aliquots were subjected to molecular diagnosis. The prevalence of S. stercoralis and hookworm infection was 21.9% and 52.0%, respectively, whilst co-infections were detected in 35 (13.7%) participants. The diagnostic agreement between real-time PCR and microscopy was excellent when both KAP and BM tested positive for S. stercoralis, but was considerably lower when only one microscopic technique was positive. The sensitivity of KAP, BM and real-time PCR for detection of S. stercoralis as compared to a combination of all diagnostic techniques was 21.4%, 37.5% and 76.8%, respectively. The inter-laboratory agreement of S. stercoralis-specific PCR was substantial (κ=0.63, preal-time PCR and stool microscopy shows high accuracy for S. stercoralis diagnosis. Besides high sensitivity, PCR may also enhance specificity by reducing microscopic misdiagnosis of morphologically similar helminth larvae (i.e. hookworm and S. stercoralis) in settings where both helminth species co-exist.

  12. Scientific Opinion on the substantiation of a health claim related to ?native chicory inulin? and maintenance of normal defecation by increasing stool frequency pursuant to Article 13.5 of Regulation (EC No 1924/2006

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    EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA

    2015-01-01

    Full Text Available Following an application from BENEO-Orafti S.A., submitted pursuant to Article 13.5 of Regulation (EC No 1924/2006 via the Competent Authority of Belgium, the Panel on Dietetic Products, Nutrition and Allergies (NDA was asked to deliver an opinion on the scientific substantiation of a health claim related to “native chicory inulin” and maintenance of normal defecation by increasing stool frequency. The food constituent that is a subject of a claim is “native chicory inulin”. The Panel considers that “native chicory inulin”, a non-fractionated mixture of monosaccharides (< 10%, disaccharides, inulin-type fructans and inulin extracted from chicory, with a mean DP ≥ 9, is sufficiently characterised in relation to the claimed effect. The Panel considers that maintenance of normal defecation by increasing stool frequency (provided that it does not result in diarrhoea is a beneficial physiological effect. Six studies involving 86 subjects consistently showed that consumption of “native chicory inulin” at an amount of at least 12 g/day increases stool frequency. The Panel also notes the plausible mechanisms by which inulin and inulin-type fructans in “native chicory inulin” could exert the claimed effect. The Panel concludes that a cause and effect relationship has been established between the consumption of “native chicory inulin” and maintenance of normal defecation by increasing stool frequency. The following wording reflects the scientific evidence: “Chicory inulin contributes to maintenance of normal defecation by increasing stool frequency”. In order to obtain the claimed effect, 12 g of “native chicory inulin” should be consumed daily.

  13. Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis.

    Science.gov (United States)

    Huang, Shichu; Do, Jaephil; Mahalanabis, Madhumita; Fan, Andy; Zhao, Lei; Jepeal, Lisa; Singh, Satish K; Klapperich, Catherine M

    2013-01-01

    In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have combined a nearly electricity-free nucleic acid extraction process with an electricity-free isothermal amplification assay to detect the presence of Clostridium difficile (C. difficile) DNA in the stool of infected patients. We used helicase-dependent isothermal amplification (HDA) to amplify the DNA in a low-cost, thermoplastic reaction chip heated with a pair of commercially available toe warmers, while using a simple Styrofoam insulator. DNA was extracted from known positive and negative stool samples. The DNA extraction protocol utilized an air pressure driven solid phase extraction device run using a standard bicycle pump. The simple heater setup required no electricity or battery and was capable of maintaining the temperature at 65°C±2°C for 55 min, suitable for repeatable HDA amplification. Experiments were performed to explore the adaptability of the system for use in a range of ambient conditions. When compared to a traditional centrifuge extraction protocol and a laboratory thermocycler, this disposable, no power platform achieved approximately the same lower limit of detection (1.25×10(-2) pg of C. difficile DNA) while requiring much less raw material and a fraction of the lab infrastructure and cost. This proof of concept study could greatly impact the accessibility of molecular assays for applications in global health.

  14. Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Bergström, Anders; Licht, Tine Rask

    2012-01-01

    Freezing stool samples prior to DNA extraction and downstream analysis is widely used in metagenomic studies of the human microbiota but may affect the inferred community composition. In this study, DNA was extracted either directly or following freeze storage of three homogenized human fecal...... samples using three different extraction methods. No consistent differences were observed in DNA yields between extractions on fresh and frozen samples; however, differences were observed between extraction methods. Quantitative PCR analysis was subsequently performed on all DNA samples using six...... different primer pairs targeting 16S rRNA genes of significant bacterial groups, and the community composition was evaluated by comparing specific ratios of the calculated abundances. In seven of nine cases, the Firmicutes to Bacteroidetes 16S rRNA gene ratio was significantly higher in fecal samples...

  15. Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Bergström, Anders; Licht, Tine Rask

    Freezing stool samples prior to DNA extraction and downstream analysis is widely used in metagenomic studies of the human microbiota but may affect the inferred community composition. In this study DNA was extracted either directly or following freeze storage of three homogenized human fecal...... samples using three different extraction methods. No consistent differences were observed in DNA yields between extractions on fresh and frozen samples, however differences were observed between extraction methods. Quantitative PCR analysis was subsequently performed on all DNA samples using six different...... primer pairs targeting 16S rRNA genes of significant bacterial groups and the community composition was evaluated by comparing specific ratios of the calculated abundances. In seven out of nine cases the Firmicutes to Bacteroidetes 16S rRNA gene ratio was significantly higher in fecal-samples that had...

  16. Study of the Prevalence of Causative Bacterial&Protozoal Agents of in Stool Samples of 470 Gastroenteritis Patients Referring to the Nikoopour Clinic in Yazd,Iran

    Directory of Open Access Journals (Sweden)

    MR Sharifi

    2004-04-01

    Full Text Available Interoduction: Gasteroenteritis is one of the problems worth consideration all over the world. It is one of the important causes of mortality, especially in children < 5 years of age, in developing countries including Iran. The aim of this descriptive study was to determine the demographic conditions influencing the presence of causative bacteria and protozoa, followed by antibiograms of isolated bacteria from stool samples of patients with gasteroenteritis referring to Nikoopour Clinic in the city of Yazd, Iran from 1998 – 2001. Materials and method: A total of 470 samples were microbiologically examined by direct method, culture and then antibiogramed. In order to isolate the possible bacteria, differential and selected media were used. Also, wet – mount technique was applied for detection of protozoa. Results: Results revealed that 272 samples (57.9% were infected by pathogenic bacteria or protozoa. 138 (50.8% pathogenic specimens were from male patients and the remaining 134(49.3% were from female patients. Isolated species were: Enteropathogenic E.coli 117(43%, Shigella 51(18.8%, Salmonella.interetidis 25(9.2%, C.jejuni 16(5.9%, Giardia lambdia 51(18.8% and Amoebae spp 12(4.4%. The most commonly detected shigella species was dysenteriae, (74.5% while boydii with 2% was the least common type observed in the specimens. Except shigella, all the other bacteria were more common in males than female, but insignificant statistically. In order to determine the sensitivity and/or resistance of pathogenic bacteria, antibiogram test was performed using selected antibiotic disks such as Ampicillin, Nalidixic Acid, Ciprofloxacin, Gentamycin and Sulfamethaxazole. Conclusion: Results revealed that some patients were probably infected by pathogenic factors other than bacteria or protozoa. Since all viruses and parasites are almost resistant to antibiotics and on the other hand, administration of antibiotics may lead to resistance of bacterial agents

  17. DNA nanostructure meets nanofabrication.

    Science.gov (United States)

    Zhang, Guomei; Surwade, Sumedh P; Zhou, Feng; Liu, Haitao

    2013-04-07

    Recent advances in DNA nanotechnology have made it possible to construct DNA nanostructures of almost arbitrary shapes with 2-3 nm of precision in their dimensions. These DNA nanostructures are ideal templates for bottom-up nanofabrication. This review highlights the challenges and recent advances in three areas that are directly related to DNA-based nanofabrication: (1) fabrication of large scale DNA nanostructures; (2) pattern transfer from DNA nanostructure to an inorganic substrate; and (3) directed assembly of DNA nanostructures.

