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

PubMed Central

Desneux, Jérémy; Pourcher, Anne-Marie

2014-01-01

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. PMID:24838631

Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy.

PubMed

Moen, Aina E F; Tannæs, Tone M; Vatn, Simen; Ricanek, Petr; Vatn, Morten Harald; Jahnsen, Jørgen

2016-06-28

Nucleic acid purification methods are of importance when performing microbiota studies and especially when analysing the intestinal microbiota as we here find a wide range of different microbes. Various considerations must be taken to lyse the microbial cell wall of each microbe. In the present article, we compare several tissue lysis steps and commercial purification kits, to achieve a joint RNA and DNA purification protocol for the purpose of investigating the microbiota and the microbiota-host interactions in a single colonic mucosal tissue sample. A further optimised tissue homogenisation and lysis protocol comprising mechanical bead beating, lysis buffer replacement and enzymatic treatment, in combination with the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) resulted in efficient and simultaneous purification of microbial and human RNA and DNA from a single mucosal colonic tissue sample. The present work provides a unique possibility to study RNA and DNA from the same mucosal biopsy sample, making a direct comparison between metabolically active microbes and total microbial DNA. The protocol also offers an opportunity to investigate other members of a microbiota such as viruses, fungi and micro-eukaryotes, and moreover the possibility to extract data on microbiota and host interactions from one single mucosal biopsy.

One-Step Purification of Enterocytozoon bieneusi Spores from Human Stools by Immunoaffinity Expanded-Bed Adsorption

PubMed Central

Accoceberry, Isabelle; Thellier, Marc; Datry, Annick; Desportes-Livage, Isabelle; Biligui, Sylvestre; Danis, Martin; Santarelli, Xavier

2001-01-01

An original, reliable, and reproducible method for the purification of Enterocytozoon bieneusi spores from human stools is described. We recently reported the production of a species-specific monoclonal antibody (MAb) 6E52D9 immunoglobulin G2a (IgG2a) raised against the exospore of E. bieneusi spore walls. The MAb was used as a ligand to develop an immunoaffinity matrix. The mouse IgG2a MAb was bound directly to a Streamline rProtein A adsorbent, used for expanded-bed adsorption of immunoglobulins, for optimal spatial orientation of the antibody and maximum binding efficiency of the antigen. The complex was then cross-linked covalently using dimethyl pimelimidate dihydrochloride. After incubation of the immunoaffinity matrix with filtered stool samples containing numerous E. bieneusi spores and before elution with 6 M guanidine HCl, the expansion of the adsorbent bed eliminated all the fecal contaminants. The presence of spores in the elution fractions was determined by an indirect immunofluorescence antibody test (IFAT). E. bieneusi spores were found in the elution fraction in all four experiments and were still highly antigenic as indicated by IFAT. Smears examined by light microscopy contained very clean spores with no fecal debris or background bacterial and fungal contaminants. However, spore recovery rates were relatively low: an average of 107 spores were purified per run. This technique for isolating E. bieneusi spores directly from human stool samples with a high degree of purity opens up new approaches for studying this parasite. PMID:11326019

Evaluation of SD BIOLINE H. pylori Ag rapid test against double ELISA with SD H. pylori Ag ELISA and EZ-STEP H. pylori Ag ELISA tests.

PubMed

Negash, Markos; Kassu, Afework; Amare, Bemnet; Yismaw, Gizachew; Moges, Beyene

2018-01-01

Helicobacter pylori antibody titters fall very slowly even after successful treatment. Therefore, tests detecting H. pylori antibody lack specificity and sensitivity. On the other hand, H. pylori stool antigen tests are reported as an alternative assay because of their reliability and simplicity. However, the comparative performance of H. pylori stool antigen tests for detecting the presence of the bacterium in clinical specimens in the study area is not assessed. Therefore, in this study we evaluated the performance of SD BIOLINE H. pylori Ag rapid test with reference to the commercially available EZ- STEP ELISA and SD BIOLINE H. pylori Ag ELISA tests. Stool samples were collected to analyse the diagnostic performance of SD BIOLINE H. pylori Ag rapid test kit using SD H. pylori Ag ELISA kit and EZ- STEP ELISA tests as a gold standard. Serum samples were also collected from each patient to test for the presence of H. pylori antibodies using dBest H. pylori Test Disk. Sensitivity, specificity, predictive values and kappa value are assessed. P values < 0.05 were taken statistically significant. Stool and serum samples were collected from 201 dyspeptic patients and analysed. The sensitivity, specificity, positive and negative predictive values of the SD BIOLINE H. pylori Ag rapid test were: 95.6% (95% CI, 88.8-98.8), 92.5% (95%CI, 89-94.1%), 86.7% (95% CI, 80.5-89.6), and 97.6% (95% CI, 993.9-99.3) respectively. The performance of SD BIOLINE H. pylori Ag rapid test was better than the currently available antibody test in study area. Therefore, the SD BIOLINE Ag rapid stool test could replace and be used to diagnose active H. pylori infection before the commencement of therapy among dyspeptic patients.

Effective removal of co-purified inhibitors from extracted DNA samples using synchronous coefficient of drag alteration (SCODA) technology.

PubMed

Schmedes, Sarah; Marshall, Pamela; King, Jonathan L; Budowle, Bruce

2013-07-01

Various types of biological samples present challenges for extraction of DNA suitable for subsequent molecular analyses. Commonly used extraction methods, such as silica membrane columns and phenol-chloroform, while highly successful may still fail to provide a sufficiently pure DNA extract with some samples. Synchronous coefficient of drag alteration (SCODA), implemented in Boreal Genomics' Aurora Nucleic Acid Extraction System (Boreal Genomics, Vancouver, BC), is a new technology that offers the potential to remove inhibitors effectively while simultaneously concentrating DNA. In this initial study, SCODA was tested for its ability to remove various concentrations of forensically and medically relevant polymerase chain reaction (PCR) inhibitors naturally found in tissue, hair, blood, plant, and soil samples. SCODA was used to purify and concentrate DNA from intentionally contaminated DNA samples containing known concentrations of hematin, humic acid, melanin, and tannic acid. The internal positive control (IPC) provided in the Quantifiler™ Human DNA Quantification Kit (Life Technologies, Foster City, CA) and short tandem repeat (STR) profiling (AmpFℓSTR® Identifiler® Plus PCR Amplification Kit; Life Technologies, Foster City, CA) were used to measure inhibition effects and hence purification. SCODA methodology yielded overall higher efficiency of purification of highly contaminated samples compared with the QIAquick® PCR Purification Kit (Qiagen, Valencia, CA). SCODA-purified DNA yielded no cycle shift of the IPC for each sample and yielded greater allele percentage recovery and relative fluorescence unit values compared with the QIAquick® purification method. The Aurora provided an automated, minimal-step approach to successfully remove inhibitors and concentrate DNA from challenged samples.

PURIFICATION OF ENTEROCYTOZOON BIENEUSI SPORES FROM STOOL SPECIMENS BY GRADIENT AND CELL SORTING TECHNIQUES. (R828042)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses

PubMed Central

Lim, Natalie Y. N.; Roco, Constance A.; Frostegård, Åsa

2016-01-01

Adequate comparisons of DNA and cDNA libraries from complex environments require methods for co-extraction of DNA and RNA due to the inherent heterogeneity of such samples, or risk bias caused by variations in lysis and extraction efficiencies. Still, there are few methods and kits allowing simultaneous extraction of DNA and RNA from the same sample, and the existing ones generally require optimization. The proprietary nature of kit components, however, makes modifications of individual steps in the manufacturer’s recommended procedure difficult. Surprisingly, enzymatic treatments are often performed before purification procedures are complete, which we have identified here as a major problem when seeking efficient genomic DNA removal from RNA extracts. Here, we tested several DNA/RNA co-extraction commercial kits on inhibitor-rich soils, and compared them to a commonly used phenol-chloroform co-extraction method. Since none of the kits/methods co-extracted high-quality nucleic acid material, we optimized the extraction workflow by introducing small but important improvements. In particular, we illustrate the need for extensive purification prior to all enzymatic procedures, with special focus on the DNase digestion step in RNA extraction. These adjustments led to the removal of enzymatic inhibition in RNA extracts and made it possible to reduce genomic DNA to below detectable levels as determined by quantitative PCR. Notably, we confirmed that DNase digestion may not be uniform in replicate extraction reactions, thus the analysis of “representative samples” is insufficient. The modular nature of our workflow protocol allows optimization of individual steps. It also increases focus on additional purification procedures prior to enzymatic processes, in particular DNases, yielding genomic DNA-free RNA extracts suitable for metatranscriptomic analysis. PMID:27803690

A robust ambient temperature collection and stabilization strategy: Enabling worldwide functional studies of the human microbiome

PubMed Central

Anderson, Ericka L.; Li, Weizhong; Klitgord, Niels; Highlander, Sarah K.; Dayrit, Mark; Seguritan, Victor; Yooseph, Shibu; Biggs, William; Venter, J. Craig; Nelson, Karen E.; Jones, Marcus B.

2016-01-01

As reports on possible associations between microbes and the host increase in number, more meaningful interpretations of this information require an ability to compare data sets across studies. This is dependent upon standardization of workflows to ensure comparability both within and between studies. Here we propose the standard use of an alternate collection and stabilization method that would facilitate such comparisons. The DNA Genotek OMNIgene∙Gut Stool Microbiome Kit was compared to the currently accepted community standard of freezing to store human stool samples prior to whole genome sequencing (WGS) for microbiome studies. This stabilization and collection device allows for ambient temperature storage, automation, and ease of shipping/transfer of samples. The device permitted the same data reproducibility as with frozen samples, and yielded higher recovery of nucleic acids. Collection and stabilization of stool microbiome samples with the DNA Genotek collection device, combined with our extraction and WGS, provides a robust, reproducible workflow that enables standardized global collection, storage, and analysis of stool for microbiome studies. PMID:27558918

Detection and sequencing of rotavirus among sudanese children.

PubMed

Magzoub, Magzoub Abbas; Bilal, Naser Eldin; Bilal, Jalal Ali; Alzohairy, Mohammad Abdulrahman; Elamin, Bahaeldin Khalid; Gasim, Gasim Ibrahim

2017-01-01

Diarrheal diseases are a big public health problem worldwide, particularly among developing countries. The current study was conducted to detect and characterize group A rotavirus among admitted children with gastroenteritis to the pediatric hospitals, Sudan. A total of 755 stool samples were collected from Sudanese children with less than 5 years of age presenting with acute gastroenteritis during the period from April to September 2010. Enzyme-linked immunosorbent assay (ELISA) was used to Detection of Rotavirus antigens. Ribonucleic acid (RNAs) were extracted from rotavirus-positive stool samples using (QIAamp ® Viral RNA Mini Kit). (Omniscript ® Reverse Transcription kit) was used to convert RNA to complementary Deoxyribonucleic acid (cDNA). The cDNAs were used as template for detection of VP4-P (P for Protease-sensitive) and VP7-G (G for Glycoprotein) genotyping of Rotavirus using nested PCR and sequencing. Out of the 755 stool samples from children with acute gastroenteritis, 121 were positive for rotavirus A. Among 24 samples that were sequenced; the VP7 predominant G type was G1 (83.3%), followed by G9 (16.7%). Out of these samples, only one VP4 P[8] genotype was detected. As a conclusion the VP7 predominant G type was G1, followed by G9 whereas only one VP4 genotype was detected and showed similarity to P[8] GenBank strain. It appears that the recently approved rotavirus vaccines in Sudan are well matched to the rotavirus genotypes identified in this study, though more studies are needed.

EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology.

PubMed

Moore, Simon J; Lai, Hung-En; Kelwick, Richard J R; Chee, Soo Mei; Bell, David J; Polizzi, Karen Marie; Freemont, Paul S

2016-10-21

Golden Gate cloning is a prominent DNA assembly tool in synthetic biology for the assembly of plasmid constructs often used in combinatorial pathway optimization, with a number of assembly kits developed specifically for yeast and plant-based expression. However, its use for synthetic biology in commonly used bacterial systems such as Escherichia coli has surprisingly been overlooked. Here, we introduce EcoFlex a simplified modular package of DNA parts for a variety of applications in E. coli, cell-free protein synthesis, protein purification and hierarchical assembly of transcription units based on the MoClo assembly standard. The kit features a library of constitutive promoters, T7 expression, RBS strength variants, synthetic terminators, protein purification tags and fluorescence proteins. We validate EcoFlex by assembling a 68-part containing (20 genes) plasmid (31 kb), characterize in vivo and in vitro library parts, and perform combinatorial pathway assembly, using pooled libraries of either fluorescent proteins or the biosynthetic genes for the antimicrobial pigment violacein as a proof-of-concept. To minimize pathway screening, we also introduce a secondary module design site to simplify MoClo pathway optimization. In summary, EcoFlex provides a standardized and multifunctional kit for a variety of applications in E. coli synthetic biology.

Stool metatranscriptomics: A technical guideline for mRNA stabilisation and isolation.

PubMed

Reck, Michael; Tomasch, Jürgen; Deng, Zhiluo; Jarek, Michael; Husemann, Peter; Wagner-Döbler, Irene

2015-07-04

The complex microbiome of the gut has an enormous impact on human health. Analysis of the transcriptional activity of microorganisms through mRNA sequencing (metatranscriptomics) opens a completely new window into their activity in vivo, but it is highly challenging due to numerous technical and bioinformatical obstacles. Here we present an optimized pipeline for extraction of high quality mRNA from stool samples. Comparison of three commercially available RNA extraction kits with the method of Zoetendal revealed that the Powermicrobiome Kit (MoBio) performed best with respect to RNA yield and purity. Next, the influence of the stabilization reagent during sample storage for up to 15 days was studied. RIN analysis and qRT-PCR of spiked-in and indigenous genes revealed that RNA Later preserved mRNA integrity most efficiently, while samples conserved in RNA Protect showed substantial mRNA decay. Using the optimized pipeline developed here, recovery rates for spiked-in E.coli cells expressing fluorescing proteins were 8.7-9.7% for SuperfolderGFP and 14.7-17.8% for mCherry. The mRNA of stabilized stool samples as well as of snap-frozen controls was sequenced with Illumina Hiseq, yielding on average 74 million reads per sample. PCoA analysis, taxonomic classification using Kraken and functional classification using bwa showed that the transcriptomes of samples conserved in RNA Later were unchanged for up to 6 days even at room temperature, while RNA Protect was inefficient for storage durations exceeding 24 h. However, our data indicate that RNA Later introduces a bias which is then maintained throughout storage, while RNA Protect conserved samples are initially more similar to the snap frozen controls. RNA Later conserved samples had a reduced abundance of e.g. Prevotellaceae transcripts and were depleted for e.g. COG category "Carbohydrate transport and metabolism". Since the overall similarity between all stool transcriptional profiles studied here was >0.92, these differences are unlikely to affect global comparisons, but should be taken into account when rare but critically important members of the stool microbiome are being studied.

Comparing ImmunoCard with two EIA assays for Clostridium difficile toxins.

PubMed

Chan, Edward L; Seales, Diane; Drum, Hong

2009-01-01

To compare three Clostridium difficile EIA kits for the detection of C. difficile toxins from clinical specimens. A total of 287 fresh and stored stool specimens were tested using all three assays. Stools with discrepant results were sent to a reference laboratory for tissue cytotoxin assay. Trinity Medical Center, a community hospital with network hospitals. Patients with diarrhea submitted stools for detection of C. difficile toxins. Of the 287 stool specimens, 116 were positive and 171 negative for C. difficile toxins. The sensitivity, specificity, and positive and negative predictive values of Meridian EIA assay were 99.1, 97.7, 96.6, and 99.4%; ImmunoCard were 100, 98.2, 97.5, and 100%; BioStar OIA assay were 94, 98.8, 98.2, and 96% respectively. ImmunoCardprovides the best sensitivity (100%) for C. difficile toxins A and B detection. The BioStar OIA rapid test missed seven positive stool specimens possibly due to failure to detect toxin B. ImmunoCard has slightly higher predictive values, shorter turnaround time and greater convenience compared to the Meridian EIA Assay. ImmunoCard may be cost effective not only in smaller laboratories, but also in high volume laboratories, when used on a STAT basis or single request.

Automated forensic DNA purification optimized for FTA card punches and identifiler STR-based PCR analysis.

PubMed

Tack, Lois C; Thomas, Michelle; Reich, Karl

2007-03-01

Forensic labs globally face the same problem-a growing need to process a greater number and wider variety of samples for DNA analysis. The same forensic lab can be tasked all at once with processing mixed casework samples from crime scenes, convicted offender samples for database entry, and tissue from tsunami victims for identification. Besides flexibility in the robotic system chosen for forensic automation, there is a need, for each sample type, to develop new methodology that is not only faster but also more reliable than past procedures. FTA is a chemical treatment of paper, unique to Whatman Bioscience, and is used for the stabilization and storage of biological samples. Here, the authors describe optimization of the Whatman FTA Purification Kit protocol for use with the AmpFlSTR Identifiler PCR Amplification Kit.

Stool submission by general practitioners in SW England - when, why and how? A qualitative study.

PubMed

McNulty, Cliodna A M; Lasseter, Gemma; Newby, Katie; Joshi, Puja; Yoxall, Harry; Kumaran, Kalyanaraman; O'Brien, Sarah J; Evans, Mark

2012-08-08

We know little about when and why general practitioners (GPs) submit stool specimens in patients with diarrhoea. The recent UK-wide intestinal infectious disease (IID2) study found ten GP consultations for every case reported to national surveillance. We aimed to explore what factors influence GP's decisions to send stool specimens for laboratory investigation, and what guidance, if any, informs them. We used qualitative methods that enabled us to explore opinions and ask open questions through 20 telephone interviews with GPs with a range of stool submission rates in England, and a discussion group with 24 GPs. Interviews were transcribed and subjected to content analysis. Interviews: GPs only sent stool specimens to microbiology if diarrhoea persisted for over one week, after recent travel, or the patient was very unwell. Very few had a systematic approach to determine the clinical or public health need for a stool specimen. Only two GPs specifically asked patients about blood in their stool; only half asked about recent antibiotics, or potential food poisoning, and few asked about patients' occupations. Few GPs gave patients advice on how to collect specimens.Results from interviews and discussion group in relation to guidance: All reported that the HPA stool guidance and patient collection instructions would be useful in their clinical work, but only one GP (an interviewee) had previously accessed them. The majority of GPs would value links to guidance on electronic requests. Most GPs were surprised that a negative stool report did not exclude all the common causes of IID. GPs value stool culture and laboratories should continue to provide it. Patient instructions on how to collect stool specimens should be within stool collection kits. Through readily accessible guidance and education, GPs need to be encouraged to develop a more systematic approach to eliciting and recording details in the patient's history that indicate greater risk of severe infection or public health consequences. Mild or short duration IID (under one week) due to any cause is less likely to be picked up in national surveillance as GPs do not routinely submit specimens in these cases.

Evaluation of commercial kits for dual extraction of DNA and RNA from human body fluids.

PubMed

Schweighardt, Andrew J; Tate, Courtney M; Scott, Kristina A; Harper, Kathryn A; Robertson, James M

2015-01-01

STR typing of DNA evidence can identify the donor with a high power of discrimination but cannot identify the tissue origin of a body-fluid stain. Using RNA to attribute a crime scene stain to a particular tissue may aid in reconstruction efforts. With blood from 10 donors, four DNA and RNA coextraction kits were evaluated by measuring yields and STR and mRNA profiles. T tests indicated some significant differences in kit performance. The Zymo Research ZR-Duet(™) kit performed best based on average DNA (41.4 ng) and mRNA (4.07 ng) yields and was the only kit to provide complete DNA/RNA profiles for all samples. The consistency of this kit was challenged by data from additional blood and saliva donors. Further testing is advised before a superior kit is unequivocally chosen. Stand-alone DNA or RNA purification generally offers higher yield, but coextraction may still allow successful STR profiling and tissue source identification. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Assessment of microscopic and molecular tools for the diagnosis and follow-up of cryptosporidiosis in patients at risk.

PubMed

Le Govic, Y; Guyot, K; Certad, G; Deschildre, A; Novo, R; Mary, C; Sendid, B; Viscogliosi, E; Favennec, L; Dei-Cas, E; Fréalle, E; Dutoit, E

2016-01-01

Cryptosporidiosis is an important though underreported public health concern. Molecular tools might be helpful in improving its diagnosis. In this study, ZR Fecal DNA MiniPrep™ Kit (ZR) and NucliSens® easyMAG® (EM) were compared using four Cryptosporidium-seeded feces and 29 Cryptosporidium-positive stools. Thereafter, ZR was selected for prospective evaluation of Cryptosporidium detection by 18S rDNA and LAXER quantitative PCR (qPCR) in 69 stools from 56 patients after Cryptosporidium detection by glycerin, modified Ziehl-Neelsen (ZN) and auramine-phenol (AP) stainings. The combination of any of the two extraction methods with 18S qPCR yielded adequate detection of Cryptosporidium in seeded stools, but the ZR kit showed the best performance. All 29 Cryptosporidium-positive samples were positive with 18S qPCR, after both ZR and EM extraction. However, false-negative results were found with LAXER qPCR or nested PCR. Cryptosporidiosis was diagnosed in 7/56 patients. All the microscopic methods enabled the initial diagnosis, but Cryptosporidium was detected in 12, 13, and 14 samples from these seven patients after glycerin, ZN, and AP staining respectively. Among these samples, 14 and 12 were positive with 18S and LAXER qPCR respectively. In two patients, Cryptosporidium DNA loads were found to be correlated with clinical evolution. Although little known, glycerin is a sensitive method for the initial detection of Cryptosporidium. When combined with 18S qPCR, ZR extraction, which had not been evaluated so far for Cryptosporidium, was an accurate tool for detecting Cryptosporidium and estimating the oocyst shedding in the course of infection.

Coexistence of IgM antihepatitis A virus and IgM antihepatitis E virus in acute viral hepatitis: a prospective, multicentre study in Korea.

PubMed

Jang, J-H; Jung, Y M; Kim, J S; Lee, S H; Kim, J-W; Hwang, S G; Rim, K S; Park, S J; Park, Y M; Kang, S-K; Lee, H S; Yun, H; Kim, J-H; Jeong, S-H

2011-10-01

This study investigated the clinical, serological and molecular characteristics of coexistence of both immunoglobulin M (IgM) antihepatitis A virus (HAV) and IgM antihepatitis E virus (HEV) in acute viral hepatitis using a prospective, multicentre design. Among a total of 771 symptomatic cases with acute viral hepatitis enrolled in a Korean city from September 2006 to August 2008, coexistence of IgM anti-HAV and IgM anti-HEV was found in 43 patients (A+E group; 6%), while the existence of IgM anti-HAV alone was found in 595 patients (A group; 77%) and that of IgM anti-HEV alone in 14 patients (E group; 2%). Clinical data analysis and measurement of IgM and IgG anti-HEV were performed using two different commercial kits, and HAV RNA and HEV RNA were detected in available serum or stool samples. The clinical features of the A+E group were similar to those of the A group. HAV RNA detection rates in the A+E and A group were similar, while HEV RNA was detected only in the stool samples of the E group, not in the A+E group. Comparative testing of anti-HEV using two different ELISA kits showed markedly discordant results for IgM anti-HEV positivity and consistently low positivity for IgG anti-HEV in the A+E group. Coexistence of IgM anti-HEV measured by the Genelabs ELISA kit in the setting of hepatitis A appears to yield false-positive results in nonendemic areas of HEV infection. Diagnosis of hepatitis E using IgM anti-HEV should be made with caution. © 2011 Blackwell Publishing Ltd.

A novel kit for rapid detection of Vibrio cholerae O1.

PubMed

Hasan, J A; Huq, A; Tamplin, M L; Siebeling, R J; Colwell, R R

1994-01-01

We report on the development and testing of a novel, rapid, colorimetric immunodiagnostic kit, Cholera SMART, for direct detection of the presence of Vibrio cholerae O1 in clinical specimens. Unlike conventional culture methods requiring several days to complete, the Cholera SMART kit can be used directly in the field by untrained or minimally skilled personnel to detect V. cholerae O1 in less than 15 min, without cumbersome laboratory equipment. A total of 120 clinical and environmental bacterial strains, including both O1 and non-O1 serotypes of V. cholerae isolated from samples collected from a variety of geographical regions, were tested, and positive reactions were observed only with V. cholerae O1. Also, results of a field trial in Bangladesh, employing Cholera SMART, showed 100% specificity and 96% sensitivity compared with conventional culture methods. Another field trial, in Mexico, showed that Cholera SMART was 100% in agreement with a recently described coagglutination test when 108 stool specimens were tested.

Medical Surveillance Monthly Report (MSMR). Volume 22, Number 6, June 2015

DTIC Science & Technology

2015-06-01

and then plateaued (with marked year-to-year variability) through 2010 (Figure 2). women and among those who were not deployed within the fi rst...acids were extracted with the QIAamp stool DNA kit (QIAGEN, USA). For the respiratory samples, detection and characterization of human infl uenza...research by the Walter Reed Army Institute of Research Division of Human Subjects Protection. Some of the authors are military service members. Th is

Strongyloidiasis in Canadian Far East war veterans

PubMed Central

Proctor, Eileen M.; Isaac-Renton, Judith L.; Robertson, William B.; Black, William A.

1985-01-01

A survey was done of Canadians who had been interned by the Japanese during World War II to assess the prevalence of latent infection with Strongyloides stercoralis in this group. Packages containing three mail-in kits and a questionnaire were sent to 992 men, 694 (70%) of whom responded. Larvae were found in the stool specimens of four of the respondents. Examination of stool specimens after formalin-ether concentration was the most successful method of detecting Strongyloides larvae. The Baermann concentration technique yielded negative results in all four men. Three of the four cases of strongyloidiasis were detected after sampling of three fecal specimens. In the fourth case additional specimens were requested on the basis of data derived from the questionnaire. The most frequently cited clinical manifestations were abdominal pain, weight loss, diarrhea and rashes. PMID:4052898

Improving compliance to colorectal cancer screening using blood and stool based tests in patients refusing screening colonoscopy in Germany.

PubMed

Adler, Andreas; Geiger, Sebastian; Keil, Anne; Bias, Harald; Schatz, Philipp; deVos, Theo; Dhein, Jens; Zimmermann, Mathias; Tauber, Rudolf; Wiedenmann, Bertram

2014-10-17

Despite strong recommendations for colorectal cancer (CRC) screening, participation rates are low. Understanding factors that affect screening choices is essential to developing future screening strategies. Therefore, this study assessed patient willingness to use non-invasive stool or blood based screening tests after refusing colonoscopy. Participants were recruited during regular consultations. Demographic, health, psychological and socioeconomic factors were recorded. All subjects were advised to undergo screening by colonoscopy. Subjects who refused colonoscopy were offered a choice of non-invasive tests. Subjects who selected stool testing received a collection kit and instructions; subjects who selected plasma testing had a blood draw during the office visit. Stool samples were tested with the Hb/Hp Complex Elisa test, and blood samples were tested with the Epi proColon® 2.0 test. Patients who were positive for either were advised to have a diagnostic colonoscopy. 63 of 172 subjects were compliant to screening colonoscopy (37%). 106 of the 109 subjects who refused colonoscopy accepted an alternative non-invasive method (97%). 90 selected the Septin9 blood test (83%), 16 selected a stool test (15%) and 3 refused any test (3%). Reasons for blood test preference included convenience of an office draw, overall convenience and less time consuming procedure. 97% of subjects refusing colonoscopy accepted a non-invasive screening test of which 83% chose the Septin9 blood test. The observation that participation can be increased by offering non-invasive tests, and that a blood test is the preferred option should be validated in a prospective trial in the screening setting.

The validation and utility of a quantitative one-step multiplex RT real-time PCR targeting Rotavirus A and Norovirus

PubMed Central

Dung, Tran Thi Ngoc; Phat, Voong Vinh; Nga, Tran Vu Thieu; My, Phan Vu Tra; Duy, Pham Thanh; Campbell, James I.; Thuy, Cao Thu; Hoang, Nguyen Van Minh; Van Minh, Pham; Le Phuc, Hoang; Tuyet, Pham Thi Ngoc; Vinh, Ha; Kien, Duong Thi Hue; Huy, Huynh Le Anh; Vinh, Nguyen Thanh; Nga, Tran Thi Thu; Hau, Nguyen Thi Thu; Chinh, Nguyen Tran; Thuong, Tang Chi; Tuan, Ha Manh; Simmons, Cameron; Farrar, Jeremy J.; Baker, Stephen

2013-01-01

Rotavirus (RoV) and Norovirus (NoV) are the main causes of viral gastroenteritis. Currently, there is no validated multiplex real-time PCR that can detect and quantify RoV and NoV simultaneously. The aim of the study was to develop, validate, and internally control a multiplex one-step RT real-time PCR to detect and quantify RoV and NoV in stool samples. PCR sensitivity was assessed by comparing amplification against the current gold standard, enzyme immunoassay (EIA), on stool samples from 94 individuals with diarrhea and 94 individuals without diarrhea. PCR detected 10% more RoV positive samples than EIA in stools samples from patients with diarrhea. PCR detected 23% more NoV genogroup II positive samples from individuals with diarrhea and 9% more from individuals without diarrhea than EIA, respectively. Genotyping of the PCR positive/EIA negative samples suggested the higher rate of PCR positivity, in comparison to EIA, was due to increased sensitivity, rather than nonspecific hybridization. Quantitation demonstrated that the viral loads of RoV and NoV in the stools of diarrheal patients were an order of magnitude greater than in individuals without diarrhea. This internally controlled real-time PCR method is robust, exhibits a high degree of reproducibility, and may have a greater utility and sensitivity than commercial EIA kits. PMID:23046990

Necessity of purification during bacterial DNA extraction with environmental soils

PubMed Central

Choi, Jung-Hyun

2017-01-01

Complexity and heterogeneity of soil samples have often implied the inclusion of purification steps in conventional DNA extraction for polymerase chain reaction (PCR) assays. Unfortunately the purification steps are also time and labor intensive. Therefore the necessity of DNA purification was re-visited and investigated for a variety of environmental soil samples that contained various amounts of PCR inhibitors. Bead beating and centrifugation was used as the baseline (without purification) method for DNA extraction. Its performance was compared with that of conventional DNA extraction kit (with purification). The necessity criteria for DNA purification were established with environmental soil samples. Using lysis conditions at 3000 rpm for 3 minutes with 0.1 mm glass beads, centrifugation time of 10 minutes and 1:10 dilution ratio, the baseline method outperformed conventional DNA extraction on cell seeded sand samples. Further investigation with PCR inhibitors (i.e., humic acids, clay, and magnesium [Mg]) showed that sand samples containing less than 10 μg/g humic acids and 70% clay may not require purifications. Interestingly, the inhibition pattern of Mg ion was different from other inhibitors due to the complexation interaction of Mg ion with DNA fragments. It was concluded that DNA extraction method without purification is suitable for soil samples that have less than 10 μg/g of humic acids, less than 70% clay content and less than 0.01% Mg ion content. PMID:28793754

Erythropoietin binding protein from mammalian serum

DOEpatents

Clemons, Gisela K.

1997-01-01

Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described.

Erythropoietin binding protein from mammalian serum

DOEpatents

Clemons, G.K.

1997-04-29

Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described. 11 figs.

[Sensitivity of three inmunocromathographic tests in faeces samples for Campylobacter and Salmonella detection in comparison to culture].

PubMed

Liébana-Martos, Ma del Carmen; Gutierrez, José; Riazzo, Cristina; Navarro, José Ma

2014-06-01

Introduction: Campylobacter sp. and Salmonella enterica are two of the main organisms causing gastroenteritis in our environment. Immunochromatographic tests for antigen detection performed directly on stool samples for its simplicity and rapid results may make them useful diagnostic elements in the context of primary care. During October 2012 we selected all feces in which enteropathogenic bacteria are isolated from those received for stool culture in the laboratory of Microbiology of the University Hospital Virgen de las Nieves of Granada. After standard management of faeces samples and isolation of any enteropathogen, the commercial kits: Campy Leti, Ridaquick Campylobacterscreen and Salmonella Leti were tested for simultaneous research of Campylobacter and Salmonella antigens. Sensitivity and specificity were determined. Two hundred and thirty five stool samples were received in which 8 Salmonella enterica (7 B serogroup and 1 D serogroup), 7 Campylobacter jejuni, 4 Aeromonas hydrophila and 1 Yersinia enterocolitica were isolated. Campy Leti, Ridaquick Campylobacterscreen and Salmonella Leti presented a sensitivity of 100%, 100% and 75%, respectively. Specificities corresponded to 46%, 69% and 100%, respectively. Immunocromatographic tests can be useful for a first screening of enteropathogen in primary care.

Diarrheal and Respiratory Illness Surveillance During US-RP Balikatan 2014.

PubMed

Velasco, John M; Valderamat, Maria T; Nogrado, Kathyleen; Wongstitwilairoong, Tippa; Swierczewski, Brett; Bodhidatta, Ladaporn; Lertsethtakarn, Paphavee; Klungthong, Chonticha; Fernandez, Stefan; Mason, Carl; Yoon, In-Kyu; Macareo, Louis

2015-06-01

Diarrheal and respiratory illness surveillance was conducted during the 2014 Republic of the Philippines-U.S. Exercise Balikatan in the Philippines. Seven stool and three respiratory specimens that met the inclusion criteria were collected. Diarrhea stool specimens were tested with commercial enzyme-linked immunosorbent assay kits and real-time polymerase chain reaction (PCR) for 12 viral, bacterial, and protozoan pathogens. Campylobacter, enterotoxigenic Escherichia coli (ETEC), and enteropathogenic Escherichia coli (EPEC) were detected in four of seven (57%), two of seven (29%), and four of seven (57%) specimens, respectively. There were co-infections of EPEC and ETEC in two cases and EPEC and Campylobacter spp. in one case. Respiratory samples were tested using RT-PCR. One of three samples was positive for influenza B. Laboratory-based surveillance is important in determining causative agents for illnesses experienced by military personnel during deployment. Development of vaccines for enteric diseases should be expedited to mitigate their impact on operational readiness.

Diagnostic accuracy of three monoclonal stool tests in a large series of untreated Helicobacter pylori infected patients.

PubMed

Lario, Sergio; Ramírez-Lázaro, María José; Montserrat, Antònia; Quílez, María Elisa; Junquera, Félix; Martínez-Bauer, Eva; Sanfeliu, Isabel; Brullet, Enric; Campo, Rafael; Segura, Ferran; Calvet, Xavier

2016-06-01

Immunochromatographic tests need to be improved in order to enhance their reliability. Recently, several new kits have appeared on the market. The objective was to evaluate the diagnostic accuracy of three monoclonal rapid stool tests - the new Uni-Gold™ H.pylori Antigen (Trinity Biotech, Ireland), the RAPID Hp StAR (Oxoid Ltd., UK) and the ImmunoCard STAT! HpSA (Meridian Diagnostics, USA) - for detecting H. pylori infection prior to eradication treatment. Diagnostic accuracy (sensitivity and specificity) and reliability (concordance between observers) were evaluated in 250 untreated consecutive dyspeptic patients. The gold standard for diagnosing H. pylori infection was defined as the concordance of two or more of rapid urease test (RUT), histopathology and urease breath test (UBT) or positive culture in isolation. Readings of immunochromatographic tests were performed by two different observers. Sensitivity, specificity, positive and negative predictive values and 95% confidence intervals were calculated. Sensitivity and specificity were compared using the McNemar test. The three tests showed a good correlation, with Kappa values>0.9. RAPID Hp StAR had a sensitivity of 91%-92% and a specificity ranging from 77% to 85%. Its sensitivity was higher than that of Uni-Gold™ H.pylori Antigen and ImmunoCard STAT! HpSA (p<0.01). Uni-Gold™ H.pylori Antigen kit showed a sensitivity of 83%, similar to ImmunoCard STAT! HpSA. Specificity of Uni-Gold™ H.pylori Antigen approached 90% (87-89%) and was superior to that of RAPID Hp StAR (p<0.01). Uni-Gold™ H.pylori Antigen and ImmunoCard STAT! HpSA present similar levels of diagnostic accuracy. RAPID Hp StAR was the most sensitive but less reliable of the three immunochromatographic stool tests. None are as accurate and reliable as UBT, RUT and histology. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

[Establishment of a canine slow transit constipation model and evalution of defecation, gastrointestinal transit and pathological sections].

PubMed

Zhu, D; Chen, S; Yao, S K; Li, Y M; Chen, S X

2018-06-12

Objective: To establish a canine model of slow transit constipation (STC), and to test the changes in defecation, gastrointestinal transit time and pathology sections. Methods: Baseline information was measured in 8 beagle dogs, and these dogs were randomly divided into the control group and the model group. The dogs in model group were given a diet of canned meat, as well as a combination of compound diphenoxylate and alosetron hydrochloride for 5 weeks. Dogs in control group were given normal diet with no special intervention. Stool frequency and consistency were observed and recorded daily, and the gastrointestinal transit time (GITT) were measured every week. All animals underwent the midline laparotomy and the colonic tissues were taken from the rectosigmoid colon, then investigated by light microscopy, electron microscopy, and immunohistochemistry to evaluate changes of protein gene product 9.5(PGP9.5), synaptophysin and c-kit between two groups. Results: 8 beagle dogs underwent all experiment items successfully.Both of the stool frequency and scores of stool consistency decreased in model group( F =6.568, P =0.043; F =25.954, P =0.002). GITT delayed in model group( F =42.573, P =0.001). After 5 weeks of intervention, in the model group, the myenteric neurons and interstitial cells of Cajal showed damage such as swelling of mitochondria under electron microscopy, and both of the PGP9.5 and synaptophysin integrated option density of rectosigmoid colon were decreased ( t =3.471, P =0.013; t =2.506, P =0.046)under immunohistochemistry. The c-kit integrated option density showed no statistically significant differences between two groups( t =1.709, P =0.138). Conclusions: The canine model of STC which was consistent with clinical symptoms and pathological changes was successfully established, and it can be used to observe and evaluate the therapeutic effect of electrical stimulation, surgery and so on.

Identification of bacteria in drinking and purified water during the monitoring of a typical water purification system

PubMed Central

Penna, Vessoni Thereza Christina; Martins, Silva Alzira Maria; Mazzola, Priscila Gava

2002-01-01

Background A typical purification system that provides purified water which meets ionic and organic chemical standards, must be protected from microbial proliferation to minimize cross-contamination for use in cleaning and preparations in pharmaceutical industries and in health environments. Methodology Samples of water were taken directly from the public distribution water tank at twelve different stages of a typical purification system were analyzed for the identification of isolated bacteria. Two miniature kits were used: (i) identification system (api 20 NE, Bio-Mérieux) for non-enteric and non-fermenting gram-negative rods; and (ii) identification system (BBL crystal, Becton and Dickson) for enteric and non-fermenting gram-negative rods. The efficiency of the chemical sanitizers used in the stages of the system, over the isolated and identified bacteria in the sampling water, was evaluated by the minimum inhibitory concentration (MIC) method. Results The 78 isolated colonies were identified as the following bacteria genera: Pseudomonas, Flavobacterium and Acinetobacter. According to the miniature kits used in the identification, there was a prevalence of isolation of P. aeruginosa 32.05%, P. picketti (Ralstonia picketti) 23.08%, P. vesiculares 12.82%,P. diminuta 11.54%, F. aureum 6.42%, P. fluorescens 5.13%, A. lwoffi 2.56%, P. putida 2.56%, P. alcaligenes 1.28%, P. paucimobilis 1.28%, and F. multivorum 1.28%. Conclusions We found that research was required for the identification of gram-negative non-fermenting bacteria, which were isolated from drinking water and water purification systems, since Pseudomonas genera represents opportunistic pathogens which disperse and adhere easily to surfaces, forming a biofilm which interferes with the cleaning and disinfection procedures in hospital and industrial environments. PMID:12182763

Comparison of Plasma Exosomes by Differential Ultracentrifugation and Solvent Precipitation Methods.

PubMed

Peng, Qiao; Zhang, Jing; Zhou, Gang

2018-06-01

Emerging evidence has identified that exosomes play a pivotal role in intercellular signal transmission. However, the standardized purification techniques to isolate high quality exosomes are still deficient at present. This study was to evaluate reproducibility and efficiency of differential ultracentrifugation and solvent precipitation-based kits by isolating plasma-derived exosomes from oral lichen planus patients. Morphology, exosomal biomarkers, particle size distribution, proteomic components, and protein yield of isolated exosomes were evaluated by transmission electron microscope, western blot, laser diffraction instrument, Coomassie staining, and BCA protein assay kit, respectively. TEM displayed representative cup-shaped morphology of exosomes and western blot identified exosomal biomarkers CD9 and CD63. The size distribution showed that particles by differential ultracentrifugation were mainly from 26.15 nm to 166.5 nm, while some of the particles obtained by solvent precipitation kits were larger than 1,000 nm. In addition, exosomes isolated by solvent precipitation kits showed a significantly higher amount of protein yield due to plasma albumin contamination. Both differential ultracentrifugation and precipitation based kits could successfully isolate plasma exosomes, and exosomes by differential ultracentrifugation were purer and more appropriate for further proteomic analysis.

Comparison of commercial DNA extraction kits for isolation and purification of bacterial and eukaryotic DNA from PAH-contaminated soils.

PubMed

Mahmoudi, Nagissa; Slater, Greg F; Fulthorpe, Roberta R

2011-08-01

Molecular characterization of the microbial populations of soils and sediments contaminated with polycyclic aromatic hydrocarbons (PAHs) is often a first step in assessing intrinsic biodegradation potential. However, soils are problematic for molecular analysis owing to the presence of organic matter, such as humic acids. Furthermore, the presence of contaminants, such as PAHs, can cause further challenges to DNA extraction, quantification, and amplification. The goal of our study was to compare the effectiveness of four commercial soil DNA extraction kits (UltraClean Soil DNA Isolation kit, PowerSoil DNA Isolation kit, PowerMax Soil DNA Isolation kit, and FastDNA SPIN kit) to extract pure, high-quality bacterial and eukaryotic DNA from PAH-contaminated soils. Six different contaminated soils were used to determine if there were any biases among the kits due to soil properties or level of contamination. Extracted DNA was used as a template for bacterial 16S rDNA and eukaryotic 18S rDNA amplifications, and PCR products were subsequently analyzed using denaturing gel gradient electrophoresis (DGGE). We found that the FastDNA SPIN kit provided significantly higher DNA yields for all soils; however, it also resulted in the highest levels of humic acid contamination. Soil texture and organic carbon content of the soil did not affect the DNA yield of any kit. Moreover, a liquid-liquid extraction of the DNA extracts found no residual PAHs, indicating that all kits were effective at removing contaminants in the extraction process. Although the PowerSoil DNA Isolation kit gave relatively low DNA yields, it provided the highest quality DNA based on successful amplification of both bacterial and eukaryotic DNA for all six soils. DGGE fingerprints among the kits were dramatically different for both bacterial and eukaryotic DNA. The PowerSoil DNA Isolation kit revealed multiple bands for each soil and provided the most consistent DGGE profiles among replicates for both bacterial and eukaryotic DNA.

A method suitable for DNA extraction from humus-rich soil.

PubMed

Miao, Tianjin; Gao, Song; Jiang, Shengwei; Kan, Guoshi; Liu, Pengju; Wu, Xianming; An, Yingfeng; Yao, Shuo

2014-11-01

A rapid and convenient method for extracting DNA from soil is presented. Soil DNA is extracted by direct cell lysis in the presence of EDTA, SDS, phenol, chloroform and isoamyl alcohol (3-methyl-1-butanol) followed by precipitation with 2-propanol. The extracted DNA is purified by modified DNA purification kit and DNA gel extraction kit. With this method, DNA extracted from humus-rich dark brown forest soil was free from humic substances and, therefore, could be used for efficient PCR amplification and restriction digestion. In contrast, DNA sample extracted with the traditional CTAB-based method had lower yield and purity, and no DNA could be extracted from the same soil sample with a commonly-used commercial soil DNA isolation kit. In addition, this method is time-saving and convenient, providing an efficient choice especially for DNA extraction from humus-rich soils.

Comparison of RNA extraction kits for the purification and detection of an enteric virus surrogate on green onions via RT-PCR.

PubMed

Xu, Ruoyang; Shieh, Y Carol; Stewart, Diana S

2017-01-01

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) offers a rapid and sensitive molecular method for detection of enteric viruses. Unfortunately, these assays are often hampered by the low virus titre found in foods and PCR inhibition due to matrix carryover during RNA extraction. Four commercial RNA extraction kits (Qiagen's QIAamp Viral RNA Mini and UltraSens Virus kits, MoBio UltraClean Tissue & Cells RNA Isolation kit, and Ambion MagMAX Viral RNA Isolation kit) were evaluated for their ability to extract and purify MS2 bacteriophage RNA, an enteric virus surrogate, from inoculated green onions, a food which has been associated with viral gastroenteritis outbreaks. Inoculated green onion wash concentrates and green onion pieces with and without Qiagen QIAshredder homogenization were assayed in the kit comparison. MS2 detection and PCR inhibition were evaluated using a duplex real-time RT-PCR for MS2 and an exogenous internal amplification control (IAC) assay. Without homogenization, MS2 inoculated at 40pfu/g was detected in at least 4 lots of green onion wash concentrates using the silica-membrane spin-column kits. Inhibition was a factor for the magnetic silica-based MagMAX kit, which resulted in detection of MS2 in 1 of 5. Addition of QIAshredder homogenization prior to extraction did not adversely affect the silica-membrane kit results but improved the MS2 detection by MagMAX to 5 of 5 lots. Use of a 1:10 dilution of primary RNA extracts also improved detection. The QIAamp Viral RNA Mini and MagMAX kits were further compared for detection of MS2 from green onion pieces inoculated at 20 and 5pfu/g. Using homogenization, the MagMAX kit detected 20pfu/g in only 1 of 2 green onion lots, whereas the QIAamp Viral RNA kit detected 2 of 2 lots at 5 pfu/g without homogenization. Published by Elsevier B.V.

Investigation of Rotavirus with Various Methods in Children with Acute Gastroenteritis and Determination of Its Molecular Epidemiology in Kayseri Province, Turkey.

PubMed

Artiran, Sukran; Atalay, Altay; Gökahmetoglu, Selma; Ozturk, Mehmet Adnan; Balci, Nurgul; Cakir, Nuri; Kilic, Huseyin; Durmaz, Riza

2017-03-01

In this study, the fresh stool samples from 254 children under 5 years of age with acute gastroenteritis which were delivered between October 2012 and December 2013 were collected. In the stool samples, rotavirus antigens were investigated using two different immunochromatographic methods which are routinely used at different times, namely the RIDA ® QUICK Rotavirus/Adenovirus Combi Test (R-Biopharm AG, Germany) and the Genx ® Rotavirus Test (Diamed-Lab, Turkey), in addition to the Rotavirus Ag (Stool) ELISA (DRG, Germany) kit. The results were compared with reverse transcriptase PCR (RT-PCR). When the Genx ® Rotavirus Test and RIDA ® QUICK Rotavirus/Adenovirus Combi Test immunochromatographic methods were compared with RT-PCR, their sensitivity and specificity were found as 97.1%, 100%, and 80.4%, 72%, respectively. As to the Rotavirus Ag (Stool) ELISA method, on the other hand, its sensitivity was found to be 95.1% and its specificity was 86.5%. The most common genotype was G9P[8] (40%), which was followed by the G1P[8] (18.7%) and G3P[8] (9.6%) genotypes. Consequently, it was revealed that the sensitivity of ELISA and immunochromatographic methods, which provide results in a short time and are used in the investigation of rotavirus antigen, was high and their specificity was low; further studies to determine the distribution of G and P genotypes will contribute to establishing strategies for vaccine development for rotavirus in the world. © 2016 Wiley Periodicals, Inc.

Laboratory diagnosis of amebiasis in a sample of students from southeastern Brazil and a comparison of microscopy with enzyme-linked immunosorbent assay for screening of infections with Entamoeba sp.

PubMed

Pereira, Valeriana Valadares; Conceição, Abiqueila da Silva; Maximiano, Leandro Henrique Silva; Belligoli, Leonardo de Queiroz Gomes; Silva, Eduardo Sergio da

2014-01-01

Epidemiological studies on amebiasis have been reassessed since Entamoeba histolytica and E. dispar were first recognized as distinct species. Because the morphological similarity of these species renders microscopic diagnosis unreliable, additional tools are required to discriminate between Entamoeba species. The objectives of our study were to compare microscopy with ELISA kit (IVD®) results, to diagnose E. histolytica infection, and to determine the prevalence of amebiasis in a sample of students from southeastern Brazil. In this study, diagnosis was based on microscopy due to its capacity for revealing potential cysts/trophozoites and on two commercial kits for antigen detection in stool samples. For 1,403 samples collected from students aged 6 to 14 years who were living in Divinópolis, Minas Gerais, Brazil, microscopy underestimated the number of individuals infected with E. histolytica/E. dispar (5.7% prevalence) compared with the ELISA kit (IVD®)-based diagnoses (15.7% for E. histolytica/E. dispar). A comparison of the ELISA (IVD®) and light microscopy results returned a 20% sensitivity, 97% specificity, low positive predictive value, and high negative predictive value for microscopy. An ELISA kit (TechLab®) that was specific for E. histolytica detected a 3.1% (43/1403) prevalence for E. histolytica infection. The ELISA kit (IVD®) can be used as an alternative screening tool. The high prevalence of E. histolytica infection detected in this study warrants the implementation of actions directed toward health promotion and preventive measures.

How much DNA is lost? Measuring DNA loss of short-tandem-repeat length fragments targeted by the PowerPlex 16® system using the Qiagen MinElute Purification Kit.

PubMed

Kemp, Brian M; Winters, Misa; Monroe, Cara; Barta, Jodi Lynn

2014-01-01

The success in recovering genetic profiles from aged and degraded biological samples is diminished by fundamental aspects of DNA extraction, as well as its long-term preservation, that are not well understood. While numerous studies have been conducted to determine whether one extraction method was superior to others, nearly all of them were initiated with no knowledge of the actual starting DNA quantity in the samples prior to extraction, so they ultimately compared the outcome of all methods relative to the best. Using quantitative PCR to estimate the copy count of synthetic standards before (i.e., "copies in") and after (i.e., "copies out") purification by the Qiagen MinElute PCR Purification Kit, we documented DNA loss within a pool of 16 different-sized fragments ranging from 106 to 409 bp in length, corresponding to those targeted by the PowerPlex 16 System (Promega, Madison, WI). Across all standards from 10(4) to 10(7) copies/μL, loss averaged between 21.75% and 60.56% (mean, 39.03%), which is not congruent with Qiagen's claim that 80% of 70 bp to 4 kb fragments are retained using this product (i.e., 20% loss). Our study also found no clear relationship either between DNA strand length and retention or between starting copy number and retention. This suggests that there is no molecule bias across the MinElute column membrane and highlights the need for manufacturers to clearly and accurately describe on what their claims are based, and should also encourage researchers to document DNA retention efficiencies of their own methods and protocols. Understanding how and where to reduce loss of molecules during extraction and purification will serve to generate clearer and more accurate data, which will enhance the utility of ancient and low-copy-number DNA as a tool for closing forensic cases or in reconstructing the evolutionary history of humans and other organisms.

Detection of Entamoeba histolytica/dispar in stool specimens by using enzyme-linked immunosorbent assay in the population of Jeddah City, Saudi Arabia.

PubMed

Barnawi, Abdulaziz B M; Tonkal, Abulkader M; Fouad, Mahmoud A H; Al-Braiken, Faten A

2007-04-01

This study determined the prevalence of intestinal parasites, particularly pathogenic Entamoeba sp. (E. histolytica), in patients attending three hospitals in Jeddah City, King Abdulaziz University Hospital, King Abdulaziz Hospital and King Fahad Hospital for gastro-intestinal troubles. 186 stool specimens were examined microscopically for parasites and by ELISA kit (E. histolytica II) for true E. histolytica. 83 samples (44.6%) were positive by microscopy for at least one parasite. Of which, 23 (12.4%) showed two parasites and 15 (8.1%) three parasites. Eight different parasite species were identified. The most prevalent were E. histolytica/dispar (n = 26, 31.3%) and Giardia lamblia (n = 13, 15.7%). Others were Blastocytosis hominis (n = 12, 14.5%), Entamoeba coli (n = 11, 13.3%), Trichuris trichuria (n = 8, 9.6%), Endolymax nana (n = 6, 7.2%), Hymenolepes nana (n = 4, 4.8%) and Chilomastix mesnili (n = 3, 3.6%). Only five stool samples (19%) from those identified by microscopy to contain E. histolytica/dispar, were E. histolytica positive by E. histolytica II ELISA. For the first time to the authors' knowledge the true prevalence of E. histolytica in Saudi Arabia was 2.7%. E. histolytica II ELISA proved to be a highly useful technique to differentiate pathogenic E. histolytica from non pathogenic E. dispar.

An optimized work-flow to reduce time-to-detection of carbapenemase-producing Enterobacteriaceae (CPE) using direct testing from rectal swabs.

PubMed

O'Connor, C; Kiernan, M G; Finnegan, C; O'Hara, M; Power, L; O'Connell, N H; Dunne, C P

2017-05-04

Rapid detection of patients with carbapenemase-producing Enterobacteriaceae (CPE) is essential for the prevention of nosocomial cross-transmission, allocation of isolation facilities and to protect patient safety. Here, we aimed to design a new laboratory work-flow, utilizing existing laboratory resources, in order to reduce time-to-diagnosis of CPE. A review of the current CPE testing processes and of the literature was performed to identify a real-time commercial polymerase chain reaction (PCR) assay that could facilitate batch testing of CPE clinical specimens, with adequate CPE gene coverage. Stool specimens (210) were collected; CPE-positive inpatients (n = 10) and anonymized community stool specimens (n = 200). Rectal swabs (eSwab™) were inoculated from collected stool specimens and a manual DNA extraction method (QIAamp® DNA Stool Mini Kit) was employed. Extracted DNA was then processed on the Check-Direct CPE® assay. The three step process of making the eSwab™, extracting DNA manually and running the Check-Direct CPE® assay, took <5 min, 1 h 30 min and 1 h 50 min, respectively. It was time efficient with a result available in under 4 h, comparing favourably with the existing method of CPE screening; average time-to-diagnosis of 48/72 h. Utilizing this CPE work-flow would allow a 'same-day' result. Antimicrobial susceptibility testing results, as is current practice, would remain a 'next-day' result. In conclusion, the Check-Direct CPE® assay was easily integrated into a local laboratory work-flow and could facilitate a large volume of CPE screening specimens in a single batch, making it cost-effective and convenient for daily CPE testing.

Adsorption of Selected Antibiotics to Resins in Extracorporeal Blood Purification.

PubMed

Harm, Stephan; Gruber, Anna; Gabor, Franz; Hartmann, Jens

2016-01-01

Extracorporeal blood purification systems (EBS) use specific adsorbents for the elimination of toxins and cytokines. The aim of this study was to test different adsorbents for their ability to reduce antibiotics in parallel to extracorporeal blood purification therapy. The in vitro adsorption experiments were carried out in human plasma with a newly established hydrophobic resin (Amberchrom CG161c) and adsorbents commercially available and approved in the clinics. The concentration of antibiotic was chosen equivalent to the recommended therapeutic dosage applied intravenously and was measured in plasma using ELISA test kits and high-performance liquid chromatography methods. The adsorbent that reduced all tested antibiotics in plasma close to the detection limit was the dia MARS AC250, which is an activated charcoal involved in the Molecular Adsorbents Recirculation System. For better antibiotic monitoring in sepsis treatment, further investigations have to be performed to determine the clearance rate of antibiotics by different EBS devices. © 2015 S. Karger AG, Basel.

Bacterial community in ancient permafrost alluvium at the Mammoth Mountain (Eastern Siberia).

PubMed

Brouchkov, Anatoli; Kabilov, Marsel; Filippova, Svetlana; Baturina, Olga; Rogov, Victor; Galchenko, Valery; Mulyukin, Andrey; Fursova, Oksana; Pogorelko, Gennady

2017-12-15

Permanently frozen (approx. 3.5Ma) alluvial Neogene sediments exposed in the Aldan river valley at the Mammoth Mountain (Eastern Siberia) are unique, ancient, and poorly studied permafrost environments. So far, the structure of the indigenous bacterial community has remained unknown. Use of 16S metagenomic analysis with total DNA isolation using DNA Spin Kit for Soil (MO-Bio) and QIAamp DNA Stool Mini Kit (Qiagen) has revealed the major and minor bacterial lineages in the permafrost alluvium sediments. In sum, 61 Operational Taxonomic Units (OTUs) with 31,239 reads (Qiagen kit) and 15,404 reads (Mo-Bio kit) could be assigned to the known taxa. Only three phyla, Bacteroidetes, Proteobacteria and Firmicutes, comprised >5% of the OTUs abundance and accounted for 99% of the total reads. OTUs pertaining to the top families (Chitinophagaceae, Caulobacteraceae, Sphingomonadaceae, Bradyrhizobiaceae, Halomonadaceae) held >90% of reads. The abundance of Actinobacteria was less (0.7%), whereas members of other phyla (Deinococcus-Thermus, Cyanobacteria/Chloroplast, Fusobacteria, and Acidobacteria) constituted a minor fraction of reads. The bacterial community in the studied ancient alluvium differs from other permafrost sediments, mainly by predominance of Bacteroidetes (>52%). The diversity of this preserved bacterial community has the potential to cause effects unknown if prompted to thaw and spread with changing climate. Therefore, this study elicits further reason to study how reintroduction of these ancient bacteria could affect the surrounding ecosystem, including current bacterial species. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparison of Para-Selles Bailenger/Kop-Color Fumouze, Para-Selles-Iodésine/Kop-Color II Fumouze diagnostic kits with conventional microscopic methods in identifying intestinal parasitic diseases in Senegal.

PubMed

Sow, Doudou; Dieng, Yémou; Haouchine, Djamal; Niang, Khadim; Niang, Thiane; Sylla, Khadime; Tine, Roger Clément; Ndiaye, Magatte; Ndiaye, Jean Louis; Faye, Babacar; Faye, Omar; Gaye, Oumar; Dieng, Thérèse; Izri, Arezki

2017-09-01

In the context of controlling intestinal parasites, accurate diagnosis is essential. Our objective was to evaluate the performance of new diagnostic kits compared to conventional microscopic methods in identifying intestinal parasites. Faeces collected in rural area in Senegal were subjected to several detection techniques. Thus, the sensitivity, specificity, positive and negative predictive values of new diagnostic techniques were compared to conventional merthiolate-iodine-formalin, conventional Bailenger and modified Ritchie. Furthermore, the kappa coefficient was calculated to evaluate the correlation between the new kit and those of modified Ritchie. Out of the 117 patients examined, 102 presented with a parasite, or prevalence of 87.1%. The Fumouze techniques proved to be as effective as the conventional methods in detecting flagellates and helminths with sensitivities ranging from 97 to 100%. However, conventional techniques were slightly more sensitive in identifying Endolimax nana and Blastocystis hominis . The correlation was nearly perfect (k = 0.83 and 1), respectively between Bailenger Fumouze, Iodesine Fumouze and modified Ritchie in identifying helminths while it was just acceptable (k = 0.27 and 0.28) in identifying B. hominis . The modified Ritchie technique routinely used in our laboratory remains a good diagnostic tool. However, the use of kit techniques was interesting when reading the pellet after concentration and the Colour KOP staining was a considerable contribution to the diagnosis of the vegetative forms. Therefore, it would be interesting to determine the cost of a stool test using Fumouze kit techniques to provide the most cost effective way.

An evaluation of direct PCR amplification

PubMed Central

Hall, Daniel E.; Roy, Reena

2014-01-01

Aim To generate complete DNA profiles from blood and saliva samples deposited on FTA® and non-FTA® paper substrates following a direct amplification protocol. Methods Saliva samples from living donors and blood samples from deceased individuals were deposited on ten different FTA® and non-FTA® substrates. These ten paper substrates containing body fluids were kept at room temperature for varying lengths of time ranging from one day to approximately one year. For all assays in this research, 1.2 mm punches were collected from each substrate containing one type of body fluid and amplified with reagents provided in the nine commercial polymerase chain reaction (PCR) amplification kits. The substrates were not subjected to purification reagent or extraction buffer prior to amplification. Results Success rates were calculated for all nine amplification kits and all ten substrates based on their ability to yield complete DNA profiles following a direct amplification protocol. Six out of the nine amplification kits, and four out of the ten paper substrates had the highest success rates overall. Conclusion The data show that it is possible to generate complete DNA profiles following a direct amplification protocol using both standard (non-direct) and direct PCR amplification kits. The generation of complete DNA profiles appears to depend more on the success of the amplification kit rather than the than the FTA®- or non-FTA®-based substrates. PMID:25559837

COMPARISON OF A GENUS-SPECIFIC CONVENTIONAL PCR AND A SPECIES-SPECIFIC NESTED-PCR FOR MALARIA DIAGNOSIS USING FTA COLLECTED SAMPLES FROM KINGDOM OF SAUDI ARABIA.

PubMed

Al-Harthi, Saeed A

2015-12-01

Molecular tools are increasingly accepted as the most sensitive and reliable techniques for malaria diagnosis and epidemiological surveys. Also, collection of finger prick blood spots onto filter papers is the most simple and affordable method for samples preservation and posterior molecular analysis, especially in rural endemic regions where malaria remains a major health problem. Two malaria molecular diagnostic tests, a Plasmodium genus-specific conventional PCR and a Plasmodium species-specific Nested PCR, were evaluated using DNA templates prepared from Whatman-FTA cards' dry blood spots using both, Methanol-fixation/Heat-extraction and FTA commercial purification kit. A total of 121 blood samples were collected from six Saudi south-western endemic districts both, as thick and thin films for routine microscopic screening and onto FTA cards for molecular studies. Out of the 121 samples, 75 were P. falciparum positive by at least one technique. No other species of Plasmodium were detected. P. falciparum parasites were identified in 69/75 (92%) samples by microscopic screening in health care centers. P. genus-specific PCR was able to amplify P. falciparum DNA in 41/75 (55%) and 59/75 (79%) samples using Methanol-fixation/Heat-extraction and FTA purification kit, respectively. P. species-specific Nested PCR revealed 68/75 (91%) and 75/75 (100%) positive samples using DNA templates were isolated by Methanol-fixation/Heat- extraction and FTA purification methods, respectively. The species-specific Nested PCR applied to Whatman-FTA preserved and processed blood samples represents the best alternative to classical microscopy for malaria diagnosis, particularly in epidemiological screening.

A technical assessment of the porcine ejaculated spermatozoa for a sperm-specific RNA-seq analysis.

PubMed

Gòdia, Marta; Mayer, Fabiana Quoos; Nafissi, Julieta; Castelló, Anna; Rodríguez-Gil, Joan Enric; Sánchez, Armand; Clop, Alex

2018-04-26

The study of the boar sperm transcriptome by RNA-seq can provide relevant information on sperm quality and fertility and might contribute to animal breeding strategies. However, the analysis of the spermatozoa RNA is challenging as these cells harbor very low amounts of highly fragmented RNA, and the ejaculates also contain other cell types with larger amounts of non-fragmented RNA. Here, we describe a strategy for a successful boar sperm purification, RNA extraction and RNA-seq library preparation. Using these approaches our objectives were: (i) to evaluate the sperm recovery rate (SRR) after boar spermatozoa purification by density centrifugation using the non-porcine-specific commercial reagent BoviPure TM ; (ii) to assess the correlation between SRR and sperm quality characteristics; (iii) to evaluate the relationship between sperm cell RNA load and sperm quality traits and (iv) to compare different library preparation kits for both total RNA-seq (SMARTer Universal Low Input RNA and TruSeq RNA Library Prep kit) and small RNA-seq (NEBNext Small RNA and TailorMix miRNA Sample Prep v2) for high-throughput sequencing. Our results show that pig SRR (~22%) is lower than in other mammalian species and that it is not significantly dependent of the sperm quality parameters analyzed in our study. Moreover, no relationship between the RNA yield per sperm cell and sperm phenotypes was found. We compared a RNA-seq library preparation kit optimized for low amounts of fragmented RNA with a standard kit designed for high amount and quality of input RNA and found that for sperm, a protocol designed to work on low-quality RNA is essential. We also compared two small RNA-seq kits and did not find substantial differences in their performance. We propose the methodological workflow described for the RNA-seq screening of the boar spermatozoa transcriptome. FPKM: fragments per kilobase of transcript per million mapped reads; KRT1: keratin 1; miRNA: micro-RNA; miscRNA: miscellaneous RNA; Mt rRNA: mitochondrial ribosomal RNA; Mt tRNA: mitochondrial transference RNA; OAZ3: ornithine decarboxylase antizyme 3; ORT: osmotic resistance test; piRNA: Piwi-interacting RNA; PRM1: protamine 1; PTPRC: protein tyrosine phosphatase receptor type C; rRNA: ribosomal RNA; snoRNA: small nucleolar RNA; snRNA: small nuclear RNA; SRR: sperm recovery rate; tRNA: transfer RNA.

High Prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae Detected in the Human Gut Using an Improved DNA Detection Protocol

PubMed Central

Dridi, Bédis; Henry, Mireille; El Khéchine, Amel; Raoult, Didier; Drancourt, Michel

2009-01-01

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. PMID:19759898

Development and validation of the AmpFℓSTR® Identifiler® Direct PCR Amplification Kit: a multiplex assay for the direct amplification of single-source samples.

PubMed

Wang, Dennis Y; Chang, Chien-Wei; Lagacé, Robert E; Oldroyd, Nicola J; Hennessy, Lori K

2011-07-01

The AmpFℓSTR(®) Identifiler(®) Direct PCR Amplification Kit is a new short tandem repeat multiplex assay optimized to allow the direct amplification of single-source blood and buccal samples on FTA(®) card without the need for sample purification and quantification. This multiplex assay has been validated according to the FBI/National Standards and SWGDAM guidelines. Validation results revealed that slight variations in primer concentration, master mix component concentration, and thermal cycling parameters did not affect the performance of the chemistry. The assay's sensitivity was demonstrated by amplifying known amounts of white blood cells spotted onto FTA(®) cards, and the assay's specificity was verified by establishing minimal cross-reactivity with nonhuman DNA. No effect on the age of the sample stored on the FTA(®) substrate was observed and full concordance was established in the population study. These findings of the validation study support the use of the Identifiler(®) Direct Kit for forensic standards and database samples genotyping. © 2011 American Academy of Forensic Sciences.

DNA Extraction from Protozoan Oocysts/Cysts in Feces for Diagnostic PCR

PubMed Central

2014-01-01

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. PMID:25031466

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

PubMed

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.

Purification of Single-Stranded cDNA Based on RNA Degradation Treatment and Adsorption Chromatography.

PubMed

Trujillo-Esquivel, Elías; Franco, Bernardo; Flores-Martínez, Alberto; Ponce-Noyola, Patricia; Mora-Montes, Héctor M

2016-08-02

Analysis of gene expression is a common research tool to study networks controlling gene expression, the role of genes with unknown function, and environmentally induced responses of organisms. Most of the analytical tools used to analyze gene expression rely on accurate cDNA synthesis and quantification to obtain reproducible and quantifiable results. Thus far, most commercial kits for isolation and purification of cDNA target double-stranded molecules, which do not accurately represent the abundance of transcripts. In the present report, we provide a simple and fast method to purify single-stranded cDNA, exhibiting high purity and yield. This method is based on the treatment with RNase H and RNase A after cDNA synthesis, followed by separation in silica spin-columns and ethanol precipitation. In addition, our method avoids the use of DNase I to eliminate genomic DNA from RNA preparations, which improves cDNA yield. As a case report, our method proved to be useful in the purification of single-stranded cDNA from the pathogenic fungus Sporothrix schenckii.

Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application.

PubMed

Zhong, Jian; Ye, Zhenqing; Lenz, Samuel W; Clark, Chad R; Bharucha, Adil; Farrugia, Gianrico; Robertson, Keith D; Zhang, Zhiguo; Ordog, Tamas; Lee, Jeong-Heon

2017-12-21

Chromatin immunoprecipitation-sequencing (ChIP-seq) is a widely used epigenetic approach for investigating genome-wide protein-DNA interactions in cells and tissues. The approach has been relatively well established but several key steps still require further improvement. As a part of the procedure, immnoprecipitated DNA must undergo purification and library preparation for subsequent high-throughput sequencing. Current ChIP protocols typically yield nanogram quantities of immunoprecipitated DNA mainly depending on the target of interest and starting chromatin input amount. However, little information exists on the performance of reagents used for the purification of such minute amounts of immunoprecipitated DNA in ChIP elution buffer and their effects on ChIP-seq data. Here, we compared DNA recovery, library preparation efficiency, and ChIP-seq results obtained with several commercial DNA purification reagents applied to 1 ng ChIP DNA and also investigated the impact of conditions under which ChIP DNA is stored. We compared DNA recovery of ten commercial DNA purification reagents and phenol/chloroform extraction from 1 to 50 ng of immunopreciptated DNA in ChIP elution buffer. The recovery yield was significantly different with 1 ng of DNA while similar in higher DNA amounts. We also observed that the low nanogram range of purified DNA is prone to loss during storage depending on the type of polypropylene tube used. The immunoprecipitated DNA equivalent to 1 ng of purified DNA was subject to DNA purification and library preparation to evaluate the performance of four better performing purification reagents in ChIP-seq applications. Quantification of library DNAs indicated the selected purification kits have a negligible impact on the efficiency of library preparation. The resulting ChIP-seq data were comparable with the dataset generated by ENCODE consortium and were highly correlated between the data from different purification reagents. This study provides comparative data on commercial DNA purification reagents applied to nanogram-range immunopreciptated ChIP DNA and evidence for the importance of storage conditions of low nanogram-range purified DNA. We verified consistent high performance of a subset of the tested reagents. These results will facilitate the improvement of ChIP-seq methodology for low-input applications.

Establishing a novel automated magnetic bead-based method for the extraction of DNA from a variety of forensic samples.

PubMed

Witt, Sebastian; Neumann, Jan; Zierdt, Holger; Gébel, Gabriella; Röscheisen, Christiane

2012-09-01

Automated systems have been increasingly utilized for DNA extraction by many forensic laboratories to handle growing numbers of forensic casework samples while minimizing the risk of human errors and assuring high reproducibility. The step towards automation however is not easy: The automated extraction method has to be very versatile to reliably prepare high yields of pure genomic DNA from a broad variety of sample types on different carrier materials. To prevent possible cross-contamination of samples or the loss of DNA, the components of the kit have to be designed in a way that allows for the automated handling of the samples with no manual intervention necessary. DNA extraction using paramagnetic particles coated with a DNA-binding surface is predestined for an automated approach. For this study, we tested different DNA extraction kits using DNA-binding paramagnetic particles with regard to DNA yield and handling by a Freedom EVO(®)150 extraction robot (Tecan) equipped with a Te-MagS magnetic separator. Among others, the extraction kits tested were the ChargeSwitch(®)Forensic DNA Purification Kit (Invitrogen), the PrepFiler™Automated Forensic DNA Extraction Kit (Applied Biosystems) and NucleoMag™96 Trace (Macherey-Nagel). After an extensive test phase, we established a novel magnetic bead extraction method based upon the NucleoMag™ extraction kit (Macherey-Nagel). The new method is readily automatable and produces high yields of DNA from different sample types (blood, saliva, sperm, contact stains) on various substrates (filter paper, swabs, cigarette butts) with no evidence of a loss of magnetic beads or sample cross-contamination. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Standardization of Procedures for the Preparation of (177)Lu- and (90)Y-labeled DOTA-Rituximab Based on the Freeze-dried Kit Formulation.

PubMed

Wojdowska, Wioletta; Karczmarczyk, Urszula; Maurin, Michal; Garnuszek, Piotr; Mikołajczak, Renata

2015-01-01

Rituximab when radiolabelled with (177)Lu or (90)Y has been investigated for the treatment of patients with Non-Hodgkin's Lymphoma. In this study, we optimized the preparation of antibody conjugates with chelating agent in the freeze-dried kit. It shortens procedures needed for the successful radiolabeling with lutetium-177 and yttrium-90 and assures reproducible labelling yields. Various molar ratios of Rituximab:DOTA (from 1:5 to 1:100) were used at the conjugation step and different purification method to remove unbound DOTA were investigated (size-exclusion chromatography, dialysis, ultrafiltration). The final monoclonal antibody concentration was quantified by Bradford method, and the number of DOTA molecules was determined by radiolabeling assay using (64)Cu. The specific activity of (177)Lu-DOTA-Rituximab and (90)Y-DOTA-Rituximab were optimized using various amounts of radiometal. Quality control (SE-HPLC, ITLC) and stability study were performed. An average of 4.2 ± 0.8 p-SCN-Bz-DOTA molecules could be randomly conjugated to a single molecule of Rituximab. The ultrafiltration system was the most efficient for purification and resulted in the highest recovery efficiency (77.2%). At optimized conditions the (177)Lu-DOTARituximab and (90)Y-DOTA-Rituximab were obtained with radiochemical purity >99% and specific activity ca. 600 MBq/mg. The radioimmunoconjugates were stable in human serum and 0.9% NaCl. After 72 h of incubation the radiochemical purity of (177)Lu-DOTA-Rituximab decreased to 94% but it was still more than 88% for (90)Y-DOTA-Rituximab. The radioimmunoconjugate showed stability after six months storage at 2 - 8(0)C, as a lyophilized formulation. Our study shows that Rituximab-DOTA can be efficiently radiolabeled with (177)Lu and (90)Y via p-SCN-Bn-DOTA using a freezedried kit.

