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Sample records for human stool samples

  1. Isolation of Campylobacter from human stool samples

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    S M Salim

    2014-01-01

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

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

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    Alessandro Tanca

    2015-09-01

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

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

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    Roselida Achieng Owuor

    2016-03-01

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

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

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    Eb Kia

    2011-06-01

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

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

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    Santos, Herbert J; Rivera, Windell L

    2013-10-01

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

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

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    Herbert J Santos; Windell L Rivera

    2013-01-01

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

  7. Identification of human Norovirus (HNoV in domestic pig stool samples

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    María F. Gutiérrez

    2011-08-01

    Full Text Available To determine the presence of NoVs as a possible causal zoonotic agent of acute diarrhea in pigs and humans. Materialsand methods. We collected a total of 77 samples from diarrheal children under 5 years and pigs under 2 months from La Chambatown in Tolima, Colombia. These samples were transported to the Laboratory of Virology of the Pontificia Universidad Javerianain Bogotá, and extraction with Trizol-reagent was done following the manufacturer’s instructions. After obtaining the RNA, thenext step was to perform RT-PCR for obtaining the expected amplification product of 213- bp NoVs. Finally, the positive samplesobtained in the RT-PCR were sequenced and analyzed by bioinformatics methods. Results. Six positive diarrheic samples fromchildren and a positive diarrheic sample from pigs were detected by a band of 231 bp. Five of the six positive samples in childrenand the positive pig sample were sequenced and analyzed. Conclusion. Given the close genetic relationship between pig andhuman sequences, this could be an indication of the potential existence of a common animal acting as a reservoir for human orother animal strains.

  8. 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

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    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

  9. Screening for enterotoxigenic Bacteroides fragilis in stool samples.

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

    2016-08-01

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

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

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

    2017-06-01

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

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

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

    2015-01-01

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

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

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

    1992-01-01

    The clinical significance of the fastidious organism DF-3 isolated from stool cultures is unclear. We sought to improve our understanding of this organism and to further define its association with human disease. Stool cultures for DF-3 were obtained from three sources: an ongoing study of enteric pathogens in patients infected with the human immunodeficiency virus, a screening procedure in which all stool samples submitted for Clostridium difficile toxin assay were cultured for DF-3, and sto...

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

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

    2007-07-01

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

  14. An uncooked vegan diet shifts the profile of human fecal microflora: computerized analysis of direct stool sample gas-liquid chromatography profiles of bacterial cellular fatty acids.

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    Peltonen, R; Ling, W H; Hänninen, O; Eerola, E

    1992-01-01

    The effect of an uncooked extreme vegan diet on fecal microflora was studied by direct stool sample gas-liquid chromatography (GLC) of bacterial cellular fatty acids and by quantitative bacterial culture by using classical microbiological techniques of isolation, identification, and enumeration of different bacterial species. Eighteen volunteers were divided randomly into two groups. The test group received an uncooked vegan diet for 1 month and a conventional diet of mixed Western type for the other month of the study. The control group consumed a conventional diet throughout the study period. Stool samples were collected. Bacterial cellular fatty acids were extracted directly from the stool samples and measured by GLC. Computerized analysis of the resulting fatty acid profiles was performed. Such a profile represents all bacterial cellular fatty acids in a sample and thus reflects its microflora and can be used to detect changes, differences, or similarities of bacterial flora between individual samples or sample groups. GLC profiles changed significantly in the test group after the induction and discontinuation of the vegan diet but not in the control group at any time, whereas quantitative bacterial culture did not detect any significant change in fecal bacteriology in either of the groups. The results suggest that an uncooked extreme vegan diet alters the fecal bacterial flora significantly when it is measured by direct stool sample GLC of bacterial fatty acids. PMID:1482187

  15. Development and Evaluation of a Real-Time PCR Assay for Detection and Quantification of Blastocystis Parasites in Human Stool Samples: Prospective Study of Patients with Hematological Malignancies▿

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    Poirier, Philippe; Wawrzyniak, Ivan; Albert, Aurélie; El Alaoui, Hicham; Delbac, Frédéric; Livrelli, Valérie

    2011-01-01

    Blastocystis anaerobic parasites are widespread worldwide in the digestive tract of many animal species, including humans. Epidemiological Blastocystis studies are often limited by the poor sensitivity of standard parasitological assays for its detection. This report presents a highly sensitive real-time quantitative PCR (qPCR) assay developed to detect Blastocystis parasites in stool samples. The assay targets a partial sequence of the Blastocystis small ribosomal subunit (SSU) rRNA gene, allowing subtyping (ST) of Blastocystis isolates by direct sequencing of qPCR products. This qPCR method was assessed in a prospective study of 186 patients belonging to two cohorts—a group of 94 immunocompromised patients presenting hematological malignancies and a control group of 92 nonimmunocompromised patients. Direct-light microscopy and xenic in vitro stool culture analysis showed only 29% and 52% sensitivity, respectively, compared to our qPCR assay. Of the 27 (14.5%) Blastocystis-positive patients, 8 (4%) experienced digestive symptoms. No correlation was found between symptomatic patients and immune status, parasite load, or parasite subtypes, although subtyping of all isolates revealed a high (63.0%) prevalence of ST4. Two unexpected avian subtypes were found, i.e., ST6 and ST7, which are frequently isolated in Asia but rarely present in Western countries. In conclusion, this qPCR proved by far the most sensitive of the tested methods and allowed subtype determination by direct sequencing of qPCR products. New diagnostic tools such as the qPCR are essential for evaluating the clinical relevance of Blastocystis subtypes and their role in acute or chronic digestive disorders. PMID:21177897

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

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    Teiliane Rodrigues Carneiro

    2013-12-01

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

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

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

    2013-01-01

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

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

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    Luciana I Gomes

    2009-12-01

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

  19. Real-time PCR for detection of Strongyloides stercoralis in human stool samples from Côte d'Ivoire: diagnostic accuracy, inter-laboratory comparison and patterns of hookworm co-infection.

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    Becker, Sören L; Piraisoody, Nivetha; Kramme, Stefanie; Marti, Hanspeter; Silué, Kigbafori D; Panning, Marcus; Nickel, Beatrice; Kern, Winfried V; Herrmann, Mathias; Hatz, Christoph F; N'Goran, Eliézer K; Utzinger, Jürg; von Müller, Lutz

    2015-10-01

    Human infections with the helminth species Strongyloides stercoralis encompass a wide clinical spectrum, ranging from asymptomatic carriage to life-threatening disease. The diagnosis of S. stercoralis is cumbersome and the sensitivity of conventional stool microscopy is low. New molecular tools have been developed to increase sensitivity. We compared the diagnostic accuracy of real-time PCR with microscopy for the detection of S. stercoralis and hookworm in human stool samples, and investigated the inter-laboratory agreement of S. stercoralis-specific real-time PCR in two European laboratories. Stool specimens from 256 randomly selected individuals in rural Côte d'Ivoire were examined using three microscopic techniques (i.e. Kato-Katz, Koga agar plate (KAP) and Baermann (BM)). Additionally, ethanol-fixed stool aliquots were subjected to molecular diagnosis. The prevalence of S. stercoralis and hookworm infection was 21.9% and 52.0%, respectively, whilst co-infections were detected in 35 (13.7%) participants. The diagnostic agreement between real-time PCR and microscopy was excellent when both KAP and BM tested positive for S. stercoralis, but was considerably lower when only one microscopic technique was positive. The sensitivity of KAP, BM and real-time PCR for detection of S. stercoralis as compared to a combination of all diagnostic techniques was 21.4%, 37.5% and 76.8%, respectively. The inter-laboratory agreement of S. stercoralis-specific PCR was substantial (κ=0.63, preal-time PCR and stool microscopy shows high accuracy for S. stercoralis diagnosis. Besides high sensitivity, PCR may also enhance specificity by reducing microscopic misdiagnosis of morphologically similar helminth larvae (i.e. hookworm and S. stercoralis) in settings where both helminth species co-exist.

  20. Prevalence and characteristics of verotoxin-producing Escherichia coli (VTEC) in stool samples from asymptomatic human carriers working in the meat processing industry in Switzerland.

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    Stephan, R; Ragettli, S; Untermann, F

    2000-02-01

    A total of 5590 stool samples from healthy employees in the meat industry were screened by PCR for verotoxin-producing Escherichia coli (VTEC). The PCR product of VT-encoding genes was detected in 3. 5% of the samples. Phenotypic and genotypic traits of 47 VTEC strains isolated from asymptomatic carriers were characterized. A variety of serotypes was found; one strain belonged to the serotype O157:H7. The majority of the isolates proved to be VT2-positive. Fifty-seven percent of the verotoxin-producing strains harboured the genes for one or several additional virulence associated factors, including intimin (eae, 8.5%), the 60 MDa plasmid (42.5%), enterohaemolysin (EHEC-hlyA, 38.3%), the heat-stable enterotoxin (astA, 6.4%), a serin protease (espP, 6.4%), colicin production (col D157, 12.8%) and a secretion system II (etpD, 10.6%). None of the strains was positive for a specific enzyme with catalase-peroxidase activity (katP).

  1. Dust mites in a routine clinical stool sample

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

    2014-01-01

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

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

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

    1992-02-01

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

  3. Adenoviruses of canine and human origins in stool samples from free-living pampas foxes (Lycalopex gymnocercus) and crab-eating foxes (Cerdocyon thous) in São Francisco de Paula, Rio dos Sinos basin.

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    Monteiro, G S; Fleck, J D; Kluge, M; Rech, N K; Soliman, M C; Staggemeier, R; Rodrigues, M T; Barros, M P; Heinzelmann, L S; Spilki, F R

    2015-05-01

    The spread of enteric viruses of domestic animals and human beings to wild species can be facilitated by the resistance of these viruses on the environment and their ability to be transmitted by water and contaminated food. The health status of the populations of pampas foxes Lycalopex gymnocercus) and crab-eating foxes (Cerdocyon thous) is largely unknown and the landscapes occupied by these animals in southern Brazil have been threatened by human occupation and expansion of agriculture. In this work, the search of genomes of human and canine adenoviruses in feces from these wild carnivores was used to track the dissemination of domestic animals and human pathogens to the free-living populations in a wildlife reserve located in southern Brazil. This was performed by virus-specific differential real-time polymerase chain reactions (qPCR) on stool specimens, avoiding capture and additional stress to the animals. Genus-specific conventional reverse-transcriptase PCR (RT-PCR) was complementarily performed aiming the detection of enteroviruses (EV) and rotaviruses (RV) on these same samples. HAdV genomes were found on 14 out of the 17 (82.35%) stool samples analysed, whereas CAV was found co-infecting 5 of these samples. RV genomes were detected on 7 of the 17 samples (41.18%) and all samples were negative for EV. The results point to the dispersion of HAdV and RV at a high rate to these species of South American wild carnivores, which can be an effect of growing anthropisation of the habitat of these animals.

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

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    H.A YOUSEFI

    2000-03-01

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

  5. Assessment of the prevalence and diversity of emergent campylobacteria in human stool samples using a combination of traditional and molecular methods.

    Science.gov (United States)

    Collado, Luis; Gutiérrez, Magali; González, Mario; Fernández, Heriberto

    2013-04-01

    This study aims to assess the diversity of campylobacteria (Campylobacter and Arcobacter) in human fecal samples from patients with diarrhea (n = 140) and asymptomatic controls (n = 116) in Chile, using a combination of traditional culture and molecular methods. The culture methods detected campylobacteria in 10.7% of the patients with diarrhea and in 1.7% of the controls. In contrast, the molecular methods detected campylobacteria more often than the traditional culture, with a prevalence of 25.7% and 5.2%, respectively. The traditional methods only recovered the species Campylobacter jejuni, Campylobacter coli, and Arcobacter butzleri, whereas the molecular methods additionally detected the emergent species Campylobacter concisus and Campylobacter ureolyticus.

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2014-11-01

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

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

    Directory of Open Access Journals (Sweden)

    Stefanie J Krauth

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

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

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    Salih Kuk

    2012-12-01

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

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

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    Beatriz Coronato

    2012-04-01

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

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

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    Martin Alberer

    2017-01-01

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

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

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    Anuradha Mokkapati

    2015-02-01

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

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

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    Savić Dejana

    2016-01-01

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

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

    Science.gov (United States)

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

    2014-12-01

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

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2002-01-01

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

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

    OpenAIRE

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

    2002-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2013-05-01

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

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

    Science.gov (United States)

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

    2017-02-01

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

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

    Science.gov (United States)

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

    2015-02-01

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

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Hassan Brim

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

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

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    M. N. Brahmbhatt

    2013-07-01

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

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

    Science.gov (United States)

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

    2014-09-01

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

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

    Institute of Scientific and Technical Information of China (English)

    Hassan Momtaz; Negar Souod; Hossein Dabiri; Meysam Sarshar

    2012-01-01

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

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

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    Predrag Stojanovic

    2012-03-01

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

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

    Science.gov (United States)

    Li, Brandon

    2016-09-01

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

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

    Science.gov (United States)

    Li, Brandon

    2016-01-01

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

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

    OpenAIRE

    2016-01-01

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

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

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    Yolanda TEIXEIRA

    2015-12-01

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

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

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

    2013-06-01

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

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

    NARCIS (Netherlands)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2017-05-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

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    Pedro Fernández-Soto

    2014-09-01

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

  18. Stool C difficile toxin

    Science.gov (United States)

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

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

    Science.gov (United States)

    Savin, Cyril; Leclercq, Alexandre; Carniel, Elisabeth

    2012-01-01

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

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

    Science.gov (United States)

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

    2013-12-01

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

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

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    Aline Fernandes Barry

    2009-11-01

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

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

    Science.gov (United States)

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

    2013-02-01

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

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

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    Satheeshkumar S

    2004-01-01

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

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

    Science.gov (United States)

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

    2016-06-01

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

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

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    Mehmet Burak Selek

    2013-12-01

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

  6. Detection and differentiation of Cryptosporidium by real-time polymerase chain reaction in stool samples from patients in Rio de Janeiro, Brazil

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    Roberta Flávia Ribeiro Rolando

    2012-06-01

    Full Text Available This study reports the first genetic characterisation of Cryptosporidium isolates in Brazil using real-time polymerase chain reaction (RT-PCR. A total of 1,197 faecal specimens from children and 10 specimens from human immunodeficiency virus-infected patients were collected between 1999-2010 and screened using microscopy. Forty-eight Cryptosporidium oocyst-positive isolates were identified and analysed using a generic TaqMan assay targeting the 18S rRNA to detect Cryptosporidium species and two other TaqMan assays to identify Cryptosporidium hominis and Cryptosporidium parvum. The 18S rRNA assay detected Cryptosporidium species in all 48 of the stool specimens. The C. parvum TaqMan assay correctly identified five/48 stool samples, while 37/48 stool specimens were correctly amplified in the C. hominis TaqMan assay. The results obtained in this study support previous findings showing that C. hominis infections are more prevalent than C. parvum infections in Brazil and they demonstrate that the TaqMan RT-PCR procedure is a simple, fast and valuable tool for the detection and differentiation of Cryptosporidium species.

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

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    Neelam Taneja

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

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

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    Parija Subhash C

    2007-05-01

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

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

    Science.gov (United States)

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

    2014-04-01

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

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

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    Sue Elizabeth Shajan, Mohammed Faisal Hashim, Michael A

    2014-04-01

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

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

    Science.gov (United States)

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

    2015-06-01

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

  12. Stool Tests

    Science.gov (United States)

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

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

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    Tawin Inpankaew

    2014-12-01

    Full Text Available Microscopy-based identification of eggs in stool offers simple, reliable and economical options for assessing the prevalence and intensity of hookworm infections, and for monitoring the success of helminth control programs. This study was conducted to evaluate and compare the diagnostic parameters of the Kato-Katz (KK and simple sodium nitrate flotation technique (SNF in terms of detection and quantification of hookworm eggs, with PCR as an additional reference test in stool, collected as part of a baseline cross-sectional study in Cambodia.Fecal samples collected from 205 people in Dong village, Rovieng district, Preah Vihear province, Cambodia were subjected to KK, SNF and PCR for the detection (and in case of microscopy-based methods, quantification of hookworm eggs in stool. The prevalence of hookworm detected using a combination of three techniques (gold standard was 61.0%. PCR displayed a highest sensitivity for hookworm detection (92.0% followed by SNF (44.0% and quadruple KK smears (36.0% compared to the gold standard. The overall eggs per gram feces from SNF tended to be higher than for quadruple KK and the SNF proved superior for detecting low egg burdens.As a reference, PCR demonstrated the higher sensitivity compared to SNF and the quadruple KK method for detection of hookworm in human stool. For microscopic-based quantification, a single SNF proved superior to the quadruple KK for the detection of hookworm eggs in stool, in particular for low egg burdens. In addition, the SNF is cost-effective and easily accessible in resource poor countries.

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

    Science.gov (United States)

    Inpankaew, Tawin; Schär, Fabian; Khieu, Virak; Muth, Sinuon; Dalsgaard, Anders; Marti, Hanspeter; Traub, Rebecca J; Odermatt, Peter

    2014-12-01

    Microscopy-based identification of eggs in stool offers simple, reliable and economical options for assessing the prevalence and intensity of hookworm infections, and for monitoring the success of helminth control programs. This study was conducted to evaluate and compare the diagnostic parameters of the Kato-Katz (KK) and simple sodium nitrate flotation technique (SNF) in terms of detection and quantification of hookworm eggs, with PCR as an additional reference test in stool, collected as part of a baseline cross-sectional study in Cambodia. Fecal samples collected from 205 people in Dong village, Rovieng district, Preah Vihear province, Cambodia were subjected to KK, SNF and PCR for the detection (and in case of microscopy-based methods, quantification) of hookworm eggs in stool. The prevalence of hookworm detected using a combination of three techniques (gold standard) was 61.0%. PCR displayed a highest sensitivity for hookworm detection (92.0%) followed by SNF (44.0%) and quadruple KK smears (36.0%) compared to the gold standard. The overall eggs per gram feces from SNF tended to be higher than for quadruple KK and the SNF proved superior for detecting low egg burdens. As a reference, PCR demonstrated the higher sensitivity compared to SNF and the quadruple KK method for detection of hookworm in human stool. For microscopic-based quantification, a single SNF proved superior to the quadruple KK for the detection of hookworm eggs in stool, in particular for low egg burdens. In addition, the SNF is cost-effective and easily accessible in resource poor countries.

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

    Science.gov (United States)

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

    2013-01-01

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

  16. Comparison of sensitivity and faecal egg counts of Mini-FLOTAC using fixed stool samples and Kato-Katz technique for the diagnosis of Schistosoma mansoni and soil-transmitted helminths.