  18. Comparison of a monoclonal antigen stool test (Hp StAR) with the 13C-urea breath test in monitoring Helicobacter pylori eradication therapy

    Institute of Scientific and Technical Information of China (English)

    Francesco Perri; Michele Quitadamo; Rosalba Ricciardi; Ada Piepoli; Rosa Cotugno; Annamaria Gentile; Alberto Pilotto; Angelo Andriulli

    2005-01-01

    AIM: To evaluate the agreement between a mAb-based stool test (HP StAR) and the urea breath test (UBT) in monitoring (H pylori) infection after eradication therapy.METHODS: Patients with discordant results on UBT and Hp StAR underwent endoscopy with biopsies for rapid urease test, culture, and histology to confirm H pylori status.RESULTS: Among 250 patients (50±14 years), 240 (96.0%) had concordant UBT and Hp StAR tests with a significant correlation between DOB and A values (R = 0.87; P<0.0001).The remaining 10 (4.0%) patients had discordant tests (positive Hp StAR and negative UBT) with the Hp StAR inaccurate in five cases (false positive) and UBT inaccurate in the other five cases (false negative). The "maximal expected" sensitivity, specificity, +PV, -PV, +LR, and -LR were 91%, 100%, 100%, 97.4%, ∞, and 8.2 respectively,for the UBT, and 100%, 97.4%, 91%, 100%, 38.8, and 0,respectively, for the Hp StAR. Overall accuracy for both tests was 98%.CONCLUSION: Both the UBT and the Hp StAR are equally accurate in monitoring H pylori infection. Nowadays,the choice of the "best" non-invasive H pylori test in the post-treatment setting should be done not only in terms of diagnostic accuracy but also in view of cost and local facilities.

  19. Microscopic diagnosis of sodium acetate-acetic acid-formalin-fixed stool samples for helminths and intestinal protozoa: a comparison among European reference laboratories.

    Science.gov (United States)

    Utzinger, J; Botero-Kleiven, S; Castelli, F; Chiodini, P L; Edwards, H; Köhler, N; Gulletta, M; Lebbad, M; Manser, M; Matthys, B; N'Goran, E K; Tannich, E; Vounatsou, P; Marti, H

    2010-03-01

    The present study aimed to compare the diagnostic performance of different European reference laboratories in diagnosing helminths and intestinal protozoa, using an ether-concentration method applied to sodium acetate-acetic acid-formalin (SAF)-preserved faecal samples. In total, 102 stool specimens were analysed during a cross-sectional parasitological survey in urban farming communities in Côte d'Ivoire. Five SAF-preserved faecal samples were prepared from each specimen and forwarded to the participating reference laboratories, processed and examined under a microscope adhering to a standard operating procedure (SOP). Schistosoma mansoni (cumulative prevalence: 51.0%) and hookworm (cumulative prevalence: 39.2%) were the predominant helminths. There was excellent agreement (kappa > 0.8; p protozoa were Entamoeba coli (median prevalence: 67.6%), Blastocystis hominis (median prevalence: 55.9%) and Entamoeba histolytica/Entamoeba dispar (median prevalence: 47.1%). Substantial agreement among reference laboratories was found for E. coli (kappa = 0.69), but only fair or moderate agreement was found for other Entamoeba species, Giardia intestinalis and Chilomastix mesnili. There was only poor agreement for B. hominis, Isospora belli and Trichomonas intestinalis. In conclusion, although common helminths were reliably diagnosed by European reference laboratories, there was only moderate agreement between centres for pathogenic intestinal protozoa. Continued external quality assessment and the establishment of a formal network of reference laboratories is necessary to further enhance both accuracy and uniformity in parasite diagnosis.

  20. 大便常规检验在季节性腹泻诊治中的应用%Application of stool routine examination in seasonal diarrhea

    Institute of Scientific and Technical Information of China (English)

    王海坤

    2016-01-01

    Objective:To investigate the clinical value of stool routine examination in the diagnosis of seasonal diarrhea.Methods:92 patients with diarrhea were selected.All of them were treated with routine inspection,and then assessment their test results. Results:The prevalence of shigella and escherichia have no seasonal characteristics,while salmonella and vibrio parahaemolyticus popular in summer and autumn.Conclusion:Taking routine inspection on patients with diarrhea can control the pathogenic factors and provide the basis on the diagnosis and treatment for medical personnel.%目的:探讨大便常规检验在季节性腹泻中的临床价值。方法:收治腹泻患者92例,对所有患者进行便常规检验,评估检验结果。结果:志贺菌、大肠埃希菌的流行不具有季节性特点,沙门菌和溶血性弧菌在夏秋季节流行。结论:对腹泻患者采用便常规检验,能够掌握患者的致病因素,为医护人员的诊治工作提供依据。

  1. Combination therapy with biofeedback, loperamide, and stool-bulking agents is effective for the treatment of fecal incontinence in women - a randomized controlled trial.

    Science.gov (United States)

    Sjödahl, Jenny; Walter, Susanna A; Johansson, Elin; Ingemansson, Anna; Ryn, Ann-Katrine; Hallböök, Olof

    2015-08-01

    Biofeedback and medical treatments have been extensively used for moderate fecal incontinence (FI). There is limited data comparing and combining these two treatments. The objective of this study was to evaluate the effect of biofeedback and medical treatments, separately and in combination. Sixty-four consecutive female patients, referred to a tertiary centre for FI, were included. The patients were randomized to start with either biofeedback (4-6 months) or medical treatment with loperamide and stool-bulking agents (2 months). Both groups continued with a combination of treatments, i.e. medical treatment was added to biofeedback and vice versa. A two-week prospective bowel symptom diary and anorectal physiology were evaluated at baseline, after single- and combination treatments. Fifty-seven patients completed the study. Median number of leakage episodes during two weeks decreased from 6 to 3 (p biofeedback and medical treatment is effective for symptom relief in FI. The symptom improvement was associated with improved fecal consistency, reduced urgency, and increased rectal sensory thresholds.

  2. Prevalence and characteristics of verotoxin-producing Escherichia coli (VTEC) in stool samples from asymptomatic human carriers working in the meat processing industry in Switzerland.

    Science.gov (United States)

    Stephan, R; Ragettli, S; Untermann, F

    2000-02-01

    A total of 5590 stool samples from healthy employees in the meat industry were screened by PCR for verotoxin-producing Escherichia coli (VTEC). The PCR product of VT-encoding genes was detected in 3. 5% of the samples. Phenotypic and genotypic traits of 47 VTEC strains isolated from asymptomatic carriers were characterized. A variety of serotypes was found; one strain belonged to the serotype O157:H7. The majority of the isolates proved to be VT2-positive. Fifty-seven percent of the verotoxin-producing strains harboured the genes for one or several additional virulence associated factors, including intimin (eae, 8.5%), the 60 MDa plasmid (42.5%), enterohaemolysin (EHEC-hlyA, 38.3%), the heat-stable enterotoxin (astA, 6.4%), a serin protease (espP, 6.4%), colicin production (col D157, 12.8%) and a secretion system II (etpD, 10.6%). None of the strains was positive for a specific enzyme with catalase-peroxidase activity (katP).

  3. DNA ligase I, the replicative DNA ligase.

    Science.gov (United States)

    Howes, Timothy R L; Tomkinson, Alan E

    2012-01-01

    Multiple DNA ligation events are required to join the Okazaki fragments generated during lagging strand DNA synthesis. In eukaryotes, this is primarily carried out by members of the DNA ligase I family. The C-terminal catalytic region of these enzymes is composed of three domains: a DNA binding domain, an adenylation domain and an OB-fold domain. In the absence of DNA, these domains adopt an extended structure but transition into a compact ring structure when they engage a DNA nick, with each of the domains contacting the DNA. The non-catalytic N-terminal region of eukaryotic DNA ligase I is responsible for the specific participation of these enzymes in DNA replication. This proline-rich unstructured region contains the nuclear localization signal and a PCNA interaction motif that is critical for localization to replication foci and efficient joining of Okazaki fragments. DNA ligase I initially engages the PCNA trimer via this interaction motif which is located at the extreme N-terminus of this flexible region. It is likely that this facilitates an additional interaction between the DNA binding domain and the PCNA ring. The similar size and shape of the rings formed by the PCNA trimer and the DNA ligase I catalytic region when it engages a DNA nick suggest that these proteins interact to form a double-ring structure during the joining of Okazaki fragments. DNA ligase I also interacts with replication factor C, the factor that loads the PCNA trimeric ring onto DNA. This interaction, which is regulated by phosphorylation of the non-catalytic N-terminus of DNA ligase I, also appears to be critical for DNA replication.