Lyophilized Kit for the Preparation of the PET Perfusion Agent [(68)Ga]-MAA.

PubMed

Amor-Coarasa, Alejandro; Milera, Andrew; Carvajal, Denny; Gulec, Seza; McGoron, Anthony J

2014-01-01

Rapid developments in the field of medical imaging have opened new avenues for the use of positron emitting labeled microparticles. The radioisotope used in our research was (68)Ga, which is easy to obtain from a generator and has good nuclear properties for PET imaging. Methods. Commercially available macroaggregated albumin (MAA) microparticles were suspended in sterile saline, centrifuged to remove the free albumin and stannous chloride, relyophilized, and stored for later labeling with (68)Ga. Labeling was performed at different temperatures and times. (68)Ga purification settings were also tested and optimized. Labeling yield and purity of relyophilized MAA microparticles were compared with those that were not relyophilized. Results. MAA particles kept their original size distribution after relyophilization. Labeling yield was 98% at 75°C when a (68)Ga purification system was used, compared to 80% with unpurified (68)Ga. Radiochemical purity was over 97% up to 4 hours after the labeling. The relyophilized MAA and labeling method eliminate the need for centrifugation purification of the final product and simplify the labeling process. Animal experiments demonstrated the high in vivo stability of the obtained PET agent with more than 95% of the activity remaining in the lungs after 4 hours.

Human Norovirus Detection and Production, Quantification, and Storage of Virus-Like Particles

PubMed Central

Debbink, Kari; Costantini, Veronica; Swanstrom, Jesica; Agnihothram, Sudhakar; Vinjé, Jan; Baric, Ralph

2014-01-01

Human noroviruses constitute a significant worldwide disease burden. Each year noroviruses cause over 267 million infections, deaths in over 200,000 children under the age of five, and over 50% of U.S. food borne illness. Due to the absence of a tissue culture model or small animal model to study human norovirus, virus-like particles (VLPs) and ELISA-based biological assays have been used to answer questions about norovirus evolution and immunity as well provide a potential vaccine platform. This chapter outlines the protocols on norovirus detection in stool and norovirus VLP design, production, purification, and storage using a Venezuelan equine encephalitis virus (VEE)-based VRP expression system. PMID:24510290

SYBR Green Real-Time PCR Method To Detect Clostridium botulinum Type A▿

PubMed Central

Fenicia, Lucia; Anniballi, Fabrizio; De Medici, Dario; Delibato, Elisabetta; Aureli, Paolo

2007-01-01

Botulinum toxins (BoNTs) are classically produced by Clostridium botulinum but rarely also from neurotoxigenic strains of Clostridium baratii and Clostridium butyricum. BoNT type A (BoNT/A), BoNT/B, BoNT/E, and very rarely BoNT/F are mainly responsible for human botulism. Standard microbiological methods take into consideration only the detection of C. botulinum. The presumptive identification of the toxigenic strains together with the typing of BoNT has to be performed by mouse bioassay. The development of PCR-based methods for the detection and typing of BoNT-producing clostridia would be an ideal alternative to the mouse bioassay. The objective of this study was to develop a rapid and robust real-time PCR method for detecting C. botulinum type A. Four different techniques for the extraction and purification of DNA from cultured samples were initially compared. Of the techniques used, Chelex 100, DNeasy tissue kit, InstaGene matrix DNA, and boiling, the boiling technique was significantly less efficient than the other three. These did not give statistically different results, and Chelex 100 was chosen because it was less expensive than the others. In order to eliminate any false-negative results, an internal amplification control was synthesized and included in the amplification mixture according to ISO 22174. The specificity of the method was tested against 75 strains of C. botulinum type A, 4 strains of C. botulinum type Ab, and 101 nontarget strains. The detection limit of the reaction was less than 6 × 101 copies of C. botulinum type A DNA. The robustness of the method was confirmed using naturally contaminated stool specimens to evaluate the tolerance of inhibitor substances. SYBR green real-time PCR showed very high specificity for the detection of C. botulinum types A and Ab (inclusivity and exclusivity, 100%). PMID:17369349

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

PubMed

Wagner Mackenzie, Brett; Waite, David W; Taylor, Michael W

2015-01-01

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.

Association of rotavirus strains and severity of gastroenteritis in Indian children.

PubMed

Saluja, Tarun; Dhingra, Mandeep S; Sharma, Shiv D; Gupta, Madhu; Kundu, Ritabrata; Kar, Sonali; Dutta, Ashok K; Silveira, Maria D P; Singh, Jai V; Kamath, Veena G; Chaudhary, Anurag; Rao, Venkateswara; Ravi, Mandyam D; Murthy, Kesava; Arumugam, Rajesh; Moureau, Annick; Prasad, Rajendra; Patnaik, Badri N

2017-03-04

Rotavirus is the leading cause of severe and dehydrating diarrhea in children aged under 5 years. We undertook this hospital-based surveillance study to examine the possible relationship between the severity of diarrhea and the various G-group rotaviruses circulating in India. Stool samples (n = 2,051) were systematically collected from 4,711 children aged <5 years admitted with severe acute gastroenteritis to 12 medical school centers from April 2011 to July 2012. Rotavirus testing was undertaken using a commercially available enzyme immunoassay kit for the rotavirus VP6 antigen (Premier Rotaclone Qualitative ELISA). Rotavirus positive samples were genotyped for VP7 and VP4 antigens by reverse-transcription polymerase chain reaction at a central laboratory. Of the stool samples tested for rotavirus antigen, 541 (26.4%) were positive for VP6 antigen. Single serotype infections from 377 stool samples were compared in terms of gastroenteritis severity. Among those with G1 rotavirus infection, very severe diarrhea (Vesikari score ≥ 16) was reported in 59 (33.9%) children, severe diarrhea (Vesikari score 11-15) in 104 (59.8%), moderate (Vesikari score 6-10) and mild diarrhea (Vesikari score 0-5) in 11 (6.3%). Among those with G2 infection, very severe diarrhea was reported in 26 (27.4%) children, severe diarrhea in 46 (48.4%), and moderate and mild diarrhea in 23 (24.2 %). Among those with G9 infection, very severe diarrhea was reported in 47 (54.5%) children, severe diarrhea in 29 (33.6%), and moderate and mild diarrhea in 10 (11.9%). Among those with G12 infection, very severe diarrhea was reported in 9 (40.9%) children and severe diarrhea in 13 (59.1%). The results of this study indicate some association between rotavirus serotypes and severity of gastroenteritis.

The purification step is not crucial in EIA measurements of thromboxane B2 and 11-dehydrothromboxane B2 in human plasma.

PubMed

Sadilkova, Lenka; Paluch, Zoltan; Mottlova, Jirina; Bednar, Frantisek; Alusik, Stefan

2012-01-01

Thromboxane B2 (TxB2) and particularly 11-dehydrothromboxane B2 (11-dTxB2) are widely used as prognostic risk markers of platelet activation in cardiovascular diseases. The main errors in TxB2 and 11-dTxB2 determination include either low concentrations of circulating TxB2 (1 - 2 pg/mL) and 11-dTxB2 (0.9 - 4.3 pg/mL) or rather high transiency (mean TxB2 half-life is approximately 5 minutes) as well as an incorrect pre-analytical phase set up. The aim of this study was to investigate the impact of a widely used purification step on the results of enzyme immunosorbent assay (EIA)--based measurement of the two selected thromboxanes. For the purpose of this study, 20 plasma samples (10 healthy donors, 10 patients under treatment with acetylsalicylic acid) were screened for TxB2 and 11-dTxB2 concentrations using commercial competitive EIA kits (Cayman Chemicals, Tallinn, Estonia; Neogen, Lexington, KY, USA) with or without the introduction of the purification procedure. The purification step does not significantly affect the results of EIA measurements of the two of TxA2 metabolites (TxB2, 11-dTxB2) in human plasma. The levels of TxB2 and 11-dTxB2 determined in the plasma samples were not significantly changed (p < 0.05) when the purification step was omitted compared to the purified samples. This study establishes a protocol allowing for reliable and reproducible plasma TxB2 and 11-dTxB2 EIA measurement for routine basic screening of platelet function.

Optimization of circulating cell-free DNA recovery for KRAS mutation and HPV detection in plasma.

PubMed

Mazurek, Agnieszka M; Fiszer-Kierzkowska, A; Rutkowski, T; Składowski, K; Pierzyna, M; Scieglińska, D; Woźniak, G; Głowacki, G; Kawczyński, R; Małusecka, E

2013-01-01

The precise analysis of tumour markers in blood such as circulating cell-free DNA (cfDNA) could have a significant impact in facilitating monitoring of patients after initial therapy. Although high levels of total cfDNA in plasma of cancer patients are consistently demonstrated, a low sensitivity of DNA alterations is reported. The major question regards the recovery of tumour-specific cfDNA such as KRAS mutated DNA and cancer-associated type 16 of human papillomavirus (HPV16). TaqMan technology was used for detection of KRAS mutation, HPV16 and to quantify cfDNA in blood plasma. Comparison of four different column-based commercial kits shows that the cfDNA purification carried out by the Genomic Mini AX Body Fluids kit and the QIAamp Circulating Nucleic Acid kit gave us the possibility to improve the sensitivity of detection of KRAS mutation and HPV16. The optimized method was used to follow the reduction in cancer-specific cfDNA after therapy. We found that large volume extractions with low volume of DNA eluate enabled trace amounts of tumour-specific cfDNA from cancer patients to be effectively identified. Data presented in this study facilitate detection of tumour-specific cfDNA and improve standards needed for the implementation of cfDNA technology into routine clinical practice.

Immunodiagnostic Value of Echinococcus Granulosus Recombinant B8/1 Subunit of Antigen B.

PubMed

Savardashtaki, Amir; Sarkari, Bahador; Arianfar, Farzane; Mostafavi-Pour, Zohreh

2017-06-01

Cystic echinococcosis (CE), as a chronic parasitic disease, is a major health problem in many countries. The performance of the currently available serodiagnostic tests for the diagnosis of CE is unsatisfactory. The current study aimed at sub-cloning a gene, encoding the B8/1 subunit of antigen B (AgB) from Echinococcus granulosus, using gene optimization for the immunodiagnosis of human CE. The coding sequence for AgB8/1 subunit of Echinococcus granulosus was selected from GenBank and was gene-optimized. The sequence was synthesized and inserted into pGEX-4T-1 vector. Purification was performed with GST tag affinity column. Diagnostic performance of the produced recombinant antigen, native antigen B and a commercial ELISA kit were further evaluated in an ELISA system, using a panel of sera from CE patients and controls. SDS-PAGE demonstrated that the protein of interest had a high expression level and purity after GST tag affinity purification. Western blotting verified the immunoreactivity of the produced recombinant antigen with the sera of CE patients. In an ELISA system, the sensitivity and specificity (for human CE diagnosis) of the recombinant antigen, native antigen B and commercial kit were respectively 93% and 92%, 87% and 90% and 97% and 95%. The produced recombinant antigen showed a high diagnostic value which can be recommended for serodiagnosis of CE in Iran and other CE-endemic areas. Utilizing the combination of other subunits of AgB8 would improve the performance value of the introduced ELISA system.

Factors associated with use and non-use of the Fecal Immunochemical Test (FIT) kit for Colorectal Cancer Screening in Response to a 2012 outreach screening program: a survey study.

PubMed

Gordon, Nancy P; Green, Beverly B

2015-06-11

The one-sample fecal immunochemical test (FIT) is gaining popularity for colorectal cancer (CRC) screening of average-risk people. However, uptake and annual use remain suboptimal. In 2013, we mailed questionnaires to three groups of nonHispanic White, Black, and Latino Kaiser Permanente Northern California (KPNC) members ages 52-76 who received FIT kits in 2010-2012: Continuers did the FIT all 3 years; Converts in 2012, but not 2010 or 2011; and Nonusers in none of the 3 years. The questionnaires covered social influences, perceived CRC risk, reasons for using (Continuers, Converts) or avoiding using (Nonusers) the FIT, and recommendations for improving the kit. Continuers (n = 607, response rate 67.5%), Converts (n = 317, response rate 35.6%), and Nonusers (n = 215, response rate 21.1%) did not differ in perceived risk or family history of CRC, but Nonusers were less likely than Continuers and Converts to know someone who had polyps or CRC. Continuers, Converts, and Nonusers did not differ in social network encouragement of CRC screening, but did differ in believing that it was very important that they be screened (88.3%, 68.4%, 47.7%) and that their medical team thought it very important that they be screened (88.6%, 79.9%, 53.9%). Approximately half of Continuers and Converts completed the FIT to please their doctor. Converts were less likely than Continuers to use the FIT to "make sure they were OK" (53.7% vs. 72.6%) or "protect their health" (46.1% vs. 76.4%). Nearly half of Converts completed the FIT out of guilt. Approximately half of FIT kit users suggested adding a disposable glove, extra paper, and wider-mouth tube to the kit. Nonusers' reasons for not using the FIT included discomfort, disgust, or embarrassment (59.6%); thinking it unnecessary (32.9%); fatalism/fear (15.5%); and thinking it too difficult to use (14.5%), but <10% did not want CRC screening at all. Nonusers and irregular users of the FIT are less intrinsically motivated to get CRC screening than long-term users and more averse to preparing their stool sample. Changes to the FIT kit to address discomfort and difficulty factors might improve uptake and continued use.

Development and testing of monoclonal antibody-based rapid immunodiagnostic test kits for direct detection of Vibrio cholerae O139 synonym Bengal.

PubMed

Hasan, J A; Huq, A; Nair, G B; Garg, S; Mukhopadhyay, A K; Loomis, L; Bernstein, D; Colwell, R R

1995-11-01

We report on the development and testing of two monoclonal antibody-based rapid immunodiagnostic test kits, BengalScreen, a coagglutination test, and Bengal DFA, a direct fluorescent-antibody test, for direct detection of Vibrio cholerae O139 synonym Bengal in clinical and environmental specimens. The BengalScreen test requires less than 5 min to complete and can be used in the field. Bengal DFA, being more sensitive than BengalScreen, requires only one reagent and less than 20 min for detection and enumeration of V. cholerae O139 synonym Bengal. In tests for specificity, all 40 strains of V. cholerae O139 reacted with both test kits, whereas 157 strains of heterologous species examined did not, yielding 100% specificity in this study. A field trial was conducted in with both BengalScreen and Bengal DFA, and the results were compared with those obtained by conventional culture methods. BengalScreen demonstrated a sensitivity of 95%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 94%. Results obtained by Bengal DFA, on the other hand, were 100% sensitive and 100% specific and yielded 100% positive and negative predictive values compared with culture methods. In a second evaluation, 93 stool specimens from Mexico that were negative for V. cholerae O139 by culture were also tested with both the BengalScreen and Bengal DFA kits. None of the 93 specimens were positive for V. cholerae O139 by both tests. A concentration method was optimized for screening of environmental water samples for V. cholerae O139 synonym Bengal with rapid test kits. BengalScreen results were unequivocally positive when water samples contained at least 2.0 x 10(3) CFU/ml, whereas Bengal DFA demonstrated an unequivocally positive reaction when the water sample contained at least 1.5 x 10(2) CFU/ml. When Bengal DFA was compared with conventional culture methods for enumeration of V. cholerae O139 synonym Bengal organisms, no difference was observed.

Lyophilized Kit for the Preparation of the PET Perfusion Agent [68Ga]-MAA

PubMed Central

Amor-Coarasa, Alejandro; Milera, Andrew; Gulec, Seza; McGoron, Anthony J.

2014-01-01

Rapid developments in the field of medical imaging have opened new avenues for the use of positron emitting labeled microparticles. The radioisotope used in our research was 68Ga, which is easy to obtain from a generator and has good nuclear properties for PET imaging. Methods. Commercially available macroaggregated albumin (MAA) microparticles were suspended in sterile saline, centrifuged to remove the free albumin and stannous chloride, relyophilized, and stored for later labeling with 68Ga. Labeling was performed at different temperatures and times. 68Ga purification settings were also tested and optimized. Labeling yield and purity of relyophilized MAA microparticles were compared with those that were not relyophilized. Results. MAA particles kept their original size distribution after relyophilization. Labeling yield was 98% at 75°C when a 68Ga purification system was used, compared to 80% with unpurified 68Ga. Radiochemical purity was over 97% up to 4 hours after the labeling. The relyophilized MAA and labeling method eliminate the need for centrifugation purification of the final product and simplify the labeling process. Animal experiments demonstrated the high in vivo stability of the obtained PET agent with more than 95% of the activity remaining in the lungs after 4 hours. PMID:24800071

Prevalence of Intestinal Protozoans among Schoolchildren in Suburban Areas near Yangon, Myanmar.

PubMed

Kim, Min-Jae; Jung, Bong-Kwang; Cho, Jaeeun; Kim, Deok-Gyu; Song, Hyemi; Lee, Keon-Hoon; Cho, Seon; Htoon, Thi Thi; Tin, Htay Htay; Chai, Jong-Yil

2016-06-01

Although intestinal protozoans are common etiologies of diarrhea, few studies have been conducted in Myanmar. This study planned to investigate the prevalence of Giardia lamblia, Entamoeba coli, Entamoeba histolytica, and Endolimax nana among schoolchildren and their guardians in suburban areas near Yangon, Myanmar. We performed a cross-sectional survey among schoolchildren and their guardians from 7 primary schools in South Dagon and Hlaing Thar Yar districts, Yangon, Myanmar. Stool samples were observed with a microscope after concentration technique and iodine staining. Total 821 stool samples, including 556 from schoolchildren and 265 from guardians, were examined. The median age was 6 years old for schoolchildren and 36 years old for guardians. A 53.1% of the school children and 14.6 % of the guardians were males. The overall prevalence of each intestinal protozoan species was as follows: 3.4% (28/821) for G. lamblia; 3.5% (29/821) for E. coli; 1.2% (10/821) for E. histoytica, and 3.0% for E. nana. This study showed that intestinal protozoans are common in primary schoolchildren and their guardians in suburban areas near Yangon, Myanmar. Health interventions, such as hand washing education, improvement of sanitation, and establishment of water purification systems are urgently needed in this area.

Prevalence of Intestinal Protozoans among Schoolchildren in Suburban Areas near Yangon, Myanmar

PubMed Central

Kim, Min-Jae; Jung, Bong-Kwang; Cho, Jaeeun; Kim, Deok-Gyu; Song, Hyemi; Lee, Keon-Hoon; Cho, Seon; Htoon, Thi Thi; Tin, Htay Htay; Chai, Jong-Yil

2016-01-01

Although intestinal protozoans are common etiologies of diarrhea, few studies have been conducted in Myanmar. This study planned to investigate the prevalence of Giardia lamblia, Entamoeba coli, Entamoeba histolytica, and Endolimax nana among schoolchildren and their guardians in suburban areas near Yangon, Myanmar. We performed a cross-sectional survey among schoolchildren and their guardians from 7 primary schools in South Dagon and Hlaing Thar Yar districts, Yangon, Myanmar. Stool samples were observed with a microscope after concentration technique and iodine staining. Total 821 stool samples, including 556 from schoolchildren and 265 from guardians, were examined. The median age was 6 years old for schoolchildren and 36 years old for guardians. A 53.1% of the school children and 14.6 % of the guardians were males. The overall prevalence of each intestinal protozoan species was as follows: 3.4% (28/821) for G. lamblia; 3.5% (29/821) for E. coli; 1.2% (10/821) for E. histoytica, and 3.0% for E. nana. This study showed that intestinal protozoans are common in primary schoolchildren and their guardians in suburban areas near Yangon, Myanmar. Health interventions, such as hand washing education, improvement of sanitation, and establishment of water purification systems are urgently needed in this area. PMID:27417092

Evaluating Protocols for Porcine Faecal Microbiome Recollection, Storage and DNA Extraction: from the Farm to the Lab.

PubMed

Muiños-Bühl, Anixa; González-Recio, Oscar; Muñoz, María; Óvilo, Cristina; García-Casco, Juan; Fernández, Ana I

2018-06-01

There is a growing interest in understanding the role of the gut microbiome on productive and meat quality-related traits in livestock species in order to develop new useful tools for improving pig production systems and industry. Faecal samples are analysed as a proxy of gut microbiota and here the selection of suitable protocols for faecal sampling and DNA isolation is a critical first step in order to obtain reliable results, even more to compare results obtained from different studies. The aim of the current study was to establish in a cost-effective way, using automated ribosomal intergenic spacer analysis technique, a protocol for porcine faecal sampling and storage at farm and slaughterhouse and to determine the most efficient microbiota DNA isolation kit among those most widely used. Operational Taxonomic Unit profiles were compared from Iberian pig faecal samples collected from rectum or ground, stored with liquid N 2 , room temperature or RNAlater, and processed with QIAamp DNA Stool (Qiagen), PowerFecal DNA Isolation (Mobio) or SpeedTools Tissue DNA extraction (Biotools) commercial kits. The results, focused on prokaryote sampling, based on DNA yield and quality, OTU number and Sørensen similarity Indexes, indicate that the recommended protocol for porcine faecal microbiome sampling at farm should include: the collection from porcine rectum to avoid contamination; the storage in liquid N 2 or even at room temperature, but not in RNAlater; and the isolation of microbiota DNA using PowerFecal DNA Isolation kit. These conditions provide more reliable DNA samples for further microbiome analysis.

Recombinant glycoprotein G analog for determination of specific immunoglobulins to herpes simplex virus type 2 by ELISA.

PubMed

Korshun, Ludmila; Vudmaska, Mariya; Moysa, Larissa; Kovtonjuk, Galina; Mikhalap, Svetlana; Ganova, Larissa; Spivak, Nikolay

2013-12-01

In order to the detection of type-specific IgG to herpes simplex virus type 2 (HSV-2) in human serum or plasma the recombinant analog of HSV-2 glycoprotein G (gG2) was created. To construct an expression vector the DNA fragment with a sequence identical to immunodominant regions of HSV-2 gG2 was cloned into modified vector pET28a containing of the glutation-S-transferase sequence (pET28-GST). Escherichia coli BL21 (DE3) were transformed with the recombinant plasmid. The target protein was expressed mainly in soluble form. Chromatographic purification of soluble GST-gG2 protein was performed taking into account the features of its primary structure that are 6His-tag and GST-tag. To determine the affinity constant of the specific IgG to GST-gG2 we used the method proposed by Friguet et al. (1985). The affinity constants were within the range of 10(7)-10(8)M(-1) proving their high-affinity. The purified recombinant HSV-2 antigen was used to design a diagnostic ELISA kit, which was evaluated with referent controls and standard panels of sera containing and/or not containing anti-HSV-2 IgG. Comparative evaluation of this kit and the commercially available "HSV-Type 2 IgG-ELISA" (NovaTec, Dietzenbach, Germany) kit was performed. There was no significant difference (P>0.05). It allows to use developed ELISA kit for clinical diagnosis of HSV-2 infection. Copyright © 2013 Elsevier B.V. All rights reserved.

Efficacy and safety of a polyherbal formulation in hemorrhoids

PubMed Central

Tripathi, Raakhi K.; Bolegave, Somesh S.; Shetty, Parvan A.; Uchil, Dinesh A.; Rege, Nirmala N.; Chawda, Mukesh B.; Rege, Sameer A.

2015-01-01

Background: The medical management of hemorrhoids should include an integrated approach. This integrated approach can be achieved by polyherbal formulations containing anti-inflammatory, styptics, analgesics, and laxative effect which reduce inflammation, pain, and bleeding, and increase gastro-intestinal motility and soften stools. One such polyherbal kit is “Arshkeyt™, a 7 day kit,” which consists of oral tablets and powder along with topical cream. Objective: Efficacy and safety of Arshkeyt™, a 7 day kit, a marketed polyherbal formulation was evaluated in comparison with conventional therapy practiced in surgery outpatient departments. Materials and Methods: Patients (n = 90) with hemorrhoids were randomly allocated to receive either Arshkeyt™ or standard therapy (combination of oral Isabgul powder and 2% lidocaine gel) for 14 days. Assessment on the basis of rectal symptoms and proctoscopic examination was done on day 0, 7, and 14 to derive a “composite score” which ranged from 0 to 25 by a blinded evaluator. The primary endpoint was number of patients achieving composite score 0 at the end of therapy (day 14). Inter-group analysis was done using Chi-square test. Results: On day 14, the composite score of 0 was achieved in 15 patients of Arshkeyt™ group versus 6 patients receiving standard therapy. The symptoms and signs which showed significant improvement in Arshkeyt™ group compared to standard treatment group were the tenesmus (visual analog score) score (P = 0.047), anal sphincter spasm (P = 0.0495) and a decrease in the grade of hemorrhoids (P = 0.0205) on day 14. Arshkeyt™ was also more beneficial in case of bleeding hemorrhoids as compared to nonbleeding hemorrhoids (P < 0.05). The incidence of adverse drug reactions in both groups was comparable and no patient required any treatment for the same. Conclusion: “Arshkeyt™, a 7 day kit,” was effective in the treatment of hemorrhoids and had a good safety profile. PMID:26834421

Comparison of the Liaison® Calprotectin kit with a well established point of care test (Quantum Blue - Bühlmann-Alere®) in terms of analytical performances and ability to detect relapses amongst a Crohn population in follow-up.

PubMed

Delefortrie, Quentin; Schatt, Patricia; Grimmelprez, Alexandre; Gohy, Patrick; Deltour, Didier; Collard, Geneviève; Vankerkhoven, Patrick

2016-02-01

Although colonoscopy associated with histopathological sampling remains the gold standard in the diagnostic and follow-up of inflammatory bowel disease (IBD), calprotectin is becoming an essential biomarker in gastroenterology. The aim of this work is to compare a newly developed kit (Liaison® Calprotectin - Diasorin®) and its two distinct extraction protocols (weighing and extraction device protocol) with a well established point of care test (Quantum Blue® - Bühlmann-Alere®) in terms of analytical performances and ability to detect relapses amongst a Crohn's population in follow-up. Stool specimens were collected over a six month period and were composed of control and Crohn's patients. Amongst the Crohn's population disease activity (active vs quiescent) was evaluated by gastroenterologists. A significant difference was found between all three procedures in terms of calprotectin measurements (weighing protocol=30.3μg/g (median); stool extraction device protocol=36.9μg/g (median); Quantum Blue® (median)=63; Friedman test, P value=0.05). However, a good correlation was found between both extraction methods coupled with the Liaison® analyzer and between the Quantum Blue® (weighing protocol/extraction device protocol Rs=0.844, P=0.01; Quantum Blue®/extraction device protocol Rs=0.708, P=0.01; Quantum Blue®/weighing protocol, Rs=0.808, P=0.01). Finally, optimal cut-offs (and associated negative predictive values - NPV) for detecting relapses were in accordance with above results (Quantum Blue® 183.5μg/g and NPV of 100%>extraction device protocol+Liaison® analyzer 124.5μg/g and NPV of 93.5%>weighing protocol+Liaison® analyzer 106.5μg/g and NPV of 95%). Although all three methods correlated well and had relatively good NPV in terms of detecting relapses amongst a Crohn's population in follow-up, the lack of any international standard is the origin of different optimal cut-offs between the three procedures. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Comparison of commercial RNA extraction kits and qPCR master mixes for studying gene expression in small biopsy tissue samples from the equine gastric epithelium.

PubMed

Tesena, Parichart; Korchunjit, Wasamon; Taylor, Jane; Wongtawan, Tuempong

2017-01-01

Gastric tissue biopsy and gene expression analysis are important tools for disease diagnosis and study of the physiology of the equine stomach. However, RNA extraction from gastric biopsy samples is a complex procedure because the samples contain low quantities of RNA and are contaminated with mucous protein and bacterial flora. The objectives of these studies were to compare the performance of RNA extraction methods and to investigate the sensitivity of commercial qPCR master mixes for gene expression analysis of gastric biopsy samples. Three commercial RNA extraction methods (TRIzol ™ , GENEzol ™ and MiniPrep ™ ) and four qPCR master mixes with SYBR ® green (qPCRBIO, KAPA, QuantiNova, and PerfeCTa) were compared. RNA qualification and quantitation were compared. Real-time PCR was used to compare qPCR master mixes. The results revealed that TRIzol and GENEzol obtained significantly higher yield of RNA (P<0.01) but that TRIzol had the highest contamination of protein and DNA (P<0.05). Conversely, MiniPrep resulting in a significantly higher purification of RNA (P<0.05) but provided the lowest yield of RNA (P<0.01). For PCR master mixes, KAPA was significantly (P<0.05) more sensitive than other qPCR kits for all amounts of DNA template, particularly at the lowest amount of cDNA. In conclusion, GENEzol is the best method to obtain a high RNA yield and purification and it is more cost-effective than the others as well. Regarding the qPCR master mixes, KAPA SYBR qPCR Master Mix (2x) Universal is superior to the other tested master mixes for studying gene expression in equine gastric biopsies.

Kit formulated asialoglycoprotein receptor targeting tracer based on copolymer for liver SPECT imaging.

PubMed

Liu, Chang; Guo, Zhide; Zhang, Pu; Song, Manli; Zhao, Zuoquan; Wu, Xiaowei; Zhang, Xianzhong

2014-08-01

Specific targeting of galactose-carrying molecule to ASGP-R in normal hepatocytes has been demonstrated before. In this study, galactosyl polystyrene was synthesized from controllable ratio of functional monomers and radio-labelled with (99m)Tc by formulated kit for SPECT imaging of hepatic function. p(VLA-co-VNI)(46:54) was synthesized by free-radical copolymerization initiated by AIBN, purified by dialysis, lyophilized to kit with Tricine and TPPTS as co-ligands for (99m)Tc labeling. Radiotracer (99m)Tc-p(VLA-co-VNI)(46:54)(Tricine)(TPPTS) was prepared and evaluated by in vitro stability, in vivo metabolism, ex vivo biodistribution and microSPECT/CT imaging in normal KM mice. MicroSPECT/CT and microMRI imaging were also performed in C57BL/b6 mice with xenograft hepatic carcinoma for hepatic function evaluation. (99m)Tc-p(VLA-co-VNI)(46:54)(Tricine)(TPPTS) was obtained in high radio chemical purity (RCP) (>99%) by using instant kit without further purification and excellent in vitro and in vivo stability. The result of biodistribution showed that liver had high uptake (90.49±10.68 ID%/g) at 30 min after injection and was blocked significantly by cold copolymer. MicroSPECT imaging in normal KM mice at 1h and 4h after injection showed good liver retention and targeting properties. Significant defect of activity was observed in the tumor site which was confirmed by MRI imaging. (99m)Tc-p(VLA-co-VNI)(46:54)(Tricine)(TPPTS) with lower ratio of targeting moiety has no observable effect on the specific binding affinity and liver uptake. This makes it possible to introduce more imaging units for multi-modality imaging. Furthermore, the instant kit preparation of (99m)Tc-labeling provides great potential for the evaluation of hepatocyte function in clinical application. Copyright © 2014 Elsevier Inc. All rights reserved.

Large-Scale Low-Cost NGS Library Preparation Using a Robust Tn5 Purification and Tagmentation Protocol

PubMed Central

Hennig, Bianca P.; Velten, Lars; Racke, Ines; Tu, Chelsea Szu; Thoms, Matthias; Rybin, Vladimir; Besir, Hüseyin; Remans, Kim; Steinmetz, Lars M.

2017-01-01

Efficient preparation of high-quality sequencing libraries that well represent the biological sample is a key step for using next-generation sequencing in research. Tn5 enables fast, robust, and highly efficient processing of limited input material while scaling to the parallel processing of hundreds of samples. Here, we present a robust Tn5 transposase purification strategy based on an N-terminal His6-Sumo3 tag. We demonstrate that libraries prepared with our in-house Tn5 are of the same quality as those processed with a commercially available kit (Nextera XT), while they dramatically reduce the cost of large-scale experiments. We introduce improved purification strategies for two versions of the Tn5 enzyme. The first version carries the previously reported point mutations E54K and L372P, and stably produces libraries of constant fragment size distribution, even if the Tn5-to-input molecule ratio varies. The second Tn5 construct carries an additional point mutation (R27S) in the DNA-binding domain. This construct allows for adjustment of the fragment size distribution based on enzyme concentration during tagmentation, a feature that opens new opportunities for use of Tn5 in customized experimental designs. We demonstrate the versatility of our Tn5 enzymes in different experimental settings, including a novel single-cell polyadenylation site mapping protocol as well as ultralow input DNA sequencing. PMID:29118030

Purification of adult hepatic progenitor cells using green fluorescent protein (GFP)-transgenic mice and fluorescence-activated cell sorting.

PubMed

Fujikawa, Takahisa; Hirose, Tetsuro; Fujii, Hideaki; Oe, Shoshiro; Yasuchika, Kentaro; Azuma, Hisaya; Yamaoka, Yoshio

2003-08-01

Recent advances in stem cell research have revealed that hepatic stem/progenitor cells may play an important role in liver development and regeneration. However, a lack of detectable definitive markers in viable cells has hindered their primary culture from adult livers. Enzymatically dissociated liver cells from green fluorescent protein (GFP)-transgenic mice, which express GFP highly in liver endodermal cells, were sorted by GFP expression using a fluorescence-activated cell sorter. Sorted cells were characterized, and also low-density cultured for extended periods to determine their proliferation and clonal differentiation capacities. When CD45(-)TER119(-) side-scatter(low) GFP(high) cells were sorted, alpha-fetoprotein-positive immature endoderm-characterized cells, having high growth potential, were present in this population. Clonal analysis and electron microscopic evaluation revealed that each single cell of this population could differentiate not only into hepatocytes, but also into biliary epithelial cells, showing their bilineage differentiation activity. When surface markers were analyzed, they were positive for Integrin-alpha6 and -beta1, but negative for c-Kit and Thy1.1. Combination of GFP-transgenic mice and fluorescence-activated cell sorting enabled purification of hepatic progenitor cells from adult mouse liver. Further analysis of this population may lead to purification of their human correspondence that would be an ideal cell-source candidate for regenerative medicine.

Biochemical Properties of Two Cinnamoyl Esterases Purified from a Lactobacillus johnsonii Strain Isolated from Stool Samples of Diabetes-Resistant Rats▿

PubMed Central

Lai, Kin Kwan; Lorca, Graciela L.; Gonzalez, Claudio F.

2009-01-01

Cinnamic acids (i.e., ferulic and caffeic acids) that are esterified to the vegetable cell walls should be enzymatically released to be absorbed in a mammal's intestines. A low dosage of ferulic acid in rodent diets stimulates insulin production and alleviates symptoms caused by diabetes (M. Sri Balasubashini, R. Rukkumani, and V. P. Menon, Acta Diabetol. 40:118-122, 2003). Several lactic acid bacteria are able to display ferulic acid esterase (FAE) activity, suggesting that their probiotic activity could be, in part, mediated by the slow release of ferulic acid. In the present work, we describe the isolation of one strain identified as being Lactobacillus johnsonii that displayed strong FAE activity in stool samples from diabetes-resistant biobreeding rats. These animals are genetically susceptible to becoming diabetic but do not develop the disease. By using genomic analysis coupled to protein purification and catalytic screening, we were able to purify two proteins with FAE activity. The enzymes displayed 42% sequence identity and a broad range of substrate preferences. High affinities and catalytic efficiencies toward aromatic compounds such as ethyl ferulate (Km = 20 to 60 μM) and chlorogenic acid (Km = 10 to 50 μM) were observed. The strain isolated herein as well as the enzymes studied could be potentially useful for the formulation of probiotics to ameliorate diabetes symptoms. PMID:19502437

Biochemical properties of two cinnamoyl esterases purified from a Lactobacillus johnsonii strain isolated from stool samples of diabetes-resistant rats.

PubMed

Lai, Kin Kwan; Lorca, Graciela L; Gonzalez, Claudio F

2009-08-01

Cinnamic acids (i.e., ferulic and caffeic acids) that are esterified to the vegetable cell walls should be enzymatically released to be absorbed in a mammal's intestines. A low dosage of ferulic acid in rodent diets stimulates insulin production and alleviates symptoms caused by diabetes (M. Sri Balasubashini, R. Rukkumani, and V. P. Menon, Acta Diabetol. 40:118-122, 2003). Several lactic acid bacteria are able to display ferulic acid esterase (FAE) activity, suggesting that their probiotic activity could be, in part, mediated by the slow release of ferulic acid. In the present work, we describe the isolation of one strain identified as being Lactobacillus johnsonii that displayed strong FAE activity in stool samples from diabetes-resistant biobreeding rats. These animals are genetically susceptible to becoming diabetic but do not develop the disease. By using genomic analysis coupled to protein purification and catalytic screening, we were able to purify two proteins with FAE activity. The enzymes displayed 42% sequence identity and a broad range of substrate preferences. High affinities and catalytic efficiencies toward aromatic compounds such as ethyl ferulate (K(m) = 20 to 60 microM) and chlorogenic acid (K(m) = 10 to 50 microM) were observed. The strain isolated herein as well as the enzymes studied could be potentially useful for the formulation of probiotics to ameliorate diabetes symptoms.

A multicenter prospective hospital-based surveillance to estimate the burden of rotavirus gastroenteritis in children less than five years of age in India.

PubMed

Saluja, T; Sharma, S D; Gupta, M; Kundu, R; Kar, S; Dutta, A; Silveira, M; Singh, J V; Kamath, V G; Chaudhary, A; Rao, J V; Ravi, M D; Murthy, S R K; Babji, S; Prasad, R; Gujjula, R; Rao, R; Dhingra, M S

2014-08-11

Rotavirus is the leading cause of severe, dehydrating diarrhea in children aged <5 years globally, with an estimated 25 million outpatient visits and 2 million hospitalizations attributable to rotavirus infections each year. The aim of this hospital-based surveillance was to summarize the local epidemiological and virological features of rotavirus and to estimate the disease burden in the population under surveillance in India. During the 16 months surveillance period from April 2011 through July 2012, a total of 4711 children under the age of 5 years were admitted with acute diarrhea at 12 medical centers attached to medical schools throughout India. Stool samples were randomly collected from 2051 (43.5%) subjects and were analyzed for rotavirus positivity using commercial enzyme immunoassay kit (Premier Rotaclone Qualitative Elisa) at the respective study centers. Rotavirus positive samples were genotyped for VP7 and VP4 by reverse-transcription polymerase chain reaction (RT-PCR) at a central laboratory. During the study period, maximum number of rotavirus related hospitalizations were reported from December 2011 through February 2012. Out of the 2051 stool samples tested for rotavirus, overall 541 (26.4%) samples were positive for rotavirus VP6 antigen in stool. The highest positivity was observed in the month of December, 2011 (52.5%) and lowest in the month of May, 2011 (10.3%). We found that majority of the rotavirus positive cases (69.7%) were in children <24 months of age. The most common genotypes reported were G1 (38%), G2 (18%), G9 (18%), G12 (9%) and mixed strains (17%). The results of this study confirm the significant burden of acute rotavirus gastroenteritis as a cause of hospitalizations in under five children in India. Copyright © 2014. Published by Elsevier Ltd.

Evaluation of a multiplex PCR assay for detection of cytomegalovirus in stool samples from patients with ulcerative colitis

PubMed Central

Nahar, Saifun; Iraha, Atsushi; Hokama, Akira; Uehara, Ayako; Parrott, Gretchen; Ohira, Tetsuya; Kaida, Masatoshi; Kinjo, Tetsu; Kinjo, Takeshi; Hirata, Tetsuo; Kinjo, Nagisa; Fujita, Jiro

2015-01-01

AIM: To evaluate a multiplex PCR assay for the detection of bacterial and viral enteropathogens in stool samples from patients with ulcerative colitis (UC). METHODS: We prospectively analyzed 300 individuals, including immunocompetent patients, immunocompromised patients, and patients with UC. Stool samples were collected from the recto-sigmoid region of the colon by endoscopy. The samples were qualitatively analyzed for bacterial and viral enteropathogens with a multiplex PCR assay using a Seeplex® Kit. Additional clinical and laboratory data were collected from the medical records. RESULTS: A multiplex PCR assay detected 397 pathogens (191 bacteria and 206 viruses) in 215 samples (71.7%). The most frequently detected bacteria were Escherichia coli H7, 85 (28.3%); followed by Aeromonas spp., 43 (14.3%); and Clostridium perfringens, 36 (12.0%) samples. The most prevalent viruses were Epstein-Barr virus (EBV), 90 (30.0%); followed by human herpes virus-6 (HHV-6), 53 (17.7%); and cytomegalovirus (CMV), 37 (12.3%) samples. The prevalence rate of CMV infection was significantly higher in the immunocompromised group than in the immunocompetent group (P < 0.01). CMV infection was more common in patients with UC (26/71; 36.6%) than in the immunocompetent patients excluding UC (6/188; 3.2%) (P < 0.01). CMV infection was more prevalent in UC active patients (25/58; 43.1%) than in UC inactive patients (1/13; 7.7%) (P < 0.05). Among 4 groups which defined by the UC activity and immunosuppressive drugs, the prevalence rate of CMV infection was highest in the UC active patients with immunosuppressive drugs (19/34; 55.8%). Epstein-Barr virus (EBV) infection was more common in the immunocompromised patients excluding UC (18/41; 43.9%) than in the immunocompetent patients excluding UC (47/188; 25.0%) (P < 0.05). The simultaneous presence of CMV and EBV and/or HHV6 in UC active patients (14/58; 24.1%) was greater than in immunocompromised patients excluding UC (5/41; 12.2%) (P < 0.05). CONCLUSION: The multiplex PCR assay that was used to analyze the stool samples in this study may serve as a non-invasive approach that can be used to exclude the possibility of CMV infection in patients with active UC who are treated with immunosuppressive therapy. PMID:26640344

Microchip-based cell lysis and DNA extraction from sperm cells for application to forensic analysis.

PubMed

Bienvenue, Joan M; Duncalf, Natalie; Marchiarullo, Daniel; Ferrance, Jerome P; Landers, James P

2006-03-01

The current backlog of casework is among the most significant challenges facing crime laboratories at this time. While the development of next-generation microchip-based technology for expedited forensic casework analysis offers one solution to this problem, this will require the adaptation of manual, large-volume, benchtop chemistry to small volume microfluidic devices. Analysis of evidentiary materials from rape kits where semen or sperm cells are commonly found represents a unique set of challenges for on-chip cell lysis and DNA extraction that must be addressed for successful application. The work presented here details the development of a microdevice capable of DNA extraction directly from sperm cells for application to the analysis of sexual assault evidence. A variety of chemical lysing agents are assessed for inclusion in the extraction protocol and a method for DNA purification from sperm cells is described. Suitability of the extracted DNA for short tandem repeat (STR) analysis is assessed and genetic profiles shown. Finally, on-chip cell lysis methods are evaluated, with results from fluorescence visualization of cell rupture and DNA extraction from an integrated cell lysis and purification with subsequent STR amplification presented. A method for on-chip cell lysis and DNA purification is described, with considerations toward inclusion in an integrated microdevice capable of both differential cell sorting and DNA extraction. The results of this work demonstrate the feasibility of incorporating microchip-based cell lysis and DNA extraction into forensic casework analysis.

Triple helix purification and sequencing

DOEpatents

Wang, Renfeng; Smith, Lloyd M.; Tong, Xinchun E.

1995-01-01

Disclosed herein are methods, kits, and equipment for purifying single stranded circular DNA and then using the DNA for DNA sequencing purposes. Templates are provided with an insert having a hybridization region. An elongated oligonucleotide has two regions that are complementary to the insert and the oligo is bound to a magnetic anchor. The oligo hybridizes to the insert on two sides to form a stable triple helix complex. The anchor can then be used to drag the template out of solution using a magnet. The system can purify sequencing templates, and if desired the triple helix complex can be opened up to a double helix so that the oligonucleotide will act as a primer for further DNA synthesis.

Triple helix purification and sequencing

DOEpatents

Wang, R.; Smith, L.M.; Tong, X.E.

1995-03-28

Disclosed herein are methods, kits, and equipment for purifying single stranded circular DNA and then using the DNA for DNA sequencing purposes. Templates are provided with an insert having a hybridization region. An elongated oligonucleotide has two regions that are complementary to the insert and the oligo is bound to a magnetic anchor. The oligo hybridizes to the insert on two sides to form a stable triple helix complex. The anchor can then be used to drag the template out of solution using a magnet. The system can purify sequencing templates, and if desired the triple helix complex can be opened up to a double helix so that the oligonucleotide will act as a primer for further DNA synthesis. 4 figures.

Enrichment of undifferentiated type a spermatogonia from goat testis using discontinuous percoll density gradient and differential plating.

PubMed

Heidari, Banafsheh; Gifani, Minoo; Shirazi, Abolfazl; Zarnani, Amir-Hassan; Baradaran, Behzad; Naderi, Mohammad Mehdi; Behzadi, Bahareh; Borjian-Boroujeni, Sara; Sarvari, Ali; Lakpour, Niknam; Akhondi, Mohammad Mehdi

2014-04-01

The well documented source for adult multipotent stem cells is Spermatogonial Stem Cells (SSCs). They are the foundation of spermatogenesis in the testis throughout adult life by balancing self-renewal and differentiation. The aim of this study was to assess the effect of percoll density gradient and differential plating on enrichment of undifferentiated type A spermatogonia in dissociated cellular suspension of goat testes. Additionally, we evaluated the separated fractions of the gradients in percoll and samples in differential plating at different times for cell number, viability and purification rate of goat SSCs in culture. Testicular cells were successfully isolated from one month old goat testis using two-step enzymatic digestion and followed by two purification protocols, differential plating with different times of culture (3, 4, 5, and 6 hr) and discontinuous percoll density with different gradients (20, 28, 30, and 32%). The difference of percentage of undifferentiated SSCs (PGP9.5 positive) in each method was compared using ANOVA and comparison between the highest percentage of corresponding value between two methods was carried out by t-test using Sigma Stat (ver. 3.5). The highest PGP9.5 (94.6±0.4) and the lowest c-Kit positive (25.1±0.7) in Percoll method was significantly (p ≤ 0.001) achieved in 32% percoll gradient. While the corresponding rates in differential plating method for the highest PGP9.5 positive cells (81.3±1.1) and lowest c-Kit (17.1±1.4) was achieved after 5 hr culturing (p < 0.001). The enrichment of undifferentiated type A spermatogonia using Percoll was more efficient than differential plating method (p < 0.001). Percoll density gradient and differential plating were efficient and fast methods for enrichment of type A spermatogonial stem cells from goat testes.

Enrichment of Undifferentiated Type A Spermatogonia from Goat Testis Using Discontinuous Percoll Density Gradient and Differential Plating

PubMed Central

Heidari, Banafsheh; Gifani, Minoo; Shirazi, Abolfazl; Zarnani, Amir-Hassan; Baradaran, Behzad; Naderi, Mohammad Mehdi; Behzadi, Bahareh; Borjian-Boroujeni, Sara; Sarvari, Ali; Lakpour, Niknam; Akhondi, Mohammad Mehdi

2014-01-01

Background The well documented source for adult multipotent stem cells is Spermatogonial Stem Cells (SSCs). They are the foundation of spermatogenesis in the testis throughout adult life by balancing self-renewal and differentiation. The aim of this study was to assess the effect of percoll density gradient and differential plating on enrichment of undifferentiated type A spermatogonia in dissociated cellular suspension of goat testes. Additionally, we evaluated the separated fractions of the gradients in percoll and samples in differential plating at different times for cell number, viability and purification rate of goat SSCs in culture. Methods Testicular cells were successfully isolated from one month old goat testis using two-step enzymatic digestion and followed by two purification protocols, differential plating with different times of culture (3, 4, 5, and 6 hr) and discontinuous percoll density with different gradients (20, 28, 30, and 32%). The difference of percentage of undifferentiated SSCs (PGP9.5 positive) in each method was compared using ANOVA and comparison between the highest percentage of corresponding value between two methods was carried out by t-test using Sigma Stat (ver. 3.5). Results The highest PGP9.5 (94.6±0.4) and the lowest c-Kit positive (25.1±0.7) in Percoll method was significantly (p ≤ 0.001) achieved in 32% percoll gradient. While the corresponding rates in differential plating method for the highest PGP9.5 positive cells (81.3±1.1) and lowest c-Kit (17.1±1.4) was achieved after 5 hr culturing (p < 0.001). The enrichment of undifferentiated type A spermatogonia using Percoll was more efficient than differential plating method (p < 0.001). Conclusion Percoll density gradient and differential plating were efficient and fast methods for enrichment of type A spermatogonial stem cells from goat testes. PMID:24834311

Investigation of Free-Standing Plasmonic Mesoporous Ag/CMK-8-Nafion Composite Membrane for the Removal of Organic Pollutants with 254-nm UV Irradiation

NASA Astrophysics Data System (ADS)

Tseng, Chuan Ming; Chen, Hsin Liang; Lai, Sz Nian; Chen, Ming Shiung; Peng, Chien Jung; Li, Chia Jui; Hung, Wei Hsuan

2017-05-01

"Carbon-based material" has demonstrated a great potential on water purification due to its strong physical adsorption to organic pollutants in the water. Three-dimensional cubic ordered mesoporous carbon (CMK-8), one of the well-known ordered mesoporous carbons, was prepared by using nanocasting method with mesoporous silica (KIT-6) as the template. In this study, CMK-8 blended with Nafion polymer to form a free-standing mesoporous CMK-8-Nafion composite membrane. The synthesis of high crystallinity CMK-8 was characterized by X-ray diffraction (XRD) and transmission electron microscopy (TEM). More than 80% methyl orange (MO) removal efficiency was observed under 254-nm UV irradiation after 120 min. Ninety-two percent recycling performance was remained after four recycling tests, which indicated a reliable servicing lifetime for the water purification. Furthermore, an additional layer of plasmonic silver nanoparticles (Ag NPs) was integrated into this CMK-8-Nafion membrane for higher pollutant removal efficiency, attributing from the generation of plasmon-resonance hot electrons from Ag NPs. A 4-in. CMK-8-Nafion composite membrane was also fabricated for the demonstration of potential large-scale utilization.

Residual matrix from different separation techniques impacts exosome biological activity.

PubMed

Paolini, Lucia; Zendrini, Andrea; Di Noto, Giuseppe; Busatto, Sara; Lottini, Elisabetta; Radeghieri, Annalisa; Dossi, Alessandra; Caneschi, Andrea; Ricotta, Doris; Bergese, Paolo

2016-03-24

Exosomes are gaining a prominent role in research due to their intriguing biology and several therapeutic opportunities. However, their accurate purification from body fluids and detailed physicochemical characterization remain open issues. We isolated exosomes from serum of patients with Multiple Myeloma by four of the most popular purification methods and assessed the presence of residual contaminants in the preparations through an ad hoc combination of biochemical and biophysical techniques - including Western Blot, colloidal nanoplasmonics, atomic force microscopy (AFM) and scanning helium ion microscopy (HIM). The preparations obtained by iodixanol and sucrose gradients were highly pure. To the contrary, those achieved with limited processing (serial centrifugation or one step precipitation kit) resulted contaminated by a residual matrix, embedding the exosomes. The contaminated preparations showed lower ability to induce NfkB nuclear translocation in endothelial cells with respect to the pure ones, probably because the matrix prevents the interaction and fusion of the exosomes with the cell membrane. These findings suggest that exosome preparation purity must be carefully assessed since it may interfere with exosome biological activity. Contaminants can be reliably probed only by an integrated characterization approach aimed at both the molecular and the colloidal length scales.

Validation of a modified Bristol stool scale - inter-rater reliability amongst pediatric gastroenterologists

USDA-ARS?s Scientific Manuscript database

Stool form and changes in stool form are important criteria in both clinical practice and clinical research. However, descriptions of stool form from both patients and physicians alike may be subjective and objective measurements of stool form are not well developed. Although the Bristol stool scale...

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

PubMed

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 tests was 99.4 and 98.3 %, respectively. DNA sequencing results of 19 microscopically confirmed cases were strictly identical (concordance 100 %) in both methods, and ST2:6, ST3:8, ST4:3, and ST6:2 were the detected subtypes. Among the 230 fecal samples, the most predominant subtypes were ST3, ST2, ST4, and ST1 by both FS and DSSFP methods. Concordance of DNA sequencing results obtained from the two methods was noted to be 90.7 %. To our knowledge, this is the first study that demonstrates DNA extraction from DSSFP is more sensitive and effective than the FS method for diagnosis of Blastocystis spp. and their subtypes by PCR and DNA sequencing.

Gastrointestinal Bleeding: MedlinePlus Health Topic

MedlinePlus

... looks like coffee grounds Black or tarry stool Dark blood mixed with stool Signs of bleeding in ... lower digestive tract include Black or tarry stool Dark blood mixed with stool Stool mixed or coated ...

Early Detection of Neonatal Cholestasis: Inadequate Assessment of Stool Color by Parents and Primary Healthcare Doctors.

PubMed

Witt, Mauri; Lindeboom, Jeanet; Wijnja, Corry; Kesler, Anneke; Keyzer-Dekker, Claudia M G; Verkade, Henkjan J; Hulscher, Jan B F

2016-02-01

Early diagnosis and surgery (< 60 days of age) improve outcomes in children with biliary atresia. Only 56% of patients undergo timely surgery in the Netherlands. Lack of acquaintance with symptoms such as discolored stools might underlie this delay. We analyzed whether Dutch parents, youth healthcare doctors, or general practitioners recognized discolored stools and evaluated the effect of the Infant Stool Color Card (ISCC) on recognizing discolored stools. We asked 100 parents, 33 youth healthcare doctors, and 50 general practitioners to classify photographs of stools as "normal" or "abnormal." Subsequently, we asked whether parents would seek medical help and doctors would refer the patient for medical investigation. Finally, parents scored stools using the ISCC. Two-third of both parents and youth healthcare doctors recognized all discolored stools. Only half of them would seek medical help for all discolored stools resp. refer patient for medical investigation. Only one-third of the general practitioners recognized all discolored stools and would refer for medical investigation for all discolored stools. Using the ISCC, the percentage of parents recognizing all discolored stool increased from 66 to 87% (p < 0.01). Neither parents nor youth healthcare doctors nor general practitioners reliably recognize discolored stool. The ISCC is an effective screening method for discolored stool. Our data indicate that the ISCC should be accompanied by unequivocal advices regarding referral for medical investigation upon detection of discolored stools. Georg Thieme Verlag KG Stuttgart · New York.

Diagnosis of Clostridium difficile-associated disease: examination of multiple algorithms using toxin EIA, glutamate dehydrogenase EIA and loop-mediated isothermal amplification.

PubMed

Bamber, A I; Fitzsimmons, K; Cunniffe, J G; Beasor, C C; Mackintosh, C A; Hobbs, G

2012-01-01

The laboratory diagnosis of Clostridium difficile infection (CDI) needs to be accurate and timely to ensure optimal patient management, infection control and reliable surveillance. Three methods are evaluated using 810 consecutive stool samples against toxigenic culture: CDT TOX A/B Premier enzyme immunoassay (EIA) kit (Meridian Bioscience, Europe), Premier EIA for C. difficile glutamate dehydrogenase (GDH) (Meridian Bioscience, Europe) and the Illumigene kit (Meridian Bioscience, Europe), both individually and within combined testing algorithms. The study revealed that the CDT TOX A/B Premier EIA gave rise to false-positive and false-negative results and demonstrated poor sensitivity (56.47%), compared to Premier EIA for C. difficile GDH (97.65%), suggesting this GDH EIA can be a useful negative screening method. Results for the Illumigene assay alone showed sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) of 91.57%, 98.07%, 99.03% and 84.44%, respectively. A two-stage algorithm using Premier EIA for C. difficile GDH/Illumigene assay yielded superior results compared with other testing algorithms (91.57%, 98.07%, 99.03% and 84.44%, respectively), mirroring the Illumigene performance. However, Illumigene is approximately half the cost of current polymerase chain reaction (PCR) methods, has a rapid turnaround time and requires no specialised skill base, making it an attractive alternative to assays such as the Xpert C. difficile assay (Cepheid, Sunnyvale, CA). A three-stage algorithm offered no improvement and would hamper workflow.

All-in-One Nanowire-Decorated Multifunctional Membrane for Rapid Cell Lysis and Direct DNA Isolation

PubMed Central

2015-01-01

This paper describes a handheld device that uses an all-in-one membrane for continuous mechanical cell lysis and rapid DNA isolation without the assistance of power sources, lysis reagents, and routine centrifugation. This nanowire-decorated multifunctional membrane was fabricated to isolate DNA by selective adsorption to silica surface immediately after disruption of nucleus membranes by ultrasharp tips of nanowires for a rapid cell lysis, and it can be directly assembled with commercial syringe filter holders. The membrane was fabricated by photoelectrochemical etching to create microchannel arrays followed by hydrothermal synthesis of nanowires and deposition of silica. The proposed membrane successfully purifies high-quality DNA within 5 min, whereas a commercial purification kit needs more than an hour. PMID:25420232

Observations on the epidemiology of rotavirus infection among hospitalized children younger than 5 years in 2 Ukrainian hospitals, 2007-2015.

PubMed

Chernyshova, Liudmyla I; Radionova, Nataliya M; Demchyshyna, Iryna V; Kotlik, Liudmyla S; Sadkova, Oleksandra B; Samoilovich, Elena O; Semeiko, Galina V; Daniels, Danni S; Cohen, Adam L; Aliabadi, Negar

2017-11-29

Acute gastroenteritis remains a burden among children under 5 years of age. Ukraine joined the World Health Organization's Global Rotavirus Surveillance Network in 2006, with a goal of providing accurate rotavirus burden data to aid policy makers in planning for rotavirus vaccine introduction. This analysis describes rotavirus epidemiology among Ukrainian children enrolled in Kyiv and Odesa, two large Ukrainian cities. Children 0-59 months of age hospitalized for acute gastroenteritis at 2 sentinel sites in Kyiv and Odesa were enrolled into the active, prospective surveillance program. In Odesa, the surveillance period was during 2007-2015 and in Kyiv, it was during 2011-2015. Acute gastroenteritis was defined as 3 or more episodes of diarrhea per day during a 24 h period, with symptom duration before hospitalization not exceeding 7 days. Guardians of enrolled children completed a questionnaire including demographic, clinical and treatment information. Each child provided a stool specimen within 2 days of hospitalization. Stools were tested for rotavirus using ProSpecT™ Rotavirus Kit (Oxoid Ltd., Great Britain), and positive specimens were genotyped. Descriptive data are reported, as well as comparison of demographic, clinical and treatment data among rotavirus positive and negative children. During July 2007-June 2015, 12,350 children were enrolled in the surveillance programs and had stool specimens collected and tested for rotavirus. Overall, rotavirus infection was diagnosed in 5412/12350 (44%) of children, 929/1734 (54%) of those in Kyiv and 4483/10616 (42%) in Odesa. Rotavirus infections peaked during the winter months. Children with rotavirus acute gastroenteritis displayed more severe clinical symptoms than those without rotavirus. Predominant genotypes identified included G1P[8], G2P[4], G3 P[8], G4 P[8] and G9 P[8]. Active surveillance of acute gastroenteritis in hospitalized children younger 5 years in two large Ukrainian cities reveals a significant burden of rotavirus infection. These data provide scientific justification for incorporating rotavirus vaccines into the Ukrainian national immunization schedule. Published by Elsevier Ltd.

Evaluation of a new multiplex PCR assay (ParaGENIE G-Amoeba Real-Time PCR kit) targeting Giardia intestinalis, Entamoeba histolytica and Entamoeba dispar/Entamoeba moshkovskii from stool specimens: evidence for the limited performances of microscopy-based approach for amoeba species identification.

PubMed

Morio, F; Valot, S; Laude, A; Desoubeaux, G; Argy, N; Nourrisson, C; Pomares, C; Machouart, M; Le Govic, Y; Dalle, F; Botterel, F; Bourgeois, N; Cateau, E; Leterrier, M; Jeddi, F; Gaboyard, M; Le Pape, P

2018-02-15

Besides the potential to identify a wide variety of gastrointestinal parasites, microscopy remains the reference standard in clinical microbiology for amoeba species identification and, especially when coupled with adhesin detection, to discriminate the pathogenic Entamoeba histolytica from its sister but non-pathogenic species Entamoeba dispar/Entamoeba moshkovskii. However, this approach is time-consuming, requires a high-level of expertise that can be jeopardized considering the low prevalence of gastrointestinal parasites in non-endemic countries. Here, we evaluated the CE-IVD-marked multiplex PCR (ParaGENIE G-Amoeba, Ademtech) targeting E. histolytica and E. dispar/E. moshkovskii and Giardia intestinalis. This evaluation was performed blindly on a reference panel of 172 clinical stool samples collected prospectively from 12 laboratories and analysed using a standardized protocol relying on microscopy (and adhesin detection by ELISA for the detection of E. histolytica) including G. intestinalis (n = 37), various amoeba species (n = 55) including E. dispar (n = 15), E. histolytica (n = 5), as well as 17 other gastrointestinal parasites (n = 80), and negative samples (n = 37). This new multiplex PCR assay offers fast and reliable results with appropriate sensitivity and specificity for the detection of G. intestinalis and E. dispar/E. moshkovskii from stools (89.7%/96.9% and 95%/100%, respectively). Detection rate and specificity were greatly improved by the PCR assay, highlighting several samples misidentified by microscopy, including false-negative and false-positive results for both E. dispar/E. moshkovskii and E. histolytica. Given the clinical relevance of amoeba species identification, microbiologists should be aware of the limitations of using an algorithm relying on microscopy coupled with adhesin detection by ELISA. Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Combination of culture, antigen and toxin detection, and cytotoxin neutralization assay for optimal Clostridium difficile diagnostic testing

PubMed Central

Alfa, Michelle J; Sepehri, Shadi

2013-01-01

BACKGROUND: There has been a growing interest in developing an appropriate laboratory diagnostic algorithm for Clostridium difficile, mainly as a result of increases in both the number and severity of cases of C difficile infection in the past decade. A C difficile diagnostic algorithm is necessary because diagnostic kits, mostly for the detection of toxins A and B or glutamate dehydrogenase (GDH) antigen, are not sufficient as stand-alone assays for optimal diagnosis of C difficile infection. In addition, conventional reference methods for C difficile detection (eg, toxigenic culture and cytotoxin neutralization [CTN] assays) are not routinely practiced in diagnostic laboratory settings. OBJECTIVE: To review the four-step algorithm used at Diagnostic Services of Manitoba sites for the laboratory diagnosis of toxigenic C difficile. RESULT: One year of retrospective C difficile data using the proposed algorithm was reported. Of 5695 stool samples tested, 9.1% (n=517) had toxigenic C difficile. Sixty per cent (310 of 517) of toxigenic C difficile stools were detected following the first two steps of the algorithm. CTN confirmation of GDH-positive, toxin A- and B-negative assays resulted in detection of an additional 37.7% (198 of 517) of toxigenic C difficile. Culture of the third specimen, from patients who had two previous negative specimens, detected an additional 2.32% (12 of 517) of toxigenic C difficile samples. DISCUSSION: Using GDH antigen as the screening and toxin A and B as confirmatory test for C difficile, 85% of specimens were reported negative or positive within 4 h. Without CTN confirmation for GDH antigen and toxin A and B discordant results, 37% (195 of 517) of toxigenic C difficile stools would have been missed. Following the algorithm, culture was needed for only 2.72% of all specimens submitted for C difficile testing. CONCLUSION: The overview of the data illustrated the significance of each stage of this four-step C difficile algorithm and emphasized the value of using CTN assay and culture as parts of an algorithm that ensures accurate diagnosis of toxigenic C difficile. PMID:24421808

Magneto-capillary valve for integrated purification and enrichment of nucleic acids and proteins.

PubMed

den Dulk, Remco C; Schmidt, Kristiane A; Sabatté, Gwénola; Liébana, Susana; Prins, Menno W J

2013-01-07

We describe the magneto-capillary valve (MCV) technology, a flexible approach for integrated biological sample preparation within the concept of stationary microfluidics. Rather than moving liquids in a microfluidic device, discrete units of liquid are present at fixed positions in the device and magnetic particles are actuated between the fluids. The MCV concept is characterized by the use of two planar surfaces at a capillary mutual distance, with specific features to confine the fluids by capillary forces, and the use of a gas or a phase-change material separating the stationary aqueous liquids. We have studied the physics of magneto-capillary valving by quantifying the magnetic force as a function of time and position, which reveals the balance of magnetic, capillary and frictional forces in the system. By purification experiments with a fluorescent tracer we have measured the amount of co-transported liquid, which is a key parameter for efficient purification. To demonstrate the versatility of the technology, several MCV device architectures were tested in a series of biological assays, showing the purification and enrichment of nucleic acids and proteins. Target recovery comparable to non-miniaturized commercial kits was observed for the extraction of DNA from human cells in buffer, using a device architecture with patterned air valves. Experiments using an enrichment module and patterned air valves demonstrate a 40-fold effective enrichment of DNA in buffer. DNA was also successfully purified from blood plasma using paraffin phase-change valves. Finally, the enrichment of a protein biomarker (prostate-specific antigen) using geometrical air valves resulted in a 7-fold increase of detection signal. The MCV technology is versatile, offers extensive freedom for the design of fully integrated systems, and is expected to be manufacturable in a cost-effective way. We conclude that the MCV technology can become an important enabling technology for point-of-care systems with sample in-result out performance.

Production and purification of polyclonal antibody against F(ab')2 fragment of human immunoglobulin G

PubMed Central

Nasiri, Hadi; Valedkarimi, Zahra; Aghebati-Maleki, Leili; Abdolalizadeh, Jalal; Kazemi, Tohid; Esparvarinha, Mojghan; Majidi, Jafar

2017-01-01

Antibodies are essential tools of biomedical and biochemical researches. Polyclonal antibodies are produced against different epitopes of antigens. Purified F(ab')2 can be used for animal’s immunization to produce polyclonal antibodies. Human immunoglobulin G (IgG) was purified by ion exchange chromatography method. In all stages verification method of the purified antibodies was sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified IgG was digested by pepsin enzyme and F(ab')2 fragment was purified by gel filtration separation method. For production of polyclonal antibody, rabbit was immunized by purified F(ab')2 and antibody production was investigated by enzyme-linked immunosorbent assay. Purified anti-IgG F(ab')2 was conjugated with fluorescein isothiocyanate. Ion exchange chromatography purification yielded 38 mg of human IgG antibody. The results of SDS-PAGE in reduced and non-reduced conditions showed bands with 25-30 kDa molecular weight (MW) and 50-kDa respectively and a distinct band with 150 kDa MW. The results of non-reduced SDS-PAGE for determining the purity of F(ab')2 fragment showed one band in 90 kDa and a band in 150 kDa MW position. Purification by Ion exchange chromatography method resulted about 12 mg rabbit polyclonal antibody. Flow cytometry showed generated polyclonal antibody had an acceptable activity compared to commercial antibody. Taking together, purified IgG F(ab')2 and polyclonal anti-IgG F(ab')2 are useful tools in biomedical and biochemical researches and diagnostic kits. PMID:29326789

Production and purification of polyclonal antibody against F(ab')2 fragment of human immunoglobulin G.

PubMed

Nasiri, Hadi; Valedkarimi, Zahra; Aghebati-Maleki, Leili; Abdolalizadeh, Jalal; Kazemi, Tohid; Esparvarinha, Mojghan; Majidi, Jafar

2017-01-01

Antibodies are essential tools of biomedical and biochemical researches. Polyclonal antibodies are produced against different epitopes of antigens. Purified F(ab') 2 can be used for animal's immunization to produce polyclonal antibodies. Human immunoglobulin G (IgG) was purified by ion exchange chromatography method. In all stages verification method of the purified antibodies was sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified IgG was digested by pepsin enzyme and F(ab') 2 fragment was purified by gel filtration separation method. For production of polyclonal antibody, rabbit was immunized by purified F(ab') 2 and antibody production was investigated by enzyme-linked immunosorbent assay. Purified anti-IgG F(ab') 2 was conjugated with fluorescein isothiocyanate. Ion exchange chromatography purification yielded 38 mg of human IgG antibody. The results of SDS-PAGE in reduced and non-reduced conditions showed bands with 25-30 kDa molecular weight (MW) and 50-kDa respectively and a distinct band with 150 kDa MW. The results of non-reduced SDS-PAGE for determining the purity of F(ab') 2 fragment showed one band in 90 kDa and a band in 150 kDa MW position. Purification by Ion exchange chromatography method resulted about 12 mg rabbit polyclonal antibody. Flow cytometry showed generated polyclonal antibody had an acceptable activity compared to commercial antibody. Taking together, purified IgG F(ab') 2 and polyclonal anti-IgG F(ab') 2 are useful tools in biomedical and biochemical researches and diagnostic kits.

Teaching older adults by adapting for aging changes.

PubMed

Weinrich, S P; Weinrich, M C; Boyd, M D; Atwood, J; Cervenka, B

1994-12-01

Few teaching programs are geared to meet the special learning needs of the elderly. This pilot study used a quasi-experimental pretest-posttest design to measure the effect of the Adaptation for Aging Changes (AAC) Method on fecal occult blood screening (FOBS) at meal sites for the elderly in the South. The AAC Method uses techniques that adjust the presentation to accommodate for normal aging changes and includes a demonstration of the procedure for collection of the stool blood test, memory reminders of the date to return the stool blood test, and written materials adapted to the 5th grade reading level. In addition, actual practice of the FOBS with the use of peanut butter was added to the AAC Method, making it the AAC with Practice Method (AACP) in two sites. The American Cancer Society's colorectal cancer educational slide-tape show served as the basis for all of the methods. Hemoccult II kits were distributed at no cost to the participants. Descriptive statistics, chi 2, and logistic regressions were used to analyze data from 135 Council on Aging meal sites' participants. The average age of the participants was 72 years; the average educational level was 8th grade; over half the sample was African-American; and half of the participants had incomes below the poverty level. Results support a significant increase in participation in FOBS in participants taught by the AACP Method [chi 2 (1, n = 56) = 5.34, p = 0.02; odds ratio = 6.2]. This research provides support for teaching that makes adaptations for aging changes, especially adaptations that include actual practice of the procedure.

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

PubMed

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.

21 CFR 868.6700 - Anesthesia stool.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Anesthesia stool. 868.6700 Section 868.6700 Food... DEVICES ANESTHESIOLOGY DEVICES Miscellaneous § 868.6700 Anesthesia stool. (a) Identification. An anesthesia stool is a device intended for use as a stool for the anesthesiologist in the operating room. (b...

21 CFR 868.6700 - Anesthesia stool.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Anesthesia stool. 868.6700 Section 868.6700 Food... DEVICES ANESTHESIOLOGY DEVICES Miscellaneous § 868.6700 Anesthesia stool. (a) Identification. An anesthesia stool is a device intended for use as a stool for the anesthesiologist in the operating room. (b...

21 CFR 868.6700 - Anesthesia stool.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Anesthesia stool. 868.6700 Section 868.6700 Food... DEVICES ANESTHESIOLOGY DEVICES Miscellaneous § 868.6700 Anesthesia stool. (a) Identification. An anesthesia stool is a device intended for use as a stool for the anesthesiologist in the operating room. (b...

21 CFR 868.6700 - Anesthesia stool.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Anesthesia stool. 868.6700 Section 868.6700 Food... DEVICES ANESTHESIOLOGY DEVICES Miscellaneous § 868.6700 Anesthesia stool. (a) Identification. An anesthesia stool is a device intended for use as a stool for the anesthesiologist in the operating room. (b...

21 CFR 868.6700 - Anesthesia stool.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Anesthesia stool. 868.6700 Section 868.6700 Food... DEVICES ANESTHESIOLOGY DEVICES Miscellaneous § 868.6700 Anesthesia stool. (a) Identification. An anesthesia stool is a device intended for use as a stool for the anesthesiologist in the operating room. (b...

Isolation of PCR quality microbial community DNA from heavily contaminated environments.

PubMed

Gunawardana, Manjula; Chang, Simon; Jimenez, Abraham; Holland-Moritz, Daniel; Holland-Moritz, Hannah; La Val, Taylor P; Lund, Craig; Mullen, Madeline; Olsen, John; Sztain, Terra A; Yoo, Jennifer; Moss, John A; Baum, Marc M

2014-07-01

Asphalts, biochemically degraded oil, contain persistent, water-soluble compounds that pose a significant challenge to the isolation of PCR quality DNA. The adaptation of existing DNA purification protocols and commercial kits proved unsuccessful at overcoming this hurdle. Treatment of aqueous asphalt extracts with a polyamide resin afforded genomic microbial DNA templates that could readily be amplified by PCR. Physicochemically distinct asphalt samples from five natural oil seeps successfully generated the expected 291 bp amplicons targeting a region of the 16S rRNA gene, illustrating the robustness of the method. DNA recovery yields were in the 50-80% range depending on how the asphalt sample was seeded with exogenous DNA. The scope of the new method was expanded to include soil with high humic acid content. DNA from soil samples spiked with a range of humic acid concentrations was extracted with a commercial kit followed by treatment with the polyamide resin. The additional step significantly improved the purity of the DNA templates, especially at high humic acid concentrations, based on qPCR analysis of the bacterial 16S rRNA genes. The new method has the advantages of being inexpensive, simple, and rapid and should provide a valuable addition to protocols in the field of petroleum and soil microbiology. Copyright © 2014 Elsevier B.V. All rights reserved.

Dried Blood Spot RNA Transcriptomes Correlate with Transcriptomes Derived from Whole Blood RNA.