    Science.gov (United States)

    Coulibaly, Jean T; Ouattara, Mamadou; Becker, Sören L; Lo, Nathan C; Keiser, Jennifer; N'Goran, Eliézer K; Ianniello, Davide; Rinaldi, Laura; Cringoli, Giuseppe; Utzinger, Jürg

    2016-12-01

    Accurate diagnostic tools for human helminthiasis are crucial for epidemiological surveys, improved patient management, and evaluation of community-based intervention studies. However, the diagnosis of intestinal schistosomiasis and soil-transmitted helminthiasis heavily relies on stool microscopy using the Kato-Katz technique, which has a low sensitivity. The Mini-FLOTAC method is an alternative microscopy-based technique, but its diagnostic performance using sodium acetate-acetic acid-formalin-(SAF)-fixed stool specimens has not been validated. The fixation of stool samples for later examination in a laboratory may reduce logistical and financial barriers of prevalence surveys by not requiring field laboratories. We compared the diagnostic accuracy of the Kato-Katz technique using fresh stool samples with the Mini-FLOTAC technique, employing matched stool samples that were fixed in SAF. Three consecutive stool samples from 149 school-aged children in Côte d'Ivoire were subjected to quintuplicate Kato-Katz thick smears examined on the same day. From the remaining stool, approximately 2g was fixed in 10ml of SAF for about 3 months, and then subjected to the Mini-FLOTAC method, using two flotation solutions (FS2 and FS7). The combined results of multiple Kato-Katz and Mini-FLOTAC readings revealed prevalences of Schistosoma mansoni, Trichuris trichiura and hookworm of 99.3%, 72.5% and 7.4%, respectively. Employing a Bayesian latent class analysis to estimate the true sensitivity of the diagnostic approaches, the sensitivity of Mini-FLOTAC using FS2 was 50.1% (95% Bayesian credible interval (BCI): 30.9-70.2%) for hookworm and 68.0% (95% BCI: 34.9-93.5%) for T. trichiura. When applying Mini-FLOTAC using FS7, the sensitivity was 89.9% (95% BCI: 86.9-97.4%) for S. mansoni, 37.2% (95% BCI: 17.2-60.6%) for hookworm and 67.7% (95% BCI: 33.0-93.0%) for T. trichiura. The specificity ranged from 80.1-95.0% in all Mini-FLOTAC tests. Mini-FLOTAC revealed higher arithmetic mean

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

    KAUST Repository

    Roperch, Jean-Pierre

    2015-05-29

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

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

    Science.gov (United States)

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

    2015-02-01

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

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

    NARCIS (Netherlands)

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

    2007-01-01

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

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

    NARCIS (Netherlands)

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

    2007-01-01

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

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

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    Stefanie Knopp

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

  2. Systematic application of multiplex PCR enhances the detection of bacteria, parasites, and viruses in stool samples.

    Science.gov (United States)

    McAuliffe, Gary N; Anderson, Trevor P; Stevens, Mary; Adams, Jacqui; Coleman, Robyn; Mahagamasekera, Patalee; Young, Sheryl; Henderson, Tom; Hofmann, Maria; Jennings, Lance C; Murdoch, David R

    2013-08-01

    To determine whether systematic testing of faecal samples with a broad range multiplex PCR increases the diagnostic yield in patients with diarrhoea compared with conventional methods and a clinician initiated testing strategy. 1758 faecal samples from 1516 patients with diarrhoea submitted to two diagnostic laboratories were tested for viral, bacterial, and parasitic pathogens by Fast-Track Diagnostics multiplex real-time PCR kits and conventional diagnostic tests. Multiplex PCR detected pathogens in 530 samples (30%): adenovirus (51, 3%), astrovirus (95, 5%), norovirus (172, 10%), rotavirus (3, 0.2%), Campylobacter jejuni/coli (85, 5%), Salmonella spp. (22, 1%), Clostridium difficile (72, 4%), entero-haemorrhagic Escherichia coli (21, 1%), Cryptosporidium spp. (3, 0.2%), Entamoeba histolytica (1, 0.1%), and Giardia lamblia (59, 3%). In contrast, conventional testing detected a pathogen in 324 (18%) samples. Using a systematic approach to the diagnosis of gastroenteritis improved diagnostic yield. This enhanced detection with PCR was achieved by a combination of improved detection of individual pathogens and detection of pathogens not requested or unable to be tested by conventional tests. This approach also allowed earlier identification for most pathogens and created a workflow which is likely to adapt well for many diagnostic laboratories. Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

  3. 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

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    Wakita Takaji

    2009-12-01

    Full Text Available Abstract Background In the global eradication program for poliomyelitis, the laboratory diagnosis plays a critical role by isolating poliovirus (PV from the stool samples of acute flaccid paralysis (AFP cases. In this study, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP system for a rapid and highly sensitive detection of enterovirus including PV to identify stool samples positive for enterovirus including PV. Methods A primer set was designed for RT-LAMP to detect enterovirus preferably those with PV-like 5'NTRs of the viral genome. The sensitivity of RT-LAMP system was evaluated with prototype strains of enterovirus. Detection of enterovirus from stool extracts was examined by using RT-LAMP system. Results We detected at least 400 copies of the viral genomes of PV(Sabin strains within 90 min by RT-LAMP with the primer set. This RT-LAMP system showed a preference for Human enterovirus species C (HEV-C strains including PV, but exhibited less sensitivity to the prototype strains of HEV-A and HEV-B (detection limits of 7,400 to 28,000 copies. Stool extracts, from which PV, HEV-C, or HEV-A was isolated in the cell culture system, were mostly positive by RT-LAMP method (positive rates of 15/16 (= 94%, 13/14 (= 93%, and 4/4 (= 100%, respectively. The positive rate of this RT-LAMP system for stool extracts from which HEV-B was isolated was lower than that of HEV-C (positive rate of 11/21 (= 52%. In the stool samples, which were negative for enterovirus isolation by the cell culture system, we found that two samples were positive for RT-LAMP (positive rates of 2/38 (= 5.3%. In these samples, enterovirus 96 was identified by sequence analysis utilizing a seminested PCR system. Conclusions RT-LAMP system developed in this study showed a high sensitivity comparable to that of the cell culture system for the detection of PV, HEV-A, and HEV-C, but less sensitivity to HEV-B. This RT-LAMP system would be useful for the

  4. Efficiency of Direct Microscopy of Stool Samples Using an Antigen-Specific Adhesin Test for Entamoeba Histolytica

    Science.gov (United States)

    İrvem, Arzu; Özdil, Kamil; Çalışkan, Zuhal; Yücel, Muhterem

    2016-01-01

    Background: E. histolytica is among the common causes of acute gastroenteritis. The pathogenic species E. histolytica and the nonpathogenic species E. dispar cannot be morphologically differentiated, although correct identification of these protozoans is important for treatment and public health. In many laboratories, the screening of leukocytes, erythrocytes, amoebic cysts, trophozoites and parasite eggs is performed using Native-Lugol’s iodine for pre-diagnosis. Aims: In this study, we aimed to investigate the frequency of E. histolytica in stool samples collected from 788 patients residing in the Anatolian region of İstanbul who presented with gastrointestinal complaints. We used the information obtained to evaluate the effectiveness of microscopic examinations when used in combination with the E. histolytica adhesin antigen test. Study Design: Retrospective cross-sectional study Methods: Preparations of stool samples stained with Native-Lugol’s iodine were evaluated using the E. histolytica adhesin test and examined using standard light microscopy at ×40 magnification. Pearson’s Chi-square and Fisher’s exact tests were used for statistical analysis. Logistic regression analysis was used for multivariate analysis. Results: Of 788 samples, 38 (4.8%) were positive for E. histolytica adhesin antigens. When evaluated together with the presences of erythrocytes, leukocytes, cysts, and trophozoites, respectively, using logistic regression analysis, leukocyte positivity was significantly higher. The odds ratio of leukocyte positivity increased adhesin test-positivity by 2,530-fold (95% CI=1.01–6.330). Adhesin test-positivity was significant (p=0.047). Conclusion: In line with these findings, the consistency between the presence of cysts and erythrocytes and adhesin test-positivity was found to be highly significant, but that of higher levels of leukocytes was found to be discordant. It was concluded that leukocytes and trophozoites were easily misjudged

  5. Automatic Recognition of Human Parasite Cysts on Microscopic Stools Images using Principal Component Analysis and Probabilistic Neural Network

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    Beaudelaire Saha Tchinda

    2015-09-01

    Full Text Available Parasites live in a host and get its food from or at the expensive of that host. Cysts represent a form of resistance and spread of parasites. The manual diagnosis of microscopic stools images is time-consuming and depends on the human expert. In this paper, we propose an automatic recognition system that can be used to identify various intestinal parasite cysts from their microscopic digital images. We employ image pixel feature to train the probabilistic neural networks (PNN. Probabilistic neural networks are suitable for classification problems. The main novelty is the use of features vectors extracted directly from the image pixel. For this goal, microscopic images are previously segmented to separate the parasite image from the background. The extracted parasite is then resized to 12x12 image features vector. For dimensionality reduction, the principal component analysis basis projection has been used. 12x12 extracted features were orthogonalized into two principal components variables that consist the input vector of the PNN. The PNN is trained using 540 microscopic images of the parasite. The proposed approach was tested successfully on 540 samples of protozoan cysts obtained from 9 kinds of intestinal parasites.

  6. Comparison of three concentration methods for the recovery of canine intestinal parasites from stool samples.

    Science.gov (United States)

    Katagiri, S; Oliveira-Sequeira, T C G

    2010-10-01

    The aim of present study was to compare the efficiency of a commercial assay and two conventional methods for fecal concentration in detecting canine gastrointestinal parasites. Fecal samples from 254 dogs were processed by centrifugation-sedimentation (CS), centrifugation-flotation (CF) and a commercial assay for fecal concentration (TF-test). The following parasites were detected: Ancylostoma (37.8%), Giardia (16.9%), Toxocara canis (8.7%), Trichuris vulpis (7.1%), Isospora (3.5%), and Sarcocystis (2.7%). The calculated analytical sensitivity indicated that CF was more accurate (Pindividual diagnosis and epidemiological investigation.

  7. Cyclospora cayetanensis in sputum and stool samples Cyclospora cayetanensis em amostra de escarro

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    Angela Beatriz DI GLIULLO

    2000-04-01

    Full Text Available We report the observation of acid-fast Cyclospora cayetanensis oocysts in a sputum sample. The patient, a 60 year-old, HIV negative man, was successfully treated for pulmonary tuberculosis during 1997. On February 1998, he was admitted to our center due to loss of weight, cough with purulent expectoration, dysphonia and a radiological picture of pulmonary fibrosis. Bacilloscopic study of sputum (negative for acid-fast bacilli stained with Ziehl-Neelsen technique showed large (8-10 µm spherical, acid-fast Cyclospora cayetanensis oocysts. No other pathogens were isolated on cultures from this sample or from laryngeal biopsy. Serial parasitologic studies showed C. cayetanensis and also eggs of Trichuris trichiura, Ascaris lumbricoides and Hymenolepis nana and of Entamoeba coli cysts. The patient lives in the outskirts of Buenos Aires in a brick-made house with potable water and works as builder of sewers. He travelled in several occasions to the rural area of province of Tucumán which has poor sanitary conditions. C. cayetanensis is an emergent agent of diarrhea and as far as we know this is the first time the parasite is observed in respiratory samples.Comunicamos a observação de grandes oocistos (8-10 µm de diâmetro esféricos, ácido-álcool-resistentes de Cyclospora cayetanensis em amostra de escarro corada com a técnica de Ziehl-Neelsen. Na amostra não foram observados nem cultivados outros agentes patogênicos. Trata-se de um paciente do sexo masculino, 60 anos de idade, HIV (-, tratado previamente para tuberculose pulmonar (1997. Em fevereiro de 1998 apresentou-se em nosso hospital com perda de peso, tosse com expectoração purulenta, disfonia e imagens radiológicas de fibrose pulmonar. As culturas das amostras de escarro e da biopsia de laringe foram negativas. O exame parasitológico seriado de fezes mostrou ovos de Ascaris lumbricoides, Hymenolepis nana e Trichuris trichiura e cistos de Entamoeba coli. O paciente mora nos

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

    Science.gov (United States)

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

    2015-12-01

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

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

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    Maria Cristina Carvalho do Espírito-Santo

    2012-10-01

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

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

    NARCIS (Netherlands)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

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

    NARCIS (Netherlands)

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

    2013-01-01

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

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

    Science.gov (United States)

    2013-01-01

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

  14. Repeated stool sampling and use of multiple techniques enhance the sensitivity of helminth diagnosis: a cross-sectional survey in southern Lao People's Democratic Republic.

    Science.gov (United States)

    Sayasone, Somphou; Utzinger, Jürg; Akkhavong, Kongsap; Odermatt, Peter

    2015-01-01

    Intestinal parasitic infections are common in Lao People's Democratic Republic (Lao PDR). We investigated the accuracy of the Kato-Katz (KK) technique in relation to varying stool sampling efforts, and determined the effect of the concurrent use of a quantitative formalin-ethyl acetate concentration technique (FECT) for helminth diagnosis and appraisal of concomitant infections. The study was carried out between March and May 2006 in Champasack province, southern Lao PDR. Overall, 485 individuals aged ≥6 months who provided three stool samples were included in the final analysis. All stool samples were subjected to the KK technique. Additionally, one stool sample per individual was processed by FECT. Diagnosis was done under a light microscope by experienced laboratory technicians. Analysis of three stool samples with KK plus a single FECT was considered as diagnostic 'gold' standard and resulted in prevalence estimates of hookworm, Opisthorchis viverrini, Ascaris lumbricoides, Trichuris trichiura and Schistosoma mekongi infection of 77.9%, 65.0%, 33.4%, 26.2% and 24.3%, respectively. As expected, a single KK and a single FECT missed a considerable number of infections. While our diagnostic 'gold' standard produced similar results than those obtained by a mathematical model for most helminth infections, the 'true' prevalence predicted by the model for S. mekongi (28.1%) was somewhat higher than after multiple KK plus a single FECT (24.3%). In the current setting, triplicate KK plus a single FECT diagnosed helminth infections with high sensitivity. Hence, such a diagnostic approach might be utilised for generating high-quality baseline data, assessing anthelminthic drug efficacy and rigorous monitoring of community interventions.

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

    Directory of Open Access Journals (Sweden)

    Hiroyuki Kubota

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

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

    Science.gov (United States)

    Futera, Zdeněk; Burda, Jaroslav V

    2014-07-15

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

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

    Science.gov (United States)

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

    2013-01-01

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

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

    NARCIS (Netherlands)

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

    2013-01-01

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

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

    NARCIS (Netherlands)

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

    2013-01-01

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

  20. [Evaluation of Two Methods (Nativ-Lugol Preparation and Enzyme-Linked Immunosorbent Assay) for Detection of Entamoeba histolytica in Stool Samples].

    Science.gov (United States)

    Alver, Oktay; Topaç, Tuncay; Töre, Okan

    2015-09-01

    This study aims to compare the performance of Native-Lugol examination and EIA Antigen Detection Test using stool samples obtained from patients diagnosed as clinical gastroenteritis and submitted to the Parasitology Laboratory in Uludağ University between January 2010 and February 2011. The stool samples taken from 116 patients and sent to the laboratory of parasitology from various clinics including outpatient services have been investigated using Native-Lugol examination and EIA Antigen Detection Kit (Wampole® E. histolytica II Techlab®, Inc., Blacksburg, Virginia) methods on all the samples. In one of 116 stool samples (%0,86), E. histolytica/E. dispar cysts and/or trophozoites were detected by using direct microscobic (nativ-lugol) method. E. histolytica specific antigen was detected in 34 (29.3%) out of the sample set, and the patients were given adequate treatment. The highest rate of E. histolytica specific antigen positivity were observed in 11-19 age group. On account of the fact that the sensitivity of direct microscopy is quite low, it is concluded that, from the viewpoint of preventing the amebiasis suspected patients from false diagnosis and hence from receiving inadequate treatment, the use of the ELISA method is more appropriate and advantageous, as it is cost effective and does not require highly qualified staff.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Luigi Vezzulli

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

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

    Science.gov (United States)

    Lass, Anna; Karanis, Panagiotis; Korzeniewski, Krzysztof

    2017-08-01

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

  5. Bloody or tarry stools

    Science.gov (United States)

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

  6. Isolation and Characterization of Escherichia coli Sequence Type 131 and Other Antimicrobial-Resistant Gram-Negative Bacilli from Clinical Stool Samples from Veterans.

    Science.gov (United States)

    Mohamed, Muhanad; Clabots, Connie; Porter, Stephen B; Thuras, Paul; Johnson, James R

    2016-08-01

    Emerging multidrug-resistant (MDR) Gram-negative bacilli (GNB), including Escherichia coli sequence type 131 (ST131) and its resistance-associated H30 subclone, constitute an ever-growing public health threat. Their reservoirs and transmission pathways are incompletely defined. To assess diarrheal stools as a potential reservoir for ST131-H30 and other MDR GNB, we cultured 100 clinical stool samples from a Veterans Affairs Medical Center clinical laboratory (October to December 2011) for fluoroquinolone- and extended-spectrum cephalosporin (ESC)-resistant E. coli and other GNB, plus total E. coli We then characterized selected resistant and susceptible E. coli isolates by clonal group, phylogenetic group, virulence genotype, and pulsotype and screened all isolates for antimicrobial resistance. Overall, 79 of 100 stool samples yielded GNB (52 E. coli; 48 other GNB). Fifteen samples yielded fluoroquinolone-resistant E. coli (10 were ST131, of which 9 were H30), 6 yielded ESC-resistant E. coli (2 were ST131, both non-H30), and 31 yielded susceptible E. coli (1 was ST131, non-H30), for 13 total ST131-positive samples. Fourteen non-E. coli GNB were ESC resistant, and three were fluoroquinolone resistant. Regardless of species, almost half (46%) of the fluoroquinolone-resistant and/or ESC-resistant non-E. coli GNB were resistant to at least three drug classes. Fecal ST131 isolates closely resembled reference clinical ST131 isolates according to virulence genotypes and pulsed-field gel electrophoresis (PFGE) profiles. Thus, a substantial minority (30%) of veterans with diarrhea who undergo stool testing excrete antibiotic-resistant GNB, including E. coli ST131. Consequently, diarrhea may pose transmission risks for more than just diarrheal pathogens and may help disseminate clinically relevant ST131 strains and other MDR GNB within hospitals and the community. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  7. Evaluation of factors affecting real-time PCR performance for diagnosis of Entamoeba histolytica and Entamoeba dispar in clinical stool samples.