  4. Avaliação de dois métodos de extração de DNA de material parafinado para amplificação em PCR Evaluation of two methods of DNA extraction from paraffin-embedded material for PCR amplification

    Directory of Open Access Journals (Sweden)

    Luciana Estevam Simonato

    2007-04-01

    Full Text Available INTRODUÇÃO: Diversos métodos para extração de DNA a partir de tecidos biológicos inclusos em parafina encontram-se descritos na literatura, sendo o sucesso desse procedimento de grande importância para a realização de métodos moleculares de diagnóstico empregando a reação em cadeia da polimerase (PCR. OBJETIVO: Este estudo avaliou dois métodos de extração de DNA de material parafinado, visando à amplificação do DNA genômico pela técnica da PCR. MATERIAL E MÉTODO: Foram utilizadas 35 amostras de casos de carcinoma epidermóide de assoalho bucal diagnosticados e tratados no Centro de Oncologia Bucal da Universidade Estadual Paulista (Unesp. Os métodos de extração de DNA avaliados incluíram: 1. digestão com proteinase K seguida por purificação com Chelex 100® (BioRad; e 2. sistema QIAamp DNA minikit® (Qiagen. O DNA obtido foi quantificado por espectrofotometria e amplificado pela técnica da PCR, utilizando-se oligonucleotídeos iniciadores para betaglobina. RESULTADOS: A concentração de DNA obtido do material extraído com o primeiro método apresentou média de 120,62 ng/µl com razão entre as leituras das absorbâncias 260/280 variando de 0,8 a 1,41. Para as amostras extraídas com o segundo procedimento, o rendimento médio foi de 67,38 ng/µl, no entanto a razão 260/280 variou entre 1,11 e 2,53. O material foi submetido à PCR e, das 35 amostras extraídas com cada método, respectivamente, 29 e 30 apresentaram sinal positivo. CONCLUSÃO: Os dois métodos utilizados para obtenção de DNA de material parafinado apresentaram desempenho semelhante, revelando que ambos têm potencial para auxiliar na prática da biologia molecular diagnóstica, assim como no estudo diagnóstico retrospectivo em material parafinizado.BACKGROUND: There are several methods for DNA extraction from formalin-fixed paraffin-embedded tissues reported in the literature. High success rate on this procedure is important for the use of

  5. Asosiasi Keragaman Lokus DNA Mikrosatelit DRB3 Gen Bola dengan Berat Badan Induk dan Berat Lahir Pedet pada Sapi Bali

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    Ni Nyoman Trinayani

    2013-11-01

    Full Text Available Normal 0 false false false EN-US X-NONE X-NONE Genomic selection has a high potency to improve the genetic quality of Bali cattle. In effort to find out a molecular marker or DNA segment which is linked with a certain phenotype, we examined the association between BoLa-DRB3 microsatellite polymorphism and dam body weight and birth weight of calves in Bali cattle. A total of 40 dams from Abuan Village, the District of Susut was investigated. The total DNA was extracted with QIAamp DNA blood mini kit. The locus was amplified using PCR technique with a 54OC annealing temperature.. Alleles were separated through PAGE 6%, and visualized with silver staining. We found 10 alleles which their length varied from 145 bp to 233 bp. Allele 195 had the highest frequency that was 35.0%. The HE, HO, PIC were 0.821, 0.875, and 0.791 consecutively. Statistical analysis showed that the polymorphism of DRB3 microsatellite had a marginally significant association (p < 0.1 to the dam body weight. However, it had no significant association to the birth weight of the calves. The results of the research reflect that the DRB3-BoLA gene has a weak potency in controlling the body weight. /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; text-align:justify; line-height:150%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin;}

  6. Sperm DNA oxidative damage and DNA adducts

    Science.gov (United States)

    Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Lin, Wen-Yi

    2015-01-01

    The objective of this study was to investigate DNA damage and adducts in sperm from coke oven workers who have been exposed to polycyclic aromatic hydrocarbons. A longitudinal study was conducted with repeated measurements during spermatogenesis. Coke-oven workers (n=112) from a coke-oven plant served the PAH-exposed group, while administrators and security personnel (n=67) served the control. Routine semen parameters (concentration, motility, vitality, and morphology) were analyzed simultaneously; the assessment of sperm DNA integrity endpoints included DNA fragmentation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo). The degree of sperm DNA fragmentation was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and sperm chromatin structure assay (SCSA). The PAH-exposed group had a significant increase in bulky DNA adducts and 8-oxo-dGuo compared to the control subjects (Ps = 0.002 and 0.045, respectively). Coke oven workers' percentages of DNA fragmentation and denaturation from the PAH-exposed group were not significantly different from those of the control subjects (Ps = 0.232 and 0.245, respectively). Routine semen parameters and DNA integrity endpoints were not correlated. Concentrations of 8-oxo-dGuo were positively correlated with percentages of DNA fragmentation measured by both TUNEL and SCSA (Ps = 0.045 and 0.034, respectively). However, the concentrations of 8-oxo-dGuo and percentages of DNA fragmentation did not correlate with concentrations of bulky DNA adducts. In summary, coke oven workers with chronic exposure to PAHs experienced decreased sperm DNA integrity. Oxidative stress could contribute to the degree of DNA fragmentation. Bulky DNA adducts may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Monitoring sperm DNA integrity is recommended as a part of the process of assessing the impact of occupational and environmental toxins on

  7. Synthesis of DNA

    Science.gov (United States)

    Mariella, Jr., Raymond P.

    2008-11-18

    A method of synthesizing a desired double-stranded DNA of a predetermined length and of a predetermined sequence. Preselected sequence segments that will complete the desired double-stranded DNA are determined. Preselected segment sequences of DNA that will be used to complete the desired double-stranded DNA are provided. The preselected segment sequences of DNA are assembled to produce the desired double-stranded DNA.

  8. 大便常规检查在小儿内科作为入院常规检查的价值%Value of stool routine examination as pediatric hospitalized routine examination

    Institute of Scientific and Technical Information of China (English)

    宋钊; 马沛然; 汪翼; 王玉林

    2013-01-01

    Objective: To investigate the necessity of stool exam as a routine exam in pediatric. Methods : By retrospective analysis 2019 hospitalized children, the positive rate, unchecked rate of stool sample, the relationship between gastrointestinal symptom and stool routine examination were studied. Results: Parent reject rate was 18. 57% , and 6. 19% of all examed stool samples was positive. The positive rate of children under 5 years old was higher than that of children over 5 years old. The stool exam positive rate of children with digestive tract symptoms was 41. 08% (76/185) , and that of children without digestive tract symptoms was 1. 17% (15/1285) . More and more parents refused to stool routine examination as the number of hospitalization is increased. Eggs, dysenter y, amebic trophozoites and cysts and colonic entamoeba trophozoites and cysts were not detected by stool examina tion. Conclusion: Because the living standard is improved, everybody understand more about the disease and the situation of children common diseases is more complicated. So it was not necessary for routine stool as as pediatric hospitalized routine examination for each hospitalized children. Only checkfor the sick children with gastrointestinal symptoms is enough to reduce costs and the heavy workloads of the medical staff.%目的:探讨大便常规检查继续作为小儿内科常规检查是否必要.方法:回顾性分析2 019例住院患儿的病例资料,统计大便常规阳性率、大便常规未检查率、消化系统症状及体征与大便常规检查结果的关系.结果:大便检查总体结果阳性率为6.19%,家长拒绝率为18.57%.5岁以下患儿大便常规检查阳性率高于5岁以上患儿,有消化道症状和体征患儿的大便常规检查结果阳性率为41.08% (76/185),无消化道症状和体征患儿的大便常规检查结果阳性率为1.17% (15/1 285).随着患儿住院次数的增多,家长拒绝大便常规检查比例升高.虫卵、痢疾

  9. Prevalence of Entamoeba histolytica -like cysts compared to E. histolytica antigens detected by ELISA in the stools of 600 patients from three socioeconomic communities in the Metropolitan City of Lahore, Pakistan.

    Science.gov (United States)

    Alam, Muhammad Azhar; Maqbool, Azhar; Nazir, Muhammad Mudasser; Lateef, Muhammad; Khan, Muhammad Sarwar; Ahmed, Atif Nisar; Ziaullah, M; Lindsay, David S

    2015-04-01

    Amoebiasis, caused by Entamoeba histolytica , has a worldwide distribution and is of public health significance in many developing countries. It has a fecal-oral transmission cycle and is most prevalent in developing countries in regions where substandard sanitary conditions exist due to poverty. Little is known about the epidemiology of E. histolytica infection and its presence in different socioeconomic communities in developing countries. We undertook the present study in the city of Lahore, Pakistan, and our prediction was that the prevalence of E. histolytica -like cysts and E. histolytica stool antigen would be lower in patients from upper socioeconomic levels than in individuals from middle or lower socioeconomic levels. We investigated the prevalence of E. histolytica in humans from 3 socioeconomic communities in territories of Lahore, Pakistan. Six hundred fecal samples were collected and examined using both microscopy (triple fecal test) to detect cysts of E. histolytica -like amoeba and ELISA (stool antigen ELISA) to demonstrate diagnostic stool antigens of E. histolytica . Samples were from individuals living under conditions deemed to be upper socioeconomic class (n = 287), middle socioeconomic class (n = 172), and lower socioeconomic class (n = 141). The total prevalence of positive samples was 22.5% (135/600) by triple test and 16.8% (101/600) by stool antigen ELISA in the 600 fecal samples. Statistically, significant (P histolytica antigens than were samples from the other 3 age groups, and that prevalence was significantly higher (P < 0.05) in the summer than in the other 3 seasons. These results highlight the importance of surveillance of this relatively ignored pathogen in this developing metropolitan city in Pakistan.

  10. [Recommendations for the use of faecal microbiota transplantation "stool transplantation": consensus of the Austrian Society of Gastroenterology and Hepatology (ÖGGH) in cooperation with the Austrian Society of Infectious Diseases and Tropical Medicine].