PubMed

Reust, Mary J; Lee, Myung Hee; Xiang, Jenny; Zhang, Wei; Xu, Dong; Batson, Tatiana; Zhang, Tuo; Downs, Jennifer A; Dupnik, Kathryn M

2018-05-01

Obtaining RNA from clinical samples collected in resource-limited settings can be costly and challenging. The goals of this study were to 1) optimize messenger RNA extraction from dried blood spots (DBS) and 2) determine how transcriptomes generated from DBS RNA compared with RNA isolated from blood collected in Tempus tubes. We studied paired samples collected from eight adults in rural Tanzania. Venous blood was collected on Whatman 903 Protein Saver cards and in tubes with RNA preservation solution. Our optimal DBS RNA extraction used 8 × 3-mm DBS punches as the starting material, bead beater disruption at maximum speed for 60 seconds, extraction with Illustra RNAspin Mini RNA Isolation kit, and purification with Zymo RNA Concentrator kit. Spearman correlations of normalized gene counts in DBS versus whole blood ranged from 0.887 to 0.941. Bland-Altman plots did not show a trend toward over- or under-counting at any gene size. We report a method to obtain sufficient RNA from DBS to generate a transcriptome. The DBS transcriptome gene counts correlated well with whole blood transcriptome gene counts. Dried blood spots for transcriptome studies could be an option when field conditions preclude appropriate collection, storage, or transport of whole blood for RNA studies.

Stool fatty acid soaps, stool consistency and gastrointestinal tolerance in term infants fed infant formulas containing high sn-2 palmitate with or without oligofructose: a double-blind, randomized clinical trial.

PubMed

Nowacki, Joyce; Lee, Hung-Chang; Lien, Reyin; Cheng, Shao-Wen; Li, Sung-Tse; Yao, Manjiang; Northington, Robert; Jan, Ingrid; Mutungi, Gisella

2014-11-05

Formula-fed (FF) infants often have harder stools and higher stool concentrations of fatty acid soaps compared to breastfed infants. Feeding high sn-2 palmitate or the prebiotic oligofructose (OF) may soften stools, reduce stool soaps, and decrease fecal calcium loss. We investigated the effect of high sn-2 palmitate alone and in combination with OF on stool palmitate soap, total soap and calcium concentrations, stool consistency, gastrointestinal (GI) tolerance, anthropometrics, and hydration in FF infants. This double-blind trial randomized 165 healthy term infants 25-45 days old to receive Control formula (n = 54), formula containing high sn-2 palmitate (sn-2; n = 56), or formula containing high sn-2 palmitate plus 3 g/L OF (sn-2+OF; n = 55). A non-randomized human milk (HM)-fed group was also included (n = 55). The primary endpoint, stool composition, was determined after 28 days of feeding, and was assessed using ANOVA accompanied by pairwise comparisons. Stool consistency, GI tolerance and hydration were assessed at baseline, day 14 (GI tolerance only) and day 28. Infants fed sn-2 had lower stool palmitate soaps compared to Control (P = 0.0028); while those fed sn-2+OF had reduced stool palmitate soaps compared to both Control and sn-2 (both P < 0.0001). Stool total soaps and calcium were lower in the sn-2+OF group than either Control (P < 0.0001) or sn-2 (P < 0.0001). The HM-fed group had lower stool palmitate soaps, total soaps and calcium (P < 0.0001 for each comparison) than all FF groups. The stool consistency score of the sn-2+OF group was lower than Control and sn-2 (P < 0.0001), but higher than the HM-fed group (P < 0.0001). GI tolerance was similar and anthropometric z-scores were <0.2 SD from the WHO growth standards in all groups, while urinary hydration markers were within normal range for all FF infants. Increasing sn-2 palmitate in infant formula reduces stool palmitate soaps. A combination of high sn-2 palmitate and OF reduces stool palmitate soaps, total soaps and calcium, while promoting softer stools. This study was registered on http://www.clinicaltrials.gov: number NCT02031003.

Quantitative Detection and Genotyping of Helicobacter pylori from Stool using Droplet Digital PCR Reveals Variation in Bacterial Loads that Correlates with cagA Virulence Gene Carriage.

PubMed

Talarico, Sarah; Safaeian, Mahboobeh; Gonzalez, Paula; Hildesheim, Allan; Herrero, Rolando; Porras, Carolina; Cortes, Bernal; Larson, Ann; Fang, Ferric C; Salama, Nina R

2016-08-01

Epidemiologic studies of the carcinogenic stomach bacterium Helicobacter pylori have been limited by the lack of noninvasive detection and genotyping methods. We developed a new stool-based method for detection, quantification, and partial genotyping of H. pylori using droplet digital PCR (ddPCR), which allows for increased sensitivity and absolute quantification by PCR partitioning. Stool-based ddPCR assays for H. pylori 16S gene detection and cagA virulence gene typing were tested using a collection of 50 matched stool and serum samples from Costa Rican volunteers and 29 H. pylori stool antigen-tested stool samples collected at a US hospital. The stool-based H. pylori 16S ddPCR assay had a sensitivity of 84% and 100% and a specificity of 100% and 71% compared to serology and stool antigen tests, respectively. The stool-based cagA genotyping assay detected cagA in 22 (88%) of 25 stools from CagA antibody-positive individuals and four (16%) of 25 stools from CagA antibody-negative individuals from Costa Rica. All 26 of these samples had a Western-type cagA allele. Presence of serum CagA antibodies was correlated with a significantly higher load of H. pylori in the stool. The stool-based ddPCR assays are a sensitive, noninvasive method for detection, quantification, and partial genotyping of H. pylori. The quantitative nature of ddPCR-based H. pylori detection revealed significant variation in bacterial load among individuals that correlates with presence of the cagA virulence gene. These stool-based ddPCR assays will facilitate future population-based epidemiologic studies of this important human pathogen. © 2015 John Wiley & Sons Ltd.

Direct Y-STR amplification of body fluids deposited on commonly found crime scene substrates.

PubMed

Dargay, Amanda; Roy, Reena

2016-04-01

Body fluids detected on commonly found crime scene substrates require extraction, purification and quantitation of DNA prior to amplification and generation of short tandem repeat (STR) DNA profiles. In this research Y-STR profiles were generated via direct amplification of blood and saliva deposited on 12 different substrates. These included cigarette butts, straws, grass, leaves, woodchips and seven different types of fabric. After depositing either 0.1 μL of blood or 0.5 μL of saliva, each substrate containing the dry body fluid stain was punched using a Harris 1.2 mm micro-punch. Each of these punched substrates, a total of 720 samples, containing minute amount of blood or saliva was either amplified directly without any pre-treatment, or was treated with one of the four washing reagents or buffer. In each of these five experimental groups the substrates containing the body fluid remained in the amplification reagent during the thermal cycling process. Each sample was amplified with the three direct Y-STR amplification kits; AmpFℓSTR(®) Yfiler(®) Direct, Yfiler(®) Plus Amplification Kits and the PowerPlex(®) Y23 System. Complete and concordant Y-STR profiles were successfully obtained from most of these 12 challenging crime scene objects when the stains were analyzed by at least one of the five experimental groups. The reagents and buffer were interchangeable among the three amplification kits, however, pre-treatment with these solutions did not appear to enhance the quality or the number of the full profiles generated with direct amplification. This study demonstrates that blood and saliva deposited on these simulated crime scene objects can be amplified directly. Copyright © 2016 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Bristol Stool Form Scale reliability and agreement decreases when determining Rome III stool form designations

USDA-ARS?s Scientific Manuscript database

Rater reproducibility of the Bristol Stool Form Scale (BSFS), which categorizes stools into one of seven types, is unknown. We sought to determine reliability and agreement by individual stool type and when responses are categorized by Rome III clinical designation as normal or abnormal (constipatio...

Microfluidic reactors for visible-light photocatalytic water purification assisted with thermolysis

PubMed Central

Wang, Ning; Tan, Furui; Wan, Li; Wu, Mengchun

2014-01-01

Photocatalytic water purification using visible light is under intense research in the hope to use sunlight efficiently, but the conventional bulk reactors are slow and complicated. This paper presents an integrated microfluidic planar reactor for visible-light photocatalysis with the merits of fine flow control, short reaction time, small sample volume, and long photocatalyst durability. One additional feature is that it enables one to use both the light and the heat energy of the light source simultaneously. The reactor consists of a BiVO4-coated glass as the substrate, a blank glass slide as the cover, and a UV-curable adhesive layer as the spacer and sealant. A blue light emitting diode panel (footprint 10 mm × 10 mm) is mounted on the microreactor to provide uniform irradiation over the whole reactor chamber, ensuring optimal utilization of the photons and easy adjustments of the light intensity and the reaction temperature. This microreactor may provide a versatile platform for studying the photocatalysis under combined conditions such as different temperatures, different light intensities, and different flow rates. Moreover, the microreactor demonstrates significant photodegradation with a reaction time of about 10 s, much shorter than typically a few hours using the bulk reactors, showing its potential as a rapid kit for characterization of photocatalyst performance. PMID:25584117

Comparison of strategies for the isolation of PCR-compatible, genomic DNA from a municipal biogas plants.

PubMed

Weiss, Agnes; Jérôme, Valérie; Freitag, Ruth

2007-06-15

The goal of the project was the extraction of PCR-compatible genomic DNA representative of the entire microbial community from municipal biogas plant samples (mash, bioreactor content, process water, liquid fertilizer). For the initial isolation of representative DNA from the respective lysates, methods were used that employed adsorption, extraction, or precipitation to specifically enrich the DNA. Since no dedicated method for biogas plant samples was available, preference was given to kits/methods suited to samples that resembled either the bioreactor feed, e.g. foodstuffs, or those intended for environmental samples including wastewater. None of the methods succeeded in preparing DNA that was directly PCR-compatible. Instead the DNA was found to still contain considerable amounts of difficult-to-remove enzyme inhibitors (presumably humic acids) that hindered the PCR reaction. Based on the isolation method that gave the highest yield/purity for all sample types, subsequent purification was attempted by agarose gel electrophoresis followed by electroelution, spermine precipitation, or dialysis through nitrocellulose membrane. A combination of phenol/chloroform extraction followed by purification via dialysis constituted the most efficient sample treatment. When such DNA preparations were diluted 1:100 they did no longer inhibit PCR reactions, while they still contained sufficient genomic DNA to allow specific amplification of specific target sequences.

Stool Tests

MedlinePlus

... 2014 More on this topic for: Parents Teens E. Coli Giardiasis Yersiniosis Stool Test: Bacteria Culture Stool Test: ... Stool Test: Ova and Parasites (O&P) Diarrhea E. Coli View more About Us Contact Us Partners Editorial ...

"Lull pgm System" for Autologous Fat Grafting: A Simple Closed System with Minimal Equipment and No Extra Cost.

PubMed

Morselli, Paolo Giovanni; Micai, Alessandro; Giorgini, Federico Armando

2016-08-01

The "Lull pgm system" is a closed system for purifying harvested fat. It processes the collected tissue safely without any additional cost. The system was conceived by referring to the targets described in the literature with the aim of creating a simple system that guarantees a high standard of purification and requires minimal equipment that is available in every operating room. Cost must be always considered: even the most prosperous hospitals must keep within tight annual budgets. "Lull" can be used instead of expensive devices or disposable kits, without substantially increasing the operating time. The system has been used in clinical practice for many plastic reconstructive procedures and has obtained positive results and patient satisfaction, and no contraindications or disadvantages have been observed.

Stool Color: When to Worry

MedlinePlus

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 ...

Bismuth-based quadruple Helicobacter pylori eradication regimen alters the composition of gut microbiota.

PubMed

Yildiz, Serap Süzük; Yalinay, Meltem; Karakan, Tarkan

2018-06-01

Microbiota is a dynamic system showing individual differences in both the number and species of microorganisms. Dietary habits, lifestyle, age, genetic predisposition of the host and use of antibiotics are effective on microbiota. The aim of our research was to carry out a quantitative comparison of Bifidobacterium spp, Bacteroides fragilis, Lactobacillus spp, Akkermansia mucinophilia and Faecalibacterium prausnitzii, important bacterial microbiota species, before and after antibiotic therapy treated with tetracycline and metronidazole in patients who are diagnosed as positive for Helicobacter pylori (HP), and to determine the effects of antibiotic use on the microbiota. Eighteen HP-positive patients were enrolled in this study. A special extraction kit (QIAmp DNA Stool Mini Kit, QIAgen, Germany) was used for the DNA isolation procedure. Primers specific to the 16S rRNA region of the bacteria included in the study were used for the amplification of the target region. All the bacteria were subjected to real-time quantification procedure with PCR method on RotorGene® 20 device (Qiagen, Germany). According to quantification before and after antibiotic use in patients receiving HP treatment, statistically significant decreases were observed in Bifidobacterium spp (p=0.001), B. fragilis (p=0.001), Lactobacillus spp (p=0.001), A. mucinophilia (p=0.001) and F. prausnitzii (p=0.001). We were unable to identify B. fragilis in the microbiota of five patients after treatment. Based on the data obtained, it can be concluded that antibiotics used to treat HP can prepare the ground that could result in dysbiosis in microbiota.

Are the definitions for chronic diarrhoea adequate? Evaluation of two different definitions in patients with chronic diarrhoea

PubMed Central

Abrahamsson, Hasse; Bajor, Antal; Kilander, Anders; Sadik, Riadh; Sjövall, Henrik; Simrén, Magnus

2015-01-01

Background The classical definition of chronic diarrhoea is ≥3 defecations/day, with a stool weight of more than 200 g and duration of ≥4 weeks. However, with this definition many patients with substantial symptoms and pathology will be excluded from further investigations. As a consequence other definitions have been proposed, mainly based on evaluation of the stool form. Objective To evaluate the accuracy of the classic criteria for diarrhoea in comparison with a definition based on stool consistency, using the Bristol Stool Form Scale. Methods All patients were investigated with laboratory tests, upper and lower gastrointestinal endoscopy with biopsies, and SeHCAT test. They were asked to complete a diary recording stool frequency and consistency during a week, as well as other gastrointestinal symptoms (pain, bloating and gas). Results One hundred and thirty-nine subjects were eligible for analysis. Ninety-one had an organic cause of diarrhoea. Fifty-three patients had ≥3 loose stools/day, whereas 86 reported <3 stools/day. Ninety had a median stool consistency that was mushy or loose and 49 had harder stools. A higher proportion of subjects with an organic cause of their diarrhoea compared with subjects with a functional bowel disorder had ≥3 loose stools/day, 43/91 (47%) vs. 10/48 (21%) (p < 0.01). Similarly, more subjects with an organic cause of their diarrhoea versus patients with a functional bowel disorder had a median stool consistency that was mushy or watery, 73/91 (80%) vs. 17/48 (35%), p < 0.0001. When diarrhoea was defined according to stool form, more patients were classified correctly as having a functional disorder or organic disorder, compared with the classical definition (p < 0.05). Conclusion Loose stools defined according to the Bristol Stool Form scale seem to be the best predictor of having an organic cause of the diarrhoea. PMID:26279847

Comparative evaluation of in-house manual, and commercial semi-automated and automated DNA extraction platforms in the sample preparation of human stool specimens for a Salmonella enterica 5'-nuclease assay.

PubMed

Schuurman, Tim; de Boer, Richard; Patty, Rachèl; Kooistra-Smid, Mirjam; van Zwet, Anton

2007-12-01

In the present study, three methods (NucliSens miniMAG [bioMérieux], MagNA Pure DNA Isolation Kit III Bacteria/Fungi [Roche], and a silica-guanidiniumthiocyanate {Si-GuSCN-F} procedure for extracting DNA from stool specimens were compared with regard to analytical performance (relative DNA recovery and down stream real-time PCR amplification of Salmonella enterica DNA), stability of the extracted DNA, hands-on time (HOT), total processing time (TPT), and costs. The Si-GuSCN-F procedure showed the highest analytical performance (relative recovery of 99%, S. enterica real-time PCR sensitivity of 91%) at the lowest associated costs per extraction (euro 4.28). However, this method did required the longest HOT (144 min) and subsequent TPT (176 min) when processing 24 extractions. Both miniMAG and MagNA Pure extraction showed similar performances at first (relative recoveries of 57% and 52%, S. enterica real-time PCR sensitivity of 85%). However, when difference in the observed Ct values after real-time PCR were taken into account, MagNA Pure resulted in a significant increase in Ct value compared to both miniMAG and Si-GuSCN-F (with on average +1.26 and +1.43 cycles). With regard to inhibition all methods showed relatively low inhibition rates (< 4%), with miniMAG providing the lowest rate (0.7%). Extracted DNA was stable for at least 1 year for all methods. HOT was lowest for MagNA Pure (60 min) and TPT was shortest for miniMAG (121 min). Costs, finally, were euro 4.28 for Si-GuSCN, euro 6.69 for MagNA Pure and euro 9.57 for miniMAG.

Two-step glutamate dehydrogenase antigen real-time polymerase chain reaction assay for detection of toxigenic Clostridium difficile.

PubMed

Goldenberg, S D; Cliff, P R; Smith, S; Milner, M; French, G L

2010-01-01

Current diagnosis of Clostridium difficile infection (CDI) relies upon detection of toxins A/B in stool by enzyme immunoassay [EIA(A/B)]. This strategy is unsatisfactory because it has a low sensitivity resulting in significant false negatives. We investigated the performance of a two-step algorithm for diagnosis of CDI using detection of glutamate dehydrogenase (GDH). GDH-positive samples were tested for C. difficile toxin B gene (tcdB) by polymerase chain reaction (PCR). The performance of the two-step protocol was compared with toxin detection by the Meridian Premier EIA kit in 500 consecutive stool samples from patients with suspected CDI. The reference standard among samples that were positive by either EIA(A/B) or GDH testing was culture cytotoxin neutralisation (culture/CTN). Thirty-six (7%) of 500 samples were identified as true positives by culture/CTN. EIA(A/B) identified 14 of the positive specimens with 22 false negatives and two false positives. The two-step protocol identified 34 of the positive samples with two false positives and two false negatives. EIA(A/B) had a sensitivity of 39%, specificity of 99%, positive predictive value of 88% and negative predictive value of 95%. The two-step algorithm performed better, with corresponding values of 94%, 99%, 94% and 99% respectively. Screening for GDH before confirmation of positives by PCR is cheaper than screening all specimens by PCR and is an effective method for routine use. Current EIA(A/B) tests for CDI are of inadequate sensitivity and should be replaced; however, this may result in apparent changes in CDI rates that would need to be explained in national surveillance statistics. Copyright 2009 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.

Comparison of stool microbiota compositions, stool alpha1-antitrypsin and calprotectin concentrations, and diarrhoeal morbidity of Indonesian infants fed breast milk or probiotic/prebiotic-supplemented formula.

PubMed

Oswari, Hanifah; Prayitno, Lamtorogung; Dwipoerwantoro, Pramita G; Firmansyah, Agus; Makrides, Maria; Lawley, Blair; Kuhn-Sherlock, Barbara; Cleghorn, Geoffrey; Tannock, Gerald W

2013-12-01

The composition of faecal microbiota of babies is known to be influenced by diet. Faecal calprotectin and α1-antitrypsin concentrations may be associated with mucosal permeability and inflammation. We aimed to assess whether there was any difference after consumption of a probiotic/prebiotic formula on faecal microbiota composition, calprotectin and α1-antitrypsin levels, and diarrhoea in comparison with breast milk-fed Indonesian infants. One hundred sixty infants, 2 to 6 weeks old, were recruited to the study. They were either breastfed or formula fed (80 per group). Faecal samples were collected at recruitment and 3 months later. Bacterial groups characteristic of the human faecal microbiota were quantified in faeces by quantitative polymerase chain reaction. Calprotectin and α1-antitrypsin concentrations were measured using commercial kits. Details of diarrhoeal morbidity were documented and rated for severity. The compositions of the faecal microbiota of formula-fed compared with breast milk-fed children were similar except that the probiotic strain Bifidobacterium animalis subsp. lactis DR10 was more abundant after 3 months consumption of the formula. Alpha1-antitrypsin levels were higher in breastfed compared with formula-fed infants. The occurrence of diarrhoea did not differ between the groups of babies. Feeding Indonesian babies with a probiotic/prebiotic formula did not produce marked differences in the composition of the faecal microbiota in comparison with breast milk. Detrimental effects of formula feeding on biomarkers of mucosal health were not observed. © 2013 The Authors. Journal of Paediatrics and Child Health © 2013 Paediatrics and Child Health Division (Royal Australasian College of Physicians).

Schistosoma mansoni and HIV infection in a Ugandan population with high HIV and helminth prevalence.

PubMed

Sanya, Richard E; Muhangi, Lawrence; Nampijja, Margaret; Nannozi, Victoria; Nakawungu, Prossy Kabuubi; Abayo, Elson; Webb, Emily L; Elliott, Alison M

2015-09-01

Recent reports suggest that Schistosoma infection may increase the risk of acquiring human immunodeficiency virus (HIV). We used data from a large cross-sectional study to investigate whether Schistosoma mansoni infection is associated with increased HIV prevalence. We conducted a household survey of residents in island fishing communities in Mukono district, Uganda, between October 2012 and July 2013. HIV status was assessed using rapid test kits. Kato-Katz (KK) stool tests and urine-circulating cathodic antigen (CCA) were used to test for Schistosoma infection. Multivariable logistic regression, allowing for the survey design, was used to investigate the association between S. mansoni infection and HIV infection. Data from 1412 participants aged 13 years and older were analysed (mean age 30.3 years, 45% female). The prevalence of HIV was 17.3%. Using the stool Kato-Katz technique on a single sample, S. mansoni infection was detected in 57.2% (719/1257) of participants; urine CCA was positive in 73.8% (478/650) of those tested. S. mansoni infection was not associated with HIV infection. [KK (aOR = 1.04; 95% CI: 0.74-1.47, P = 0.81), CCA (aOR = 1.53; 95% CI: 0.78-3.00, P = 0.19)]. The median S. mansoni egg count per gram was lower in the HIV-positive participants (P = 0.005). These results add to the evidence that S. mansoni has little effect on HIV transmission, but may influence egg excretion. © 2015 The Authors. Tropical Medicine & International Health Published by John Wiley & Sons Ltd.

A dental stool with chest support reduces lower back muscle activation.

PubMed

Tran, Viet; Turner, Reid; MacFadden, Andrew; Cornish, Stephen M; Esliger, Dale; Komiyama, Kunio; Chilibeck, Philip D

2016-09-01

Activation of back musculature during work tasks leads to fatigue and potential injury. This is especially prevalent in dentists who perform much of their work from a seated position. We examined the use of an ergonomic dental stool with mid-sternum chest support for reducing lower back muscle activation. Electromyography of lower back extensors was assessed from 30 dental students for 20 s during three conditions in random order: (a) sitting upright at 90° of hip flexion on a standard stool, (b) leaning forward at 80° of hip flexion on a standard stool, and (c) leaning forward at 80° of hip flexion while sitting on an ergonomic stool. Muscular activity of the back extensors was reduced when using the ergonomic stool compared to the standard stool, by 33-50% (p < 0.01). This suggests a potential musculoskeletal benefit with use of a dental stool with mid-sternum chest support.

Assessment of the best N(3-) donors in preparation of [M(N)(PNP)]-based (M=(99m)Tc-; (188)Re) target-specific radiopharmaceuticals: Comparison among succinic dihydrazide (SDH), N-methyl-S-methyl dithiocarbazate (HDTCZ) and PEGylated N-methyl-S-methyl dithiocarbazate (HO2C-PEG600-DTCZ).

PubMed

Carta, Davide; Jentschel, Christian; Thieme, Stefan; Salvarese, Nicola; Morellato, Nicolò; Refosco, Fiorenzo; Ruzza, Paolo; Bergmann, Ralf; Pietzsch, Hans-Jurgen; Bolzati, Cristina

2014-08-01

Succinic dihydrazide (SDH), N-methyl-S-methyl dithiocarbazate (HDTCZ) and PEGylated N-methyl-S-methyl dithiocarbazate (HO2C-PEG600-DTCZ) are nitrido nitrogen atom donors employed for the preparation of nitride [M(N)]-complexes (M=(99m)Tc and (188)Re). This study aims to compare the capability and the efficiency of these three N(3-) group donors, in the preparation of [M(N)PNP]-based target-specific compounds (M=(99m)Tc, (188)Re; PNP=aminodiphosphine). For this purpose, three different kit formulations (SDH kit; HO2C-PEG600-DTCZ kit; HDTCZ kit) were assembled and used in the preparation of [M(N)(cys~)(PNP3)](0/+) complexes (cys~=cysteine derivate ligands). For each formulation, the radiochemical yield (RCY) of the [M(N)(~cys)(PNP3)] compounds, was determined by HPLC. The deviation of the percentage of RCY, due to changes in concentration of the N(3-) donors and of the exchanging ligand, was determined. For (99m)Tc, data clearly show that HDTCZ is the most efficient donor of N(3-); however, SDH is the most suitable nitrido nitrogen atom donor for the preparation of [(99m)Tc(N)(PNP)]-based target-specific agents with high specific activity. When HO2C-PEG600-DTCZ or HDTCZ are used in N(3-) donation, high amounts of the exchanging ligand (10(-4)M) were required for the formation of the final complex in acceptable yield. The possibility to use microgram amounts of HDTCZ also in [(188)Re(N)] preparation (0.050mg) reduces its ability to compete in ligand exchange reactions, minimizing the quantity of chelators required to obtain the final complex in high yield. This finding can be exploit for increasing the radiolabeling efficiency in [(188)Re(N)]-radiopharmaceutical preparations compared to the previously reported HDTCZ-based procedure, notwithstanding a purification process could be necessary to improve the specific activity of the complexes. Copyright © 2014 Elsevier Inc. All rights reserved.

Performance Characteristics and Comparison of Abbott and artus Real-Time Systems for Hepatitis B Virus DNA Quantification ▿

PubMed Central

Ismail, Ashrafali M.; Sivakumar, Jayashree; Anantharam, Raghavendran; Dayalan, Sujitha; Samuel, Prasanna; Fletcher, Gnanadurai J.; Gnanamony, Manu; Abraham, Priya

2011-01-01

Virological monitoring of hepatitis B virus (HBV) DNA is critical to the management of HBV infection. With several HBV DNA quantification assays available, it is important to use the most efficient testing system for virological monitoring. In this study, we evaluated the performance characteristics and comparability of three HBV DNA quantification systems: Abbott HBV real-time PCR (Abbott PCR), artus HBV real-time PCR with QIAamp DNA blood kit purification (artus-DB), and artus HBV real-time PCR with the QIAamp DSP virus kit purification (artus-DSP). The lower limits of detection of these systems were established against the WHO international standards for HBV DNA and were found to be 1.43, 82, and 9 IU/ml, respectively. The intra-assay and interassay coefficients of variation of plasma samples (1 to 6 log10 IU/ml) ranged between 0.05 to 8.34% and 0.16 to 3.48% for the Abbott PCR, 1.53 to 26.85% and 0.50 to 12.89% for artus-DB, and 0.29 to 7.42% and 0.94 to 3.01% for artus-DSP, respectively. Ninety HBV clinical samples were used for comparison of assays, and paired quantitative results showed strong correlation by linear regression analysis (artus-DB with Abbott PCR, r = 0.95; Abbott PCR with artus-DSP, r = 0.97; and artus-DSP with artus-DB, r = 0.94). Bland-Altman analysis showed a good level of agreement for Abbott PCR and artus-DSP, with a mean difference of 0.10 log10 IU/ml and limits of agreement of −0.91 to 1.11 log10 IU/ml. No genotype-specific bias was seen in all three systems for HBV genotypes A, C, and D, which are predominant in this region. This finding illustrates that the Abbott real-time HBV and artus-DSP systems show more comparable performance than the artus-DB system, meeting the current guidelines for assays to be used in the management of hepatitis B. PMID:21795507

Performance characteristics and comparison of Abbott and artus real-time systems for hepatitis B virus DNA quantification.

PubMed

Ismail, Ashrafali M; Sivakumar, Jayashree; Anantharam, Raghavendran; Dayalan, Sujitha; Samuel, Prasanna; Fletcher, Gnanadurai J; Gnanamony, Manu; Abraham, Priya

2011-09-01

Virological monitoring of hepatitis B virus (HBV) DNA is critical to the management of HBV infection. With several HBV DNA quantification assays available, it is important to use the most efficient testing system for virological monitoring. In this study, we evaluated the performance characteristics and comparability of three HBV DNA quantification systems: Abbott HBV real-time PCR (Abbott PCR), artus HBV real-time PCR with QIAamp DNA blood kit purification (artus-DB), and artus HBV real-time PCR with the QIAamp DSP virus kit purification (artus-DSP). The lower limits of detection of these systems were established against the WHO international standards for HBV DNA and were found to be 1.43, 82, and 9 IU/ml, respectively. The intra-assay and interassay coefficients of variation of plasma samples (1 to 6 log(10) IU/ml) ranged between 0.05 to 8.34% and 0.16 to 3.48% for the Abbott PCR, 1.53 to 26.85% and 0.50 to 12.89% for artus-DB, and 0.29 to 7.42% and 0.94 to 3.01% for artus-DSP, respectively. Ninety HBV clinical samples were used for comparison of assays, and paired quantitative results showed strong correlation by linear regression analysis (artus-DB with Abbott PCR, r = 0.95; Abbott PCR with artus-DSP, r = 0.97; and artus-DSP with artus-DB, r = 0.94). Bland-Altman analysis showed a good level of agreement for Abbott PCR and artus-DSP, with a mean difference of 0.10 log(10) IU/ml and limits of agreement of -0.91 to 1.11 log(10) IU/ml. No genotype-specific bias was seen in all three systems for HBV genotypes A, C, and D, which are predominant in this region. This finding illustrates that the Abbott real-time HBV and artus-DSP systems show more comparable performance than the artus-DB system, meeting the current guidelines for assays to be used in the management of hepatitis B.

Rotaviral and bacterial gastroenteritis in children during winter: an evaluation of physician ordering patterns.

PubMed

Chemaly, Roy F; Yen-Lieberman, Belinda; Schindler, Sue A; Goldfarb, Johanna; Hall, Gerri S; Procop, Gary W

2003-09-01

Identification of the agents of infectious diarrhea may facilitate appropriate therapy and prevent inappropriate antibiotic use. To better define the etiology of infectious diarrhea for children <12 years in our community and to study the ordering patterns of physicians. We reviewed test results of stool specimens from children <12 years old at our institution (CCF) and those submitted through our reference laboratory for rotavirus enzyme immunoassay (REIA) and stool cultures for a 7-month period (11/1/00-6/1/01). For CCF patients, REIA and stool cultures for usual bacterial enteric pathogens (BEP) were performed, regardless of the test ordered (i.e. REIA alone, stool culture alone or both). We compared the results with the orders placed to determine if requests for rotavirus alone or bacterial stool culture alone missed BEP or rotavirus, respectively. Overall, REIAs were performed on 81% (538/661) of stool specimens, with 37% positive. Stool cultures were performed on 62% (408/661) of stool specimens, with 4.4% positive. Stool specimens (280) from CCF pediatric patients were evaluated for both rotavirus and BEP. Some 42% of REIA and 23% of stool cultures were ordered as single tests, while both tests were ordered for 35% of the patients. Of the REIA ordered alone, 34% were positive for rotavirus; however, 2.5% of these contained BEP that would have been missed. Of the stool cultures that were ordered alone, 8% were positive; however, 19% of these contained rotavirus that would have been missed. When both tests were ordered, 22% contained rotavirus and 2% contained BEP. Both rotavirus and bacterial enteric infections were missed with selective viral versus bacterial specific ordering patterns. A rotaviral screen prior to stool culture may be useful for children with diarrhea during the winter months.

Purifying, Separating, and Concentrating Cells From a Sample Low in Biomass

NASA Technical Reports Server (NTRS)

Benardini, James N.; LaDuc, Myron T.; Diamond, Rochelle

2012-01-01

Frequently there is an inability to process and analyze samples of low biomass due to limiting amounts of relevant biomaterial in the sample. Furthermore, molecular biological protocols geared towards increasing the density of recovered cells and biomolecules of interest, by their very nature, also concentrate unwanted inhibitory humic acids and other particulates that have an adversarial effect on downstream analysis. A novel and robust fluorescence-activated cell-sorting (FACS)-based technology has been developed for purifying (removing cells from sampling matrices), separating (based on size, density, morphology), and concentrating cells (spores, prokaryotic, eukaryotic) from a sample low in biomass. The technology capitalizes on fluorescent cell-sorting technologies to purify and concentrate bacterial cells from a low-biomass, high-volume sample. Over the past decade, cell-sorting detection systems have undergone enhancements and increased sensitivity, making bacterial cell sorting a feasible concept. Although there are many unknown limitations with regard to the applicability of this technology to environmental samples (smaller cells, few cells, mixed populations), dogmatic principles support the theoretical effectiveness of this technique upon thorough testing and proper optimization. Furthermore, the pilot study from which this report is based proved effective and demonstrated this technology capable of sorting and concentrating bacterial endospore and bacterial cells of varying size and morphology. Two commercial off-the-shelf bacterial counting kits were used to optimize a bacterial stain/dye FACS protocol. A LIVE/DEAD BacLight Viability and Counting Kit was used to distinguish between the live and dead cells. A Bacterial Counting Kit comprising SYTO BC (mixture of SYTO dyes) was employed as a broad-spectrum bacterial counting agent. Optimization using epifluorescence microscopy was performed with these two dye/stains. This refined protocol was further validated using varying ratios and mixtures of cells to ensure homogenous staining compared to that of individual cells, and were utilized for flow analyzer and FACS labeling. This technology focuses on the purification and concentration of cells from low-biomass spacecraft assembly facility samples. Currently, purification and concentration of low-biomass samples plague planetary protection downstream analyses. Having a capability to use flow cytometry to concentrate cells out of low-biomass, high-volume spacecraft/ facility sample extracts will be of extreme benefit to the fields of planetary protection and astrobiology. Successful research and development of this novel methodology will significantly increase the knowledge base for designing more effective cleaning protocols, and ultimately lead to a more empirical and true account of the microbial diversity present on spacecraft surfaces. Refined cleaning and an enhanced ability to resolve microbial diversity may decrease the overall cost of spacecraft assembly and/or provide a means to begin to assess challenging planetary protection missions.

Quantitative profiling of CpG island methylation in human stool for colorectal cancer detection.

PubMed

Elliott, Giles O; Johnson, Ian T; Scarll, Jane; Dainty, Jack; Williams, Elizabeth A; Garg, D; Coupe, Amanda; Bradburn, David M; Mathers, John C; Belshaw, Nigel J

2013-01-01

The aims of this study were to investigate the use of quantitative CGI methylation data from stool DNA to classify colon cancer patients and to relate stool CGI methylation levels to those found in corresponding tissue samples. We applied a quantitative methylation-specific PCR assay to determine CGI methylation levels of six genes, previously shown to be aberrantly methylated during colorectal carcinogenesis. Assays were performed on DNA from biopsies of "normal" mucosa and stool samples from 57 patients classified as disease-free, adenoma, or cancer by endoscopy, and in tumour tissue from cancer patients. Additionally, CGI methylation was analysed in stool DNA from an asymptomatic population of individuals covering a broad age range (mean = 47 ± 24 years) CGI methylation levels in stool DNA were significantly higher than in DNA from macroscopically normal mucosa, and a significant correlation between stool and mucosa was observed for ESR1 only. Multivariate statistical analyses using the methylation levels of each CGI in stool DNA as a continuous variable revealed a highly significant (p = 0.003) classification of cancer vs. non-cancer (adenoma + disease-free) patients (sensitivity = 65 %, specificity = 81 %). CGI methylation profiling of stool DNA successfully identified patients with cancer despite the methylation status of CGIs in stool DNA not generally reflecting those in DNA from the colonic mucosa.

Mitochondrial and Y-chromosomal profile of the Kazakh population from East Kazakhstan

PubMed Central

Tarlykov, Pavel V.; Zholdybayeva, Elena V.; Akilzhanova, Ainur R.; Nurkina, Zhannur M.; Sabitov, Zhaxylyk M.; Rakhypbekov, Tolebay K.; Ramanculov, Erlan M.

2013-01-01

Aim To study the genetic relationship of Kazakhs from East Kazakhstan to other Eurasian populations by examining paternal and maternal DNA lineages. Methods Whole blood samples were collected in 2010 from 160 unrelated healthy Kazakhs residing in East Kazakhstan. Genomic DNA was extracted with Wizard® genomic DNA Purification Kit. Nucleotide sequence of hypervariable segment I of mitochondrial DNA (mtDNA) was determined and analyzed. Seventeen Y-short tandem repeat (STR) loci were studied in 67 samples with the AmpFiSTR Y-filer PCR Amplification Kit. In addition, mtDNA data for 2701 individuals and Y-STR data for 677 individuals were retrieved from the literature for comparison. Results There was a high degree of genetic differentiation on the level of mitochondrial DNA. The majority of maternal lineages belonged to haplogroups common in Central Asia. In contrast, Y-STR data showed very low genetic diversity, with the relative frequency of the predominant haplotype of 0.612. Conclusion The results revealed different migration patterns in the population sample, showing there had been more migration among women. mtDNA genetic diversity in this population was equivalent to that in other Central Asian populations. Genetic evidence suggests the existence of a single paternal founder lineage in the population of East Kazakhstan, which is consistent with verbal genealogical data of the local tribes. PMID:23444242

Impact of palm olein in infant formulas on stool consistency and frequency: a meta-analysis of randomized clinical trials

PubMed Central

Lasekan, John B.; Hustead, Deborah S.; Masor, Marc; Murray, Robert

2017-01-01

ABSTRACT Background: Meta-analysis studies have documented that palm olein (PALM) predominant formulas reduce calcium and fat absorption, and bone mineralization in infants, but none have been documented for stool consistency and frequency. Objective: The study objective was to conduct a meta-analysis of published randomized clinical trials (RCTs) on the effect of PALM-based formulas on stool consistency and frequency in infants. Design: A literature search was conducted in BIOSIS Previews®, Embase®, Embase® Alert, MEDLINE® and Cochrane databases. PALM-based RCTs with available stool outcomes were selected and meta-analyzed. Mean rank stool consistency (MRSC, primary outcome) and stool frequency (secondary outcome) were compared between infants fed PALM-based and PALM-free formulas (NoPALM), using random effects model. Results: Nine out of identified16 studies were meta-analyzed. The mean MRSC (scale of 1 = watery to 5 = hard) in the NoPALM-fed infants was lower (softer stools) compared to the PALM-fed infants (mean difference ‒0.355, 95% Confidence Interval [CI] of ‒0.472 to ‒0.239, p < 0.001). Difference for stool frequency was not significant (p = 0.613). Conclusion: Meta-analysis of RCTs indicated that NoPALM-fed infants have significantly softer stools but similar stool frequencies versus PALM-fed infants, despite differences in study types and design. Future meta-analysis could benefit from including comparison with human milk-fed infants. PMID:28659741

The effect of step stool use and provider height on CPR quality during pediatric cardiac arrest: A simulation-based multicentre study.

PubMed

Cheng, Adam; Lin, Yiqun; Nadkarni, Vinay; Wan, Brandi; Duff, Jonathan; Brown, Linda; Bhanji, Farhan; Kessler, David; Tofil, Nancy; Hecker, Kent; Hunt, Elizabeth A

2018-01-01

We aimed to explore whether a) step stool use is associated with improved cardiopulmonary resuscitation (CPR) quality; b) provider adjusted height is associated with improved CPR quality; and if associations exist, c) determine whether just-in-time (JIT) CPR training and/or CPR visual feedback attenuates the effect of height and/or step stool use on CPR quality. We analysed data from a trial of simulated cardiac arrests with three study arms: No intervention; CPR visual feedback; and JIT CPR training. Step stool use was voluntary. We explored the association between 1) step stool use and CPR quality, and 2) provider adjusted height and CPR quality. Adjusted height was defined as provider height + 23 cm (if step stool was used). Below-average height participants were ≤ gender-specific average height; the remainder were above average height. We assessed for interaction between study arm and both adjusted height and step stool use. One hundred twenty-four subjects participated; 1,230 30-second epochs of CPR were analysed. Step stool use was associated with improved compression depth in below-average (female, p=0.007; male, p<0.001) and above-average (female, p=0.001; male, p<0.001) height providers. There is an association between adjusted height and compression depth (p<0.001). Visual feedback attenuated the effect of height (p=0.025) on compression depth; JIT training did not (p=0.918). Visual feedback and JIT training attenuated the effect of step stool use (p<0.001) on compression depth. Step stool use is associated with improved compression depth regardless of height. Increased provider height is associated with improved compression depth, with visual feedback attenuating the effects of height and step stool use.

IBS Patients Show Frequent Fluctuations between Loose/Watery and Hard/Lumpy Stools: Implications for Treatment

PubMed Central

Palsson, Olafur S.; Baggish, Jeffrey S.; Turner, Marsha J.; Whitehead, William E.

2013-01-01

Aims To determine how variable stool consistency is in patients with irritable bowel (IBS) and assess the relationship between stool consistency and gastrointestinal symptoms. Methods Individuals with a physician diagnosis of IBS were recruited by advertisement. Enrollment questionnaires included the Rome III Diagnostic Questionnaire and IBS Symptom Severity Scale. Then 185 patients meeting Rome criteria for IBS rated the consistency (using the Bristol Stool Scale) of each bowel movement (BM) for 90 days and whether the BM was accompanied by pain, urgency, or soiling. Each night they transferred BM ratings from a paper diary to an internet form and also reported the average daily intensity of abdominal pain, bloating, bowel habit dissatisfaction, and life interference of bowel symptoms. Only the longest sequence of consecutive days of diary data was used in analysis (average of 73 days). Results Patients were 89% female with average age 36.6 years. 78% had both loose/watery and hard/lumpy stools; the average was 3 fluctuations between these extremes per month. The proportion of loose/watery stools correlated r=.78 between the first and second months and the proportion of hard/lumpy stools correlated r=.85 between months. Loose/watery stools were associated with more BM-related pain, urgency, and soiling than hard/lumpy or normal stools; however, IBS-C patients had significantly more BM-unrelated abdominal pain, bloating, dissatisfaction with bowel habits, and life interference than IBS-D. Questionnaires overestimated the frequency of abnormal stool consistency and gastrointestinal symptoms compared to diaries. Conclusions Stool consistency varies greatly within individuals. However, stool patterns are stable within an individual from month to month. The paradoxical findings of greater symptom severity after individual loose/watery BMs vs. greater overall symptom severity in IBS-C implies different physiological mechanisms for symptoms in constipation compared to diarrhea. Daily symptom monitoring is more sensitive and reliable than a questionnaire. PMID:22068664

Modular development of a prototype point of care molecular diagnostic platform for sexually transmitted infections.

PubMed

Branavan, Manoharanehru; Mackay, Ruth E; Craw, Pascal; Naveenathayalan, Angel; Ahern, Jeremy C; Sivanesan, Tulasi; Hudson, Chris; Stead, Thomas; Kremer, Jessica; Garg, Neha; Baker, Mark; Sadiq, Syed T; Balachandran, Wamadeva

2016-08-01

This paper presents the design of a modular point of care test platform that integrates a proprietary sample collection device directly with a microfluidic cartridge. Cell lysis, within the cartridge, is conducted using a chemical method and nucleic acid purification is done on an activated cellulose membrane. The microfluidic device incorporates passive mixing of the lysis-binding buffers and sample using a serpentine channel. Results have shown extraction efficiencies for this new membrane of 69% and 57% compared to the commercial Qiagen extraction method of 85% and 59.4% for 0.1ng/µL and 100ng/µL salmon sperm DNA respectively spiked in phosphate buffered solution. Extraction experiments using the serpentine passive mixer cartridges incorporating lysis and nucleic acid purification showed extraction efficiency around 80% of the commercial Qiagen kit. Isothermal amplification was conducted using thermophillic helicase dependant amplification and recombinase polymerase amplification. A low cost benchtop real-time isothermal amplification platform has been developed capable of running six amplifications simultaneously. Results show that the platform is capable of detecting 1.32×10(6) of sample DNA through thermophillic helicase dependant amplification and 1×10(5) copy numbers Chlamydia trachomatis genomic DNA within 10min through recombinase polymerase nucleic acid amplification tests. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Large Scale Generation and Characterization of Anti-Human CD34 Monoclonal Antibody in Ascetic Fluid of Balb/c Mice

PubMed Central

Aghebati Maleki, Leili; Majidi, Jafar; Baradaran, Behzad; Abdolalizadeh, Jalal; Kazemi, Tohid; Aghebati Maleki, Ali; Sineh sepehr, Koushan

2013-01-01

Purpose: Monoclonal antibodies or specific antibodies are now an essential tool of biomedical research and are of great commercial and medical value. The purpose of this study was to produce large scale of monoclonal antibody against CD34 in order to diagnostic application in leukemia and purification of human hematopoietic stem/progenitor cells. Methods: For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human CD34 were injected into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. 5 ml ascitic fluid was harvested from each mouse in two times. Evaluation of mAb titration was assessed by ELISA method. The ascitic fluid was examined for class and subclasses by ELISA mouse mAb isotyping Kit. mAb was purified from ascitic fluid by affinity chromatography on Protein A-Sepharose. Purity of monoclonal antibody was monitored by SDS -PAGE and the purified monoclonal antibody was conjugated with FITC. Results: Monoclonal antibodies with high specificity and sensitivity against human CD34 by hybridoma technology were prepared. The subclass of antibody was IgG1 and its light chain was kappa. Conclusion: The conjugated monoclonal antibody could be a useful tool for isolation, purification and characterization of human hematopoietic stem cells. PMID:24312838

Production, purification and utilization of biogas as fuel for internal combustion engine

NASA Astrophysics Data System (ADS)

Hernandez, Noel M.; Villanueva, Eliseo P.

2018-03-01

This study attempts to modify a 4-cylinder gasoline engine to run with a purified compressed biogas as substitute for fossil fuels. Water scrubbing method was used as the easiest purification technique to remove CO2 and iron filing for H2S. The pressurized raw biogas was fed in a low cost made portable floating type gas holder with volume capacity of 0.74 m3. The purified biogas was compressed using a reciprocating compressor through a two stage series of enrichment and moisture removal process using activated alumina into the steel cylinder to improve the quality of the methane content. The enriched biogas was filled in the LPG tank for 20 minutes at 10 bars at an average of 73.67% CH4 with no traces of H2S as storage for engine utilization. The modification involved the installation and mounting of LPG conversion kit. A comparative analysis of the performance and combustion characteristics of the engine was evaluated separately with gasoline and purified compressed biogas using electro-dynamometer as variable loads. The findings show that power output deterioration in compressed biogas was mainly due to high percentage of CO2 and other gases impurities. It also shows that because of the calorific value of biogas, the thermal efficiency is lesser than that of gasoline. It implies that the overall engine performance can be improved by removing undesirable gases in the mixture.

Camel as a transboundary vector for emerging exotic Salmonella serovars.

PubMed

Ghoneim, Nahed H; Abdel-Moein, Khaled A; Zaher, Hala

2017-05-01

The current study was conducted to shed light on the role of imported camels as a transboundary vector for emerging exotic Salmonella serovars. Fecal samples were collected from 206 camels directly after slaughtering including 25 local camels and 181 imported ones as well as stool specimens were obtained from 50 slaughterhouse workers at the same abattoir. The obtained samples were cultured while Salmonella serovars were identified through Gram's stain films, biochemical tests and serotyping with antisera kit. Moreover, the obtained Salmonella serovars were examined by PCR for the presence of invA and stn genes. The overall prevalence of Salmonella serovars among the examined camels was 8.3%. Stn gene was detected in the vast majority of exotic strains (11/14) 78.6% including emerging serovars such as Salmonella Saintpaul, S. Chester, S. Typhimurium whereas only one isolate from local camels carried stn gene (1/3) 33.3%. On the other hand, none of the examined humans yielded positive result. Our findings highlight the potential role of imported camels as a transboundary vector for exotic emerging Salomenella serovars.

An iPhone application using a novel stool color detection algorithm for biliary atresia screening.

PubMed

Hoshino, Eri; Hayashi, Kuniyoshi; Suzuki, Mitsuyoshi; Obatake, Masayuki; Urayama, Kevin Y; Nakano, Satoshi; Taura, Yasuyuki; Nio, Masaki; Takahashi, Osamu

2017-10-01

The stool color card has been the primary tool for identifying acholic stools in infants with biliary atresia (BA), in several countries. However, BA stools are not always acholic, as obliteration of the bile duct occurs gradually. This study aims to introduce Baby Poop (Baby unchi in Japanese), a free iPhone application, employing a detection algorithm to capture subtle differences in colors, even with non-acholic BA stools. The application is designed for use by caregivers of infants aged approximately 2 weeks-1 month. Baseline analysis to determine optimal color parameters predicting BA stools was performed using logistic regression (n = 50). Pattern recognition and machine learning processes were performed using 30 BA and 34 non-BA images. Additional 5 BA and 35 non-BA pictures were used to test accuracy. Hue, saturation, and value (HSV) were the preferred parameter for BA stool identification. A sensitivity and specificity were 100% (95% confidence interval 0.48-1.00 and 0.90-1.00, respectively) even among a collection of visually non-acholic, i.e., pigmented BA stools and relatively pale-colored non-BA stools. Results suggest that an iPhone mobile application integrated with a detection algorithm is an effective and convenient modality for early detection of BA, and potentially for other related diseases.

[When should a stool culture be done in adults with nosocomial diarrhea?].

PubMed

Mathieu, Alexandre; Tachet, Anne; Pariente, Alexandre

2005-01-29

To assess the diagnostic efficacy, cost and possible corrective measure of the indications for routine stool cultures in nosocomial diarrhoea in adults. A retrospective study over a 10-month period of 660 standard stool cultures, 256 of which were conducted after the 3rd day of hospitalisation, conducted in 528 patients at the hospital centre in Pau. The positivity rate of the stool cultures was of 26/336 patients (7.7%), and of 37/404 examinations (9%) within the first three days of hospitalisation, versus 2/192 patients (1%) and 3/256 examinations (1%) after the 3rd day of hospitalisation (p<0.05). In 83 patients a stool culture was repeated, and was only positive in one patient with an initially negative culture. If a stool culture had not been performed after the 3rd day, 2 infections would not have been diagnosed (1 salmonella and 1 K. oxytoca) and 256 stool cultures could have been economised (estimated cost: 6,144 euro). Moreover, by eliminating repeated stool cultures, 3 infections would not have been diagnosed (2 salmonella, and 1 K. oxytoca) and 321 stool cultures would have been avoided (estimated cost: 7,704 euro). If the stool cultures had been conducted after the 3rd day of hospitalisation only in those aged over 64 with comorbidity, immunosuppression or within the context of an epidemic, no false negative would have been observed and 149 stool cultures would have been economised (estimated cost: 3,576 euro). The positivity rate of the search for C. difficile, only conducted on explicit request from the practitioners, was of 5/23 (22%) and 4/28 (14%) before and after the 3rd day of hospitalisation (non-significant difference). Restriction of standard stool cultures after the 3rd day of hospitalisation to patients aged over 64 with comorbidity, to the immunodepressed, and within an epidemic context would economise around 4,300 euro per month in a medium-sized general hospital. No systematic restriction should be applied to the search for C. difficile.

Increasing butyrate concentration in the distal colon by accelerating intestinal transit

PubMed Central

Lewis, S; Heaton, K

1997-01-01

Background—Populations at low risk of colonic cancer consume large amounts of fibre and starch and pass acid, bulky stools. One short chain fatty acid (SCFA), butyrate, is the colon's main energy source and inhibits malignant transformation in vitro. 
Aim—To test the hypothesis that altering colonic transit rate alters colonic pH and the SCFA content of the stools. 
Patients—Thirteen healthy adults recruited by advertisement. 
Methods—Volunteers consumed, in turn, wheat bran, senna and loperamide, each for nine days with a two week washout period between study periods, dietary intake being unchanged. Before, and in the last four days of each intervention, whole gut transit time (WGTT), defaecation frequency, stool form, stool β-glucuronidase activity, stool pH, stool SCFA concentrations and intracolonic pH (using a radiotelemetry capsule for continuous monitoring) were assessed. 
Results—WGTT decreased, stool output and frequency increased with wheat bran and senna, vice versa with loperamide. The pH was similar in the distal colon and stool. Distal colonic pH fell with wheat bran and senna and tended to increase with loperamide. Faecal SCFA concentrations, including butyrate, increased with senna and fell with loperamide. With wheat bran the changes were non-significant, possibly because of the short duration of the study. Baseline WGTT correlated with faecal SCFA concentration (r=−0.511, p=0.001), with faecal butyrate (r=−0.577, p<0.001) and with distal colonic pH (r=0.359, p=0.029). 
Conclusion—Bowel transit rate is a determinant of stool SCFA concentration including butyrate and distal colonic pH. This may explain the inter-relations between colonic cancer, dietary fibre intake, stool output, and stool pH. 

 Keywords: bowel cancer; colonic pH; fibre; intestinal transit; pH; short chain fatty acids PMID:9301506

Stool DNA Test

MedlinePlus

... in the United States. Why it's done Stool DNA testing is intended to screen for colon cancer or ... and poses no risks. How you prepare Stool DNA testing requires no preparation. You can eat and drink ...

Prevalence of multi-drug resistant organisms in stool of paediatric patients with acute leukaemia and correlation with blood culture positivity: A single institution experience.

PubMed

Shankar, Krupa; Radhakrishnan, Venkatraman; Vijayakumar, Varalakskmi; Ramamoorthy, Jaikumar; Ganesan, Prasanth; Dhanushkodi, Manikandan; Ganesan, T S; Sagar, T G

2018-01-01

Multi-drug resistant (MDR) bacteria are associated with increased morbidity and mortality in children with acute leukaemia. The present study was conducted to assess the prevalence of MDR bacteria in stool cultures of patients with acute leukaemia at presentation to the hospital. The results were then correlated with blood cultures when patients developed septicaemia. The study involved analysis of case records of patients with newly diagnosed acute leukaemia less than 18 years of age treated at our centre from January 2015 to December 2015. Stool cultures were sent within 72 hr of hospital admission and blood cultures were sent when clinically indicated. MDR was defined as resistance to at least one antibiotic in three or more following antimicrobial groups: cephalosporins, β-lactam/β-lactamase inhibitor, carbapenems, fluoroquinolones and aminoglycosides. The analysis included 85 patients with acute leukaemia, among whom 48 of 85 (56%) patients had positive stool cultures and 42 of 85 (50%) patients were positive for MDR bacteria. Blood cultures were positive in 13 of 48 patients (27%, seven MDR and six non-MDR) with positive stool cultures and three of 37 patients (8%, one MDR and two non-MDR) with negative stool cultures (P = 0.01). The concordance between stool and blood culture for similar organism was 61%. There were seven deaths in 48 stool culture positive patients and two deaths in 37 stool culture negative patients. This study shows the high prevalence of MDR bacteria in newly diagnosed children with acute leukaemia. Colonisation with MDR bacteria in stools is associated with increased positivity of blood cultures and mortality. © 2017 Wiley Periodicals, Inc.

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

PubMed

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.

Trypsin and chymotrypsin in stool

MedlinePlus

... the stool. You can catch the stool on plastic wrap that is loosely placed over the toilet bowl ... child wears a diaper, line the diaper with plastic wrap. Place the plastic wrap so that urine and ...

Anatomic Problems of the Lower GI Tract

MedlinePlus

... then changes waste from liquid to a solid matter called stool. Stool passes from the colon to ... include abdominal tenderness, swelling, or bloating bloody or dark-red stools constipation—a condition in which a ...

Differences in gut microbiota associated with age, sex, and stool consistency in healthy Japanese subjects.

PubMed

Takagi, Tomohisa; Naito, Yuji; Inoue, Ryo; Kashiwagi, Saori; Uchiyama, Kazuhiko; Mizushima, Katsura; Tsuchiya, Saeko; Dohi, Osamu; Yoshida, Naohisa; Kamada, Kazuhiro; Ishikawa, Takeshi; Handa, Osamu; Konishi, Hideyuki; Okuda, Kayo; Tsujimoto, Yoshimasa; Ohnogi, Hiromu; Itoh, Yoshito

2018-06-20

Human gut microbiota is involved in host health and disease development. Investigations of age-related and sex-related alterations in gut microbiota are limited, and the association between stool consistency and gut microbiota has not been fully investigated. We investigated gut microbiota differences related to age, sex, and stool consistency in healthy Japanese subjects. Two-hundred and seventy-seven healthy Japanese subjects aged 20-89 years were enrolled. Fecal samples were obtained to analyze the gut microbiome. We evaluated the association between stool consistency [Bristol stool scale (BSS)] and gut microbiota. Although there were significant differences in the microbial structure between males and females, the α-diversity of gut microbiota showed no difference between males and females or among age groups. There were significant increases in genera Prevotella, Megamonas, Fusobacterium, and Megasphaera and Bifidobacterium, Ruminococcus, and Akkermansia in males and females, respectively. The ratio of hard stools (BSS types 1 and 2) was higher in females; the ratio of loose stools (BSS type 6) was higher in males. No younger male had BSS type 1 or type 2. Fusobacterium in males was significantly higher in the loose consistency group, and Oscillospira was significantly higher in the hard consistency group in males; Campylobacter, SMB53, and Turicibacter were significantly higher in the hard consistency group in females. Several changes in gut microbiota were associated with age and sex. Stool consistency and gut microbiota associations emphasized the importance of stool consistency assessments to understand intestinal function.

Stool antigen immunodetection for diagnosis of Giardia duodenalis infection in human subjects with HIV and cancer.

PubMed

Nooshadokht, Maryam; Kalantari-Khandani, Behjat; Sharifi, Iraj; Kamyabi, Hossein; Liyanage, Namal P M; Lagenaur, Laurel A; Kagnoff, Martin F; Singer, Steven M; Babaei, Zahra; Solaymani-Mohammadi, Shahram

2017-10-01

Human infection with the protozoan parasite Giardia duodenalis is one the most common parasitic diseases worldwide. Higher incidence rates of giardiasis have been reported from human subjects with multiple debilitating chronic conditions, including hypogammaglobulinemia and common variable immunodeficiency (CVID). In the current study, stool specimens were collected from 199 individuals diagnosed with HIV or cancer and immunocompetent subjects. The sensitivity of microscopy-based detection on fresh stool preparations, trichrome staining and stool antigen immunodetection for the diagnosis of G. duodenalis were 36%, 45.5% and 100%, respectively when compared with a highly sensitive stool-based PCR method as the gold standard. Further multilocus molecular analyses using glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) loci demonstrated that the AI genotype of G. duodenalis was the most prevalent, followed by the AII genotype and mixed (AI+B) infections. We concluded that stool antigen immunodetection-based immunoassays and stool-based PCR amplification had comparable sensitivity and specificity for the diagnosis of G. duodenalis infections in these populations. Stool antigen detection-based diagnostic modalities are rapid and accurate and may offer alternatives to conventional microscopy and PCR-based diagnostic methods for the diagnosis of G. duodenalis in human subjects living with HIV or cancer. Copyright © 2017. Published by Elsevier B.V.

Identification of Skeletal Remains of Communist Armed Forces Victims During and After World War II: Combined Y-chromosome Short Tandem Repeat (STR) and MiniSTR Approach

PubMed Central

Marjanović, Damir; Durmić-Pašić, Adaleta; Kovačević, Lejla; Avdić, Jasna; Džehverović, Mirela; Haverić, Sanin; Ramić, Jasmin; Kalamujić, Belma; Bilela, Lada Lukić; Škaro, Vedrana; Projić, Petar; Bajrović, Kasim; Drobnič, Katja; Davoren, Jon; Primorac, Dragan

2009-01-01

Aim To report on the use of STR, Y-STRs, and miniSTRs typing methods in the identification of victims of revolutionary violence and crimes against humanity committed by the Communist Armed Forces during and after World War II in which bodies were exhumed from mass and individual graves in Slovenia. Methods Bone fragments and teeth were removed from human remains found in several small and closely located hidden mass graves in the Škofja Loka area (Lovrenska Grapa and Žolšče) and 2 individual graves in the Ljubljana area (Podlipoglav), Slovenia. DNA was isolated using the Qiagen DNA extraction procedure optimized for bone and teeth. Some DNA extracts required additional purification, such as N-buthanol treatment. The QuantifilerTM Human DNA Quantification Kit was used for DNA quantification. Initially, PowerPlex 16 kit was used to simultaneously analyze 15 short tandem repeat (STR) loci. The PowerPlex S5 miniSTR kit and AmpFℓSTR® MiniFiler PCR Amplification Kit was used for additional analysis if preliminary analysis yielded weak partial or no profiles at all. In 2 cases, when the PowerPlex 16 profiles indicated possible relatedness of the remains with reference samples, but there were insufficient probabilities to call the match to possible male paternal relatives, we resorted to an additional analysis of Y-STR markers. PowerPlex® Y System was used to simultaneously amplify 12 Y-STR loci. Fragment analysis was performed on an ABI PRISM 310 genetic analyzer. Matching probabilities were estimated using the DNA-View software. Results Following the Y-STR analysis, 1 of the “weak matches” previously obtained based on autosomal loci, was confirmed while the other 1 was not. Combined standard STR and miniSTR approach applied to bone samples from 2 individual graves resulted in positive identifications. Finally, using the same approach on 11 bone samples from hidden mass grave Žološče, we were able to obtain 6 useful DNA profiles. Conclusion The results of this study, in combination with previously obtained results, demonstrate that Y-chromosome testing and miniSTR methodology can contribute to the identification of human remains of victims of revolutionary violence from World War II. PMID:19480024

Cloning, optimization of induction conditions and purification of Mycobacterium tuberculosis Rv1733c protein expressed in Escherichia coli

PubMed Central

Ashayeri-Panah, Mitra; Eftekhar, Fereshteh; Kazemi, Bahram; Joseph, Joan

2017-01-01

Background and Objectives: Rv1733c is a latency antigen from Mycobacterium tuberculosis, a probable integral-membrane protein with promiscuous T-cell and B-cell epitopes, making it a potential vaccine candidate against tuberculosis. This study aimed to clone and optimize the expression of recombinant Rv1733c in Escherichia coli for purification. Materials and Methods: Chemically synthesized rv1733c coding sequence was cloned in pET-23a(+) followed by transforming E. coli BL21 (DE3) cells. To evaluate the induction conditions for optimized expression, factorial design of experiments was employed using four different media as well as four levels of isopropyl-b-D-thiogalactopyranosid [IPTG] concentration and duration of induction. The recombinant protein was then purified using a His-tag purification kit and detected through western blotting. Results: Recombinant Rv1733c (> 24 kDa) was expressed and accumulated in the cytoplasm of the E. coli cells. Medium composition showed the most significant effect on the yield of the recombinant protein (P = 0.000). The highest yield of recombinant Rv1733c occurred in the presence of 0.4 mM of IPTG in Terrific Broth medium (containing 1.2% tryptone, 2.4% yeast extract, 72 mM K 2 HPO 4 , 17 mM KH 2 PO 4 and 0.4% glycerol) after 10 h at 37°C. Under these conditions, the expression level was around 0.5 g/L of culture medium. Purified Rv1733c was detected by an anti-polyhistidine antibody and a tuberculosis patient’s serum. Systematic optimization of induction conditions gave us high yield of recombinant polyhistidine-tagged Rv1733c in E. coli which was successfuly purified. Conclusion: We believe that the purified Rv1733c recombinant protein from M. tuberculosis might be a good candidate for vaccine production against tuberculosis. PMID:29213997

Evaluation of a fecal immunochemistry test prior to colonoscopy for outpatients with various indications.

PubMed

Szilagyi, Andrew; Xue, Xiaoqing

2017-01-01

Stool tests can predict advanced neoplasms prior to colonoscopy. Results of immunochemical stool tests to predict findings at colonoscopy for various indications are less often reported. We compared pre-colonoscopy stool tests with findings in patients undergoing colonoscopy for different indications. Charts of patients undergoing elective or semi-urgent colonoscopy were reviewed. Comparison of adenoma detection rates and pathological findings was made between prescreened and non-prescreened, and between stool-positive and stool-negative cases. Demographics, quality of colonoscopy, and pathological findings were recorded. Odds ratios (ORs) and 95% confidence intervals (CIs) were assessed. Statistical significance was accepted at p ≤0.05. Charts of 325 patients were reviewed. Among them, stool tests were done on 144 patients: 114 were negative and 30 were positive. Findings were similar in the pretest and non-pretest groups. Detection of advanced adenomas per patient was higher in the stool-positive group compared to the stool-negative group (23.4% vs 3.5%, p =0.0016, OR =7.6 [95% CI: 2-29.3]). Five advanced adenomas (without high-grade dysplasia or adenocarcinoma) and several cases of multiple adenomas were missed in the negative group. Sensitivity and specificity for advanced polyps was 63.6% and 82.7%, respectively. The negative predictive value was 96.5%. Male gender was independently predictive of any adenoma. The stool immunochemical test best predicted advanced neoplasms and had a high negative predictive value in this small cohort. Whether this test can be applied to determine the need for colonoscopy in groups other than average risk would require more studies.

Assessment of commonly used pediatric stool scales: a pilot study.

PubMed

Saps, M; Nichols-Vinueza, D; Dhroove, G; Adams, P; Chogle, A

2013-01-01

The Bristol Stool Form Scale (BSFS) and a modified child-friendly version (M-BSFS) are frequently used in clinical practice and research. These scales have not been validated in children. 3-D stool scale models may be better adapted to the child's development. To assess the usefulness of the BSFS, M-BSFS, and a newly developed 3-D stool scale in children. Fifty children were asked to rank the picture cards of the BSFS and 3-D models from hardest to softest and to match the pictures with descriptors for each stool type. Thirty percent of the children appropriately characterized the stools as hard, loose, or normal using the BSFS vs. 36.6% with the 3-D model (p=0.27). Appropriate correlation of stools as hard, loose, or normal consistency using the BSFS vs. the 3-D model by age group was: 6 to 11-year-olds, 27.5% vs. 33.3% (p=0.58) and 12 to 17-year-olds, 32.1% vs. 39.5% (p=0.41). Thirty-three percent correlated the BSFS pictures with the correct BSFS words, 46% appropriately correlated with the M-BSFS words, and 46% correlated the 3-D stool models with the correct wording. The BSFS and M-BSFS that are widely used as stool assessment instruments are not user-friendly for children. The 3-D model was not found to be better than the BSFS and the M-BSFS. Copyright © 2013 Asociación Mexicana de Gastroenterología. Published by Masson Doyma México S.A. All rights reserved.

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

PubMed Central

2011-01-01

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 < 0.02). There was no significant change in the stool's fat, protein or carbohydrate content. Parental impressions of constipation were improved with the decrease in stool hardness (100% with hard stools at baseline, 10% after 2 weeks), pain with defecation (90% at baseline, 10% after 2 weeks), and the requirement for rectal stimulation to achieve defecation (70% at baseline, 30% after 2 weeks, p < 0.001 for all three indicators). 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. PMID:21595890

Development of an automated modular system for the synthesis of [11C]acetate.

PubMed

Felicini, Chiara; Någren, Kjell; Berton, Andrea; Pascali, Giancarlo; Salvadori, Piero Alberto

2010-12-01

Carboxylation reactions offer a straightforward method for the synthesis of carbon-11 labelled carboxylic acids. Among these, the preparation of carbon-11 (C)-acetate is receiving increasing attention because of diagnostic applications in oncology in addition to its well-established use as a probe for myocardial oxidative metabolism. Although a number of dedicated modules are commercially available, the development of the synthesis on flexible platforms would be beneficial to widen the number of tracers, in particular for preclinical assessment and testing. In this study, the carboxylation reaction was implemented for the synthesis of sodium 1-[C]acetate after the classic route of carboxylation of methylmagnesium chloride by [C]carbon dioxide, followed by the acidic hydrolysis, purification and sterile filtration. This was performed using a commercially available kit of preassembled hardware units and fully compatible components of radiochemistry automation (VarioSystem). The system proved be to highly versatile and inexpensive and allowed a quick translation of the radiochemistry project into a working system even by less experienced personnel, because of predefined interfaces between electronic parts and operating software (preloaded on a laptop and included in the kit). The automatic module proved to be a simple and reliable system for the production of 1-[C]acetate that was prepared in 24 min (total synthesis time) with stable radiochemical yields (20% nondecay corrected) and high radiochemical purity (>97%). The module is used routinely to produce 1-[C]acetate for preclinical studies and is being implemented for the production of the labelled fatty acids.

Comparison of reverse transcription-quantitative polymerase chain reaction methods and platforms for single cell gene expression analysis.

PubMed

Fox, Bridget C; Devonshire, Alison S; Baradez, Marc-Olivier; Marshall, Damian; Foy, Carole A

2012-08-15

Single cell gene expression analysis can provide insights into development and disease progression by profiling individual cellular responses as opposed to reporting the global average of a population. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the "gold standard" for the quantification of gene expression levels; however, the technical performance of kits and platforms aimed at single cell analysis has not been fully defined in terms of sensitivity and assay comparability. We compared three kits using purification columns (PicoPure) or direct lysis (CellsDirect and Cells-to-CT) combined with a one- or two-step RT-qPCR approach using dilutions of cells and RNA standards to the single cell level. Single cell-level messenger RNA (mRNA) analysis was possible using all three methods, although the precision, linearity, and effect of lysis buffer and cell background differed depending on the approach used. The impact of using a microfluidic qPCR platform versus a standard instrument was investigated for potential variability introduced by preamplification of template or scaling down of the qPCR to nanoliter volumes using laser-dissected single cell samples. The two approaches were found to be comparable. These studies show that accurate gene expression analysis is achievable at the single cell level and highlight the importance of well-validated experimental procedures for low-level mRNA analysis. Copyright © 2012 Elsevier Inc. All rights reserved.

DNA Identification of Skeletal Remains from World War II Mass Graves Uncovered in Slovenia

PubMed Central

Marjanović, Damir; Durmić-Pašić, Adaleta; Bakal, Narcisa; Haverić, Sanin; Kalamujić, Belma; Kovačević, Lejla; Ramić, Jasmin; Pojskić, Naris; Škaro, Vedrana; Projić, Petar; Bajrović, Kasim; Hadžiselimović, Rifat; Drobnič, Katja; Huffine, Ed; Davoren, Jon; Primorac, Dragan

2007-01-01

Aim To present the joint effort of three institutions in the identification of human remains from the World War II found in two mass graves in the area of Škofja Loka, Slovenia. Methods The remains of 27 individuals were found in two small and closely located mass graves. The DNA was isolated from bone and teeth samples using either standard phenol/chloroform alcohol extraction or optimized Qiagen DNA extraction procedure. Some recovered samples required the employment of additional DNA purification methods, such as N-buthanol treatment. QuantifilerTM Human DNA Quantification Kit was used for DNA quantification. PowerPlex 16 kit was used to simultaneously amplify 15 short tandem repeat (STR) loci. Matching probabilities were estimated using the DNA View program. Results Out of all processed samples, 15 remains were fully profiled at all 15 STR loci. The other 12 profiles were partial. The least successful profile included 13 loci. Also, 69 referent samples (buccal swabs) from potential living relatives were collected and profiled. Comparison of victims' profile against referent samples database resulted in 4 strong matches. In addition, 5 other profiles were matched to certain referent samples with lower probability. Conclusion Our results show that more than 6 decades after the end of the World War II, DNA analysis may significantly contribute to the identification of the remains from that period. Additional analysis of Y-STRs and mitochondrial DNA (mtDNA) markers will be performed in the second phase of the identification project. PMID:17696306

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

PubMed

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.

Optimal screening and donor management in a public stool bank.

PubMed

Kazerouni, Abbas; Burgess, James; Burns, Laura J; Wein, Lawrence M

2015-12-17

Fecal microbiota transplantation is an effective treatment for recurrent Clostridium difficile infection and is being investigated as a treatment for other microbiota-associated diseases. To facilitate these activities, an international public stool bank has been created, which screens donors and processes stools in a standardized manner. The goal of this research is to use mathematical modeling and analysis to optimize screening and donor management at the stool bank. Compared to the current policy of screening active donors every 60 days before releasing their quarantined stools for sale, costs can be reduced by 10.3 % by increasing the screening frequency to every 36 days. In addition, the stool production rate varies widely across donors, and using donor-specific screening, where higher producers are screened more frequently, also reduces costs, as does introducing an interim (i.e., between consecutive regular tests) stool test for just rotavirus and C. difficile. We also derive a donor release (i.e., into the system) policy that allows the supply to approximately match an exponentially increasing deterministic demand. More frequent screening, interim screening for rotavirus and C. difficile, and donor-specific screening, where higher stool producers are screened more frequently, are all cost-reducing measures. If screening costs decrease in the future (e.g., as a result of bringing screening in house), a bottleneck for implementing some of these recommendations may be the reluctance of donors to undergo serum screening more frequently than monthly.

Maintenance of Pain in Children With Functional Abdominal Pain.

PubMed

Czyzewski, Danita I; Self, Mariella M; Williams, Amy E; Weidler, Erica M; Blatz, Allison M; Shulman, Robert J

2016-03-01

A significant proportion of children with functional abdominal pain develop chronic pain. Identifying clinical characteristics predicting pain persistence is important in targeting interventions. We examined whether child anxiety and/or pain-stooling relations were related to maintenance of abdominal pain frequency and compared the predictive value of 3 methods for assessing pain-stooling relations (ie, diary, parent report, child report). Seventy-six children (7-10 years old at baseline) who presented for medical treatment of functional abdominal pain were followed up 18 to 24 months later. Baseline anxiety and abdominal pain-stooling relations based on pain and stooling diaries and child- and parent questionnaires were examined in relationship to the persistence of abdominal pain frequency. Children's baseline anxiety was not related to persistence of pain frequency. Children who, however, displayed irritable bowel syndrome (IBS) symptoms at baseline maintained pain frequency at follow-up, whereas in children in whom there was no relationship between pain and stooling, pain frequency decreased. Pain and stool diaries and parent report of pain-stooling relations were predictive of pain persistence but child-report questionnaires were not. The presence of IBS symptoms in school-age children with functional abdominal pain appears to predict persistence of abdominal pain over time, whereas anxiety does not. Prospective pain and stooling diaries and parent report of IBS symptoms were predictors of pain maintenance, but child report of symptoms was not.