    Science.gov (United States)

    Forsell, Joakim; Koskiniemi, Satu; Hedberg, Ida; Edebro, Helén; Evengård, Birgitta; Granlund, Margareta

    2015-09-01

    Although PCR offers the potential for sensitive detection of parasites there are several pitfalls for optimal performance, especially when DNA is extracted from a complex sample material such as stool. With the aid of a sensitive inhibitor control in a duplex real-time PCR (qPCR) for identification of Entamoeba histolytica and Entamoeba dispar we have evaluated factors that influenced the performance of the qPCR and have suggested a rationale to be used in the analysis of clinical samples. Pre-PCR processing was found to be of outmost importance for an optimal amplification since inhibitors caused false-negative results when higher amounts of sample were used. Stool sampling with a flocked swab (ESwab, Copan), yielding on average 173 mg, gave positive qPCR results in samples with cysts of E. dispar that were negative in serially diluted stool samples. The degree of inhibition found varied between samples and was not an on-off phenomenon. Even low-grade inhibition, shown as an increase of two cycles in the qPCR for the inhibitor control, could lead to false negativity in samples with low amounts of parasites. Lack of amplification in the qPCR due to inhibition could be overcome by dilution of the extracted DNA by 1/10-1/20. We also describe the use of guanidinium thiocyanate buffer for transport and storage of samples as well as a time-saving semi-automated DNA extraction method in an Arrow instrument (Nordiag) preceded by bead beating.

  8. Comparison of methods for detection of Blastocystis infection in routinely submitted stool samples, and also in IBS/IBD Patients in Ankara, Turkey.

    Directory of Open Access Journals (Sweden)

    Funda Dogruman-Al

    Full Text Available BACKGROUND: This study compared diagnostic methods for identifying Blastocystis in stool samples, and evaluated the frequency of detection of Blastocystis in patients with irritable bowel syndrome (IBS and inflammatory bowel disease (IBD. RESULTS AND DISCUSSION: From a set of 105 stool specimens submitted for routine parasitological analysis, 30 were identified as positive for Blastocystis by the culture method. From that group of 30 positives, Lugol's stain, trichrome staining, and an immunofluorescence assay identified 11, 15, and 26 samples as positive respectively. Using culture as a standard, the sensitivity of Lugol's stain was 36.7%, trichrome staining was 50%, and the IFA stain was 86.7%. The specificity of Lugol's stain was 91%, trichrome staining was 100%, and the IFA stain was 97.3%. In the group of 27 IBS and IBD patients, using all methods combined, we detected Blastocystis in 67% (18/27 of the patients. Blastocystis was detected in 33% (2/6 of IBD patients and 76% (16/21 of IBS patients. For comparison, trichrome staining alone, the method most frequently used in many countries, would have only identified Blastocystis infection in 29% (6/21 of the IBS patients. No parasitic co-infections were identified in the IBS/IBD patients. Most Blastocystis-positive IBS/IBD patients were over 36 with an average length of illness of 4.9 years. CONCLUSIONS: Most IBS patients in this study were infected with Blastocystis. IFA staining may be a useful alternative to stool culture, especially if stool specimens have been chemically preserved.

  9. Improvement in the detection of enteric protozoa from clinical stool samples using the automated urine sediment analyzer sediMAX(®) 2 compared to sediMAX(®) 1.

    Science.gov (United States)

    Intra, J; Sala, M R; Falbo, R; Cappellini, F; Brambilla, P

    2017-01-01

    Detection of intestinal parasites from fecal samples is routinely performed by direct wet mount examination. This method requires skilled personnel, and it is time consuming. The aim of this work is to demonstrate the usefulness of the newer automated urinary sediment analyser sediMAX 2 for a fast detection of intestinal protozoa in stool samples. A total of 700 consecutively preserved samples consisting of 70 positives and 630 negatives were analyzed. SediMAX 2 takes digital images of each sediment sample, and analysis was conducted using a dilution of stool specimens, allowing determination of typical morphology. Compared to manual microscopy, sediMAX 2 showed sensitivity and specificity of 100 % in the detection of intestinal parasites, as also recently demonstrated for sediMAX 1. However, all clinically important human protozoa were detected using only 15 images for each specimen, compared to 30 images required in sediMAX 1 analysis. Moreover, changing manually the focus, it is possible to carry out a discrimination between morphologically identical Entamoeba complex members, including the pathogenic E. histolytica and the non-pathogenic E. dispar, E. moshkovskii and E. Bangladeshi, from the non-pathogenic Entamoeba coli based on the number of nuclei present in the cells. This study presents sediMAX 2 as an automatic aid to traditional microscopy.

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

    DEFF Research Database (Denmark)

    Inpankaew, Tawin; Schär, Fabian; Khieu, Virak

    2014-01-01

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

  11. Microbiota-Gut-Brain Axis: Yeast Species Isolated from Stool Samples of Children with Suspected or Diagnosed Autism Spectrum Disorders and In Vitro Susceptibility Against Nystatin and Fluconazole.

    Science.gov (United States)

    Kantarcioglu, A Serda; Kiraz, Nuri; Aydin, Ahmet

    2016-02-01

    Autism spectrum disorder (ASD) is a general term for a group of complex neurodevelopmental disorders of brain development that limits a person's ability to function normally. Etiology has not been clearly defined up to date. However, gut microbiota and the bidirectional communication between the gastrointestinal tract and brain, the so-called microbiota-gut-brain axis, are hypothesized, which may be involved in the etiology of several mental disorders. Recent reports suggest that Candida, particularly Candida albicans, growth in intestines may cause lower absorption of carbohydrates and minerals and higher toxin levels which are thought to contribute autistic behaviors. The aim of this study was to identify the 3-year deposited yeasts isolated from stool samples of children with diagnosed or suspected ASD and to determine in vitro activity of nystatin and fluconazole against these isolates using Clinical Laboratory Standards Institute M27-A3 guidelines. A 17-year retrospective assessment was also done using our laboratory records. Among the species identified, intrinsically fluconazole-resistant Candida krusei (19.8 %) and Candida glabrata (14.8 %) with elevated MICs were remarkable. Overall, C. albicans (57.4 %) was the most commonly isolated species in 17 years. The species identification and/or antifungal susceptibility tests have to be performed using the strain isolated from stool sample, to select the appropriate antifungal agent, if antimycotic therapy is needed.

  12. Toxin Profile, Biofilm Formation, and Molecular Characterization of Emetic Toxin-Producing Bacillus cereus Group Isolates from Human Stools.

    Science.gov (United States)

    Oh, Su Kyung; Chang, Hyun-Joo; Choi, Sung-Wook; Ok, Gyeongsik; Lee, Nari

    2015-11-01

    Emetic toxin-producing Bacillus cereus group species are an important problem, because the staple food for Korean is grains such as rice. In this study, we determined the prevalence (24 of 129 isolates) of emetic B. cereus in 36,745 stool samples from sporadic food-poisoning cases in Korea between 2007 and 2008. The toxin gene profile, toxin production, and biofilm-forming ability of the emetic B. cereus isolates were investigated. Repetitive element sequence polymorphism polymerase chain reaction fingerprints (rep-PCR) were also used to assess the intraspecific biodiversity of these isolates. Emetic B. cereus was present in 0.07% of the sporadic food-poisoning cases. The 24 emetic isolates identified all carried the nheABC and entFM genes and produced NHE enterotoxin. However, they did not have hemolysin BL toxin or related genes. A relationship between biofilm formation and toxin production was not observed in this study. The rep-PCR fingerprints of the B. cereus isolates were not influenced by the presence of toxin genes, or biofilm-forming ability. The rep-PCR assay discriminated emetic B. cereus isolates from nonemetic isolates, even if this assay did not perfectly discriminate these isolates. Further study on emetic isolates possessing a high degree of diversity may be necessary to evaluate the performance of the subtyping assay to discriminate emetic and nonemetic B. cereus isolates and could provide a more accurate indication of the risk from B. cereus strains.

  13. Study of the Prevalence of Causative Bacterial&Protozoal Agents of in Stool Samples of 470 Gastroenteritis Patients Referring to the Nikoopour Clinic in Yazd,Iran

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    MR Sharifi

    2004-04-01

    Full Text Available Interoduction: Gasteroenteritis is one of the problems worth consideration all over the world. It is one of the important causes of mortality, especially in children < 5 years of age, in developing countries including Iran. The aim of this descriptive study was to determine the demographic conditions influencing the presence of causative bacteria and protozoa, followed by antibiograms of isolated bacteria from stool samples of patients with gasteroenteritis referring to Nikoopour Clinic in the city of Yazd, Iran from 1998 – 2001. Materials and method: A total of 470 samples were microbiologically examined by direct method, culture and then antibiogramed. In order to isolate the possible bacteria, differential and selected media were used. Also, wet – mount technique was applied for detection of protozoa. Results: Results revealed that 272 samples (57.9% were infected by pathogenic bacteria or protozoa. 138 (50.8% pathogenic specimens were from male patients and the remaining 134(49.3% were from female patients. Isolated species were: Enteropathogenic E.coli 117(43%, Shigella 51(18.8%, Salmonella.interetidis 25(9.2%, C.jejuni 16(5.9%, Giardia lambdia 51(18.8% and Amoebae spp 12(4.4%. The most commonly detected shigella species was dysenteriae, (74.5% while boydii with 2% was the least common type observed in the specimens. Except shigella, all the other bacteria were more common in males than female, but insignificant statistically. In order to determine the sensitivity and/or resistance of pathogenic bacteria, antibiogram test was performed using selected antibiotic disks such as Ampicillin, Nalidixic Acid, Ciprofloxacin, Gentamycin and Sulfamethaxazole. Conclusion: Results revealed that some patients were probably infected by pathogenic factors other than bacteria or protozoa. Since all viruses and parasites are almost resistant to antibiotics and on the other hand, administration of antibiotics may lead to resistance of bacterial agents

  14. Differential Diagnosis of Entamoeba spp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain Reaction

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    Thiago dos Santos Gomes

    2014-01-01

    Full Text Available Amoebiasis, a disease caused by Entamoeba histolytica, is usually diagnosed by microscopic examination, which does not differentiate the morphologically identical species of the E. histolytica/E. dispar complex. Furthermore, morphologically similar species such as Entamoeba hartmanni contribute to misidentification. Therefore, there is a need for more sensitive and specific methods. This study standardized a multiplex real-time PCR system for E. histolytica and E. dispar and a single real-time PCR for E. hartmanni. The multiplex protocol detected up to 0.0143 pg of E. histolytica DNA and 0.5156 pg of E. dispar DNA, and the average melting temperature (Tm was 73°C and 70°C, respectively. For E. hartmanni, the Tm was 73°C and the amplification was successful down to 0.03 fg of plasmid DNA. Negative controls and other intestinal parasites presented no amplification. Among the 48 samples tested, E. dispar DNA was detected in 37; none exhibited E. histolytica DNA and 11 were negative in the multiplex protocol. In 4 of these 11 samples, however, E. hartmanni DNA was amplified. SYBR Green is demonstrated to be an interesting option and these combined PCR reactions can improve laboratory diagnosis of amoebiasis in developing countries.

  15. Differential diagnosis of Entamoeba spp. in clinical stool samples using SYBR green real-time polymerase chain reaction.

    Science.gov (United States)

    Gomes, Thiago Dos Santos; Garcia, Mariana Coimbra; de Souza Cunha, Flavia; Werneck de Macedo, Heloisa; Peralta, José Mauro; Peralta, Regina Helena Saramago

    2014-01-01

    Amoebiasis, a disease caused by Entamoeba histolytica, is usually diagnosed by microscopic examination, which does not differentiate the morphologically identical species of the E. histolytica/E. dispar complex. Furthermore, morphologically similar species such as Entamoeba hartmanni contribute to misidentification. Therefore, there is a need for more sensitive and specific methods. This study standardized a multiplex real-time PCR system for E. histolytica and E. dispar and a single real-time PCR for E. hartmanni. The multiplex protocol detected up to 0.0143 pg of E. histolytica DNA and 0.5156 pg of E. dispar DNA, and the average melting temperature (T(m)) was 73 °C and 70 °C, respectively. For E. hartmanni, the T(m) was 73 °C and the amplification was successful down to 0.03 fg of plasmid DNA. Negative controls and other intestinal parasites presented no amplification. Among the 48 samples tested, E. dispar DNA was detected in 37; none exhibited E. histolytica DNA and 11 were negative in the multiplex protocol. In 4 of these 11 samples, however, E. hartmanni DNA was amplified. SYBR Green is demonstrated to be an interesting option and these combined PCR reactions can improve laboratory diagnosis of amoebiasis in developing countries.

  16. Differential Diagnosis of Entamoeba spp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain Reaction

    Science.gov (United States)

    Gomes, Thiago dos Santos; Garcia, Mariana Coimbra; de Souza Cunha, Flavia; Peralta, José Mauro; Peralta, Regina Helena Saramago

    2014-01-01

    Amoebiasis, a disease caused by Entamoeba histolytica, is usually diagnosed by microscopic examination, which does not differentiate the morphologically identical species of the E. histolytica/E. dispar complex. Furthermore, morphologically similar species such as Entamoeba hartmanni contribute to misidentification. Therefore, there is a need for more sensitive and specific methods. This study standardized a multiplex real-time PCR system for E. histolytica and E. dispar and a single real-time PCR for E. hartmanni. The multiplex protocol detected up to 0.0143 pg of E. histolytica DNA and 0.5156 pg of E. dispar DNA, and the average melting temperature (Tm) was 73°C and 70°C, respectively. For E. hartmanni, the Tm was 73°C and the amplification was successful down to 0.03 fg of plasmid DNA. Negative controls and other intestinal parasites presented no amplification. Among the 48 samples tested, E. dispar DNA was detected in 37; none exhibited E. histolytica DNA and 11 were negative in the multiplex protocol. In 4 of these 11 samples, however, E. hartmanni DNA was amplified. SYBR Green is demonstrated to be an interesting option and these combined PCR reactions can improve laboratory diagnosis of amoebiasis in developing countries. PMID:24693242

  17. Microscopic diagnosis of sodium acetate-acetic acid-formalin-fixed stool samples for helminths and intestinal protozoa: a comparison among European reference laboratories.

    Science.gov (United States)

    Utzinger, J; Botero-Kleiven, S; Castelli, F; Chiodini, P L; Edwards, H; Köhler, N; Gulletta, M; Lebbad, M; Manser, M; Matthys, B; N'Goran, E K; Tannich, E; Vounatsou, P; Marti, H

    2010-03-01

    The present study aimed to compare the diagnostic performance of different European reference laboratories in diagnosing helminths and intestinal protozoa, using an ether-concentration method applied to sodium acetate-acetic acid-formalin (SAF)-preserved faecal samples. In total, 102 stool specimens were analysed during a cross-sectional parasitological survey in urban farming communities in Côte d'Ivoire. Five SAF-preserved faecal samples were prepared from each specimen and forwarded to the participating reference laboratories, processed and examined under a microscope adhering to a standard operating procedure (SOP). Schistosoma mansoni (cumulative prevalence: 51.0%) and hookworm (cumulative prevalence: 39.2%) were the predominant helminths. There was excellent agreement (kappa > 0.8; p protozoa were Entamoeba coli (median prevalence: 67.6%), Blastocystis hominis (median prevalence: 55.9%) and Entamoeba histolytica/Entamoeba dispar (median prevalence: 47.1%). Substantial agreement among reference laboratories was found for E. coli (kappa = 0.69), but only fair or moderate agreement was found for other Entamoeba species, Giardia intestinalis and Chilomastix mesnili. There was only poor agreement for B. hominis, Isospora belli and Trichomonas intestinalis. In conclusion, although common helminths were reliably diagnosed by European reference laboratories, there was only moderate agreement between centres for pathogenic intestinal protozoa. Continued external quality assessment and the establishment of a formal network of reference laboratories is necessary to further enhance both accuracy and uniformity in parasite diagnosis.

  18. Stool Color: When to Worry

    Science.gov (United States)

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

  19. Opisthorchis viverrini: Detection by polymerase chain reaction (PCR) in human stool samples

    Digital Repository Service at National Institute of Oceanography (India)

    Umesha, K.R.; SanathKumar; Parvathi, A.; Duenngai, K.; Sithithaworn, P.; Karunasagar, Indrani; Karunasagar, Iddya

    Journal of Tropical Medicine and Public Health 22 (Suppl.), 174–178. Duenngai, K., Sithithaworn, P., Rudrappa, U.K., Karunasagar, I., Laha, T., Stensvold, C.R., Strandgaard, H., Johansen, M.V., 2008. Improvement of PCR for detection of Opisthrochis... and lecithodendriid trematodes. Southeast Asian Journal of Tropical Medicine and Public Health 22, 623–630. Kobayashi, J., Vannachone, B., Sato, Y., Manivong, K., Nambanya, S., Inthakone, S., 2000. An epidemiological study on Opisthorchis viverrini infection in Lao...

  20. Simultaneous detection of Entamoeba histolytica/dispar, Giardia duodenalis and cryptosporidia by immunochromatographic assay in stool samples from patients living in the Greater Cairo Region, Egypt.

    Science.gov (United States)

    Banisch, Dagmar M; El-Badry, Ayman; Klinnert, Jorge V; Ignatius, Ralf; El-Dib, Nadia

    2015-08-01

    Gastrointestinal infection due to intestinal parasites is an enormous health problem in developing countries and its reliable diagnosis is demanding. Therefore, this study aimed at evaluating a commercially available immunochromatographic assay (ICA) for the detection of cryptosporidia, Giardia duodenalis, and Entamoeba histolytica/dispar for its usefulness in the Greater Cairo Region, Egypt. Stool samples of 104 patients who presented between October 2012 and March 2013 with gastrointestinal symptoms or for the exclusion of parasites at Kasr-Al-Ainy University Medical School were examined by light microscopy of wet mounts and the triple ICA. Microscopy revealed in 20% of the patients [95% confidence interval (CI), 13.5-29.0%] parasites with Hymenolepis nana, E. histolytica/dispar and Blastocystis hominis being the most frequent ones, but was not able to detect G. duodenalis and cryptosporidia, whereas ICA was positive in 21% (95% CI, 14.3-30.0%) and detected E. histolytica/dispar in 12.5% (95% CI, 7.3-20.4%), cryptosporidia in 6.7% (95% CI, 3.1-13.5%) and G. duodenalis in 15.4% (95% CI, 9.6-23.6%) of the patients. Detection of one or more pathogens was associated with access to water retrieved from a well or pump (p = 0.01). Patients between 20 and 29 years of age (p = 0.08) and patients with symptoms of 5 days or longer (p = 0.07) tended to have a higher risk to be infected than patients of other age groups or with shorter-lasting symptoms. In conclusion, the ICA was easy to perform and timesaving. Importantly, it enabled the detection of cryptosporidia, which cannot be found microscopically in unstained smears, demonstrated a higher sensitivity for the detection of G. duodenalis than microscopy, and was more specific for distinguishing E. histolytica/dispar from apathogenic amoeba.