    Science.gov (United States)

    Kump, P K; Krause, R; Steininger, C; Gröchenig, H P; Moschen, A; Madl, C; Novacek, G; Allerberger, F; Högenauer, C

    2014-12-01

    The intestinal microbiota has a pivotal role in the maintenance of health of the human organism, especially in the defense against pathogenic microorganisms. Alterations in the microbiota, also termed dysbiosis, seem to be involved in the pathogenesis of a variety of intestinal and extraintestinal diseases. Fecal microbiota transplantation (FMT), also known as stool transplantation, is a therapeutic procedure aiming at restoring an altered intestinal microbiota by administration of stool microorganisms from a healthy donor into the intestinal tract of a patient. FMT is most commonly used for recurrent forms of Clostridium difficile infections (CDI). There are currently many cohort studies in a large number of patients and a randomized controlled trial showing a dramatic effect of FMT for this indication. Therefore FMT is recommended by international medical societies for the treatment of recurrent CDI with high scientific evidence. Other potential indications are the treatment of fulminant CDI or the treatment of inflammatory bowel diseases. In the practical utilization of FMT there are currently several open questions regarding the screening of stool donors, the processing of stool and the mode of FMT application. Different modes of FMT application have been described, the application into the colon has to be preferred due to less reported side effects than the application into the upper gastrointestinal tract. So far only very few side effects due to FMT have been reported, nevertheless the use and risks of FMT are currently intensely debated in the medical community. This consensus report of the Austrian society of gastroenterology and hepatology (ÖGGH) in cooperation with the Austrian society of infectious diseases and tropical medicine provides instructions for physicians who want to use FMT which are based on the current medical literature.

  11. Método rápido para la observación de Cryptosporidium en heces Rapid method for detection of cryptosporidium in stools

    Directory of Open Access Journals (Sweden)

    Graciela Barona

    1993-02-01

    Full Text Available Entre agosto de 1990 y diciembre de 1991 se examinaron 120 muestras de materia fecal de niños o adultos que consultaron por diarrea, sugestiva de ser causada por Cryptosporidium spp. En todos los casos se realizó la coloración con Lugol-Nigroslna, que proponemos, y se hizo la confirmación con la de Ziehl Neelsen modificada, pese a su limitación de teñir con el mismo patrón de coloración el Cryptosporldium y estructuras diferentes a él. En 20 (16.6% muestras (12 de niños y 8 de adultos se identificaron ooquistes de Cryptosporldlum spp y todas se confirmaron como positivas por la coloración de Ziehl Neelsen modificada. Dado que no siempre es fácil la observación de parásitos de poca prevalencia sugerimos esta coloración como ensayo de rutina porque ayuda a distinguir los ooquistes de Cryptosporidium y mejora la observación de todos los protozoarios.

    We examined 120 stool specimens from patients with diarrheal disease, suspected of being infected with Cryptosporidium. Preliminary observation was made with a Lugol-Nigrosine stain and confirmation with modified Ziehl-Neelsen. Twenty specimens (12 from children and 8 from adults (16.6% were positive for Cryptosporidium oocysts andevery one of them was confirmed with ZN stain. Since It may be difficult to detect low-prevalence parasites we suggest routine use of Lugol-Nigrosine which is useful for the detection of Cryptosporidium as well as of other protozoa.

  12. Simultaneous detection of Entamoeba histolytica/dispar, Giardia duodenalis and cryptosporidia by immunochromatographic assay in stool samples from patients living in the Greater Cairo Region, Egypt.

    Science.gov (United States)

    Banisch, Dagmar M; El-Badry, Ayman; Klinnert, Jorge V; Ignatius, Ralf; El-Dib, Nadia

    2015-08-01

    Gastrointestinal infection due to intestinal parasites is an enormous health problem in developing countries and its reliable diagnosis is demanding. Therefore, this study aimed at evaluating a commercially available immunochromatographic assay (ICA) for the detection of cryptosporidia, Giardia duodenalis, and Entamoeba histolytica/dispar for its usefulness in the Greater Cairo Region, Egypt. Stool samples of 104 patients who presented between October 2012 and March 2013 with gastrointestinal symptoms or for the exclusion of parasites at Kasr-Al-Ainy University Medical School were examined by light microscopy of wet mounts and the triple ICA. Microscopy revealed in 20% of the patients [95% confidence interval (CI), 13.5-29.0%] parasites with Hymenolepis nana, E. histolytica/dispar and Blastocystis hominis being the most frequent ones, but was not able to detect G. duodenalis and cryptosporidia, whereas ICA was positive in 21% (95% CI, 14.3-30.0%) and detected E. histolytica/dispar in 12.5% (95% CI, 7.3-20.4%), cryptosporidia in 6.7% (95% CI, 3.1-13.5%) and G. duodenalis in 15.4% (95% CI, 9.6-23.6%) of the patients. Detection of one or more pathogens was associated with access to water retrieved from a well or pump (p = 0.01). Patients between 20 and 29 years of age (p = 0.08) and patients with symptoms of 5 days or longer (p = 0.07) tended to have a higher risk to be infected than patients of other age groups or with shorter-lasting symptoms. In conclusion, the ICA was easy to perform and timesaving. Importantly, it enabled the detection of cryptosporidia, which cannot be found microscopically in unstained smears, demonstrated a higher sensitivity for the detection of G. duodenalis than microscopy, and was more specific for distinguishing E. histolytica/dispar from apathogenic amoeba.

  13. Providing Quantitative Information and a Nudge to Undergo Stool Testing in a Colorectal Cancer Screening Decision Aid: A Randomized Clinical Trial.

    Science.gov (United States)

    Schwartz, Peter H; Perkins, Susan M; Schmidt, Karen K; Muriello, Paul F; Althouse, Sandra; Rawl, Susan M

    2017-08-01

    Guidelines recommend that patient decision aids should provide quantitative information about probabilities of potential outcomes, but the impact of this information is unknown. Behavioral economics suggests that patients confused by quantitative information could benefit from a "nudge" towards one option. We conducted a pilot randomized trial to estimate the effect sizes of presenting quantitative information and a nudge. Primary care patients (n = 213) eligible for colorectal cancer screening viewed basic screening information and were randomized to view (a) quantitative information (quantitative module), (b) a nudge towards stool testing with the fecal immunochemical test (FIT) (nudge module), (c) neither a nor b, or (d) both a and b. Outcome measures were perceived colorectal cancer risk, screening intent, preferred test, and decision conflict, measured before and after viewing the decision aid, and screening behavior at 6 months. Patients viewing the quantitative module were more likely to be screened than those who did not ( P = 0.012). Patients viewing the nudge module had a greater increase in perceived colorectal cancer risk than those who did not ( P = 0.041). Those viewing the quantitative module had a smaller increase in perceived risk than those who did not ( P = 0.046), and the effect was moderated by numeracy. Among patients with high numeracy who did not view the nudge module, those who viewed the quantitative module had a greater increase in intent to undergo FIT ( P = 0.028) than did those who did not. The limitations of this study were the limited sample size and single healthcare system. Adding quantitative information to a decision aid increased uptake of colorectal cancer screening, while adding a nudge to undergo FIT did not increase uptake. Further research on quantitative information in decision aids is warranted.

  14. Interactions of the "piano-stool" [ruthenium(II) (eta6-arene)(en)CL]+ complexes with water and nucleobases; ab initio and DFT study.

    Science.gov (United States)

    Futera, Zdenek; Klenko, Julia; Sponer, Judit E; Sponer, Jirí; Burda, Jaroslav V

    2009-09-01

    Piano stool ruthenium complexes of the composition [Ru(II)(eta6-arene)(en)Cl](+/2+) (en = ethylenediamine) represent an emerging class of cisplatin-analogue anticancer drug candidates. In this study, we use computational quantum chemistry to characterize the structure, stability and reactivity of these compounds. All these structures were optimized at DFT(B3LYP)/6-31G(d) level and their single point properties were determined by the MP2/6-31++G(2df,2pd) method. Thermodynamic parameters and rate constants were determined for the aquation process, as a replacement of the initial chloro ligand by water and subsequent exchange reaction of aqua ligand by nucleobases. The computations were carried out at several levels of DFT and ab initio theories (B3LYP, MP2 and CCSD) utilizing a range of bases sets (from 6-31G(d) to aug-cc-pVQZ). Excellent agreement with experimental results for aquation process was obtained at the CCSD level and reasonable match was achieved also with the B3LYP/6-31++G(2df,2pd) method. This level was used also for nucleobase-water exchange reaction where a smaller rate constant for guanine exchange was found in comparison with adenine. Although adenine follows a simple replacement mechanism, guanine complex passes by a two-step mechanism. At first, Ru-O6(G) adduct is formed, which is transformed through a chelate TS2 to the Ru-N7(G) final complex. In case of guanine, the exchange reaction is more favorable thermodynamically (releasing in total by about 8 kcal/mol) but according to our results, the rate constant for guanine substitution is slightly smaller than the analogous constant in adenine case when reaction course from local minimum is considered. Copyright 2008 Wiley Periodicals, Inc.