Genotyping of Plant and Animal Samples without Prior DNA Purification

PubMed Central

Chum, Pak Y.; Haimes, Josh D.; André, Chas P.; Kuusisto, Pia K.; Kelley, Melissa L.

2012-01-01

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors. PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size. PMID:23051689

Rapid Diagnosis of Diarrhea Caused by Shigella sonnei Using Dipsticks; Comparison of Rectal Swabs, Direct Stool and Stool Culture

PubMed Central

Duran, Claudia; Nato, Faridabano; Dartevelle, Sylvie; Thi Phuong, Lan Nguyen; Taneja, Neelam; Ungeheuer, Marie Noëlle; Soza, Guillermo; Anderson, Leslie; Benadof, Dona; Zamorano, Agustín; Diep, Tai The; Nguyen, Truong Quang; Nguyen, Vu Hoang; Ottone, Catherine; Bégaud, Evelyne; Pahil, Sapna; Prado, Valeria; Sansonetti, Philippe; Germani, Yves

2013-01-01

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 106 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. PMID:24278267

A randomised, double-blind study of polyethylene glycol 4000 and lactulose in the treatment of constipation in children

PubMed Central

2014-01-01

Background Chronic constipation is frequent in children. The objective of this study is to compare the efficacy and safety of PEG 4000 and lactulose for the treatment of chronic constipation in young children. Methods This randomised, double-blind study enrolled 88 young children aged 12 to 36 months, who were randomly assigned to receive lactulose (3.3 g per day) or PEG 4000 (8 g per day) for four weeks. The primary efficacy variable was stool frequency during the fourth week of treatment. Secondary outcomes were the number and frequency of subjective symptoms associated with defecation at each visit. Results Stool frequency was comparable in the two groups at baseline (lactulose: 0.7 ± 0.5; PEG 4000: 0.5 ± 0.55). Mean stool frequency increased from 0.70 ± 0.50 stools/day at baseline to 0.80 ± 0.41 at Week 4 in the lactulose group and from 0.50 ± 0.55 to 1.10 ± 0.55 stools/day in the PEG 4000 group. A significant difference was observed in the adjusted mean change from baseline, which was 0.15 stools/day in the lactulose group and 0.51 stools/day in the PEG 4000 group, with a least-squares mean difference of 0.36 stools/day [95% CI: 0.16 to 0.56]. With respect to secondary outcome variables, stool consistency and ease of stool passage improved more in the PEG 4000 group (p = 0.001). The incidence of adverse events was similar in both groups, the majority of which were mild. Conclusions PEG 4000 has superior efficacy to lactulose for the treatment of chronic constipation in young children and is well tolerated. Trial registration US National Institute of Health Clinical Trials database; study NCT00255372 first registered 17th November 2005. PMID:24943105

Fetal exposures and perinatal influences on the stool microbiota of premature infants

PubMed Central

Chernikova, Diana A.; Koestler, Devin C.; Hoen, Anne Gatewood; Housman, Molly L.; Hibberd, Patricia L.; Moore, Jason H.; Morrison, Hilary G.; Sogin, Mitchell L.; Ul-Abideen, Muhammad Zain; Madan, Juliette C.

2015-01-01

Objective To test the hypothesis that maternal complications significantly affect gut colonization patterns in very low birth weight infants. Methods 49 serial stool samples were obtained weekly from 9 extremely premature infants enrolled in a prospective longitudinal study. Sequencing of the bacterial 16S rRNA gene from stool samples was performed to approximate the intestinal microbiome. Linear mixed effects models were used to evaluate relationships between perinatal complications and intestinal microbiome development. Results Subjects with prenatal exposure to a non-sterile intrauterine environment, i.e. PPPROM and chorioamnionitis exposure, were found to have a relatively higher abundance of potentially pathogenic bacteria in the stool across all time points compared to subjects without those exposures, irrespective of exposure to postnatal antibiotics. Compared with those delivered by Caesarean section, vaginally delivered subjects were found to have significantly lower diversity of stool microbiota across all time points, with lower abundance of many genera, most in the family Enterobacteriaceae. Conclusions We identified persistently increased potential pathogen abundance in the developing stool microbiota of subjects exposed to a non-sterile uterine environment. Maternal complications appear to significantly influence the diversity and bacterial composition of the stool microbiota of premature infants, with findings persisting over time. PMID:25394613

Fermented Fiber Supplements Are No Better Than Placebo for a Laxative Effect.

PubMed

McRorie, Johnson W; Chey, William D

2016-11-01

Misconceptions about the effects of dietary fiber and 'functional' fiber on stool parameters and constipation persist in the literature. A comprehensive literature review was conducted with the use of the Scopus and PubMed scientific databases to identify and objectively assess well-controlled clinical studies that evaluated the effects of fiber on stool parameters and constipation. The totality of well-controlled randomized clinical studies show that, to exert a laxative effect, fiber must: (1) resist fermentation to remain intact throughout the large bowel and present in stool, and (2) significantly increase stool water content and stool output, resulting in soft/bulky/easy-to-pass stools. Poorly fermented insoluble fiber (e.g., wheat bran) remains as discreet particles which can mechanically irritate the gut mucosa, stimulating water & mucous secretion if the particles are sufficiently large/coarse. For soluble fibers, some have no effect on viscosity (e.g., inulin, wheat dextrin) while others form high viscosity gels (e.g., β-glucan, psyllium). If the soluble fiber is readily fermented, whether non-viscous or gel-forming, it has no effect on stool output or stool water content, and has no laxative effect. In contrast, a non-fermented, gel-forming soluble fiber (e.g., psyllium) retains its gelled nature and high water-holding capacity throughout the large bowel, resulting in soft/bulky/easy-to-pass stools. When considering a recommendation for a fiber supplement regimen to treat and/or prevent constipation, it is important to consider which fibers have the physical characteristics to exert a laxative effect, and which fiber supplements have rigorous clinical evidence of a significant benefit in patients with constipation.

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

PubMed

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 use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Patterns of detection of Strongyloides stercoralis in stool specimens: implications for diagnosis and clinical trials.

PubMed Central

Dreyer, G; Fernandes-Silva, E; Alves, S; Rocha, A; Albuquerque, R; Addiss, D

1996-01-01

Reported efficacies of drugs used to treat Strongyloides stercoralis infection vary widely. Because diagnostic methods are insensitive, therapeutic trials generally require multiple negative posttreatment stool specimens as evidence of drug efficacy. However, only a single positive stool specimen is usually required for study enrollment. To determine the reproducibility of detection of S. stercoralis larvae in the stool, 108 asymptomatic infected men submitted 25 g of fresh stool once a week for eight consecutive weeks for examination by the Baermann technique. During the 8-week study, 239 (27.7%) of 864 stool specimens were positive for S. stercoralis. Rates of detection of larvae in the stool specimens ranged from eight of eight specimens in 3 (2.8%) men to none of eight specimens in 36 (33.3%) men. Of 43 men for whom S. stercoralis was detected in at least two of the first four stool specimens, only 1 (2.3%) man tested negative on all of the next four specimens. In comparison, of 29 men who had detectable larvae in only one of the first four specimens, 22 (75.9%) tested negative on all of the next four samples. Thus, if these 29 men had been enrolled in a therapeutic trial between the first and second sets of four specimens, the efficacy of a drug with no activity against this parasite would have been estimated to be 76%. These data suggest that patterns of S. stercoralis detection vary widely among infected persons and that intermittent larval shedding can lead to inflated estimates of drug efficacy. Before a patient is entered in a clinical trial of drug efficacy, four consecutive stool specimens should be examined for S. stercoralis; only persons with two or more positive specimens should be enrolled. PMID:8880521

Stool Microbiome and Metabolome Differences between Colorectal Cancer Patients and Healthy Adults

PubMed Central

Weir, Tiffany L.; Manter, Daniel K.; Sheflin, Amy M.; Barnett, Brittany A.; Heuberger, Adam L.; Ryan, Elizabeth P.

2013-01-01

In this study we used stool profiling to identify intestinal bacteria and metabolites that are differentially represented in humans with colorectal cancer (CRC) compared to healthy controls to identify how microbial functions may influence CRC development. Stool samples were collected from healthy adults (n = 10) and colorectal cancer patients (n = 11) prior to colon resection surgery at the University of Colorado Health-Poudre Valley Hospital in Fort Collins, CO. The V4 region of the 16s rRNA gene was pyrosequenced and both short chain fatty acids and global stool metabolites were extracted and analyzed utilizing Gas Chromatography-Mass Spectrometry (GC-MS). There were no significant differences in the overall microbial community structure associated with the disease state, but several bacterial genera, particularly butyrate-producing species, were under-represented in the CRC samples, while a mucin-degrading species, Akkermansia muciniphila, was about 4-fold higher in CRC (p<0.01). Proportionately higher amounts of butyrate were seen in stool of healthy individuals while relative concentrations of acetate were higher in stools of CRC patients. GC-MS profiling revealed higher concentrations of amino acids in stool samples from CRC patients and higher poly and monounsaturated fatty acids and ursodeoxycholic acid, a conjugated bile acid in stool samples from healthy adults (p<0.01). Correlative analysis between the combined datasets revealed some potential relationships between stool metabolites and certain bacterial species. These associations could provide insight into microbial functions occurring in a cancer environment and will help direct future mechanistic studies. Using integrated “omics” approaches may prove a useful tool in identifying functional groups of gastrointestinal bacteria and their associated metabolites as novel therapeutic and chemopreventive targets. PMID:23940645

Importance of the regiospecific distribution of long-chain saturated fatty acids on gut comfort, fat and calcium absorption in infants.

PubMed

Petit, Valérie; Sandoz, Laurence; Garcia-Rodenas, Clara L

2017-06-01

Gastrointestinal tolerance and fat and calcium (Ca) absorption are different between breast-fed (BF) and formula-fed (FF) infants. Certain components and/or structural particularities in human milk (HM), can contribute to favorable outcomes in BF infants. In HM, the long-chain saturated fatty acid (LCSFA) palmitic acid has a different stereospecific distribution (sn-2 position) compared to most infant formula (IF) (primarily sn-1, 3 positions), which may contribute to unfavorable outcomes. Evidence suggests palmitic acid is important in the formation of stool FA-mineral (or FA-Ca) soaps, associated with harder stools in FF infants. Partial replacement by structured palmitic acid-rich triacylglycerols (TAGs) promotes palmitic acid absorption. However, evidence for stool softening, improved fat absorption and reduced Ca excretion in stools is inconsistent. IFs with less palmitic acid can improve fat and Ca absorption, and stool consistency. The presence of other LCSFAs (myristic and stearic acids) in sn-1, 3 positions may also contribute to reduced absorption of fat and Ca, and stool hardness, in FF infants. Nevertheless, little attention has been given to modifying these other LCSFAs in IF. We review literature comparing the effect of HM and IF with different lipid compositions on stool patterns and/or fat and Ca absorption in healthy, term infants. Based on available data, we estimate a maximum level for sn-1, 3 LCSFAs of 13% of TAGs, under which fat and Ca absorption and stool consistency are improved. IF designed according to this threshold could efficiently improve nutrient absorption and stool patterns in healthy infants who cannot be breast-fed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Expression Analysis of Previously Verified Fecal and Plasma Dow-regulated MicroRNAs (miR-4478, 1295-3p, 142-3p and 26a-5p), in FFPE Tissue Samples of CRC Patients.

PubMed

Ghanbari, Reza; Rezasoltani, Sama; Hashemi, Javad; Mohamadkhani, Ashraf; Tahmasebifar, Arash; Arefian, Ehsan; Mobarra, Naser; Asadi, Jahanbakhsh; Nazemalhosseini Mojarad, Ehsan; Yazdani, Yaghoub; Knuutila, Sakari; Malekzadeh, Reza

2017-02-01

Colorectal cancer (CRC) is one of the most common causes of cancer-related mortality worldwide. Early diagnosis of this neoplasm is critical and may reduce patients' mortality. MicroRNAs are small non-coding RNA molecules whose expression pattern can be altered in various diseases such as CRC. In this study, we evaluated the expression levels of miR-142-3p, miR-26a-5p (their reduced expression in plasma samples of CRC patients was previously confirmed), miR-4478 and miR-1295-3p (their reduced expression in stool samples of CRC patients was previously confirmed) in tissue samples of CRC patients in comparison to healthy subjects. To achieve this purpose, total RNA including small RNA was extracted from 53 CRC and 35 normal subjects' Formalin-fixed, Paraffin-embedded (FFPE) tissue samples using the miRNeasy FFPE Mini Kit. The expression levels of these four selected miRNAs were measured using quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR). We found that the expression levels of miR-4478 and miR-1295b-3p (two previously down-regulated fecal miRNAs) were significantly decreased in FFPE samples of CRC patients compared to healthy controls. On the other hand, no significant differences were seen in expression levels of miR-142-3p and miR-26a-5p (two previously down-regulated circulating miRNAs) in FFPE samples between these two groups. Regarding current findings, it may be concluded that to diagnose CRC patients based on the miRNAs approach, stool samples are more likely preferable to plasma samples; nevertheless, additional studies with more samples are needed to confirm the results.

Production of Monoclonal Antibody Against Excretory-Secretory Antigen of Fasciola hepatica and Evaluation of Its Efficacy in the Diagnosis of Fascioliasis.

PubMed

Abdolahi Khabisi, Samaneh; Sarkari, Bahador; Moshfe, Abdolali; Jalali, Sedigheh

2017-02-01

Parasitological methods are not helpful for the diagnosis of fascioliasis in acute and invasive periods of the disease. Detection of coproantigens seems to be a suitable alternative approach in the diagnosis of fascioliasis. The present study aimed to develop a reliable antigen detection system, using monoclonal antibodies raised against excretory-secretory (ES) antigen of Fasciola hepatica, for the diagnosis of fascioliasis. Fasciola adult worms were collected from the bile ducts of infected animals. Species of the fluke was determined by polymerase chain reaction-restriction fragment length polymorphism (RFLP-PCR). ES antigen of F. hepatica was prepared. For production of monoclonal antibodies, mice were immunized with ES antigens of F. hepatica. Spleen cells from the immunized mice were fused with NS-1 myeloma cells, using polyethylene glycol. Hybridoma cells secreting specific antibody were expanded and cloned by limiting dilution. Moreover, polyclonal antibody was produced against F. hepatica ES antigen in rabbits. A capture enzyme-linked immunosorbent assay (ELISA) system, using produced monoclonal antibody, was designed and stool samples of infected animals along with control samples were tested by the system. The capture ELISA detected the coproantigen in 27 of 30 (90%) parasitologically confirmed fascioliasis cases, while 4 of 39 (10.25%) samples infected with other parasitic infections showed a positive reaction in this system. No positive reactivity was found with healthy control samples. Accordingly, sensitivity of 90% and specificity of 94.2% were obtained for the capture ELISA system. The results were compared with those obtained with commercial BIO-X ELISA, and a very good (kappa = 0.9) agreement was found between the commercial kit and the developed capture ELISA. Findings of this study showed that the produced monoclonal antibody has appropriate performance for the detection of Fasciola coproantigen in stool samples and can be appropriately used for the diagnosis of fascioliasis.

Maintenance of Pain in Children with Functional Abdominal Pain

PubMed Central

Czyzewski, Danita I.; Self, Mariella M.; Williams, Amy E.; Weidler, Erica M.; Blatz, Allison M.; Shulman, Robert J.

2015-01-01

Objectives A significant proportion of children with functional abdominal pain develop chronic pain. Identifying clinical characteristics predicting pain persistence is important in targeting interventions. We examined whether child anxiety and/or pain-stooling relations were related to maintenance of abdominal pain frequency and compared the predictive value of three methods for assessing pain-stooling relations (i.e., diary, parent report, child report). Methods Seventy-six children (7–10-years-old at baseline) who presented for medical treatment of functional abdominal pain were followed up 18–24 months later. Baseline anxiety and abdominal pain-stooling relations based on pain and stooling diaries and child- and parent-questionnaires were examined in relationship to the persistence of abdominal pain frequency. Results Children’s baseline anxiety was not related to persistence of pain frequency. However, children who displayed irritable bowel syndrome (IBS) symptoms at baseline maintained pain frequency at follow-up, whereas in children in whom there was no relationship between pain and stooling, pain frequency decreased. Pain and stool diaries and parent report of pain-stooling relations were predictive of pain persistence but child-report questionnaires were not. Conclusions The presence of IBS symptoms in school age children with functional abdominal pain appears to predict persistence of abdominal pain over time, while anxiety does not. Prospective pain and stooling diaries and parent report of IBS symptoms were predictors of pain maintenance, but child report of symptoms was not. PMID:26301615

Stool microbiome and metabolome differences between colorectal cancer patients and healthy adults

USDA-ARS?s Scientific Manuscript database

In this study we used stool profiling to identify intestinal bacteria and metabolites that are differentially represented in humans with colorectal cancer (CRC) compared to healthy controls to identify how microbial functions may influence CRC development. Stool samples were collected from healthy a...

Fetal exposures and perinatal influences on the stool microbiota of premature infants.

PubMed

Chernikova, Diana A; Koestler, Devin C; Hoen, Anne Gatewood; Housman, Molly L; Hibberd, Patricia L; Moore, Jason H; Morrison, Hilary G; Sogin, Mitchell L; Zain-Ul-Abideen, Muhammad; Madan, Juliette C

2016-01-01

To test the hypothesis that maternal complications significantly affect gut colonization patterns in very low birth weight infants. Forty-nine serial stool samples were obtained weekly from nine extremely premature infants enrolled in a prospective longitudinal study. Sequencing of the bacterial 16S rRNA gene from stool samples was performed to approximate the intestinal microbiome. Linear mixed effects models were used to evaluate relationships between perinatal complications and intestinal microbiome development. Subjects with prenatal exposure to a non-sterile intrauterine environment, i.e. prolonged preterm premature rupture of membranes (PPPROM) and chorioamnionitis exposure, were found to have a relatively higher abundance of potentially pathogenic bacteria in the stool across all time points compared to subjects without those exposures, irrespective of exposure to postnatal antibiotics. Compared with those delivered by Caesarean section, vaginally delivered subjects were found to have significantly lower diversity of stool microbiota across all time points, with lower abundance of many genera, most in the family Enterobacteriaceae. We identified persistently increased potential pathogen abundance in the developing stool microbiota of subjects exposed to a non-sterile uterine environment. Maternal complications appear to significantly influence the diversity and bacterial composition of the stool microbiota of premature infants, with findings persisting over time.

Comparison of ELISA and Microscopy for detection of Cryptosporidium in stool

PubMed Central

Sharma, Madhu; Chaudhary, Uma; Yadav, Aparna

2014-01-01

Background: Cryptosporidiosis, a diarrheal disease caused by the protozoan parasite Cryptosporidium spp. has become recognized as one of the most common causes of water borne diseases in humans. Aims and Objectives: To compare the sensitivity of ELISA and Microscopy for detection of Cryptosporidium in stool samples Materials and Methods: The study was conducted in the Department of Microbiology of PT. B.D. Sharma PGIMS Rohtak, between January 2011 to june 2011 on 50 stool samples, which were processed for detection of cryptosporidial antigen by ELISA and detection of cysts by microscopy (Modified Ziehl and Nelsen staining). Study and Design: This was a prospective study conducted in the Department of Microbiology in PT. BD Sharma, PGIMS, Rohtak, India. Result: Out of total, 50 stool samples eighteen (36%) samples were found positive for Cryptosporidium cysts by microscopy in comparison to 3(6%) stool samples which were found positive for cryptosporidial antigen by ELISA. Samples found positive with ELISA were also positive with microscopy. Sensitivity, specificity, positive predictive value and negative predictive value for ELISA was 16.7%, 100%, 100% and 68% respectively. Conclusion: The study concludes that stool microscopic Modified acid fast staining is more sensitive method than ELISA for detection of Cryptosporidium in stool samples but the specificity of ELISA was more than microscopy. PMID:25584216

Stool frequency recording in severe acute malnutrition ('StoolSAM'); an agreement study comparing maternal recall versus direct observation using diapers.

PubMed

Voskuijl, Wieger; Potani, Isabel; Bandsma, Robert; Baan, Anne; White, Sarah; Bourdon, Celine; Kerac, Marko

2017-06-07

Approximately 50% of the deaths of children under the age of 5 can be attributed to undernutrition, which also encompasses severe acute malnutrition (SAM). Diarrhoea is strongly associated with these deaths and is commonly diagnosed solely based on stool frequency and consistency obtained through maternal recall. This trial aims to determine whether this approach is equivalent to a 'directly observed method' in which a health care worker directly observed stool frequency using diapers in hospitalised children with complicated SAM. This study was conducted at 'Moyo' Nutritional Rehabilitation Unit, Queen Elizabeth Central Hospital, Malawi. Participants were children aged 5-59 months admitted with SAM. We compared 2 days of stool frequency data obtained with next-day maternal-recall versus a 'gold standard' in which a health care worker observed stool frequency every 2 h using diapers. After study completion, guardians were asked their preferred method and their level of education. We found poor agreement between maternal recall and the 'gold standard' of directly observed diapers. The sensitivity to detect diarrhoea based on maternal recall was poor, with only 75 and 56% of diarrhoea cases identified on days 1 and 2, respectively. However, the specificity was higher with more than 80% of children correctly classified as not having diarrhoea. On day 1, the mean stool frequency difference between the two methods was -0.17 (SD; 1.68) with limits of agreement (of stool frequency) of -3.55 and 3.20 and, similarly on day 2, the mean difference was -0.2 (SD; 1.59) with limits of agreement of -3.38 and 2.98. These limits extend beyond the pre-specified 'acceptable' limits of agreement (±1.5 stool per day) and indicate that the 2 methods are non-equivalent. The higher the stool frequency, the more discrepant the two methods were. Most primary care givers strongly preferred using diapers. This study shows lack of agreement between the assessment of stool frequency in SAM patients using maternal recall and direct observation of diapers. When designing studies, one should consider using diapers to determining diarrhoea incidence/prevalence in SAM patients especially when accuracy is essential. ISRCTN11571116 (registered 29/11/2013).

Simultaneous identification of DNA and RNA viruses present in pig faeces using process-controlled deep sequencing.

PubMed

Sachsenröder, Jana; Twardziok, Sven; Hammerl, Jens A; Janczyk, Pawel; Wrede, Paul; Hertwig, Stefan; Johne, Reimar

2012-01-01

Animal faeces comprise a community of many different microorganisms including bacteria and viruses. Only scarce information is available about the diversity of viruses present in the faeces of pigs. Here we describe a protocol, which was optimized for the purification of the total fraction of viral particles from pig faeces. The genomes of the purified DNA and RNA viruses were simultaneously amplified by PCR and subjected to deep sequencing followed by bioinformatic analyses. The efficiency of the method was monitored using a process control consisting of three bacteriophages (T4, M13 and MS2) with different morphology and genome types. Defined amounts of the bacteriophages were added to the sample and their abundance was assessed by quantitative PCR during the preparation procedure. The procedure was applied to a pooled faecal sample of five pigs. From this sample, 69,613 sequence reads were generated. All of the added bacteriophages were identified by sequence analysis of the reads. In total, 7.7% of the reads showed significant sequence identities with published viral sequences. They mainly originated from bacteriophages (73.9%) and mammalian viruses (23.9%); 0.8% of the sequences showed identities to plant viruses. The most abundant detected porcine viruses were kobuvirus, rotavirus C, astrovirus, enterovirus B, sapovirus and picobirnavirus. In addition, sequences with identities to the chimpanzee stool-associated circular ssDNA virus were identified. Whole genome analysis indicates that this virus, tentatively designated as pig stool-associated circular ssDNA virus (PigSCV), represents a novel pig virus. The established protocol enables the simultaneous detection of DNA and RNA viruses in pig faeces including the identification of so far unknown viruses. It may be applied in studies investigating aetiology, epidemiology and ecology of diseases. The implemented process control serves as quality control, ensures comparability of the method and may be used for further method optimization.

Fecal weight, colon cancer risk, and dietary intake of nonstarch polysaccharides (dietary fiber)

PubMed

Cummings, J H; Bingham, S A; Heaton, K W; Eastwood, M A

1992-12-01

Low fecal weight and slow bowel transit time are thought to be associated with bowel cancer risk, but few published data defining bowel habits in different communities exist. Therefore, data on stool weight were collected from 20 populations in 12 countries to define this risk more accurately, and the relationship between stool weight and dietary intake of nonstarch polysaccharides (NSP) (dietary fiber) was quantified. In 220 healthy U.K. adults undertaking careful fecal collections, median daily stool weight was 106 g/day (men, 104 g/day; women, 99 g/day; P = 0.02) and whole-gut transit time was 60 hours (men, 55 hours; women, 72 hours; P = 0.05); 17% of women, but only 1% of men, passed < 50 g stool/day. Data from other populations of the world show average stool weight to vary from 72 to 470 g/day and to be inversely related to colon cancer risk (r = -0.78). Meta-analysis of 11 studies in which daily fecal weight was measured accurately in 26 groups of people (n = 206) on controlled diets of known NSP content shows a significant correlation between fiber intake and mean daily stool weight (r = 0.84). Stool weight in many Westernized populations is low (80-120 g/day), and this is associated with increased colon cancer risk. Fecal output is increased by dietary NSP. Diets characterized by high NSP intake (approximately 18 g/day) are associated with stool weights of 150 g/day and should reduce the risk of bowel cancer.

Culturing Stool Specimens for Campylobacter spp., Pennsylvania, USA

PubMed Central

M’ikanatha, Nkuchia M.; Dettinger, Lisa A.; Perry, Amanda; Rogers, Paul; Reynolds, Stanley M.

2012-01-01

In 2010, we surveyed 176 clinical laboratories in Pennsylvania regarding stool specimen testing practices for enteropathogens, including Campylobacter spp. Most (96.3%) routinely test for Campylobacter spp. In 17 (15.7%), a stool antigen test is the sole method for diagnosis. We recommend that laboratory practice guidelines for Campylobacter spp. testing be developed. PMID:22377086

Prevalence of Clostridium perfringens toxin in patients suspected of having antibiotic-associated diarrhea.

PubMed

Kim, Young Jin; Kim, Si Hyun; Ahn, Junggu; Cho, Soongmoon; Kim, Dongchun; Kim, Kwanghyun; Lee, Heegun; Son, Hyunwoo; Lee, Hee Joo; Yong, Dongeun; Choi, Jun Yong; Kim, Hye Ran; Shin, Jeong Hwan

2017-12-01

Although Clostridium perfringens has been reported as a cause of antibiotic-associated diarrhea (AAD), it is uncommon to detect this pathogen in clinical microbiology laboratories in Korea. The aim of this study was to investigate the prevalence of C. perfringens toxin in patients suspected of having AAD. A total of 135 stool specimens submitted to a clinical microbiology laboratory for C. difficile toxin assay were tested. We tried to detect both C. difficile and C. perfringens toxins using the Seeplex Diarrhea ACE Detection kit (Seegene, Seoul, Korea). We evaluated the prevalence of 10 bacteria and 5 viruses. A total of 40 Clostridium spp. were detected in 34 specimens (29.6%). The C. perfringens toxin was detected in 14 of 135 specimens (10.4%), while C. difficile toxin was detected in 26 specimens (19.3%). Other bacteria and viruses, including 8 Aeromonas spp., were detected in 15 specimens. All tests were negative in 92 of the 135 specimens (68.1%). Clostridium perfringens toxin is relatively common, and we should consider the possibility of its presence in patients suspected of having AAD, especially if C. difficile tests are negative. Copyright © 2017 Elsevier Ltd. All rights reserved.

I. POLIOMYELITIC VIRUS IN HUMAN STOOLS.

PubMed

Trask, J D; Paul, J R; Vignec, A J

1940-05-31

1. The detection of the virus of poliomyelitis in 10 stools from 8 individuals is reported. All were in relation to epidemic poliomyelitis and 7 of them represented well recognized forms of the disease. The positive stools were distributed among 56 specimens collected from 53 persons in the first 4 weeks of illness. 2. The ease of detection of virus was directly related to the non-paralytic type of disease and inversely related to the age of the patients. 3. The negative results with stools employed for controls gives point to the use of the fecal examinations as an epidemiological tool. 4. The stability of the virus in feces has been demonstrated by successful mailing of samples over long distances and during the heat of summer. 5. At least one infective dose per gram of fecal material was extracted from one stool.

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

PubMed Central

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 applied for effective diagnosis of strongyloidiasis in a clinical setting. PMID:27415764

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.

PubMed

Fernández-Soto, Pedro; Sánchez-Hernández, Alicia; Gandasegui, Javier; Bajo Santos, Cristina; López-Abán, Julio; Saugar, José María; Rodríguez, Esperanza; Vicente, Belén; Muro, Antonio

2016-07-01

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. 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. 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 applied for effective diagnosis of strongyloidiasis in a clinical setting.

The role of gut microbiota in fetal methylmercury exposure: Insights from a pilot study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rothenberg, Sarah E.; Keiser, Sharon; Ajami, Nadim J.

The mechanisms by which gut microbiota contribute to methylmercury metabolism remain unclear. Among a cohort of pregnant mothers, the main objectives of our pilot study were to determine 1) associations between gut microbiota and mercury concentrations in biomarkers (stool, hair and cord blood) and 2) the contributions of gut microbial mercury methylation/demethylation to stool methylmercury. Moreover, for pregnant women (36-39 weeks gestation, n=17) donated hair and stool specimens, and cord blood was collected for a subset (n=7). The diversity of gut microbiota was determined using 16S rRNA gene profiling (n=17). For 6 stool samples with highest/lowest methylmercury concentrations, metagenomic wholemore » genome shotgun sequencing was employed to search for one mercury methylation gene (hgcA), and two mer operon genes involved in methylmercury detoxification (merA and merB). There were seventeen bacterial genera that were significantly correlated (increasing or decreasing) with stool methylmercury, stool inorganic mercury, or hair total mercury; however, aside from one genus, there was no overlap between biomarkers. No definitive matches for hgcA or merB, while merA were detected at low concentrations in all six samples. Proportional differences in stool methylmercury were not likely attributed to gut microbiota through methylation/demethylation. Gut microbiota potentially altered methylmercury metabolism using indirect pathways.« less

The role of gut microbiota in fetal methylmercury exposure: Insights from a pilot study

DOE PAGES

Rothenberg, Sarah E.; Keiser, Sharon; Ajami, Nadim J.; ...

2016-02-01

The mechanisms by which gut microbiota contribute to methylmercury metabolism remain unclear. Among a cohort of pregnant mothers, the main objectives of our pilot study were to determine 1) associations between gut microbiota and mercury concentrations in biomarkers (stool, hair and cord blood) and 2) the contributions of gut microbial mercury methylation/demethylation to stool methylmercury. Moreover, for pregnant women (36-39 weeks gestation, n=17) donated hair and stool specimens, and cord blood was collected for a subset (n=7). The diversity of gut microbiota was determined using 16S rRNA gene profiling (n=17). For 6 stool samples with highest/lowest methylmercury concentrations, metagenomic wholemore » genome shotgun sequencing was employed to search for one mercury methylation gene (hgcA), and two mer operon genes involved in methylmercury detoxification (merA and merB). There were seventeen bacterial genera that were significantly correlated (increasing or decreasing) with stool methylmercury, stool inorganic mercury, or hair total mercury; however, aside from one genus, there was no overlap between biomarkers. No definitive matches for hgcA or merB, while merA were detected at low concentrations in all six samples. Proportional differences in stool methylmercury were not likely attributed to gut microbiota through methylation/demethylation. Gut microbiota potentially altered methylmercury metabolism using indirect pathways.« less

Comparison of routine and unprepped CT colonography augmented by low fiber diet and stool tagging: a pilot study.

PubMed

Dachman, Abraham H; Dawson, Damien O; Lefere, Philippe; Yoshida, Hiro; Khan, Nasreen U; Cipriani, Nicole; Rubin, David T

2007-01-01

We performed a pilot study examining the feasibility of a new unprepped CT colonography (CTC) strategy: low fiber diet and tagging (unprepped) vs. low fiber diet, tagging and a magnesium citrate cleansing preparation (prepped). Prior reports of tagging were limited in that the residual stool was neither measured and stratified by size nor did prior reports subjectively evaluate the ease of interpretation by a reader experienced in interpreting CTC examinations. Prospective randomized to unprepped n = 14 and prepped n = 14. Colonic segments were subjectively evaluated for residual stool that would potentially interfere with interpretation. Scores were given in the following categories: percentage of residual stool that was touching or nearly touching mucosa, the largest piece of retained stool, effectiveness of tagging, height of residual fluid, degree of distention, ease of interpretation, and reading time. Ease of the CT read (scale where 4 = optimal read) averaged 1.3 for the unprepped group and 2.3 for the prepped group. The mean read time averaged 17.5 min for unprepped and 17.9 min for prepped. The degree of distention (scale where 4 = well distended) averaged 3.7 for unprepped and 3.6 for prepped. Supine and prone images combined, the unprepped group had 160 segments with stool; prepped group had 58 segments. The amount of stool covering the mucosa in all segments averaged 1.6 (33%-66% coverage) in the unprepped group and 0.35 (<33% mucosal coverage) in the prepped group. The mean size of the largest piece of stool was 33.67 mm for unprepped and 4.01 mm for prepped. Percentage of tagged stool was not significantly different between the groups (range of 94-98%). The height of residual fluid averaged 8.37 mm for unprepped and 13.4 mm for prepped. Three polyps in three patients were found during optical colonoscopy (OC) in the unprepped group (5, 6, and 10 mm), none of which were prospectively detected at CTC. Three polyps in three patients were detected during OC in the prepped group (5, 10, and 15 mm), two of which were prospectively detected at CTC. Two false-positive lesions were observed at CTC in one patient in the prepped group. There was more stool in the unprepped group and while this factor did not slow down the reading time, it made the examination subjectively harder to interpret and likely caused the three polyps in this group to be missed. We conclude that a truly unprepped strategy that leaves significant residual stool, even if well tagged, is not desirable.

Effects of Prebiotics and Synbiotics on Functional Constipation.

PubMed

Yu, Ting; Zheng, Yong-Ping; Tan, Jia-Cheng; Xiong, Wen-Jie; Wang, Yun; Lin, Lin

2017-03-01

The objective was to determine the effects of prebiotics and synbiotics on adults with functional constipation (FC). Medline, Embase and the Cochrane Library were searched for literature published up to February 2015. We selected randomized controlled trials (RCTs) that reported administration of prebiotics or synbiotics to adults with FC. The end points included stool frequency, stool consistency and other symptoms related to constipation. Mean differences (MD) or standard mean differences (SMD) were used for continuous outcomes and risk ratios for discontinuous outcomes using a random-effects model. The Cochrane Risk of Bias Tool was used to determine the quality of the trials. Funnel plots and Egger's test were used to analyze for publication bias. We included 5 RCTs involving 199 patients who were administered prebiotics and 8 RCTs involving 825 patients who were administered synbiotics. Prebiotics increased weekly stool frequency (MD: 1.01bowel movements/week, 95% CI: 0.04-1.99) and improved stool consistency (SMD: -0.59, 95% CI: -1.16 to -0.02). Subgroup analysis showed specific effects for galacto-oligosaccharides on stool frequency, consistency, ease of defecation and abdominal pain. Synbiotics significantly improved stool frequency (MD: 1.15bowel movements/week, 95% CI: 0.58-1.71), consistency (SMD: 0.63, 95% CI: 0.33-0.92) and reduced whole-gut transit time (MD: 13.52, 95% CI: -26.56 to -0.49) in patients with FC. Subgroup analysis showed specific effects for fructo-oligosaccharides and probiotic combinations on stool frequency, consistency, straining defecation and bloating. Galacto-oligosaccharides and synbiotics made up of fructo-oligosaccharides with probiotic combinations may improve stool frequency, consistency and some other symptoms related to constipation. Copyright © 2017 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.

Reliability of a new technique for the determination of vitamin B12 absorption in children: single stool sample test--a double isotope technique

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hjelt, K.

1986-03-01

The fractional vitamin B12 absorption (FAB12) was determined in 39 patients with various gastrointestinal diseases by a double-isotope technique, employing a single stool sample test (SSST), as well as a complete stool collection. The age of the patients ranged from 2.5 months to 16.2 years (mean 5.0 years). The test dose was administered orally and consisted of 0.5-4.5 micrograms of /sup 57/CoB12 (approximately 0.05 microCi), carmine powder, and 2 mg /sup 51/CrCl/sub 3/ (approximately 1.25 microCi) as the inabsorbable tracer. The wholebody radiation to a 1-year-old child averaged only 20 mrad. The stool and napkin was collected and homogenized bymore » addition of 300 ml chromium sulfuric acid. A 300-ml sample of the homogenized stool and napkin, as well as 300 ml chromium sulfuric acid (75% v/v) containing the standards, were counted in a broad-based well counter. The FAB12 determined by SSST employing the stool with the highest content of /sup 51/Cr (which corresponded to the most carmine-colored stool) correlated closely to the FAB12 based on complete stool collection (r = 0.98, n = 39, p less than 0.001). The reproducibility of FAB12 determined by SSST was assessed from double assays in 19 patients. For a mean value of 12%, the SD was 3%, which corresponded to a coefficient of variation (CV) of 25%. The excretion of /sup 57/Co and /sup 51/Cr in the urine was examined in six patients with moderate to severe mucosal damage and was found to be low.« less

Oral diosmectite reduces stool output and diarrhea duration in children with acute watery diarrhea.

PubMed

Dupont, Christophe; Foo, Jimmy Lee Kok; Garnier, Philippe; Moore, Nicholas; Mathiex-Fortunet, Hèlène; Salazar-Lindo, Eduardo

2009-04-01

Diosmectite is a clay used to treat children with acute watery diarrhea. However, its effects on stool output reduction, the key outcome for pediatric antidiarrheal drugs, have not been shown. Two parallel, double-blind studies of diosmectite efficacy on stool reduction were conducted in children 1 to 36 months old in Peru (n = 300) and Malaysia (n = 302). Inclusion criteria included 3 or more watery stools per day for less than 72 hours and weight/height ratios of 0.8 or greater. Exclusion criteria were the need for intravenous rehydration, gross blood in stools, fever higher than 39 degrees C, or current treatment with antidiarrheal or antibiotic medications. Rotavirus status was determined. Diosmectite dosage was 6 g/day (children 1-12 months old) or 12 g/day (children 13-36 months old), given for at least 3 days, followed by half doses until complete recovery. Patients were assigned randomly to groups given diosmectite or placebo, in addition to oral rehydration solution (World Health Organization). Children in each study had comparable average ages and weights. The frequencies of rotavirus infection were 22% in Peru and 12% in Malaysia. Similar amounts of oral rehydration solution were given to children in the diosmectite and placebo groups. Stool output was decreased significantly by diosmectite in both studies, especially among rotavirus-positive children. In pooled data, children had a mean stool output of 94.5 +/- 74.4 g/kg of body weight in the diosmectite group versus 104.1 +/- 94.2 g/kg in the placebo group (P = .002). Diarrhea duration was reduced by diosmectite, which was well tolerated. These results show that diosmectite significantly decreased stool output in children with acute watery diarrhea, especially those who were rotavirus-positive.

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

PubMed Central

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

2017-01-01

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. PMID:28643618

Rectal and Naris Swabs: Practical and Informative Samples for Analyzing the Microbiota of Critically Ill Patients.

PubMed

Bansal, Saumya; Nguyen, Jenny P; Leligdowicz, Aleksandra; Zhang, Yu; Kain, Kevin C; Ricciuto, Daniel R; Coburn, Bryan

2018-06-27

Commensal microbiota are immunomodulatory, and their pathological perturbation can affect the risk and outcomes of infectious and inflammatory diseases. Consequently, the human microbiota is an emerging diagnostic and therapeutic target in critical illness. In this study, we compared four sample types-rectal, naris, and antecubital swabs and stool samples-for 16S rRNA gene microbiota sequencing in intensive care unit (ICU) patients. Stool samples were obtained in only 31% of daily attempts, while swabs were reliably obtained (≥97% of attempts). Swabs were compositionally distinct by anatomical site, and rectal swabs identified within-patient temporal trends in microbiota composition. Rectal swabs from ICU patients demonstrated differences from healthy stool similar to those observed in comparing stool samples from ICU patients to those from the same healthy controls. Rectal swabs are a useful complement to other sample types for analysis of the intestinal microbiota in critical illness, particularly when obtaining stool may not be feasible or practical. IMPORTANCE Perturbation of the microbiome has been correlated with various infectious and inflammatory diseases and is common in critically ill patients. Stool is typically used to sample the microbiota in human observational studies; however, it is often unavailable for collection from critically ill patients, reducing its utility as a sample type to study this population. Our research identified alternatives to stool for sampling the microbiota during critical illness. Rectal and naris swabs were practical alternatives for use in these patients, as they were observed to be more reliably obtained than stool, were suitable for culture-independent analysis, and successfully captured within- and between-patient microbiota differences. Copyright © 2018 Bansal et al.

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

PubMed

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.

Novel methylation panel for the early detection of colorectal tumors in stool DNA.

PubMed

Azuara, Daniel; Rodriguez-Moranta, Francisco; de Oca, Javier; Soriano-Izquierdo, Antonio; Mora, Josefina; Guardiola, Jordi; Biondo, Sebastiano; Blanco, Ignacio; Peinado, Miguel Angel; Moreno, Victor; Esteller, Manel; Capellá, Gabriel

2010-07-01

Previous studies showed that the assessment of promoter hypermethylation of a limited number of genes in tumor biopsies may identify the majority of colorectal tumors. This study aimed to assess the clinical usefulness of a panel of methylation biomarkers in stool DNA in the identification of colorectal tumors, using methylation-specific melting curve analysis (MS-MCA), a technique that simultaneously analyzes all cytosine-phosphate-guanine (CpG) residues within a promoter. The promoter methylation status of 4 tumor-related genes (RARB2, p16INK4a, MGMT, and APC) was analyzed in DNA stool samples and corresponding tissues in an initial set of 12 patients with newly diagnosed primary colorectal carcinomas and 20 patients with newly diagnosed colorectal adenomas, using methylation-specific polymerase chain reaction. Results were replicated in a set of 82 patients (20 healthy subjects, 16 patients with inflammatory bowel disease (IBD), 20 patients with adenomas, and 26 patients with carcinomas), using MS-MCA analyses. In the initial set, >or= 1 positive methylation marker was detected in the stools of 9 of 12 patients (75%) with carcinomas and 12 of 20 patients (60%) with adenomas, with no false-positive results. Stool analyses missed 7 methylated lesions (25%). In the replication set, stool DNA testing detected 16 of 26 carcinomas (62%) and 8 of 20 adenomas (40%). The MS-MCAs missed 14 methylated tumors (37%). No aberrant methylation was evident in healthy subjects, but the RARB2 marker was positive in 2 of 15 stool samples (13%) of patients with IBD. Analysis via MS-MCA of a panel of methylation markers in stool DNA may offer a good alternative in the early, noninvasive detection of colorectal tumors.

Production and Characterization of Monoclonal Antibody against Recombinant Virus Coat Protein CP42.

PubMed

Shibaei, Naeimeh; Majidi, Jafar; Razavi, Khadijeh; Karkhane, Ali Asghar; Sokhandan-Bashir, Nemat; Aghebati-Maleki, Leili

2017-02-01

There are many studies related to the production of a ELISA kit for diagnosing virus infections. However, production of most kits depends on purification of whole virus particles, which involves the use of costly equipment and reagents. The purpose of this study was to check out if the anti-CP42 antibodies could be used as a diagnostic assay for detection of Grapevine fanleaf Virus (GFLV). In this study, recombinant GFLV coat protein gene related to selected antigenic determinants was inserted into pET-28a bacterial expression vector and the construct (pET-28a CP42) was cloned into E. coli strain (DE3). Expressed protein was verified with western blotting assay by the use of commercially available anti-GFLV antibody. The recombinant protein was purified using nickel-nitrilotriacetic acid (Ni-NTA) resin. Balb/c mice were immunized with purified protein and splenocytes of hyperimmunized mice were fused with murine myeloma Sp2/0 cells. Positive hybridomas were selected by ELISA using CP42 as coating antigen. The results showed that monoclonal antibody (MAb) specific to CP42 has been successfully generated. Efficiency of produced antibody was analyzed by ELISA and western blotting assay using some confirmed grapevine samples. The infection was confirmed previously based on morphological features and ELISA assay, performed using commercial anti-GFLV antibody. The monoclonal antibody reacted with antigen in ELISA and immunoblot method. Our results demonstrated that anti recombinant CP42 monoclonal antibodies are able to diagnose whole virus in infected grapevine sample using ELISA test.

Recombinase polymerase amplification-based assay to diagnose Giardia in stool samples.

PubMed

Crannell, Zachary Austin; Cabada, Miguel Mauricio; Castellanos-Gonzalez, Alejandro; Irani, Ayesha; White, Arthur Clinton; Richards-Kortum, Rebecca

2015-03-01

Giardia duodenalis is one of the most commonly identified parasites in stool samples. Although relatively easy to treat, giardiasis can be difficult to detect as it presents similar to other diarrheal diseases. Here, we present a recombinase polymerase amplification-based Giardia (RPAG) assay to detect the presence of Giardia in stool samples. The RPAG assay was characterized on the bench top using stool samples spiked with Giardia cysts where it showed a limit-of-detection nearly as low as the gold standard polymerase chain reaction assay. The RPAG assay was then tested in the highlands of Peru on 104 stool samples collected from the surrounding communities where it showed 73% sensitivity and 95% specificity against a polymerase chain reaction and microscopy composite gold standard. Further improvements in clinical sensitivity will be needed for the RPAG assay to have clinical relevance. © The American Society of Tropical Medicine and Hygiene.

Recombinase Polymerase Amplification-Based Assay to Diagnose Giardia in Stool Samples

PubMed Central

Crannell, Zachary Austin; Cabada, Miguel Mauricio; Castellanos-Gonzalez, Alejandro; Irani, Ayesha; White, Arthur Clinton; Richards-Kortum, Rebecca

2015-01-01

Giardia duodenalis is one of the most commonly identified parasites in stool samples. Although relatively easy to treat, giardiasis can be difficult to detect as it presents similar to other diarrheal diseases. Here, we present a recombinase polymerase amplification-based Giardia (RPAG) assay to detect the presence of Giardia in stool samples. The RPAG assay was characterized on the bench top using stool samples spiked with Giardia cysts where it showed a limit-of-detection nearly as low as the gold standard polymerase chain reaction assay. The RPAG assay was then tested in the highlands of Peru on 104 stool samples collected from the surrounding communities where it showed 73% sensitivity and 95% specificity against a polymerase chain reaction and microscopy composite gold standard. Further improvements in clinical sensitivity will be needed for the RPAG assay to have clinical relevance. PMID:25510713

In Vivo Test for Chemical Induction of Micronucleated Polychromatic Erythrocytes in Mouse Bone Marrow Cells. Test Article: Dimethylamine-2-2ethyl azide (DMAZ)

DTIC Science & Technology

2008-06-25

Hunched Back Unusual Body Secretions - Nasal Discharge - Lacrimation - Salivation - Bloody Stool - Dianhea Abnormal Behavior - Convulsions...WetGroin - Hunched Back Unusual Body Secretions - Nasal Discharge - Lacrimation - Salivation - Bloody Stool - Diarrhea Abnormal Behavior...Ataxia - Piloerection - WetGrain - Hunched Back Unusual Body Secretions - Nasal Discharge - Lacrimation - Salivation - Bloody Stool

Exocrine and endocrine pancreatic function in 21 patients suffering from autoimmune pancreatitis before and after steroid treatment.

PubMed

Frulloni, Luca; Scattolini, Chiara; Katsotourchi, Anna Maria; Amodio, Antonio; Gabbrielli, Armando; Zamboni, Giuseppe; Benini, Luigi; Vantini, Italo

2010-01-01

Autoimmune pancreatitis (AIP) responds rapidly and dramatically to steroid therapy. The aim of this study was to evaluate pancreatic exocrine and endocrine function in patients suffering from AIP both before and after steroid therapy. Fecal elastase 1 and diabetes were evaluated before steroid therapy and within 1 month of its suspension in 21 patients (13 males and 8 females, mean age 43 +/- 16.5 years) diagnosed as having AIP between 2006 and 2008. At clinical onset, fecal elastase 1 was 107 +/- 126 microg/g stool. Thirteen patients (62%) showed severe pancreatic insufficiency (<100 microg/g stool), 4 (19%) had mild insufficiency (100-200 microg/g stool), while 4 (19%) had normal pancreatic function (>200 microg/g stool). Before steroids, diabetes was diagnosed in 5 patients (24%), all of whom had very low levels of fecal elastase 1 (<19 microg/g stool). Following steroids, fecal elastase 1 increased in all patients (237 +/- 193 microg/g stool) and observed levels were significantly higher than those seen before steroids (p = 0.001). Patients suffering from AIP display exocrine and/or endocrine pancreatic insufficiency at clinical onset. These insufficiencies improve after steroid therapy. Copyright 2010 S. Karger AG, Basel.

Development of a polymerase chain reaction applicable to rapid and sensitive detection of Clonorchis sinensis eggs in human stool samples

PubMed Central

Cho, Pyo Yun; Na, Byoung-Kuk; Mi Choi, Kyung; Kim, Jin Su; Cho, Shin-Hyeong; Lee, Won-Ja; Lim, Sung-Bin; Cha, Seok Ho; Park, Yun-Kyu; Pak, Jhang Ho; Lee, Hyeong-Woo; Hong, Sung-Jong; Kim, Tong-Soo

2013-01-01

Microscopic examination of eggs of parasitic helminths in stool samples has been the most widely used classical diagnostic method for infections, but tiny and low numbers of eggs in stool samples often hamper diagnosis of helminthic infections with classical microscopic examination. Moreover, it is also difficult to differentiate parasite eggs by the classical method, if they have similar morphological characteristics. In this study, we developed a rapid and sensitive polymerase chain reaction (PCR)-based molecular diagnostic method for detection of Clonorchis sinensis eggs in stool samples. Nine primers were designed based on the long-terminal repeat (LTR) of C. sinensis retrotransposon1 (CsRn1) gene, and seven PCR primer sets were paired. Polymerase chain reaction with each primer pair produced specific amplicons for C. sinensis, but not for other trematodes including Metagonimus yokogawai and Paragonimus westermani. Particularly, three primer sets were able to detect 10 C. sinensis eggs and were applicable to amplify specific amplicons from DNA samples purified from stool of C. sinensis-infected patients. This PCR method could be useful for diagnosis of C. sinensis infections in human stool samples with a high level of specificity and sensitivity. PMID:23916334

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

PubMed

Alberer, Martin; Schlenker, Nicklas; Bauer, Malkin; Helfrich, Kerstin; Mengele, Carolin; Löscher, Thomas; Nothdurft, Hans Dieter; Bretzel, Gisela; Beissner, Marcus

2017-01-01

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.

RAPD analysis of the genetic diversity of mango (Mangifera indica) germplasm in Brazil.

PubMed

Souza, I G B; Valente, S E S; Britto, F B; de Souza, V A B; Lima, P S C

2011-12-14

We evaluated genetic variability of mango (Mangifera indica) accessions maintained in the Active Germplasm Bank of Embrapa Meio-Norte in Teresina, Piauí, Brazil, using RAPDs. Among these accessions, 35 originated from plantings in Brazil, six from the USA and one from India. Genomic DNA, extracted from leaf material using a commercial purification kit, was subjected to PCR with the primers A01, A09, G03, G10, N05, and M16. Fifty-five polymorphic loci were identified, with mean of 9.16 ± 3.31 bands per primer and 100% polymorphism. Application of unweighted pair group method using arithmetic average cluster analysis demonstrated five genotypic groups among the accessions examined. The genotypes Rosa 41, Rosa 48 and Rosa 49 were highly similar (94% similarity), whereas genotypes Sensation and Rosa 18 were the most divergent (only 7% similarity). The mango accessions were found to have considerable genetic variability, demonstrating the importance of analyzing each genotype in a collection in order to efficiently maintain the germplasm collection.

Detection of porcine DNA in gelatine and gelatine-containing processed food products-Halal/Kosher authentication.

PubMed

Demirhan, Yasemin; Ulca, Pelin; Senyuva, Hamide Z

2012-03-01

A commercially available real-time PCR, based on a multi-copy target cytochrome b (cyt b) using porcine specific primers, has been validated for the Halal/Kosher authentication of gelatine. Extraction and purification of DNA from gelatine were successfully achieved using the SureFood® PREP Animal system, and real-time PCR was carried out using SureFood® Animal ID Pork Sens kit. The minimum level of adulteration that could be detected was 1.0% w/w for marshmallows and gum drops. A small survey was undertaken of processed food products such as gum drops, marshmallows and Turkish delight, believed to contain gelatine. Of fourteen food products from Germany, two samples were found to contain porcine gelatine, whereas of twenty-nine samples from Turkey twenty-eight were negative. However, one product from Turkey contained porcine DNA and thus was not Halal, and neither was the use of porcine gelatine indicated on the product label. Copyright © 2011 Elsevier Ltd. All rights reserved.

Mass spectrometric detection of siRNA in plasma samples for doping control purposes.

PubMed

Kohler, Maxie; Thomas, Andreas; Walpurgis, Katja; Schänzer, Wilhelm; Thevis, Mario

2010-10-01

Small interfering ribonucleic acid (siRNA) molecules can effect the expression of any gene by inducing the degradation of mRNA. Therefore, these molecules can be of interest for illicit performance enhancement in sports by affecting different metabolic pathways. An example of an efficient performance-enhancing gene knockdown is the myostatin gene that regulates muscle growth. This study was carried out to provide a tool for the mass spectrometric detection of modified and unmodified siRNA from plasma samples. The oligonucleotides are purified by centrifugal filtration and the use of an miRNA purification kit, followed by flow-injection analysis using an Exactive mass spectrometer to yield the accurate masses of the sense and antisense strands. Although chromatography and sensitive mass spectrometric analysis of oligonucleotides are still challenging, a method was developed and validated that has adequate sensitivity (limit of detection 0.25-1 nmol mL(-1)) and performance (precision 11-21%, recovery 23-67%) for typical antisense oligonucleotides currently used in clinical studies.

Pancreatic Reference Set Application- Ann Killary-MD Anderson (2012) — EDRN Public Portal

Cancer.gov

ELISA assays. We propose to screen a two gene panel (TNC/TFPI) from functional genomic pathways approaches, by ELISA assays, in a collection of blinded plasma samples with the PCCG. In subsequent years, we will add to this panel additional candidates identified in the parent EDRN grant that are in the migration signature network or the 20q amplicon. For ELISA assays, we will utilize commercially available kits. Plasma levels of the predominant isoform of TNC, TNC-large variant (TNC-L) will be determined using a Human Tenascin-C Large (HMV) (FNIII-B) ELISA kit (IBL-America, Minneapolis, MN), which detects human TNC high molecular weight variant by sandwich ELISA. Plasma TFPI levels will be determined using a commercially available Quantikine Human TFPI ELISA kit (DTFP10) (R&D; Systems, Inc. Minneapolis, MN), which detects predominantly free TFPI and a very small percentage of LDL and HDL-bound TFPI by sandwich ELISA. Additional ELISA assays will be added in years 2-3 using ingenuity network analysis to identify secreted proteins that interact with migration signature proteins identified using functional genomic approaches. In addition, Dr. Sen’s lab has identified candidate biomarkers mapping with the 20q amplicon and which are both amplified and overexpressed as well as are secreted proteins. In years 2-3, we will examine all candidate markers by ELISA to determine 20q pathway markers increase the sensitivity and specificity of the 3p panel. Validation of a 4 miR Panel. For assaying the levels of miRs in plasma, qRT-PCR assays will be performed, as described (9). For the selected panel of miRs, we propose to develop a high throughput qRT-PCR assay by adapting our published method to a 96 well format where the critical steps of purification, such as LiCl precipitation and DNAse treatments will be done in successive steps in the same plates. In years 2-3, additional miRs that target both the 20q amplicon genes as well as miRs that target migration genes in Dr. Killary’

Shared and Distinct Features of Human Milk and Infant Stool Viromes

PubMed Central

Pannaraj, Pia S.; Ly, Melissa; Cerini, Chiara; Saavedra, Monica; Aldrovandi, Grace M.; Saboory, Abdul A.; Johnson, Kevin M.; Pride, David T.

2018-01-01

Infants acquire many of their microbes from their mothers during the birth process. The acquisition of these microbes is believed to be critical in the development of the infant immune system. Bacteria also are transmitted to the infant through breastfeeding, and help to form the microbiome of the infant gastrointestinal (GI) tract; it is unknown whether viruses in human milk serve to establish an infant GI virome. We examined the virome contents of milk and infant stool in a cohort of mother-infant pairs to discern whether milk viruses colonize the infant GI tract. We observed greater viral alpha diversity in milk than in infant stool, similar to the trend we found for bacterial communities from both sites. When comparing beta diversity, viral communities were mostly distinguishable between milk and infant stool, but each was quite distinct from adult stool, urine, and salivary viromes. There were significant differences in viral families in the infant stool (abundant bacteriophages from the family Siphoviridae) compared to milk (abundant bacteriophages from the family Myoviridae), which may reflect significant differences in the bacterial families identified from both sites. Despite the differences in viral taxonomy, we identified a significant number of shared viruses in the milk and stool from all mother-infant pairs. Because of the significant proportion of bacteriophages transmitted in these mother-infant pairs, we believe the transmission of milk phages to the infant GI tract may help to shape the infant GI microbiome. PMID:29910789

Albendazole Stimulates the Excretion of Strongyloides stercoralis Larvae in Stool Specimens and Enhances Sensitivity for Diagnosis of Strongyloidiasis▿

PubMed Central

Anamnart, Witthaya; Pattanawongsa, Attarat; Intapan, Pewpan Maleewong; Maleewong, Wanchai

2010-01-01

We succeeded in stimulation of excretion of Strongyloides stercoralis larvae in stool by oral administration of a single dose of 400 mg albendazole to strongyloidiasis patients. This result overcame the false-negative results of stool examination due to low larval numbers. Stool samples were collected from 152 asymptomatic strongyloidiasis patients in the morning, prior to eating. After breakfast, they were given a dose of 400 mg albendazole, and stool samples were collected the following morning. Agar plate culture (APC), modified formalin-ether concentration technique (MFECT), and direct-smear (DS) methods were used to examine stool specimens within 3 h after defecation. The results before and after albendazole was taken were compared. All APCs that were positive became negative after albendazole administration, while MFECT showed a 1.4- to 18.0-fold increase in larval numbers in 97.4% (148/152) of the samples. The DSs were positive in 3 out of 3 smears at a larval number of ≥45 larvae per g (lpg) of stool, and in 1or 2 out of 3 smears at a larval number between 35 and 44 lpg. At a larval number of <35 lpg, the DS became negative. Interestingly 90.5% (19/21) of the samples that were negative by all methods before albendazole administration became positive by MFECT after the treatment. Thus, MFECT can be effectively used for diagnosis of strongyloidiasis with prior administration of albendazole to the subject. PMID:20844212

Use of Occult Blood Detection Cards for Real-Time PCR-Based Diagnosis of Schistosoma Mansoni Infection

PubMed Central

Schunk, Mirjam; Kebede Mekonnen, Seleshi; Wondafrash, Beyene; Mengele, Carolin; Fleischmann, Erna; Herbinger, Karl-Heinz; Verweij, Jaco J.; Geldmacher, Christof; Bretzel, Gisela; Löscher, Thomas; Zeynudin, Ahmed

2015-01-01

Background In Schistosoma mansoni infection, diagnosis and control after treatment mainly rely on parasitological stool investigations which are laborious and have limited sensitivity. PCR methods have shown equal or superior sensitivity but preservation and storage methods limit their use in the field. Therefore, the use of occult blood detection cards (fecal cards) for easy sampling and storage of fecal samples for further PCR testing was evaluated in a pilot study. Methodology Stool specimens were collected in a highly endemic area for S. mansoni in Ethiopia and submitted in an investigator-blinded fashion to microscopic examination by Kato-Katz thick smear as well as to real-time PCR using either fresh frozen stool samples or stool smears on fecal cards which have been stored at ambient temperature for up to ten months. Principal Findings Out of 55 stool samples, 35 were positive by microscopy, 33 and 32 were positive by PCR of frozen samples and of fecal card samples, respectively. When microscopy was used as diagnostic “gold standard”, the sensitivity of PCR on fresh stool was 94.3% (95%-CI: 86.6; 100) and on fecal cards 91.4% (95%-CI: 82.2; 100). Conclusions The use of fecal cards proved to be a simple and useful method for stool collection and prolonged storage prior to PCR based diagnosis of S. mansoni infection. This technique may be a valuable approach for large scale surveillance and post treatment assessments PMID:26360049

Use of Occult Blood Detection Cards for Real-Time PCR-Based Diagnosis of Schistosoma Mansoni Infection.

PubMed

Schunk, Mirjam; Kebede Mekonnen, Seleshi; Wondafrash, Beyene; Mengele, Carolin; Fleischmann, Erna; Herbinger, Karl-Heinz; Verweij, Jaco J; Geldmacher, Christof; Bretzel, Gisela; Löscher, Thomas; Zeynudin, Ahmed

2015-01-01

In Schistosoma mansoni infection, diagnosis and control after treatment mainly rely on parasitological stool investigations which are laborious and have limited sensitivity. PCR methods have shown equal or superior sensitivity but preservation and storage methods limit their use in the field. Therefore, the use of occult blood detection cards (fecal cards) for easy sampling and storage of fecal samples for further PCR testing was evaluated in a pilot study. Stool specimens were collected in a highly endemic area for S. mansoni in Ethiopia and submitted in an investigator-blinded fashion to microscopic examination by Kato-Katz thick smear as well as to real-time PCR using either fresh frozen stool samples or stool smears on fecal cards which have been stored at ambient temperature for up to ten months. Out of 55 stool samples, 35 were positive by microscopy, 33 and 32 were positive by PCR of frozen samples and of fecal card samples, respectively. When microscopy was used as diagnostic "gold standard", the sensitivity of PCR on fresh stool was 94.3% (95%-CI: 86.6; 100) and on fecal cards 91.4% (95%-CI: 82.2; 100). The use of fecal cards proved to be a simple and useful method for stool collection and prolonged storage prior to PCR based diagnosis of S. mansoni infection. This technique may be a valuable approach for large scale surveillance and post treatment assessments.

49 CFR 173.161 - Chemical kits and first aid kits.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Chemical kits and first aid kits. 173.161 Section... Class 7 § 173.161 Chemical kits and first aid kits. (a) Chemical kits and First aid kits must conform to... 10 kg. (b) Chemical kits and First aid kits are excepted from the specification packaging...

Fe3O4@SiO2@KIT-6 as an Efficient and Reusable Catalyst for the Synthesis of Novel Derivatives of 3,3'-((Aryl-1-phenyl-1H-pyrazol-4- yl)methylene)bis (1H-indole).

PubMed

Nikpassand, Mohammad; Fekri, Leila Zare; Nabatzadeh, Mozhdeh

2017-01-01

Korea advanced institute of science and technology cubic ordered mesoporous silica (KIT-6 mesoporous) silica coated magnetite nanoparticles, is an effective, ecobenign and recyclable catalyst for the electrophilic substitution reactions of indoles with various synthetized aldehydes to afford the corresponding novel diindolylmethanes in high yields and short reaction times. The catalyst can be recovered and reused without loss of activity. The work-up of the reaction consists of a simple separation, followed by concentration of the crude product and purification. The present methodology offers several advantages such as aqueous media, excellent yields, simple procedure, mild conditions and reduced environmental consequences. All of synthesized compounds are new and were characterized by IR, NMR and elemental analyses. A mixture of synthetized pyrazolecarbaldehydes [24] (2.0mmol), indole (4.0mmol) and Fe3O4@SiO2@KIT-6 (0.04mmol) and H2O (10mL) were stirred at room temperature for the required reaction time according to Table 2. After completion of the reaction, the product was solved in CHCl3 (3×10 mL) and insoluble catalyst was removed by filtration in the presence of an efficient magnetic bar. The organic phase including the product and CHCl3 was evaporated under vacuum. The resulting crude material was purified by recrystallization from EtOH to afford pure products. As part of our on interest for the development of efficient and environmentally friendly procedures for the synthesis of heterocyclic and pharmaceutical compounds, an efficient, facile and aqueous media was introduced for the synthesis of novel derivatives of diindolylmethanes containing pyrazole moiety. A variety of synthetized aldehyde compounds reacted smoothly with indoles to produce diindolylmethanes under these reaction conditions. The electron deficiency and the nature of the substituents on the aromatic ring show some effects on this conversion. All the reactions were run with catalytic amounts of catalysts. The best results gained with 0.04mmol of Fe3O4@SiO2@KIT-6 as a strong Lewis acid at room temperature in aqueous media. In conclusion, we have investigated the synthesized KIT-6 mesoporous silica coated magnetite nanoparticles (MMNPs) as a mild and efficient catalyst for the synthesis of novel diindolylmethanes coupled with pyrazole moiety in aqua media. The simplicity, easy workup together with the use of inexpensive, environmentally friendly and reusable catalyst, is the notable features of this catalytic procedure. To the best of our knowledge, this is the first report for the synthesis of a new library of diindolylmethane compound bearing pyrazole moiety that enhance the biological and pharmacological activity. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

49 CFR 173.161 - Chemical kits and first aid kits.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 2 2012-10-01 2012-10-01 false Chemical kits and first aid kits. 173.161 Section... Class 7 § 173.161 Chemical kits and first aid kits. (a) Applicability. Chemical kits and first aid kits... assigned to the chemical kit and first aid kit as a whole must be the most stringent packing group assigned...

Selective vs. nonselective media and direct plating vs. enrichment technique in isolation of Vibrio cholerae: recommendations for clinical laboratories.

PubMed

Rennels, M B; Levine, M M; Daya, V; Angle, P; Young, C

1980-09-01

The occurrence of human cholera along the Gulf of Mexico and the isolation of Vibrio cholerae O1 from the Gulf and Chesapeake Bay make it imperative that microbiology laboratories along estuaries develop the capabilities to culture for these pathogens. In attempts to devise a simplified but efficient culture procedure, a selective medium, thiosulfate-citrate-bile salts-sucrose (TCBS) agar, was compared with a nonselective medium, gelatin agar (GA), and the utility of enrichment was examined. TCBS agar detected 99% of the stools found to be positive by all techniques combined, whereas GA identified only 80%. Of acute diarrheal stools, 96% were positive on direct plating, whereas only 66% of formed stools containing V. cholerae were detected by direct plating. Stools from patients with acute diarrhea can be plated directly into TCBS agar alone; stools from persons shedding low numbers of organisms (such as contacts, carriers, or patients receiving antibiotics) should be incubated first in an enrichment broth and then on TCBS agar.

Pelvic Floor Muscle Exercise for Paediatric Functional Constipation.

PubMed

Farahmand, Fatemeh; Abedi, Aidin; Esmaeili-Dooki, Mohammad Reza; Jalilian, Rozita; Tabari, Sanaz Mehrabani

2015-06-01

Functional constipation (FC) is one of the most common gastrointestinal problems among children. This study was designed to investigate the effectiveness of pelvic floor muscle exercise on treatment of FC. In this study which was conducted in Children's Medical Center, children with a diagnosis of FC (aged 4-18 y) who did not respond to medical treatment, performed sessions of pelvic floor muscle exercise at home twice a day for 8 wk. Frequency of defecation, overall improvement of constipation, stool withholding, painful defecation and stool consistency were measured at the final week of the intervention compared to baseline. Forty children (16 males, 24 females mean age 5.6±1.03 y) completed the 8-wk exercise program. Subjective overall improvement of the symptoms was present in 36 patients (90%). The changes in stool frequency, stool diameter and consistency were statistically significant. However, there were no statistically significant differences in the stool withholding, fecal impaction, fecal incontinence and painful defecation. Pelvic floor muscle exercise is an effective non-pharmacologic treatment for Paediatric FC.

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

PubMed

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.

Diagnostic Yield of Routine Enteropathogenic Stool Tests in Pediatric Ulcerative Colitis.

PubMed

Ihekweazu, Faith D; Ajjarapu, Avanthi; Kellermayer, Richard

2015-01-01

It can be important to exclude infectious etiologies prior to adjusting immunosuppressive therapy in patients with ulcerative colitis (UC) exacerbation. We sought to determine the diagnostic yield of routine infectious stool studies in pediatric UC patients. We conducted a retrospective review of 152 pediatric UC patients at Texas Children's Hospital between January 2003 and December 2009. The patient records were followed through July 2014. The number and type of infectious stool studies performed and the results of those were collected. Three hundred fifty-four diagnostic stool tests were conducted for Clostridium difficile; 13.6% were positive. Two hundred twenty stool bacterial cultures were performed, and 1.8% were positive, all growing non-typhoid Salmonella. One of 13 (7.7%) Adenovirus PCR tests was positive. Two of 152 examinations (1.3%) for Ova and Parasites were positive. No stool tests for viral culture, viral particles, Yersinia or Rotavirus were positive. Clostridium difficile infection is common in pediatric UC, and routine screening during flares is strongly recommended. Other bacterial and parasitic infections routinely tested for are uncommon, but Salmonella may be a potentially important attribute to disease exacerbations in select patients. In patients without co-morbid conditions, the utility of performing non-specific fecal viral tests is questionable. © 2015 by the Association of Clinical Scientists, Inc.

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

PubMed Central

Schlenker, Nicklas; Bauer, Malkin; Helfrich, Kerstin; Mengele, Carolin; Löscher, Thomas; Nothdurft, Hans Dieter; Bretzel, Gisela; Beissner, Marcus

2017-01-01

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. PMID:28408937

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.

PubMed

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.

The gut microbiota, bile acids and their correlation in primary sclerosing cholangitis associated with inflammatory bowel disease.

PubMed

Torres, J; Palmela, C; Brito, H; Bao, X; Ruiqi, H; Moura-Santos, P; Pereira da Silva, J; Oliveira, A; Vieira, C; Perez, K; Itzkowitz, S H; Colombel, J F; Humbert, L; Rainteau, D; Cravo, M; Rodrigues, C M; Hu, J

2018-02-01

Patients with primary sclerosing cholangitis associated with inflammatory bowel disease (PSC-IBD) have a very high risk of developing colorectal neoplasia. Alterations in the gut microbiota and/or gut bile acids could account for the increase in this risk. However, no studies have yet investigated the net result of cholestasis and a potentially altered bile acid pool interacting with a dysbiotic gut flora in the inflamed colon of PSC-IBD. The aim of this study was to compare the gut microbiota and stool bile acid profiles, as well as and their correlation in patients with PSC-IBD and inflammatory bowel disease alone. Thirty patients with extensive colitis (15 with concomitant primary sclerosing cholangitis) were prospectively recruited and fresh stool samples were collected. The microbiota composition in stool was profiled using bacterial 16S rRNA sequencing. Stool bile acids were assessed by high-performance liquid chromatography tandem mass spectrometry. The total stool bile acid pool was significantly reduced in PSC-IBD. Although no major differences were observed in the individual bile acid species in stool, their overall combination allowed a good separation between PSC-IBD and inflammatory bowel disease. Compared with inflammatory bowel disease alone, PSC-IBD patients demonstrated a different gut microbiota composition with enrichment in Ruminococcus and Fusobacterium genus compared with inflammatory bowel disease. At the operational taxonomic unit level major shifts were observed within the Firmicutes (73%) and Bacteroidetes phyla (17%). Specific microbiota-bile acid correlations were observed in PSC-IBD, where 12% of the operational taxonomic units strongly correlated with stool bile acids, compared with only 0.4% in non-PSC-IBD. Patients with PSC-IBD had distinct microbiota and microbiota-stool bile acid correlations as compared with inflammatory bowel disease. Whether these changes are associated with, or may predispose to, an increased risk of colorectal neoplasia needs to be further clarified.

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

PubMed

Meurs, Lynn; Brienen, Eric; Mbow, Moustapha; Ochola, Elizabeth A; Mboup, Souleymane; Karanja, Diana M S; Secor, W Evan; Polman, Katja; van Lieshout, Lisette

2015-01-01

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 neglected tropical diseases.

Stool C difficile toxin

MedlinePlus

... 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 ( ...

Evaluation of a New Selective Medium, BD BBL CHROMagar MRSA II, for Detection of Methicillin-Resistant Staphylococcus aureus in Stool Specimens ▿

PubMed Central

Havill, Nancy L.; Boyce, John M.