  1. Recombinase Polymerase Amplification Compared to Real-Time Polymerase Chain Reaction Test for the Detection of Fasciola hepatica in Human Stool.

    Science.gov (United States)

    Cabada, Miguel M; Malaga, Jose L; Castellanos-Gonzalez, Alejandro; Bagwell, Kelli A; Naeger, Patrick A; Rogers, Hayley K; Maharsi, Safa; Mbaka, Maryann; White, A Clinton

    2017-02-08

    Fasciola hepatica is the most widely distributed trematode infection in the world. Control efforts may be hindered by the lack of diagnostic capacity especially in remote endemic areas. Polymerase chain reaction (PCR)-based methods offer high sensitivity and specificity but require expensive technology. However, the recombinase polymerase amplification (RPA) is an efficient isothermal method that eliminates the need for a thermal cycler and has a high deployment potential to resource-limited settings. We report on the characterization of RPA and PCR tests to detect Fasciola infection in clinical stool samples with low egg burdens. The sensitivity of the RPA and PCR were 87% and 66%, respectively. Both tests were 100% specific showing no cross-reactivity with trematode, cestode, or nematode parasites. In addition, RPA and PCR were able to detect 47% and 26% of infections not detected by microscopy, respectively. The RPA adapted to a lateral flow platform was more sensitive than gel-based detection of the reaction products. In conclusion, the Fasciola RPA is a highly sensitive and specific test to diagnose chronic infection using stool samples. The Fasciola RPA lateral flow has the potential for deployment to endemic areas after further characterization. © The American Society of Tropical Medicine and Hygiene.

  2. Occurrence of Listeria species in meat, chicken products and human stools in Assiut city, Egypt with PCR use for rapid identification of Listeria monocytogenes

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    Ashraf Mohamed Abd El-Malek

    Full Text Available The present research was conducted to check the presence of Listeria spp. in some meat and chicken products purchased from retail supermarkets in Assiut (Egypt. A total of 100 samples including 25 samples each of minced frozen beef, luncheon, frozen chicken legs and frozen chicken breast fillets were collected over a 7-month period between January and July 2009 and analyzed for the presence of Listeria spp. In addition, 28 stool cultures examined for Listeria spp. from hospitalized children resident in Assiut Pediatric University Hospital with diarrhea or fever. Out of the total 100 meat samples examined, Listeria spp. were detected in 8 (32% of minced frozen beef, 8 (32% of luncheon, 13 (52% of frozen chicken leg and 14 (56% of frozen chicken fillet samples analyzed, respectively. Regarding the examined 28 stool cultures from hospitalized children with underlying disease in Assiut Univ. hospital, 2 (7.14% were found positive for Listeria spp. For identification of L. monocytogenes using polymerase chain reaction (PCR, two primers were selected to detect 217-pb fragment ofthe prfA (transcriptional activator of the virulence factor gene for L. monocytogenes. 13 selected Listeria isolates displayed beta-haemolysis on sheep blood agar and positive CAMP test were further identified using PCR. PCR results showed that L. monocytogenes were confirmed in one of minced imported frozen meat examined, two of luncheon samples and two of frozen chicken legs with the total incidence of 5 isolates (5% from the total 100 examined food samples. This suggests the presence of a significant public health hazard linked to the consumption of these meat and chicken products sold in Assiut city contaminated with L. monocytogenes. The public health significance of these pathogens as well as recommended sanitary measures was discussed. [Veterinary World 2010; 3(8.000: 353-359

  3. Detection of enteropathogens associated with travelers’ diarrhea using a multiplex Luminex-based assay performed on stool samples smeared on Whatman FTA Elute cards

    Science.gov (United States)

    Lalani, Tahaniyat; Tisdale, Michele D; Maguire, Jason D; Wongsrichanalai, Chansuda; Riddle, Mark S; Tribble, David R

    2015-01-01

    We evaluated the limits of detection (LoD) for an 11-plex PCR-Luminex assay performed on Whatman FTA Elute cards smeared with stool containing pathogens associated with travelers’ diarrhea. LoDs ranged between 102-105 CFU, PFU or cysts/g for most pathogens except Cryptosporidium. Campylobacter and norovirus LoD increased with prolonged storage of cards. PMID:26072151

  4. Rapid isolation of gluten-digesting bacteria from human stool and saliva by using gliadin-containing plates

    Science.gov (United States)

    Sarantopoulos, Christos; Ongchangco, Deryn; Sry, Jeremy; Cesario, Thomas

    2014-01-01

    The number of individuals with gluten intolerance has increased dramatically over the last years. To date, the only therapy for gluten intolerance is the complete avoidance of dietary gluten. To sustain a strictly gluten-free diet, however, is very challenging. Therefore, there is need for a non-dietary therapy. Any such treatment must appreciate that the immunogenic part of gluten are gliadin peptides which are poorly degraded by the enzymes of the gastrointestinal tract. Probiotic therapy and oral enzyme therapy containing gluten-degrading bacteria (GDB) and their gliadin-digesting enzymes are possible new approaches for the treatment of gluten intolerance, however effectively isolating GDB for these treatments is problematic. The goal of this study was to develop an easy technique to isolate GDB rapidly and efficiently with the hope it might lead to newer ways of developing either probiotics or traditional medicines to treat gluten intolerance. Several researchers have already isolated successfully GDB by using gluten minimal or limited agar plates. Although these plates can be used to isolate bacteria which can tolerate gluten, further assays are needed to investigate if the same bacteria can also digest gluten. The agar plates we developed can detect bacteria which cannot only tolerate gluten but are able to digest it as well. Therefore, we were able to combine two steps into one step. Using such technologies, we were able to isolate five GDB from saliva and stool, and identified three bacterial reference strains with gluten-degrading activity. The technique we developed to isolate bacteria with gluten-degrading activity is fast, effective, and easy to use. The GDB isolated by our technology could have potential as part of a probiotic or enzymatic therapy for people with gluten intolerance. PMID:25519429

  5. Rapid isolation of gluten-digesting bacteria from human stool and saliva by using gliadin-containing plates.

    Science.gov (United States)

    Berger, Martina; Sarantopoulos, Christos; Ongchangco, Deryn; Sry, Jeremy; Cesario, Thomas

    2015-07-01

    The number of individuals with gluten intolerance has increased dramatically over the last years. To date, the only therapy for gluten intolerance is the complete avoidance of dietary gluten. To sustain a strictly gluten-free diet, however, is very challenging. Therefore, there is need for a non-dietary therapy. Any such treatment must appreciate that the immunogenic part of gluten are gliadin peptides which are poorly degraded by the enzymes of the gastrointestinal tract. Probiotic therapy and oral enzyme therapy containing gluten-degrading bacteria (GDB) and their gliadin-digesting enzymes are possible new approaches for the treatment of gluten intolerance, however effectively isolating GDB for these treatments is problematic. The goal of this study was to develop an easy technique to isolate GDB rapidly and efficiently with the hope it might lead to newer ways of developing either probiotics or traditional medicines to treat gluten intolerance. Several researchers have already isolated successfully GDB by using gluten minimal or limited agar plates. Although these plates can be used to isolate bacteria which can tolerate gluten, further assays are needed to investigate if the same bacteria can also digest gluten. The agar plates we developed can detect bacteria which cannot only tolerate gluten but are able to digest it as well. Therefore, we were able to combine two steps into one step. Using such technologies, we were able to isolate five GDB from saliva and stool, and identified three bacterial reference strains with gluten-degrading activity. The technique we developed to isolate bacteria with gluten-degrading activity is fast, effective, and easy to use. The GDB isolated by our technology could have potential as part of a probiotic or enzymatic therapy for people with gluten intolerance.

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

    Science.gov (United States)

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

    2015-01-01

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

  7. Comparison of the Triage Micro Parasite Panel and Microscopy for the Detection of Entamoeba histolytica/Entamoeba dispar, Giardia lamblia, and Cryptosporidium parvum in Stool Samples Collected in Kenya

    Directory of Open Access Journals (Sweden)

    Brett Swierczewski

    2012-01-01

    Full Text Available Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum are three of the most important parasitic causes of acute diarrhea worldwide. Laboratory diagnosis of these parasites is usually done by ova and parasite examination (O&P examination via microscopy. The sensitivity and specificity of O&P examination varies among laboratories and can be labor intensive and time consuming. The Triage Micro Parasite Panel (BioSite, San Diego, California is an enzyme immunoassay kit that can detect E. histolytica/E. dispar, G. lamblia, and C. parvum simultaneously using fresh or frozen stool. The present study evaluated the Triage Micro Parasite Panel in detecting E. histolytica/E. dispar, G. lamblia, and C. parvum compared to O&P examination in 266 stool samples collected at medical facilities in Kenya. The sensitivity and specificity results for the Triage Micro Parasite Panel were: for E. histolytica/E. dispar: 100%, 100%, G. lamblia: 100%, 100% and C. parvum: 73%, 100%. There was no evidence of cross reactivity using the kit with other parasites identified in the stool specimens. These results indicate that the Triage Micro Parasite Panel is a highly sensitive kit that can be used for screening purposes in large scale studies or outbreak investigations or as a possible alternative to O&P examination.

  8. Estimating the sensitivity and specificity of Kato-Katz stool examination technique for detection of hookworms, Ascaris lumbricoides and Trichuris trichiura infections in humans in the absence of a 'gold standard'.

    Science.gov (United States)

    Tarafder, M R; Carabin, H; Joseph, L; Balolong, E; Olveda, R; McGarvey, S T

    2010-03-15

    The accuracy of the Kato-Katz technique in identifying individuals with soil-transmitted helminth (STH) infections is limited by day-to-day variation in helminth egg excretion, confusion with other parasites and the laboratory technicians' experience. We aimed to estimate the sensitivity and specificity of the Kato-Katz technique to detect infection with Ascaris lumbricoides, hookworm and Trichuris trichiura using a Bayesian approach in the absence of a 'gold standard'. Data were obtained from a longitudinal study conducted between January 2004 and December 2005 in Samar Province, the Philippines. Each participant provided between one and three stool samples over consecutive days. Stool samples were examined using the Kato-Katz technique and reported as positive or negative for STHs. In the presence of measurement error, the true status of each individual is considered as latent data. Using a Bayesian method, we calculated marginal posterior densities of sensitivity and specificity parameters from the product of the likelihood function of observed and latent data. A uniform prior distribution was used (beta distribution: alpha=1, beta=1). A total of 5624 individuals provided at least one stool sample. One, two and three stool samples were provided by 1582, 1893 and 2149 individuals, respectively. All STHs showed variation in test results from day to day. Sensitivity estimates of the Kato-Katz technique for one stool sample were 96.9% (95% Bayesian Credible Interval [BCI]: 96.1%, 97.6%), 65.2% (60.0%, 69.8%) and 91.4% (90.5%, 92.3%), for A. lumbricoides, hookworm and T. trichiura, respectively. Specificity estimates for one stool sample were 96.1% (95.5%, 96.7%), 93.8% (92.4%, 95.4%) and 94.4% (93.2%, 95.5%), for A. lumbricoides, hookworm and T. trichiura, respectively. Our results show that the Kato-Katz technique can perform with reasonable accuracy with one day's stool collection for A. lumbricoides and T. trichiura. Low sensitivity of the Kato-Katz for detection

  9. Performance of TechLab C. DIFF QUIK CHEK and TechLab C. DIFFICILE TOX A/B II for the detection of Clostridium difficile in stool samples.

    Science.gov (United States)

    Reyes, Romina C; John, Michael A; Ayotte, Diane L; Covacich, Alexia; Milburn, Susan; Hussain, Zafar

    2007-09-01

    Two membrane-bound enzyme immunoassays by TechLab, Blacksburg, VA, were evaluated and compared with the Triage Micro C. difficile Panel (Biosite Diagnostics, San Diego, CA), with culture, and with cytotoxic assay. The TechLab panels were C. DIFF QUIK CHEK (QC-GDH) and C. DIFFICILE TOX A/B II (QC-toxinA/B), which detect glutamate dehydrogenase (GDH) and Clostridium difficile toxins A and B, respectively. The Triage Panel detects GDH (TR-GDH) and toxin A (TR-toxinA). Stool samples were inoculated onto CCFA plates (Q-Labs, Quebec, Canada) after alcohol shock, and suspected colonies were identified by the MicroScreen C. difficile latex slide agglutination test (Microgen Bioproducts, Surrey, UK). TR-GDH, TR-toxinA, QC-GDH, and QC-toxinA/B tests were performed according to the manufacturers' instructions on all the samples. Samples positive for GDH or culture but negative for TR-toxinA and QC-toxinA/B were further tested by cytotoxin assay (CTA). CTA was also performed on samples that caused blackening of the Triage Micro C. difficile Panel. A total of 313 of 401 stool samples were negative for GDH and toxins (78%). Eighty-eight samples were positive either for GDH or culture or both. Thirteen of these could not be evaluated for C. difficile-associated diarrhea (CDAD) because CTA test was not performed. Toxin/s was detected at least by one method in 46 (11.8%) of 388 samples that were positive for culture or GDH and were considered diagnostic of CDAD. The QC-GDH was more sensitive than culture and TR-GDH for the detection of C. difficile. However, in 18GDH-positive samples positive for either of the Triage or TechLab immunoassays, the culture remained negative. Ten (2%) results of the Triage immunoassays could not be evaluated because of discoloration of the panels. QC-GDH (93.5%) was more sensitive for detecting the presence of toxin-producing C. difficile than TR-GDH (79.5%). TR-toxinA was more specific for detecting the presence of toxin-producing C. difficile than QC

  10. Prevalence, Pathogenesis, Antibiotic Susceptibility Profiles, and In-vitro Activity of Selected Medicinal Plants Against Aeromonas Isolates from Stool Samples of Patients in the Venda Region of South Africa

    Science.gov (United States)

    Obi, C.L.; Ramalivhana, J.; Samie, A.; Igumbor, E.O.

    2007-01-01

    The prevalence, pathogenic indices, such as haemolytic and haemagglutinating activities, antibiograms, and in-vitro activities of local medicinal plants against Aeromonas isolates in Vhembe district of Limpopo province, South Africa, were studied using standard microbiological methods. In total, 309 diarrhoeic stool samples were collected from patients attending five health centres in the region during December 2004–May 2005. Aeromonas species were identified using the API 20E system. The haemagglutinating and haemolytic activities of isolates on human, sheep, pig and chicken red blood cells were investigated. Antibiotic susceptibility profiles of the isolates to several antibiotics and in-vitro activity of local medicinal plants were also ascertained using previously-reported schemes. Results showed that 104 (33.6%) of the 309 samples were positive for Aeromonas species, of which 89 (85.6%) were Aeromonas hydrophila, 12 (11.5%) A. sobria, and three (2.9%) A. caviae. All strains of A. hydrophila and A. caviae produced haemolysis on sheep blood, while eight of the 12 A. sobria strains were haemolytic on sheep blood. The haemolytic activities of the isolates were variable on other red blood cells tested. High level of resistance was observed to amoxicillin and ampicillin, followed by cefuroxime (79%), chloramphenicol (74%), and erythromycin (65%). The carbapenems were the most active drugs with only 7% resistance to meropenem and 11% to imipenem. About 12% of the isolates were resistant to ciprofloxacin. The extracts of three of seven medicinal plants tested showed inhibitory activity against all Aeromonas isolates; these included acetone and hexane extracts of Pterocarpus angolensis, Syzygium cordatum, and Zornia milneana. The results suggest a high prevalence of Aeromonas species in the region. The isolates demonstrated multiple resistant profiles to different antibiotics tested. Some local medicinal plants were inhibitory to Aeromonas isolates, indicating a

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

    DEFF Research Database (Denmark)

    Inpankaew, Tawin; Schär, Fabian; Khieu, Virak;

    2014-01-01

    BACKGROUND: Microscopy-based identification of eggs in stool offers simple, reliable and economical options for assessing the prevalence and intensity of hookworm infections, and for monitoring the success of helminth control programs. This study was conducted to evaluate and compare the diagnost...

  12. Development of a thermostabilized, one-step, nested, tetraplex PCR assay for simultaneous identification and differentiation of Entamoeba species, Entamoeba histolytica and Entamoeba dispar from stool samples.

    Science.gov (United States)

    Foo, Phiaw Chong; Chan, Yean Yean; See Too, Wei Cun; Tan, Zi Ning; Wong, Weng Kin; Lalitha, Pattabhiraman; Lim, Boon Huat

    2012-09-01

    Entamoeba histolytica is the only Entamoeba species that causes amoebiasis in humans. Approximately 50 million people are infected, with 100, 000 deaths annually in endemic countries. Molecular diagnosis of Entamoeba histolytica is important to differentiate it from the morphologically identical Entamoeba dispar to avoid unnecessary medication. Conventional molecular diagnostic tests require trained personnel, cold-chain transportation and/or are storage-dependent, which make them user-unfriendly. The aim of this study was to develop a thermostabilized, one-step, nested, tetraplex PCR assay for the detection of Entamoeba histolytica, Entamoeba dispar and Entamoeba species in cold-chain-free and ready-to-use form. The PCR test was designed based on the Entamoeba small subunit rRNA (SSU-rRNA) gene, which detects the presence of any Entamoeba species, and simultaneously can be used to differentiate Entamoeba histolytica from Entamoeba dispar. In addition, a pair of primers was designed to serve as an internal amplification control to help identify inhibitors in the samples. All PCR reagents together with the designed primers were thermostabilized by lyophilization and were stable at 24 °C for at least 6 months. The limit of detection of the tetraplex PCR was found to be 39 pg DNA or 1000 cells for Entamoeba histolytica and 78 pg DNA or 1000 cells for Entamoeba dispar, and the specificity was 100 %. In conclusion, this cold-chain-free, thermostabilized, one-step, nested, multiplex PCR assay was found to be efficacious in differentiating Entamoeba histolytica from other non-pathogenic Entamoeba species.