  15. Stool Test: H. Pylori Antigen

    Science.gov (United States)

    ... Development Infections Diseases & Conditions Pregnancy & Baby Nutrition & Fitness Emotions & Behavior School & Family Life First Aid & Safety Doctors & ... placed in tiny vials. Specific chemicals and a color developer are added. At the end of the ...

  16. Using stool antigen to screen for Helicobacter pylori in immigrants and refugees from high prevalence countries is relatively cost effective in reducing the burden of gastric cancer and peptic ulceration.

    Directory of Open Access Journals (Sweden)

    Thomas R Schulz

    Full Text Available OBJECTIVES: Refugees and immigrants from developing countries settling in industrialised countries have a high prevalence of Helicobacter pylori (H. pylori. Screening these groups for H. pylori and use of eradication therapy to reduce the future burden of gastric cancer and peptic ulcer disease is not currently recommended in most countries. We investigated whether a screening and eradication approach would be cost effective in high prevalence populations. METHODS: Nine different screening and follow-up strategies for asymptomatic immigrants from high H. pylori prevalence areas were compared with the current approach of no screening. Cost effectiveness comparisons assumed population prevalence's of H. pylori of 25%, 50% or 75%. The main outcome measure was the net cost for each cancer prevented for each strategy. Total costs of each strategy and net costs including savings from reductions in ulcers and gastric cancer were also calculated. RESULTS: Stool antigen testing with repeat testing after treatment was the most cost effective approach relative to others, for each prevalence value. The net cost per cancer prevented with this strategy was US$111,800 (assuming 75% prevalence, $132,300 (50% and $193,900 (25%. A test and treat strategy using stool antigen remained relatively cost effective, even when the prevalence was 25%. CONCLUSIONS: H. pylori screening and eradication can be an effective strategy for reducing rates of gastric cancer and peptic ulcers in high prevalence populations and our data suggest that use of stool antigen testing is the most cost effective approach.

  17. Comparison of the Triage Micro Parasite Panel and Microscopy for the Detection of Entamoeba histolytica/Entamoeba dispar, Giardia lamblia, and Cryptosporidium parvum in Stool Samples Collected in Kenya

    Directory of Open Access Journals (Sweden)

    Brett Swierczewski

    2012-01-01

    Full Text Available Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum are three of the most important parasitic causes of acute diarrhea worldwide. Laboratory diagnosis of these parasites is usually done by ova and parasite examination (O&P examination via microscopy. The sensitivity and specificity of O&P examination varies among laboratories and can be labor intensive and time consuming. The Triage Micro Parasite Panel (BioSite, San Diego, California is an enzyme immunoassay kit that can detect E. histolytica/E. dispar, G. lamblia, and C. parvum simultaneously using fresh or frozen stool. The present study evaluated the Triage Micro Parasite Panel in detecting E. histolytica/E. dispar, G. lamblia, and C. parvum compared to O&P examination in 266 stool samples collected at medical facilities in Kenya. The sensitivity and specificity results for the Triage Micro Parasite Panel were: for E. histolytica/E. dispar: 100%, 100%, G. lamblia: 100%, 100% and C. parvum: 73%, 100%. There was no evidence of cross reactivity using the kit with other parasites identified in the stool specimens. These results indicate that the Triage Micro Parasite Panel is a highly sensitive kit that can be used for screening purposes in large scale studies or outbreak investigations or as a possible alternative to O&P examination.

  18. Molecular DNA switches and DNA chips

    Science.gov (United States)

    Sabanayagam, Chandran R.; Berkey, Cristin; Lavi, Uri; Cantor, Charles R.; Smith, Cassandra L.

    1999-06-01

    We present an assay to detect single-nucleotide polymorphisms on a chip using molecular DNA switches and isothermal rolling- circle amplification. The basic principle behind the switch is an allele-specific oligonucleotide circularization, mediated by DNA ligase. A DNA switch is closed when perfect hybridization between the probe oligonucleotide and target DNA allows ligase to covalently circularize the probe. Mismatches around the ligation site prevent probe circularization, resulting in an open switch. DNA polymerase is then used to preferentially amplify the closed switches, via rolling-circle amplification. The stringency of the molecular switches yields 102 - 103 fold discrimination between matched and mismatched sequences.

  19. Application of DNA hybridization techniques in the assessment of diarrheal disease among refugess in Thailand. [Shigella; Escherichia coli; Campylobacter; Cryptosporidium

    Energy Technology Data Exchange (ETDEWEB)

    Taylor, D.N.; Echeverria, P.; Pitarangsi, C.; Seriwatana, J.; Sethabutr, O.; Bodhidatta, L.; Brown, C.; Herrmann, J.E.; Blacklow, N.R.

    1988-01-01

    The epidemiology and etiology of acute diarrheal disease were determined in a Hmong refugee camp on the Thai-Laotian border from April 11 to May 14, 1985. DNA hybridization techniques were used to detect Shigella species, enteroinvasive Escherichia coli, and enterotoxigenic E. coli. A monoclonal enzyme-linked immunosorbent assay was used to detect rotavirus, and standard microbiology was used to detect other enteropathogens. The age-specific diarrheal disease rates were 47 episodes per month per 1000 children less than five years old and 113 episodes per month per 1000 children less than one year old. Rotavirus, enterotoxigenic E. coli, Campylobacter, and Cryptosporidium were the predominant pathogens in children less than two years old. The DNA probe hybridized with 94% of 31 specimens identified as enterotoxigenic E. coli by the standard assays and with none of the specimens in which the standard assays were negative. The probe for Shigella and enteroinvasive E. coli hybridized in eight of 10 stools that contained Shigella and four of 314 stools from which Shigella and enteroinvasive E. coli were not isolated. The use of DNA probes allows specimens to be collected in remote areas with a minimum amount of equipment and technical expertise so that they can be easily transported to a central laboratory for further processing.

  20. Discovery of a novel circular single-stranded DNA virus from porcine faeces.

    Science.gov (United States)

    Sikorski, Alyssa; Argüello-Astorga, Gerardo R; Dayaram, Anisha; Dobson, Renwick C J; Varsani, Arvind

    2013-01-01

    A large number of novel single-stranded DNA (ssDNA) viruses have been characterised from various environmental sources in the last 5 years. The bulk of these have been from faecal sources, and faecal sampling is an ideal non-invasive pathogen sampling method. We characterised a novel ssDNA from a porcine faecal sample from Cass Basin of the South Island of New Zealand. The novel viral genome has two large open reading frames (ORFs), which are bidirectionally transcribed and separated by intergenic regions. The largest ORF has some degree of similarity (DNA virus (PigSCV), whereas the second-largest ORF has high similarity to the putative replication-associated protein (Rep) of ChiSCV (~50 %) and bovine stool-associated circular DNA virus (BoSCV; ~30 %). Based on genome architecture, location of putative stem-loop like elements, and maximum-likelihood phylogenetic analysis of the gene encoding the Rep protein, the novel isolate belongs to the same family of ssDNA viruses as ChiSCV and BoSCV.

  1. Increased DNA amplification success of non-invasive genetic samples by successful removal of inhibitors from faecal samples collected in the field

    DEFF Research Database (Denmark)

    Hebert, Louise; Darden, Safi K.; Pedersen, Bo Vest;

    2011-01-01

    The use of non-invasive genetic sampling (NGS) is becoming increasingly important in the study of wild animal populations. Obtaining DNA from faecal samples is of particular interest because faeces can be collected without deploying sample capture devices. However, PCR amplification of DNA...... extracted from faeces is problematic because of high concentrations of inhibitors. Here we present a method for increasing the successful application of donor DNA extracted from faecal samples through inhibitor reduction. After standard extraction with a DNA stool kit we used a ‘Concentrated Chelex...... Treatment’ (CCT) that increased the amplification success from 31.7 to 61.4% of loci. Our results suggest that darker supernatant and samples with more precipitate contain more inhibitors than lighter samples and samples with little or no precipitate. We expect the use of this technique to have wide...

  2. Infant Stool Color Card Screening Helps Reduce the Hospitalization Rate and Mortality of Biliary Atresia: A 14-Year Nationwide Cohort Study in Taiwan.