2010-01-01

We compared the recovery of methicillin-resistant Staphylococcus aureus (MRSA) on a new selective chromogenic agar, BD BBL CHROMagar MRSA II (CMRSAII), to that on traditional culture media with 293 stool specimens. The recovery of MRSA was greater on the CMRSAII agar. Screening of stool samples can identify patients who were previously unknown carriers of MRSA. PMID:20392908

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

PubMed

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%, 1/2 in 20%, 1/3 in 13%, 1 to 4 per month in 30%. The goal of inspection was "routine" in 54%, and devoted to track down any pathological condition ("self examination") in 46%. Eighty percent of the subjects considered having no shame or specific reticence and only 17% of them, had some interrogations concerning the real rational of such an inspection. BSC is a useful tool widely used in routine practice, helping to the diagnosis of constipation and the control of the different therapeutic strategies. There is no psychological barriers or metaphysics inconveniences for its use. But it seems legitimate to understand the hidden reasons of such a behavior with unconscious purposes reflecting the intimal nature of the humans. 3. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Comparison of stool collection on site versus at home in a population-based study : feasibility and participants' preference in Pretest 2 of the German National Cohort.

PubMed

Schultze, A; Akmatov, M K; Andrzejak, M; Karras, N; Kemmling, Y; Maulhardt, A; Wieghold, S; Ahrens, W; Günther, K; Schlenz, H; Krause, G; Pessler, F

2014-11-01

For certain laboratory investigations it is necessary to obtain native stool samples and process them within a narrow time window at the point of contact or a nearby laboratory. However, it is not known whether it is feasible to obtain stool samples from asymptomatic individuals during an appointment in a study center (SC). We therefore compared participants' preference, feasibility and acceptance of stool sample collection during the appointment at the study center (on-site sampling) to collection at home after the appointment. The study was conducted at two sites in Northern Germany (Bremen, n = 156; Hannover, n = 147) during the Pretest 2 phase of the German National Cohort (GNC), drawing upon a randomly selected population supplemented by a small convenience sample. In the study center, the participants were given the choice to provide a stool sample during the appointment or to collect a sample later at home and return it by mail. In all, 303 of the 351 participants (86 %) of Pretest 2 at these sites participated in this feasibility study. Only 7.9 % (24/303) of the participants chose on-site collection, whereas 92 % (279/303) chose at-home collection. There were significant differences between the two study sites in that 14 % (21/147) of participants in Hannover and 2 % (3/156) of participants in Bremen chose on-site collection. Compliance was high in both groups, as 100 % (24/24) and 98 % (272/279) of participants in the on-site and at-home groups, respectively, provided complete samples. Both methods were highly accepted, as 92 % of the participants in each group (22/24 and 227/248) stated that stool collection at the respective site was acceptable. When given a choice, most participants in this population-based study preferred home collection of stool samples to collection in the study center. Thus, native stool samples for immediate processing in the study center may potentially be obtained only from a subpopulation of participants, which may lead to selection bias. Home collection, on the other hand, proved to be a highly feasible method for studies that do not require freshly collected native stool.

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

PubMed

Franciscovich, Amy; Vaidya, Dhananjay; Doyle, Joe; Bolinger, Josh; Capdevila, Montserrat; Rice, Marcus; Hancock, Leslie; Mahr, Tanya; Mogul, Douglas B

2015-01-01

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 settings. PoopMD may be a valuable tool to help parents identify acholic stools in the perinatal period, and provide guidance as to whether additional evaluation with their pediatrician is indicated. PoopMD may improve outcomes for children with BA.

DNA methylation of phosphatase and actin regulator 3 detects colorectal cancer in stool and complements FIT.

PubMed

Bosch, Linda J W; Oort, Frank A; Neerincx, Maarten; Khalid-de Bakker, Carolina A J; Terhaar sive Droste, Jochim S; Melotte, Veerle; Jonkers, Daisy M A E; Masclee, Ad A M; Mongera, Sandra; Grooteclaes, Madeleine; Louwagie, Joost; van Criekinge, Wim; Coupé, Veerle M H; Mulder, Chris J; van Engeland, Manon; Carvalho, Beatriz; Meijer, Gerrit A

2012-03-01

Using a bioinformatics-based strategy, we set out to identify hypermethylated genes that could serve as biomarkers for early detection of colorectal cancer (CRC) in stool. In addition, the complementary value to a Fecal Immunochemical Test (FIT) was evaluated. Candidate genes were selected by applying cluster alignment and computational analysis of promoter regions to microarray-expression data of colorectal adenomas and carcinomas. DNA methylation was measured by quantitative methylation-specific PCR on 34 normal colon mucosa, 71 advanced adenoma, and 64 CRC tissues. The performance as biomarker was tested in whole stool samples from in total 193 subjects, including 19 with advanced adenoma and 66 with CRC. For a large proportion of these series, methylation data for GATA4 and OSMR were available for comparison. The complementary value to FIT was measured in stool subsamples from 92 subjects including 44 with advanced adenoma or CRC. Phosphatase and Actin Regulator 3 (PHACTR3) was identified as a novel hypermethylated gene showing more than 70-fold increased DNA methylation levels in advanced neoplasia compared with normal colon mucosa. In a stool training set, PHACTR3 methylation showed a sensitivity of 55% (95% CI: 33-75) for CRC and a specificity of 95% (95% CI: 87-98). In a stool validation set, sensitivity reached 66% (95% CI: 50-79) for CRC and 32% (95% CI: 14-57) for advanced adenomas at a specificity of 100% (95% CI: 86-100). Adding PHACTR3 methylation to FIT increased sensitivity for CRC up to 15%. PHACTR3 is a new hypermethylated gene in CRC with a good performance in stool DNA testing and has complementary value to FIT.

Dietary glutamine and oral antibiotics each improve indexes of gut barrier function in rat short bowel syndrome.

PubMed

Tian, Junqiang; Hao, Li; Chandra, Prakash; Jones, Dean P; Willams, Ifor R; Gewirtz, Andrew T; Ziegler, Thomas R

2009-02-01

Short bowel syndrome (SBS) is associated with gut barrier dysfunction. We examined effects of dietary glutamine (GLN) or oral antibiotics (ABX) on indexes of gut barrier function in a rat model of SBS. Adult rats underwent a 60% distal small bowel + proximal colonic resection (RX) or bowel transection (TX; control). Rats were pair fed diets with or without l-GLN for 20 days after operation. Oral ABX (neomycin, metronidazole, and polymyxin B) were given in some RX rats fed control diet. Stool secretory immunoglobulin A (sIgA) was measured serially. On day 21, mesenteric lymph nodes (MLN) were cultured for gram-negative bacteria. IgA-positive plasma cells in jejunum, stool levels of flagellin- and lipopolysaccharide (LPS)-specific sIgA, and serum total, anti-flagellin- and anti-LPS IgG levels were determined. RX caused gram-negative bacterial translocation to MLN, increased serum total and anti-LPS IgG and increased stool total sIgA. After RX, dietary GLN tended to blunt bacterial translocation to MLN (-29%, P = NS) and significantly decreased anti-LPS IgG levels in serum, increased both stool and jejunal mucosal sIgA and increased stool anti-LPS-specific IgA. Oral ABX eliminated RX-induced bacterial translocation, significantly decreased total and anti-LPS IgG levels in serum, significantly decreased stool total IgA and increased stool LPS-specific IgA. Partial small bowel-colonic resection in rats is associated with gram-negative bacterial translocation from the gut and a concomitant adaptive immune response to LPS. These indexes of gut barrier dysfunction are ameliorated or blunted by administration of dietary GLN or oral ABX, respectively. Dietary GLN upregulates small bowel sIgA in this model.

Radotinib induces high cytotoxicity in c-KIT positive acute myeloid leukemia cells.

PubMed

Heo, Sook-Kyoung; Noh, Eui-Kyu; Kim, Jeong Yi; Jo, Jae-Cheol; Choi, Yunsuk; Koh, SuJin; Baek, Jin Ho; Min, Young Joo; Kim, Hawk

2017-06-05

Previously, we reported that radotinib, a BCR-ABL1 tyrosine kinase inhibitor, induced cytotoxicity in acute myeloid leukemia (AML) cells. However, the effects of radotinib in the subpopulation of c-KIT-positive AML cells were unclear. We observed that low-concentration radotinib had more potent cytotoxicity in c-KIT-positive cells than c-KIT-negative cells from AML patients. To address this issue, cell lines with high c-KIT expression, HEL92.1.7, and moderate c-KIT expression, H209, were selected. HEL92.1.7 cells were grouped into intermediate and high c-KIT expression populations. The cytotoxicity of radotinib against the HEL92.1.7 cell population with intermediate c-KIT expression was not different from that of the population with high c-KIT expression. When H209 cells were grouped into c-KIT expression-negative and c-KIT expression-positive populations, radotinib induced cytotoxicity in the c-KIT-positive population, but not the c-KIT-negative population. Thus, radotinib induces cytotoxicity in c-KIT-positive cells, regardless of the c-KIT expression intensity. Therefore, radotinib induces significant cytotoxicity in c-KIT-positive AML cells, suggesting that radotinib is a potential target agent for the treatment of c-KIT-positive malignancies including AML. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of an Efficient Entire-Capsid-Coding-Region Amplification Method for Direct Detection of Poliovirus from Stool Extracts

PubMed Central

Kilpatrick, David R.; Nakamura, Tomofumi; Burns, Cara C.; Bukbuk, David; Oderinde, Soji B.; Oberste, M. Steven; Kew, Olen M.; Pallansch, Mark A.; Shimizu, Hiroyuki

2014-01-01

Laboratory diagnosis has played a critical role in the Global Polio Eradication Initiative since 1988, by isolating and identifying poliovirus (PV) from stool specimens by using cell culture as a highly sensitive system to detect PV. In the present study, we aimed to develop a molecular method to detect PV directly from stool extracts, with a high efficiency comparable to that of cell culture. We developed a method to efficiently amplify the entire capsid coding region of human enteroviruses (EVs) including PV. cDNAs of the entire capsid coding region (3.9 kb) were obtained from as few as 50 copies of PV genomes. PV was detected from the cDNAs with an improved PV-specific real-time reverse transcription-PCR system and nucleotide sequence analysis of the VP1 coding region. For assay validation, we analyzed 84 stool extracts that were positive for PV in cell culture and detected PV genomes from 100% of the extracts (84/84 samples) with this method in combination with a PV-specific extraction method. PV could be detected in 2/4 stool extract samples that were negative for PV in cell culture. In PV-positive samples, EV species C viruses were also detected with high frequency (27% [23/86 samples]). This method would be useful for direct detection of PV from stool extracts without using cell culture. PMID:25339406

Malabsorption

MedlinePlus

... that of other children of similar age and gender. This is called failure to thrive . The child ... deficiency Secretin stimulation test Small bowel biopsy Stool culture or culture of small intestine aspirate Stool fat ...

Identification of human remains from the Second World War mass graves uncovered in Bosnia and Herzegovina

PubMed Central

Marjanović, Damir; Hadžić Metjahić, Negra; Čakar, Jasmina; Džehverović, Mirela; Dogan, Serkan; Ferić, Elma; Džijan, Snježana; Škaro, Vedrana; Projić, Petar; Madžar, Tomislav; Rod, Eduard; Primorac, Dragan

2015-01-01

Aim To present the results obtained in the identification of human remains from World War II found in two mass graves in Ljubuški, Bosnia and Herzegovina. Methods Samples from 10 skeletal remains were collected. Teeth and femoral fragments were collected from 9 skeletons and only a femoral fragment from 1 skeleton. DNA was isolated from bone and teeth samples using an optimized phenol/chloroform DNA extraction procedure. All samples required a pre-extraction decalcification with EDTA and additional post-extraction DNA purification using filter columns. Additionally, DNA from 12 reference samples (buccal swabs from potential living relatives) was extracted using the Qiagen DNA extraction method. QuantifilerTM Human DNA Quantification Kit was used for DNA quantification. PowerPlex ESI kit was used to simultaneously amplify 15 autosomal short tandem repeat (STR) loci, and PowerPlex Y23 was used to amplify 23 Y chromosomal STR loci. Matching probabilities were estimated using a standard statistical approach. Results A total of 10 samples were processed, 9 teeth and 1 femoral fragment. Nine of 10 samples were profiled using autosomal STR loci, which resulted in useful DNA profiles for 9 skeletal remains. A comparison of established victims' profiles against a reference sample database yielded 6 positive identifications. Conclusion DNA analysis may efficiently contribute to the identification of remains even seven decades after the end of the World War II. The significant percentage of positively identified remains (60%), even when the number of the examined possible living relatives was relatively small (only 12), proved the importance of cooperation with the members of the local community, who helped to identify the closest missing persons’ relatives and collect referent samples from them. PMID:26088850

Optimisation of DNA extraction from the crustacean Daphnia

PubMed Central

Athanasio, Camila Gonçalves; Chipman, James K.; Viant, Mark R.

2016-01-01

Daphnia are key model organisms for mechanistic studies of phenotypic plasticity, adaptation and microevolution, which have led to an increasing demand for genomics resources. A key step in any genomics analysis, such as high-throughput sequencing, is the availability of sufficient and high quality DNA. Although commercial kits exist to extract genomic DNA from several species, preparation of high quality DNA from Daphnia spp. and other chitinous species can be challenging. Here, we optimise methods for tissue homogenisation, DNA extraction and quantification customised for different downstream analyses (e.g., LC-MS/MS, Hiseq, mate pair sequencing or Nanopore). We demonstrate that if Daphnia magna are homogenised as whole animals (including the carapace), absorbance-based DNA quantification methods significantly over-estimate the amount of DNA, resulting in using insufficient starting material for experiments, such as preparation of sequencing libraries. This is attributed to the high refractive index of chitin in Daphnia’s carapace at 260 nm. Therefore, unless the carapace is removed by overnight proteinase digestion, the extracted DNA should be quantified with fluorescence-based methods. However, overnight proteinase digestion will result in partial fragmentation of DNA therefore the prepared DNA is not suitable for downstream methods that require high molecular weight DNA, such as PacBio, mate pair sequencing and Nanopore. In conclusion, we found that the MasterPure DNA purification kit, coupled with grinding of frozen tissue, is the best method for extraction of high molecular weight DNA as long as the extracted DNA is quantified with fluorescence-based methods. This method generated high yield and high molecular weight DNA (3.10 ± 0.63 ng/µg dry mass, fragments >60 kb), free of organic contaminants (phenol, chloroform) and is suitable for large number of downstream analyses. PMID:27190714

Identification of human remains from the Second World War mass graves uncovered in Bosnia and Herzegovina.

PubMed

Marjanović, Damir; Hadžić Metjahić, Negra; Čakar, Jasmina; Džehverović, Mirela; Dogan, Serkan; Ferić, Elma; Džijan, Snježana; Škaro, Vedrana; Projić, Petar; Madžar, Tomislav; Rod, Eduard; Primorac, Dragan

2015-06-01

To present the results obtained in the identification of human remains from World War II found in two mass graves in Ljubuški, Bosnia and Herzegovina. Samples from 10 skeletal remains were collected. Teeth and femoral fragments were collected from 9 skeletons and only a femoral fragment from 1 skeleton. DNA was isolated from bone and teeth samples using an optimized phenol/chloroform DNA extraction procedure. All samples required a pre-extraction decalcification with EDTA and additional post-extraction DNA purification using filter columns. Additionally, DNA from 12 reference samples (buccal swabs from potential living relatives) was extracted using the Qiagen DNA extraction method. QuantifilerTM Human DNA Quantification Kit was used for DNA quantification. PowerPlex ESI kit was used to simultaneously amplify 15 autosomal short tandem repeat (STR) loci, and PowerPlex Y23 was used to amplify 23 Y chromosomal STR loci. Matching probabilities were estimated using a standard statistical approach. A total of 10 samples were processed, 9 teeth and 1 femoral fragment. Nine of 10 samples were profiled using autosomal STR loci, which resulted in useful DNA profiles for 9 skeletal remains. A comparison of established victims' profiles against a reference sample database yielded 6 positive identifications. DNA analysis may efficiently contribute to the identification of remains even seven decades after the end of the World War II. The significant percentage of positively identified remains (60%), even when the number of the examined possible living relatives was relatively small (only 12), proved the importance of cooperation with the members of the local community, who helped to identify the closest missing persons' relatives and collect referent samples from them.

Detection of Viable Cryptosporidium parvum in Soil by Reverse Transcription–Real-Time PCR Targeting hsp70 mRNA ▿

PubMed Central

Liang, Zhanbei; Keeley, Ann

2011-01-01

Extraction of high-quality mRNA from Cryptosporidium parvum is a key step in PCR detection of viable oocysts in environmental samples. Current methods for monitoring oocysts are limited to water samples; therefore, the goal of this study was to develop a rapid and sensitive procedure for Cryptosporidium detection in soil samples. The efficiencies of five RNA extraction methods were compared (mRNA extraction with the Dynabeads mRNA Direct kit after chemical and physical sample treatments, and total RNA extraction methods using the FastRNA Pro Soil-Direct, PowerSoil Total RNA, E.Z.N.A. soil RNA, and Norgen soil RNA purification kits) for the direct detection of Cryptosporidium with oocyst-spiked sandy, loamy, and clay soils by using TaqMan reverse transcription-PCR. The study also evaluated the presence of inhibitors by synthesis and incorporation of an internal positive control (IPC) RNA into reverse transcription amplifications, used different facilitators (bovine serum albumin, yeast RNA, salmon DNA, skim milk powder, casein, polyvinylpyrrolidone, sodium hexametaphosphate, and Salmonella enterica serovar Typhi) to mitigate RNA binding on soil components, and applied various treatments (β-mercaptoethanol and bead beating) to inactivate RNase and ensure the complete lysis of oocysts. The results of spiking studies showed that Salmonella cells most efficiently relieved binding of RNA. With the inclusion of Salmonella during extraction, the most efficient mRNA method was Dynabeads, with a detection limit of 6 × 102 oocysts g−1 of sandy soil. The most efficient total RNA method was PowerSoil, with detection limits of 1.5 × 102, 1.5 × 103, and 1.5 × 104 C. parvum oocysts g−1 soil for sandy, loamy, and clay samples, respectively. PMID:21803904

Techni-kits and Techni-kit Building Systems

NASA Technical Reports Server (NTRS)

Callender, E. D.; Hartsough, C.; Morris, R. V.; Yamamoto, Y.

1985-01-01

Techni-kits consists of theories, methods, standards and computer based tools that assist in design of information-intensive systems. Techni-kit "building system" is techni-kit that builds techni-kits.

Mathematical inference on helminth egg counts in stool and its applications in mass drug administration programmes to control soil-transmitted helminthiasis in public health.

PubMed

Levecke, Bruno; Anderson, Roy M; Berkvens, Dirk; Charlier, Johannes; Devleesschauwer, Brecht; Speybroeck, Niko; Vercruysse, Jozef; Van Aelst, Stefan

2015-03-01

In the present study, we present a hierarchical model based on faecal egg counts (FECs; expressed in eggs per 1g of stool) in which we first describe the variation in FECs between individuals in a particular population, followed by describing the variance due to counting eggs under a microscope separately for each stool sample. From this general framework, we discuss how to calculate a sample size for assessing a population mean FEC and the impact of an intervention, measured as reduction in FECs, for any scenario of soil-transmitted helminth (STH) epidemiology (the intensity and aggregation of FECs within a population) and diagnostic strategy (amount of stool examined (∼sensitivity of the diagnostic technique) and examination of individual/pooled stool samples) and on how to estimate prevalence of STH in the absence of a gold standard. To give these applications the most wide relevance as possible, we illustrate each of them with hypothetical examples. Copyright © 2015 Elsevier Ltd. All rights reserved.

Rice solution and World Health Organization solution by gastric infusion for high stool output diarrhea.

PubMed

Mota-Hernández, F; Bross-Soriano, D; Pérez-Ricardez, M L; Velásquez-Jones, L

1991-08-01

We sought to determine the efficacy of three different types of treatment in children with acute diarrhea who, during the oral rehydration period, had high stool output (greater than 10 mL/kg per hour). Sixty-six children, aged 1 to 18 months, with an average stool output of 22.6 mL/kg per hour were randomly distributed into three groups: group 1 received a rice flour solution, group 2 received the World Health Organization rehydration solution by gastric infusion, and group 3 continued to receive this solution orally. In all three groups, a decrease in stool output was observed, with the higher decrease observed in group 1 patients. Such a decrease facilitated rehydration of all 22 patients in group 1 (100%) in 3.3 +/- 1.5 hours, 16 (73%) in group 2 in 4.3 +/- 2.1 hours, and 15 (69%) in group 3 in 4.9 +/- 2.0 hours. No complications were observed. These data indicate that the rice flour solution is effective in children with high stool output diarrhea.

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

PubMed

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 < 0.001). When the 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 < 0.001) means that any alteration in the infant datasets has a proportionally larger effect. The commercial storage vials appear to be suitable for high diversity microbiota samples, but may be less appropriate for lower diversity samples. Differences between fresh and stored samples mean that where storage is unavoidable, a consistent storage regime should be used. We would recommend extraction ideally within the first week of storage.

Preservation Methods Differ in Fecal Microbiome Stability, Affecting Suitability for Field Studies.

PubMed

Song, Se Jin; Amir, Amnon; Metcalf, Jessica L; Amato, Katherine R; Xu, Zhenjiang Zech; Humphrey, Greg; Knight, Rob

2016-01-01

Immediate freezing at -20°C or below has been considered the gold standard for microbiome preservation, yet this approach is not feasible for many field studies, ranging from anthropology to wildlife conservation. Here we tested five methods for preserving human and dog fecal specimens for periods of up to 8 weeks, including such types of variation as freeze-thaw cycles and the high temperature fluctuations often encountered under field conditions. We found that three of the methods-95% ethanol, FTA cards, and the OMNIgene Gut kit-can preserve samples sufficiently well at ambient temperatures such that differences at 8 weeks are comparable to differences among technical replicates. However, even the worst methods, including those with no fixative, were able to reveal microbiome differences between species at 8 weeks and between individuals after a week, allowing meta-analyses of samples collected using various methods when the effect of interest is expected to be larger than interindividual variation (although use of a single method within a study is strongly recommended to reduce batch effects). Encouragingly for FTA cards, the differences caused by this method are systematic and can be detrended. As in other studies, we strongly caution against the use of 70% ethanol. The results, spanning 15 individuals and over 1,200 samples, provide our most comprehensive view to date of storage effects on stool and provide a paradigm for the future studies of other sample types that will be required to provide a global view of microbial diversity and its interaction among humans, animals, and the environment. IMPORTANCE Our study, spanning 15 individuals and over 1,200 samples, provides our most comprehensive view to date of storage and stabilization effects on stool. We tested five methods for preserving human and dog fecal specimens for periods of up to 8 weeks, including the types of variation often encountered under field conditions, such as freeze-thaw cycles and high temperature fluctuations. We show that several cost-effective methods provide excellent microbiome stability out to 8 weeks, opening up a range of field studies with humans and wildlife that would otherwise be cost-prohibitive.

Nutritional status, intestinal parasite infection and allergy among school children in northwest Ethiopia.

PubMed

Amare, Bemnet; Ali, Jemal; Moges, Beyene; Yismaw, Gizachew; Belyhun, Yeshambel; Gebretsadik, Simon; Woldeyohannes, Desalegn; Tafess, Ketema; Abate, Ebba; Endris, Mengistu; Tegabu, Desalegn; Mulu, Andargachew; Ota, Fusao; Fantahun, Bereket; Kassu, Afework

2013-01-12

Parasitic infections have been shown to have deleterious effects on host nutritional status. In addition, although helmintic infection can modulate the host inflammatory response directed against the parasite, a causal association between helminths and allergy remains uncertain. The present study was therefore designed to evaluate the relationship between nutritional status, parasite infection and prevalence of allergy among school children. A cross sectional study was performed involving school children in two elementary schools in Gondar, Ethiopia. Nutritional status of these children was determined using anthropometric parameters (weight-for-age, height-for-age and BMI-for-age). Epi-Info software was used to calculate z-scores. Stool samples were examined using standard parasitological procedures. The serum IgE levels were quantified by total IgE ELISA kit following the manufacturer's instruction. A total of 405 children (with mean age of 12.09.1 ± 2.54 years) completed a self-administered allergy questionnaire and provided stool samples for analysis. Overall prevalence of underweight, stunting and thinness/wasting was 15.1%, 25.2%, 8.9%, respectively. Of the total, 22.7% were found to be positive for intestinal parasites. The most prevalent intestinal parasite detected was Ascaris lumbricoides (31/405, 7.6%). There was no statistically significant association between prevalence of malnutrition and the prevalence of parasitic infections. Median total serum IgE level was 344 IU/ml (IQR 117-2076, n=80) and 610 IU/ml (143-1833, n=20), respectively, in children without and with intestinal parasite infection (Z=-0.198, P>0.8). The prevalence of self reported allergy among the subset was 8%. IgE concentration was not associated either with the presence of parasitic infection or history of allergy. The prevalence of malnutrition, intestinal parasitism and allergy was not negligible in this population. In addition, there was no significant association between the prevalence of allergy and their nutritional status, and parasite infection. Further research prospective observational and intervention studies are required to address the question of causality between nutritional factors, parasites, and allergy.

Nutritional status, intestinal parasite infection and allergy among school children in Northwest Ethiopia

PubMed Central

2013-01-01

Background Parasitic infections have been shown to have deleterious effects on host nutritional status. In addition, although helmintic infection can modulate the host inflammatory response directed against the parasite, a causal association between helminths and allergy remains uncertain. The present study was therefore designed to evaluate the relationship between nutritional status, parasite infection and prevalence of allergy among school children. Methods A cross sectional study was performed involving school children in two elementary schools in Gondar, Ethiopia. Nutritional status of these children was determined using anthropometric parameters (weight-for-age, height-for-age and BMI-for-age). Epi-Info software was used to calculate z-scores. Stool samples were examined using standard parasitological procedures. The serum IgE levels were quantified by total IgE ELISA kit following the manufacturer’s instruction. Result A total of 405 children (with mean age of 12.09.1 ± 2.54 years) completed a self-administered allergy questionnaire and provided stool samples for analysis. Overall prevalence of underweight, stunting and thinness/wasting was 15.1%, 25.2%, 8.9%, respectively. Of the total, 22.7% were found to be positive for intestinal parasites. The most prevalent intestinal parasite detected was Ascaris lumbricoides (31/405, 7.6%). There was no statistically significant association between prevalence of malnutrition and the prevalence of parasitic infections. Median total serum IgE level was 344 IU/ml (IQR 117–2076, n = 80) and 610 IU/ml (143–1833, n = 20), respectively, in children without and with intestinal parasite infection (Z = −0.198, P > 0.8). The prevalence of self reported allergy among the subset was 8%. IgE concentration was not associated either with the presence of parasitic infection or history of allergy. Conclusion The prevalence of malnutrition, intestinal parasitism and allergy was not negligible in this population. In addition, there was no significant association between the prevalence of allergy and their nutritional status, and parasite infection. Further research prospective observational and intervention studies are required to address the question of causality between nutritional factors, parasites, and allergy. PMID:23311926

Evaluation of Immunochromatographic Test for Dual Detection of Noroviruses and Group A Rotaviruses in Stool Samples.

PubMed

Khamrin, Pattara; Kumthip, Kattareeya; Thongprachum, Aksara; Takanashi, Sayaka; Okitsu, Shoko; Maneekarn, Niwat; Hayakawa, Satoshi; Ushijima, Hiroshi

2018-05-01

Viral enteric infections, such as noroviruses and rotaviruses are considered as the major causes of acute gastroenteritis in young children and elderly people all over the world. These viruses are responsible for numerous outbreaks of nonbacterial gastroenteritis in several settings such as hospitals, day care centers, nursing homes, and restaurants. Therefore, a rapid and sensitive diagnostic tool for virus detections could be helpful for disease control. A new immunochromatographic test for the dual detection of noroviruses and group A rotaviruses was evaluated by using specimens that were known to be positive for noroviruses and rotaviruses, as well as other diarrheal viruses. The sensitivity of detections for noroviruses and group A rotaviruses were 89.5% and 100%, respectively, while the specificity was 100% for both viruses. This immunochromatographic test was reactive to several norovirus and group A rotavirus genotypes (NoV GII.2, GII.3, GII.4, GII.6, GII.7, GII.17 and RVA G1P[8], G2P[4], G3P[8], G8P[8], G9P[8]). The speed and ease of use of the immunochromatographic test makes this kit particularly attractive as an alternative method for the detections of noroviruses and group A rotaviruses in the primary care unit.

[Determination of prevalence and etiology of anemia during pregnancy in southern Benin, in conjunction with revision of national management policy].

PubMed

Koura, K G; Briand, V; Massougbodji, A; Chippaux, J P; Cot, M; Garcia, A

2011-02-01

Anemia during pregnancy is a serious public health problem. Control requires identification of the underlying etiology. The objective of this study carried out in conjunction with revision of the national policy for the protection of pregnant women in Benin was to determine the prevalence and etiology of anemia. From October 2006 to April 2007, 300 pregnant women were examined at two maternities in Ouidah, Benin. Sociodemographic and environmental characteristics, dietary data, behavioral practices, and history of malaria infection during pregnancy were collected. Blood and stool samples were tested for the presence of malaria parasites and intestinal worms respectively. Hemoglobin and ferritinemia levels were also determined. The prevalence of anemia (Hb < 11 g/dL) was 65.7% while that of malaria and intestinal worms was 4.3% and 8% respectively. Iron deficiency was not found. A borderline significant correlation was found between helminthiasis and anemia. No correlation was found between anemia and malaria. These findings indicate that kits progressively introduced by the health system during the study period provided relatively effective care. This study demonstrates a high prevalence of moderate anemia during pregnancy and suggests that it is mainly due to intestinal helminthiasis. These findings underline the importance of preventive antihelminthic treatment during pregnancy.

Detection by PCR of Enteroviruses in Cerebrospinal Fluid during a Summer Outbreak of Aseptic Meningitis in Switzerland

PubMed Central

Gorgievski-Hrisoho, Meri; Schumacher, Jean-Daniel; Vilimonovic, Nevenka; Germann, Daniel; Matter, Lukas

1998-01-01

Enteroviruses (EV) are among the most common causes of aseptic meningitis. Standard diagnostic techniques are often too slow and lack sensitivity to be of clinical relevance. EV RNA can be detected within 5 h by a commercially available reverse transcription-PCR (RT-PCR) test kit. Cerebrospinal fluid (CSF) samples from 68 patients presenting with aseptic meningitis during a summer outbreak in Switzerland were examined in parallel with cell culture and commercial RT-PCR. RT-PCR was positive in all 16 CSF specimens positive by cell culture (100%). In addition, 42 of 52 (80%) CSF samples negative by cell culture were PCR positive. In 26 of these 42 (62%) patients, viral culture from other sites (throat swab or stool) was also positive. The CSF virus culture took 3 to 7 days to become positive. Echovirus 30 was the type most often isolated in this outbreak. The sensitivity of CSF RT-PCR based on clinical diagnosis during this aseptic meningitis outbreak in patients with negative bacterial culture results was 85%, i.e., considerably higher than the sensitivity of CSF virus culture (24%). We conclude that this commercial RT-PCR assay allows a positive diagnosis with minimal delay and may thus influence clinical decisions. PMID:9705364

[Laboratory diagnosis of lymphocytic meningitis].

PubMed

Marí, José María Navarro; Ruiz, Mercedes Pérez; Anza, Diego Vicente

2010-01-01

Lymphocytic meningitis, mainly those with an acute and benign course, are caused by viruses. In our area, the most commonly involved agents are enteroviruses, herpes simplex, varicella zoster and Toscana viruses. Nucleic acids amplification techniques (NAAT) are the methods of choice to diagnose viral meningitis from cerebrospinal fluid (CSF) samples. They are more rapid and sensitive, and indeed, they are not influenced by the viability of the virus in the clinical specimen as traditional methods are. The development of commercial equipments, the degree of automation, and the use of real-time polymerase chain reaction (PCR) systems are the most important premises to choose the molecular method in each laboratory. Recently, commercial kits of real-time PCR are available for the detection of enteroviruses and herpesviruses, which are the most frequently viruses involved in meningitis. Although NAAT from the clinical sample have replaced cell culture for diagnostic purposes, the combination of both methods remain useful. When the detection of the causal agent from the CSF sample is not possible, other specimens (pharyngeal exudates, stools) or serological methods can be used. Serology is the reference method for meningitis caused by West Nile virus and lymphocytic choriomeningitis virus, which are less frequently detected in our area. 2010 Elsevier España S.L. All rights reserved.

Liver Panel

MedlinePlus

... may be present before symptoms such as jaundice , dark urine, light-colored stools, itching ( pruritus ), nausea, fatigue, ... or pain Jaundice (yellowing of eyes or skin) Dark urine, light-colored stool Itching ( pruritus ) Diarrhea Usually ...

Aeromonas isolates from human diarrheic stool and groundwater compared by pulsed-field gel electrophoresis.

PubMed

Borchardt, Mark A; Stemper, Mary E; Standridge, Jon H

2003-02-01

Gastrointestinal infections of Aeromonas species are generally considered waterborne; for this reason, Aeromonas hydrophila has been placed on the United States Environmental Protection Agency Contaminant Candidate List of emerging pathogens in drinking water. In this study, we compared pulsed-field gel electrophoresis patterns of Aeromonas isolates from stool specimens of patients with diarrhea with Aeromonas isolates from patients' drinking water. Among 2,565 diarrheic stool specimens submitted to a Wisconsin clinical reference laboratory, 17 (0.66%) tested positive for Aeromonas. Groundwater isolates of Aeromonas were obtained from private wells throughout Wisconsin and the drinking water of Aeromonas-positive patients. The analysis showed that the stool and drinking water isolates were genetically unrelated, suggesting that in this population Aeromonas gastrointestinal infections were not linked with groundwater exposures.

Aeromonas Isolates from Human Diarrheic Stool and Groundwater Compared by Pulsed-Field Gel Electrophoresis

PubMed Central

Stemper, Mary E.; Standridge, Jon H.

2003-01-01

Gastrointestinal infections of Aeromonas species are generally considered waterborne; for this reason, Aeromonas hydrophila has been placed on the United States Environmental Protection Agency Contaminant Candidate List of emerging pathogens in drinking water. In this study, we compared pulsed-field gel electrophoresis patterns of Aeromonas isolates from stool specimens of patients with diarrhea with Aeromonas isolates from patients’ drinking water. Among 2,565 diarrheic stool specimens submitted to a Wisconsin clinical reference laboratory, 17 (0.66%) tested positive for Aeromonas. Groundwater isolates of Aeromonas were obtained from private wells throughout Wisconsin and the drinking water of Aeromonas-positive patients. The analysis showed that the stool and drinking water isolates were genetically unrelated, suggesting that in this population Aeromonas gastrointestinal infections were not linked with groundwater exposures. PMID:12603994

A decrease in ubiquitination and resulting prolonged life-span of KIT underlies the KIT overexpression-mediated imatinib resistance of KIT mutation-driven canine mast cell tumor cells.

PubMed

Kobayashi, Masato; Kuroki, Shiori; Kurita, Sena; Miyamoto, Ryo; Tani, Hiroyuki; Tamura, Kyoichi; Bonkobara, Makoto

2017-10-01

Overexpression of KIT is one of the mechanisms that contributes to imatinib resistance in KIT mutation-driven tumors. Here, the mechanism underlying this overexpression of KIT was investigated using an imatinib-sensitive canine mast cell tumor (MCT) line CoMS, which has an activating mutation in KIT exon 11. A KIT-overexpressing imatinib-resistant subline, rCoMS1, was generated from CoMS cells by their continuous exposure to increasing concentrations of imatinib. Neither a secondary mutation nor upregulated transcription of KIT was detected in rCoMS1 cells. A decrease in KIT ubiquitination, a prolonged KIT life-span, and KIT overexpression were found in rCoMS1 cells. These events were suppressed by withdrawal of imatinib and were re-induced by re‑treatment with imatinib. These findings suggest that imatinib elicited overexpression of KIT via suppression of its ubiquitination. These results also indicated that imatinib-induced overexpression of KIT in rCoMS1 cells was not a permanently acquired feature but was a reversible response of the cells. Moreover, the pan deubiquitinating enzyme inhibitor PR619 prevented imatinib induction of KIT overexpression, suggesting that the imatinib-induced decrease in KIT ubiquitination could be mediated by upregulation and/or activation of deubiquitinating enzyme(s). It may be possible that a similar mechanism of KIT overexpression underlies the acquisition of imatinib resistance in some human tumors that are driven by KIT mutation.

Optimized small molecule antibody labeling efficiency through continuous flow centrifugal diafiltration.

PubMed

Cappione, Amedeo; Mabuchi, Masaharu; Briggs, David; Nadler, Timothy

2015-04-01

Protein immuno-detection encompasses a broad range of analytical methodologies, including western blotting, flow cytometry, and microscope-based applications. These assays which detect, quantify, and/or localize expression for one or more proteins in complex biological samples, are reliant upon fluorescent or enzyme-tagged target-specific antibodies. While small molecule labeling kits are available with a range of detection moieties, the workflow is hampered by a requirement for multiple dialysis-based buffer exchange steps that are both time-consuming and subject to sample loss. In a previous study, we briefly described an alternative method for small-scale protein labeling with small molecule dyes whereby all phases of the conjugation workflow could be performed in a single centrifugal diafiltration device. Here, we expand on this foundational work addressing functionality of the device at each step in the workflow (sample cleanup, labeling, unbound dye removal, and buffer exchange/concentration) and the implications for optimizing labeling efficiency. When compared to other common buffer exchange methodologies, centrifugal diafiltration offered superior performance as measured by four key parameters (process time, desalting capacity, protein recovery, retain functional integrity). Originally designed for resin-based affinity purification, the device also provides a platform for up-front antibody purification or albumin carrier removal. Most significantly, by exploiting the rapid kinetics of NHS-based labeling reactions, the process of continuous diafiltration minimizes reaction time and long exposure to excess dye, guaranteeing maximal target labeling while limiting the risks associated with over-labeling. Overall, the device offers a simplified workflow with reduced processing time and hands-on requirements, without sacrificing labeling efficiency, final yield, or conjugate performance. Copyright © 2015 Elsevier B.V. All rights reserved.

FACS-based isolation, propagation and characterization of mouse embryonic cardiomyocytes based on VCAM-1 surface marker expression.

PubMed

Pontén, Annica; Walsh, Stuart; Malan, Daniela; Xian, Xiaojie; Schéele, Susanne; Tarnawski, Laura; Fleischmann, Bernd K; Jovinge, Stefan

2013-01-01

Purification of cardiomyocytes from the embryonic mouse heart, embryonic stem (ES) or induced pluripotent stem cells (iPS) is a challenging task and will require specific isolation procedures. Lately the significance of surface markers for the isolation of cardiac cell populations with fluorescence activated cell sorting (FACS) has been acknowledged, and the hunt for cardiac specific markers has intensified. As cardiomyocytes have traditionally been characterized by their expression of specific transcription factors and structural proteins, and not by specific surface markers, this constitutes a significant bottleneck. Lately, Flk-1, c-kit and the cellular prion protein have been reported to specify cardiac progenitors, however, no surface markers have so far been reported to specify a committed cardiomyocyte. Herein show for the first time, that embryonic cardiomyocytes can be isolated with 98% purity, based on their expression of vascular cell adhesion molecule-1 (VCAM-1). The FACS-isolated cells express phenotypic markers for embryonic committed cardiomyocytes but not cardiac progenitors. An important aspect of FACS is to provide viable cells with retention of functionality. We show that VCAM-1 positive cardiomyocytes can be isolated with 95% viability suitable for in vitro culture, functional assays or expression analysis. In patch-clamp experiments we provide evidence of functionally intact cardiomyocytes of both atrial and ventricular subtypes. This work establishes that cardiomyocytes can be isolated with a high degree of purity and viability through FACS, based on specific surface marker expression as has been done in the hematopoietic field for decades. Our FACS protocol represents a significant advance in which purified populations of cardiomyocytes may be isolated and utilized for downstream applications, such as purification of ES-cell derived cardiomyocytes.

Production of first generation adenoviral vectors for preclinical protocols: amplification, purification and functional titration.

PubMed

Armendáriz-Borunda, Juan; Bastidas-Ramírez, Blanca Estela; Sandoval-Rodríguez, Ana; González-Cuevas, Jaime; Gómez-Meda, Belinda; García-Bañuelos, Jesús

2011-11-01

Gene therapy represents a promising approach in the treatment of several diseases. Currently, the ideal vector has yet to be designed; though, adenoviral vectors (Ad-v) have provided the most utilized tool for gene transfer due principally to their simple production, among other specific characteristics. Ad-v viability represents a critical variable that may be affected by storage or shipping conditions and therefore it is advisable to be assessed previously to protocol performance. The present work is unique in this matter, as the complete detailed process to obtain Ad-v of preclinical grade is explained. Amplification in permissive HEK-293 cells, purification in CsCl gradients in a period of 10 h, spectrophotometric titration of viral particles (VP) and titration of infectious units (IU), yielding batches of AdβGal, AdGFP, AdHuPA and AdMMP8, of approximately 10¹³-10¹⁴ VP and 10¹²-10¹³ IU were carried out. In vivo functionality of therapeutic AdHuPA and AdMMP8 was evidenced in rats presenting CCl₄-induced fibrosis, as more than 60% of fibrosis was eliminated in livers after systemic delivery through iliac vein in comparison with irrelevant AdβGal. Time required to accomplish the whole Ad-v production steps, including IU titration was 20 to 30 days. We conclude that production of Ad-v following standard operating procedures assuring vector functionality and the possibility to effectively evaluate experimental gene therapy results, leaving aside the use of high-cost commercial kits or sophisticated instrumentation, can be performed in a conventional laboratory of cell culture. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

FACS-Based Isolation, Propagation and Characterization of Mouse Embryonic Cardiomyocytes Based on VCAM-1 Surface Marker Expression

PubMed Central

Pontén, Annica; Walsh, Stuart; Malan, Daniela; Xian, Xiaojie; Schéele, Susanne; Tarnawski, Laura; Fleischmann, Bernd K.; Jovinge, Stefan

2013-01-01

Purification of cardiomyocytes from the embryonic mouse heart, embryonic stem (ES) or induced pluripotent stem cells (iPS) is a challenging task and will require specific isolation procedures. Lately the significance of surface markers for the isolation of cardiac cell populations with fluorescence activated cell sorting (FACS) has been acknowledged, and the hunt for cardiac specific markers has intensified. As cardiomyocytes have traditionally been characterized by their expression of specific transcription factors and structural proteins, and not by specific surface markers, this constitutes a significant bottleneck. Lately, Flk-1, c-kit and the cellular prion protein have been reported to specify cardiac progenitors, however, no surface markers have so far been reported to specify a committed cardiomyocyte. Herein show for the first time, that embryonic cardiomyocytes can be isolated with 98% purity, based on their expression of vascular cell adhesion molecule-1 (VCAM-1). The FACS-isolated cells express phenotypic markers for embryonic committed cardiomyocytes but not cardiac progenitors. An important aspect of FACS is to provide viable cells with retention of functionality. We show that VCAM-1 positive cardiomyocytes can be isolated with 95% viability suitable for in vitro culture, functional assays or expression analysis. In patch-clamp experiments we provide evidence of functionally intact cardiomyocytes of both atrial and ventricular subtypes. This work establishes that cardiomyocytes can be isolated with a high degree of purity and viability through FACS, based on specific surface marker expression as has been done in the hematopoietic field for decades. Our FACS protocol represents a significant advance in which purified populations of cardiomyocytes may be isolated and utilized for downstream applications, such as purification of ES-cell derived cardiomyocytes. PMID:24386094

Constipation and catharsis.

PubMed Central

Thompson, W. G.

1976-01-01

Constipation is endemic in the Western world. Stool consistence and associated symptoms are more important than stool frequency. The patient's attitude towards his bowel habit is also important. Exclusion of "organic" disease, reassurance and discussion of normal variations of bowel habit are necessary first steps in treatment. Adquate bulk in the stool must be ensured, either through diet or bulking agnets, and bowel retraining attempted. Laxatives should be reserved for episodes of constipation following enforced bedrest, or as a preparation for diagnostic procedures. Long-term use should be avoided. PMID:1268778

Bloody or tarry stools

MedlinePlus

... small intestine Diverticulosis (abnormal pouches in the colon) Hemorrhoids (common cause of bright red blood) Inflammatory bowel ... have an exam even if you think that hemorrhoids are causing the blood in your stool. In ...

Diagnosis of Parasitic Diseases

MedlinePlus

... that cause diarrhea, loose or watery stools, cramping, flatulence (gas) and other abdominal illness. CDC recommends that ... that cause diarrhea, loose or watery stools, cramping, flatulence (gas) and other abdominal illness. This test is ...

Flushable reagent stool blood test

MedlinePlus

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 ...

Multiple Myeloma Symptoms

MedlinePlus

... as walking or swimming, can also be helpful. Diarrhea Diarrhea is defined as frequent (more than three per ... loose or watery stools. If you experience severe diarrhea (more than six loose stools per day for ...

Impact of serum-derived bovine immunoglobulin/protein isolate therapy on irritable bowel syndrome and inflammatory bowel disease: a survey of patient perspective

PubMed Central

Shaw, Audrey L; Tomanelli, Adam; Bradshaw, Timothy P; Petschow, Bryon W; Burnett, Bruce P

2017-01-01

Background Patients with irritable bowel syndrome (IBS) or inflammatory bowel disease (IBD) commonly experience diarrhea, abdominal pain, bloating, and urgency. These symptoms significantly compromise the patient’s quality of life (QoL) by limiting participation in normal daily activities and adversely affect work productivity and performance. Purpose The aim of this study was to understand from the patient’s perspective how oral serum-derived bovine immunoglobulin/protein isolate (SBI) impacts bowel habits, management of condition, and basic QoL. Methods A 1-page questionnaire was distributed randomly to >14,000 patients who were prescribed SBI (EnteraGam®) for relevant intended uses. The survey was designed to collect data related to the influence of IBS or IBD on daily life activities and the impact of SBI usage on daily stool frequency, management of their condition, and QoL. Patient-reported responses were analyzed using a paired t-test to compare mean change in daily stool output and descriptive statistics for continuous variables. Results A total of 1,377 patients returned the surveys. Results from 595 surveys were analyzed with a focus on patients with IBS or IBD who had provided numeric responses regarding daily stool frequency. Respondents with IBS who reported having a normal stool frequency (≤4 stools per day) increased from 35% prior to using SBI to 91% while using SBI. A similar change toward normal stool frequency was reported by IBD respondents. Mean daily stool numbers decreased for respondents in the combined IBS and IBD groups (P=0.0001) from 6.5±4.3 before SBI to 2.6±1.9 following SBI use. The majority of respondents agreed strongly or very strongly that SBI helped them manage their condition (66.9%) and helped them return to the activities they enjoyed (59.1%). Conclusion Results from this patient survey suggest that SBI use can lead to clinically relevant decreases in daily stool frequency in patients with IBS or IBD along with improvements in the overall management of their condition and aspects of QoL. PMID:28615929

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.

PubMed

Arita, Minetaro; Ling, Hua; Yan, Dongmei; Nishimura, Yorihiro; Yoshida, Hiromu; Wakita, Takaji; Shimizu, Hiroyuki

2009-12-16

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. 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. 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. 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 direct detection of enterovirus from the stool extracts.

Oncogenic signaling by Kit tyrosine kinase occurs selectively on the Golgi apparatus in gastrointestinal stromal tumors

PubMed Central

Obata, Y; Horikawa, K; Takahashi, T; Akieda, Y; Tsujimoto, M; Fletcher, J A; Esumi, H; Nishida, T; Abe, R

2017-01-01

Gastrointestinal stromal tumors (GISTs) are caused by gain-of-function mutations in the Kit receptor tyrosine kinase. Most primary GIST patients respond to the Kit inhibitor imatinib, but this drug often becomes ineffective because of secondary mutations in the Kit kinase domain. The characteristic intracellular accumulation of imatinib-sensitive and -resistant Kit protein is well documented, but its relationship to oncogenic signaling remains unknown. Here, we show that in cancer tissue from primary GIST patients as well as in cell lines, mutant Kit accumulates on the Golgi apparatus, whereas normal Kit localizes to the plasma membrane (PM). In imatinib-resistant GIST with a secondary Kit mutation, Kit localizes predominantly on the Golgi apparatus. Both imatinib-sensitive and imatinib-resistant Kit (Kit(mut)) become fully auto-phosphorylated only on the Golgi and only if in a complex-glycosylated form. Kit(mut) accumulates on the Golgi during the early secretory pathway, but not after endocytosis. The aberrant kinase activity of Kit(mut) prevents its export from the Golgi to the PM. Furthermore, Kit(mut) on the Golgi signals and activates the phosphatidylinositol 3-kinase–Akt (PI3K–Akt) pathway, signal transducer and activator of transcription 5 (STAT5), and the Mek–Erk pathway. Blocking the biosynthetic transport of Kit(mut) to the Golgi from the endoplasmic reticulum inhibits oncogenic signaling. PM localization of Kit(mut) is not required for its signaling. Activation of Src-family tyrosine kinases on the Golgi is essential for oncogenic Kit signaling. These results suggest that the Golgi apparatus serves as a platform for oncogenic Kit signaling. Our study demonstrates that Kit(mut)’s pathogenicity is related to its mis-localization, and may offer a new strategy for treating imatinib-resistant GISTs. PMID:28192400

Protein kinase C-δ-mediated recycling of active KIT in colon cancer.

PubMed

Park, Misun; Kim, Won Kyu; Song, Meiying; Park, Minhee; Kim, Hyunki; Nam, Hye Jin; Baek, Sung Hee; Kim, Hoguen

2013-09-15

Abnormal signaling through receptor tyrosine kinase (RTK) moieties is important in tumorigenesis and drug targeting of colorectal cancers. Wild-type KIT (WT-KIT), a RTK that is activated upon binding with stem cell factor (SCF), is highly expressed in some colon cancers; however, little is known about the functional role of SCF-dependent KIT activation in colon cancer pathogenesis. We aimed to elucidate the conditions and roles of WT-KIT activation in colon cancer tumorigenesis. Colorectal cancers with KIT expression were characterized by immunoblotting and immunohistochemistry. The biologic alterations after KIT-SCF binding were analyzed with or without protein kinase C (PKC) activation. We found that WT-KIT was expressed in a subset of colon cancer cell lines and was activated by SCF, leading to activation of downstream AKT and extracellular signal-regulated kinase (ERK) signaling pathways. We also showed that KIT expression gradually decreased, after prolonged SCF stimulation, due to lysosomal degradation. Degradation of WT-KIT after SCF binding was significantly rescued when PKC was activated. We also showed the involvement of activated PKC-δ in the recycling of WT-KIT. We further showed that a subset of colorectal cancers exhibit expressions of both WT-KIT and activated PKC-δ and that expression of KIT is correlated with poor patient survival (P = 0.004). Continuous downstream signal activation after KIT-SCF binding is accomplished through PKC-δ-mediated recycling of KIT. This sustained KIT activation may contribute to tumor progression in a subset of colon cancers with KIT expression and might provide the rationale for a therapeutic approach targeting KIT. ©2013 AACR.

Creatinine

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Rotavirus antigen test

MedlinePlus

... stool samples. You can catch the stool on plastic wrap that is loosely placed over the toilet bowl ... young children wearing diapers, line the diaper with plastic wrap. Position the plastic wrap to prevent urine and ...

Giardiasis (For Parents)

MedlinePlus

... their stool. The stool can then contaminate public water supplies, community swimming pools, and water sources like lakes ... normal amounts of chlorine used to purify community water supplies, and can live for more than 2 months ...

Cardiac c-Kit Biology Revealed by Inducible Transgenesis.

PubMed

Gude, Natalie A; Firouzi, Fareheh; Broughton, Kathleen M; Ilves, Kelli; Nguyen, Kristine P; Payne, Christina R; Sacchi, Veronica; Monsanto, Megan M; Casillas, Alexandria R; Khalafalla, Farid G; Wang, Bingyan J; Ebeid, David E; Alvarez, Roberto; Dembitsky, Walter P; Bailey, Barbara A; van Berlo, Jop; Sussman, Mark A

2018-06-22

Biological significance of c-Kit as a cardiac stem cell marker and role(s) of c-Kit+ cells in myocardial development or response to pathological injury remain unresolved because of varied and discrepant findings. Alternative experimental models are required to contextualize and reconcile discordant published observations of cardiac c-Kit myocardial biology and provide meaningful insights regarding clinical relevance of c-Kit signaling for translational cell therapy. The main objectives of this study are as follows: demonstrating c-Kit myocardial biology through combined studies of both human and murine cardiac cells; advancing understanding of c-Kit myocardial biology through creation and characterization of a novel, inducible transgenic c-Kit reporter mouse model that overcomes limitations inherent to knock-in reporter models; and providing perspective to reconcile disparate viewpoints on c-Kit biology in the myocardium. In vitro studies confirm a critical role for c-Kit signaling in both cardiomyocytes and cardiac stem cells. Activation of c-Kit receptor promotes cell survival and proliferation in stem cells and cardiomyocytes of either human or murine origin. For creation of the mouse model, the cloned mouse c-Kit promoter drives Histone2B-EGFP (enhanced green fluorescent protein; H2BEGFP) expression in a doxycycline-inducible transgenic reporter line. The combination of c-Kit transgenesis coupled to H2BEGFP readout provides sensitive, specific, inducible, and persistent tracking of c-Kit promoter activation. Tagging efficiency for EGFP+/c-Kit+ cells is similar between our transgenic versus a c-Kit knock-in mouse line, but frequency of c-Kit+ cells in cardiac tissue from the knock-in model is 55% lower than that from our transgenic line. The c-Kit transgenic reporter model reveals intimate association of c-Kit expression with adult myocardial biology. Both cardiac stem cells and a subpopulation of cardiomyocytes express c-Kit in uninjured adult heart, upregulating c-Kit expression in response to pathological stress. c-Kit myocardial biology is more complex and varied than previously appreciated or documented, demonstrating validity in multiple points of coexisting yet heretofore seemingly irreconcilable published findings. © 2018 American Heart Association, Inc.

Advantage of using a home-made ELISA kit for detection of Helicobacter pylori infection over commercially imported kits.

PubMed

Mohammadi, M; Talebkhan, Y; Khalili, G; Mahboudi, F; Massarrat, S; Zamaninia, L; Oghalaei, A

2008-01-01

To evaluate a home-made ELISA kit for detection of Helicobacter pylori (Hp) infection and comparison of its immunologic criteria with those of foreign commercial kits. A home-made IgG ELISA kit was developed using soluble antigenic fractions of Hp proteins. Confirmed sera were tested and serological criteria were evaluated through assessment of 199 serum samples. The accuracy, sensitivity and specificity values of home-made kit were 92, 92 and 90.4%, respectively. These immunologic criteria for Trinity kit were 95.2, 95.2 and 95% in comparison with IBL kit (91.3, 92.2 and 88.5%), BIOHIT kit (72.4, 41.6 and 94.1%) and HelicoBlot2.1 (94.2, 93.4 and 100%). Kappa agreement assessment demonstrated that two of the imported ELISA kits had fair to moderate agreement with the home-made kit while the other one had a poor agreement value. Apart from comparable values between the home-made kit and the most efficient imported kit (Trinity) there was significant cost benefit. Therefore, we recommend the home-made kit as a suitable substitution for detection of Hp infection in the Iranian population.

Toxoplasmosis Testing

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Helicobacter pylori Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

VMA Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Vitamin A Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

B Vitamins Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Celiac Disease Tests

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Plasma Free Metanephrines

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Dengue Fever Testing

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Lactose Tolerance Tests

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Uric Acid Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Comprehensive Metabolic Panel

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

5-HIAA Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Phosphorus Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Peritoneal Fluid Analysis

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Serotonin Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Throat Culture

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

TB Screening Tests

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

PTH Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Blood Typing

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

AMA Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Progesterone Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Gram Stain

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Amebiasis (For Parents)

MedlinePlus

... the stool. When infected stool contaminates food or water supplies, amebiasis can spread quickly to many people at once. This is especially true in developing countries where drinking water may be contaminated. Amebiasis can also be spread ...

Bowel Movement

MedlinePlus

A bowel movement is the last stop in the movement of food through your digestive tract. Your stool passes out of ... what you eat and drink. Sometimes a bowel movement isn't normal. Diarrhea happens when stool passes ...

Straight Poop on Kids and Diarrhea

MedlinePlus

... home remedies for diarrhea like boiled milk or rice water. “Sports drinks are also not recommended for ... of 102 degrees or higher stools that are black and tarry stools containing blood or pus Blood ...

Stools - floating

MedlinePlus

... diagnosis. Alternative Names Floating stools Images Lower digestive anatomy References Schiller LR, Sellin JH. Diarrhea. In: Feldman M, Friedman LS, Brandt LJ, eds. Sleisenger and Fordtran's Gastrointestinal and Liver Disease . 10th ed. Philadelphia, PA: Elsevier Saunders; 2016: ...

Biliary atresia

MedlinePlus

... weight normally for the first month. After that point, the baby will lose weight and become irritable, and will have worsening jaundice. Other symptoms may include: Dark urine Enlarged spleen Floating stools Foul-smelling stools Pale or clay-colored ...

Performance evaluation of direct saline stool microscopy, Formol ether concentration and Kato Katz diagnostic methods for intestinal parasitosis in the absence of gold standard methods.

PubMed

Hailu, Tadesse; Abera, Bayeh

2015-07-01

The parasite load within the sample and the amount of sample taken during examination greatly compromise the sensitivity of direct saline stool microscopy. A cross-sectional study was conducted in March 2011 in Bahir Dar city among 778 fresh single stool samples to evaluate the performance of direct saline (DS), Kato Katz (KK) and Formol ether concentration (FEC) methods against the 'Gold' standard. Among 778 stool samples from school age children, the highest prevalence of intestinal parasites was recorded by FEC (55.1%). The sensitivity of DS, FEC and KK were 61.1%, 92.3% and 58.7%, respectively. FEC is more sensitive than DS and KK. Hence, use of the latter is preferred. © The Author(s) 2015.

Beta-glucuronidase and Beta-glucosidase activity in stool specimens of children with inflammatory bowel disease.

PubMed

Mroczyńska, Marta; Galecka, Miroslawa; Szachta, Patrycja; Kamoda, Dorota; Libudzisz, Zdzislawa; Roszak, Dorota

2013-01-01

The aim of the study was to analyze the differences in the activity of beta-glucuronidase and beta-glucosidase in stool specimens of children with Inflammatory Bowel Diseases (IBD) and healthy subjects. The disease activity was determined according to the PCDAI scale (Crohn disease) and Truelove-Witts scale (Ulcerative colitis). Enzyme activity was determined by spectrophotometry. There was a correlation between the level of beta - glucosidase activity in stool and patient's age in the group of healthy controls, but not in the IBD group. beta-glucosidase activity in IBD and healthy subjects stool specimens did not differ significantly. The activity of beta-glucuronidase in children with IBD was two times lower than in the healthy group and was correlated with age in children with IBD, but not in the group of healthy ones.

Effects of cereal fiber on bowel function: A systematic review of intervention trials

PubMed Central

de Vries, Jan; Miller, Paige E; Verbeke, Kristin

2015-01-01

AIM: To comprehensively review and quantitatively summarize results from intervention studies that examined the effects of intact cereal dietary fiber on parameters of bowel function. METHODS: A systematic literature search was conducted using PubMed and EMBASE. Supplementary literature searches included screening reference lists from relevant studies and reviews. Eligible outcomes were stool wet and dry weight, percentage water in stools, stool frequency and consistency, and total transit time. Weighted regression analyses generated mean change (± SD) in these measures per g/d of dietary fiber. RESULTS: Sixty-five intervention studies among generally healthy populations were identified. A quantitative examination of the effects of non-wheat sources of intact cereal dietary fibers was not possible due to an insufficient number of studies. Weighted regression analyses demonstrated that each extra g/d of wheat fiber increased total stool weight by 3.7 ± 0.09 g/d (P < 0.0001; 95%CI: 3.50-3.84), dry stool weight by 0.75 ± 0.03 g/d (P < 0.0001; 95%CI: 0.69-0.82), and stool frequency by 0.004 ± 0.002 times/d (P = 0.0346; 95%CI: 0.0003-0.0078). Transit time decreased by 0.78 ± 0.13 h per additional g/d (P < 0.0001; 95%CI: 0.53-1.04) of wheat fiber among those with an initial transit time greater than 48 h. CONCLUSION: Wheat dietary fiber, and predominately wheat bran dietary fiber, improves measures of bowel function. PMID:26269686

Development of an efficient entire-capsid-coding-region amplification method for direct detection of poliovirus from stool extracts.

PubMed

Arita, Minetaro; Kilpatrick, David R; Nakamura, Tomofumi; Burns, Cara C; Bukbuk, David; Oderinde, Soji B; Oberste, M Steven; Kew, Olen M; Pallansch, Mark A; Shimizu, Hiroyuki

2015-01-01

Laboratory diagnosis has played a critical role in the Global Polio Eradication Initiative since 1988, by isolating and identifying poliovirus (PV) from stool specimens by using cell culture as a highly sensitive system to detect PV. In the present study, we aimed to develop a molecular method to detect PV directly from stool extracts, with a high efficiency comparable to that of cell culture. We developed a method to efficiently amplify the entire capsid coding region of human enteroviruses (EVs) including PV. cDNAs of the entire capsid coding region (3.9 kb) were obtained from as few as 50 copies of PV genomes. PV was detected from the cDNAs with an improved PV-specific real-time reverse transcription-PCR system and nucleotide sequence analysis of the VP1 coding region. For assay validation, we analyzed 84 stool extracts that were positive for PV in cell culture and detected PV genomes from 100% of the extracts (84/84 samples) with this method in combination with a PV-specific extraction method. PV could be detected in 2/4 stool extract samples that were negative for PV in cell culture. In PV-positive samples, EV species C viruses were also detected with high frequency (27% [23/86 samples]). This method would be useful for direct detection of PV from stool extracts without using cell culture. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Evaluation of the kinase domain of c-KIT in canine cutaneous mast cell tumors

PubMed Central

Webster, Joshua D; Kiupel, Matti; Yuzbasiyan-Gurkan, Vilma

2006-01-01

Background Mutations in the c-KIT proto-oncogene have been implicated in the progression of several neoplastic diseases, including gastrointestinal stromal tumors and mastocytosis in humans, and cutaneous mast cell tumors (MCTs) in canines. Mutations in human mastocytosis patients primarily occur in c-KIT exon 17, which encodes a portion of its kinase domain. In contrast, deletions and internal tandem duplication (ITD) mutations are found in the juxtamembrane domain of c-KIT in approximately 15% of canine MCTs. In addition, ITD c-KIT mutations are significantly associated with aberrant KIT protein localization in canine MCTs. However, some canine MCTs have aberrant KIT localization but lack ITD c-KIT mutations, suggesting that other mutations or other factors may be responsible for aberrant KIT localization in these tumors. Methods In order to characterize the prevalence of mutations in the phospho-transferase portion of c-KIT's kinase domain in canine MCTs exons 16–20 of 33 canine MCTs from 33 dogs were amplified and sequenced. Additionally, in order to determine if mutations in c-KIT exon 17 are responsible for aberrant KIT localization in MCTs that lack juxtamembrane domain c-KIT mutations, c-KIT exon 17 was amplified and sequenced from 18 canine MCTs that showed an aberrant KIT localization pattern but did not have ITD c-KIT mutations. Results No mutations or polymorphisms were identified in exons 16–20 of any of the MCTs examined. Conclusion In conclusion, mutations in the phospho-transferase portion of c-KIT's kinase domain do not play an important role in the progression of canine cutaneous MCTs, or in the aberrant localization of KIT in canine MCTs. PMID:16579858

Adult cardiac stem cells are multipotent and robustly myogenic: c-kit expression is necessary but not sufficient for their identification.

PubMed

Vicinanza, Carla; Aquila, Iolanda; Scalise, Mariangela; Cristiano, Francesca; Marino, Fabiola; Cianflone, Eleonora; Mancuso, Teresa; Marotta, Pina; Sacco, Walter; Lewis, Fiona C; Couch, Liam; Shone, Victoria; Gritti, Giulia; Torella, Annalaura; Smith, Andrew J; Terracciano, Cesare Mn; Britti, Domenico; Veltri, Pierangelo; Indolfi, Ciro; Nadal-Ginard, Bernardo; Ellison-Hughes, Georgina M; Torella, Daniele

2017-12-01

Multipotent adult resident cardiac stem cells (CSCs) were first identified by the expression of c-kit, the stem cell factor receptor. However, in the adult myocardium c-kit alone cannot distinguish CSCs from other c-kit-expressing (c-kit pos ) cells. The adult heart indeed contains a heterogeneous mixture of c-kit pos cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kit pos cells has generated confusion and controversy about the existence and role of CSCs in the adult heart. Here, to unravel CSC identity within the heterogeneous c-kit-expressing cardiac cell population, c-kit pos cardiac cells were separated through CD45-positive or -negative sorting followed by c-kit pos sorting. The blood/endothelial lineage-committed (Lineage pos ) CD45 pos c-kit pos cardiac cells were compared to CD45 neg (Lineage neg /Lin neg ) c-kit pos cardiac cells for stemness and myogenic properties in vitro and in vivo. The majority (~90%) of the resident c-kit pos cardiac cells are blood/endothelial lineage-committed CD45 pos CD31 pos c-kit pos cells. In contrast, the Lin neg CD45 neg c-kit pos cardiac cell cohort, which represents ⩽10% of the total c-kit pos cells, contain all the cardiac cells with the properties of adult multipotent CSCs. These characteristics are absent from the c-kit neg and the blood/endothelial lineage-committed c-kit pos cardiac cells. Single Lin neg c-kit pos cell-derived clones, which represent only 1-2% of total c-kit pos myocardial cells, when stimulated with TGF-β/Wnt molecules, acquire full transcriptome and protein expression, sarcomere organisation, spontaneous contraction and electrophysiological properties of differentiated cardiomyocytes (CMs). Genetically tagged cloned progeny of one Lin neg c-kit pos cell when injected into the infarcted myocardium, results in significant regeneration of new CMs, arterioles and capillaries, derived from the injected cells. The CSC's myogenic regenerative capacity is dependent on commitment to the CM lineage through activation of the SMAD2 pathway. Such regeneration was not apparent when blood/endothelial lineage-committed c-kit pos cardiac cells were injected. Thus, among the cardiac c-kit pos cell cohort only a very small fraction has the phenotype and the differentiation/regenerative potential characteristics of true multipotent CSCs.

Pathologic Stimulus Determines Lineage Commitment of Cardiac C-kit+ Cells.

PubMed

Chen, Zhongming; Zhu, Wuqiang; Bender, Ingrid; Gong, Wuming; Kwak, Il-Youp; Yellamilli, Amritha; Hodges, Thomas J; Nemoto, Natsumi; Zhang, Jianyi; Garry, Daniel J; van Berlo, Jop H

2017-12-12

Although cardiac c-kit + cells are being tested in clinical trials, the circumstances that determine lineage differentiation of c-kit + cells in vivo are unknown. Recent findings suggest that endogenous cardiac c-kit + cells rarely contribute cardiomyocytes to the adult heart. We assessed whether various pathological stimuli differentially affect the eventual cell fates of c-kit + cells. We used single-cell sequencing and genetic lineage tracing of c-kit + cells to determine whether various pathological stimuli would result in different fates of c-kit + cells. Single-cell sequencing of cardiac CD45 - c-kit + cells showed innate heterogeneity, indicative of the existence of vascular and mesenchymal c-kit + cells in normal hearts. Cardiac pressure overload resulted in a modest increase in c-kit-derived cardiomyocytes, with significant increases in the numbers of endothelial cells and fibroblasts. Doxorubicin-induced acute cardiotoxicity did not increase c-kit-derived endothelial cell fates but instead induced cardiomyocyte differentiation. Mechanistically, doxorubicin-induced DNA damage in c-kit + cells resulted in expression of p53. Inhibition of p53 blocked cardiomyocyte differentiation in response to doxorubicin, whereas stabilization of p53 was sufficient to increase c-kit-derived cardiomyocyte differentiation. These results demonstrate that different pathological stimuli induce different cell fates of c-kit + cells in vivo. Although the overall rate of cardiomyocyte formation from c-kit + cells is still below clinically relevant levels, we show that p53 is central to the ability of c-kit + cells to adopt cardiomyocyte fates, which could lead to the development of strategies to preferentially generate cardiomyocytes from c-kit + cells. © 2017 American Heart Association, Inc.

Evaluation of Performance and Potential Clinical Impact of ProSpecT Shiga Toxin Escherichia coli Microplate Assay for Detection of Shiga Toxin-Producing E. coli in Stool Samples

PubMed Central

Gavin, Patrick J.; Peterson, Lance R.; Pasquariello, Anna C.; Blackburn, Joanna; Hamming, Mark G.; Kuo, Kuo J.; Thomson, Richard B.

2004-01-01

Shiga toxin-producing Escherichia coli bacteria (STEC) are emerging pathogens capable of producing sporadic and epidemic diarrhea, hemorrhagic colitis, and potentially life-threatening hemolytic-uremic syndrome. Although the presence of E. coli O157 can be readily detected in stool by sorbitol-MacConkey agar culture (SMAC), STEC non-O157 serotypes cannot. In contrast to culture, testing for the presence of Shiga toxins 1 and 2 in stool detects both O157 and non-O157 STEC serotypes capable of causing disease. Over two consecutive summers, we evaluated the performance of the ProSpecT Shiga toxin E. coli Microplate assay (Alexon-Trend, Ramsey, Minn.), an enzyme immunoassay for the detection of Shiga toxins 1 and 2, on all stools submitted for culture of enteric pathogens, and the potential clinical impact of Shiga toxin detection. Twenty-nine stool specimens were STEC positive by ProSpecT assay. Twenty-seven of 29 STEC-positive isolates were confirmed by SMAC and serotyping or by a second enzyme immunoassay and PCR (positive predictive value, 93%). Thirteen of 27 confirmed Shiga toxin-producing strains were serotype O157. The remaining 14 strains represented 8 other serotypes. The ProSpecT assay was 100% sensitive and specific for detection of E. coli O157 in stool (7 of 7) compared to SMAC. In addition, the ProSpecT assay detected twice as many STEC as SMAC. Fifty-two percent of confirmed STEC-positive stools were nonbloody. Thus, in our population, screening strategies that test only visibly bloody stools for STEC would miss a majority of cases. Eleven (41%) STEC-positive patients were hospitalized, and eight (30%) developed severe disease (two developed hemolytic-uremic syndrome, and six developed hemorrhagic colitis). Prior to detection of STEC infection, seven (26%) and eight patients (30%) underwent unnecessary diagnostic procedures or received potentially deleterious empirical treatment, respectively. We propose that establishing a specific diagnosis of STEC may have prevented these potentially harmful interventions. We conclude that the ProSpecT assay is sensitive and specific for the detection of Shiga toxins 1 and 2 in stool and has potentially significant clinical impact for the individual patient and public health. Shiga toxin assays should be considered for routine use in settings where prevalence of STEC disease warrants testing. PMID:15071021

Survey of gastrointestinal reactions to foods in adults in relation to atopy, presence of mucus in the stools, swelling of joints and arthralgia in patients with gastrointestinal reactions to foods.

PubMed

Bengtsson, U; Hanson, L A; Ahlstedt, S

1996-12-01

Food intolerance in adults is mostly associated with vague symptoms and not clearly related to atopy and food allergy. A combination of different pathogenetic mechanisms may be responsible for the symptoms. The aim of this study was to describe patients with a history of food-related gastrointestinal symptoms in relation to the presence of mucus in the stools, joint swelling and arthralgia and to determine whether or not there is an association between the presence of these parameters, atopic disease and the presence of immune complexes in serum. Fifty-eight patients consecutively referred to our clinic with food-related gastrointestinal symptoms were investigated. Thirty-five patients (60%) had mucus in their stools, 24 patients (41%) complained about joint swelling and 41 patients (71%) had arthralgia. There were no correlations between these parameters and atopy according to Phadiatope test or skin prick test (SPT). No correlations were found between the occurrence of mucus in the stools, arthralgia and joint swelling. There were significantly higher levels of circulating immune complexes in patients with a history of arthralgia compared with patients with no such history (P < 0.03) and the number of individuals with the presence of such immune complexes was higher among the patients with arthralgia than among the patients without. In general the patients did not relate the exposure to certain foods to symptoms like joint swelling, arthralgia and presence of mucus in the stools. However, there were significant positive correlations between food-related gastrointestinal symptoms in the following instances: chocolate-induced gastrointestinal symptoms and mucus in the stools (P = 0.006), vegetable-induced gastrointestinal symptoms and mucus in the stools (P = 0.002) and meat-induced gastrointestinal symptoms and mucus in the stools (P = 0.003). In a group of individuals without food-related symptoms investigated separately, a very low frequency of mucus in the stools, joint swelling and arthralgia was seen (none, two and three individuals of the 20 subjects, respectively). Of 41 patients with immediate onset of gastrointestinal symptoms, 20 were atopic according to Phadiatope and SPT. Of 11 patients with late onset of symptoms 10 were negative in Phadiatope and SPT (P < 0.05). The most frequently involved foods were fruits, vegetables, milk, fish and meat. The results suggest the involvement of different inflammatogenic mechanisms in food-related gastrointestinal symptoms.

Stool-based biomarkers of interstitial cystitis/bladder pain syndrome.