  13. Flushable reagent stool blood test

    Science.gov (United States)

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

  14. Optimized microbial DNA extraction from diarrheic stools

    Directory of Open Access Journals (Sweden)

    Donatin Emilie

    2012-12-01

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

  15. Community Laboratory Testing for Cryptosporidium: Multicenter Study Retesting Public Health Surveillance Stool Samples Positive for Cryptosporidium by Rapid Cartridge Assay with Direct Fluorescent Antibody Testing

    Science.gov (United States)

    Roellig, Dawn M.; Yoder, Jonathan S.; Madison-Antenucci, Susan; Robinson, Trisha J.; Van, Tam T.; Collier, Sarah A.; Boxrud, Dave; Monson, Timothy; Bates, Leigh Ann; Blackstock, Anna J.; Shea, Shari; Larson, Kirsten; Xiao, Lihua; Beach, Michael

    2017-01-01

    Cryptosporidium is a common cause of sporadic diarrheal disease and outbreaks in the United States. Increasingly, immunochromatography-based rapid cartridge assays (RCAs) are providing community laboratories with a quick cryptosporidiosis diagnostic method. In the current study, the Centers for Disease Control and Prevention (CDC), the Association of Public Health Laboratories (APHL), and four state health departments evaluated RCA-positive samples obtained during routine Cryptosporidium testing. All samples underwent “head to head” re-testing using both RCA and direct fluorescence assay (DFA). Community level results from three sites indicated that 54.4% (166/305) of Meridian ImmunoCard STAT! positives and 87.0% (67/77) of Remel Xpect positives were confirmed by DFA. When samples were retested by RCA at state laboratories and compared with DFA, 83.3% (155/186) of Meridian ImmunoCard STAT! positives and 95.2% (60/63) of Remel Xpect positives were confirmed. The percentage of confirmed community results varied by site: Minnesota, 39.0%; New York, 63.9%; and Wisconsin, 72.1%. The percentage of confirmed community results decreased with patient age; 12.5% of community positive tests could be confirmed by DFA for patients 60 years of age or older. The percentage of confirmed results did not differ significantly by sex, storage temperature, time between sample collection and testing, or season. Findings from this study demonstrate a lower confirmation rate of community RCA positives when compared to RCA positives identified at state laboratories. Elucidating the causes of decreased test performance in order to improve overall community laboratory performance of these tests is critical for understanding the epidemiology of cryptosporidiosis in the United States (US). PMID:28085927

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

    Science.gov (United States)

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

    2015-03-01

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

  17. Whole-genome sequencing of Campylobacter jejuni isolated from Danish routine human stool samples reveals surprising degree of clustering

    DEFF Research Database (Denmark)

    Joensen, K G; Kuhn, K G; Müller, L

    2017-01-01

    ; one which had not been identified through the existing surveillance system. CONCLUSIONS: Using WGS, we show that Campylobacter case clustering and even outbreaks appear to occur more frequently than previously assumed, providing important new insight into the relatively poorly understood epidemiology...... of the most important cause of bacterial gastroenteritis in the industrialized world....

  18. The prevalence of Giardia infection in dogs and cats, a systematic review and meta-analysis of prevalence studies from stool samples.

    Science.gov (United States)

    Bouzid, Maha; Halai, Kapil; Jeffreys, Danielle; Hunter, Paul R

    2015-01-30

    Giardia has a wide range of host species and is a common cause of diarrhoeal disease in humans and animals. Companion animals are able to transmit a range of zoonotic diseases to their owners including giardiasis, but the size of this risk is not well known. The aim of this study was to analyse giardiasis prevalence rates in dogs and cats worldwide using a systematic search approach. Meta-analysis enabled to describe associations between Giardia prevalence and various confounding factors. Pooled prevalence rates were 15.2% (95% CI 13.8-16.7%) for dogs and 12% (95% CI 9.2-15.3%) for cats. However, there was very high heterogeneity between studies. Meta-regression showed that the diagnostic method used had a major impact on reported prevalence with studies using ELISA, IFA and PCR reporting prevalence rates between 2.6 and 3.7 times greater than studies using microscopy. Conditional negative binomial regression found that symptomatic animals had higher prevalence rates ratios (PRR) than asymptomatic animals 1.61 (95% CI 1.33-1.94) in dogs and 1.94 (95% CI 1.47-2.56) in cats. Giardia was much more prevalent in young animals. For cats >6 months, PRR=0.47 (0.42-0.53) and in dogs of the same age group PRR=0.36 (0.32-0.41). Additionally, dogs kept as pets were less likely to be positive (PRR=0.56 (0.41-0.77)) but any difference in cats was not significant. Faecal excretion of Giardia is common in dogs and slightly less so in cats. However, the exact rates depend on the diagnostic method used, the age and origin of the animal. What risk such endemic colonisation poses to human health is still unclear as it will depend not only on prevalence rates but also on what assemblages are excreted and how people interact with their pets. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  19. Multiplex Real-Time PCR Assay Targeting Eight Parasites Customized to the Korean Population: Potential Use for Detection in Diarrheal Stool Samples from Gastroenteritis Patients

    Science.gov (United States)

    Won, Eun Jeong; Kim, Soo Hyun; Kee, Seung Jung; Shin, Jong Hee; Suh, Soon Pal; Chai, Jong Yil; Ryang, Dong Wook; Shin, Myung Geun

    2016-01-01

    Intestinal parasitic diseases occur worldwide and can cause diarrhea or gastroenteritis; however, their diagnosis is quite difficult, especially in low-endemism countries. We developed a multiplex real-time PCR assay for detection of eight intestinal parasites and prospectively evaluated it for patients with gastroenteritis. The assay targeted Cryptosporidium parvum, Giardia lamblia, Entamoeba histolytica, Blastocystis hominis, Dientamoeba fragilis, Clonorchis sinensis, Metagonimus yokogawai, and Gymnophalloides seoi. Performance characteristics were evaluated based on recovery after DNA extraction, analytical sensitivity, specificity, reproducibility, cross-reactivity, and interference characteristics. Clinical performance was validated against microscopy on 123 diarrheal samples. The assay demonstrated strong correlations between DNA concentrations and Ct values (R2, 0.9924–0.9998), and had a high PCR efficiency (83.3%–109.5%). Polymerase chain reactions detected as few as 10–30 copies of genomic DNA, and coefficient of variance was 0–7%. There was no cross-reactivity to the other 54 microorganisms tested. Interference occurred only in presence of high concentrations of erythrocytes or leukocytes. This assay had a higher correct identification rate (100.0% vs. 90.2%) and lower incorrect ID rate (0.0% vs. 9.8%) when compared to microscopy. Overall, this assay showed a higher sensitivity (100.0%; 95% confidence interval [CI] of 80.5–100.0) than microscopy (29.4%; 95% CI 10.31–55.96), and the specificity levels were comparable for both methods (100.0%; 95% CI 96.58–100.0). This newly developed multiplex real-time PCR assay offers a potential use for detecting intestinal parasitic pathogens customized to the Korean population. PMID:27861635

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

    Directory of Open Access Journals (Sweden)

    Ganem Donald

    2009-06-01

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

  1. A tool kit for quantifying eukaryotic rRNA gene sequences from human microbiome samples.

    Science.gov (United States)

    Dollive, Serena; Peterfreund, Gregory L; Sherrill-Mix, Scott; Bittinger, Kyle; Sinha, Rohini; Hoffmann, Christian; Nabel, Christopher S; Hill, David A; Artis, David; Bachman, Michael A; Custers-Allen, Rebecca; Grunberg, Stephanie; Wu, Gary D; Lewis, James D; Bushman, Frederic D

    2012-07-03

    Eukaryotic microorganisms are important but understudied components of the human microbiome. Here we present a pipeline for analysis of deep sequencing data on single cell eukaryotes. We designed a new 18S rRNA gene-specific PCR primer set and compared a published rRNA gene internal transcribed spacer (ITS) gene primer set. Amplicons were tested against 24 specimens from defined eukaryotes and eight well-characterized human stool samples. A software pipeline https://sourceforge.net/projects/brocc/ was developed for taxonomic attribution, validated against simulated data, and tested on pyrosequence data. This study provides a well-characterized tool kit for sequence-based enumeration of eukaryotic organisms in human microbiome samples.

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

    Directory of Open Access Journals (Sweden)

    Claudia Duran

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

  3. One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq® in stool samples

    Directory of Open Access Journals (Sweden)

    Rashi Gautam

    2016-01-01

    Full Text Available Background. Group A rotavirus (RVA infection is the major cause of acute gastroenteritis (AGE in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq® rotavirus strains along with an internal processing control (Xeno or MS2 RNA. Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12 and VP4 (P[4], P[6] and P[8] genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT and amplification (PCR steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 q

  4. One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples.

    Science.gov (United States)

    Gautam, Rashi; Mijatovic-Rustempasic, Slavica; Esona, Mathew D; Tam, Ka Ian; Quaye, Osbourne; Bowen, Michael D

    2016-01-01

    Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8-100% sensitivity, 99.7-100% specificity, 85-95% efficiency and a limit of detection of 4-60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81-92% efficiency and limit of detection of 150-600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98

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

    Science.gov (United States)

    Amarenco, G

    2014-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Gharavi, MJ. (PhD

    2015-01-01

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

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

    Science.gov (United States)

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

    2013-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Mehmet Sinan Dal

    2011-03-01

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

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

    Science.gov (United States)

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

    2013-12-01

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

  10. Evaluation of proper height for squatting stool.

    Science.gov (United States)

    Jung, Hwa S; Jung, Hyung-Shik

    2008-05-01

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

  11. Stool Testing for Colorectal Cancer Screening.

    Science.gov (United States)

    Robertson, Douglas J; Imperiale, Thomas F

    2015-10-01

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

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

    Science.gov (United States)

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

    2017-05-01

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

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

    Science.gov (United States)

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

    2016-04-01

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

  14. Pregnancy Constipation: Are Stool Softeners Safe?

    Science.gov (United States)

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

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

    Directory of Open Access Journals (Sweden)

    Keramettin Yanık

    2011-09-01

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

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

    Science.gov (United States)

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

    2013-01-25

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

  17. Evaluation of ergonomic dental stools through clinical simulation.

    Science.gov (United States)

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

    2000-01-01

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

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

    OpenAIRE

    Dal, Tuba

    2015-01-01

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

  19. [Phenotypic characterization and distribution of Yersinia in human and environmental samples].

    Science.gov (United States)

    Javier Castillo, F; Larraz, V; Asunción Lafarga, M; Navarro, M; Gómez-Lus, R

    1994-01-01

    The distribution of species and phenotypes of Yersinia isolated from environmental samples over an eight year period are compared to that of stool cultures obtained from patients of the same geographical location (Zaragoza, Spain). The number of samples and the percentage contamination were as follows: wastewater 362, 67.4%, freshwater 523, 13.4%, raw food 607, 24.5% and cooked food 1134, 7.9%. Yersinia enterocolitica was isolated significantly more frequently than other species in wastewater, while Yersinia intermedia was the most significant species found in freshwater. Significant differences between the percentage isolates of identified species in raw and cooked foods were not found. Fifteen different serogroups were identified from faeces, thirteen of which were also isolated from environmental samples. Three serogroups of Y. enterocolitica associated with human disease were isolated from the patients faeces as follows: O:3, 145 cases; O:8, 3 cases and O:5,27, 1 case. A low proportion were isolated from food: O:3, 3 strains; O:8, 2 strains and O:5,27, 5 strains. Only one isolate from serogroup O:3 was obtained from freshwater.

  20. Impact of Different Fecal Processing Methods on Assessments of Bacterial Diversity in the Human Intestine

    Science.gov (United States)

    Hsieh, Yu-Hsin; Peterson, Courtney M.; Raggio, Anne; Keenan, Michael J.; Martin, Roy J.; Ravussin, Eric; Marco, Maria L.

    2016-01-01

    The intestinal microbiota are integral to understanding the relationships between nutrition and health. Therefore, fecal sampling and processing protocols for metagenomic surveys should be sufficiently robust, accurate, and reliable to identify the microorganisms present. We investigated the use of different fecal preparation methods on the bacterial community structures identified in human stools. Complete stools were collected from six healthy individuals and processed according to the following methods: (i) randomly sampled fresh stool, (ii) fresh stool homogenized in a blender for 2 min, (iii) randomly sampled frozen stool, and (iv) frozen stool homogenized in a blender for 2 min, or (v) homogenized in a pneumatic mixer for either 10, 20, or 30 min. High-throughput DNA sequencing of the 16S rRNA V4 regions of bacterial community DNA extracted from the stools showed that the fecal microbiota remained distinct between individuals, independent of processing method. Moreover, the different stool preparation approaches did not alter intra-individual bacterial diversity. Distinctions were found at the level of individual taxa, however. Stools that were frozen and then homogenized tended to have higher proportions of Faecalibacterium, Streptococcus, and Bifidobacterium and decreased quantities of Oscillospira, Bacteroides, and Parabacteroides compared to stools that were collected in small quantities and not mixed prior to DNA extraction. These findings indicate that certain taxa are at particular risk for under or over sampling due to protocol differences. Importantly, homogenization by any method significantly reduced the intra-individual variation in bacteria detected per stool. Our results confirm the robustness of fecal homogenization for microbial analyses and underscore the value of collecting and mixing large stool sample quantities in human nutrition intervention studies. PMID:27812352

  1. Impact of Different Fecal Processing Methods on Assessments of Bacterial Diversity in the Human Intestine

    Directory of Open Access Journals (Sweden)

    Yu-Hsin Hsieh

    2016-10-01

    Full Text Available The intestinal microbiota are integral to understanding the relationships between nutrition and health. Therefore, fecal sampling and processing protocols for metagenomic surveys should be sufficiently robust, accurate, and reliable to identify the microorganisms present. We investigated the use of different fecal preparation methods on the bacterial community structures identified in human stools. Complete stools were collected from six healthy individuals and processed according to the following methods: (i randomly sampled fresh stool, (ii fresh stool homogenized in a blender for 2 min, (iii randomly sampled frozen stool, and (iv frozen stool homogenized in a blender for 2 min or (v homogenized in a pneumatic mixer for either 10, 20, or 30 min. High-throughput DNA sequencing of the 16S rRNA V4 regions of bacterial community DNA extracted from the stools showed that the fecal microbiota remained distinct between individuals, independent of processing method. Moreover, the different stool preparation approaches did not alter intra-individual bacterial diversity. Distinctions were found at the level of individual taxa, however. Stools that were frozen and then homogenized tended to have higher proportions of Faecalibacterium, Streptococcus, and Bifidobacterium and decreased quantities of Oscillospira, Bacteroides, and Parabacteroides compared to stools that were collected in small quantities and not mixed prior to DNA extraction. These findings indicate that certain taxa are at particular risk for under or over sampling due to protocol differences. Importantly, homogenization by any method significantly reduced the intra-individual variation in bacteria detected per stool. Our results confirm the robustness of fecal homogenization for microbial analyses and underscore the value of collecting and mixing large stool sample quantities in human nutrition intervention studies.

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

    OpenAIRE

    Arita, Minetaro

    2013-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2015-11-19

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

  5. Dielectric characterisation of human tissue samples

    NARCIS (Netherlands)

    Rossum, W.L. van; Nennie, F.; Deiana, D.; Veen, A.J. van der; Monni, S.

    2014-01-01

    The electrical properties of tissues samples are required for investigation and simulation purposes in biomedical applications of EM sensors. While available open literature mostly deals with ex-vivo characterization of isolated tissues, knowledge on dielectric properties of these tissues in their o

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

    Science.gov (United States)

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

    2016-02-16

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

  7. Pattern designation of PCBs in human samples

    Energy Technology Data Exchange (ETDEWEB)

    Wolff, M.S.; Fischbein, A.; Rosenman, K.D.; Levin, S.M.

    1986-03-01

    In order to asses the nature of PCB exposures in humans, statistical measures of PCB patterns in blood serum (as Aroclor 1254 or 1260) were made in 348 cases, representing several exposed and non-exposed groups. Although the cases were not representative of any population, most (252/348) had an Arcolor 1260 pattern, with evidence that PCB congeners in blood serum were usually derived from both Aroclor 1254 and 1260. The method is readily applied to routine packed column gc analysis.

  8. Lactobacillus plantarum PADA FESES INDIVIDU DEWASA SEHAT YANG MENGONSUMSI Lactobacillus plantarum IS-10506 DARI DADIH [Lactobacillus plantarum in Stool of Apparently Healthy Adults Consuming Lactobacillus plantarum IS-10506 from Dadih

    Directory of Open Access Journals (Sweden)

    Azmier Adib*

    2013-12-01

    Full Text Available A placebo double blind pre-post human study was conducted in apparently healthy adults. There were two treatment groups consisting of Group A and B representing probiotic and placebo group, respectively. Twenty four participants were randomly assigned, each supplemented with either placebo or probiotic Lactobacillus plantarum IS-10506. The micro encapsulated powder was given at a dose of 2.6x1010 CFU/day for 21 consecutive days. Stool samples were collected before and after the supplementation. The fresh stool samples were analyzed for the viability of Lactobacillus sp. by conventional plate count method in MRS agar. Some stool samples were kept frozen to be analyzed by using real time PCR to trace back the availability of Lactobacillus plantarum with species specific primer. The Lactobacillus sp. in stools of healthy adults given microencapsulated probiotic Lactobacillus plantarum IS-10506 powder was significantly more than those who consumed microencapsulated placebo powder. Molecular detection by qPCR confirmed the availability of Lactobacillus plantarum in fecal samples of the probiotic group after given the supplementation for 21 days. The molecular detection validation confirmed that probiotic Lactobacillus plantarum was available in the fecal samples of the probiotic group of healthy adults. However, the availability and viability of Lactobacillus plantarum were not consistently found in the intestinal tract.

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

    Directory of Open Access Journals (Sweden)

    Ranin Bechara

    2016-12-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

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

    Science.gov (United States)

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

    2016-11-14

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

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

    Institute of Scientific and Technical Information of China (English)

    宋美茹; 姚萍; 张跃新

    2012-01-01

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

  13. Forensic Identification of Human Blood: comparison of two one-step presumptive tests for blood screening of crime scene samples.

    Directory of Open Access Journals (Sweden)

    Ana Flávia Belchior Andrade

    2014-08-01

    Full Text Available Blood is the most common body fluid found at crime scenes. One-step presumptive tests have been designed as a rapid immunological test for the qualitative detection of human hemoglobin in stool samples (faecal occult blood their usefulness for forensic purposes has been demonstrated before. In this study we compare Hexagon OBTI kit and FOB One-step Bioeasy kit sensitivity in the analysis of diluted blood samples. With Hexagon OBTI, positive test results are achieved in whole blood dilutions up to 1:1.000. Sensitivity decreased with aged samples, if samples were not stored under low temperatures regardless of which presumptive test is used. Whole blood tests must take into consideration that “hook” effect may interfere. Comparing both tests, OBTI Hexagon Kit is more sensible to detect diluted blood, showing a wider detection window in all conditions. This is interesting when analyzing forensic samples as forensic analysts usually do not know about the history of the analyzed sample before its collection.