    Science.gov (United States)

    Lee, Min; Chen, Solomon Chih-Cheng; Yang, Hsin-Yi; Huang, Jui-Hua; Yeung, Chun-Yan; Lee, Hung-Chang

    2016-03-01

    Biliary atresia (BA) is a significant liver disease in children. Since 2004, Taiwan has implemented a national screening program that uses an infant stool color card (SCC) for the early detection of BA. The purpose of this study was to examine the outcomes of BA cases before and after the launch of this screening program. The objectives of this study were to evaluate the rates of hospitalization, liver transplantation (LT), and mortality of BA cases before and after the program, and to examine the association between the hospitalization rate and survival outcomes.This was a population-based cohort study. BA cases born during 1997 to 2010 were identified from the Taiwan National Health Insurance Research Database. Sex, birth date, hospitalization date, LT, and death data were collected and analyzed. The hospitalization rate by 2 years of age (Hosp/2yr) was calculated to evaluate its association with the outcomes of LT or death.Among 513 total BA cases, 457 (89%) underwent the Kasai procedure. Of these, the Hosp/2yr was significantly reduced from 6.0 to 6.9/case in the earlier cohort (1997-2004) to 4.9 to 5.3/case in the later cohort (2005-2010). This hospitalization rate reduction was followed by a reduction in mortality from 26.2% to 15.9% after 2006. The Cox proportional hazards model showed a significant increase in the risk for both LT (hazard ratio [HR] = 1.14, 95% confidence interval [CI] = 1.10-1.18) and death (HR = 1.05, 95% CI = 1.01-1.08) for each additional hospitalization. A multivariate logistic regression model found that cases with a Hosp/2yr >6 times had a significantly higher risk for both LT (adjusted odds ratio [aOR] = 4.35, 95% CI = 2.82-6.73) and death (aOR = 1.75, 95% CI = 1.17-2.62).The hospitalization and mortality rates of BA cases in Taiwan were significantly and coincidentally reduced after the launch of the SCC screening program. There was a significant association between the hospitalization rate and final

  3. Comparison of three methods for total DNA extraction from intestinal microflora of the fowl%鸡肠道菌群总DNA三种提取方法的比较

    Institute of Scientific and Technical Information of China (English)

    卢龙娣; 江胜滔; 林跃鑫

    2009-01-01

    Objective To screen a method that could provide a higher quality DNA template for molecular analysis of composition of intestinal microflora. Method Repeated freeze-thaw method, lysozyme method and E.N.Z.A Stool DNA Kit method were used to extract total DNA from intestinal microflora of the fowl. The quality of DNA extracted by the three methods was compared based on the concentration, purity, 16S DNA PCR products and length polymorphism of ERIC-PCR products. Result Total DNA extracted by any of the three methods could be used to 16S DNA amplification. But the result of ERIC-PCR of DNA extracted by the last two methods showed more microflora diversities. Conclusion The quality of total DNA extracted by E.N.Z.A Stool DNA Kit method and lysozyme method were better than that of repeated freeze-thaw method, so can both be used to study molecular ecology of intestinal microflora.%目的 比较不同方法提取鸡肠道菌群总DNA的差异, 为分子方法分析肠道菌群组成提供质量较高的DNA模板.方法 采用反复冻融法、酶裂解法和试剂盒法(E.N.Z.A Stool DNA Kit)来提取鸡肠道菌群的总DNA,并根据DNA浓度及纯度、16S DNA扩增产物和ERIC-PCR产物所反映的片段多态性4个指标,对这3种方法提取的DNA质量进行比较.结果 3种方法均能提取DNA,所得DNA都可以用于16S DNA的扩增,但后2种方法所得DNA的ERIC-PCR结果能反映出更高的菌群多样性.结论 试剂盒法和酶裂解法所提取的DNA质量好,适合用于肠道菌群的分子生态研究.

  4. DNA fragmentation in apoptosis

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Cleavage of chromosomal DNA into oligonucleosomal size fragments is an integral part of apoptosis. Elegant biochemical work identified the DNA fragmentation factor (DFF) as a major apoptotic endonuclease for DNA fragmentation in vitro. Genetic studies in mice support the importance of DFF in DNA fragmentation and possibly in apoptosis in vivo. Recent work also suggests the existence of additional endonucleases for DNA degradation. Understanding the roles of individual endonucleases in apoptosis, and how they might coordinate to degrade DNA in different tissues during normal development and homeostasis, as well as in various diseased states, will be a major research focus in the near future.

  5. ex vivo DNA assembly

    Directory of Open Access Journals (Sweden)

    Adam B Fisher

    2013-10-01

    Full Text Available Even with decreasing DNA synthesis costs there remains a need for inexpensive, rapid and reliable methods for assembling synthetic DNA into larger constructs or combinatorial libraries. Advances in cloning techniques have resulted in powerful in vitro and in vivo assembly of DNA. However, monetary and time costs have limited these approaches. Here, we report an ex vivo DNA assembly method that uses cellular lysates derived from a commonly used laboratory strain of Escherichia coli for joining double-stranded DNA with short end homologies embedded within inexpensive primers. This method concurrently shortens the time and decreases costs associated with current DNA assembly methods.

  6. Techniques for minimizing the effects of PCR inhibitors in the chytridiomycosis assay.

    Science.gov (United States)

    Kosch, T A; Summers, K

    2013-03-01

    Chytridiomycosis is an amphibian disease of global conservation concern that is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Since the discovery of Bd in 1998, several methods have been used for detection of Bd; among these polymerase chain reaction (PCR) from skin swabs is accepted as the best method due to its noninvasiveness, high sensitivity and ease of use. However, PCR is not without problems - to be successful, this technique is dependent upon the presence of nondegraded DNA template and reaction contents that are free from inhibitors. Here, we report on an investigation of several techniques aimed at improving the reliability of the Bd PCR assay by minimizing the effects of humic acid (HA), a potent PCR inhibitor. We compared the effectiveness of four DNA extraction kits (DNeasy, QIAamp DNA Stool, PowerLyzer Power Soil and PrepMan Ultra) and four PCR methods (Amplitaq Gold, bovine serum albumin, PowerClean DNA Clean-up and inhibitor resistant Taq Polymerase). The results of this and previous studies indicate that chytridiomycosis studies that use PCR methods for disease detection may be significantly underestimating the occurrence of Bd. Our results suggest that to minimize the inhibitory effects of HA, DNeasy should be used for sample DNA extraction and Amplitaq Gold with bovine serum albumin should be used for the Bd PCR assay. We also outline protocols tested, show the results of our methods comparisons and discuss the pros and cons of each method.

  7. [Biases on community structure during DNA extraction of shrimp intestinal microbiota revealed by high-throughput sequencing].

    Science.gov (United States)

    Wen, Chongqing; He, Yaoyao; Xue, Ming; Liang, Huafang; Dong, Junde

    2016-01-04

    High-throughput sequencing technology is increasingly applied in intestinal microbiota of aquatic animals including shrimp. However, there is a lack of standard method or kit for DNA isolation from shrimp intestinal microbiota, and little is known about the effectiveness and biases regarding DNA extraction based on high-throughput sequencing. The aim of this study was to study the biases of different DNA extraction kits on community structure of shrimp intestinal microbiota through high-throughput sequencing, and to better understand the structure and composition of bacterial flora associated with healthy Litopenaeus vannamei. We extracted the total DNA of intestinal microbiota from L. vannamei with three commercial kits designed for DNA extraction from bacteria, stool and tissue (Omega, USA). DNA quality was evaluated based on the absorbance ratios of 260/280 nm by NanoDrop, while DNA concentration was quantified using PicoGreen. Then Illumina MiSeq high-throughput sequencing was used to examine the intestinal bacterial communities following PCR amplification of 16S rDNA V4 region. The yield and purity of the DNA from the Bacterial Kit (SIB) were superior to those from the Stool Kit (SIS), whereas the DNA from Tissue Kit (SIT) presented too small amount to be amplified efficiently. The average sequence reads obtained from SIB and SIS samples were 52151 ± 5085 and 55296 ± 5147 respectively. After resampling at the same depth of 46800 reads, the operational taxonomic unit (OTU) number and Shannon diversity index of SIS samples were significantly higher than those of SIB samples. By contrast, the reproducibility of OTU among SIB replicates was higher than that among SIS replicates. The dominant phyla of SIS and SIB samples were identical, including Proteobacteria, Firmicutes, Bacteroidetes, Planctomycetes, Actinobacteria, and Cyanobacteria. However, the relative abundances of almost all the dominant groups at various taxonomic levels differed greatly between these

  8. Less is More--Optimization of DNA Extraction from Canine Feces.

    Science.gov (United States)

    Lindquist, Christina D; Wictum, Elizabeth J

    2016-01-01

    Although most DNA crime laboratories may not encounter fecal samples often, they are a familiar sample type in non-human forensic laboratories due to their prevalence in the environment. Fecal matter can be challenging due to low numbers of nucleated cells and the presence of inhibitors that impede amplification success. Sampling location (internal vs. external), sampling quantity (10-200 mg), and various extraction protocols (silica matrix, bead beating, and clean-up column) were evaluated to maximize DNA yield. The greatest yield of intact DNA was obtained using a modified silica matrix extraction protocol (VGL-Fecal) on 30-50 mg of fecal matter collected from the external surface of a stool that had been dried for 24 h. This optimized sampling and extraction protocol was applied to a pilot study where DNA yield and genotyping success were evaluated. By optimizing our collection, sampling, and extraction procedures, a reliable method for maximizing the yield of canine fecal DNA was developed. © 2015 American Academy of Forensic Sciences.