PubMed

Braundmeier-Fleming, A; Russell, Nathan T; Yang, Wenbin; Nas, Megan Y; Yaggie, Ryan E; Berry, Matthew; Bachrach, Laurie; Flury, Sarah C; Marko, Darlene S; Bushell, Colleen B; Welge, Michael E; White, Bryan A; Schaeffer, Anthony J; Klumpp, David J

2016-05-18

Interstitial cystitis/bladder pain syndrome (IC) is associated with significant morbidity, yet underlying mechanisms and diagnostic biomarkers remain unknown. Pelvic organs exhibit neural crosstalk by convergence of visceral sensory pathways, and rodent studies demonstrate distinct bacterial pain phenotypes, suggesting that the microbiome modulates pelvic pain in IC. Stool samples were obtained from female IC patients and healthy controls, and symptom severity was determined by questionnaire. Operational taxonomic units (OTUs) were identified by16S rDNA sequence analysis. Machine learning by Extended Random Forest (ERF) identified OTUs associated with symptom scores. Quantitative PCR of stool DNA with species-specific primer pairs demonstrated significantly reduced levels of E. sinensis, C. aerofaciens, F. prausnitzii, O. splanchnicus, and L. longoviformis in microbiota of IC patients. These species, deficient in IC pelvic pain (DIPP), were further evaluated by Receiver-operator characteristic (ROC) analyses, and DIPP species emerged as potential IC biomarkers. Stool metabolomic studies identified glyceraldehyde as significantly elevated in IC. Metabolomic pathway analysis identified lipid pathways, consistent with predicted metagenome functionality. Together, these findings suggest that DIPP species and metabolites may serve as candidates for novel IC biomarkers in stool. Functional changes in the IC microbiome may also serve as therapeutic targets for treating chronic pelvic pain.

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

PubMed Central

2011-01-01

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. PMID:21875444

von Willebrand Factor Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

ADH (Antidiuretic Hormone) Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Anti-Müllerian Hormone

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

C-Reactive Protein (CRP) Test

MedlinePlus

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GGT (Gamma-Glutamyl Transferase) Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Catecholamines, Plasma and Urine Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

PT and INR Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Pleural Fluid Analysis Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

T3 (Triiodothyronine) Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Tips on Blood Testing

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

CEA (Carcinoembryonic Antigen) Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Ketorolac

MedlinePlus

... heartburn, vomit that is bloody or looks like coffee grounds, blood in the stool, or black and tarry stools.Ketorolac may cause kidney failure. Tell your doctor if you have kidney or liver disease, if you have had severe vomiting or diarrhea ...

Accidental Bowel Leakage

MedlinePlus

... Women with a bowel control problem may leak gas or liquid or solid stool. Other symptoms may include the ... which can lead to leakage of solid or liquid stool (feces) or gas. Anoscopy: An exam of the anus using an ...

Receptor tyrosine kinase (c-Kit) inhibitors: a potential therapeutic target in cancer cells

PubMed Central

Abbaspour Babaei, Maryam; Kamalidehghan, Behnam; Saleem, Mohammad; Huri, Hasniza Zaman; Ahmadipour, Fatemeh

2016-01-01

c-Kit, a receptor tyrosine kinase, is involved in intracellular signaling, and the mutated form of c-Kit plays a crucial role in occurrence of some cancers. The function of c-Kit has led to the concept that inhibiting c-Kit kinase activity can be a target for cancer therapy. The promising results of inhibition of c-Kit for treatment of cancers have been observed in some cancers such as gastrointestinal stromal tumor, acute myeloid leukemia, melanoma, and other tumors, and these results have encouraged attempts toward improvement of using c-Kit as a capable target for cancer therapy. This paper presents the findings of previous studies regarding c-Kit as a receptor tyrosine kinase and an oncogene, as well as its gene targets and signaling pathways in normal and cancer cells. The c-Kit gene location, protein structure, and the role of c-Kit in normal cell have been discussed. Comprehending the molecular mechanism underlying c-Kit-mediated tumorogenesis is consequently essential and may lead to the identification of future novel drug targets. The potential mechanisms by which c-Kit induces cellular transformation have been described. This study aims to elucidate the function of c-Kit for future cancer therapy. In addition, it has c-Kit inhibitor drug properties and their functions have been listed in tables and demonstrated in schematic pictures. This review also has collected previous studies that targeted c-Kit as a novel strategy for cancer therapy. This paper further emphasizes the advantages of this approach, as well as the limitations that must be addressed in the future. Finally, although c-Kit is an attractive target for cancer therapy, based on the outcomes of treatment of patients with c-Kit inhibitors, it is unlikely that Kit inhibitors alone can lead to cure. It seems that c-Kit mutations alone are not sufficient for tumorogenesis, but do play a crucial role in cancer occurrence. PMID:27536065

Validation of reference materials for uranium radiochronometry in the frame of nuclear forensic investigations

DOE PAGES

Varga, Z.; Mayer, K.; Bonamici, C. E.; ...

2015-05-11

The results of a joint effort by expert nuclear forensic laboratories in the area of age dating of uranium, i.e. the elapsed time since the last chemical purification of the material are presented and discussed. Completely separated uranium materials of known production date were distributed among the laboratories, and the samples were dated according to routine laboratory procedures by the measurement of the ²²⁰Th/²³⁴U ratio. The measurement results were in good agreement with the known production date showing that the concept for preparing uranium age dating reference material based on complete separation is valid. Detailed knowledge of the laboratory proceduresmore » used for uranium age dating allows the identification of possible improvements in the current protocols and the development of improved practice in the future. The availability of age dating reference materials as well as the evolvement of the age dating best-practice protocol will increase the relevance and applicability of age dating as part of the tool-kit available for nuclear forensic investigations.« less

Facile Recovery of Individual High-Molecular-Weight, Low-Copy-Number Natural Plasmids for Genomic Sequencing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Williams, L.E.; Detter, C,; Barrie, K.

2006-06-01

Sequencing of the large (>50 kb), low-copy-number (<5 per cell) plasmids that mediate horizontal gene transfer has been hindered by the difficulty and expense of isolating DNA from individual plasmids of this class. We report here that a kit method previously devised for purification of bacterial artificial chromosomes (BACs) can be adapted for effective preparation of individual plasmids up to 220 kb from wild gram-negative and gram-positive bacteria. Individual plasmid DNA recovered from less than 10 ml of Escherichia coli, Staphylococcus, and Corynebacterium cultures was of sufficient quantity and quality for construction of highcoverage libraries, as shown by sequencing fivemore » native plasmids ranging in size from 30 kb to 94 kb. We also report recommendations for vector screening to optimize plasmid sequence assembly, preliminary annotation of novel plasmid genomes, and insights on mobile genetic element biology derived from these sequences. Adaptation of this BAC method for large plasmid isolation removes one major technical hurdle to expanding our knowledge of the natural plasmid gene pool.« less

An Optimized DNA Analysis Workflow for the Sampling, Extraction, and Concentration of DNA obtained from Archived Latent Fingerprints.

PubMed

Solomon, April D; Hytinen, Madison E; McClain, Aryn M; Miller, Marilyn T; Dawson Cruz, Tracey

2018-01-01

DNA profiles have been obtained from fingerprints, but there is limited knowledge regarding DNA analysis from archived latent fingerprints-touch DNA "sandwiched" between adhesive and paper. Thus, this study sought to comparatively analyze a variety of collection and analytical methods in an effort to seek an optimized workflow for this specific sample type. Untreated and treated archived latent fingerprints were utilized to compare different biological sampling techniques, swab diluents, DNA extraction systems, DNA concentration practices, and post-amplification purification methods. Archived latent fingerprints disassembled and sampled via direct cutting, followed by DNA extracted using the QIAamp® DNA Investigator Kit, and concentration with Centri-Sep™ columns increased the odds of obtaining an STR profile. Using the recommended DNA workflow, 9 of the 10 samples provided STR profiles, which included 7-100% of the expected STR alleles and two full profiles. Thus, with carefully selected procedures, archived latent fingerprints can be a viable DNA source for criminal investigations including cold/postconviction cases. © 2017 American Academy of Forensic Sciences.

Validation of reference materials for uranium radiochronometry in the frame of nuclear forensic investigations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Varga, Z.; Mayer, K.; Bonamici, C. E.

The results of a joint effort by expert nuclear forensic laboratories in the area of age dating of uranium, i.e. the elapsed time since the last chemical purification of the material are presented and discussed. Completely separated uranium materials of known production date were distributed among the laboratories, and the samples were dated according to routine laboratory procedures by the measurement of the ²²⁰Th/²³⁴U ratio. The measurement results were in good agreement with the known production date showing that the concept for preparing uranium age dating reference material based on complete separation is valid. Detailed knowledge of the laboratory proceduresmore » used for uranium age dating allows the identification of possible improvements in the current protocols and the development of improved practice in the future. The availability of age dating reference materials as well as the evolvement of the age dating best-practice protocol will increase the relevance and applicability of age dating as part of the tool-kit available for nuclear forensic investigations.« less

Protein Crystallization.

PubMed

McPherson, Alexander

2017-01-01

Protein crystallization was discovered by chance nearly 200 years ago and was developed in the late nineteenth century as a powerful purification tool, and a demonstration of chemical purity. The crystallization of proteins, nucleic acids, and large biological complexes, such as viruses, depends on the creation of a solution that is supersaturated in the macromolecule, but exhibits conditions that do not significantly perturb its natural state. Supersaturation is produced through the addition of mild precipitating agents such as neutral salts or polymers, and by manipulation of various parameters that include temperature, ionic strength, and pH. Also important in the crystallization process are factors that can affect the structural state of the macromolecule, such as metal ions, inhibitors, cofactors, or other conventional small molecules. A variety of approaches have been developed that combine the spectrum of factors that effect and promote crystallization, and among the most widely used are vapor diffusion, dialysis, batch, and liquid-liquid diffusion. Successes in macromolecular crystallization have multiplied rapidly in recent years due to the advent of practical, easy-to-use screening kits, and the application of laboratory robotics.

Construction, expression, purification and biotin labeling of a single recombinant multi-epitope antigen for double-antigen sandwich ELISA to detect hepatitis C virus antibody.

PubMed

He, Jing; Xiu, Bingshui; Wang, Guohua; Chen, Kun; Feng, Xiaoyan; Song, Xiaoguo; Zhu, Cuixia; Yang, Xiqin; Bai, Guanzhong; Ling, Shigan; Zhang, Heqiu

2011-08-01

Based on B cell epitope predictions, a recombinant antigen with multiple epitopes from four Hepatitis C Virus fragments (C, NS3, NS4 and NS5) were engineered. The recombinant gene was then highly expressed in E. coli. The non-modified and C-terminal-modified recombinant proteins were used for coating and biotin labeling, respectively, to establish the double-antigen sandwich ELISA. Ten positive reference samples confirmed by the CHIRON RIBA HCV 3.0 SIA kit were detected positive, Forty one plasma samples were positive among samples from 441 volunteers, which indicated that the recombinant antigen could readily react well with plasma HCV antibody. As critical reagents of double-antigen sandwich ELISA, the recombinant multi-epitope antigen and the C-terminal-modified and biotin-conjugated antigen show good antigenicity. In this study, we provide a simple approach to produce multiple epitopes within one recombinant protein in order to avoid the costly expression of less-effective pools of multiple proteins, which is the conventional strategy of diagnostic antigen production for HCV antibody detection.

Effect of dietary fiber on constipation: A meta analysis

PubMed Central

Yang, Jing; Wang, Hai-Peng; Zhou, Li; Xu, Chun-Fang

2012-01-01

AIM: To investigate the effect of dietary fiber intake on constipation by a meta-analysis of randomized controlled trials (RCTs). METHODS: We searched Ovid MEDLINE (from 1946 to October 2011), Cochrane Library (2011), PubMed for articles on dietary fiber intake and constipation using the terms: constipation, fiber, cellulose, plant extracts, cereals, bran, psyllium, or plantago. References of important articles were searched manually for relevant studies. Articles were eligible for the meta-analysis if they were high-quality RCTs and reported data on stool frequency, stool consistency, treatment success, laxative use and gastrointestinal symptoms. The data were extracted independently by two researchers (Yang J and Wang HP) according to the described selection criteria. Review manager version 5 software was used for analysis and test. Weighted mean difference with 95%CI was used for quantitative data, odds ratio (OR) with 95%CI was used for dichotomous data. Both I2 statistic with a cut-off of ≥ 50% and the χ2 test with a P value < 0.10 were used to define a significant degree of heterogeneity. RESULTS: We searched 1322 potential relevant articles, 19 of which were retrieved for further assessment, 14 studies were excluded for various reasons, five studies were included in the analysis. Dietary fiber showed significant advantage over placebo in stool frequency (OR = 1.19; 95%CI: 0.58-1.80, P < 0.05). There was no significant difference in stool consistency, treatment success, laxative use and painful defecation between the two groups. Stool frequency were reported by five RCTs, all results showed either a trend or a significant difference in favor of the treatment group, number of stools per week increased in treatment group than in placebo group (OR = 1.19; 95%CI: 0.58-1.80, P < 0.05), with no significant heterogeneity among studies (I2= 0, P = 0.77). Four studies evaluated stool consistency, one of them presented outcome in terms of percentage of hard stool, which was different from others, so we included the other three studies for analysis. Two studies reported treatment success. There was significant heterogeneity between the studies (P < 0.1, I2 > 50%). Three studies reported laxative use, quantitative data was shown in one study, and the pooled analysis of the other two studies showed no significant difference between treatment and placebo groups in laxative use (OR = 1.07; 95%CI 0.51-2.25), and no heterogeneity was found (P = 0.84, I2= 0). Three studies evaluated painful defecation: one study presented both quantitative and dichotomous data, the other two studies reported quantitative and dichotomous data separately. We used dichotomous data for analysis. CONCLUSION: Dietary fiber intake can obviously increase stool frequency in patients with constipation. It does not obviously improve stool consistency, treatment success, laxative use and painful defecation. PMID:23326148

SHP-1 Binds and Negatively Modulates the c-Kit Receptor by Interaction with Tyrosine 569 in the c-Kit Juxtamembrane Domain

PubMed Central

Kozlowski, Maya; Larose, Louise; Lee, Fai; Le, Duc Mingh; Rottapel, Robert; Siminovitch, Katherine A.

1998-01-01

The SH2 domain-containing SHP-1 tyrosine phosphatase has been shown to negatively regulate a broad spectrum of growth factor- and cytokine-driven mitogenic signaling pathways. Included among these is the cascade of intracellular events evoked by stem cell factor binding to c-Kit, a tyrosine kinase receptor which associates with and is dephosphorylated by SHP-1. Using a series of glutathione S-transferase (GST) fusion proteins containing either tyrosine-phosphorylated segments of the c-Kit cytosolic region or the SH2 domains of SHP-1, we have shown that SHP-1 interacts with c-Kit by binding selectively to the phosphorylated c-Kit juxtamembrane region and that the association of c-Kit with the larger of the two SHP-1 isoforms may be mediated through either the N-terminal or C-terminal SHP-1 SH2 domain. The results of binding assays with mutagenized GST-Kit juxtamembrane fusion proteins and competitive inhibition assays with phosphopeptides encompassing each c-Kit juxtamembrane region identified the tyrosine residue at position 569 as the major site for binding of SHP-1 to c-Kit and suggested that tyrosine 567 contributes to, but is not required for, this interaction. By analysis of Ba/F3 cells retrovirally transduced to express c-Kit receptors, phenylalanine substitution of c-Kit tyrosine residue 569 was shown to be associated with disruption of c-Kit–SHP-1 binding and induction of hyperproliferative responses to stem cell factor. Although phenylalanine substitution of c-Kit tyrosine residue 567 in the Ba/F3–c-Kit cells did not alter SHP-1 binding to c-Kit, the capacity of a second c-Kit-binding tyrosine phosphatase, SHP-2, to associate with c-Kit was markedly reduced, and the cells again showed hyperproliferative responses to stem cell factor. These data therefore identify SHP-1 binding to tyrosine 569 on c-Kit as an interaction pivotal to SHP-1 inhibitory effects on c-Kit signaling, but they indicate as well that cytosolic protein tyrosine phosphatases other than SHP-1 may also negatively regulate the coupling of c-Kit engagement to proliferation. PMID:9528781

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

PubMed Central

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

2018-01-01

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 < 0.001). PE was strongly associated with dietary fibre intake (adjusted regression model; unstandardised B −1.44 (95% CI −1.62, −1.24) g/day, p < 0.001). PE had a lower percentage of fibre from vegetables compared with NPE (8.9% vs 15.7%, respectively, p < 0.001). There was an association between PE and usually having 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. PMID:26879221

Detection of KRAS G12D in colorectal cancer stool by droplet digital PCR

PubMed Central

Olmedillas-López, Susana; Lévano-Linares, Dennis César; Alexandre, Carmen Laura Aúz; Vega-Clemente, Luz; Sánchez, Edurne León; Villagrasa, Alejandro; Ruíz-Tovar, Jaime; García-Arranz, Mariano; García-Olmo, Damián

2017-01-01

AIM To assess KRAS G12D mutation detection by droplet digital PCR (ddPCR) in stool-derived DNA from colorectal cancer (CRC) patients. METHODS In this study, tumor tissue and stool samples were collected from 70 patients with stage I-IV CRC diagnosed by preoperative biopsy. KRAS mutational status was determined by pyrosequencing analysis of DNA obtained from formalin-fixed paraffin-embedded (FFPE) tumor tissues. The KRAS G12D mutation was then analyzed by ddPCR in FFPE tumors and stool-derived DNA from patients with this point mutation. Wild-type (WT) tumors, as determined by pyrosequencing, were included as controls; analysis of FFPE tissue and stool-derived DNA by ddPCR was performed for these patients as well. RESULTS Among the total 70 patients included, KRAS mutations were detected by pyrosequencing in 32 (45.71%), whereas 38 (54.29%) had WT tumors. The frequency of KRAS mutations was higher in left-sided tumors (11 located in the right colon, 15 in the left, and 6 in the rectum). The predominant point mutation was KRAS G12D (14.29%, n = 10), which was more frequent in early-stage tumors (I-IIA, n = 7). In agreement with pyrosequencing results, the KRAS G12D mutation was detected by ddPCR in FFPE tumor-derived DNA, and only a residual number of mutated copies was found in WT controls. The KRAS G12D mutation was also detected in stool-derived DNA in 80% of all fecal samples from CRC patients with this point mutation. CONCLUSION ddPCR is a reliable and sensitive method to analyze KRAS G12D mutation in stool-derived DNA from CRC patients, especially at early stages. This non-invasive approach is potentially applicable to other relevant biomarkers for CRC management. PMID:29093617

Distribution of rotavirus genotypes associated with acute diarrhoea in Zimbabwean children less than five years old before and after rotavirus vaccine introduction.

PubMed

Mukaratirwa, Arnold; Berejena, Chipo; Nziramasanga, Pasipanodya; Ticklay, Ismail; Gonah, Archebald; Nathoo, Kusum; Manangazira, Portia; Mangwanya, Douglas; Marembo, Joan; Mwenda, Jason M; Weldegebriel, Goitom; Seheri, Mapaseka; Tate, Jacqueline E; Yen, Catherine; Parashar, Umesh; Mujuru, Hilda

2018-04-05

Sentinel surveillance for diarrhoea is important to monitor changes in rotavirus epidemiological trends and circulating genotypes among children under 5 years before and after vaccine introduction. The Zimbabwe Ministry of Health and Child Care introduced rotavirus vaccine in national immunization program in May 2014. Active hospital-based surveillance for diarrhoea was conducted at 3 sentinel sites from 2008 to 2016. Children aged less than 5 years, who presented with acute gastroenteritis as a primary illness and who were admitted to a hospital ward or treated at the emergency unit, were enrolled and had a stool specimen collected and tested for rotavirus by enzyme immunoassay (EIA). Genotyping of positive stools was performed using reverse-transcription polymerase chain reaction and genotyping assays. Pre-vaccine introduction, 10% of all positive stool specimens were genotyped and all adequate positive stools were genotyped post-vaccine introduction. During the pre-vaccine period, a total of 6491 acute gastroenteritis stools were collected, of which 3016 (46%) tested positive for rotavirus and 312 (10%) of the rotavirus positive stools were genotyped. During the post-vaccine period, a total of 3750 acute gastroenteritis stools were collected, of which 937 (25%) tested positive for rotavirus and 784 (84%) were genotyped. During the pre-vaccine introduction the most frequent genotype was G9P[8] (21%) followed by G2P[4] (12%), G1P[8] (6%), G2P[6] (5%), G12P[6] (4%), G9P[6] (3%) and G8P[4] (3%). G1P[8] (30%) was most dominant two years after vaccine introduction followed by G9P[6] (20%), G2P[4] (15%), G9P[8] (11%) and G1P[6] (4%). The decline in positivity rate is an indication of early vaccine impact. Diversity of circulating strains underscores the importance of continued monitoring and strain surveillance after vaccine introduction. Copyright © 2018. Published by Elsevier Ltd.

Xpert MTB/RIF diagnosis of childhood tuberculosis from sputum and stool samples in a high TB-HIV-prevalent setting.

PubMed

Orikiriza, Patrick; Nansumba, Margaret; Nyehangane, Dan; Bastard, Mathieu; Mugisha, Ivan Taremwa; Nansera, Denis; Mwanga-Amumpaire, Juliet; Boum, Yap; Kumbakumba, Elias; Bonnet, Maryline

2018-05-08

The Xpert MTB/RIF assay is a major advance for diagnosis of tuberculosis (TB) in high-burden countries but is limited in children by their difficulty to produce sputum. We investigated TB in sputum and stool from children with the aim of improving paediatric TB diagnosis. A prospective cohort of children with presumptive TB, provided two sputum or induced sputum at enrolment in a regional referral hospital in Uganda. Stool was collected from those started on TB treatment. All specimen were tested for Xpert MTB/RIF, mycobacteria growth indicator tube (MGIT), Lowenstein Jensen cultures and microscopy (except stool). We compared TB detection between age categories and assessed the performance of Xpert MTB/RIF in sputum and stool. Of the 392 children enrolled, 357 (91.1%) produced at least one sputum sample. Sputum culture yield was 13/357 (3.6%): 3/109 (2.6%), 3/89 (3.2%), 3/101 (2.6%) and 4/44 (8.2%) among children of < 2, 2-5, ≥ 5-10 and > 10 years, respectively (p = 0.599). Xpert MTB/RIF yield was 14/350 (4.0%): 3/104 (2.9%), 4/92 (4.3%), 3/88 (2.9%) and 4/50 (.0%), respectively (p = 0.283). Sensitivity and specificity of Xpert MTB/RIF in sputum against sputum culture were 90.9% (95% CI 58.7-99.8) and 99.1% (99.1-99.8). In stool, it was 55.6% (21.2-86.3) and 98.2% (98.2-100) against Xpert MTB/RIF and culture in sputum. Only a minority of children had microbiologically confirmed TB with a higher proportion in children above 10 years. Although sensitivity of Xpert MTB/RIF in stool was low, with good optimization, it might be a good alternative to sputum in children.

Diagnosis of amphimeriasis by LAMPhimerus assay in human stool samples long-term storage onto filter paper

PubMed Central

Calvopiña, Manuel; Buendía-Sánchez, María; López-Abán, Julio; Vicente, Belén; Muro, Antonio

2018-01-01

Amphimeriasis, a fish-borne zoonotic disease caused by the liver fluke Amphimerus spp., has recently been reported as an emerging disease affecting an indigenous Ameridian group, the Chachi, living in Ecuador. The only method for diagnosing amphimeriasis was the microscopic detection of eggs from the parasite in patients' stool samples with very low sensitivity. Our group developed an ELISA technique for detection of anti-Amphimerus IgG in human sera and a molecular method based on LAMP technology (named LAMPhimerus) for specific and sensitive parasite DNA detection. The LAMPhimerus method showed to be much more sensitive than classical parasitological methods for amphimeriasis diagnosis using human stool samples for analysis. The objective of this work is to demonstrate the feasibility of using dried stool samples on filter paper as source of DNA in combination with the effectiveness of our previously designed LAMPhimerus assay for successfully Amphimerus sp. detection in clinical stool samples. A total of 102 untreated and undiluted stool samples collected from Chachi population were spread as thin layer onto common filter paper for easily transportation to our laboratory and stored at room temperature for one year until DNA extraction. When LAMPhimerus method was applied for Amphimerus sp. DNA detection, a higher number of positive results was detected (61/102; 59.80%) in comparison to parasitological methods (38/102; 37.25%), including 28/61 (45.90%) microscopy-confirmed Amphimerus sp. infections. The diagnostic parameters for the sensitivity and specificity werecalculated for our LAMPhimerus assay, which were 79.17% and 65.98%, respectively. We demonstrate, for the first time, that common filter paper is useful for easy collection and long-term storage of human stool samples for later DNA extraction and molecular analysis of human-parasitic trematode eggs. This simple, economic and easily handling method combined with the specific and sensible LAMPhimerus assay has the potential to beused as an effective molecular large-scale screening test for amphimeriasis-endemic areas. PMID:29444135

Gut microbial and short-chain fatty acid profiles in adults with chronic constipation before and after treatment with lubiprostone.

PubMed

Kang, Dae-Wook; DiBaise, John K; Ilhan, Zehra Esra; Crowell, Michael D; Rideout, Jai Ram; Caporaso, J Gregory; Rittmann, Bruce E; Krajmalnik-Brown, Rosa

2015-06-01

Identifying specific gut microorganisms associated with chronic constipation may be useful for diagnostic and therapeutic purposes. The objective of this study was to evaluate whether or not the gut microbial community of constipated subjects had specific microbial signatures and to assess the effects of lubiprostone treatment on the gut microbial community. Stool diaries, breath H2 and CH4 levels, and stool samples were collected from ten healthy subjects and nine patients meeting the Rome III criteria for chronic functional constipation. Constipated subjects received lubiprostone for four weeks, during which stool diaries were maintained. Stool samples were evaluated for gut microbial communities using pyrosequencing and quantitative real-time PCR (qPCR) targeting 16S-rRNA gene, along with concentrations of short-chain fatty acids (SCFAs) using high-performance liquid chromatography. Prior to treatment, gut microbial profiles were similar between constipated subjects and healthy subjects, while iso-butyrate levels were significantly higher in constipated subjects compared with healthy subjects. Despite increases in stool frequency and improvements in consistency after lubiprostone treatment, gut microbial profiles and community diversity after treatment showed no significant change compared to before treatment. While we did not observe a significant difference in either breath methane or archaeal abundance between the stool samples of healthy and constipated subjects, we confirmed a strong correlation between archaeal abundance measured by qPCR and the amount of methane gas exhaled in the fasting breath. Butyrate levels, however, were significantly higher in the stool samples of constipated subjects after lubiprostone treatment, suggesting that lubiprostone treatment had an effect on the net accumulation of SCFAs in the gut. In conclusion, lubiprostone treatment improved constipation symptoms and increased levels of butyrate without substantial modification of the gut microbial structure. Copyright © 2015 Elsevier Ltd. All rights reserved.

[Serotype identification and antibiotic susceptibility of Shiga toxin-producing Escherichia coli in the Weishan area in Shandong Province, China].

PubMed

Shao, C C; Hu, B; Bi, Z W; Kou, Z Q; Fang, M; Chen, B L; Bi, Z Q

2017-01-06

Objective: To determine the serotypes and drug resistance profiles of Shiga toxin-producing Escherichia coli (STEC) in animal stools from the Weishan area in Shandong Province, China. To provide the basis for further study. Methods: Five hundred animal stool samples (from pigs, cattle, sheep, dogs and birds) were collected from the Weishan area and STEC strains were isolated from these samples. Strains were serotyped by a serum agglutination test, and their drug resistance profiles were determined through antimicrobial sensitivity experiments. In this study, PCR was used to detect tetracycline resistance genes ( tetA , tetB , tetC , tetD ) and beta-lactam resistance genes ( blaSHV -1, blaCTX - M , blaTEM ). Results: Sixteen strains of STEC were isolated from animal stool samples. Thirteen strains were isolated from pig stool samples, two from bovine stool samples and one from a sheep stool sample. Two of the strains were identified as E. coli O157:H7, and other 14 strains were non-O157 STEC of different serotypes. Antimicrobial sensitivity experiments showed that 15 of the strains were multidrug resistant. The rates of resistance were as follows: nalidixic acid (12/16 strains), sulfisoxazole (11/16), trimethoprim and sulphame-thoxazole (11/16), doxycycline (9/16), azithromycin (9/16), tetracycline (9/16), chloramphenicol (8/16) and streptomycin (8/16). Therefore, nalidixic acid showed the highest rate of resistance among the strains, followed by trimethoprim and sulphame-thoxazole, and sulfisoxazole. Resistance to cefepime or imipenem was not detected. In total, three types of drug resistance genes ( tetA , tetB and tetC ) were detected among the 16 strains. Conclusion: The results showed that STEC strains isolated from animals in the Weishan area were of a range of serotypes. The 16 strains of STEC isolated from animal stools in this area were resistant to a number of antibiotics, with many strains displaying multidrug resistance.

A SPECTRUM OF PATHOGENIC AND NON-PATHOGENIC INTESTINAL PARASITES IN PRE-EMPLOYMENT MEDICAL CHECK-UP FOR WORKERS AND THEIR FAMILIES

PubMed Central

Koshak, Emad A.; Zakai, Haytham A.

2003-01-01

Introduction: Stool analysis plays an important role in pre-employment tests for the screening of intestinal parasites in new workers. Objective: to explore the spectrum of intestinal parasites in stool samples of workers and their families during the pre-employment tests over a one-year period at King Abdulaziz University Hospital (KAUH). Methods: Subjects were selected sequentially from routine single stool analysis forms labeled for pre-employment tests. Stool specimens were examined using the formalin ether technique at the parasitology laboratory at KAUH. Results: Two hundred and ninety two different stool samples of the workers and their families were studied. Their ages ranged from 3 to 72 year old (mean 32 ± 8.5 SD) and females formed 58.6% of the number. Intestinal parasites were detected in 161 workers (55%). The prevalence of intestinal parasites in Saudi workers was significantly lower than non-Saudi nationals, 15.8% versus 57.9% (p<0.001). Of all the positive cases, pathogenic intestinal parasites were found in 40 % of them and the commonest were Trichuris trichuria (39.1%), Hookworm (34.2%), Entamoeba histolytica (16.1%). Non-pathogenic parasites were found in 19.5% and the commonest were Blastocystis hominis (34.8%), Endolimax nana (29.8%), Entamoeba coli (15.5%). One type of parasite was found in 75 (46.6%) and multiple different parasites were found in 86 (53.4%). There was a high significant correlation between the detection of pathogenic and non-pathogenic parasites (p<0.001). Conclusion: Infestation of stools with pathogenic and non-pathogenic intestinal parasites is a common finding in more than half of the new workers and their families. The correlation between non-pathogenic and pathogenic parasites reflects mutual risk factors, and their potential hazards cannot be overlooked. Effective stool screening and eradication strategies for intestinal parasites in new workers should be rigorously enforced. PMID:23011980

Stool consistency and stool frequency are excellent clinical markers for adequate colon preparation after polyethylene glycol 3350 cleansing protocol: a prospective clinical study in children.

PubMed

Safder, Shaista; Demintieva, Yulia; Rewalt, Mary; Elitsur, Yoram

2008-12-01

Colon preparation for a colonoscopy in children is a difficult task because of the unpalatable taste and large volume of cleansing solution that needs to be consumed to ensure a clean colon. Consequently, an unprepared colon frequently occurs in routine practices, which causes early termination and a repeated procedure. (1) To assess the effectiveness of polyethylene glycol solution (PEG 3350) in preparing the colon of children scheduled for a colonoscopy and (2) to investigate clinical markers associated with an adequate colon preparation before a colonoscopy. A total of 167 children scheduled for a colonoscopy. In a prospective study, children scheduled for a colonoscopy were given PEG 3350 solution (1.5 g/kg per day, up to 100 g/d) over a 4-day preparation period. Each day, a simple questionnaire that documents the amount of liquid consumed, adverse effects, and the number and consistency of stool was completed by the parents. After a colonoscopy procedure, the colon preparation was assigned a number grade. The data were later assessed and were compared to determine the association between the grade of cleansing and the frequency and/or consistency of stool during preparation. Colon preparation was completed in 149 children, 133 of whom were adequately prepared. Inadequate preparation was found in 16 children; the procedure was terminated prematurely in 2 of these patients because of unacceptable conditions. No significant adverse effects were noted. A number of >or=5 stools/d, and liquid stool consistency in the last 2 days of preparation were associated with adequate colon preparation. PEG 3350 solution is safe, efficacious, and tolerable for children. Stool frequency and consistency in the last 2 days of preparation were excellent markers (positive predictive value 91%-95%), which predict an adequately clean colon before a colonoscopy in children.

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

PubMed

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 (p<0.05) compared to non-IBS and non-IBS Blasto group. The level of IL-3 in were seen to be significantly higher in than IBS Blasto group and IBS group (p<0.05) compared to non-IBS. Meanwhile, the IL-5 levels were significantly higher in IBS Blasto group (p<0.05) compared to non-IBS and non-IBS Blasto group. This study implicates that detecting Blastosystis by PCR method using colonic aspirate samples during colonoscopy, suggests that this may be a better method for sample collection due to the parasite's irregular shedding in Blastocystis-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.

Urine Albumin and Albumin/ Creatinine Ratio

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Total Protein and Albumin/Globulin Ratio Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Stool guaiac test

MedlinePlus

... guaiac test; gFOBT; Guaiac smear test; Fecal occult blood test - guaiac smear; Stool occult blood test - guaiac smear ... This test detects blood in the digestive tract. It may be done if: You are being screened or tested for colon cancer You ...

Tailbone trauma - aftercare

MedlinePlus

... or naproxen (Aleve, Naprosyn, and others). You can buy these medicines without a prescription. DO NOT use these medicines ... of fluids to avoid constipation. Use stool softener medicine if needed. You can buy stool softeners at the drugstore. Activity As your ...

Comparison of three DNA extraction kits to establish maximum yield and quality of coral-associated microbial DNA

USGS Publications Warehouse

Baker, Erin J.; Kellogg, Christina A.

2014-01-01

Coral microbiology is an expanding field, yet there is no standard DNA extraction protocol. Although many researchers depend on commercial extraction kits, no specific kit has been optimized for use with coral samples. Both soil and plant DNA extraction kits from MO BIO Laboratories, Inc., have been used by many research groups for this purpose. MO BIO recently replaced their PowerPlant® kit with an improved PowerPlantPro kit, but it was unclear how these changes would affect the kit’s use with coral samples. In order to determine which kit produced the best results, we conducted a comparison between the original PowerPlant kit, the new PowerPlantPro kit, and an alternative kit, PowerSoil, using samples from several different coral genera. The PowerPlantPro kit had the highest DNA yields, but the lack of 16S rRNA gene amplification in many samples suggests that much of the yield may be coral DNA rather than microbial DNA. The most consistent positive amplifications came from the PowerSoil kit.

Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product.

PubMed Central

Furitsu, T; Tsujimura, T; Tono, T; Ikeda, H; Kitayama, H; Koshimizu, U; Sugahara, H; Butterfield, J H; Ashman, L K; Kanayama, Y

1993-01-01

The c-kit proto-oncogene encodes a receptor tyrosine kinase. Binding of c-kit ligand, stem cell factor (SCF) to c-kit receptor (c-kitR) is known to activate c-kitR tyrosine kinase, thereby leading to autophosphorylation of c-kitR on tyrosine and to association of c-kitR with substrates such as phosphatidylinositol 3-kinase (PI3K). In a human mast cell leukemia cell line HMC-1, c-kitR was found to be constitutively phosphorylated on tyrosine, activated, and associated with PI3K without the addition of SCF. The expression of SCF mRNA transcript in HMC-1 cells was not detectable by means of PCR after reverse transcription (RT-PCR) analysis, suggesting that the constitutive activation of c-kitR was ligand independent. Sequencing of whole coding region of c-kit cDNA revealed that c-kit genes of HMC-1 cells were composed of a normal, wild-type allele and a mutant allele with two point mutations resulting in intracellular amino acid substitutions of Gly-560 for Val and Val-816 for Asp. Amino acid sequences in the regions of the two mutations are completely conserved in all of mouse, rat, and human c-kit. In order to determine the causal role of these mutations in the constitutive activation, murine c-kit mutants encoding Gly-559 and/or Val-814, corresponding to human Gly-560 and/or Val-816, were constructed by site-directed mutagenesis and expressed in a human embryonic kidney cell line, 293T cells. In the transfected cells, both c-kitR (Gly-559, Val-814) and c-kitR (Val-814) were abundantly phosphorylated on tyrosine and activated in immune complex kinase reaction in the absence of SCF, whereas tyrosine phosphorylation and activation of c-kitR (Gly-559) or wild-type c-kitR was modest or little, respectively. These results suggest that conversion of Asp-816 to Val in human c-kitR may be an activating mutation and responsible for the constitutive activation of c-kitR in HMC-1 cells. Images PMID:7691885

Distinct cellular properties of oncogenic KIT receptor tyrosine kinase mutants enable alternative courses of cancer cell inhibition

PubMed Central

Shi, Xiarong; Sousa, Leiliane P.; Mandel-Bausch, Elizabeth M.; Tome, Francisco; Reshetnyak, Andrey V.; Hadari, Yaron; Schlessinger, Joseph; Lax, Irit

2016-01-01

Large genomic sequencing analysis as part of precision medicine efforts revealed numerous activating mutations in receptor tyrosine kinases, including KIT. Unfortunately, a single approach is not effective for inhibiting cancer cells or treating cancers driven by all known oncogenic KIT mutants. Here, we show that each of the six major KIT oncogenic mutants exhibits different enzymatic, cellular, and dynamic properties and responds distinctly to different KIT inhibitors. One class of KIT mutants responded well to anti-KIT antibody treatment alone or in combination with a low dose of tyrosine kinase inhibitors (TKIs). A second class of KIT mutants, including a mutant resistant to imatinib treatment, responded well to a combination of TKI with anti-KIT antibodies or to anti-KIT toxin conjugates, respectively. We conclude that the preferred choice of precision medicine treatments for cancers driven by activated KIT and other RTKs may rely on clear understanding of the dynamic properties of oncogenic mutants. PMID:27482095

Megakaryocytes compensate for Kit insufficiency in murine arthritis.

PubMed

Cunin, Pierre; Penke, Loka R; Thon, Jonathan N; Monach, Paul A; Jones, Tatiana; Chang, Margaret H; Chen, Mary M; Melki, Imene; Lacroix, Steve; Iwakura, Yoichiro; Ware, Jerry; Gurish, Michael F; Italiano, Joseph E; Boilard, Eric; Nigrovic, Peter A

2017-05-01

The growth factor receptor Kit is involved in hematopoietic and nonhematopoietic development. Mice bearing Kit defects lack mast cells; however, strains bearing different Kit alleles exhibit diverse phenotypes. Herein, we investigated factors underlying differential sensitivity to IgG-mediated arthritis in 2 mast cell-deficient murine lines: KitWsh/Wsh, which develops robust arthritis, and KitW/Wv, which does not. Reciprocal bone marrow transplantation between KitW/Wv and KitWsh/Wsh mice revealed that arthritis resistance reflects a hematopoietic defect in addition to mast cell deficiency. In KitW/Wv mice, restoration of susceptibility to IgG-mediated arthritis was neutrophil independent but required IL-1 and the platelet/megakaryocyte markers NF-E2 and glycoprotein VI. In KitW/Wv mice, platelets were present in numbers similar to those in WT animals and functionally intact, and transfer of WT platelets did not restore arthritis susceptibility. These data implicated a platelet-independent role for the megakaryocyte, a Kit-dependent lineage that is selectively deficient in KitW/Wv mice. Megakaryocytes secreted IL-1 directly and as a component of circulating microparticles, which activated synovial fibroblasts in an IL-1-dependent manner. Transfer of WT but not IL-1-deficient megakaryocytes restored arthritis susceptibility to KitW/Wv mice. These findings identify functional redundancy among Kit-dependent hematopoietic lineages and establish an unanticipated capacity of megakaryocytes to mediate IL-1-driven systemic inflammatory disease.

Evaluation of a chromogenic culture medium for isolation of Clostridium difficile within 24 hours.

PubMed

Perry, John D; Asir, Kerry; Halimi, Diane; Orenga, Sylvain; Dale, Joanne; Payne, Michelle; Carlton, Ruth; Evans, Jim; Gould, F Kate

2010-11-01

Rapid and effective methods for the isolation of Clostridium difficile from stool samples are desirable to obtain isolates for typing or to facilitate accurate diagnosis of C. difficile-associated diarrhea. We report on the evaluation of a prototype chromogenic medium (ID C. difficile prototype [IDCd]) for isolation of C. difficile. The chromogenic medium was compared using (i) 368 untreated stool samples that were also inoculated onto CLO medium, (ii) 339 stool samples that were subjected to alcohol shock and also inoculated onto five distinct selective agars, and (iii) standardized suspensions of 10 C. difficile ribotypes (untreated and alcohol treated) that were also inoculated onto five distinct selective agars. Two hundred thirty-six isolates of C. difficile were recovered from 368 untreated stool samples, and all but 1 of these strains (99.6%) were recovered on IDCd within 24 h, whereas 74.6% of isolates were recovered on CLO medium after 48 h. Of 339 alcohol-treated stool samples cultured onto IDCd and five other selective agars, C. difficile was recovered from 218 samples using a combination of all media. The use of IDCd allowed recovery of 96.3% of isolates within 24 h, whereas 51 to 83% of isolates were recovered within 24 h using the five other media. Finally, when they were challenged with pure cultures, all 10 ribotypes of C. difficile generated higher colony counts on IDCd irrespective of alcohol pretreatment or duration of incubation. We conclude that IDCd is an effective medium for isolation of C. difficile from stool samples within 24 h.

Randomized, double-blind, placebo-controlled trial of oral docusate in the management of constipation in hospice patients.

PubMed

Tarumi, Yoko; Wilson, Mitchell P; Szafran, Olga; Spooner, G Richard

2013-01-01

The stool softener docusate is widely used in the management of constipation in hospice patients. There is little experimental evidence to support this practice, and no randomized trials have been conducted in the hospice setting. To assess the efficacy of docusate in hospice patients. This was a 10-day, prospective, randomized, double-blind, placebo-controlled trial of docusate and sennosides vs. placebo and sennosides in hospice patients in Edmonton, Alberta. Patients were included if they were age 18 years or older, able to take oral medications, did not have a gastrointestinal stoma, and had a Palliative Performance Scale score of 20% or more. The primary outcome measures were stool frequency, volume, and consistency. Secondary outcomes were patient perceptions of bowel movements (difficulty and completeness of evacuation) and bowel-related interventions. A total of 74 patients were randomized into the study (35 to the docusate group and 39 to the placebo group). There were neither significant differences between the groups in stool frequency, volume, or consistency, nor in difficulty or completeness of evacuation. On the Bristol Stool Form Scale, more patients in the placebo group had Type 4 (smooth and soft) and Type 5 (soft blobs) stool, whereas in the docusate group, more had Type 3 (sausage like) and Type 6 (mushy) stool (P=0.01). There was no significant benefit of docusate plus sennosides compared with placebo plus sennosides in managing constipation in hospice patients. Docusate use should be considered on an individual basis. Copyright © 2013 U.S. Cancer Pain Relief Committee. Published by Elsevier Inc. All rights reserved.

Multiplex PCR detection of Cryptosporidium sp, Giardia lamblia and Entamoeba histolytica directly from dried stool samples from Guinea-Bissauan children with diarrhoea.

PubMed

Mero, Sointu; Kirveskari, Juha; Antikainen, Jenni; Ursing, Johan; Rombo, Lars; Kofoed, Poul-Erik; Kantele, Anu

2017-09-01

In developing countries, diarrhoea is the most common cause of death for children under five years of age, with Giardia lamblia, Cryptosporidium and Entamoeba histolytica as the most frequent pathogenic parasites. Traditional microscopy for stool parasites has poor sensitivity and specificity, while new molecular methods may provide more accurate diagnostics. In poor regions with sample storage hampered by uncertain electricity supply, research would benefit from a method capable of analysing dried stools. A real-time multiplex PCR method with internal inhibition control was developed for detecting Giardia lamblia, Cryptosporidium hominis/parvum and Entamoeba histolytica directly from stool specimens. Applicability to dried samples was checked by comparing with fresh ones in a small test material. Finally, the assay was applied to dried specimens collected from Guinea-Bissauan children with diarrhoea. The PCR's analytical sensitivity limit was 0.1 ng/ml for G. lamblia DNA, 0.01 ng/ml for E. histolytica DNA and 0.1 ng/ml for Cryptosporidium sp. In the test material, the assay performed similarly with fresh and dried stools. Of the 52 Guinea-Bissauan samples, local microscopy revealed a parasite in 15%, while PCR detected 62% positive for at least one parasite: 44% of the dried samples had Giardia, 23% Cryptosporidium and 0% E. histolytica. Our new multiplex real-time PCR for protozoa presents a sensitive method applicable to dried samples. As proof of concept, it worked well on stools collected from Guinea-Bissauan children with diarrhoea. It provides an epidemiological tool for analysing dried specimens from regions poor in resources.

Difference in Ulex europaeus agglutinin I-binding activity of decay-accelerating factor detected in the stools of patients with colorectal cancer and ulcerative colitis.

PubMed

Okazaki, Hiroaki; Mizuno, Motowo; Nasu, Junichirou; Makidono, Chiho; Hiraoka, Sakiko; Yamamoto, Kazuhide; Okada, Hiroyuki; Fujita, Teizo; Tsuji, Takao; Shiratori, Yasushi

2004-03-01

Expression of decay-accelerating factor (DAF, CD55), a complement-regulatory glycoprotein, is enhanced in colorectal-cancer (CC) cells and colonic epithelium in ulcerative colitis (UC), and stools from these patients contain increased amounts of DAF. Carbohydrate chains of glycoproteins are often altered during malignant transformation or inflammation. In this study, we investigated whether DAF molecules in patients with CC and those with UC differ with respect to oligosaccharide side chains. We analyzed DAF in stools and homogenates of colonic-tissue specimens obtained from patients with CC or UC using solid-phase enzyme-linked assay and Western blotting for reactivity with the lectins Ulex europaeus agglutinin I (UEA-I), wheat-germ agglutinin, peanut agglutinin, and concanavalin A. UEA-I bound to DAF in stools from patients with UC but not in that from the stools of CC patients, as demonstrated on the solid-phase enzyme-linked assay (P <.05, Mann-Whitney U test) and Western blotting. Binding of UEA-I was specifically inhibited by the addition of fucose. The difference in UEA-I reactivity with DAF was observed also in colonic-tissue homogenates from patients with UC and those with CC. DAF expressed in the mucosa and excreted into the stools of UC patients is different from that expressed in CC with regard to UEA-I reactivity. Future studies should be directed toward determining whether a qualitatively unique isoform of DAF is present, of which sugar chains are specific to CC in UC patients.

Multiplex real time PCR panels to identify fourteen colonization factors of enterotoxigenic Escherichia coli (ETEC).

PubMed

Liu, Jie; Silapong, Sasikorn; Jeanwattanalert, Pimmada; Lertsehtakarn, Paphavee; Bodhidatta, Ladaporn; Swierczewski, Brett; Mason, Carl; McVeigh, Annette L; Savarino, Stephen J; Nshama, Rosemary; Mduma, Esto; Maro, Athanasia; Zhang, Jixian; Gratz, Jean; Houpt, Eric R

2017-01-01

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood diarrhea in low income countries and in travelers to those areas. Inactivated enterotoxins and colonization factors (CFs) are leading vaccine candidates, therefore it is important to determine the prevailing CF types in different geographic locations and populations. Here we developed real time PCR (qPCR) assays for 14 colonization factors, including the common vaccine targets. These assays, along with three enterotoxin targets (STh, STp, and LT) were formulated into three 5-plex qPCR panels, and validated on 120 ETEC isolates and 74 E. coli colony pools. The overall sensitivity and specificity was 99% (199/202) and 99% (2497/2514), respectively, compared to the CF results obtained with conventional PCR. Amplicon sequencing of discrepant samples revealed that the qPCR was 100% accurate. qPCR panels were also performed on nucleic acid extracted from stool and compared to the results of the ETEC isolates or E. coli colony pools cultured from them. 95% (105/110) of the CF detections in the cultures were confirmed in the stool. Additionally, direct testing of stool yielded 30 more CF detections. Among 74 randomly selected E. coli colony pools with paired stool, at least one CF was detected in 63% (32/51) of the colony pools while at least one CF was detected in 78% (47/60) of the stool samples (P = NS). We conclude that these ETEC CF assays can be used on both cultures and stool samples to facilitate better understanding of CF distribution for ETEC epidemiology and vaccine development.

[Overdiagnosis of amebiasis in children with dysentery].

PubMed

Beltramino, Juan Carlos; Sosa, Horacio; Gamba, Natalia; Busquets, Natalia; Navarro, Lucas; Virgolini, Stella; Ricardo, Omar

2009-12-01

There are morphologically identical amebaes, but with differences that can distinguish them; one as pathogenic: Entamoeba histolytica, and the other: Entamoeba dispar, as inoffensive. That brought the new hypothesis that many of the cases treated as amebiasis, weren't so. To identify E. hystolitica in patients with dysentery, supposed to be caused by amebae. Transversal and observational study performed between March 2005 and November 2007 in the city of Santa Fe, Argentina. Stools from children aged 2 months to 15 years-old with dysentery and direct exams with E. hystolitica/ dispar, were studied with ELISA to detect the adhesin of E. histolytica (adhesin Eh). Permanent stains for amebae were done as well as stool cultures. Clinical data were charted. 75 children were studied; 35 were male and 40, female, with a median age of 3 years-old. All of them presented diarrhea with leucocyte, 73% macroscopic blood on stool and 27% detectable on the microscope. Elisa Eh was positive in 21; 3 cases had hematophagous trophozoites. In 15 stool cultures were found: S. flexneri S2 type in 5 cases. Other parasites: 6 (Blastocystis homini 5). In 54 adhesin Eh was negative, 19% of the coulouring detected E. dispar. From 44 stool cultures: S. flexneri S2 type was detected in 13, Shigella sp in 1, C jejuni 5, other: 3. Other parasites: 12 (Blastocystis homini 9). In this group of children with "amebic dysentery", half of them developed invasive bacteriae and only 28% had E. histolytica on stools; that means that the prevalence of positive cases in the population could be 18% to 38% [CI 95% (0.179; 0.381)].

Seroprevalence of human fascioliasis in Van province, Turkey.

PubMed

Taş Cengiz, Zeynep; Yılmaz, Hasan; Dülger, Ahmet Cumhur; Akdeniz, Hayrettin; Karahocagil, Mustafa Kasım; Çiçek, Mutalip

2015-05-01

Fasciola hepatica is a rare zoonotic parasite that infects the liver of many mammals including humans. The aim of this study was to determine the seroprevalence of fascioliasis in Van province by ELISA (antibody detection) on the assumption that not all cases could be detected by stool examination alone. A total of randomly selected 1,600 patients, directed from affiliated outpatient clinics to Yüzüncü Yıl University Medical Faculty Parasitology Laboratory, were enrolled in the study. Their mean age was 44.44±19.00 years. Blood samples were collected from all the patients, and their stool samples were examined. For the stool examination, native-lugol and sedimentation (in formalin-ethyl acetate) methods were employed. ELISA for F. hepatica was performed on the blood samples from all patients. Seropositive patients were treated with triclabendazole. F. hepatica was detected by ELISA in 89 (5.6%) of the 1,600 patients, but eggs were identified on the stool examination in only 29 (1.8%) patients. The prevalence of F. hepatica was higher in females (7.2%) than in males (4.2%) and was higher in the ≥36-year age group (6.7%) than in the ≤35-year age group (4.4%). Abdominal pain (93.3%), fatigue (88.8%), and weight loss (69.7%) were the most common symptoms. Eosinophilia was present in 89.9% of the patients. All seropositive patients had a history of eating raw aquatic plants. Stool examination alone is not sufficient to diagnose F. hepatica. Serological tests such as ELISA must be used together with stool examination.

Evaluation of a Chromogenic Culture Medium for Isolation of Clostridium difficile within 24 Hours ▿

PubMed Central

Perry, John D.; Asir, Kerry; Halimi, Diane; Orenga, Sylvain; Dale, Joanne; Payne, Michelle; Carlton, Ruth; Evans, Jim; Gould, F. Kate

2010-01-01

Rapid and effective methods for the isolation of Clostridium difficile from stool samples are desirable to obtain isolates for typing or to facilitate accurate diagnosis of C. difficile-associated diarrhea. We report on the evaluation of a prototype chromogenic medium (ID C. difficile prototype [IDCd]) for isolation of C. difficile. The chromogenic medium was compared using (i) 368 untreated stool samples that were also inoculated onto CLO medium, (ii) 339 stool samples that were subjected to alcohol shock and also inoculated onto five distinct selective agars, and (iii) standardized suspensions of 10 C. difficile ribotypes (untreated and alcohol treated) that were also inoculated onto five distinct selective agars. Two hundred thirty-six isolates of C. difficile were recovered from 368 untreated stool samples, and all but 1 of these strains (99.6%) were recovered on IDCd within 24 h, whereas 74.6% of isolates were recovered on CLO medium after 48 h. Of 339 alcohol-treated stool samples cultured onto IDCd and five other selective agars, C. difficile was recovered from 218 samples using a combination of all media. The use of IDCd allowed recovery of 96.3% of isolates within 24 h, whereas 51 to 83% of isolates were recovered within 24 h using the five other media. Finally, when they were challenged with pure cultures, all 10 ribotypes of C. difficile generated higher colony counts on IDCd irrespective of alcohol pretreatment or duration of incubation. We conclude that IDCd is an effective medium for isolation of C. difficile from stool samples within 24 h. PMID:20739493

Kinetics of Poliovirus Shedding following Oral Vaccination as Measured by Quantitative Reverse Transcription-PCR versus Culture

PubMed Central

Begum, Sharmin; Uddin, Md Jashim; Platts-Mills, James A.; Liu, Jie; Kirkpatrick, Beth D.; Chowdhury, Anwarul H.; Jamil, Khondoker M.; Haque, Rashidul; Petri, William A.; Houpt, Eric R.

2014-01-01

Amid polio eradication efforts, detection of oral polio vaccine (OPV) virus in stool samples can provide information about rates of mucosal immunity and allow estimation of the poliovirus reservoir. We developed a multiplex one-step quantitative reverse transcription-PCR (qRT-PCR) assay for detection of OPV Sabin strains 1, 2, and 3 directly in stool samples with an external control to normalize samples for viral quantity and compared its performance with that of viral culture. We applied the assay to samples from infants in Dhaka, Bangladesh, after the administration of trivalent OPV (tOPV) at weeks 14 and 52 of life (on days 0 [pre-OPV], +4, +11, +18, and +25 relative to vaccination). When 1,350 stool samples were tested, the sensitivity and specificity of the quantitative PCR (qPCR) assay were 89 and 91% compared with culture. A quantitative relationship between culture+/qPCR+ and culture−/qPCR+ stool samples was observed. The kinetics of shedding revealed by qPCR and culture were similar. qPCR quantitative cutoffs based on the day +11 or +18 stool samples could be used to identify the culture-positive shedders, as well as the long-duration or high-frequency shedders. Interestingly, qPCR revealed that a small minority (7%) of infants contributed the vast majority (93 to 100%) of the total estimated viral excretion across all subtypes at each time point. This qPCR assay for OPV can simply and quantitatively detect all three Sabin strains directly in stool samples to approximate shedding both qualitatively and quantitatively. PMID:25378579

Signal transduction by normal isoforms and W mutant variants of the Kit receptor tyrosine kinase.

PubMed

Reith, A D; Ellis, C; Lyman, S D; Anderson, D M; Williams, D E; Bernstein, A; Pawson, T

1991-09-01

Germline mutations at the Dominant White Spotting (W) and Steel (Sl) loci have provided conclusive genetic evidence that c-kit mediated signal transduction pathways are essential for normal mouse development. We have analysed the interactions of normal and mutant W/c-kit gene products with cytoplasmic signalling proteins, using transient c-kit expression assays in COS cells. In addition to the previously identified c-kit gene product (Kit+), a second normal Kit isoform (KitA+) containing an in-frame insertion, Gly-Asn-Asn-Lys, within the extracellular domain, was detected in murine mast cell cultures and mid-gestation placenta. Both Kit+ and KitA+ isoforms showed increased autophosphorylation and enhanced association with phosphatidylinositol (PI) 3' kinase and PLC gamma 1, when stimulated with recombinant soluble Steel factor. No association or increase in phosphorylation of GAP and two GAP-associated proteins, p62 and p190, was observed. The two isoforms had distinct activities in the absence of exogenous soluble Steel factor; Kit+, but not KitA+, showed constitutive tyrosine phosphorylation that was accompanied by a low constitutive level of association with PI-3' kinase and PLC gamma 1. Introduction of the point substitutions associated with W37 (Glu582----Lys) or W41 (Val831----Met) mutant alleles into c-kit expression constructs abolished (W37) or reduced (W41) the Steel factor-induced association of the Kit receptor with signalling proteins in a manner proportional to the overall severity of the corresponding W mutant phenotype. These data suggest a diversity of normal Kit signalling pathways and indicate that W mutant phenotypes result from primary defects in the Kit receptor that affect its interaction with cytoplasmic signalling proteins.

Effect of a fermented milk containing Bifidobacterium lactis DN-173010 on Chinese constipated women.

PubMed

Yang, Yue-Xin; He, Mei; Hu, Gang; Wei, Jie; Pages, Philippe; Yang, Xian-Hua; Bourdu-Naturel, Sophie

2008-10-28

To investigate the effect of a fermented milk containing Bifidobacterium lactis DN-173010 and yogurt strains (BIO(R)) on adult women with constipation in Beijing. A total of 135 adult females with constipation were randomly allocated to consume for 2 wk either 100 g of the test fermented milk or 100 g of an acidified milk containing non-living bacteria (control). Stool frequency, defecation condition scores, stool consistency and food intake were recorded at baseline and after 1 and 2 wk in an intention-to-treat population of 126 subjects. In parallel, safety evaluation parameters were performed. At baseline, no differences were found between groups. Following consumption of test product, stool frequency was significantly increased after 1 wk (3.5 +/- 1.5 vs 2.4 +/- 0.6, P < 0.01) and 2 wk (4.1 +/- 1.7 vs 2.4 +/- 0.6, P < 0.01), vs baseline. Similarly, after 1 and 2 wk, of test product consumption, defecation condition (1.1 +/- 0.9 vs 1.9 +/- 1.2, P < 0.01 and 0.8 +/- 1.0 vs 1.9 +/- 1.2, P < 0.01, respectively) and stool consistency (1.0 +/- 0.8 vs 1.5 +/- 1.1, P < 0.01 and 0.6 +/- 0.8 vs 1.5 +/- 1.1, P < 0.01, respectively) were significantly improved. Compared with the control group, stool frequency was also significantly increased (3.5 +/- 1.5 vs 2.5 +/- 0.9, P < 0.01 and 4.1 +/- 1.7 vs 2.6 +/- 1.0, P < 0.01, respectively), and defecation condition (1.1 +/- 0.9 vs 1.6 +/- 1.1, P < 0.01 and 0.8 +/- 1.0 vs 1.6 +/- 1.1, P < 0.01, respectively) and stool consistency (1.0 +/- 0.8 vs 1.4 +/- 1.0, P < 0.05 and 0.6 +/- 0.8 vs 1.3 +/- 1.0, P < 0.01, respectively) significantly decreased after 1 and 2 wk of product consumption. During the same period, food intake did not change between the two groups, and safety parameters of the subjects were within normal ranges. This study suggests a beneficial effect of a fermented milk containing B. lactis DN-173010 on stool frequency, defecation condition and stool consistency in adult women with constipation constipated women after 1 and 2 wk of consumption.

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

PubMed Central

2009-01-01

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 direct detection of enterovirus from the stool extracts. PMID:20015403

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'.

PubMed

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 of hookworm infection may be related to rapid degeneration of delicate hookworm eggs with time. (c) 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Germline mutations of KIT in gastrointestinal stromal tumor (GIST) and mastocytosis.

PubMed

Ke, Hengning; Kazi, Julhash U; Zhao, Hui; Sun, Jianmin

2016-01-01

Somatic mutations of KIT are frequently found in mastocytosis and gastrointestinal stromal tumor (GIST), while germline mutations of KIT are rare, and only found in few cases of familial GIST and mastocytosis. Although ligand-independent activation is the common feature of KIT mutations, the phenotypes mediated by various germline KIT mutations are different. Germline KIT mutations affect different tissues such as interstitial cells of Cajal (ICC), mast cells or melanocytes, and thereby lead to GIST, mastocytosis, or abnormal pigmentation. In this review, we summarize germline KIT mutations in familial mastocytosis and GIST and discuss the possible cellular context dependent transforming activity of KIT mutations.

The challenges of lean manufacturing implementation in kitting assembly

NASA Astrophysics Data System (ADS)

Fansuri, A. F. H.; Rose, A. N. M.; Nik Mohamed, N. M. Z.; Ahmad, H.

2017-10-01

Literature studies shows that lean manufacturing goes way back with the original founder Eli Whitney in year 1799. The main purpose of lean manufacturing is to identify and eliminate waste in production. The application of lean manufacturing can be carried out in any industrial processes with regards to the understanding of lean principles, theories and practices. Kitting is one of the important aspects in a successful production. The continuous supply of materials from store to production has to be systematic and able to achieve lean standard for it to be successful. The objective of this paper is to review the implementation of lean manufacturing in kitting assembly. Previous papers show that, the implementation of lean manufacturing in kitting assembly may be beneficial to the organization such as reduce in space occupancy, part shortages, lead time and manpower. Based on previous research, some industries may tend to change between kitting and line stocking which are due to lack of understanding when implementing kitting and causes longer lead time and materials overflow in store. With a proper understanding on what to kit, where to kit, how to kit, why to kit and who kits the material with a standardised process flow may ensure the success of kitting.

No GIST-type c-kit gain of function mutations in neuroblastic tumours

PubMed Central

Korja, M; Finne, J; Salmi, T T; Haapasalo, H; Tanner, M; Isola, J

2005-01-01

Aims: Neuroblastic tumours (NTs) have been shown to respond to imatinib treatment in vivo and in vitro, possibly via inactivating the c-kit receptor. The purpose of this study was to identify gastrointestinal stromal tumour (GIST)-type c-kit gene associated mutations in exons 9, 11, 13, and 17 in NTs to recognise a subset of tumours that would probably respond to imatinib treatment. Methods: Expression of the c-kit protein was detected immunohistochemically in a total of 37 archival paraffin wax embedded NTs using polyclonal rabbit antihuman c-kit antibody. After immunohistochemistry, c-kit gene associated chromosomal mutations in all cases of NT were detected with denaturing high performance liquid chromatography (HPLC). Results: Denaturing HLPC analysis did not reveal GIST-type mutations in four immunohistochemically detected c-kit positive or in 33 c-kit negative NTs. Conclusions: c-kit receptor expression and GIST-type c-kit gene mutations are rare events in NTs. Oncogenic activation of c-kit in NTs presumably differs from that of GISTs, which may influence their responsiveness to imatinib treatment. Whether c-kit has an essential role in the pathogenesis of NTs remains to be investigated. PMID:15976348

Pim1 kinase regulates c-Kit gene translation.

PubMed

An, Ningfei; Cen, Bo; Cai, Houjian; Song, Jin H; Kraft, Andrew; Kang, Yubin

2016-01-01

Receptor tyrosine kinase, c-Kit (CD117) plays a pivotal role in the maintenance and expansion of hematopoietic stem/progenitor cells (HSPCs). Additionally, over-expression and/or mutational activation of c-Kit have been implicated in numerous malignant diseases including acute myeloid leukemia. However, the translational regulation of c-Kit expression remains largely unknown. We demonstrated that loss of Pim1 led to specific down-regulation of c-Kit expression in HSPCs of Pim1 -/- mice and Pim1 -/- 2 -/- 3 -/- triple knockout (TKO) mice, and resulted in attenuated ERK and STAT3 signaling in response to stimulation with stem cell factor. Transduction of c-Kit restored the defects in colony forming capacity seen in HSPCs from Pim1 -/- and TKO mice. Pharmacologic inhibition and genetic modification studies using human megakaryoblastic leukemia cells confirmed the regulation of c-Kit expression by Pim1 kinase: i.e., Pim1-specific shRNA knockdown down-regulated the expression of c-Kit whereas overexpression of Pim1 up-regulated the expression of c-Kit. Mechanistically, inhibition or knockout of Pim1 kinase did not affect the transcription of c-Kit gene. Pim1 kinase enhanced c-Kit 35 S methionine labeling and increased the incorporation of c-Kit mRNAs into the polysomes and monosomes, demonstrating that Pim1 kinase regulates c-Kit expression at the translational level. Our study provides the first evidence that Pim1 regulates c-Kit gene translation and has important implications in hematopoietic stem cell transplantation and cancer treatment.

Temozolomide

MedlinePlus

... medical treatment: unusual bleeding or bruising red or black, tarry stools pink, red, or dark brown urine coughing up or vomiting blood or material ... the following: unusual bleeding or bruising red or black, tarry stools pink, red, or dark brown urine coughing up or vomiting blood or material ...

In vitro growth of Vibrio cholerae in cholera stool fluid leads to differential expression of virulence factors.

PubMed

Sánchez, J; Castillo, G; Medrano, A I; Martinez-Palomo, A; Rodríguez, M H

1995-01-01

We report on the physiological response of Vibrio cholerae upon growth on bacteria-free intestinal fluids prepared from feces of individuals in the acute phase of cholera. Sterilized stool fluids supported growth of V. cholerae to reach 0.3-0.4 O.D. units (600 nm) at 37 degrees C. Scanning electron microscopy showed vibrios to be slender and elongated as compared to bacteria in synthetic media. Growth in stool fluid apparently induced expression of several immunoreactive proteins using cholera convalescent sera. Supernatants of fluid-grown vibrios had undetectable cholera toxin (CT) concentrations. Soluble hemagglutinins and soluble proteases were much less reduced when compared to cultures in Syncase or AKI media while cell-associated mannose-sensitive hemagglutinin (MSHA) was expressed at good levels. Lack of production of CT in fluid devoid of tissue may be due to absence of stimulating elements in intact intestine. Alternatively, culturing V. cholerae in stool fluid might resemble a late proliferation stage where downregulation of toxin might occur. Irrespectively, concomitant production of other virulence factors represents a phenomenon of differential regulation by fluid. Efforts are now underway to determine if this response depends upon factors in stool fluid acting through known genetic regulatory cascades or other. Attempts are also geared to identify fluid-induced proteins and their genes.

Clinical correlates of trichuriasis diagnosed at colonoscopy.

PubMed

Jha, Ashish Kumar; Goenka, Mahesh Kumar; Suchismita, Arya

2017-09-01

Diagnosis of Trichuris trichiura infestations is usually based on identification of barrel-shaped ova in stool, but is frequently missed on stool microscopy. We describe the clinical profile of patients in whom Trichuris infection was incidentally diagnosed at colonoscopy. In a cross-sectional study, patients with colonoscopic diagnosis of trichuriasis were enrolled from the endoscopy unit in a tertiary care center. Blood and stool samples were collected from all those who were willing to participate and provide samples. Sixty-two patients participated, with mean (SD) age of 50.5 (13.6) years and male to female ratio of 40:22. Abdominal pain (61.2%) and/or altered bowel habits (32.2%) were the most common indication for colonoscopy. Most (66.6%) of the Trichuris were located in the cecum and ascending colon. Majority of the patients had live worms, either motile or adhering to the colonic mucosa. The number of worms was single or a few (<15) in 74.2% of patients. Out of 62 patients, 16 (25.8%) had relatively heavy load of parasites. Most patients had normal colonoscopic findings (80.6%). Periappendicular and/or cecal ulcerations/erosions were the most common (16.1%) abnormalities noted. Stool examination showed parasite ova only in four (6.4%) patients. In conclusion, colonoscopy was better than stool microscopy for the diagnosis of trichuriasis in our study.

Comparative Evaluation of Three Methods (Microscopic Examination, Direct Fluorescent Antibody Assay, and Immunochromatographic Method) for the Diagnosis of Giardia intestinalis From Stool Specimens.

PubMed

Karadam, Senem Yaman; Ertuğ, Sema; Ertabaklar, Hatice

2016-03-01

The aim of this study was to compare direct microscopic examination, direct fluorescent antibody assay (DFA), and the immunochromatographic method (IK) and identify the best suitable method for the diagnosis of Giardia intestinalis. In this study, 25 stool samples that had been diagnosed as being infected with G. intestinalis using the native-Lugol and/or formol-ethyl acetate concentration method and 25 non-parasite-infected samples (the control group) were examined. After microscopic examination of stools, they were kept at -20°C for examination using DFA and IK. Stool samples were studied using DFA (CeLLabs, Crypto/Giardia-Cel IF) and IK (RIDA QUICK, Cryptosporidium/Giardia Combi Dipstick), as per the manufacturers' instructions. In our study, using the DFA method, parasites were detected in all 25 stool samples in which G. intestinalis was diagnosed by direct microscopic examination. Using the IK method, a particular band indicative of the parasite was detected in 24 samples. No parasites were detected in all 25 samples in the control group. Thus, when direct microscopic examination is taken as reference, the senstivity and specificity of DFA for the diagnosis of G. intestinalis were found to be 100% each, while those of IK were found to be 96% and 100%, respectively.

Diagnostic value of secreted frizzled-related protein 2 gene promoter hypermethylation in stool for colorectal cancer: A meta-analysis.

PubMed

Zhou, Zhiran; Zhang, Huitian; Lei, Yunxia

2016-10-01

To evaluate the diagnostic value of secreted frizzled-related protein 2 (SFRP2) gene promoter hypermethylation in stool for colorectal cancer (CRC). Open published diagnostic study of SFRP2 gene promoter hypermethylation in stool for CRC detection was electronic searched in the databases of PubMed, EMBASE, Cochrane Library, Web of Science, and China National Knowledge Infrastructure. The data of true positive, false positive false negative, and true negative identified by stool SFRP2 gene hypermethylation was extracted and pooled for diagnostic sensitivity, specificity, and summary receiver operating characteristic (SROC) curve. According to the inclusion and exclusion criteria, we finally included nine publications with 792 cases in the meta-analysis. Thus, the diagnostic sensitivity was aggregated through random effect model. The pooled sensitivity was 0.82 with the corresponding 95% confidence interval (95% CI) of 0.79-0.85; the pooled specificity and its corresponding 95% CI were 0.47 and 0.40-0.53 by the random effect model; we pooled the SROC curve by sensitivity versus specificity according to data published in the nine studies. The area under the SROC curve was 0.70 (95% CI: 0.65-0.73). SFRP2 gene promoter hypermethylation in stool can was a potential biomarker for CRC diagnosis with relative high sensitivity.

Excretion of Hepatitis A Virus (HAV) in Adults: Comparison of Immunologic and Molecular Detection Methods and Relationship between HAV Positivity and Infectivity in Tamarins

PubMed Central

Polish, Louis B.; Robertson, Betty H.; Khanna, Bhawna; Krawczynski, Krzysztof; Spelbring, John; Olson, Fred; Shapiro, Craig N.

1999-01-01

Fecal excretion of hepatitis A virus (HAV) in 18 patients with HAV infection was evaluated by enzyme immunoassay (EIA) to detect viral antigen and by reverse transcription-PCR amplification followed by ethidium bromide staining (PCR-ETBr) or nucleic acid hybridization (PCR-NA) to detect viral genetic material. A gradation of sensitivity was observed in the detection of virus by the three methods. In persons who had detectable virus, serial stool samples were found to be positive by EIA for up to 24 days after the peak elevation of liver enzymes. Viral genetic material could be detected by PCR-ETBr for up to 34 days and by PCR-NA for up to 54 days after the peak elevation of liver enzymes. After intravenous inoculation of tamarins with stool suspensions categorized as highly reactive for HAV (positive by EIA, PCR-ETBr, and PCR-NA), moderately reactive (positive by PCR-ETBr and PCR-NA), or weakly reactive (positive by PCR-NA), only tamarins infected with highly reactive stool suspensions (EIA positive) developed HAV infection. We conclude that positivity of stool specimens for HAV by PCR-ETBr or PCR-NA indicates a lower potential for infectivity, compared to that of EIA-positive stools. PMID:10523563

Early kit mortality and growth in farmed mink are affected by litter size rather than nest climate.

PubMed

Schou, T M; Malmkvist, J

2017-09-01

We investigated the effects of nest box climate on early mink kit mortality and growth. We hypothesised that litters in warm nest boxes experience less hypothermia-induced mortality and higher growth rates during the 1st week of life. This study included data from 749, 1-year-old breeding dams with access to nesting materials. Kits were weighed on days 1 and 7, dead kits were collected daily from birth until day 7 after birth, and nest climate was measured continuously from days 1 to 6. We tested the influences of the following daily temperature (T) and humidity (H) parameters on the number of live-born kit deaths and kit growth: T mean, T min, T max, T var (fluctuation) and H mean. The nest microclimate experienced by the kits was buffered against the ambient climate, with higher temperatures and reduced climate fluctuation. Most (77.0%) live-born kit deaths in the 1st week occurred on days 0 and 1. Seven of 15 climate parameters on days 1 to 3 had significant effects on live-born kit mortality. However, conflicting effects among days, marginal effects and late effects indicated that climate was not the primary cause of kit mortality. Five of 30 climate parameters had significant effects on kit growth. Few and conflicting effects indicated that the climate effect on growth was negligible. One exception was that large nest temperature fluctuations on day 1 were associated with reduced deaths of live-born kit (P<0.001) and increased kit growth (P=0.003). Litter size affected kit vitality; larger total litter size at birth was associated with greater risks of kit death (P<0.001) and reduced growth (P<0.001). The number of living kits in litters had the opposite effect, as kits in large liveborn litters had a reduced risk of death (P<0.001) and those with large mean litter size on days 1 to 7 had increased growth (P=0.026). Nest box temperature had little effect on early kit survival and growth, which could be due to dams' additional maternal behaviour. Therefore, we cannot confirm that temperature is the primary reason for kit mortality, under the conditions of plenty straw access for maternal nest building. Instead, prenatal and/or parturient litter size is the primary factor influencing early kit vitality. The results indicate that the focus should be on litter size and dam welfare around the times of gestation and birth to increase early kit survival in farmed mink.