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

    Directory of Open Access Journals (Sweden)

    Eiki Yamasaki

    2015-10-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  16. Occurrence and characteristics of extended-spectrum β-lactamases producing Escherichia coli in foods of animal origin and human clinical samples in Chhattisgarh, India

    Directory of Open Access Journals (Sweden)

    Bhoomika

    2016-09-01

    Full Text Available Aim: To assess the prevalence of antimicrobial resistance producing extended-spectrum β-lactamases (ESBL (blaTEM, blaSHV, and blaCTX-M genes in Escherichia coli isolated from chicken meat, chevon meat, raw milk, and human urine and stool samples collected from tribal districts of Chhattisgarh, viz., Jagdalpur, Dantewada, Kondagaon, and Kanker. Materials and Methods: A total of 330 samples, comprising 98 chicken meat, 82 chevon meat, 90 raw milk, and 60 human urine and stool samples, were processed for isolation of E. coli. Isolates were confirmed biochemically and further tested against commonly used antibiotics to know their resistant pattern. The resistant isolates were tested for ESBL production by phenotypic method followed by characterization with molecular method using multiplex-polymerase chain reaction technique. Results: Overall 57.87% (191/330 samples were found positive for E. coli, which include 66.32% (65/98 chicken meat, 46.34% (38/82 chevon meat, 81.11% (73/90 raw milk, and 25% (15/60 human urine and stool samples. Isolates showed the highest resistance against cefotaxime (41.36% followed by oxytetracycline (34.03%, ampicillin (29.31%, cephalexin (24.60%, cefixime (16.75%, and ceftazidime (13.08%. Phenotypic method detected 10.99% (21/191 isolates as presumptive ESBL producers, however, molecular method detected 3.66% (7/191, 2.09% (4/191, and 0.00% (0/191 prevalence of blaTEM, blaCTX-M, and blaSHV, respectively. Conclusion: The present study indicates a high prevalence of E. coli in raw chicken meat, chevon meat, and milk due to poor hygienic practices. The antibiotic susceptibility test detected the presence of the resistance pattern against ESBL in E. coli isolated from raw chicken meat, chevon meat, milk, and also in human clinical samples is of great concern. The appearance of E. coli in the human food chain is alarming and requires adaptation of hygienic practices and stipulate use of antibiotics.

  17. Frequency of Blastocystis hominis and other intestinal parasites in stool samples examined at the Parasitology Laboratory of the School of Pharmaceutical Sciences at the São Paulo State University, Araraquara Freqüência de Blastocystis hominis e outros enteroparasitas em amostras de fezes examinadas no Laboratório de Parasitologia da Faculdade de Ciências Farmacêuticas da Universidade Estadual Paulista, Araraquara

    Directory of Open Access Journals (Sweden)

    Júlio César Miné

    2008-12-01

    Full Text Available Blastocystis homins is a protozoan that causes an intestinal infection known as human blastocystosis. This infection is diagnosed by means of parasitological examination of stools and by permanent staining techniques. The present study was developed to evaluate the frequency of Blastocystis hominis infection among inhabitants of the Araraquara region, State of São Paulo, and to compare different methods for investigating this protozoan in feces samples. Evaluations on 503 stool samples were performed by means of direct fresh examination and using the techniques of Faust et al., Lutz and Rugai et al. In addition, the iron hematoxylin, trichrome and modified Kinyoun staining techniques were used. Out of the 503 samples examined, 174 (34.6% were found to be positive for the presence of intestinal parasites. The most frequent protozoa and helminths were Entamoeba coli (14.6% and Strongyloides stercoralis (6.7%, respectively. Blastocystis hominis was present in 23 (4.6% fecal samples, with a predominately pasty consistency and without characterizing a condition of diarrhea. Despite the low frequency of Blastocystis hominis found in the Araraquara region, compared with other regions of Brazil, it is important to perform laboratory diagnostic tests for this protozoan. Its finding in fecal material is indicative of food and drinking water contamination. Since the transmission route for this parasite is accepted to be oral-fecal, this implies that the population needs guidance regarding hygiene and basic sanitation measures as a means for controlling health problems caused by enteroparasites.Blastocystis hominis é um protozoário, causador de infecção intestinal denominada blastocistose humana, cujo diagnóstico é realizado pelo exame coproparasitológico e por meio de técnicas de coloração permanente. Este estudo foi desenvolvido para avaliar a freqüência da infecção por Blastocystis hominis em habitantes da região de Araraquara/SP, bem

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

    Science.gov (United States)

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

    2014-07-01

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

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

    Science.gov (United States)

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

    1988-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

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

    Science.gov (United States)

    Petrof, Elaine O; Khoruts, Alexander

    2014-05-01

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

  2. Plesiomonas shigelloides in stool samples of patients in the Venda ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-11-16

    Nov 16, 2009 ... rent types of animals such as cattle, goats, swine, cats, dogs, monkeys ... genetic diversity, which will impact on epidemiological control, a study on ..... However, further studies are needed to unravel genetic profiles of resis-.

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

    Directory of Open Access Journals (Sweden)

    shahram Khademvatan

    2011-06-01

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

  4. Approach-Induced Biases in Human Information Sampling

    Science.gov (United States)

    Hunt, Laurence T.; Rutledge, Robb B.; Malalasekera, W. M. Nishantha; Kennerley, Steven W.; Dolan, Raymond J.

    2016-01-01

    Information sampling is often biased towards seeking evidence that confirms one’s prior beliefs. Despite such biases being a pervasive feature of human behavior, their underlying causes remain unclear. Many accounts of these biases appeal to limitations of human hypothesis testing and cognition, de facto evoking notions of bounded rationality, but neglect more basic aspects of behavioral control. Here, we investigated a potential role for Pavlovian approach in biasing which information humans will choose to sample. We collected a large novel dataset from 32,445 human subjects, making over 3 million decisions, who played a gambling task designed to measure the latent causes and extent of information-sampling biases. We identified three novel approach-related biases, formalized by comparing subject behavior to a dynamic programming model of optimal information gathering. These biases reflected the amount of information sampled (“positive evidence approach”), the selection of which information to sample (“sampling the favorite”), and the interaction between information sampling and subsequent choices (“rejecting unsampled options”). The prevalence of all three biases was related to a Pavlovian approach-avoid parameter quantified within an entirely independent economic decision task. Our large dataset also revealed that individual differences in the amount of information gathered are a stable trait across multiple gameplays and can be related to demographic measures, including age and educational attainment. As well as revealing limitations in cognitive processing, our findings suggest information sampling biases reflect the expression of primitive, yet potentially ecologically adaptive, behavioral repertoires. One such behavior is sampling from options that will eventually be chosen, even when other sources of information are more pertinent for guiding future action. PMID:27832071

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

    Directory of Open Access Journals (Sweden)

    Amy Franciscovich

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

  6. Human-Robot Site Survey and Sampling for Space Exploration

    Science.gov (United States)

    Fong, Terrence; Bualat, Maria; Edwards, Laurence; Flueckiger, Lorenzo; Kunz, Clayton; Lee, Susan Y.; Park, Eric; To, Vinh; Utz, Hans; Ackner, Nir

    2006-01-01

    NASA is planning to send humans and robots back to the Moon before 2020. In order for extended missions to be productive, high quality maps of lunar terrain and resources are required. Although orbital images can provide much information, many features (local topography, resources, etc) will have to be characterized directly on the surface. To address this need, we are developing a system to perform site survey and sampling. The system includes multiple robots and humans operating in a variety of team configurations, coordinated via peer-to-peer human-robot interaction. In this paper, we present our system design and describe planned field tests.

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

    Institute of Scientific and Technical Information of China (English)

    Beuy Joob; Viroj Wiwanitkit

    2014-01-01

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

  8. Whole genome detection of rotavirus mixed infections in human, porcine and bovine samples co-infected with various rotavirus strains collected from sub-Saharan Africa.

    Science.gov (United States)

    Nyaga, Martin M; Jere, Khuzwayo C; Esona, Mathew D; Seheri, Mapaseka L; Stucker, Karla M; Halpin, Rebecca A; Akopov, Asmik; Stockwell, Timothy B; Peenze, Ina; Diop, Amadou; Ndiaye, Kader; Boula, Angeline; Maphalala, Gugu; Berejena, Chipo; Mwenda, Jason M; Steele, A Duncan; Wentworth, David E; Mphahlele, M Jeffrey

    2015-04-01

    Group A rotaviruses (RVA) are among the main global causes of severe diarrhea in children under the age of 5years. Strain diversity, mixed infections and untypeable RVA strains are frequently reported in Africa. We analysed rotavirus-positive human stool samples (n=13) obtained from hospitalised children under the age of 5years who presented with acute gastroenteritis at sentinel hospital sites in six African countries, as well as bovine and porcine stool samples (n=1 each), to gain insights into rotavirus diversity and evolution. Polyacrylamide gel electrophoresis (PAGE) analysis and genotyping with G-(VP7) and P-specific (VP4) typing primers suggested that 13 of the 15 samples contained more than 11 segments and/or mixed G/P genotypes. Full-length amplicons for each segment were generated using RVA-specific primers and sequenced using the Ion Torrent and/or Illumina MiSeq next-generation sequencing platforms. Sequencing detected at least one segment in each sample for which duplicate sequences, often having distinct genotypes, existed. This supported and extended the PAGE and RT-PCR genotyping findings that suggested these samples were collected from individuals that had mixed rotavirus infections. The study reports the first porcine (MRC-DPRU1567) and bovine (MRC-DPRU3010) mixed infections. We also report a unique genome segment 9 (VP7), whose G9 genotype belongs to lineage VI and clusters with porcine reference strains. Previously, African G9 strains have all been in lineage III. Furthermore, additional RVA segments isolated from humans have a clear evolutionary relationship with porcine, bovine and ovine rotavirus sequences, indicating relatively recent interspecies transmission and reassortment. Thus, multiple RVA strains from sub-Saharan Africa are infecting mammalian hosts with unpredictable variations in their gene segment combinations. Whole-genome sequence analyses of mixed RVA strains underscore the considerable diversity of rotavirus sequences and

  9. In silico analyses of metagenomes from human atherosclerotic plaque samples

    DEFF Research Database (Denmark)

    Mitra, Suparna; Drautz-Moses, Daniela I; Alhede, Morten

    2015-01-01

    a challenge. RESULTS: To investigate microbiome diversity within human atherosclerotic tissue samples, we employed high-throughput metagenomic analysis on: (1) atherosclerotic plaques obtained from a group of patients who underwent endarterectomy due to recent transient cerebral ischemia or stroke. (2...

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

    Science.gov (United States)

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

    2016-12-01

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

  11. A DNA methylation fingerprint of 1628 human samples

    Science.gov (United States)

    Fernandez, Agustin F.; Assenov, Yassen; Martin-Subero, Jose Ignacio; Balint, Balazs; Siebert, Reiner; Taniguchi, Hiroaki; Yamamoto, Hiroyuki; Hidalgo, Manuel; Tan, Aik-Choon; Galm, Oliver; Ferrer, Isidre; Sanchez-Cespedes, Montse; Villanueva, Alberto; Carmona, Javier; Sanchez-Mut, Jose V.; Berdasco, Maria; Moreno, Victor; Capella, Gabriel; Monk, David; Ballestar, Esteban; Ropero, Santiago; Martinez, Ramon; Sanchez-Carbayo, Marta; Prosper, Felipe; Agirre, Xabier; Fraga, Mario F.; Graña, Osvaldo; Perez-Jurado, Luis; Mora, Jaume; Puig, Susana; Prat, Jaime; Badimon, Lina; Puca, Annibale A.; Meltzer, Stephen J.; Lengauer, Thomas; Bridgewater, John; Bock, Christoph; Esteller, Manel

    2012-01-01

    Most of the studies characterizing DNA methylation patterns have been restricted to particular genomic loci in a limited number of human samples and pathological conditions. Herein, we present a compromise between an extremely comprehensive study of a human sample population with an intermediate level of resolution of CpGs at the genomic level. We obtained a DNA methylation fingerprint of 1628 human samples in which we interrogated 1505 CpG sites. The DNA methylation patterns revealed show this epigenetic mark to be critical in tissue-type definition and stemness, particularly around transcription start sites that are not within a CpG island. For disease, the generated DNA methylation fingerprints show that, during tumorigenesis, human cancer cells underwent a progressive gain of promoter CpG-island hypermethylation and a loss of CpG methylation in non-CpG-island promoters. Although transformed cells are those in which DNA methylation disruption is more obvious, we observed that other common human diseases, such as neurological and autoimmune disorders, had their own distinct DNA methylation profiles. Most importantly, we provide proof of principle that the DNA methylation fingerprints obtained might be useful for translational purposes by showing that we are able to identify the tumor type origin of cancers of unknown primary origin (CUPs). Thus, the DNA methylation patterns identified across the largest spectrum of samples, tissues, and diseases reported to date constitute a baseline for developing higher-resolution DNA methylation maps and provide important clues concerning the contribution of CpG methylation to tissue identity and its changes in the most prevalent human diseases. PMID:21613409

  12. Retrospective Species Identification of Microsporidian Spores in Diarrheic Fecal Samples from Human Immunodeficiency Virus/AIDS Patients by Multiplexed Fluorescence In Situ Hybridization▿

    Science.gov (United States)

    Graczyk, Thaddeus K.; Johansson, Michael A.; Tamang, Leena; Visvesvara, Govinda S.; Moura, Laci S.; DaSilva, Alexandre J.; Girouard, Autumn S.; Matos, Olga

    2007-01-01

    In order to assess the applicability of multiplexed fluorescence in situ hybridization (FISH) assay for the clinical setting, we conducted retrospective analysis of 110 formalin-stored diarrheic stool samples from human immunodeficiency virus (HIV)/AIDS patients with intestinal microsporidiosis collected between 1992 and 2003. The multiplexed FISH assay identified microsporidian spores in 94 of 110 (85.5%) samples: 49 (52.1%) were positive for Enterocytozoon bieneusi, 43 (45.8%) were positive for Encephalitozoon intestinalis, 2 (2.1%) were positive for Encephalitozoon hellem, and 9 samples (9.6%) contained both E. bieneusi and E. intestinalis spores. Quantitative spore counts per ml of stool yielded concentration values from 3.5 × 103 to 4.4 × 105 for E. bieneusi (mean, 8.8 × 104/ml), 2.3 × 102 to 7.8 × 104 (mean, 1.5 × 104/ml) for E. intestinalis, and 1.8 × 102 to 3.6 × 102 for E. hellem (mean, 2.7 × 102/ml). Identification of microsporidian spores by multiplex FISH assay was more sensitive than both Chromotrope-2R and CalcoFluor White M2R stains; 85.5% versus 72.7 and 70.9%, respectively. The study demonstrated that microsporidian coinfection in HIV/AIDS patients with intestinal microsporidiosis is not uncommon and that formalin-stored fecal samples older than 10 years may not be suitable for retrospective analysis by techniques targeting rRNA. Multiplexed FISH assay is a reliable, quantitative fluorescence microscopy method for the simultaneous identification of E. bieneusi, E. intestinalis, and E. hellem, as well as Encephalitozoon cuniculi, spores in fecal samples and is a useful tool for assessing spore shedding intensity in intestinal microsporidiosis. The method can be used for epidemiological investigations and applied in clinical settings. PMID:17287331

  13. Groundbreaking Mars Sample Return for Science and Human Exploration

    Science.gov (United States)

    Cohen, Barbara; Draper, David; Eppler, Dean; Treiman, Allan

    2012-01-01

    Partnerships between science and human exploration have recent heritage for the Moon (Lunar Precursor Robotics Program, LPRP) and nearearth objects (Exploration Precursor Robotics Program, xPRP). Both programs spent appreciable time and effort determining measurements needed or desired before human missions to these destinations. These measurements may be crucial to human health or spacecraft design, or may be desired to better optimize systems designs such as spacesuits or operations. Both LPRP and xPRP recommended measurements from orbit, by landed missions and by sample return. LPRP conducted the Lunar Reconnaissance Orbiter (LRO) and Lunar Crater Observation and Sensing Satellite (LCROSS) missions, providing high-resolution visible imagery, surface and subsurface temperatures, global topography, mapping of possible water ice deposits, and the biological effects of radiation [1]. LPRP also initiated a landed mission to provide dust and regolith properties, local lighting conditions, assessment of resources, and demonstration of precision landing [2]. This mission was canceled in 2006 due to funding shortfalls. For the Moon, adequate samples of rocks and regolith were returned by the Apollo and Luna programs to conduct needed investigations. Many near-earth asteroids (NEAs) have been observed from the Earth and several have been more extensively characterized by close-flying missions and landings (NEAR, Hayabusa, Rosetta). The current Joint Robotic Precursor Activity program is considering activities such as partnering with the New Frontiers mission OSIRIS-Rex to visit a NEA and return a sample to the Earth. However, a strong consensus of the NEO User Team within xPRP was that a dedicated mission to the asteroid targeted by humans is required [3], ideally including regolith sample return for more extensive characterization and testing on the Earth.

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

    DEFF Research Database (Denmark)

    Mirsepasi, Hengameh; Persson, Søren; Struve, Carsten

    2014-01-01

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

  15. Sampling strategy for estimating human exposure pathways to consumer chemicals

    Directory of Open Access Journals (Sweden)

    Eleni Papadopoulou

    2016-03-01

    Full Text Available Human exposure to consumer chemicals has become a worldwide concern. In this work, a comprehensive sampling strategy is presented, to our knowledge being the first to study all relevant exposure pathways in a single cohort using multiple methods for assessment of exposure from each exposure pathway. The selected groups of chemicals to be studied are consumer chemicals whose production and use are currently in a state of transition and are; per- and polyfluorinated alkyl substances (PFASs, traditional and “emerging” brominated flame retardants (BFRs and EBFRs, organophosphate esters (OPEs and phthalate esters (PEs. Information about human exposure to these contaminants is needed due to existing data gaps on human exposure intakes from multiple exposure pathways and relationships between internal and external exposure. Indoor environment, food and biological samples were collected from 61 participants and their households in the Oslo area (Norway on two consecutive days, during winter 2013-14. Air, dust, hand wipes, and duplicate diet (food and drink samples were collected as indicators of external exposure, and blood, urine, blood spots, hair, nails and saliva as indicators of internal exposure. A food diary, food frequency questionnaire (FFQ and indoor environment questionnaire were also implemented. Approximately 2000 samples were collected in total and participant views on their experiences of this campaign were collected via questionnaire. While 91% of our participants were positive about future participation in a similar project, some tasks were viewed as problematic. Completing the food diary and collection of duplicate food/drink portions were the tasks most frequent reported as “hard”/”very hard”. Nevertheless, a strong positive correlation between the reported total mass of food/drinks in the food record and the total weight of the food/drinks in the collection bottles was observed, being an indication of accurate performance

  16. [Fusarium graminearum presence in wheat samples for human consumption].

    Science.gov (United States)

    Martinez, Mauro; Castañares, Eliana; Dinolfo, María I; Pacheco, Walter G; Moreno, María V; Stenglein, Sebastián A

    2014-01-01

    One of the most important diseases in cereal crops is Fusarium head blight, being Fusarium graminearum the main etiological agent. This fungus has the ability to produce a wide spectrum and quantity of toxins, especially deoxynivalenol (DON). During the last crop season (2012-2013) the climatic conditions favored Fusarium colonization. The objective of this work was to determine the presence of this fungus as well as the DON content in 50 wheat grain samples. Our results showed that 80% of the samples were contaminated with Fusarium graminearum. Twenty four percent (24%) of the samples contained ≥ 1μg/g DON, 26% ranged from 0,5 and 0,99μg/g, and the remaining 50% had values lower than 0,5μg/g. Correlation was found between the presence of Fusarium graminearum and DON. It is necessary to establish DON limit values in wheat grains for human consumption.