  9. Rapid microfluidic solid-phase extraction system for hyper-methylated DNA enrichment and epigenetic analysis.

    Science.gov (United States)

    De, Arpita; Sparreboom, Wouter; van den Berg, Albert; Carlen, Edwin T

    2014-09-01

    Genetic sequence and hyper-methylation profile information from the promoter regions of tumor suppressor genes are important for cancer disease investigation. Since hyper-methylated DNA (hm-DNA) is typically present in ultra-low concentrations in biological samples, such as stool, urine, and saliva, sample enrichment and amplification is typically required before detection. We present a rapid microfluidic solid phase extraction (μSPE) system for the capture and elution of low concentrations of hm-DNA (≤1 ng ml(-1)), based on a protein-DNA capture surface, into small volumes using a passive microfluidic lab-on-a-chip platform. All assay steps have been qualitatively characterized using a real-time surface plasmon resonance (SPR) biosensor, and quantitatively characterized using fluorescence spectroscopy. The hm-DNA capture/elution process requires less than 5 min with an efficiency of 71% using a 25 μl elution volume and 92% efficiency using a 100 μl elution volume.

  10. [Uracil-DNA glycosylases].

    Science.gov (United States)

    Pytel, Dariusz; Słupianek, Artur; Ksiazek, Dominika; Skórski, Tomasz; Błasiak, Janusz

    2008-01-01

    Uracil is one of four nitrogen bases, most frequently found in normal RNA. Uracyl can be found also in DNA as a result of enzymatic or non-enzymatic deamination of cytosine as well as misincorporation of dUMP instead of dTMP during DNA replication. Uracil from DNA can be removed by DNA repair enzymes with apirymidine site as an intermediate. However, if uracil is not removed from DNA a pair C:G in parental DNA can be changed into a T:A pair in the daughter DNA molecule. Therefore, uracil in DNA may lead to a mutation. Uracil in DNA, similarly to thymine, forms energetically most favorable hydrogen bonds with adenine, therefore uracil does not change the coding properties of DNA. Uracil in DNA is recognized by uracil DNA glycosylase (UDGs), which initiates DNA base excision repair, leading to removing of uracil from DNA and replacing it by thymine or cytosine, when arose as a result of cytosine deamination. Eukaryotes have at least four nuclear UDGs: UNG2, SMUG1, TDG i MBD4, while UNG1 operates in the mitochondrium. UNG2 is involved in DNA repair associated with DNA replication and interacts with PCNA and RPA proteins. Uracil can also be an intermediate product in the process of antigen-dependent antibody diversification in B lymphocytes. Enzymatic deamination of viral DNA by host cells can be a defense mechanism against viral infection, including HIV-1. UNG2, MBD4 and TDG glycosylases may cooperate with mismatch repair proteins and TDG can be involved in nucleotide excision repair system.

  11. Force induced DNA melting

    Energy Technology Data Exchange (ETDEWEB)

    Santosh, Mogurampelly; Maiti, Prabal K [Center for Condensed Matter Theory, Department of Physics, Indian Institute of Science, Bangalore-12 (India)], E-mail: santosh@physics.iisc.ernet.in, E-mail: maiti@physics.iisc.ernet.in

    2009-01-21

    When pulled along the axis, double-strand DNA undergoes a large conformational change and elongates by roughly twice its initial contour length at a pulling force of about 70 pN. The transition to this highly overstretched form of DNA is very cooperative. Applying a force perpendicular to the DNA axis (unzipping), double-strand DNA can also be separated into two single-stranded DNA, this being a fundamental process in DNA replication. We study the DNA overstretching and unzipping transition using fully atomistic molecular dynamics (MD) simulations and argue that the conformational changes of double-strand DNA associated with either of the above mentioned processes can be viewed as force induced DNA melting. As the force at one end of the DNA is increased the DNA starts melting abruptly/smoothly above a critical force depending on the pulling direction. The critical force f{sub m}, at which DNA melts completely decreases as the temperature of the system is increased. The melting force in the case of unzipping is smaller compared to the melting force when the DNA is pulled along the helical axis. In the case of melting through unzipping, the double-strand separation has jumps which correspond to the different energy minima arising due to sequence of different base pairs. The fraction of Watson-Crick base pair hydrogen bond breaking as a function of force does not show smooth and continuous behavior and consists of plateaus followed by sharp jumps.

  12. DNA damage and autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez-Rocha, Humberto; Garcia-Garcia, Aracely [Redox Biology Center and School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583 (United States); Panayiotidis, Mihalis I. [School of Community Health Sciences, University of Nevada, Reno, NV 89557 (United States); Franco, Rodrigo, E-mail: rfrancocruz2@unl.edu [Redox Biology Center and School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583 (United States)

    2011-06-03

    Both exogenous and endogenous agents are a threat to DNA integrity. Exogenous environmental agents such as ultraviolet (UV) and ionizing radiation, genotoxic chemicals and endogenous byproducts of metabolism including reactive oxygen species can cause alterations in DNA structure (DNA damage). Unrepaired DNA damage has been linked to a variety of human disorders including cancer and neurodegenerative disease. Thus, efficient mechanisms to detect DNA lesions, signal their presence and promote their repair have been evolved in cells. If DNA is effectively repaired, DNA damage response is inactivated and normal cell functioning resumes. In contrast, when DNA lesions cannot be removed, chronic DNA damage triggers specific cell responses such as cell death and senescence. Recently, DNA damage has been shown to induce autophagy, a cellular catabolic process that maintains a balance between synthesis, degradation, and recycling of cellular components. But the exact mechanisms by which DNA damage triggers autophagy are unclear. More importantly, the role of autophagy in the DNA damage response and cellular fate is unknown. In this review we analyze evidence that supports a role for autophagy as an integral part of the DNA damage response.

  13. Effect of room temperature transport vials on DNA quality and phylogenetic composition of faecal microbiota of elderly adults and infants.

    Science.gov (United States)

    Hill, Cian J; Brown, Jillian R M; Lynch, Denise B; Jeffery, Ian B; Ryan, C Anthony; Ross, R Paul; Stanton, Catherine; O'Toole, Paul W

    2016-05-10

    Alterations in intestinal microbiota have been correlated with a growing number of diseases. Investigating the faecal microbiota is widely used as a non-invasive and ethically simple proxy for intestinal biopsies. There is an urgent need for collection and transport media that would allow faecal sampling at distance from the processing laboratory, obviating the need for same-day DNA extraction recommended by previous studies of freezing and processing methods for stool. We compared the faecal bacterial DNA quality and apparent phylogenetic composition derived using a commercial kit for stool storage and transport (DNA Genotek OMNIgene GUT) with that of freshly extracted samples, 22 from infants and 20 from older adults. Use of the storage vials increased the quality of extracted bacterial DNA by reduction of DNA shearing. When infant and elderly datasets were examined separately, no differences in microbiota composition were observed due to storage. When the two datasets were combined, there was a difference according to a Wilcoxon test in the relative proportions of Faecalibacterium, Sporobacter, Clostridium XVIII, and Clostridium XlVa after 1 week's storage compared to immediately extracted samples. After 2 weeks' storage, Bacteroides abundance was also significantly different, showing an apparent increase from week 1 to week 2. The microbiota composition of infant samples was more affected than that of elderly samples by storage, with significantly higher Spearman distances between paired freshly extracted and stored samples (p microbiota profiles were analysed at the operational taxonomic unit (OTU) level, three infant datasets in the study did not cluster together, while only one elderly dataset did not. The lower microbiota diversity of the infant gut microbiota compared to the elderly gut microbiota (p microbiota samples, but may be less appropriate for lower diversity samples. Differences between fresh and stored samples mean that where storage is

  14. 不同操作平台、染料间高分辨率熔解曲线分析技术检测烘便基因突变的性能差异%High-resolution melting curve analysis in stool mutation detection: performance differences in different opera ting platforms and dyes

    Institute of Scientific and Technical Information of China (English)