The clinical significance of c-Kit mutations in metastatic oral mucosal melanoma in China.

PubMed

Ma, Xuhui; Wu, Yunteng; Zhang, Tian; Song, Hao; Jv, Houyu; Guo, Wei; Ren, Guoxin

2017-10-10

c-Kit mutations are frequently detected in mucosal melanomas, but their clinical significance in metastatic oral mucosal melanomas (OMM) remains unclear. The main purpose of this study was to investigate the clinical and pathological features of metastatic OMMs with c-Kit mutations and the efficiency of the tyrosine kinase inhibitor imatinib in treating metastatic OMMs. We found thatresidual primary lesion and neck lymph nodes could act as independent prognostic factors in metastatic OMM patients. c-Kit mutations were detected in 22 out of 139 (15.8%) metastatic OMM patients. Under chemotherapy, the overall survival (OS) of c-Kit mutant patients was significantly shorter than that of wild-type patients. The Ki67 expression was significantly higher in c-Kit mutant patients than in wild-type patients. In distant metastatic OMM patients with c-Kit mutations, the treatment with c-Kit inhibitor resulted in a better OS. In conclusion, residual primary lesion, cervical lymph nodes and c-Kit mutations act as adverse prognostic factors of metastatic OMMs. The Kit inhibitor imatinib could benefit metastatic OMM patients with c-Kit mutations.

Intratumoral CD3+ T-Lymphocytes Immunoexpression and Its Association with c-Kit, Angiogenesis, and Overall Survival in Malignant Canine Mammary Tumors

PubMed Central

Carvalho, Maria Isabel; Pires, Isabel; Dias, Marlene; Prada, Justina; Gregório, Hugo; Lobo, Luis; Queiroga, Felisbina

2015-01-01

In this study 80 malignant CMT were submitted to immunohistochemical detection of CD3, c-kit, VEGF, and CD31, together with clinicopathological parameters of tumor aggressiveness. CD3+ T-cells and c-kit overexpression revealed a positive correlation with VEGF (r = 0.503, P < 0.0001; r = 0.284, P = 0.023 for CD3 and c-kit, resp.) and CD31 (r = 0.654, P < 0.0001; r = 0.365, P = 0.003 for CD3 and c-kit, resp.). A significant association (P = 0.039) and a positive correlation (r = 0.263, P = 0.039) between CD3 and c-kit were also observed. High CD3/VEGF, c-kit/VEGF, and CD3/c-kit tumors were associated with elevated grade of malignancy (P < 0.0001 for all groups), presence of intravascular emboli (P < 0.0001 for CD3/VEGF and CD3/c-kit; P = 0.002 for c-kit/VEGF), and presence of lymph node metastasis (P < 0.0001 for all groups). Tumors with high CD3/VEGF (P = 0.006), c-kit/VEGF (P < 0.0001), and CD3/c-kit (P = 0.002) were associated with poor prognosis. Interestingly high c-kit/VEGF tumors retained their significance by multivariate analysis arising as independent prognostic factor. PMID:26346272

Temozolomide Injection

MedlinePlus

... medical treatment: unusual bleeding or bruising red or black, tarry stools pink, red, or dark brown urine coughing up or vomiting blood or material ... the following: unusual bleeding or bruising red or black, tarry stools pink, red, or dark brown urine coughing up or vomiting blood or material ...

MRI and low back pain

MedlinePlus

... back pain: Cannot pass urine or stools Cannot control your urine or stools Difficulty with walking and balance Back pain that is severe in children Fever History of cancer Other signs or symptoms of cancer Recent serious fall or injury Back pain that is very severe, ...

Warning Signs of Breastfeeding Problems

MedlinePlus

... and four stools per day, her urine is dark yellow or specked with red, or her stools are still dark rather than yellow and loose. If you or ... Academy of Pediatrics) The information contained on this Web site should not be used as a substitute ...

The value of molecular expression of KIT and KIT ligand analysed using real-time polymerase chain reaction and immunohistochemistry as a prognostic indicator for canine cutaneous mast cell tumours.

PubMed

Costa Casagrande, T A; de Oliveira Barros, L M; Fukumasu, H; Cogliati, B; Chaible, L M; Dagli, M L Z; Matera, J M

2015-03-01

This study investigated the correlation between KIT gene expression determined by immunohistochemistry and real-time polymerase chain reaction (RT-PCR) and the rate of tumour recurrence and tumour-related deaths in dogs affected with mast cell tumour (MCT). Kaplan-Meier curves were constructed to compare tumour recurrence and tumour-related death between patients. The log-rank test was used to check for significant differences between curves. KIT-I, KIT-II and KIT-III staining patterns were observed in 9 (11.11%), 50 (61.73%) and 22 (27.16%) tumours, respectively. Tumour recurrence rates and tumour-related deaths were not associated with KIT staining patterns (P = 0278, P > 0.05), KIT (P = 0.289, P > 0.05) or KIT ligand (P = 0.106, P > 0.05) gene expression. Despite the lack of association between KIT staining pattern and patient survival time, the results suggest a correlation between aberrant KIT localization and increased proliferative activity of MCTs. RT-PCR seems to be a sensible method for quantitative detection of KIT gene expression in canine MCT, although expressions levels are not correlated with prognosis. © 2013 Blackwell Publishing Ltd.

A meta-analysis of prognostic value of KIT mutation status in gastrointestinal stromal tumors

PubMed Central

Jiang, Zhiqiang; Zhang, Jian; Li, Zhi; Liu, Yingjun; Wang, Daohai; Han, Guangsen

2016-01-01

Numerous types of KIT mutations have been reported in gastrointestinal stromal tumors (GISTs); however, controversy still exists regarding their clinicopathological significance. In this study, we reviewed the publicly available literature to assess the data by a meta-analysis to characterize KIT mutations and different types of KIT mutations in prognostic prediction in patients with GISTs. Twenty-eight studies that included 4,449 patients were identified and analyzed. We found that KIT mutation status was closely correlated with size of tumors and different mitosis indexes, but not with tumor location. KIT mutation was also observed to be significantly correlated with tumor recurrence, metastasis, as well as the overall survival of patients. Interestingly, there was higher risk of progression in KIT exon 9-mutated patients than in exon 11-mutated patients. Five-year relapse-free survival (RFS) rate was significantly higher in KIT exon 11-deleted patients than in those with other types of KIT exon 11 mutations. In addition, RFS for 5 years was significantly worse in patients bearing KIT codon 557–558 deletions than in those bearing other KIT exon 11 deletions. Our results strongly support the hypothesis that KIT mutation status is another evaluable factor for prognosis prediction in GISTs. PMID:27350754

Targeting c-KIT (CD117) by dasatinib and radotinib promotes acute myeloid leukemia cell death.

PubMed

Heo, Sook-Kyoung; Noh, Eui-Kyu; Kim, Jeong Yi; Jeong, Yoo Kyung; Jo, Jae-Cheol; Choi, Yunsuk; Koh, SuJin; Baek, Jin Ho; Min, Young Joo; Kim, Hawk

2017-11-10

Dasatinib and radotinib are oral BCR-ABL tyrosine kinase inhibitors that were developed as drugs for the treatment of chronic myeloid leukemia. We report here that the c-KIT (CD117) targeting with dasatinib and radotinib promotes acute myeloid leukemia (AML) cell death, and c-KIT endocytosis is essential for triggering c-KIT-positive AML cell death by dasatinib and radotinib during the early stages. In addition, dasatinib and radotinib reduce heat shock protein 90β (HSP90β) expression and release Apaf-1 in c-KIT-positive AML cells. Finally, this activates a caspase-dependent apoptotic pathway in c-KIT-positive AML cells. Moreover, the inhibition of c-KIT endocytosis by dynamin inhibitor (DY) reversed cell viability and c-KIT expression by dasatinib and radotinib. HSP90β expression was recovered by DY in c-KIT-positive AML cells as well. Furthermore, the effect of radotinib on c-KIT and HSP90β showed the same pattern in a xenograft animal model using HEL92.1.7 cells. Therefore, dasatinib and radotinib promote AML cell death by targeting c-KIT. Taken together, these results indicate that dasatinib and radotinib treatment have a potential role in anti-leukemic therapy on c-KIT-positive AML cells.

RanBPM (RanBP9) regulates mouse c-Kit receptor level and is essential for normal development of bone marrow progenitor cells

PubMed Central

Singh, Satyendra; Klarmann, Kimberly D.; Coppola, Vincenzo; Keller, Jonathan R.; Tessarollo, Lino

2016-01-01

c-Kit is a tyrosine kinase receptor important for gametogenesis, hematopoiesis, melanogenesis and mast cell biology. Dysregulation of c-Kit function is oncogenic and its expression in the stem cell niche of a number of tissues has underlined its relevance for regenerative medicine and hematopoietic stem cell biology. Yet, very little is known about the mechanisms that control c-Kit protein levels. Here we show that the RanBPM/RanBP9 scaffold protein binds to c-Kit and is necessary for normal c-Kit protein expression in the mouse testis and subset lineages of the hematopoietic system. RanBPM deletion causes a reduction in c-Kit protein but not its mRNA suggesting a posttranslational mechanism. This regulation is specific to the c-Kit receptor since RanBPM reduction does not affect other membrane proteins examined. Importantly, in both mouse hematopoietic system and testis, RanBPM deficiency causes defects consistent with c-Kit loss of expression suggesting that RanBPM is an important regulator of c-Kit function. The finding that this regulatory mechanism is also present in human cells expressing endogenous RanBPM and c-Kit suggests a potential new strategy to target oncogenic c-Kit in malignancies. PMID:27835883

RanBPM (RanBP9) regulates mouse c-Kit receptor level and is essential for normal development of bone marrow progenitor cells.

PubMed

Puverel, Sandrine; Kiris, Erkan; Singh, Satyendra; Klarmann, Kimberly D; Coppola, Vincenzo; Keller, Jonathan R; Tessarollo, Lino

2016-12-20

c-Kit is a tyrosine kinase receptor important for gametogenesis, hematopoiesis, melanogenesis and mast cell biology. Dysregulation of c-Kit function is oncogenic and its expression in the stem cell niche of a number of tissues has underlined its relevance for regenerative medicine and hematopoietic stem cell biology. Yet, very little is known about the mechanisms that control c-Kit protein levels. Here we show that the RanBPM/RanBP9 scaffold protein binds to c-Kit and is necessary for normal c-Kit protein expression in the mouse testis and subset lineages of the hematopoietic system. RanBPM deletion causes a reduction in c-Kit protein but not its mRNA suggesting a posttranslational mechanism. This regulation is specific to the c-Kit receptor since RanBPM reduction does not affect other membrane proteins examined. Importantly, in both mouse hematopoietic system and testis, RanBPM deficiency causes defects consistent with c-Kit loss of expression suggesting that RanBPM is an important regulator of c-Kit function. The finding that this regulatory mechanism is also present in human cells expressing endogenous RanBPM and c-Kit suggests a potential new strategy to target oncogenic c-Kit in malignancies.

Changes in c-Kit expression levels during the course of radiation therapy for nasopharyngeal carcinoma.

PubMed

Jiang, Feng; Hu, Wei; Zhang, Bicheng; Xu, Jing; Shui, Yongjie; Zhou, Xiaofeng; Ren, Xiaoqiu; Chen, Xiaozhong; Shen, Li; Wei, Qichun

2016-10-01

In the era of intensity-modulated radiotherapy, distant metastasis is currently the main cause of treatment failure for nasopharyngeal carcinoma (NPC). Additional therapeutic strategies are required to control the metastasis and improve survival. One strategy is targeted therapy, for example against c-Kit. In the current study, the frequency of c-Kit expression was determined immunohistochemically in 106 NPC patients. c-Kit expression changes during the course of radiation therapy were detected in 41 cases via weekly biopsy. Twelve cases (11.3%) had c-Kit expression scores of 3 + and 16 (15.1%) had scores of 2 + . Thus, c-Kit overexpression (2 + or 3 + ) was observed in 28 (26.4%) patients. There were 35 (33.0%) and 43 (40.6%) patients with c-Kit expression scores of 1 + and 0, respectively. Furthermore, a trend of decreased c-Kit expression was observed after commencing radiotherapy according to the 41 NPC patients who were biopsied weekly. Therefore, c-Kit overexpression was identified to be common in NPC, and evaluating c-Kit as a therapeutic target for metastatic NPC via c-Kit overexpression subsequent to first line treatment may be of interest. To the best of our knowledge, the present study is the first to demonstrate a trend of decreased c-Kit expression during the course of radiotherapy.

Developing Save Your Food Kit (Sayofu Kit) to Support Inquiry, Improve Student Learning Outcomes at SMP Plus Hidayatul Mubtadiin and Public Awareness on Food Additives

NASA Astrophysics Data System (ADS)

Astutik, J.

2017-02-01

Food additives are materials that can not be separated from the lives of students and the community. Based on the preliminary questionnaire, it indicates the lack of kit supporting material additives in some schools and communities. The research objectives of this development are (1) to develop Kit experiment (SAYOFU KIT) and supplementary books to improve student learning outcomes in the classroom and public awareness on food additives (2) to describe the feasibility and potential effectiveness of SAYOFU KIT developed (3) to analyze the practice of SAYOFU KIT and benefits for students and the community. This development study uses 4-D models Thiagarajan, et al (1974). Through some stages, they are: defining, designing, developing and disseminating which involes the students and community. The developed SAYOFU KIT includes additives sample kit, borax test kit, curcumin test kit, formaldehyde test kit, modification heater to the identification of dyes and dye test paper. The study is conducted at SMP Plus Hidayatul Mubtadiin, and TKIT Al Uswah. The products are validated by experts and education practitioners. Qualitative data processing uses descriptive method, whereas quantitative data by using the N-gain. The average yield of expert validation of SAYOFU KIT with supplementary books 76.50% teacher’s book and 76.30% student’s book are eligible. The average yield of 96.81% validation of educational practitioners criteria, piloting a small group of 83.15%, and 82.89% field trials are very decent. The average yield on the student questionnaire responses SAYOFU kit and supplementary book is 87.6% with the criteria very well worth it. N-Gain 0:56 cognitive achievement with the criteria enough. The results of the public poll showed 95% feel the benefits SAYOFU kits for testing food. Based from description indicates that SAYOFU Kit developed feasible, practical, useful to support inquiry learning and improve student learning outcomes as well as public awareness of food additives.

High within-day variability of fecal calprotectin levels in patients with active ulcerative colitis: what is the best timing for stool sampling?

PubMed

Calafat, Margalida; Cabré, Eduard; Mañosa, Míriam; Lobatón, Triana; Marín, Laura; Domènech, Eugeni

2015-05-01

Fecal calprotectin (FC) is considered the best noninvasive way to assess disease activity in ulcerative colitis (UC). However, it is not known which is the more suitable moment for stool sampling in patients with increased stool frequency. The aims of this study were to assess the intraindividual variation of FC within day and to evaluate if the first bowel movement in the morning is the more suitable sample for FC measurement in patients with acute flares of UC. Patients admitted because of active UC were invited to collect samples from several bowel movements (including the first in the morning) during the same day providing their ordinal chronology. FC was measured by means of a quantitative rapid point-of-care test based on lateral flow assay immunochromatography. Eighteen patients were included for a total of 56 stool samples. Most patients had extensive UC and severe disease activity. Within-day FC values varied widely, and the median coefficient of variation was 40% (5%-114%) with a median range of variation of FC values of 3887 mg/kg (69-9946). The sample from the first stool in the morning obtained the highest individual FC within-day value in 33.3% of cases and the lowest in 38.9%. FC values widely vary between motions in patients with active UC. Stool sample collection from the first bowel movement in the morning does not ensure the highest or lowest within-day FC value. In patients with overt active UC, a single FC determination should not be used as the basis for therapeutic strategies.

Salivary Microbiota Reflects Changes in Gut Microbiota in Cirrhosis with Hepatic Encephalopathy

PubMed Central

Bajaj, Jasmohan S; Betrapally, Naga S; Hylemon, Phillip B; Heuman, Douglas M; Daita, Kalyani; White, Melanie B; Unser, Ariel; Thacker, Leroy R; Sanyal, Arun J; Kang, Dae Joong; Sikaroodi, Masoumeh; Gillevet, Patrick M

2015-01-01

Background Altered gut microbiome is associated with systemic inflammation and cirrhosis decompensation. However, the correlation of the oral microbiome with inflammation in cirrhosis is unclear. Aim Evaluate the oral microbiome in cirrhosis and compare with stool microbiome. Methods Cirrhotic outpatients [with/without hepatic encephalopathy (HE)] and controls underwent stool/saliva microbiome analysis (for composition and function) and also systemic inflammatory evaluation. 90-day liver-related hospitalizations were recorded. Salivary inflammation was studied using Th1 cytokines/secretory IgA, histatins and lysozyme in a subsequent group. Results 102 cirrhotics (43 prior-HE) and 32 age-matched controls were included. On PCO, stool and saliva microbiome clustered far apart showing differences between sites as a whole. Salivary microbiome With prior-HE, relative abundance of autochthonous families decreased while potentially pathogenic ones (Enterobacteriaceae, Enterococcaceae) increased in saliva. Endotoxin-related predicted functions were significantly higher in cirrhotic saliva. Stool microbiome Relative autochthonous taxa abundance reduced in prior-HE, along with increased Enterobacteriaceae and Enterococcaceae. Cirrhotic stool microbiota demonstrated a significantly higher correlation with systemic inflammation compared to saliva microbiota on correlation networks. Outcomes 38 patients were hospitalized within 90 days. Their salivary dysbiosis was significantly worse and predicted this outcome independent of cirrhosis severity. Salivary inflammation was studied in an additional 86 age-matched subjects (43 controls/43 cirrhotics); significantly higher IL-6/IL-1β, secretory IgA and lower lysozyme, and histatins 1 and 5 were found in cirrhotics compared to controls. Conclusions Dysbiosis, represented by reduction in autochthonous bacteria, is present in both saliva and stool in cirrhosis patients compared to controls. Cirrhotic patients have impaired salivary defenses and worse inflammation. Salivary dysbiosis was greater in cirrhotics who developed 90-day hospitalizations. These findings could represent a global mucosal-immune interface change in cirrhosis. PMID:25820757

Validation of the 3-day rule for stool bacterial tests in Japan.

PubMed

Kobayashi, Masanori; Sako, Akahito; Ogami, Toshiko; Nishimura, So; Asayama, Naoki; Yada, Tomoyuki; Nagata, Naoyoshi; Sakurai, Toshiyuki; Yokoi, Chizu; Kobayakawa, Masao; Yanase, Mikio; Masaki, Naohiko; Takeshita, Nozomi; Uemura, Naomi

2014-01-01

Stool cultures are expensive and time consuming, and the positive rate of enteric pathogens in cases of nosocomial diarrhea is low. The 3-day rule, whereby clinicians order a Clostridium difficile (CD) toxin test rather than a stool culture for inpatients developing diarrhea >3 days after admission, has been well studied in Western countries. The present study sought to validate the 3-day rule in an acute care hospital setting in Japan. Stool bacterial and CD toxin test results for adult patients hospitalized in an acute care hospital in 2008 were retrospectively analyzed. Specimens collected after an initial positive test were excluded. The positive rate and cost-effectiveness of the tests were compared among three patient groups. The adult patients were divided into three groups for comparison: outpatients, patients hospitalized for ≤3 days and patients hospitalized for ≥4 days. Over the 12-month period, 1,597 stool cultures were obtained from 992 patients, and 880 CD toxin tests were performed in 529 patients. In the outpatient, inpatient ≤3 days and inpatient ≥4 days groups, the rate of positive stool cultures was 14.2%, 3.6% and 1.3% and that of positive CD toxin tests was 1.9%, 7.1% and 8.5%, respectively. The medical costs required to obtain one positive result were 9,181, 36,075 and 103,600 JPY and 43,200, 11,333 and 9,410 JPY, respectively. The 3-day rule was validated for the first time in a setting other than a Western country. Our results revealed that the "3-day rule" is also useful and cost-effective in Japan.

New ultrasonographic evaluation of stool and/or gas distribution for treatment of chronic constipation.

PubMed

Manabe, Noriaki; Kamada, Tomoari; Hata, Jiro; Haruma, Ken

2018-03-01

The first aim of this study was to develop a new ultrasonographic method (US) to evaluate stool and/or gas distribution. The second aim was to apply this method to compare stool and/or gas distribution between healthy subjects and patients with chronic constipation and evaluate whether US parameters could be an alternative to the colonic transit time (CTT). We enrolled seven healthy volunteers (four men, three women; mean age 29.3 ± 5.2 years) who underwent US and computed tomography (CT) on the same day to evaluate the reproducibility of US results. We then enrolled 268 patients with chronic constipation (94 men, 174 women; mean age 63.3 ± 4.2 years) and 66 age- and sex-matched healthy subjects (controls). The transverse diameters of four segments of the colon [ascending (AC), transverse (TC), descending (DC), and sigmoid (SC)] and the rectum (R) were measured, and their stool and/or gas distribution was evaluated using the constipation index (CI) [AC + TC + DC + SC + R/5] and left/right (L/R) distribution [(DC + SC)/(AC + TC)]. The CTT was assessed using radiopaque markers. All healthy subjects underwent US and CT successfully, with a sufficiently high reproducibility coefficient for this method and significant correlation between the US and CT parameters. The stool and/or gas distribution evaluated by US showed a significant difference in one of the US parameters between healthy subjects and patients, and the CI was an indirect indicator for the CTT. These findings may assist physicians evaluate stool and/or gas distribution of patients with chronic constipation, which is an indirect indicator for CTT.

A case of Dipylidium caninum infection in a child from the southeastern Poland.

PubMed

Szwaja, Bogusława; Romański, Leszek; Zabczyk, Michał

2011-01-01

Dipylidium caninum is a common intestinal tapeworm of dogs, cats and foxes. However, it occasionally infects also humans. We present a case of D. caninum infection in a 2-year-old child living in the Subcarpathian province. The infection was asymptomatic in the first months. The symptoms of abdominal pains, sleep disorders, loss of appetite, hyperactivity and occasional slimy stools appeared later. Proglottids on the underwear, in water while bathing and mobile proglottids passed with the stool were also observed. Prior to appropriate diagnosis the child was treated with pyrantelum (Pyrantelum) and albendazolum (Zentel). However, proglottids were found again in the stool after a few days. We examined stool samples and perianal smears collected from the child and his family. The stool samples were tested by coproscopic methods. Direct methods (direct preparation in 0.9% sodium chloride and in Lugol's solution, Kato thick smear) and concentration methods (decantation with distilled water and Faust's zinc sulphate centrifugal flotation) were used. In the stool samples taken from the child, we observed D. caninum proglottids demonstrating lateral genital pores and many packets of eggs containing from one to a few, mostly 3 to 4 eggs. In the direct preparations in 0.9% sodium chloride and in Lugol's solution single packets with D. caninum eggs were detected. In decantation preparations many D. caninum egg packets were observed. It has to be reported that the child's mother was infected with Giardia intestinalis. Dipylidiasis in humans is a rarely encountered infection in Poland and the diagnosis may be difficult. For these reasons we reported clinical case presentation, diagnostics, treatment and epidemiology of D. caninum infection. We have shown that concentration methods such as decantation might be very helpful in the diagnosis of dipylidiasis.

Laxative effects of agarwood on low-fiber diet-induced constipation in rats.

PubMed

Kakino, Mamoru; Tazawa, Shigemi; Maruyama, Hiroe; Tsuruma, Kazuhiro; Araki, Yoko; Shimazawa, Masamitsu; Hara, Hideaki

2010-11-15

Agarwood (Aquilaria sinensis), well known as incense in Southeast Asia, has been used as a digestive in traditional medicine. We investigated the laxative effects of an ethanol extract of agarwood leaves (EEA) in a rat model of low-fiber diet-induced constipation. A set of rats was bred on a normal diet while another set was placed on a low-fiber diet to induce constipation. The laxative effect of agarwood was then investigated on both sets of rats. Pretreatment of normal rats with single dose of EEA (600 mg/kg, p.o.) significantly increased frequency and weight of stools. Also, treatments with EEA (300 and 600 mg/kg, p.o.) for 14 days caused a significant increase in stool frequency and weight. Feeding of the animals with a low-fiber diet resulted in a decrease in stool weight, frequency, and water content and also delayed carmine egestion. A single treatment with EEA (600 mg/kg) or senna (150 and 300 mg/kg) significantly increased stool frequency, weight, and water content and also accelerated carmine egestion in the model rats. Once daily administrations of EEA (150 mg/kg), for 14 days, caused a significant increase in water content of stools. The higher doses of EEA (300 and 600 mg/kg) significantly increased frequency, weight, and water content of the stools while accelerating carmine egestion in the constipated rats. Senna (150 and 300 mg/kg) produced similar effect as the higher doses of EEA but, in addition, induced severe diarrhea. These findings indicate that EEA has a laxative effect, without causing diarrhea, in a rat model of low-fiber diet-induced constipation. These findings suggest that EEA may be highly effective on constipation as a complementary medicine in humans suffering from life style-induced constipation.

Recommendations for pharmacological clinical trials in children with irritable bowel syndrome: the Rome foundation pediatric subcommittee on clinical trials.

PubMed

Saps, M; van Tilburg, M A L; Lavigne, J V; Miranda, A; Benninga, M A; Taminiau, J A; Di Lorenzo, C

2016-11-01

There is little published evidence of efficacy for the most commonly used treatments. Thus, there is an urgent need to conduct clinical trials on existing and novel therapies. In order to address these issues the Rome Foundation and members of the Pediatric Committee of the European Medicines Agency formed a subcommittee on clinical trials to develop guidelines for the design of clinical trials in children with irritable bowel syndrome (IBS). The following recommendations are based on evidence from published data when available and expert opinion. The subcommittee recommends randomized, double-blind, placebo-controlled, parallel-group, clinical trials to assess the efficacy of new drugs. The combined endpoints for abdominal pain are a decrease in intensity of at least 30% compared with baseline and to meet or exceed the Reliable Change Index (RCI) for the sample. Stool consistency is measured with the Bristol Stool Scale Form (BSFS). The subcommittee recommends as entry criteria for abdominal pain a weekly average of worst abdominal pain in past 24 h of at least 3.0 on a 0-10 point scale or at least 30 mm in 100 mm Visual Analog Scale. For stool endpoints the committee recommends an average stool consistency lower than 3 in the BSFS during the run-in period for clinical trials on IBS-C and an average stool consistency greater than 5 in the BSFS during the run-in period for clinical trials on IBS-D. Changes in stool consistency are the primary endpoints for both IBS with diarrhea (IBS-D) and IBS with constipation (IBS-C). © 2016 John Wiley & Sons Ltd.

Intestinal blood loss during cow milk feeding in older infants: quantitative measurements.

PubMed

Jiang, T; Jeter, J M; Nelson, S E; Ziegler, E E

2000-07-01

To determine the response, in terms of fecal hemoglobin excretion and clinical symptoms, of normal 9 1/2-month-old infants to being fed cow milk. Longitudinal (before-after) trial in which each infant was fed formula for 1 month (baseline) followed by 3 months during which cow milk was fed. Healthy infants living in Iowa City, Iowa, a town with a population of about 60,000. Hemoglobin concentration in spot stools, 96-hour quantitative fecal hemoglobin excretion, stool characteristics, feeding-related behaviors, and iron nutritional status. Fecal hemoglobin concentration during formula feeding (baseline) was higher than previously observed in younger infants. Nine of 31 infants responded to cow milk feeding with increased fecal hemoglobin concentration. Fecal hemoglobin concentration (mean +/- SD) of the 9 responders rose from 1,395 +/- 856 microg/g of dry stool (baseline) to 2,711 +/- 1,732 microg/g of dry stool (P=.01). The response rate (29%) was similar to that in younger infants, but the intensity of the response was much less. Quantitative hemoglobin excretion was in general agreement with estimates based on spot stool hemoglobin concentrations. Cow milk feeding was not associated with recognizable changes in stool characteristics, nor were there clinical signs related to fecal blood loss. Iron status was similar, except that after 3 months of cow milk feeding responders showed lower (P= .047) ferritin concentrations than nonresponders. Cow milk-induced blood loss is present in 9 1/2-month-old infants but is of such low intensity that its clinical significance seems questionable. Nevertheless, infants without cow milk-induced blood loss were in better iron nutritional status than infants who showed blood loss.

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.

PubMed

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. Copyright © 2014 Elsevier B.V. All rights reserved.

Digestion-resistant maltodextrin effects on colonic transit time and stool weight: a randomized controlled clinical study.

PubMed

Abellán Ruiz, María Salud; Barnuevo Espinosa, María Dolores; Contreras Fernández, Carlos J; Luque Rubia, Antonio J; Sánchez Ayllón, Francisca; Aldeguer García, Miriam; García Santamaría, Carlos; López Román, Francisco Javier

2016-12-01

Increased awareness of the importance of dietary fibre has led to increased interest in "functional" fibre components like digestion-resistant maltodextrin (RMD). This randomized, placebo-controlled, double-blind study assessed the effects of RMD in the colonic transit time (CTT) and defecation characteristics (frequency, stool volume and consistency). Sixty-six healthy adult volunteers (32 men) who did not have a daily defecation habit had a 7-day run-in period before the 21-day intervention period with RMD or placebo. CTT and segmental CTT (SCTT) were assessed by a single abdominal X-ray film taken at the end of both periods after radiopaque marker ingestion. Defecation characteristics and intestinal functions were also assessed, which were self-reported by patients. Intragroup comparisons were evaluated by Student's paired t test, Bonferroni test and Chi-square test, while time comparisons by analysis of variance (ANOVA) and time-by-treatment interaction by repeated-measures ANOVA. Fifty-seven subjects were assessed for CTT (placebo, n = 28; RMD, n = 29). In the RMD group, the total CTT, left SCTT and rectosigmoidal SCTT decreased significantly compared to baseline (p < 0.01 each; -13.3, -4.7, -8.7 h, respectively). Significant differences between groups were observed in total CTT and left SCTT. Significant time-by-treatment interaction was observed in the RMD group for stool volume (p = 0.014), increasing 56 % compared to baseline (p < 0.01), while remained unchanged in the placebo group. Stool consistency was improved only in the RMD group (p < 0.01). No adverse effects related to study products were observed. The results show that RMD improved CTT, stool volume, stool consistency and some intestinal functions in a healthy population.

A mechanistic multicentre, parallel group, randomised placebo-controlled trial of mesalazine for the treatment of IBS with diarrhoea (IBS-D).

PubMed

Lam, Ching; Tan, Wei; Leighton, Matthew; Hastings, Margaret; Lingaya, Melanie; Falcone, Yirga; Zhou, Xiaoying; Xu, Luting; Whorwell, Peter; Walls, Andrew F; Zaitoun, Abed; Montgomery, Alan; Spiller, Robin

2016-01-01

Immune activation has been reported in the mucosa of IBS patients with diarrhoea (IBS-D), and some small studies have suggested that mesalazine may reduce symptoms. We performed a double-blind, randomised placebo-controlled trial of 2 g mesalazine twice daily versus placebo for 3 months in patients with Rome III criteria IBS-D. Primary outcome was daily average stool frequency during weeks 11-12; secondary outcomes were abdominal pain, stool consistency, urgency and satisfactory relief of IBS symptoms. Participants were randomised after a 2-week baseline stool diary. All participants completed a 12-week stool diary and at the end of each week recorded the presence of 'satisfactory relief of IBS symptoms'. 136 patients with IBS-D (82 women, 54 men) were randomised, 10 patients withdrew from each group. Analysis by intention to treat showed the daily average stool frequency during weeks 11 and 12 were mean (SD), 2.8 (1.2) in mesalazine and 2.7 (1.9) in the placebo group with no significant group difference, (95% CI) 0.1 (-0.33 to 0.53), p=0.66. Mesalazine did not improve abdominal pain, stool consistency nor percentage with satisfactory relief compared with placebo during the last two-weeks follow-up. This study does not support any clinically meaningful benefit or harm of mesalazine compared with placebo in unselected patients with IBS-D. More precise subtyping based on underlying disease mechanisms is needed to allow more effective targeting of treatment in IBS. NCT01316718. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

DNA Methylation and Mutation of Small Colonic Neoplasms in Ulcerative Colitis and Crohn's Colitis: Implications for Surveillance.

PubMed

Johnson, David H; Taylor, William R; Aboelsoud, Mohammed M; Foote, Patrick H; Yab, Tracy C; Cao, Xiaoming; Smyrk, Thomas C; Loftus, Edward V; Mahoney, Douglas W; Ahlquist, David A; Kisiel, John B

2016-07-01

Stool DNA testing in patients with inflammatory bowel disease (IBD) may detect colorectal cancer and advanced precancers with high sensitivity; less is known about the presence of DNA markers in small IBD lesions, their association with metachronous neoplasia, or contribution to stool test positivity. At a single center in 2 blinded phases, we assayed methylated bone morphogenic protein 3, methylated N-Myc downstream-regulated gene 4, and mutant KRAS in DNA extracted from paraffin-embedded benign lesions, and matched control tissues of patients with IBD, who were followed for subsequent colorectal dysplasia. Stool samples from independent cases and controls with lesions <1 cm or advanced neoplasms were assayed for the same markers. Among IBD lesions (29 low-grade dysplasia, 19 serrated epithelial change, and 10 sessile serrated adenoma/polyps), the prevalence of methylation was significantly higher than in mucosae from 44 matched IBD controls (P < 0.0001 for methylated bone morphogenic protein 3 or methylated N-Myc downstream-regulated gene 4). KRAS mutations were more abundant in serrated epithelial change than all other groups (P < 0.001). Subsequent dysplasia was not associated with DNA marker levels. In stools, the sensitivity of methylated bone morphogenic protein 3 as a single marker was 60% for all lesions <1 cm, 63% for low-grade dysplasia ≥1 cm and 81% for high-grade dysplasia/colorectal cancer, all at 91% specificity (P < 0.0001). Selected DNA markers known to be present in advanced IBD neoplasia can also be detected in both tissues and stools from IBD patients with small adenomas and serrated lesions. Mutant KRAS exfoliated from serrated epithelial change lesions might raise false-positive rates. These findings have relevance to potential future applications of stool DNA testing for IBD surveillance.

Use of a Novel Real-Time PCR Assay To Detect Oral Polio Vaccine Shedding and Reversion in Stool and Sewage Samples after a Mexican National Immunization Day▿

PubMed Central

Troy, Stephanie B.; Ferreyra-Reyes, Leticia; Huang, ChunHong; Mahmud, Nadim; Lee, Yu-Jin; Canizales-Quintero, Sergio; Flaster, Harry; Báez-Saldaña, Renata; García-García, Lourdes; Maldonado, Yvonne

2011-01-01

During replication, oral polio vaccine (OPV) can revert to neurovirulence and cause paralytic poliomyelitis. In individual vaccinees, it can acquire specific revertant point mutations, leading to vaccine-associated paralytic poliomyelitis (VAPP). With longer replication, OPV can mutate into vaccine-derived poliovirus (VDPV), which causes poliomyelitis outbreaks similar to those caused by wild poliovirus. After wild poliovirus eradication, safely phasing out vaccination will likely require global use of inactivated polio vaccine (IPV) until cessation of OPV circulation. Mexico, where children receive routine IPV but where OPV is given biannually during national immunization days (NIDs), provides a natural setting to study the duration of OPV circulation in a population primarily vaccinated with IPV. We developed a real-time PCR assay to detect and distinguish revertant and nonrevertant OPV serotype 1 (OPV-1), OPV-2, and OPV-3 from RNA extracted directly from stool and sewage. Stool samples from 124 children and 8 1-liter sewage samples from Orizaba, Veracruz, Mexico, collected 6 to 13 weeks after a NID were analyzed. Revertant OPV-1 was found in stool at 7 and 9 weeks, and nonrevertant OPV-2 and OPV-3 were found in stool from two children 10 weeks after the NID. Revertant OPV-1 and nonrevertant OPV-2 and -3 were detected in sewage at 6 and 13 weeks after the NID. Our real-time PCR assay was able to detect small amounts of OPV in both stool and sewage and to distinguish nonrevertant and revertant serotypes and demonstrated that OPV continues to circulate at least 13 weeks after a NID in a Mexican population routinely immunized with IPV. PMID:21411577

[Development of surgical antibioprophylaxis kits: evaluation of the impact on prescribing habits].

PubMed

Aouizerate, P; Guizard, M

2002-01-01

In our hospital, surgical antibioprophylaxis (ATBP) was too often administered too late, thus raising the infectious risk. Antibiotic stocks of the anaesthesia department were also systematically used, instead of nominal prescriptions of these drugs. The pharmacy could neither charge antibiotics to each surgical department nor quantify and differentiate ATBP from curative antibiotic therapy. The pharmacy and anaesthesia departments therefore set out to standardize surgical ATBP, in order to adapt this treatment to each surgical indication, and particularly in the case of allergy to beta-lactamase antibiotics (second line treatment kits). Consequently, prescription forms were developed and supplied to each surgery department, as well as ATBP kits. The kits were prepared and distributed by the pharmacy, and comprised boxes containing antibiotics in sufficient quantities to respect the protocols approved by the French Society of Anaesthesia and Resuscitation (SFAR). A protocol describing prescriptions, dispensation and administration has been presented to physicians and nurses. Fifteen surgical departments were included in our study and 30 different kits were prepared. From 1998 to 2001, 5586 surgical operations required administration of a kit (second line treatment kits in 5% of cases): 1848 (33%) in visceral surgery; 764 (13.8%) in urology; 802 (14%) in orthopaedics; 13 (0.2%) in vascular and thoracic surgery; 1236 (22%) in ear-nose-throat (ENT), periodontics and ophtalmology, and 923 (17%) in gynaecology and obstetrics. 93% of filled prescriptions forms were spontaneously returned to the pharmacy, the others were obtained during the renewal of kit stocks. The cost (over 4 years) of ATBP was quantified: 157,871 F for the 15 departments included, 26,123 F in visceral surgery, 13,520 F in urology, 73,741 F in orthopaedics, 569 F in vascular surgery, 39,720 F in ENT/ophthalmology/periodontics and 4,198 F in gynaecology and obstetrics. According to the Altemeier classification, 2226 class I, 3151 class II, and 209 class III surgical operations were performed. Since the kits have been brought into use, the committee for the protection against nosocomial infections (CLIN) has observed a reduction in the incidence of post-operative infections, according to the Altemeier classification: from 1.6% to 0.5% in class I, from 6.5% to 4.3% in class II, and from 11% to 8.5% in class III. The difference was statistically significant only for classes I (p < 0.01) and II (p < 0.001), and unchanged for class III (p = 0.3). No analysis was carried out for class IV (curative treatments). Both nurses and physicians have greatly appreciated the implementation of this organization. The advantage in terms of post-operative infections, administration exhaustiveness and stock management is obvious. The prescribed kits were systematically appropriate for the surgical interventions. In orthopaedics, cefamandole was used over 24 h (188 kits) in ligament plasty and osteotomy, or for 48 h (499 kits) in prosthetic surgery; 24 amoxicillin/clavulanic acid (first line) and 9 clindamycin/gentamicin (second line) single dose kits have been prescribed in traumatic indications. In ophthalmology, kits were only prescribed in endophtalmitis (24 ofloxacin/fosfomycin single amount kits), implant replacement or cornea graft (1076 ofloxacin 24 h kits) and cataract surgery in diabetic patients (12 ofloxacin single amount kits). In ENT and periodontics, 124 surgical operations required cefazolin single dose kits. In vascular surgery, 5 pefloxacin/gentamicin 48 h kits and 1 amoxicillin/clavulanic acid 48 h kit were used in contaminated limb amputation, 1 cefamandole 48 h kit in class I surgery and 1 vancomycin 24 h kit (betalactamase antibiotic allergy); in thoracic surgery, 1 cefamandole 24 h kit was used for a thoracic wound. In visceral surgery, 9 different kits have been used, depending on the opening (class II) or not (class I) of the digestive tract. 797 cefazolin (first line) and 68 clindamycin/gentamicin (second line) single dose kits were used in class I surgery, and 689 amoxicillin/clavulanic acid single dose (SD) kits in class II surgery. Specific protocols consisted of 18 ceftriaxone/metronidazole and 48 metronidazole/gentamicin SD kits in oesophagus surgery, 11 ceftriaxone and 17 gentamicin SD kits in biliary endoscopy, 137 metronidazole SD kits in proctology and 34 amoxicillin/gentamicin 6 h kits for prevention of endocarditis. In urology, 133 cefotaxime and 20 pefloxacin/gentamicin SD kits were precribed in renal lithiasis, 102 amoxicillin/clavulanic acid SD kits in cystectomy, 27 amoxicillin/gentamicin 6 h kits in endocarditis prevention and 58 cefamandole SD kits in all other indications. In gynaecology and obstetrics, 534 cefazoline and 19 clindamycin/gentamicin (second line) SD kits were used, and 370 doxycyclin SD kits were prescribed in pregnancy termination. Some departments (orthopaedics and visceral surgery) adapted the protocols to their needs, specifically with regard to treatment duration. However, these situations were quickly corrected. A constant follow-up and update of this system, associated with routine audits, should allow the maintenance and possibly the improvement of these results, hence shortening treatment duration.

49 CFR 173.165 - Polyester resin kits.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 2 2012-10-01 2012-10-01 false Polyester resin kits. 173.165 Section 173.165... Polyester resin kits. (a) Except for transportation by aircraft, polyester resin kits consisting of a base... resin kits consisting of a base material component (Class 3, Packing Group II or III) and an activator...

Adaptation of commercial biomarker kits and proposal for 'drug development kits' to support bioanalysis: call for action.

PubMed

Islam, Rafiqul; Kar, Sumit; Islam, Clarinda; Farmen, Raymond

2018-06-01

There has been an increased use of commercial kits for biomarker measurement, commensurate with the increased demand for biomarkers in drug development. However, in most cases these kits do not meet the quality attributes for use in regulated environment. The process for adaptation of these kits can be frustrating, time consuming and resource intensive. In addition, a lack of harmonized guidance for the validation of biomarker poses a significant challenge in the adaptation of kits in a regulated environment. The purpose of this perspective is to propose a tiered approach to commercial drug development kits with clearly defined quality attributes and to demonstrate how these kits can be adapted to perform analytical validation in a regulated environment.

The Use of Kits in the Analysis of Tissue Lipids Requires Validation.

PubMed

Rider, T; LeBoeuf, R C; Tso, Patrick; Jandacek, R J

2016-04-01

The ready availability and ease of use of kits for the measurement of serum lipids has greatly facilitated these measurements. In many cases it would be convenient to use these kits in the determination of lipid concentrations in tissues. The successful application of serum kits in tissue analysis requires that two important issues be considered. First, the solvent system for the extraction of the lipids and the solvent used for analysis by the kit must be compatible with the reactions in the kit. Second, the concentration range in the analyzed solution must be within the range for which the kit is used. We report here that lipids in liver and adipose tissues may be significantly underestimated by the use of some kits. We recommend that the use of kits for tissue analysis of lipids be validated for the specific analysis.

Oncogenic activation of v-kit involves deletion of a putative tyrosine-substrate interaction site.

PubMed

Herbst, R; Munemitsu, S; Ullrich, A

1995-01-19

The transforming gene of the Hardy-Zuckerman-4 strain of feline sarcoma virus, v-kit, arose by transduction of the cellular c-kit gene, which encodes the receptor tyrosine kinase (RTK) p145c-kit. To gain insight into the molecular basis of the v-kit transforming potential, we characterized the feline c-kit by cDNA cloning. Comparison of the feline v-kit and c-kit sequences revealed, in addition to deletions of the extracellular and transmembrane domains, three additional mutations in the v-kit oncogene product: deletion of tyrosine-569 and valine-570, the exchange of aspartate at position 761 to glycine, and replacement of the C-terminal 50 amino acids by five unrelated residues. Examinations of individual v-kit mutations in the context of chimeric receptors yielded inhibitory effects for some mutants on both autophosphorylation and substrate phosphorylation functions. In contrast, deletion of tyrosine-569 and valine-570 significantly enhanced transforming and mitogenic activities of p145c-kit, while the other mutations had no significant effects. Conservation in subclass III RTKs and the identification of the corresponding residue in beta PDGF-R, Y579, as a binding site for src family tyrosine kinases suggests an important role for Y568 in kit signal regulation and the definition of its oncogenic potential. Repositioning of Y571 by an inframe two codon deletion may be the crucial alteration resulting in enhancement of v-kit oncogenic activity.

M-COPA suppresses endolysosomal Kit-Akt oncogenic signalling through inhibiting the secretory pathway in neoplastic mast cells.

PubMed

Hara, Yasushi; Obata, Yuuki; Horikawa, Keita; Tasaki, Yasutaka; Suzuki, Kyohei; Murata, Takatsugu; Shiina, Isamu; Abe, Ryo

2017-01-01

Gain-of-function mutations in Kit receptor tyrosine kinase result in the development of a variety of cancers, such as mast cell tumours, gastrointestinal stromal tumours (GISTs), acute myeloid leukemia, and melanomas. The drug imatinib, a selective inhibitor of Kit, is used for treatment of mutant Kit-positive cancers. However, mutations in the Kit kinase domain, which are frequently found in neoplastic mast cells, confer an imatinib resistance, and cancers expressing the mutants can proliferate in the presence of imatinib. Recently, we showed that in neoplastic mast cells that endogenously express an imatinib-resistant Kit mutant, Kit causes oncogenic activation of the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the signal transducer and activator of transcription 5 (STAT5) but only on endolysosomes and on the endoplasmic reticulum (ER), respectively. Here, we show a strategy for inhibition of the Kit-PI3K-Akt pathway in neoplastic mast cells by M-COPA (2-methylcoprophilinamide), an inhibitor of this secretory pathway. In M-COPA-treated cells, Kit localization in the ER is significantly increased, whereas endolysosomal Kit disappears, indicating that M-COPA blocks the biosynthetic transport of Kit from the ER. The drug greatly inhibits oncogenic Akt activation without affecting the association of Kit with PI3K, indicating that ER-localized Kit-PI3K complex is unable to activate Akt. Importantly, M-COPA but not imatinib suppresses neoplastic mast cell proliferation through inhibiting anti-apoptotic Akt activation. Results of our M-COPA treatment assay show that Kit can activate Erk not only on the ER but also on other compartments. Furthermore, Tyr568/570, Tyr703, Tyr721, and Tyr936 in Kit are phosphorylated on the ER, indicating that these five tyrosine residues are all phosphorylated before mutant Kit reaches the plasma membrane (PM). Our study provides evidence that Kit is tyrosine-phosphorylated soon after synthesis on the ER but is unable to activate Akt and also demonstrates that M-COPA is efficacious for growth suppression of neoplastic mast cells.

M-COPA suppresses endolysosomal Kit-Akt oncogenic signalling through inhibiting the secretory pathway in neoplastic mast cells

PubMed Central

Hara, Yasushi; Obata, Yuuki; Horikawa, Keita; Tasaki, Yasutaka; Suzuki, Kyohei; Murata, Takatsugu; Shiina, Isamu; Abe, Ryo

2017-01-01

Gain-of-function mutations in Kit receptor tyrosine kinase result in the development of a variety of cancers, such as mast cell tumours, gastrointestinal stromal tumours (GISTs), acute myeloid leukemia, and melanomas. The drug imatinib, a selective inhibitor of Kit, is used for treatment of mutant Kit-positive cancers. However, mutations in the Kit kinase domain, which are frequently found in neoplastic mast cells, confer an imatinib resistance, and cancers expressing the mutants can proliferate in the presence of imatinib. Recently, we showed that in neoplastic mast cells that endogenously express an imatinib-resistant Kit mutant, Kit causes oncogenic activation of the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the signal transducer and activator of transcription 5 (STAT5) but only on endolysosomes and on the endoplasmic reticulum (ER), respectively. Here, we show a strategy for inhibition of the Kit-PI3K-Akt pathway in neoplastic mast cells by M-COPA (2-methylcoprophilinamide), an inhibitor of this secretory pathway. In M-COPA-treated cells, Kit localization in the ER is significantly increased, whereas endolysosomal Kit disappears, indicating that M-COPA blocks the biosynthetic transport of Kit from the ER. The drug greatly inhibits oncogenic Akt activation without affecting the association of Kit with PI3K, indicating that ER-localized Kit-PI3K complex is unable to activate Akt. Importantly, M-COPA but not imatinib suppresses neoplastic mast cell proliferation through inhibiting anti-apoptotic Akt activation. Results of our M-COPA treatment assay show that Kit can activate Erk not only on the ER but also on other compartments. Furthermore, Tyr568/570, Tyr703, Tyr721, and Tyr936 in Kit are phosphorylated on the ER, indicating that these five tyrosine residues are all phosphorylated before mutant Kit reaches the plasma membrane (PM). Our study provides evidence that Kit is tyrosine-phosphorylated soon after synthesis on the ER but is unable to activate Akt and also demonstrates that M-COPA is efficacious for growth suppression of neoplastic mast cells. PMID:28403213

Long-term Shedding of Influenza A Virus in Stool of Immunocompromised Child

PubMed Central

Mix, Samantha; Rowe, Judy; Ikemoto, Sheryl; Baron, Ellen J.

2010-01-01

In immunocompromised patients, influenza infection may progress to prolonged viral shedding from the respiratory tract despite antiviral therapy. We describe chronic influenza A virus infection in an immunocompromised child who had prolonged shedding of culturable influenza virus in stool. PMID:20587197

6. MOBILE LAUNCHER SIDE 4, SHOWING MILK STOOL AND LUT. ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

6. MOBILE LAUNCHER SIDE 4, SHOWING MILK STOOL AND LUT. PROTRUSION ON UPPER RIGHT HAND SIDE OF LUT IS SWING ARM NINE WHICH PROVIDED ACCESS TO CAPSULE OF LAUNCH VEHICLE WHILE ON LAUNCHER. - Mobile Launcher One, Kennedy Space Center, Titusville, Brevard County, FL

[Rice water with and without electrolytes in diarrhea with a high stool output].

PubMed

Mota-Hernández, F; Posadas-Tello, N M; Rodríguez-Leyva, G

1993-12-01

The objective of the study was to determine the efficacy and safety of two rice-based oral rehydration solutions, with and without added electrolyte in children presenting acute diarrheal dehydration with high stool output (> 10 mL/kg/h) during a two-hour rehydration period. Twenty-two patients of one to 18 months old were recruited and randomly distributed into two groups: group A received the rice-based solution without electrolytes, and group B received the rice-based solution with electrolytes. A stool output diminishing was observed in both groups and rehydration was achieved in 4.0 +/- 0.9 hours in 21 patients from group A and in 4.6 +/- 0.9 hours in 13 patients group group B. There was not a statistically significant difference between the groups regarding the laboratory results. The rice-based oral rehydration solution without added electrolytes was useful for rehydration of children presenting high stool output, after administering the WHO/ORS recommended formula during a two-hour period.

Results of a 6-month survey of stool cultures for Escherichia coli O157:H7.

PubMed

Marshall, W F; McLimans, C A; Yu, P K; Allerberger, F J; Van Scoy, R E; Anhalt, J P

1990-06-01

Escherichia coli O157:H7 is a recently recognized enteric pathogen that causes acute hemorrhagic colitis. Although the infection is usually self-limited, it may be complicated by hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. At our institution, stool specimens are now routinely cultured for this organism. To determine the prevalence of E. coli O157:H7-associated diarrhea in our patient population, we surveyed all submitted stool cultures for 6 months for this organism. Specimens were screened for non-sorbitol fermenting E. coli and confirmed by slide-agglutination and immobilization testing. Of 2,164 specimens, 10 yielded E. coli O157:H7. It was the fourth most common bacterial stool pathogen found. Bloody diarrhea and abdominal pain were the most common symptoms of the infected patients. E. coli O157:H7 causes sporadic infections in our patient population and should be considered in the differential diagnosis of acute hemorrhagic colitis.

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

PubMed

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 < 0.001). PE was strongly associated with dietary fibre intake (adjusted regression model; unstandardised B -1.44 (95% CI -1.62, -1.24) g/day, p < 0.001). PE had a lower percentage of fibre from vegetables compared with NPE (8.9% vs 15.7%, respectively, p < 0.001). There was an association between PE and usually having 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. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Randomized, double-blind, placebo-controlled clinical trial of loperamide plus simethicone versus loperamide alone and simethicone alone in the treatment of acute diarrhea with gas-related abdominal discomfort.

PubMed

Hanauer, Stephen B; DuPont, Herbert L; Cooper, Kimberly M; Laudadio, Charles

2007-05-01

To compare efficacy and tolerability of a loperamide/simethicone (LOP/SIM) combination product with that of loperamide (LOP) alone, simethicone (SIM) alone, and placebo (PBO) for acute nonspecific diarrhea with gas-related abdominal discomfort. In this multicenter, double-blind, 48-h study, patients were randomly assigned to receive two tablets, each containing either LOP/SIM 2 mg/125 mg (n = 121), LOP 2 mg (n = 120), SIM 125 mg (n = 123), or PBO (n = 121), followed by one tablet after each unformed stool, up to four tablets in any 24-h period. The primary outcome measures were time to last unformed stool and time to complete relief of gas-related abdominal discomfort. For time to last unformed stool, an unformed stool after a 24-h period of formed stools or no stools was considered a continuance of the original episode (stricter definition) or a new episode (alternate definition). A total of 483 patients were included in the intent-to-treat analysis. The median time to last unformed stool for LOP/SIM (7.6 h) was significantly shorter than that of LOP (11.5 h), SIM (26.0 h), and PBO (29.4 h) (p < or = 0.0232 in comparison with survival curves) using the alternate definition; it was numerically but not significantly shorter than that of LOP (p = 0.0709) and significantly shorter than that of SIM and PBO (p = 0.0001) using the stricter definition. LOP/SIM-treated patients had a shorter time to complete relief of gas-related abdominal discomfort than patients who received either ingredient alone or placebo (all p = 0.0001). Few patients reported adverse events in the four treatment groups, none of which were serious in nature. Potential study limitations include the ability to generalize study results to the population at large, variability in total dose consumed, and subjectivity of patient diary data. LOP/SIM was well-tolerated and more efficacious than LOP alone, SIM alone, or placebo for acute nonspecific diarrhea and gas-related abdominal discomfort.

Dietary Milk-Fat-Globule Membrane Affects Resistance to Diarrheagenic Escherichia coli in Healthy Adults in a Randomized, Placebo-Controlled, Double-Blind Study.

PubMed

Ten Bruggencate, Sandra J; Frederiksen, Pernille D; Pedersen, Simon M; Floris-Vollenbroek, Esther G; Lucas-van de Bos, Elly; van Hoffen, Els; Wejse, Peter L

2016-02-01

The milk-fat-globule membrane (MFGM) contains phospholipids and membrane glycoproteins that have been shown to affect pathogen colonization and gut barrier integrity. In the present study, we determined whether commercial heat-treated MFGM can increase resistance to diarrheagenic Escherichia coli. A randomized, placebo-controlled, double-blind, 4-wk parallel-intervention study was conducted in healthy adults. Participants were randomly assigned to a milk protein concentrate rich in MFGM [10 g Lacprodan PL-20 (Arla Foods Ingredients Group P/S), twice daily; n = 30; MFGM group) or a control [10 g Miprodan 30 (sodium caseinate), twice daily; n = 28]. After 2 wk, participants were orally challenged with live, attenuated diarrheagenic E. coli (10(10) colony-forming units). Primary outcomes were infection-induced diarrhea and fecal diarrheagenic E. coli excretion. Secondary outcomes were gastrointestinal symptoms [Gastrointestinal Symptom Rating Scale (GSRS)], stool frequency, and stool consistency (Bristol Stool Scale). Diarrheagenic E. coli resulted in increased fecal output, lower relative fecal dry weight, increased fecal E. coli numbers, and an increase in stool frequency and gastrointestinal complaints at day 1 after challenge. MFGM significantly decreased the E. coli-induced changes in reported stool frequency (1.1 ± 0.1 stools/d in the MFGM group; 1.6 ± 0.2 stools/d in the control group; P = 0.04) and gastrointestinal complaints at day 2 (1.1 ± 0.5 and 2.5 ± 0.6 GSRS scores in the MFGM and control groups, respectively; P = 0.05). MFGM did not affect fecal wet weight and E. coli excretion at day 2 after challenge. The attenuated diarrheagenic E. coli strain transiently induced mild symptoms of a food-borne infection, with complete recovery of reported clinical symptoms within 2 d. The present diarrheagenic E. coli challenge trial conducted in healthy adults indicates that a milk concentrate rich in natural, bioactive phospho- and sphingolipids from the MFGM may improve in vivo resistance to diarrheagenic E. coli. This trial was registered at clinicaltrials.gov as NCT01800396. © 2016 American Society for Nutrition.

Prevalence of intestinal parasites in breeding kennel dogs in Japan.

PubMed

Itoh, Naoyuki; Kanai, Kazutaka; Kimura, Yuya; Chikazawa, Seishiro; Hori, Yasutomo; Hoshi, Fumio

2015-03-01

The present study is the first to show overall prevalences of intestinal parasites among breeding kennel dogs in Japan. A total of 573 fresh fecal samples were collected from dogs at 12 breeding kennels. Giardia-specific coproantigen was examined by ELISA kit (SNAP(®) Giardia, IDEXX Laboratories, Inc., Maine, USA). Other intestinal parasites were determined microscopically using the formalin-ethyl acetate sedimentation technique. Overall prevalences of two genera of protists, Giardia spp. and Cystoisospora spp., were 25.7 and 1.2 %, respectively. The prevalence of helminthes was recorded as: Toxocara canis 0.2 %, Toxascaris leonina 0.9 %, Ancylostoma caninum 0.2 %, Trichuris vulpis 2.1 %, and Spirometra erinacei 0.4 %. According to age categories, Giardia spp., Cystoisospora spp., and T. leonina in <1-year-old dogs were significantly more prevalent than in ≥ 1-year-old dogs (61.0 vs. 19.8 %, P < 0.0001; 7.3 vs. 0.2 %, P < 0.0001; and 4.9 vs. 0.2 %, P < 0.001; respectively). With respect to fecal condition, the prevalences of T. leonina and T. vulpis were significantly higher in unformed stool dogs than in formed ones (2.4 vs. 0 %, P < 0.01, and 4.3 vs. 0.8 %, P < 0.05, respectively). In all of the breeding kennels except for one kennel, intestinal parasite infections were found at the high prevalent, ranging from 16.0 to 70.0 %.

A national survey of home-based care kits for palliative HIV/AIDS care in South Africa.

PubMed

Mabude, Z A; Beksinska, M E; Ramkissoon, A; Wood, S; Folsom, M

2008-09-01

The objective of this study was to assess home-based care (HBC) kits and programs in South Africa to evaluate the feasibility of scaling up kit production and distribution. South African HBC organizations received structured questionnaires; key informant interviews and a literature review were completed to assess systems for production, distribution and supply of HBC kits. Meetings with stakeholders were held in two Provinces to share and analyze the study findings. The study team distributed questionnaires to 466 organizations and conducted interviews with representatives from 45 organizations, the Provincial Department of Health (DoH) and manufacturers of kits. All identifiable HBC organizations in South Africa were included in the survey. As a result 215 HBC organizations returned questionnaires; including non-governmental organizations (56%), community-based organizations (32%) and organizations affiliated with government health departments. Two types of kits were available: a home kit and a professional kit. The demand for HBC kits exceeded availability, kit contents and availability varied considerably and the supply chain was irregular. Kit production and distribution systems were fragmented. Replenishment of kit items was problematic. End-users are mostly caregivers who have not received adequate training on their use. The study shows that substantial work has been done by HBC organizations in South Africa to respond to the need for palliative care supplies within resource constraints. The growing demand for kits exceeds the supply. There is a need to improve the supply chain management of HBC kits, strengthen referral systems and links between community-based organizations and government departments, expand training opportunities for care givers, and develop monitoring and evaluation systems.

Single-cell analysis of the fate of c-kit-positive bone marrow cells

NASA Astrophysics Data System (ADS)

Czarna, Anna; Sanada, Fumihiro; Matsuda, Alex; Kim, Junghyun; Signore, Sergio; Pereira, João D.; Sorrentino, Andrea; Kannappan, Ramaswamy; Cannatà, Antonio; Hosoda, Toru; Rota, Marcello; Crea, Filippo; Anversa, Piero; Leri, Annarosa

2017-10-01

The plasticity of c-kit-positive bone marrow cells (c-kit-BMCs) in tissues different from their organ of origin remains unclear. We tested the hypothesis that c-kit-BMCs are functionally heterogeneous and only a subgroup of these cells possesses cardiomyogenic potential. Population-based assays fall short of identifying the properties of individual stem cells, imposing on us the introduction of single cell-based approaches to track the fate of c-kit-BMCs in the injured heart; they included viral gene-tagging, multicolor clonal-marking and transcriptional profiling. Based on these strategies, we report that single mouse c-kit-BMCs expand clonally within the infarcted myocardium and differentiate into specialized cardiac cells. Newly-formed cardiomyocytes, endothelial cells, fibroblasts and c-kit-BMCs showed in their genome common sites of viral integration, providing strong evidence in favor of the plasticity of a subset of BMCs expressing the c-kit receptor. Similarly, individual c-kit-BMCs, which were infected with multicolor reporters and injected in infarcted hearts, formed cardiomyocytes and vascular cells organized in clusters of similarly colored cells. The uniform distribution of fluorescent proteins in groups of specialized cells documented the polyclonal nature of myocardial regeneration. The transcriptional profile of myogenic c-kit-BMCs and whole c-kit-BMCs was defined by RNA sequencing. Genes relevant for engraftment, survival, migration, and differentiation were enriched in myogenic c-kit-BMCs, a cell subtype which could not be assigned to a specific hematopoietic lineage. Collectively, our findings demonstrate that the bone marrow comprises a category of cardiomyogenic, vasculogenic and/or fibrogenic c-kit-positive cells and a category of c-kit-positive cells that retains an undifferentiated state within the damaged heart.

Single-cell analysis of the fate of c-kit-positive bone marrow cells.

PubMed

Czarna, Anna; Sanada, Fumihiro; Matsuda, Alex; Kim, Junghyun; Signore, Sergio; Pereira, João D; Sorrentino, Andrea; Kannappan, Ramaswamy; Cannatà, Antonio; Hosoda, Toru; Rota, Marcello; Crea, Filippo; Anversa, Piero; Leri, Annarosa

2017-01-01

The plasticity of c-kit-positive bone marrow cells (c-kit-BMCs) in tissues different from their organ of origin remains unclear. We tested the hypothesis that c-kit-BMCs are functionally heterogeneous and only a subgroup of these cells possesses cardiomyogenic potential. Population-based assays fall short of identifying the properties of individual stem cells, imposing on us the introduction of single cell-based approaches to track the fate of c-kit-BMCs in the injured heart; they included viral gene-tagging, multicolor clonal-marking and transcriptional profiling. Based on these strategies, we report that single mouse c-kit-BMCs expand clonally within the infarcted myocardium and differentiate into specialized cardiac cells. Newly-formed cardiomyocytes, endothelial cells, fibroblasts and c-kit-BMCs showed in their genome common sites of viral integration, providing strong evidence in favor of the plasticity of a subset of BMCs expressing the c-kit receptor. Similarly, individual c-kit-BMCs, which were infected with multicolor reporters and injected in infarcted hearts, formed cardiomyocytes and vascular cells organized in clusters of similarly colored cells. The uniform distribution of fluorescent proteins in groups of specialized cells documented the polyclonal nature of myocardial regeneration. The transcriptional profile of myogenic c-kit-BMCs and whole c-kit-BMCs was defined by RNA sequencing. Genes relevant for engraftment, survival, migration, and differentiation were enriched in myogenic c-kit-BMCs, a cell subtype which could not be assigned to a specific hematopoietic lineage. Collectively, our findings demonstrate that the bone marrow comprises a category of cardiomyogenic, vasculogenic and/or fibrogenic c-kit-positive cells and a category of c-kit-positive cells that retains an undifferentiated state within the damaged heart.

In-frame Val216-Ser217 deletion of KIT in mild piebaldism causes aberrant secretion and SCF response.

PubMed

Hattori, Mai; Ishikawa, Osamu; Oikawa, Daisuke; Amano, Hiroo; Yasuda, Masahito; Kaira, Kyoichi; Ishida-Yamamoto, Akemi; Nakano, Hajime; Sawamura, Daisuke; Terawaki, Shin-Ichi; Wakamatsu, Kaori; Tokunaga, Fuminori; Shimizu, Akira

2018-03-21

Piebaldism is a pigmentary disorder characterized by a white forelock and depigmented patches. Although the loss-of-function mutations in the KIT gene underlie the disease, the intracellular dynamics of the mutant KIT are largely unknown. We herein report a Japanese family with piebaldism in which the affected members showed a mild phenotype. The objective of this study is to investigate the functions and intracellular dynamics of the mutant KIT protein. We performed genetic analyses of the KIT gene using peripheral blood cells. We analyzed the intracellular localization of the mutant KIT protein in HEK293T cells transfected with wild-type (Wt) and/or mutant KIT genes. Immunoprecipitation analyses, immunoblotting and immunofluorescence studies were performed using antibodies against KIT and downstream signaling proteins. Glycosidase digestion analysis was performed to clarify the intracellular localization of KIT protein. A genetic analysis revealed a novel heterozygous mutation c.645_650delTGTGTC which results in the in-frame deletion of Val 216 and Ser 217 in the extracellular domain of KIT. Immunoprecipitation analyses confirmed that the wild and mutant KIT formed a heterodimer after treatment with stem cell factor (SCF); however, the phosphorylation of the downstream signaling factors was decreased. In an immunofluorescence study, the mutant KIT accumulated predominantly in the endoplasmic reticulum (ER) and was sparsely expressed on the cell surface. A glycosidase digestion study revealed that the mutant KIT is predominantly localized in the ER. These data reveal an aberrant function and intracellular localization of mutant KIT protein in piebaldism. Copyright © 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Loss of c-Kit function impairs arteriogenesis in a mouse model of hindlimb ischemia.

PubMed

Hernandez, Diana R; Artiles, Adriana; Duque, Juan C; Martinez, Laisel; Pinto, Mariana T; Webster, Keith A; Velazquez, Omaida C; Vazquez-Padron, Roberto I; Lassance-Soares, Roberta M

2018-04-01

Arteriogenesis is a process whereby collateral vessels remodel usually in response to increased blood flow and/or wall stress. Remodeling of collaterals can function as a natural bypass to alleviate ischemia during arterial occlusion. Here we used a genetic approach to investigate possible roles of tyrosine receptor c-Kit in arteriogenesis. Mutant mice with loss of c-Kit function (Kit W/W-v ), and controls were subjected to hindlimb ischemia. Blood flow recovery was evaluated pre-, post-, and weekly after ischemia. Foot ischemic damage and function were assessed between days 1 to 14 post-ischemia while collaterals remodeling were measured 28 days post-ischemia. Both groups of mice also were subjected to wild type bone marrow cells transplantation 3 weeks before hindlimb ischemia to evaluate possible contributions of defective bone marrow c-Kit expression on vascular recovery. Kit W/W-v mice displayed impaired blood flow recovery, greater ischemic damage and foot dysfunction after ischemia compared to controls. Kit W/W-v mice also demonstrated impaired collateral remodeling consistent with flow recovery findings. Because arteriogenesis is a biological process that involves bone marrow-derived cells, we investigated which source of c-Kit signaling (bone marrow or vascular) plays a major role in arteriogenesis. Kit W/W-v mice transplanted with bone marrow wild type cells exhibited similar phenotype of impaired blood flow recovery, greater tissue ischemic damage and foot dysfunction as nontransplanted Kit W/W-v mice. This study provides evidence that c-Kit signaling is required during arteriogenesis. Also, it strongly suggests a vascular role for c-Kit signaling because rescue of systemic c-Kit activity by bone marrow transplantation did not augment the functional recovery of Kit W/W-v mouse hindlimbs. Copyright © 2017 Elsevier Inc. All rights reserved.

Membrane estrogen receptor alpha is an important modulator of bone marrow C-Kit+ cells mediated cardiac repair after myocardial infarction

PubMed Central

Su, Feng; Zhang, Wentian; Liu, Jianfang

2015-01-01

It has been validated that c-kit positive (c-kit+) cells in infarcted myocardium are from bone marrow (BM). Given the recent study that in the heart, estrogen receptor alpha (ERα) is involved in adaptive mechanisms by supporting cardiomyocytes survival via post-infarct cardiac c-kit+ cells, we tested a novel hypothesis that membrane ERα (mERа) supports survival of BM c-kit+ cells and enhance protective paracrine function for cardiac repair. Our data showed that myocardial infarction (MI) leads to an increase in c-kit+ first in bone marrow and then specifically within the infarcted myocardium. Also up-regulated mERа in post-infarct BM c-kit+ cells was found in day 3 post MI. In vitro co-culture system, mERа+ enhances the beneficial effects of BM c-kit+ cells by increasing their viability and reducing apoptosis. Post-infarct c-kit+ mERа+ cells population expresses predominant ERα and holds self-renewal as well as cardiac differentiation potentials after MI. In vivo, BM c-kit+ cells reduced infarct size, fibrosis and improved cardiac function. In conclusion, BM c-kit+ mERа+ exerted significantly cardiac protection after MI. A potential important implication of this study is that the manipulation of BM c-kit+ stem cells with ERа-dependent fashion may be helpful in recovering functional performance after cardiac tissue injury. PMID:26191121

Direct engagement of the PI3K pathway by mutant KIT dominates oncogenic signaling in gastrointestinal stromal tumor.

PubMed

Bosbach, Benedikt; Rossi, Ferdinand; Yozgat, Yasemin; Loo, Jennifer; Zhang, Jennifer Q; Berrozpe, Georgina; Warpinski, Katherine; Ehlers, Imke; Veach, Darren; Kwok, Andrew; Manova, Katia; Antonescu, Cristina R; DeMatteo, Ronald P; Besmer, Peter

2017-10-03

Gastrointestinal stromal tumors (GISTs) predominantly harbor activating mutations in the receptor tyrosine kinase KIT. To genetically dissect in vivo the requirement of different signal transduction pathways emanating from KIT for tumorigenesis, the oncogenic Kit V558Δ mutation was combined with point mutations abrogating specific phosphorylation sites on KIT. Compared with single-mutant Kit V558Δ/+ mice, double-mutant Kit V558Δ;Y567F/Y567F knock-in mice lacking the SRC family kinase-binding site on KIT (pY567) exhibited attenuated MAPK signaling and tumor growth. Surprisingly, abrogation of the PI3K-binding site (pY719) in Kit V558Δ;Y719F/Y719F mice prevented GIST development, although the interstitial cells of Cajal (ICC), the cells of origin of GIST, were normal. Pharmacologic inhibition of the PI3K pathway in tumor-bearing Kit V558Δ/+ mice with the dual PI3K/mTOR inhibitor voxtalisib, the pan-PI3K inhibitor pilaralisib, and the PI3K-alpha-restricted inhibitor alpelisib each diminished tumor proliferation. The addition of the MEK inhibitor PD-325901 or binimetinib further decreased downstream KIT signaling. Moreover, combining PI3K and MEK inhibition was effective against imatinib-resistant Kit V558Δ;T669I/+ tumors.

Direct engagement of the PI3K pathway by mutant KIT dominates oncogenic signaling in gastrointestinal stromal tumor

PubMed Central

Bosbach, Benedikt; Rossi, Ferdinand; Yozgat, Yasemin; Loo, Jennifer; Zhang, Jennifer Q.; Berrozpe, Georgina; Warpinski, Katherine; Ehlers, Imke; Kwok, Andrew; Manova, Katia; Antonescu, Cristina R.; DeMatteo, Ronald P.; Besmer, Peter

2017-01-01

Gastrointestinal stromal tumors (GISTs) predominantly harbor activating mutations in the receptor tyrosine kinase KIT. To genetically dissect in vivo the requirement of different signal transduction pathways emanating from KIT for tumorigenesis, the oncogenic KitV558Δ mutation was combined with point mutations abrogating specific phosphorylation sites on KIT. Compared with single-mutant KitV558Δ/+ mice, double-mutant KitV558Δ;Y567F/Y567F knock-in mice lacking the SRC family kinase-binding site on KIT (pY567) exhibited attenuated MAPK signaling and tumor growth. Surprisingly, abrogation of the PI3K-binding site (pY719) in KitV558Δ;Y719F/Y719F mice prevented GIST development, although the interstitial cells of Cajal (ICC), the cells of origin of GIST, were normal. Pharmacologic inhibition of the PI3K pathway in tumor-bearing KitV558Δ/+ mice with the dual PI3K/mTOR inhibitor voxtalisib, the pan-PI3K inhibitor pilaralisib, and the PI3K-alpha–restricted inhibitor alpelisib each diminished tumor proliferation. The addition of the MEK inhibitor PD-325901 or binimetinib further decreased downstream KIT signaling. Moreover, combining PI3K and MEK inhibition was effective against imatinib-resistant KitV558Δ;T669I/+ tumors. PMID:28923937

21 CFR 868.1100 - Arterial blood sampling kit.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Arterial blood sampling kit. 868.1100 Section 868...) MEDICAL DEVICES ANESTHESIOLOGY DEVICES Diagnostic Devices § 868.1100 Arterial blood sampling kit. (a) Identification. An arterial blood sampling kit is a device, in kit form, used to obtain arterial blood samples...