  17. Prevalence of Entamoeba histolytica -like cysts compared to E. histolytica antigens detected by ELISA in the stools of 600 patients from three socioeconomic communities in the Metropolitan City of Lahore, Pakistan.

    Science.gov (United States)

    Alam, Muhammad Azhar; Maqbool, Azhar; Nazir, Muhammad Mudasser; Lateef, Muhammad; Khan, Muhammad Sarwar; Ahmed, Atif Nisar; Ziaullah, M; Lindsay, David S

    2015-04-01

    Amoebiasis, caused by Entamoeba histolytica , has a worldwide distribution and is of public health significance in many developing countries. It has a fecal-oral transmission cycle and is most prevalent in developing countries in regions where substandard sanitary conditions exist due to poverty. Little is known about the epidemiology of E. histolytica infection and its presence in different socioeconomic communities in developing countries. We undertook the present study in the city of Lahore, Pakistan, and our prediction was that the prevalence of E. histolytica -like cysts and E. histolytica stool antigen would be lower in patients from upper socioeconomic levels than in individuals from middle or lower socioeconomic levels. We investigated the prevalence of E. histolytica in humans from 3 socioeconomic communities in territories of Lahore, Pakistan. Six hundred fecal samples were collected and examined using both microscopy (triple fecal test) to detect cysts of E. histolytica -like amoeba and ELISA (stool antigen ELISA) to demonstrate diagnostic stool antigens of E. histolytica . Samples were from individuals living under conditions deemed to be upper socioeconomic class (n = 287), middle socioeconomic class (n = 172), and lower socioeconomic class (n = 141). The total prevalence of positive samples was 22.5% (135/600) by triple test and 16.8% (101/600) by stool antigen ELISA in the 600 fecal samples. Statistically, significant (P histolytica antigens than were samples from the other 3 age groups, and that prevalence was significantly higher (P < 0.05) in the summer than in the other 3 seasons. These results highlight the importance of surveillance of this relatively ignored pathogen in this developing metropolitan city in Pakistan.

  18. Variation in the limit-of-detection of the ProSpecT Campylobacter microplate enzyme immunoassay in stools spiked with emerging Campylobacter species.

    Science.gov (United States)

    Bojanić, Krunoslav; Midwinter, Anne Camilla; Marshall, Jonathan Craig; Rogers, Lynn Elizabeth; Biggs, Patrick Jon; Acke, Els

    2016-08-01

    Campylobacter enteritis in humans is primarily associated with C. jejuni/coli infection. The impact of other Campylobacter spp. is likely to be underestimated due to the bias of culture methods towards Campylobacter jejuni/coli diagnosis. Stool antigen tests are becoming increasingly popular and appear generally less species-specific. A review of independent studies of the ProSpecT® Campylobacter Microplate enzyme immunoassay (EIA) developed for C. jejuni/coli showed comparable diagnostic results to culture methods but the examination of non-jejuni/coli Campylobacter spp. was limited and the limit-of-detection (LOD), where reported, varied between studies. This study investigated LOD of EIA for Campylobacter upsaliensis, Campylobacter hyointestinalis and Campylobacter helveticus spiked in human stools. Multiple stools and Campylobacter isolates were used in three different concentrations (10(4)-10(9)CFU/ml) to reflect sample heterogeneity. All Campylobacter species evaluated were detectable by EIA. Multivariate analysis showed LOD varied between Campylobacter spp. and faecal consistency as fixed effects and individual faecal samples as random effects. EIA showed excellent performance in replicate testing for both within and between batches of reagents, in agreement between visual and spectrophotometric reading of results, and returned no discordance between the bacterial concentrations within independent dilution test runs (positive results with lower but not higher concentrations). This study shows how limitations in experimental procedures lead to an overestimation of consistency and uniformity of LOD for EIA that may not hold under routine use in diagnostic laboratories. Benefits and limitations for clinical practice and the influence on estimates of performance characteristics from detection of multiple Campylobacter spp. by EIA are discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Estimating Sampling Selection Bias in Human Genetics: A Phenomenological Approach

    Science.gov (United States)

    Risso, Davide; Taglioli, Luca; De Iasio, Sergio; Gueresi, Paola; Alfani, Guido; Nelli, Sergio; Rossi, Paolo; Paoli, Giorgio; Tofanelli, Sergio

    2015-01-01

    This research is the first empirical attempt to calculate the various components of the hidden bias associated with the sampling strategies routinely-used in human genetics, with special reference to surname-based strategies. We reconstructed surname distributions of 26 Italian communities with different demographic features across the last six centuries (years 1447–2001). The degree of overlapping between "reference founding core" distributions and the distributions obtained from sampling the present day communities by probabilistic and selective methods was quantified under different conditions and models. When taking into account only one individual per surname (low kinship model), the average discrepancy was 59.5%, with a peak of 84% by random sampling. When multiple individuals per surname were considered (high kinship model), the discrepancy decreased by 8–30% at the cost of a larger variance. Criteria aimed at maximizing locally-spread patrilineages and long-term residency appeared to be affected by recent gene flows much more than expected. Selection of the more frequent family names following low kinship criteria proved to be a suitable approach only for historically stable communities. In any other case true random sampling, despite its high variance, did not return more biased estimates than other selective methods. Our results indicate that the sampling of individuals bearing historically documented surnames (founders' method) should be applied, especially when studying the male-specific genome, to prevent an over-stratification of ancient and recent genetic components that heavily biases inferences and statistics. PMID:26452043

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

    Science.gov (United States)

    Trabelsi, Sonia; Aouinet, Amira; Khaled, Samira

    2012-06-01

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

  1. Blautia massiliensis sp. nov., isolated from a fresh human fecal sample and emended description of the genus Blautia.

    Science.gov (United States)

    Durand, Guillaume A; Pham, Thao; Ndongo, Sokhna; Traore, Sory Ibrahima; Dubourg, Grégory; Lagier, Jean-Christophe; Michelle, Caroline; Armstrong, Nicholas; Fournier, Pierre-Edouard; Raoult, Didier; Million, Matthieu

    2017-02-01

    The strain GD9(T) is the type strain of the newly proposed species Blautia massiliensis sp. nov., belonging to the family Lachnospiraceae. It was isolated from a fresh stool sample collected from a healthy human using the culturomics strategy. Cells are Gram-negative rods, oxygen intolerant, non-motile and non-spore forming. The 16S rRNA gene sequencing showed that strain GD9(T) was closely related to Blautia luti, with a 97.8% sequence similarity. Major fatty acids were C14:0 (19.8%) and C16:0 (53.2%). Strain GD9(T) exhibits a genome of 3,717,339 bp that contains 3,346 protein-coding genes and 81 RNAs genes including 63 tRNAs. The features of this organism are described here, with its complete genome sequence and annotation. Compared with other Blautia species which are Gram positive, the strain was Gram negative justifying an emended description of the genus Blautia.

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

    Directory of Open Access Journals (Sweden)

    M Leonor Fernández-Murga

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

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

    Science.gov (United States)

    Fernández-Murga, M Leonor; Sanz, Yolanda

    2016-01-01

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

  4. Recognition of human face based on improved multi-sample

    Institute of Scientific and Technical Information of China (English)

    LIU Xia; LI Lei-lei; LI Ting-jun; LIU Lu; ZHANG Ying

    2009-01-01

    In order to solve the problem caused by variation illumination in human face recognition, we bring forward a face recognition algorithm based on the improved muhi-sample. In this algorithm, the face image is processed with Retinex theory, meanwhile, the Gabor filter is adopted to perform the feature extraction. The experimental results show that the application of Retinex theory improves the recognition accuracy, and makes the algorithm more robust to the variation illumination. The Gabor filter is more effective and accurate for extracting more useable facial local features. It is proved that the proposed algorithm has good recognition accuracy and it is stable under variation illumination.

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

    Science.gov (United States)

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

    2016-09-01

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

  6. Human papillomavirus detection from human immunodeficiency virus-infected Colombian women's paired urine and cervical samples.

    Science.gov (United States)

    Munoz, Marina; Camargo, Milena; Soto-De Leon, Sara C; Sanchez, Ricardo; Parra, Diana; Pineda, Andrea C; Sussmann, Otto; Perez-Prados, Antonio; Patarroyo, Manuel E; Patarroyo, Manuel A

    2013-01-01

    Infection, coinfection and type-specific human papillomavirus (HPV) distribution was evaluated in human immunodeficiency virus (HIV)-positive women from paired cervical and urine samples. Paired cervical and urine samples (n = 204) were taken from HIV-positive women for identifying HPV-DNA presence by using polymerase chain reaction (PCR) with three generic primer sets (GP5+/6+, MY09/11 and pU1M/2R). HPV-positive samples were typed for six high-risk HPV (HR-HPV) (HPV-16, -18, -31, -33, -45 and -58) and two low-risk (LR-HPV) (HPV-6/11) types. Agreement between paired sample results and diagnostic performance was evaluated. HPV infection prevalence was 70.6% in cervical and 63.2% in urine samples. HPV-16 was the most prevalent HPV type in both types of sample (66.7% in cervical samples and 62.0% in urine) followed by HPV-31(47.2%) in cervical samples and HPV-58 (35.7%) in urine samples. There was 55.4% coinfection (infection by more than one type of HPV) in cervical samples and 40.2% in urine samples. Abnormal Papanicolau smears were observed in 25.3% of the women, presenting significant association with HPV-DNA being identified in urine samples. There was poor agreement of cervical and urine sample results in generic and type-specific detection of HPV. Urine samples provided the best diagnosis when taking cytological findings as reference. In conclusion including urine samples could be a good strategy for ensuring adherence to screening programs aimed at reducing the impact of cervical cancer, since this sample is easy to obtain and showed good diagnostic performance.

  7. Human papillomavirus detection from human immunodeficiency virus-infected Colombian women's paired urine and cervical samples.

    Directory of Open Access Journals (Sweden)

    Marina Munoz

    Full Text Available Infection, coinfection and type-specific human papillomavirus (HPV distribution was evaluated in human immunodeficiency virus (HIV-positive women from paired cervical and urine samples. Paired cervical and urine samples (n = 204 were taken from HIV-positive women for identifying HPV-DNA presence by using polymerase chain reaction (PCR with three generic primer sets (GP5+/6+, MY09/11 and pU1M/2R. HPV-positive samples were typed for six high-risk HPV (HR-HPV (HPV-16, -18, -31, -33, -45 and -58 and two low-risk (LR-HPV (HPV-6/11 types. Agreement between paired sample results and diagnostic performance was evaluated. HPV infection prevalence was 70.6% in cervical and 63.2% in urine samples. HPV-16 was the most prevalent HPV type in both types of sample (66.7% in cervical samples and 62.0% in urine followed by HPV-31(47.2% in cervical samples and HPV-58 (35.7% in urine samples. There was 55.4% coinfection (infection by more than one type of HPV in cervical samples and 40.2% in urine samples. Abnormal Papanicolau smears were observed in 25.3% of the women, presenting significant association with HPV-DNA being identified in urine samples. There was poor agreement of cervical and urine sample results in generic and type-specific detection of HPV. Urine samples provided the best diagnosis when taking cytological findings as reference. In conclusion including urine samples could be a good strategy for ensuring adherence to screening programs aimed at reducing the impact of cervical cancer, since this sample is easy to obtain and showed good diagnostic performance.

  8. Spectrophotometric assay of creatinine in human serum sample

    Directory of Open Access Journals (Sweden)

    Avinash Krishnegowda

    2017-05-01

    Full Text Available A new spectrophotometric method for the analysis of creatinine concentration in human serum samples is developed. The method explores the oxidation of p-methylamino phenol sulfate (Metol in the presence of copper sulfate and creatinine which yields an intense violet colored species with maximum absorbance at 530 nm. The calibration graph of creatinine by fixed time assay ranged from 4.4 to 620 μM. Recovery of creatinine in human serum samples varied from 101% to 106%. Limit of detection and limit of quantification were 0.145 μM and 0.487 μM respectively. Sandell’s sensitivity was 0.112 μg cm−2 and molar absorptivity was 0.101 × 104 L mol−1 cm−1. Within day precision was 2.5–4.8% and day-to-day precision range was 3.2–7.8%. The robustness and ruggedness of the method expressed in RSD values ranged from 0.78% to 2.12% and 1.32% to 3.46% respectively, suggesting that the developed method was rugged. This method provides good sensitivity and is comparable to standard Jaffe’s method with comparatively less interference from foreign substances.

  9. Rapid extraction and preservation of genomic DNA from human samples.

    Science.gov (United States)

    Kalyanasundaram, D; Kim, J-H; Yeo, W-H; Oh, K; Lee, K-H; Kim, M-H; Ryew, S-M; Ahn, S-G; Gao, D; Cangelosi, G A; Chung, J-H

    2013-02-01

    Simple and rapid extraction of human genomic DNA remains a bottleneck for genome analysis and disease diagnosis. Current methods using microfilters require cumbersome, multiple handling steps in part because salt conditions must be controlled for attraction and elution of DNA in porous silica. We report a novel extraction method of human genomic DNA from buccal swab and saliva samples. DNA is attracted onto a gold-coated microchip by an electric field and capillary action while the captured DNA is eluted by thermal heating at 70 °C. A prototype device was designed to handle four microchips, and a compatible protocol was developed. The extracted DNA using microchips was characterized by qPCR for different sample volumes, using different lengths of PCR amplicon, and nuclear and mitochondrial genes. In comparison with a commercial kit, an equivalent yield of DNA extraction was achieved with fewer steps. Room-temperature preservation for 1 month was demonstrated for captured DNA, facilitating straightforward collection, delivery, and handling of genomic DNA in an environment-friendly protocol.

  10. FANTOM5 CAGE profiles of human and mouse samples

    KAUST Repository

    Noguchi, Shuhei

    2017-08-29

    In the FANTOM5 project, transcription initiation events across the human and mouse genomes were mapped at a single base-pair resolution and their frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled with single-molecule sequencing. Approximately three thousands of samples, consisting of a variety of primary cells, tissues, cell lines, and time series samples during cell activation and development, were subjected to a uniform pipeline of CAGE data production. The analysis pipeline started by measuring RNA extracts to assess their quality, and continued to CAGE library production by using a robotic or a manual workflow, single molecule sequencing, and computational processing to generate frequencies of transcription initiation. Resulting data represents the consequence of transcriptional regulation in each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE profiles, approximately 200,000 and 150,000 peaks for the human and mouse genomes, were identified and annotated to provide precise location of known promoters as well as novel ones, and to quantify their activities.

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

  12. Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

    Science.gov (United States)

    Vidal, Samuel J.; Quinn, S. Aidan; de la Iglesia-Vicente, Janis; Bonal, Dennis M.; Rodriguez-Bravo, Veronica; Firpo-Betancourt, Adolfo; Cordon-Cardo, Carlos; Domingo-Domenech, Josep

    2014-01-01

    The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice. PMID:24686446

  13. Phylogenetic analysis of Escherichia coli strains isolated from human samples

    Directory of Open Access Journals (Sweden)

    Abdollah Derakhshandeh

    2013-12-01

    Full Text Available Escherichia coli (E. coli is a normal inhabitant of the gastrointestinal tract of vertebrates, including humans. Phylogenetic analysis has shown that E. coli is composed of four main phylogenetic groups (A, B1, B2 and D. Group A and B1 are generally associated with commensals, whereas group B2 is associated with extra-intestinal pathotypes. Most enteropathogenic isolates, however, are assigned to group D. In the present study, a total of 102 E. coli strains, isolated from human samples, were used. Phylogenetic grouping was done based on the Clermont triplex PCR method using primers targeted at three genetic markers, chuA, yjaA and TspE4.C2. Group A contained the majority of the collected isolates (69 isolates, 67.64%, followed by group B2 (18 isolates, 17.64% and D (15 isolates, 14.7% and no strains were found to belong to group B1. The distribution of phylogenetic groups in our study suggests that although the majority of strains were commensals, the prevalence of enteropathogenic and extra-intestinal pathotypes was noteworthy. Therefore, the role of E. coli in human infections including diarrhea, urinary tract infections and meningitis should be considered.

  14. Molecular Detection and Identification of Zoonotic Microspor-idia Spore in Fecal Samples of Some Animals with Close-Con-tact to Human

    Directory of Open Access Journals (Sweden)

    Zeinab ASKARI

    2015-10-01

    Full Text Available Background: Microsporidia species are obligatory intracellular agents that can in­fect all major animal groups including mammals, birds, fishes and insects. Whereas world­wide human infection reports are increasing, the cognition of sources of infec­tion particularly zoonotic transmission could be helpful. We aimed to detect zoono­tic microsporidia spore in fecal samples from some animals with close – contact to human.Methods: Overall, 142 fecal samples were collected from animals with closed-con­tact to human, during 2012-2013. Trichrome – blue staining were performed and DNA was then extracted from samples, identified positive, microscopically. Nested PCR was also carried out with primers targeting SSU rRNA gene and PCR products were sequenced.Results: From 142 stool samples, microsporidia spores have been observed microscopi­cally in 15 (10.56% samples. En. cuniculi was found in the faces of 3 (15% small white mice and 1 (10% laboratory rabbits(totally 2.81%. Moreover, E. bieneusi was detected in 3 (10% samples of sheep, 2 (5.12% cattle, 1 (10% rabbit, 3 (11.53% cats and 2 (11.76% ownership dogs (totally 7.74%. Phylogenetic analysis showed interesting data. This is the first study in Iran, which identified E. bieneusi and En. Cuniculi in fecal samples of laboratory animals with close – contact to human as well as domesticated animal and analyzed them in phylogenetic tree. Conclusion: E. bieneusi is the most prevalent microsporidia species in animals. Our results can also alert us about potentially zoonotic transmission of microsporidiosis.