    李丙生; 许岸高; 甘爱华; 李琛; 余志金; 张晓慧

    2011-01-01

    目的 探讨在不同操作平台、染料间高分辨率熔解曲线分析(HRM)技术检测粪便基因突变的性能差异.方法 收集合格的粪便125份,其中大肠癌40份,晚期腺瘤35份,正常对照50份,在LightCycler 480与Rotor-Gene 6000平台设备上,分别使用Resolight或Eva Green染料,检测K-ras基因突变率的差异,所有样品均行测序.结果 测序发现,大肠癌组、晚期腺瘤组、正常对照组K-ras基因突变率分别是40%、31.4%、0%.在LightCycler 480平台,用Resolight/Eva Green染料突变检出率分别为40%/45%、31.4%/34.2%、0%/0%.在Rotor-Gene 6000平台,用Resolight/Eva Green染料突变检出率分别为42.5%/47.5%、34.2%/37.1%、0%/0%.在各组中,不同操作平台、染料间突变检出率差异无统计学意义(P>0.05).HRM检测粪便基因突变敏感度达100%.结论 HRM在不同操作平台、染料间的突变检出率差异无显著性.%Objective To evaluate the performance of high- resolution melting curve analysis ( HRM ) in detection of fecal mutations on different operating platforms and dyes. Methods A total of 125 qualified stool samples were collected from different subjects, including patients with colorectal cancer ( n = 40 ) and advanced adenoma ( n = 35 ), and normal controls ( n = 50 ). On both Light - Cycler 480 and Rotor - Gene 6000 platforms, Resolight or Eva Green dye was used in detection of K - ras mutation. DNA sequencing was applied as gold standard. Results DNA sequencing showed that K - ras gene mutation rates were 40, 31. 4 and 0% in colorectal cancer, advanced adenomas, and normal controls, respectively. On Light -Cycler 480 platform with Resolight/EvaGreen dye, K-ras gene mutation rates were 40%/45% , 31. 4%/34. 2% , and 0%/0% ; whereas in the Rotor-Gene 6000 platform with Resolight/EvaGreen dye, K -ras gene mutation rates were 42. 5%/47. 5% , 34. 2%/37. 1% , and 0%/0% . In each group, there was no significant difference among different operating platforms with different

  15. DNA tagged microparticles

    Energy Technology Data Exchange (ETDEWEB)

    Farquar, George Roy; Leif, Roald N; Wheeler, Elizabeth

    2015-05-05

    A simulant that includes a carrier and DNA encapsulated in the carrier. Also a method of making a simulant including the steps of providing a carrier and encapsulating DNA in the carrier to produce the simulant.

  16. Modeling DNA Replication.

    Science.gov (United States)

    Bennett, Joan

    1998-01-01

    Recommends the use of a model of DNA made out of Velcro to help students visualize the steps of DNA replication. Includes a materials list, construction directions, and details of the demonstration using the model parts. (DDR)

  17. DNA computing models

    CERN Document Server

    Ignatova, Zoya; Zimmermann, Karl-Heinz

    2008-01-01

    In this excellent text, the reader is given a comprehensive introduction to the field of DNA computing. The book emphasizes computational methods to tackle central problems of DNA computing, such as controlling living cells, building patterns, and generating nanomachines.

  18. Forensic DNA and bioinformatics

    National Research Council Canada - National Science Library

    Bianchi, Lucia; Liò, Pietro

    The field of forensic science is increasingly based on biomolecular data and many European countries are establishing forensic databases to store DNA profiles of crime scenes of known offenders and apply DNA testing...

  19. DNA tagged microparticles

    Science.gov (United States)

    Farquar, George Roy; Leif, Roald N; Wheeler, Elizabeth

    2015-05-05

    A simulant that includes a carrier and DNA encapsulated in the carrier. Also a method of making a simulant including the steps of providing a carrier and encapsulating DNA in the carrier to produce the simulant.

  20. Click chemistry with DNA

    OpenAIRE

    El-Sagheer, Afaf H.; Brown, Tom

    2010-01-01

    The advent of click chemistry has led to an influx of new ideas in the nucleic acids field. The copper catalysed alkyne–azide cycloaddition (CuAAC) reaction is the method of choice for DNA click chemistry due to its remarkable efficiency. It has been used to label oligonucleotides with fluorescent dyes, sugars, peptides and other reporter groups, to cyclise DNA, to synthesise DNA catenanes, to join oligonucleotides to PNA, and to produce analogues of DNA with modified nucleobases and backbone...

  1. Application effects of quality control circle in quality control before analysis of stool and urine specimen%品管圈在大小便标本分析前质量控制中的应用效果

    Institute of Scientific and Technical Information of China (English)

    徐丽雅

    2016-01-01

    目的:通过品管圈活动及管理工具的运用,提高临床大小便标本分析前质量,降低标本收集及送检缺陷率的发生。方法2013年5月成立7人小组的品管圈,通过选定主题、制定活动计划、解析问题、拟定对策、实施措施等步骤,运用质量管理工具对大小便标本分析前存在的诸多原因进行分析,制定流程改善系统,并比较改善前后效果。结果本科室活动改善前大小便标本分析前标本缺陷发生率为1.69%,实施品管圈活动改善后缺陷发生率降至0.62%,完成目标值设定。结论通过品管圈活动干预可有效降低科室大小便标本缺陷发生率,提高送检质量。%Objective To improve the quality control before analysis of stool and urine specimen and to reduce the rate of defect in specimen collection and being submitted for examination by activities of quality control circle and management tools. Methods The quality control circle composed of 7 persons which was established in May 2013. Various reasons existed before analysis of stool and urine specimen were discussed with quality management tools by the process including selecting the topic, drawing up an activity program, explaining problems, generating responses, drafting countermeasures, implementing measures and so on. The system to improve the process was developed. Effects were compared before and after the improvement. Results The rate of defect before analysis of stool and urine specimen was 1. 69% before improvement and was 0. 62% after implementing activities of quality control circle with achieving target values. Conclusions The quality control circle activities can effectively reduce the defect rate of stool and urine specimen and the quality of being submitted for examination.

  2. Real-Time TaqMan PCR Assay for the Detection of Heat-Labile and Heat-Stable Enterotoxin Genes in a Geographically Diverse Collection of Enterotoxigenic Escherichia coli Strains and Stool Specimens.

    Science.gov (United States)

    Pattabiraman, Vaishnavi; Parsons, Michele B; Bopp, Cheryl A

    2016-04-01

    Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrhea in children under the age of 5 years in developing countries and are the leading bacterial agent of traveler's diarrhea in persons traveling to these countries. ETEC strains secrete heat-labile (LT) and/or heat-stable (ST) enterotoxins that induce diarrhea by causing water and electrolyte imbalance. We describe the validation of a real-time TaqMan PCR (RT-PCR) assay to detect LT, ST1a, and ST1b enterotoxin genes in E. coli strains and in stool specimens. We validated LT/ST1b duplex and ST1a single-plex RT-PCR assay using a conventional PCR assay as a gold standard with 188 ETEC strains and 42 non-ETEC strains. We validated LT/ST1b duplex and ST1a single-plex RT-PCR assay in stool specimens (n = 106) using traditional culture as the gold standard. RT- PCR assay sensitivities for LT, ST1a, and ST1b detection in strains were 100%, 100%, and 98%; specificities were 95%, 98%, and 99%, and Pearson correlation coefficient r was 0.9954 between RT-PCR assay and the gold standard. In stool specimens, RT-PCR assay sensitivities for LT, ST1a, and ST1b detection were 97%, 100%, and 97%; and specificities were 99%, 94%, and 97%. Pearson correlation coefficient r was 0.9975 between RT-PCR results in stool specimens and the gold standard. Limits of detection of LT, ST1a, and ST1b by RT-PCR assay were 0.1 to1.0 pg/μL and by conventional PCR assay were 100 to1000 pg/μL. The accuracy, rapidity and sensitivity of this RT-PCR assay is promising for ETEC detection in public health/clinical laboratories and for laboratories in need of an independent method to confirm results of other culture independent diagnostic tests.

  3. Scientific Opinion on the substantiation of a health claim related to “native chicory inulin” and maintenance of normal defecation by increasing stool frequency pursuant to Article 13.5 of Regulation (EC) No 1924/2006

    DEFF Research Database (Denmark)

    Tetens, Inge

    Following an application from BENEO-Orafti S.A., submitted pursuant to Article 13.5 of Regulation (EC) No 1924/2006 via the Competent Authority of Belgium, the Panel on Dietetic Products, Nutrition and Allergies (NDA) was asked to deliver an opinion on the scientific substantiation of a health...... the claimed effect. The Panel concludes that a cause and effect relationship has been established between the consumption of “native chicory inulin” and maintenance of normal defecation by increasing stool frequency. The following wording reflects the scientific evidence: “Chicory inulin contributes...

  4. Replicating animal mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    Emily A. McKinney

    2013-01-01

    Full Text Available The field of mitochondrial DNA (mtDNA replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading-and lagging-strand synthesis (resembling bacterial genome replication and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS. The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase y, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase. Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells.

  5. Three-Dimensional DNA Nanostructures Assembled from DNA Star Motifs.

    Science.gov (United States)

    Tian, Cheng; Zhang, Chuan

    2017-01-01

    Tile-based DNA self-assembly is a promising method in DNA nanotechnology and has produced a wide range of nanostructures by using a small set of unique DNA strands. DNA star motif, as one of DNA tiles, has been employed to assemble varieties of symmetric one-, two-, three-dimensional (1, 2, 3D) DNA nanostructures. Herein, we describe the design principles, assembly methods, and characterization methods of 3D DNA nanostructures assembled from the DNA star motifs.