40 CFR 745.88 - Recognized test kits.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Recognized test kits. 745.88 Section... Renovation § 745.88 Recognized test kits. (a) Effective June 23, 2008, EPA recognizes the test kits that have... publicizes its recognition of the first test kit that meets both the negative response and positive response...

21 CFR 868.1100 - Arterial blood sampling kit.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Arterial blood sampling kit. 868.1100 Section 868...) MEDICAL DEVICES ANESTHESIOLOGY DEVICES Diagnostic Devices § 868.1100 Arterial blood sampling kit. (a) Identification. An arterial blood sampling kit is a device, in kit form, used to obtain arterial blood samples...

21 CFR 868.1100 - Arterial blood sampling kit.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Arterial blood sampling kit. 868.1100 Section 868...) MEDICAL DEVICES ANESTHESIOLOGY DEVICES Diagnostic Devices § 868.1100 Arterial blood sampling kit. (a) Identification. An arterial blood sampling kit is a device, in kit form, used to obtain arterial blood samples...

21 CFR 868.1100 - Arterial blood sampling kit.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Arterial blood sampling kit. 868.1100 Section 868...) MEDICAL DEVICES ANESTHESIOLOGY DEVICES Diagnostic Devices § 868.1100 Arterial blood sampling kit. (a) Identification. An arterial blood sampling kit is a device, in kit form, used to obtain arterial blood samples...

21 CFR 868.1100 - Arterial blood sampling kit.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Arterial blood sampling kit. 868.1100 Section 868...) MEDICAL DEVICES ANESTHESIOLOGY DEVICES Diagnostic Devices § 868.1100 Arterial blood sampling kit. (a) Identification. An arterial blood sampling kit is a device, in kit form, used to obtain arterial blood samples...

46 CFR 121.710 - First-aid kits.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 4 2012-10-01 2012-10-01 false First-aid kits. 121.710 Section 121.710 Shipping COAST... SYSTEMS AND EQUIPMENT Miscellaneous § 121.710 First-aid kits. A vessel must carry either a first-aid kit approved under approval series 160.041 or a kit with equivalent contents and instructions. For equivalent...

19 CFR 122.132 - Sealing of aircraft liquor kits.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 19 Customs Duties 1 2010-04-01 2010-04-01 false Sealing of aircraft liquor kits. 122.132 Section... OF THE TREASURY AIR COMMERCE REGULATIONS Aircraft Liquor Kits § 122.132 Sealing of aircraft liquor kits. (a) Sealing required. Aircraft liquor kits shall be sealed on board the aircraft by crewmembers...

46 CFR 184.710 - First-aid kits.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 7 2012-10-01 2012-10-01 false First-aid kits. 184.710 Section 184.710 Shipping COAST... CONTROL AND MISCELLANEOUS SYSTEMS AND EQUIPMENT Miscellaneous § 184.710 First-aid kits. A vessel must carry either a first-aid kit approved under approval series 160.041 or a kit with equivalent contents...

21 CFR 870.1350 - Catheter balloon repair kit.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Catheter balloon repair kit. 870.1350 Section 870... repair kit. (a) Identification. A catheter balloon repair kit is a device used to repair or replace the balloon of a balloon catheter. The kit contains the materials, such as glue and balloons, necessary to...

21 CFR 870.1350 - Catheter balloon repair kit.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Catheter balloon repair kit. 870.1350 Section 870... repair kit. (a) Identification. A catheter balloon repair kit is a device used to repair or replace the balloon of a balloon catheter. The kit contains the materials, such as glue and balloons, necessary to...

46 CFR 184.710 - First-aid kits.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 7 2013-10-01 2013-10-01 false First-aid kits. 184.710 Section 184.710 Shipping COAST... CONTROL AND MISCELLANEOUS SYSTEMS AND EQUIPMENT Miscellaneous § 184.710 First-aid kits. A vessel must carry either a first-aid kit approved under approval series 160.041 or a kit with equivalent contents...

46 CFR 121.710 - First-aid kits.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 4 2014-10-01 2014-10-01 false First-aid kits. 121.710 Section 121.710 Shipping COAST... SYSTEMS AND EQUIPMENT Miscellaneous § 121.710 First-aid kits. A vessel must carry either a first-aid kit approved under approval series 160.041 or a kit with equivalent contents and instructions. For equivalent...

19 CFR 122.132 - Sealing of aircraft liquor kits.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 19 Customs Duties 1 2011-04-01 2011-04-01 false Sealing of aircraft liquor kits. 122.132 Section... OF THE TREASURY AIR COMMERCE REGULATIONS Aircraft Liquor Kits § 122.132 Sealing of aircraft liquor kits. (a) Sealing required. Aircraft liquor kits shall be sealed on board the aircraft by crewmembers...

21 CFR 870.1350 - Catheter balloon repair kit.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Catheter balloon repair kit. 870.1350 Section 870... repair kit. (a) Identification. A catheter balloon repair kit is a device used to repair or replace the balloon of a balloon catheter. The kit contains the materials, such as glue and balloons, necessary to...

46 CFR 121.710 - First-aid kits.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 4 2011-10-01 2011-10-01 false First-aid kits. 121.710 Section 121.710 Shipping COAST... SYSTEMS AND EQUIPMENT Miscellaneous § 121.710 First-aid kits. A vessel must carry either a first-aid kit approved under approval series 160.041 or a kit with equivalent contents and instructions. For equivalent...

46 CFR 184.710 - First-aid kits.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 7 2014-10-01 2014-10-01 false First-aid kits. 184.710 Section 184.710 Shipping COAST... CONTROL AND MISCELLANEOUS SYSTEMS AND EQUIPMENT Miscellaneous § 184.710 First-aid kits. A vessel must carry either a first-aid kit approved under approval series 160.041 or a kit with equivalent contents...

46 CFR 121.710 - First-aid kits.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 4 2013-10-01 2013-10-01 false First-aid kits. 121.710 Section 121.710 Shipping COAST... SYSTEMS AND EQUIPMENT Miscellaneous § 121.710 First-aid kits. A vessel must carry either a first-aid kit approved under approval series 160.041 or a kit with equivalent contents and instructions. For equivalent...

19 CFR 122.132 - Sealing of aircraft liquor kits.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 19 Customs Duties 1 2013-04-01 2013-04-01 false Sealing of aircraft liquor kits. 122.132 Section... OF THE TREASURY AIR COMMERCE REGULATIONS Aircraft Liquor Kits § 122.132 Sealing of aircraft liquor kits. (a) Sealing required. Aircraft liquor kits shall be sealed on board the aircraft by crewmembers...

19 CFR 122.132 - Sealing of aircraft liquor kits.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 19 Customs Duties 1 2012-04-01 2012-04-01 false Sealing of aircraft liquor kits. 122.132 Section... OF THE TREASURY AIR COMMERCE REGULATIONS Aircraft Liquor Kits § 122.132 Sealing of aircraft liquor kits. (a) Sealing required. Aircraft liquor kits shall be sealed on board the aircraft by crewmembers...

46 CFR 184.710 - First-aid kits.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 7 2011-10-01 2011-10-01 false First-aid kits. 184.710 Section 184.710 Shipping COAST... CONTROL AND MISCELLANEOUS SYSTEMS AND EQUIPMENT Miscellaneous § 184.710 First-aid kits. A vessel must carry either a first-aid kit approved under approval series 160.041 or a kit with equivalent contents...

19 CFR 122.132 - Sealing of aircraft liquor kits.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 19 Customs Duties 1 2014-04-01 2014-04-01 false Sealing of aircraft liquor kits. 122.132 Section... OF THE TREASURY AIR COMMERCE REGULATIONS Aircraft Liquor Kits § 122.132 Sealing of aircraft liquor kits. (a) Sealing required. Aircraft liquor kits shall be sealed on board the aircraft by crewmembers...

DEMONSTRATION BULLETIN: CLOR-N-SOIL PCB TEST KIT L2000 PCB/CHLORIDE ANALYZER - DEXSIL CORP.

EPA Science Inventory

DEXSIL CORP(Environmental Test Kits)The Dexsil Corporation (Dexsil) produces two test kits that detect polychlorinated biphenyls (PCB) in soil: the Dexsil Clor-N-Soil PCB Screening Kit, and the Dexsil L2000 PCB/Chloride Analyzer. The Dexsil Clor-N-Soil PCB Screening Kit extr...

46 CFR 184.710 - First-aid kits.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 7 2010-10-01 2010-10-01 false First-aid kits. 184.710 Section 184.710 Shipping COAST... CONTROL AND MISCELLANEOUS SYSTEMS AND EQUIPMENT Miscellaneous § 184.710 First-aid kits. A vessel must carry either a first-aid kit approved under approval series 160.041 or a kit with equivalent contents...

Human Volunteer Studies with Campylobacter jejuni

DTIC Science & Technology

1993-01-01

having volunteers ingest a gelatin erage of approximately five liquid stools and 0.5 string device (Entero-Test; Health Development, liter total stool...vaccine. Infect. Irn- tract Al-l12666 from the National Institute of Allergy mun. 4J:515-522. and Infectious Diseases. 9. Llor, H. Persona) communicatio

Effects of fructo-oligosaccharide-supplemented infant cereal: a double-blind, randomized trial.

PubMed

Moore, Nancy; Chao, Cewin; Yang, Li-Ping; Storm, Heidi; Oliva-Hemker, Maria; Saavedra, Jose M

2003-09-01

Fructo-oligosaccharides (FOS) may have potential benefits, since they exhibit many soluble dietary fibre-like properties. Foods currently available for weaning infants are generally low in fibre content and lack these potential benefits. Data documenting tolerance of FOS in weaning foods are greatly lacking. Our present objective was to evaluate the tolerance and gastrointestinal effects of FOS-supplemented infant cereal used as a daily addition to the diet of healthy infants. Healthy infants were randomly assigned to receive either 0.75 g FOS per serving of cereal or placebo for 28 d. The primary outcome of interest was gastrointestinal tolerance, which was assessed by daily parental reporting of functional variables for 28 d, including stool patterns and signs and symptoms of gastrointestinal tolerance. Secondary outcomes were also measured including: cereal intake (g cereal and g FOS/d), stool pH, changes in anthropometric measurements and adverse events. The study population included a total of fifty-six infants, age range 16.2-46.2 weeks with a mean age of 32.5 (sd 8.9) weeks; twenty-nine infants were randomized to the control group (age 31.8 (sd 9.0) weeks) and twenty-seven to the FOS-supplemented group (34.7 (sd 8.9) weeks). Average daily total intake per infant and average intake per serving were similar in both groups. Average FOS consumption was 0.74 (sd 0.39) g/d and as high as 3.00 g/d. Stool consistency was less likely to be described as 'hard', and more likely to be described as 'soft' or 'loose', in the FOS v. control group. The mean number of stools per infant was 1.99 (sd 0.62) per d in the FOS-supplemented group compared with 1.58 (sd 0.66) in the control group (P=0.02). There were no differences between the groups in reporting for crying, spitting-up or colic. No differences were found for stool pH. FOS-supplements added to cereal were well tolerated in doses of up to 3.00 g/d. FOS consumption led to more regular and softer stools, without diarrhoea, as well as less-reported frequency of symptoms associated with constipation such as hard stools or skipped days without stool. The present study is one of few studies documenting tolerance to increased fibre intake in the form of FOS as part of a weaning food.

Analysis of DNA Methylation at Specific Loci in Stool Samples Detects Colorectal Cancer and High-grade Dysplasia in Patients with Inflammatory Bowel Disease.

PubMed

Kisiel, John B; Klepp, Pasquale; Allawi, Hatim T; Taylor, William R; Giakoumopoulos, Maria; Sander, Tamara; Yab, Tracy C; Moum, Bjorn A; Lidgard, Graham P; Brackmann, Stephan; Mahoney, Douglas W; Roseth, Arne; Ahlquist, David A

2018-05-15

Patients with inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), are at increased risk for colorectal cancer (CRC). Analyses of DNA methylation patterns in stool samples have been reported to detect CRC in patients with IBD. We sought to validate these findings in larger cohorts and assess the accuracy of analysis of DNA methylation patterns in stool for detection of CRC and high-grade dysplasia (HGD) normalized to methylation level at ZDHHC1. We obtained buffered, frozen stool samples from a United States case-control study and from 2 European surveillance cohorts (referral or population based) of patients with chronic UC (n=248), CD (n=82), indeterminate colitis (n=2), or IBD with primary sclerosing cholangitis (n=38). Stool samples were collected before bowel preparation for colonoscopy or at least 1 week after colonoscopy. Among the study samples, stools from individuals with IBD but without neoplasia were used as controls (n=291). DNA was isolated from stool, exposed to bisulfite, and then assayed by multiplex quantitative allele-specific real-time target and signal amplification. We analyzed methylation levels of BMP3, NDRG4, VAV3, and SFMBT2 relative to the methylation level of ZDHHC1, and compared these between patients with CRC or HGD and controls. Levels of methylation at BMP3 and VAV3, relative to ZDHHC1 methylation, identified patients with CRC and HGD with an area under curve value of 0.91 (95% CI, 0.77-1.00). Methylation levels at specific promotor regions of these genes identified 11 of the 12 patients with CRC and HGD, with 92% sensitivity (95% CI, 60%-100%) and 90% specificity (95% CI, 86%-93%). The proportion of false-positive results did not differ significantly among the case-control, referral cohort, and population cohort studies (P=.60) when the 90% specificity cut-off from the whole sample set was applied. In an analysis of stool samples from 3 independent studies, of 332 patients with IBD, we associated levels of methylation at 2 genes (BMP3 and VAV3), relative to level of methylation at ZDHHC1, with detection of CRC and HGD. These methylation patterns identified patients with CRC and HGD with more than 90% specificity, and might be used in CRC surveillance. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

Insulin-like growth factor-1-mediated regulation of miR-193a expression promotes the migration and proliferation of c-kit-positive mouse cardiac stem cells.

PubMed

Sun, Yuning; Xu, Rongfeng; Huang, Jia; Yao, Yuyu; Pan, Xiaodong; Chen, Zhongpu; Ma, Genshan

2018-02-21

C-kit-positive cardiac stem cells (CSCs) have been shown to be a promising candidate treatment for myocardial infarction and heart failure. Insulin-like growth factor (IGF)-1 is an anabolic growth hormone that regulates cellular proliferation, differentiation, senescence, and death in various tissues. Although IGF-1 promotes the migration and proliferation of c-kit-positive mouse CSCs, the underlying mechanism remains unclear. Cells were isolated from adult mouse hearts, and c-kit-positive CSCs were separated using magnetic beads. The cells were cultured with or without IGF-1, and c-kit expression was measured by Western blotting. IGF-1 induced CSC proliferation and migration, as measured through Cell Counting Kit-8 (CCK-8) and Transwell assays, respectively. The miR-193a expression was measured by quantitative real-time PCR (qPCR) assays. IGF-1 enhanced c-kit expression in c-kit-positive CSCs. The activities of the phosphoinositol 3-kinase (PI3K)/AKT signaling pathway and DNA methyltransferases (DNMTs) were enhanced, and their respective inhibitors LY294002 and 5-azacytidine (5-AZA) blunted c-kit expression. Based on the results of quantitative real-time PCR (qPCR) assays, the expression of miR-193a, which is embedded in a CpG island, was down-regulated in the IGF-1-stimulated group and negatively correlated with c-kit expression, whereas c-kit-positive CSCs infected with lentivirus carrying micro-RNA193a displayed reduced c-kit expression, migration and proliferation. IGF-1 upregulated c-kit expression in c-kit-positive CSCs resulting in enhanced CSC proliferation and migration by activating the PI3K/AKT/DNMT signaling pathway to epigenetically silence miR-193a, which negatively modifies the c-kit expression level.

Investigation of c-KIT and Ki67 expression in normal, preneoplastic and neoplastic canine prostate.

PubMed

Fonseca-Alves, Carlos Eduardo; Kobayashi, Priscilla Emiko; Palmieri, Chiara; Laufer-Amorim, Renée

2017-12-06

c-KIT expression has been related to bone metastasis in human prostate cancer, but whether c-KIT expression can be similarly classified in canine prostatic tissue is unknown. This study assessed c-KIT and Ki67 expression in canine prostate cancer (PC). c-KIT gene and protein expression and Ki67 expression were evaluated in forty-four canine prostatic tissues by immunohistochemistry, RT-qPCR and western blot. Additionally, we have investigated c-KIT protein expression by immunoblotting in two primary canine prostate cancer cell lines. Eleven normal prostates, 12 proliferative inflammatory atrophy (PIA) prostates, 18 PC, 3 metastatic lesions and two prostate cancer cell cultures (PC1 and PC2) were analysed. The prostatic tissue exhibited varying degrees of membranous, cytoplasmic or membranous/cytoplasmic c-KIT staining. Four normal prostates, 4 PIA and 5 prostatic carcinomas showed positive c-KIT expression. No c-KIT immunoexpression was observed in metastases. Canine prostate cancer and PIA samples contained a higher number of Ki67-positive cells compared to normal samples. The median relative quantification (RQ) for c-KIT expression in normal, PIA and prostate cancer and metastatic samples were 0.6 (0.1-2.5), 0.7 (0.09-2.1), 0.7 (0.09-5.1) and 0.1 (0.07-0.6), respectively. A positive correlation between the number of Ki67-positive cells and c-KIT transcript levels was observed in prostate cancer samples. In the cell line, PC1 was negative for c-KIT protein expression, while PC2 was weakly positive. The present study identified a strong correlation between c-KIT expression and proliferative index, suggesting that c-KIT may influence cell proliferation. Therefore, c-KIT heterogeneous protein expression among the samples (five positive and thirteen negative prostate cancer samples) indicates a personalized approach for canine prostate cancer.

Evaluation of BBL™ Sensi-Discs™ and FTA® cards as sampling devices for detection of rotavirus in stool samples

PubMed Central

Tam, Ka Ian; Esona, Mathew D.; Williams, Alice; Ndze, Valentine N.; Boula, Angeline; Bowen, Michael D.

2015-01-01

Rotavirus is the most important cause of severe childhood gastroenteritis worldwide. Rotavirus vaccines are available and rotavirus surveillance is carried out to assess vaccination impact. In surveillance studies, stool samples are stored typically at 4°C or frozen to maintain sample quality. Uninterrupted cold storage is a problem in developing countries because of power interruptions. Cold-chain transportation of samples from collection sites to testing laboratories is costly. In this study, we evaluated the use of BBL™ Sensi-Discs™ and FTA® cards for storage and transportation of samples for virus isolation, EIA, and RT-PCR testing. Infectious rotavirus was recovered after 30 days of storage on Sensi-Discs™ at room temperature. We were able to genotype 98–99% of samples stored on Sensi-Discs™ and FTA® cards at temperatures ranging from −80°C to 37°C up to 180 days. A field sampling test using samples prepared and shipped from Cameroon, showed that both matrices yielded 100% genotyping success compared with whole stool and Sensi-Discs™ demonstrated 95% concordance with whole stool in EIA testing. The utilization of BBL™ Sensi-Discs™ and FTA® cards for stool sample storage and shipment has the potential to have great impact on global public health by facilitating surveillance and epidemiological investigations of rotavirus strains worldwide at a reduced cost. PMID:26022083

Prevalence of Clostridium difficile colonization among healthcare workers

PubMed Central

2013-01-01

Background Clostridium difficile infection (CDI) has increased to epidemic proportions in recent years. The carriage of C. difficile among healthy adults and hospital inpatients has been established. We sought to determine whether C. difficile colonization exists among healthcare workers (HCWs) in our setting. Methods A point prevalence study of stool colonization with C. difficile among doctors, nurses and allied health staff at a large regional teaching hospital in Geelong, Victoria. All participants completed a short questionnaire and all stool specimens were tested by Techlab® C.diff Quik Check enzyme immunoassay followed by enrichment culture. Results Among 128 healthcare workers, 77% were female, of mean age 43 years, and the majority were nursing staff (73%). Nineteen HCWs (15%) reported diarrhoea, and 12 (9%) had taken antibiotics in the previous six weeks. Over 40% of participants reported having contact with a patient with known or suspected CDI in the 6 weeks before the stool was collected. C. difficile was not isolated from the stool of any participants. Conclusion Although HCWs are at risk of asymptomatic carriage and could act as a reservoir for transmission in the hospital environment, with the use of a screening test and culture we were unable to identify C. difficile in the stool of our participants in a non-outbreak setting. This may reflect potential colonization resistance of the gut microbiota, or the success of infection prevention strategies at our institution. PMID:24090343

Cumin Extract for Symptom Control in Patients with Irritable Bowel Syndrome: A Case Series

PubMed Central

Agah, Shahram; Taleb, Amir Mehdi; Moeini, Reyhane; Gorji, Narjes; Nikbakht, Hajar

2013-01-01

BACKGROUND Irritable bowel syndrome is one of the most common gastrointestinal disorders Characterized by chronic abdominal pain, altered bowel habits or changes in stool consistency. Unfortunately, no specific treatments for relieving IBS symptoms have been suggested yet. This pilot study was conducted to evaluate the efficacy of the Cumin extract, a kind of herbal used in the treatment of gastrointestinal disorders like bloating, and other symptoms of IBS. METHODS Fifty seven patients with IBS (according to the ROME II diagnostic criteria) with no nay other accompanying illness enrolled in study. Patients were advised to discontinue their other treatments during the study course, then 20 drops per day of Cumin essential oil was administered for included patients. IBS-associated symptoms including abdominal pain, nausea, painful defection, presence of mucosa in stool, changes in stool consistency and defecation frequency were evaluated using a questionnairebefore treatment, 2 and 4 weeks after beginning treatment and 2 and 4 weeks after stopping treatment. RESULTS Abdominal pain, bloating, incomplete defecation, fecal urgency and presence of mucus discharge in stool were statistically significant decreased during and after treatment with Cumin extract. Stool consistency and defecation frequency were also both statistically significant improved in patients with constipation dominant pattern of IBS. CONCLUSION Cumin extract can be effective in improving all IBS symptoms. Considering its low cost and easy availability Cumin administration in patients with IBS may have economic benefits. PMID:24829694

Relating Stool Microbial Metabolite Levels, Inflammatory Markers and Dietary Behaviors to Screening Colonoscopy Findings in a Racially/Ethnically Diverse Patient Population.

PubMed

Bridges, Kristina M; Diaz, Francisco J; Wang, Zhiwen; Ahmed, Ishfaq; Sullivan, Debra K; Umar, Shahid; Buckles, Daniel C; Greiner, K Allen; Hester, Christina M

2018-02-26

Colorectal cancer (CRC) is the third leading cause of cancer death for both men and women in the United States, yet it is treatable and preventable. African Americans have higher incidence of CRC than other racial/ethnic groups, however, it is unclear whether this disparity is primarily due to environmental or biological factors. Short chain fatty acids (SCFAs) are metabolites produced by bacteria in the colon and are known to be inversely related to CRC progression. The aim of this study is to investigate how stool SCFA levels, markers of inflammation in stool and dietary intake relate to colonoscopy findings in a diverse patient population. Stool samples from forty-eight participants were analyzed for SCFA levels and inflammatory markers (lysozyme, secretory IgA, lactoferrin). Additionally, participants completed the National Cancer Institute's Diet History Questionnaire II (DHQ II) to report dietary intake over the past year. Subsequently, the majority of participants underwent screening colonoscopy. Our results showed that African Americans had higher total levels of SCFAs in stool than other racial/ethnic groups, significantly lower intake of non-starchy vegetables and similar inflammatory marker expression and colonoscopy outcomes, compared to others. This work is an initial exploration into the biological and clinical factors that may ultimately inform personalized screening approaches and clinical decision-making to improve colorectal cancer disparities for African Americans.

A High Level of Intestinal Alkaline Phosphatase Is Protective Against Type 2 Diabetes Mellitus Irrespective of Obesity.

PubMed

Malo, Madhu S

2015-12-01

Mice deficient in intestinal alkaline phosphatase (IAP) develop type 2 diabetes mellitus (T2DM). We hypothesized that a high level of IAP might be protective against T2DM in humans. We determined IAP levels in the stools of 202 diabetic patients and 445 healthy non-diabetic control people. We found that compared to controls, T2DM patients have approx. 50% less IAP (mean +/- SEM: 67.4 +/- 3.2 vs 35.3 +/- 2.5 U/g stool, respectively; p < 0.000001) indicating a protective role of IAP against T2DM. Multiple logistic regression analyses showed an independent association between the IAP level and diabetes status. With each 25 U/g decrease in stool IAP, there is a 35% increased risk of diabetes. The study revealed that obese people with high IAP (approx. 65 U/g stool) do not develop T2DM. Approx. 65% of the healthy population have < 65.0 U/g stool IAP, and predictably, these people might have 'the incipient metabolic syndrome', including 'incipient diabetes', and might develop T2DM and other metabolic disorders in the near future. In conclusion, high IAP levels appear to be protective against diabetes irrespective of obesity, and a 'temporal IAP profile' might be a valuable tool for predicting 'the incipient metabolic syndrome', including 'incipient diabetes'.

Clinical value of dual-isotope fat absorption test system (FATS) using glycerol (/sup 125/I)trioleate and glycerol (/sup 75/Se)triether

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lembcke, B.; Loesler, A.C.; Caspary, W.F.

1986-08-01

In order to delineate the clinical value of a dual-isotope fat absorption test system (FATS) using glycerol (/sup 75/Se)triether as lipid-phase marker and glycerol (/sup 125/I)trioleate as the test lipid, fecal isotope ratios from single stools (and a 72-hr stool homogenate) were compared to quantitative fecal fat excretion. The study included 11 patients without and 24 patients with steatorrhea. With a figure of 0.8% as the upper limit of normal, the test was a reliable indicator of steatorrhea with 87.5% sensitivity and 81.8% specificity; efficiency was 85.7%. Related to a prevalence of steatorrhea of 45.9% as the mean value ofmore » 1269 consecutive 72-hr specimens investigated for steatorrhea during 1978-1982, the positive (negative) predictive value of the FATS is 80.3% (87.2%). With 2% as the upper limit of normal, no false positive results ensued. It is concluded that a two-step interpretation of the FATS (0.8% limit and 2% limit) may be regarded a valid qualitative index for steatorrhea. The FATS isotope ratio using single stools correlated well with FATS ratios in the 72-hr stool homogenates (r = 0.97). FATS therefore allows a convenient estimate of steatorrhea from measuring single stools. As a quantitative measure of fecal fat excretion, the FATS is unreliable.« less

Evaluation of BBL™ Sensi-Discs™ and FTA® cards as sampling devices for detection of rotavirus in stool samples.

PubMed

Tam, Ka Ian; Esona, Mathew D; Williams, Alice; Ndze, Valantine N; Boula, Angeline; Bowen, Michael D

2015-09-15

Rotavirus is the most important cause of severe childhood gastroenteritis worldwide. Rotavirus vaccines are available and rotavirus surveillance is carried out to assess vaccination impact. In surveillance studies, stool samples are stored typically at 4°C or frozen to maintain sample quality. Uninterrupted cold storage is a problem in developing countries because of power interruptions. Cold-chain transportation of samples from collection sites to testing laboratories is costly. In this study, we evaluated the use of BBL™ Sensi-Discs™ and FTA(®) cards for storage and transportation of samples for virus isolation, EIA, and RT-PCR testing. Infectious rotavirus was recovered after 30 days of storage on Sensi-Discs™ at room temperature. We were able to genotype 98-99% of samples stored on Sensi-Discs™ and FTA(®) cards at temperatures ranging from -80°C to 37°C up to 180 days. A field sampling test using samples prepared and shipped from Cameroon, showed that both matrices yielded 100% genotyping success compared with whole stool and Sensi-Discs™ demonstrated 95% concordance with whole stool in EIA testing. The utilization of BBL™ Sensi-Discs™ and FTA(®) cards for stool sample storage and shipment has the potential to have great impact on global public health by facilitating surveillance and epidemiological investigations of rotavirus strains worldwide at a reduced cost. Published by Elsevier B.V.

The effects of Bifidobacterium animalis ssp. lactis B94 on gastrointestinal wellness in adults with Prader-Willi syndrome: study protocol for a randomized controlled trial.

PubMed

Alyousif, Zainab; Miller, Jennifer L; Sandoval, Mariana Y; MacPherson, Chad W; Nagulesapillai, Varuni; Dahl, Wendy J

2018-04-27

Constipation is a frequent problem in adults with Prader-Willi syndrome. Certain probiotics have been shown to improve transit and gastrointestinal symptoms of adults with functional constipation. The aim of this study is to determine the effect of daily consumption of Bifidobacterium animalis ssp. lactis B94 (B. lactis B94) on stool frequency, stool form, and gastrointestinal symptoms in adults with Prader-Willi syndrome. Adults with Prader-Willi syndrome (18-75 years old, n = 36) will be recruited and enrolled in a 20-week, randomized, double-blind, placebo-controlled, crossover study. Study subjects will be randomized to B. lactis B94 or placebo each for a 4-week period, preceded by a 4-week baseline and followed by 4-week washouts. Subjects will complete daily records of stool frequency and stool form (a proxy of transit time). Dietary intake data also will be collected. Stools, one in each period, will be collected for exploratory microbiota analyses. To our knowledge, this is the first randomized controlled trial evaluating the effectiveness of B. lactis in adults with Prader-Willi syndrome. The results of this study will provide evidence of efficacy for future clinical trials in patient populations with constipation. ClinicalTrials.gov ( NCT03277157 ). Registered on 08 September 2017.

Relating Stool Microbial Metabolite Levels, Inflammatory Markers and Dietary Behaviors to Screening Colonoscopy Findings in a Racially/Ethnically Diverse Patient Population

PubMed Central

Bridges, Kristina M.; Diaz, Francisco J.; Wang, Zhiwen; Ahmed, Ishfaq; Sullivan, Debra K.; Umar, Shahid; Buckles, Daniel C.; Greiner, K. Allen; Hester, Christina M.

2018-01-01

Colorectal cancer (CRC) is the third leading cause of cancer death for both men and women in the United States, yet it is treatable and preventable. African Americans have higher incidence of CRC than other racial/ethnic groups, however, it is unclear whether this disparity is primarily due to environmental or biological factors. Short chain fatty acids (SCFAs) are metabolites produced by bacteria in the colon and are known to be inversely related to CRC progression. The aim of this study is to investigate how stool SCFA levels, markers of inflammation in stool and dietary intake relate to colonoscopy findings in a diverse patient population. Stool samples from forty-eight participants were analyzed for SCFA levels and inflammatory markers (lysozyme, secretory IgA, lactoferrin). Additionally, participants completed the National Cancer Institute’s Diet History Questionnaire II (DHQ II) to report dietary intake over the past year. Subsequently, the majority of participants underwent screening colonoscopy. Our results showed that African Americans had higher total levels of SCFAs in stool than other racial/ethnic groups, significantly lower intake of non-starchy vegetables and similar inflammatory marker expression and colonoscopy outcomes, compared to others. This work is an initial exploration into the biological and clinical factors that may ultimately inform personalized screening approaches and clinical decision-making to improve colorectal cancer disparities for African Americans. PMID:29495356

Efficacy and optimal dose of daily polyethylene glycol 3350 for treatment of constipation and encopresis in children.

PubMed

Pashankar, D S; Bishop, W P

2001-09-01

To determine efficacy, safety, and optimal dose of a laxative, polyethylene glycol (PEG) 3350, in children with chronic constipation. Children with chronic constipation (n = 24) were treated with PEG for 8 weeks at an initial dose of 1 g/kg/d. The dose was adjusted every 3 days as required to achieve 2 soft stools per day. A diary was kept to monitor dose, stool frequency and consistency, soiling, and other symptoms. Stool consistency was rated from 1 (hard) to 5 (watery). Subjects were examined for fecal retention. The Student t test and the Fisher exact test were used for data analysis. All 20 children who completed the study found PEG to be palatable and were satisfied with the treatment. There were no significant adverse effects. Weekly stool frequency increased from 2.3 +/- 0.4 to 16.9 +/- 1.6 (P <.0001) during treatment and stool consistency from 1.2 +/- 0.1 to 3.3 +/- 0.1 (P <.0001). In 9 children with soiling, weekly soiling events declined from 10.0 +/- 2.4 to 1.3 +/- 0.7 (P =.003). The mean effective dose was 0.84 g/kg/d (range, 0.27-1.42 g/kg/d). Daily administration of PEG at a mean dose of 0.8 g/kg is an effective, safe, and palatable treatment for constipation.

Disruption of c-Kit Signaling in Kit(W-sh/W-sh) Growing Mice Increases Bone Turnover.

PubMed

Lotinun, Sutada; Krishnamra, Nateetip

2016-08-16

c-Kit tyrosine kinase receptor has been identified as a regulator of bone homeostasis. The c-Kit loss-of-function mutations in WBB6F1/J-Kit(W/W-v) mice result in low bone mass. However, these mice are sterile and it is unclear whether the observed skeletal phenotype is secondary to a sex hormone deficiency. In contrast, C57BL/6J-Kit(W-sh)/(W-sh) (W(sh)/W(sh)) mice, which carry an inversion mutation affecting the transcriptional regulatory elements of the c-Kit gene, are fertile. Here, we showed that W(sh)/W(sh) mice exhibited osteopenia with elevated bone resorption and bone formation at 6- and 9-week-old. The c-Kit W(sh) mutation increased osteoclast differentiation, the number of committed osteoprogenitors, alkaline phosphatase activity and mineralization. c-Kit was expressed in both osteoclasts and osteoblasts, and c-Kit expression was decreased in W(sh)/W(sh)osteoclasts, but not osteoblasts, suggesting an indirect effect of c-Kit on bone formation. Furthermore, the osteoclast-derived coupling factor Wnt10b mRNA was increased in W(sh)/W(sh) osteoclasts. Conditioned medium from W(sh)/W(sh) osteoclasts had elevated Wnt10b protein levels and induced increased alkaline phosphatase activity and mineralization in osteoblast cultures. Antagonizing Wnt10b signaling with DKK1 or Wnt10b antibody inhibited these effects. Our data suggest that c-Kit negatively regulates bone turnover, and disrupted c-Kit signaling couples increased bone resorption with bone formation through osteoclast-derived Wnt 10 b.

Exacerbated experimental autoimmune encephalomyelitis in mast-cell-deficient Kit W-sh/W-sh mice.

PubMed

Piconese, Silvia; Costanza, Massimo; Musio, Silvia; Tripodo, Claudio; Poliani, Pietro L; Gri, Giorgia; Burocchi, Alessia; Pittoni, Paola; Gorzanelli, Andrea; Colombo, Mario P; Pedotti, Rosetta

2011-04-01

Mast cell (MC)-deficient c-Kit mutant Kit(W/W-v) mice are protected against experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, suggesting a detrimental role for MCs in this disease. To further investigate the role of MCs in EAE, we took advantage of a recently characterized model of MC deficiency, Kit(W-sh/W-sh). Surprisingly, we observed that myelin oligodendrocyte glycoprotein (MOG)(35-55)-induced chronic EAE was exacerbated in Kit(W-sh/W-sh) compared with Kit(+/+) mice. Kit(W-sh/W-sh) mice showed more inflammatory foci in the central nervous system (CNS) and increased T-cell response against myelin. To understand whether the discrepant results obtained in Kit(W-sh/W-sh) and in Kit(W/W-v) mice were because of the different immunization protocols, we induced EAE in these two strains with varying doses of MOG(35-55) and adjuvants. Although Kit(W-sh/W-sh) mice exhibited exacerbated EAE under all immunization protocols, Kit(W/W-v) mice were protected from EAE only when immunized with high, but not low, doses of antigen and adjuvants. Kit(W-sh/W-sh) mice reconstituted systemically, but not in the CNS, with bone marrow-derived MCs still developed exacerbated EAE, indicating that protection from disease could be exerted by MCs mainly in the CNS, and/or by other cells possibly dysregulated in Kit(W-sh/W-sh) mice. In summary, these data suggest to reconsider MC contribution to EAE, taking into account the variables of using different experimental models and immunization protocols.

Discovery of amido-benzisoxazoles as potent c-Kit inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kunz, Roxanne K.; Rumfelt, Shannon; Chen, Ning

2010-01-12

Deregulation of the receptor tyrosine kinase c-Kit is associated with an increasing number of human diseases, including certain cancers and mast cell diseases. Interference of c-Kit signaling with multi-kinase inhibitors has been shown clinically to successfully treat gastrointestinal stromal tumors and mastocytosis. Targeted therapy of c-Kit activity may provide therapeutic advantages against off-target effects for non-oncology applications. A new structural class of c-Kit inhibitors is described, including in vitro c-Kit potency, kinase selectivity, and the observed binding mode.

Ultrastructural study of relationships between c-kit immunoreactive interstitial cells and other cellular elements in the human colon.

PubMed

Mazzia, C; Porcher, C; Julé, Y; Christen, M O; Henry, M

2000-05-01

C-kit immunocytochemistry was performed on ultrathin sections of human distal colon. Our attention was focused on relationships between c-kit immunoreactive interstitial cells (c-kit ICs) and muscular cells and nervous elements located in the external muscular layers of the colonic wall. C-kit ICs established membrane apposition with both nerve fibers and smooth muscle cells of, respectively, the longitudinal and circular muscle layers, the myenteric area, and the extremus submucosus plexus. C-kit ICs also surrounded the external submucosus plexus and established membrane appositions with nerve elements located inside the myenteric ganglia. These membrane appositions were observed either at the level of the c-kit IC bodies or at that of their cytoplasmic processes. In some cases, membrane appositions were observed concomitantly between the c-kit ICs, nerve fibers, and smooth muscle cells. In all the regions studied, the c-kit ICs were also found to be located in the close vicinity of blood vessels and to have established close contacts with non-immunoreactive fibroblast-like cells. The results of the present study shed essential light on the relationships of c-kit ICs with the neighboring muscle cells and nerve elements, and confirm that the intercalated c-kit ICs well fit with the so-called "interstitial cells of Cajal".

SRC-like adaptor protein 2 (SLAP2) is a negative regulator of KIT-D816V-mediated oncogenic transformation.

PubMed

Rupar, Kaja; Moharram, Sausan A; Kazi, Julhash U; Rönnstrand, Lars

2018-04-23

KIT is a receptor tyrosine kinase (RTK) involved in several cellular processes such as regulation of proliferation, survival and differentiation of early hematopoietic cells, germ cells and melanocytes. Activation of KIT results in phosphorylation of tyrosine residues in the receptor, and recruitment of proteins that mediate downstream signaling and also modulate receptor signaling. Here we show that the SRC-like adaptor protein 2 (SLAP2) binds to wild-type KIT in a ligand-dependent manner and is furthermore found constitutively associated with the oncogenic mutant KIT-D816V. Peptide fishing analysis mapped pY568 and pY570 as potential SLAP2 association sites in KIT, which overlaps with the SRC binding sites in KIT. Expression of SLAP2 in cells expressing the transforming mutant KIT-D816V led to reduced cell viability and reduced colony formation. SLAP2 also partially blocked phosphorylation of several signal transduction molecules downstream of KIT such as AKT, ERK, p38 and STAT3. Finally, SLAP2 expression enhanced ubiquitination of KIT and its subsequent degradation. Taken together, our data demonstrate that SLAP2 negatively modulates KIT-D816V-mediated transformation by enhancing degradation of the receptor.

Characterization of various types of mast cells derived from model mice of familial gastrointestinal stromal tumors with KIT-Asp818Tyr mutation

PubMed Central

Kajimoto, Noriko; Nakai, Norihiro; Ohkouchi, Mizuka; Hashikura, Yuka; Liu-Kimura, Ning-Ning; Isozaki, Koji; Hirota, Seiichi

2015-01-01

Sporadic mast cell neoplasms and gastrointestinal stromal tumors (GISTs) often have various types of somatic gain-of-function mutations of the c-kit gene which encodes a receptor tyrosine kinase, KIT. Several types of germline gain-of-function mutations of the c-kit gene have been detected in families with multiple GISTs. All three types of model mice for the familial GISTs with germline c-kit gene mutations at exon 11, 13 or 17 show development of GIST, while they are different from each other in skin mast cell number. Skin mast cell number in the model mice with exon 17 mutation was unchanged compared to the corresponding wild-type mice. In the present study, we characterized various types of mast cells derived from the model mice with exon 17 mutation (KIT-Asp818Tyr) corresponding to human familial GIST case with human KIT-Asp820Tyr to clarify the role of the c-kit gene mutation in mast cells. Bone marrow-derived cultured mast cells (BMMCs) derived from wild-type mice, heterozygotes and homozygotes were used for the experiments. Immortalized BMMCs, designated as IMC-G4 cells, derived from BMMCs of a homozygote during long-term culture were also used. Ultrastructure, histamine contents, proliferation profiles and phosphorylation of various signaling molecules in those cells were examined. In IMC-G4 cells, presence of additional mutation(s) of the c-kit gene and effect of KIT inhibitors on both KIT autophosphorylation and cell proliferation were also analyzed. We demonstrated that KIT-Asp818Tyr did not affect ultrastructure and proliferation profiles but did histamine contents in BMMCs. IMC-G4 cells had an additional novel c-kit gene mutation of KIT-Tyr421Cys which is considered to induce neoplastic transformation of mouse mast cells and the mutation appeared to be resistant to a KIT inhibitor of imatinib but sensitive to another KIT inhibitor of nilotinib. IMC-G4 cells might be a useful mast cell line to investigate mast cell biology. PMID:26722383

The effect of inactivated Lactobacillus LB fermented culture medium on symptom severity: observational investigation in 297 patients with diarrhea-predominant irritable bowel syndrome.

PubMed

Tarrerias, A L; Costil, V; Vicari, F; Létard, J C; Adenis-Lamarre, P; Aisène, A; Batistelli, D; Bonnaud, G; Carpentier, S; Dalbiès, P; Ecuer, S; Etienne, J; Fantoli, M; Grunberg, B; Lannoy, P; Lapuelle, J; Margulies, A; Neumeier, M; Rouillon, J M; Schmets, L; Pingannaud, M P; Coulom, P; Kholer, F; Canard, J M

2011-01-01

Little is known about the intensity of symptoms of diarrhea-predominant IBS (IBS-D) or the consequences of the disease on patients' health-related quality of life (HRQOL). This observational investigation assessed the symptoms (abdominal pain, bloating, number of stools per day, and stool consistency), impact on HRQOL, and consequence on anal continence in 297 patients with IBS-D before and after 1 month of probiotic treatment with Lacteol (inactivated Lactobacillus LB plus fermented culture medium). Functional assessment using a standardized visual analogue scale in order to quantify abdominal pain, bloating, and quality of life before and after 1 month of treatment with 2 capsules/day of Lacteol. The number of symptomatic days per week, number of stools, consistency of stools, secondary fecal incontinence rate, and potential trigger effect of food were quantified. A χ2 test was used to compare qualitative data and the variance of quantitative criteria was analyzed. The pain score decreased from 4.46±0.15 on a scale of 0-10 before treatment to 2.8±0.14 after treatment (p<0.0001). Bloating decreased from 4.49±0.18 to 2.5±0.15 on a scale of 0-10 (p<0.0001). The HRQOL score, which is inversely correlated with quality of life, decreased from 5.99±0.14 to 3.92±0.16 (p<0.0001). In this cohort study, the fecal incontinence rate secondary to diarrhea was clearly higher than that of the general population: 18% versus a prevalence of 9-10%, according to different studies. The mean number of stools per week decreased from 17.59 to 12.83 after treatment (p<0.0001). Before treatment, 54% of patients had watery stools and 46% had smooth stools; at the end of treatment, only 18.5% of patients still had watery stools, and 34% had normal stools. 52% of patients attributed their symptoms to their diet: 34% to vegetables, 29% to fruit, 15% to milk, 15% to fat, 6% to peppers and spices, and 4% to sugar. This observational investigation shed new light on patients with IBS-D, the HRQOL of which is altered by a fecal incontinence rate twice as high as that of the general population. Correlation with diet is confirmed by 1 out of 2 patients reporting poor tolerance of fiber and dairy products. Nutritional management should thus be part of these patients' treatment. Inactivated Lactobacillus LB plus fermented culture medium is a probiotic drug that has been used by physicians for a long time to treat patients with diarrhea. Strongly concentrated, it has no side effects and seems to help these patients. Due to a strong placebo effect in patients with this pathology, however, a controlled study is necessary to confirm this result. Copyright © 2011 S. Karger AG, Basel.

46 CFR 121.710 - First-aid kits.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 4 2010-10-01 2010-10-01 false First-aid kits. 121.710 Section 121.710 Shipping COAST... SYSTEMS AND EQUIPMENT Miscellaneous § 121.710 First-aid kits. A vessel must carry either a first-aid kit... kits, the contents must be stowed in a suitable, watertight container that is marked “First-Aid Kit”. A...

Genetic lineage tracing identifies in situ Kit-expressing cardiomyocytes

PubMed Central

Liu, Qiaozhen; Yang, Rui; Huang, Xiuzhen; Zhang, Hui; He, Lingjuan; Zhang, Libo; Tian, Xueying; Nie, Yu; Hu, Shengshou; Yan, Yan; Zhang, Li; Qiao, Zengyong; Wang, Qing-Dong; Lui, Kathy O; Zhou, Bin

2016-01-01

Cardiac cells marked by c-Kit or Kit, dubbed cardiac stem cells (CSCs), are in clinical trials to investigate their ability to stimulate cardiac regeneration and repair. These studies were initially motivated by the purported cardiogenic activity of these cells. Recent lineage tracing studies using Kit promoter to drive expression of the inducible Cre recombinase showed that these CSCs had highly limited cardiogenic activity, inadequate to support efficient cardiac repair. Here we reassess the lineage tracing data by investigating the identity of cells immediately after Cre labeling. Our instant lineage tracing approach identifies Kit-expressing cardiomyocytes, which are labeled immediately after tamoxifen induction. In combination with long-term lineage tracing experiments, these data reveal that the large majority of long-term labeled cardiomyocytes are pre-existing Kit-expressing cardiomyocytes rather than cardiomyocytes formed de novo from CSCs. This study presents a new interpretation for the contribution of Kit+ cells to cardiomyocytes and shows that Kit genetic lineage tracing over-estimates the cardiogenic activity of Kit+ CSCs. PMID:26634606

Oncogenic Kit signals on endolysosomes and endoplasmic reticulum are essential for neoplastic mast cell proliferation

PubMed Central

Obata, Yuuki; Toyoshima, Shota; Wakamatsu, Ei; Suzuki, Shunichi; Ogawa, Shuhei; Esumi, Hiroyasu; Abe, Ryo

2014-01-01

Kit is a receptor-type tyrosine kinase found on the plasma membrane. It can transform mast cells through activating mutations. Here, we show that a mutant Kit from neoplastic mast cells from mice, Kit(D814Y), is permanently active and allows cells to proliferate autonomously. It does so by activating two signalling pathways from different intracellular compartments. Mutant Kit from the cell surface accumulates on endolysosomes through clathrin-mediated endocytosis, which requires Kit’s kinase activity. Kit(D814Y) is constitutively associated with phosphatidylinositol 3-kinase, but the complex activates Akt only on the cytoplasmic surface of endolysosomes. It resists destruction because it is under-ubiquitinated. Kit(D814Y) also appears in the endoplasmic reticulum soon after biosynthesis, and there, can activate STAT5 aberrantly. These mechanisms of oncogenic signalling are also seen in rat and human mast cell leukemia cells. Thus, oncogenic Kit signalling occurs from different intracellular compartments, and the mutation acts by altering Kit trafficking as well as activation. PMID:25493654

Structural Basis for Activation of the Receptor Tyrosine Kinase KIT by Stem Cell Factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuzawa,S.; Opatowsky, Y.; Zhang, Z.

2007-01-01

Stem Cell Factor (SCF) initiates its multiple cellular responses by binding to the ectodomain of KIT, resulting in tyrosine kinase activation. We describe the crystal structure of the entire ectodomain of KIT before and after SCF stimulation. The structures show that KIT dimerization is driven by SCF binding whose sole role is to bring two KIT molecules together. Receptor dimerization is followed by conformational changes that enable lateral interactions between membrane proximal Ig-like domains D4 and D5 of two KIT molecules. Experiments with cultured cells show that KIT activation is compromised by point mutations in amino acids critical for D4-D4more » interaction. Moreover, a variety of oncogenic mutations are mapped to the D5-D5 interface. Since key hallmarks of KIT structures, ligand-induced receptor dimerization, and the critical residues in the D4-D4 interface, are conserved in other receptors, the mechanism of KIT stimulation unveiled in this report may apply for other receptor activation.« less

Ladders and Easels and Stools, Oh My!

ERIC Educational Resources Information Center

Greenman, Geri

2002-01-01

Describes an art lesson for high school seniors in an intermediate oil painting class. Explains that the teacher set up a still life with a ladder, easel, and a stool with a white background to capture the shadows. States that the students painted their still life in oil. (CMK)

Colorectal cancer screening: The role of the noninvasive options.

PubMed

Dickerson, Lisa; Varcak, Susan Combs

2016-09-01

Recommended screening options for colorectal cancer are divided into noninvasive stool-based options, and invasive procedure-based options. Because multiple screening strategies are effective, efforts to reduce deaths from colorectal cancer should focus on maximizing the number of patients who are screened. This article reviews noninvasive stool-based screening options.

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

PubMed

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.

Prevalence and transmission of antimicrobial resistance among Aeromonas populations from a duckweed aquaculture based hospital sewage water recycling system in Bangladesh.

PubMed

Rahman, Mokhlasur; Huys, Geert; Kühn, Inger; Rahman, Motiur; Möllby, Roland

2009-10-01

In order to investigate the influence of a duckweed aquaculture based hospital sewage water recycling plant on the prevalence and dissemination of antibiotic resistance, we made use of an existing collection of 1,315 Aeromonas isolates that were previously typed by the biochemical fingerprinting PhP-AE system. In these treatment plant, hospital raw sewage water is first collected in a settlement pond (referred to as sewage water in this study) and is then transferred to a lagoon, where the duckweed (Lemnaceae) is grown (referred to as lagoon). The duckweed is harvested and used as feed for the fish in a separate pond (referred to as fish pond). From this collection, representatives of 288 PhP types were subjected to antibiotic susceptibility testing for eight antimicrobials by broth microdilution method. The overall resistance rates among Aeromonas isolates from the treatment plant were highest for ampicillin (87%) and erythromycin (79%) followed by cephalothin (58%), nalidixic acid (52%), streptomycin (51%), tetracycline (31%), chloramphenicol (13%) and gentamicin (8%). A significantly lower prevalence of antibiotic resistance was found in Aeromonas from environmental control water, patient stool samples, duckweed and fish compared to sewage water isolates. The prevalence of resistance in the sewage water was not significantly reduced compared to the lagoon water and fish pond. Throughout the treatment system, the frequencies of resistant strains were found to diminish during the sewage water purification process, i.e. in the lagoon where sewage water is used to grow the duckweed. However, the frequency of resistant strains again increased in the fish pond where sewage grown duckweed is used for aquaculture. Among the selected isolates, two multiresistant clonal groups of Aeromonas caviae HG4 were identified that exhibited indistinguishable PhP and amplified fragment length polymorphism fingerprints and shared a common plasmid of approximately 5 kb. Representatives of both groups were recovered from almost every part of the sewage treatment plant but not in the control ponds nor in human samples, which suggests that specific multiresistant Aeromonas clones are able to persist and spread throughout the entire purification process.

Effect of planecta and ROSE™ on the frequency characteristics of blood pressure-transducer kits.

PubMed

Fujiwara, Shigeki; Kawakubo, Yoshifumi; Mori, Satoshi; Tachihara, Keiichi; Toyoguchi, Izumi; Yokoyama, Takeshi

2015-12-01

Pressure-transducer kits have frequency characteristics such as natural frequency and damping coefficient, which affect the monitoring accuracy. The aim of the present study was to investigate the effect of planecta ports and a damping device (ROSE™, Argon Medical Devices, TX, USA) on the frequency characteristics of pressure-transducer kits. The FloTrac sensor kit (Edwards Lifesciences, CA, USA) and the DTXplus transducer kit (Argon Medical Devices) were prepared with planecta ports, and their frequency characteristics were tested with or without ROSE™. The natural frequency and damping coefficient of each kit were obtained using frequency characteristics analysis software and evaluated by plotting them on the Gardner's chart. By inserting a planecta port, the natural frequency markedly decreased in both the FloTrac sensor kit (from 40 to 22 Hz) and the DTXplus transducer kit (from 35 to 22 Hz). In both kits with one planecta port, the damping coefficient markedly increased by insertion of ROSE™ from 0.2 to 0.5, optimising frequency characteristics. In both kits with two planecta ports, however, the natural frequency decreased from 22 to 12 Hz. The damping coefficient increased from 0.2 to 0.8 by insertion of ROSE™; however, optimisation was not achieved even by ROSE™ insertion. Planecta ports decrease the natural frequency of the kit. ROSE™ is useful to optimise the frequency characteristics in the kits without or with one planecta port. However, optimisation is difficult with two or more planecta ports, even with the ROSE™ device.

KIT Mutations Are Common in Testicular Seminomas

PubMed Central

Kemmer, Kathleen; Corless, Christopher L.; Fletcher, Jonathan A.; McGreevey, Laura; Haley, Andrea; Griffith, Diana; Cummings, Oscar W.; Wait, Cecily; Town, Ajia; Heinrich, Michael C.

2004-01-01

Expression of KIT tyrosine kinase is critical for normal germ cell development and is observed in the majority of seminomas. Activating mutations in KIT are common in gastrointestinal stromal tumors and mastocytosis. In this study we examined the frequency and spectrum of KIT mutations in 54 testicular seminomas, 1 ovarian dysgerminoma and 37 non-seminomatous germ cell tumors (NSGCT). Fourteen seminomas (25.9%) contained exon 17 point mutations including D816V (6 cases), D816H (3 cases), Y823D (2 cases), and single examples of Y823C, N822K, and T801I. No KIT mutations were found in the ovarian dysgerminoma or the NSGCTs. In transient transfection assays, mutant isoforms D816V, D816H, Y823D, and N822K were constitutively phosphorylated in the absence of the natural ligand for KIT, stem cell factor (SCF). In contrast, activation of T801I and wild-type KIT required SCF. Mutants N822K and Y823D were inhibited by imatinib mesylate (Gleevec, previously STI571) whereas D816V and D816H were both resistant to imatinib mesylate. Biochemical evidence of KIT activation, as assessed by KIT phosphorylation and KIT association with phosphatidylinositol (PI) 3-kinase in tumor cell lysates, was largely confined to seminomas with a genomic KIT mutation. These findings suggest that activating KIT mutations may contribute to tumorigenesis in a subset of seminomas, but are not involved in NSGCT. PMID:14695343

FES kinase participates in KIT-ligand induced chemotaxis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Voisset, Edwige, E-mail: Edwige.Voisset@inserm.fr; Institut Paoli-Calmettes, Marseille; Universite de la Mediterranee, Aix-Marseille II

2010-02-26

FES is a cytoplasmic tyrosine kinase activated by several membrane receptors, originally identified as a viral oncogene product. We have recently identified FES as a crucial effector of oncogenic KIT mutant receptor. However, FES implication in wild-type KIT receptor function was not addressed. We report here that FES interacts with KIT and is phosphorylated following activation by its ligand SCF. Unlike in the context of oncogenic KIT mutant, FES is not involved in wild-type KIT proliferation signal, or in cell adhesion. Instead, FES is required for SCF-induced chemotaxis. In conclusion, FES kinase is a mediator of wild-type KIT signalling implicatedmore » in cell migration.« less

Telescience Resource Kit

NASA Technical Reports Server (NTRS)

Schneider, Michelle

2003-01-01

This viewgraph representation provides an overview of the Telescience Resource Kit. The Telescience Resource Kit is a pc-based telemetry and command system that will be used by scientists and engineers to monitor and control experiments located on-board the International Space Station (ISS). Topics covered include: ISS Payload Telemetry and Command Flow, kit computer applications, kit telemetry capabilities, command capabilities, and training/testing capabilities.

Hotspot Mutations in KIT Receptor Differentially Modulate Its Allosterically Coupled Conformational Dynamics: Impact on Activation and Drug Sensitivity

PubMed Central

Chauvot de Beauchêne, Isaure; Allain, Ariane; Panel, Nicolas; Laine, Elodie; Trouvé, Alain; Dubreuil, Patrice; Tchertanov, Luba

2014-01-01

Receptor tyrosine kinase KIT controls many signal transduction pathways and represents a typical allosterically regulated protein. The mutation-induced deregulation of KIT activity impairs cellular physiological functions and causes serious human diseases. The impact of hotspots mutations (D816H/Y/N/V and V560G/D) localized in crucial regulatory segments, the juxtamembrane region (JMR) and the activation (A-) loop, on KIT internal dynamics was systematically studied by molecular dynamics simulations. The mutational outcomes predicted in silico were correlated with in vitro and in vivo activation rates and drug sensitivities of KIT mutants. The allosteric regulation of KIT in the native and mutated forms is described in terms of communication between the two remote segments, JMR and A-loop. A strong correlation between the communication profile and the structural and dynamical features of KIT in the native and mutated forms was established. Our results provide new insight on the determinants of receptor KIT constitutive activation by mutations and resistance of KIT mutants to inhibitors. Depiction of an intra-molecular component of the communication network constitutes a first step towards an integrated description of vast communication pathways established by KIT in physiopathological contexts. PMID:25079768

KIT pathway alterations in mucosal melanomas of the vulva and other sites.

PubMed

Omholt, Katarina; Grafström, Eva; Kanter-Lewensohn, Lena; Hansson, Johan; Ragnarsson-Olding, Boel K

2011-06-15

A significant proportion of mucosal melanomas contain alterations in KIT. The aim of this study was to characterize the pattern of KIT, NRAS, and BRAF mutations in mucosal melanomas at specific sites and to assess activation of the KIT downstream RAF/MEK/extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/AKT pathways in mucosal melanoma specimens. Seventy-one primary mucosal melanomas from various sites were studied. Mutation analysis was done by DNA sequencing. Expression of KIT, phosphorylated (p)-ERK, and p-AKT was evaluated by immunohistochemistry. KIT mutations were detected in 35% (8 of 23) of vulvar, 9% (2 of 22) of anorectal, 7% (1 of 14) of nasal cavity, and 20% (1 of 5) of penile melanomas. No KIT mutations were found in 7 vaginal melanomas. The difference in KIT mutation frequency between vulvar and nonvulvar cases was statistically significant (P = 0.014). The overall frequencies of NRAS and BRAF mutations were 10% and 6%, respectively. Notably, vaginal melanomas showed a NRAS mutation rate of 43%. KIT gene amplification (≥4 copies), as assessed by quantitative real-time PCR, was observed in 19% of cases. KIT expression was associated with KIT mutation status (P < 0.001) and was more common in vulvar than nonvulvar tumors (P = 0.016). Expression of p-ERK and p-AKT was observed in 42% and 59% of tumors, respectively, and occurred irrespective of KIT/NRAS/BRAF mutation status. NRAS mutation was associated with worse overall survival in univariate analysis. Results show that KIT mutations are more common in vulvar melanomas than other types of mucosal melanomas and that both the RAF/MEK/ERK and PI3K/AKT pathways are activated in mucosal melanoma specimens. ©2011 AACR.

Imatinib for melanomas harboring mutationally activated or amplified KIT arising on mucosal, acral, and chronically sun-damaged skin.

PubMed

Hodi, F Stephen; Corless, Christopher L; Giobbie-Hurder, Anita; Fletcher, Jonathan A; Zhu, Meijun; Marino-Enriquez, Adrian; Friedlander, Philip; Gonzalez, Rene; Weber, Jeffrey S; Gajewski, Thomas F; O'Day, Steven J; Kim, Kevin B; Lawrence, Donald; Flaherty, Keith T; Luke, Jason J; Collichio, Frances A; Ernstoff, Marc S; Heinrich, Michael C; Beadling, Carol; Zukotynski, Katherine A; Yap, Jeffrey T; Van den Abbeele, Annick D; Demetri, George D; Fisher, David E

2013-09-10

Amplifications and mutations in the KIT proto-oncogene in subsets of melanomas provide therapeutic opportunities. We conducted a multicenter phase II trial of imatinib in metastatic mucosal, acral, or chronically sun-damaged (CSD) melanoma with KIT amplifications and/or mutations. Patients received imatinib 400 mg once per day or 400 mg twice per day if there was no initial response. Dose reductions were permitted for treatment-related toxicities. Additional oncogene mutation screening was performed by mass spectroscopy. Twenty-five patients were enrolled (24 evaluable). Eight patients (33%) had tumors with KIT mutations, 11 (46%) with KIT amplifications, and five (21%) with both. Median follow-up was 10.6 months (range, 3.7 to 27.1 months). Best overall response rate (BORR) was 29% (21% excluding nonconfirmed responses) with a two-stage 95% CI of 13% to 51%. BORR was significantly greater than the hypothesized null of 5% and statistically significantly different by mutation status (7 of 13 or 54% KIT mutated v 0% KIT amplified only). There were no statistical differences in rates of progression or survival by mutation status or by melanoma site. The overall disease control rate was 50% but varied significantly by KIT mutation status (77% mutated v 18% amplified). Four patients harbored pretreatment NRAS mutations, and one patient acquired increased KIT amplification after treatment. Melanomas that arise on mucosal, acral, or CSD skin should be assessed for KIT mutations. Imatinib can be effective when tumors harbor KIT mutations, but not if KIT is amplified only. NRAS mutations and KIT copy number gain may be mechanisms of therapeutic resistance to imatinib.

Facilitators and Barriers to Naloxone Kit Use Among Opioid-Dependent Patients Enrolled in Medication Assisted Therapy Clinics in North Carolina.

PubMed

Khatiwoda, Prasana; Proeschold-Bell, Rae Jean; Meade, Christina S; Park, Lawrence P; Proescholdbell, Scott

2018-01-01

BACKGROUND Naloxone-an opioid antagonist that reverses the effects of opioids-is increasingly being distributed in non-medical settings. We sought to identify the facilitators of, and barriers to, opioid users using naloxone kits in North Carolina. METHODS In 2015, we administered a 15-item survey to a convenience sample of 100 treatment seekers at 4 methadone/buprenorphine Medication Assisted Therapy (MAT) clinics in North Carolina. RESULTS Seventy-four percent of participants reported having ever gotten a naloxone kit; this percentage was higher for females (81%) than males (63%) ( P = .06). The primary reason given for not having a kit was not knowing where to get one. Only 6% had heard of kits from the media and only 5% received one from a medical provider. Among kit recipients, 56% of both females and males reported mostly or sometimes carrying the kit, with additional participants reporting always. Reasons for not carrying a kit were no longer being around drugs, forgetting it, and the kit being too large. Men discussed the difficulties of carrying the naloxone kits, which are currently too large to fit in a pocket. Ninety-four percent of naloxone users reported intending to call emergency services in case of an overdose emergency. LIMITATIONS Study limitations included a small sample, participants limited to MAT clinics, and a predominantly white sample. CONCLUSIONS MAT treatment seekers reported a willingness to carry and use naloxone kits. Education, outreach, media, and medical providers need to promote naloxone kits. A smaller kit may increase the likelihood of men carrying one. ©2018 by the North Carolina Institute of Medicine and The Duke Endowment. All rights reserved.

Identification of a population of cells with hematopoietic stem cell properties in mouse aorta-gonad-mesonephros cultures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nobuhisa, Ikuo; Ohtsu, Naoki; Okada, Seiji

The aorta-gonad-mesonephros (AGM) region is a primary source of definitive hematopoietic cells in the midgestation mouse embryo. In cultures of dispersed AGM regions, adherent cells containing endothelial cells are observed first, and then non-adherent hematopoietic cells are produced. Here we report on the characterization of hematopoietic cells that emerge in the AGM culture. Based on the expression profiles of CD45 and c-Kit, we defined three cell populations: CD45{sup low} c-Kit{sup +} cells that had the ability to form hematopoietic cell colonies in methylcellulose media and in co-cultures with stromal cells; CD45{sup low} c-Kit{sup -} cells that showed a granulocyte morphology;more » CD45{sup high} c-Kit{sup low/-} that exhibited a macrophage morphology. In co-cultures of OP9 stromal cells and freshly prepared AGM cultures, CD45{sup low} c-Kit{sup +} cells from the AGM culture had the abilities to reproduce CD45{sup low} c-Kit{sup +} cells and differentiate into CD45{sup low} c-Kit{sup -} and CD45{sup high} c-Kit{sup low/-} cells, whereas CD45{sup low} c-Kit{sup -} and CD45{sup high} c-Kit{sup low/-} did not produce CD45{sup low} c-Kit{sup +} cells. Furthermore, CD45{sup low} c-Kit{sup +} cells displayed a long-term repopulating activity in adult hematopoietic tissue when transplanted into the liver of irradiated newborn mice. These results indicate that CD45{sup low} c-Kit{sup +} cells from the AGM culture have the potential to reconstitute multi-lineage hematopoietic cells.« less

Evaluation of four commercial quantitative real-time PCR kits with inhibited and degraded samples.

PubMed

Holmes, Amy S; Houston, Rachel; Elwick, Kyleen; Gangitano, David; Hughes-Stamm, Sheree

2018-05-01

DNA quantification is a vital step in forensic DNA analysis to determine the optimal input amount for DNA typing. A quantitative real-time polymerase chain reaction (qPCR) assay that can predict DNA degradation or inhibitors present in the sample prior to DNA amplification could aid forensic laboratories in creating a more streamlined and efficient workflow. This study compares the results from four commercial qPCR kits: (1) Investigator® Quantiplex® Pro Kit, (2) Quantifiler® Trio DNA Quantification Kit, (3) PowerQuant® System, and (4) InnoQuant® HY with high molecular weight DNA, low template samples, degraded samples, and DNA spiked with various inhibitors.The results of this study indicate that all kits were comparable in accurately predicting quantities of high quality DNA down to the sub-picogram level. However, the InnoQuant(R) HY kit showed the highest precision across the DNA concentration range tested in this study. In addition, all kits performed similarly with low concentrations of forensically relevant PCR inhibitors. However, in general, the Investigator® Quantiplex® Pro Kit was the most tolerant kit to inhibitors and provided the most accurate quantification results with higher concentrations of inhibitors (except with salt). PowerQuant® and InnoQuant® HY were the most sensitive to inhibitors, but they did indicate significant levels of PCR inhibition. When quantifying degraded samples, each kit provided different degradation indices (DI), with Investigator® Quantiplex® Pro indicating the largest DI and Quantifiler® Trio indicating the smallest DI. When the qPCR kits were paired with their respective STR kit to genotype highly degraded samples, the Investigator® 24plex QS and GlobalFiler® kits generated more complete profiles when the small target concentrations were used for calculating input amount.

Aberrant expressions of c-KIT and DOG-1 in mucinous and nonmucinous colorectal carcinomas and relation to clinicopathologic features and prognosis.

PubMed

Foda, Abd Al-Rahman Mohammad; Mohamed, Mie Ali

2015-10-01

c-KIT and DOG-1 are 2 highly expressed proteins in gastrointestinal stromal tumors. Few studies had investigated c-KIT, but not DOG-1, expression in colorectal carcinoma (CRC). This study aims to investigate expressions of c-KIT and DOG-1 in colorectal mucinous carcinoma and nonmucinous carcinoma using manual tissue microarray technique. In this work, we studied tumor tissue specimens from 150 patients with colorectal mucinous (MA) and nonmucinous adenocarcinoma (NMA). High-density manual tissue microarrays were constructed using modified mechanical pencil tip technique, and immunohistochemistry for c-KIT and DOG-1 was done. We found that aberrant c-KIT expression was detected in 12 cases (8%); 6 cases (4%) showed strong expression. Aberrant DOG-1 expression was detected in 15 cases (10%); among them, only 4 cases (2.7%) showed strong expression. Nonmucinous adenocarcinoma showed a significantly high expression of c-KIT, but not DOG-1, than MA. Aberrant c-KIT and DOG-1 expressions were significantly unrelated but were associated with excessive microscopic abscess formation. Neither c-KIT nor DOG-1 expression showed a significant impact on disease-free survival or overall survival. In conclusion, aberrant c-KIT and DOG-1 expressions in CRC are rare events, either in NMA or MA. Nonmucinous adenocarcinoma showed a significantly higher expression of c-KIT, but not DOG-1, than MA. The expressions of both in CRC are significantly unrelated but are associated with microscopic abscess formation. Neither c-KIT nor DOG-1 expression showed a significant impact on disease-free survival or overall survival. So, c-KIT and DOG-1 immunostaining is not a cost-effective method of identifying patients with CRC who may benefit from treatment with tyrosine kinase inhibitors. Copyright © 2015 Elsevier Inc. All rights reserved.

Molecular Alterations of KIT Oncogene in Gliomas

PubMed Central

Gomes, Ana L.; Reis-Filho, Jorge S.; Lopes, José M.; Martinho, Olga; Lambros, Maryou B. K.; Martins, Albino; Schmitt, Fernando; Pardal, Fernando; Reis, Rui M.

2007-01-01

Gliomas are the most common and devastating primary brain tumours. Despite therapeutic advances, the majority of gliomas do not respond either to chemo or radiotherapy. KIT, a class III receptor tyrosine kinase (RTK), is frequently involved in tumourigenic processes. Currently, KIT constitutes an attractive therapeutic target. In the present study we assessed the frequency of KIT overexpression in gliomas and investigated the genetic mechanisms underlying KIT overexpression. KIT (CD117) immunohistochemistry was performed in a series of 179 gliomas of various grades. KIT activating gene mutations (exons 9, 11, 13 and 17) and gene amplification analysis, as defined by chromogenic in situ hybridization (CISH) and quantitative real-time PCR (qRT-PCR) were performed in CD117 positive cases. Tumour cell immunopositivity was detected in 15.6% (28/179) of cases, namely in 25% (1/4) of pilocytic astrocytomas, 25% (5/20) of diffuse astrocytomas, 20% (1/5) of anaplastic astrocytomas, 19.5% (15/77) of glioblastomas and one third (3/9) of anaplastic oligoastrocytomas. Only 5.7% (2/35) of anaplastic oligodendrogliomas showed CD117 immunoreactivity. No association was found between tumour CD117 overexpression and patient survival. In addition, we also observed CD117 overexpression in endothelial cells, which varied from 0–22.2% of cases, being more frequent in high-grade lesions. No KIT activating mutations were identified. Interestingly, CISH and/or qRT-PCR analysis revealed the presence of KIT gene amplification in 6 glioblastomas and 2 anaplastic oligoastrocytomas, corresponding to 33% (8/24) of CD117 positive cases. In conclusion, our results demonstrate that KIT gene amplification rather than gene mutation is a common genetic mechanism underlying KIT expression in subset of malignant gliomas. Further studies are warranted to determine whether glioma patients exhibiting KIT overexpression and KIT gene amplification may benefit from therapy with anti-KIT RTK inhibitors. PMID:17726262

A phase 2 trial of dasatinib in patients with locally advanced or stage IV mucosal, acral, or vulvovaginal melanoma: A trial of the ECOG-ACRIN Cancer Research Group (E2607).

PubMed

Kalinsky, Kevin; Lee, Sandra; Rubin, Krista M; Lawrence, Donald P; Iafrarte, Anthony J; Borger, Darell R; Margolin, Kim A; Leitao, Mario M; Tarhini, Ahmad A; Koon, Henry B; Pecora, Andrew L; Jaslowski, Anthony J; Cohen, Gary I; Kuzel, Timothy M; Lao, Christopher D; Kirkwood, John M

2017-07-15

KIT-directed tyrosine kinase inhibitors such as imatinib have demonstrated benefits in KIT-mutant (KIT+) mucosal, acral, vulvovaginal, and chronically sun-damaged (CSD) melanoma. Dasatinib has superior preclinical activity in comparison with other tyrosine kinase inhibitors against cells with the most common KIT mutation, exon 11 L576P . The ECOG-ACRIN E2607 trial assessed dasatinib in patients with these melanoma subtypes. Patients received 70 mg of oral dasatinib twice daily. The primary objective for this 2-stage phase 2 trial was response rate. Stage I was open to KIT+ and wild-type KIT (KIT-) mucosal, acral, and CSD melanoma (n = 57). Stage II accrued only KIT+ tumors (n = 30). To enrich the trial for KIT+ tumors, vulvovaginal melanoma was added, and CSD melanoma was removed from eligibility. Secondary objectives included progression-free survival (PFS), overall survival (OS), and safety. From May 2009 to December 2010, the first stage enrolled 57 patients. Among the evaluable patients, 3 of 51 (5.9%) achieved a partial response: all were KIT-. Stage II closed early because of slow accrual (November 2011 to December 2015). In stage II, 4 of 22 evaluable patients (18.2%) had a partial response; the median duration was 4.2 months. The median PFS was 2.1 months (n = 73; 95% confidence interval [CI], 1.5-2.9 months). The median OS was 7.5 months (95% CI, 6.0-11.9 months). In exploratory analyses, no differences were seen in PFS or OS with the KIT status or subtype. Dasatinib was discontinued because of adverse events in 9 of 75 patients (12%). The dasatinib response rate among KIT+ melanoma patients was low. In view of its clinical activity, it is recommended that imatinib remain the KIT tyrosine kinase inhibitor of choice for unresectable KIT+ melanoma. Cancer 2017;123:2688-97. © 2017 American Cancer Society. © 2017 American Cancer Society.