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

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    Faridabano Nato

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

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

    OpenAIRE

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

    2007-01-01

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

  17. Rapid detection of clostridium difficile in human stool by real-time fluorescence PCR%实时荧光PCR快速检测粪便中艰难梭菌方法

    Institute of Scientific and Technical Information of China (English)

    邵景东; 吴琳; 王毅谦; 傅春玲; 吴福平

    2011-01-01

    Objective: To develop a real - time fluorescence PCR assay for rapid detection of Clostridium difficile. Methods: The special tpi gene of C. difficile were amplified through designing special primers and TaqMan probes within the conserved and specific regions for this gene. In this way, a rapid and stable method of real - time PCR assay for the detection of C. difficile standard bacterial concentration with 106 -10 cfu/ml was established. The specificity and sensitivity of PCR were also analyzed. By adding standard culture fluid in blank fecal sample,the sensitivity and interference of the method was evaluated. Results: The detection limits of pure culture in the real - time PCR assay were 10 CFU/ml. The detection limit for C. difficile in artificially contaminated fecal sample was 103 CFU/ml. Conclusion: These results indicated that the real -time PCR method for C. difficile detection was rapid, high in specificity and sensitivity and suitable for the detection of C. difficile in fecal.%目的:建立实时荧光PCR快速检测艰难梭菌的方法.方法:以艰难梭菌磷酸丙糖异构酶(tpi)基因的保守序列为模板设计和合成特异性引物和荧光标记探针,建立实时荧光PCR检测体系,通过检测含有艰难梭菌标准菌株浓度为10(6)-10 CFU/ml的细菌培养物及加标模拟样本进行敏感性分析,并对其特异性和干扰性进行评价.结果:该方法只对艰难梭菌进行特异性扩增,其他常见的病原菌均不能扩增;整个检测过程只需要2h,对艰难梭菌菌悬液可检测至10 CFU/ml细菌,对加标粪便样本可检测至1000 CFU/ml细菌.结论:本研究建立的实时荧光PCR检测艰难梭菌方法具有快速、特异、敏感性高等优点,能实现对艰难梭菌的快速检测.

  18. Calicivirus from novel recovirus genogroup in human diarrhea, Bangladesh

    NARCIS (Netherlands)

    S.L. Smits (Saskia); M. Rahman (Mustafizur); C.M.E. Schapendonk (Claudia); M. van Leeuwen (Marije); S.M. Faruque (Shah M.); B.L. Haagmans (Bart); H.P. Endtz (Hubert); A.D.M.E. Osterhaus (Albert)

    2012-01-01

    textabstractTo identify unknown human viruses in the enteric tract, we examined 105 stool specimens from patients with diarrhea in Bangladesh. A novel calicivirus was identified in a sample from 1 patient and subsequently found in samples from 5 other patients. Phylogenetic analyses classified this

  19. Calicivirus from Novel Recovirus Genogroup in Human Diarrhea, Bangladesh

    Science.gov (United States)

    Rahman, Mustafizur; Schapendonk, Claudia M.E.; van Leeuwen, Marije; Faruque, Abu S.G.; Haagmans, Bart L.; Endtz, Hubert P.; Osterhaus, Albert D.M.E.

    2012-01-01

    To identify unknown human viruses in the enteric tract, we examined 105 stool specimens from patients with diarrhea in Bangladesh. A novel calicivirus was identified in a sample from 1 patient and subsequently found in samples from 5 other patients. Phylogenetic analyses classified this virus within the proposed genus Recovirus. PMID:22709854

  20. Monoclonal antibody-based dipstick assay: a reliable field applicable technique for diagnosis of Schistosoma mansoni infection using human serum and urine samples.

    Science.gov (United States)

    Demerdash, Zeinab; Mohamed, Salwa; Hendawy, Mohamed; Rabia, Ibrahim; Attia, Mohy; Shaker, Zeinab; Diab, Tarek M

    2013-02-01

    A field applicable diagnostic technique, the dipstick assay, was evaluated for its sensitivity and specificity in diagnosing human Schistosoma mansoni infection. A monoclonal antibody (mAb) against S. mansoni adult worm tegumental antigen (AWTA) was employed in dipstick and sandwich ELISA for detection of circulating schistosome antigen (CSA) in both serum and urine samples. Based on clinical and parasitological examinations, 60 S. mansoni-infected patients, 30 patients infected with parasites other than schistosomiasis, and 30 uninfected healthy individuals were selected. The sensitivity and specificity of dipstick assay in urine samples were 86.7% and 90.0%, respectively, compared to 90.0% sensitivity and 91.7% specificity of sandwich ELISA. In serum samples, the sensitivity and specificity were 88.3% and 91.7% for dipstick assay vs. 91.7% and 95.0% for sandwich ELISA, respectively. The diagnostic efficacy of dipstick assay in urine and serum samples was 88.3% and 90.0%, while it was 90.8% and 93.3% for sandwich ELISA, respectively. The diagnostic indices of dipstick assay and ELISA either in serum or in urine were statistically comparable (P>0.05). In conclusion, the dipstick assay offers an alternative simple, rapid, non-invasive technique in detecting CSA or complement to stool examinations especially in field studies.

  1. Yersinia pseudotuberculosis in Italy. Attempted recovery from 37,666 samples.

    Science.gov (United States)

    Chiesa, C; Pacifico, L; Nanni, F; Renzi, A M; Ravagnan, G

    1993-01-01

    From 1981 to 1991, 37,666 human, animal, food and environmental samples were cultured for Yersinia pseudotuberculosis using direct plating methods and/or cold enhancement techniques. Despite an intensive surveillance and adequate culture methods, Y. pseudotuberculosis was isolated from stools of 0.05% (5/9,720) of patients with acute enteritis, and alimentary tracts of 0.1% (10/6,849) of apparently healthy animals. No Y. pseudotuberculosis strains were recovered from stools of 4,726 health controls nor from the appendices (656), mesenteric lymph nodes (84), and stools (421) of 656 patients operated for suspected appendicitis. Of the 10,842 food and 4,368 environmental samples, none yielded positive cultures for Y. pseudotuberculosis.

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

    Directory of Open Access Journals (Sweden)

    Chua Kaw Yan

    2011-08-01

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

  3. Giardia duodenalis in Damascus, Syria: Identification of Giardia genotypes in a sample of human fecal isolates using polymerase chain reaction and restriction fragment length polymorphism analyzing method.

    Science.gov (United States)

    Skhal, Dania; Aboualchamat, Ghalia; Al Nahhas, Samar

    2016-02-01

    Giardia duodenalis is a common gastrointestinal parasite that infects humans and many other mammals. It is most prevalent in many developing and industrialized countries. G. duodenalis is considered to be a complex species. While no morphological distinction among different assemblages exist, it can be genetically differentiated into eight major assemblages: A to H. The aim of this study was to determine the genetic heterogeneity of G. duodenalis in human isolates (a study conducted for the first time in Syria). 40 fecal samples were collected from three different hospitals during the hot summer season of 2014. Extraction of genomic DNA from all Giardia positive samples (based on a microscopic examination) was performed using QIAamp DNA Stool Mini Kit. β-giardin gene was used to differentiate between different Giardia assemblages. The 514 bp fragment was amplified using the Polymerase Chain Reaction method, followed by digestion in HaeIII restriction enzyme. Our result showed that genotype A was more frequent than genotype B, 27/40 (67.5%); 4/40 (10%) respectively. A mixed genotype of A+B was only detected in 9 isolates (22.5%). This is the first molecular study performed on G. duodenalis isolates in Syria in order to discriminate among the different genotypes. Further expanded studies using more genes are needed to detect and identify the Giardia parasite at the level of assemblage and sub-assemblage.

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

    Directory of Open Access Journals (Sweden)

    Lynn Meurs

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

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

    Science.gov (United States)

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

    2015-04-01

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

  6. Distribution of human waste samples in relation to sizing waste processing in space

    Science.gov (United States)

    Parker, Dick; Gallagher, S. K.

    1992-01-01

    Human waste processing for closed ecological life support systems (CELSS) in space requires that there be an accurate knowledge of the quantity of wastes produced. Because initial CELSS will be handling relatively few individuals, it is important to know the variation that exists in the production of wastes rather than relying upon mean values that could result in undersizing equipment for a specific crew. On the other hand, because of the costs of orbiting equipment, it is important to design the equipment with a minimum of excess capacity because of the weight that extra capacity represents. A considerable quantity of information that had been independently gathered on waste production was examined in order to obtain estimates of equipment sizing requirements for handling waste loads from crews of 2 to 20 individuals. The recommended design for a crew of 8 should hold 34.5 liters per day (4315 ml/person/day) for urine and stool water and a little more than 1.25 kg per day (154 g/person/day) of human waste solids and sanitary supplies.

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

    Science.gov (United States)

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

    2017-01-01

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

  8. Megaselia scalaris causing human intestinal myiasis in Egypt.

    Science.gov (United States)

    Mazayad, Said A M; Rifaat, Manal M A

    2005-04-01

    Megaselia scalaris is a worldwide distributed insect of medical importance. In a laboratory-based study, stool samples with undefined maggot infestation were examined and the presence of M. scalaris maggots was confirmed. Binocular stereo-microscopy was used for identification of the maggots. Larvae were allowed to develop into adults onto a human stool culture. The larvae and the emerged flies were identified using standard keys. This may be the first report of M. scalaris as a causative agent of human myiasis in Egypt. Details of the third instar larva, pupa and adults were given.

  9. Fecal specimens preparation methods for PCR diagnosis of human taeniosis

    Directory of Open Access Journals (Sweden)

    Nunes Cáris Maroni

    2006-01-01

    Full Text Available Sample preparation and DNA extraction protocols for DNA amplification by PCR, which can be applied in human fecal samples for taeniasis diagnosis, are described. DNA extracted from fecal specimens with phenol/chloroform/isoamilic alcohol and DNAzol® reagent had to be first purified to generate fragments of 170 pb and 600 pb by HDP2-PCR. This purification step was not necessary with the use of QIAmp DNA stool mini kit®. Best DNA extraction results were achieved after eggs disruption with glass beads, either with phenol/chloroform/isoamilic alcohol, DNAzol® reagent or QIAmp DNA stool mini kit®.

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

    Institute of Scientific and Technical Information of China (English)

    鄢盛恺; 林其燧; 宋耀虹; 王树琴

    2003-01-01

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

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

    Directory of Open Access Journals (Sweden)

    MM Dallal

    2015-11-01

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

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

    Science.gov (United States)

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

    2015-06-02

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

  13. Diagnostic accuracy and applicability of a PCR system for the detection of Schistosoma mansoni DNA in human urine samples from an endemic area.

    Directory of Open Access Journals (Sweden)

    Martin Johannes Enk

    Full Text Available Schistosomiasis caused by Schistosoma mansoni, one of the most neglected human parasitoses in Latin America and Africa, is routinely confirmed by microscopic visualization of eggs in stool. The main limitation of this diagnostic approach is its lack of sensitivity in detecting individual low worm burdens and consequently data on infection rates in low transmission settings are little reliable. According to the scientific literature, PCR assays are characterized by high sensitivity and specificity in detecting parasite DNA in biological samples. A simple and cost effective extraction method for DNA of Schistosoma mansoni from urine samples in combination with a conventional PCR assay was developed and applied in an endemic area. This urine based PCR system was tested for diagnostic accuracy among a population of a small village in an endemic area, comparing it to a reference test composed of three different parasitological techniques. The diagnostic parameters revealed a sensitivity of 100%, a specificity of 91.20%, positive and negative predictive values of 86.25% and 100%, respectively, and a test accuracy of 94.33%. Further statistical analysis showed a k index of 0.8806, indicating an excellent agreement between the reference test and the PCR system. Data obtained from the mouse model indicate the infection can be detected one week after cercariae penetration, opening a new perspective for early detection and patient management during this stage of the disease. The data indicate that this innovative PCR system provides a simple to handle and robust diagnostic tool for the detection of S. mansoni DNA from urine samples and a promising approach to overcome the diagnostic obstacles in low transmission settings. Furthermore the principals of this molecular technique, based on the examination of human urine samples may be useful for the diagnosis of other neglected tropical diseases that can be detected by trans-renal DNA.

  14. Hanging drop cultures of human testis and testis cancer samples

    DEFF Research Database (Denmark)

    Jørgensen, Anne; Young, J; Nielsen, J E

    2014-01-01

    limited by the lack of experimental models. The aim of this study was to establish an experimental tissue culture model to maintain normal and malignant germ cells within their niche and allow investigation of treatment effects. METHODS: Human testis and testis cancer specimens from orchidectomies were...

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

    Science.gov (United States)

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

    2015-02-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  17. Characterizing healthy samples for studies of human cognitive aging

    OpenAIRE

    Geldmacher, David S.; Levin, Bonnie E.; Wright, Clinton B.

    2012-01-01

    Characterizing the cognitive declines associated with aging, and differentiating them from the effects of disease in older adults, are important goals for human neuroscience researchers. This is also an issue of public health urgency in countries with rapidly aging populations. Progress toward understanding cognitive aging is complicated by numerous factors. Researchers interested in cognitive changes in healthy older adults need to consider these complexities when they design and interpre...

  18. Heterogeneity of Campylobacter species isolated from serial stool specimens of Egyptian children using pulsed field gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Atef M. El-Gendy

    2013-03-01

    Full Text Available Background: The genus Campylobacter spp. is a common cause of human acute bacteria lenteritis and travellers’ diarrhoea worldwide.Objective: To determine whether multiple serial isolations of Campylobacter spp., when obtained from a single child, represented the same or a different organism.Methods: In a birth cohort study conducted in Egypt, numerous children showed serial isolations of Campylobacter spp. Of these, 13 children were selected from different households based on the successive isolation of six or more Campylobacter isolates from stool samples.Results: Eighty isolates were recovered and identified as either Campylobacter coli (n = 25 or Campylobacter jejuni (n = 55. Pulsed-field gel electrophoresis (PFGE revealed the presence of 38 unique C. jejuni and 24 C. coli profiles at a similarity level of ≥ 90%. Although no seriallyidentical isolates were detected in six children, others demonstrated at least one identical couple of isolates; all identified serially between one to six weeks. Two children demonstrated > 80% similar couples of isolates that appeared seven months apart. PFGE could be a useful tool for differentiating reinfection, relapse and convalescent excretion phases.Conclusion: Our data suggest that Campylobacter infection in children is a complex process; children are exposed to multiple species in endemic environments and strains of the same bacterium appear to be shed serially between one to six weeks after the first exposure. Isolates that persisted for longer periods were relatively less similar, as shown from the results of this study.

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

    Science.gov (United States)

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

    2015-10-01

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

  20. Sampling Based Trajectory Planning for Robots in Dynamic Human Environments

    DEFF Research Database (Denmark)

    Svenstrup, Mikael

    2010-01-01

    Open-ended human environments, such as pedestrian streets, hospital corridors, train stations etc., are places where robots start to emerge. Hence, being able to plan safe and natural trajectories in these dynamic environments is an important skill for future generations of robots. In this work...... method for selecting the best trajectory in the RRT, according to the cost of traversing a potential field. Furthermore the RRT expansion is enhanced to direct the search and account for the kinodynamic robot constraints. A model predictive control (MPC) approach is taken to accommodate...

  1. Sampling

    CERN Document Server

    Thompson, Steven K

    2012-01-01

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

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

    Science.gov (United States)

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

    2017-01-05

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

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

    Institute of Scientific and Technical Information of China (English)

    Tahereh Falsafi; Raha Favaedi; Fatemeh Mahjoub; Mehri Najafi

    2009-01-01

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

  4. [Analysis of human tissue samples for volatile fire accelerants].

    Science.gov (United States)

    Treibs, Rudolf

    2014-01-01

    In police investigations of fires, the cause of a fire and the fire debris analysis regarding traces of fire accelerants are important aspects for forensic scientists. Established analytical procedures were recently applied to the remains of fire victims. When examining lung tissue samples, vapors inhaled from volatile ignitable liquids could be identified and differentiated from products of pyrolysis caused by the fire. In addition to the medico-legal results this evidence allowed to draw conclusions as to whether the fire victim was still alive when the fire started.

  5. Human papillomavirus self-sampling for screening nonattenders

    DEFF Research Database (Denmark)

    Lam, Janni Uyen Hoa; Rebolj, Matejka; Ejegod, Ditte Møller

    2017-01-01

    In organized cervical screening programs, typically 25% of the invited women do not attend. The Copenhagen Self-sampling Initiative (CSi) aimed to gain experiences on participation among screening nonattenders in the Capital Region of Denmark. Here, we report on the effectiveness of different...... region of Denmark were identified via the organized national invitation module. Screening history was obtained via the nationwide pathology registry. Twenty-four thousand women were invited, and as an alternative to the regular communication platforms (letter and phone), women could request a home test...... via a mobile-friendly webpage. Instruction material and video-animation in several languages were made available online. Chi-square test was used to test differences. Out of all invited, 31.7% requested a home test, and 20% returned it to the laboratory. In addition, 10% were screened at the physician...

  6. Reducing sample complexity of polyclonal human autoantibodies by chromatofocusing.

    Science.gov (United States)

    Hagemann, Sascha; Faude, Alexander; Rabenstein, Monika; Balzer-Geldsetzer, Monika; Nölker, Carmen; Bacher, Michael; Dodel, Richard

    2010-08-15

    Chromatofocusing was performed in order to separate a polyclonal antigen-specific mixture of human immunoglobulins (IgGs) that would then allow for further analyses of as few different IgGs as possible. Because polyclonal IgGs only differ by amino acid sequence and possible post-translational modifications but not by molecular weight, we chose chromatofocusing for protein separation by different isoelectric points. We isolated antigen-specific IgGs from commercially available intravenous immunoglobulins (IVIG) using a combination of affinity- and size exclusion-chromatography and in order to reduce the complexity of the starting material IVIG was then replaced by single-donor plasmapheresis material. Using two-dimensional gel electrophoresis (2-DE), we observed a clear decrease in the number of different light and heavy chains in the chromatofocusing peak as compared to the starting material. In parallel, we monitored slight problems with the selected peak in isoelectric focusing as the first dimension of 2-DE, displayed in by the less proper focusing of the spots. When we tested whether IgGs were binding to their specific antigen after chromatofocusing, we were able to show that they were still in native conformation. In conclusion, we showed that chromatofocusing can be used as a first step in the analysis of mixtures of very similar proteins, e.g. polyclonal IgG preparations, in order to minimize the amount of different proteins in separated fractions in a reproducible way. Copyright 2010 Elsevier B.V. All rights reserved.

  7. Intestinal parasites and genotyping of Giardia duodenalis in children: first report of genotype B in isolates from human clinical samples in Mexico

    OpenAIRE

    Julio César Torres-Romero; Antonio de Jesus Euan-Canto; Namibya Benito-González; Nayely Padilla-Montaño; Claribel Huchin-Chan; Julio Lara-Riegos; Roberto Cedillo-Rivera

    2014-01-01

    Giardia duodenalis is one of the most prevalent enteroparasites in children. This parasite produces several clinical manifestations. The aim of this study was to determine the prevalence of genotypes of G. duodenalis causing infection in a region of southeastern Mexico. G. duodenalis cysts were isolated (33/429) from stool samples of children and molecular genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, targeting the trioseph...

  8. Assessment of human exposure to airborne fungi in agricultural confinements: personal inhalable sampling versus stationary sampling.

    Science.gov (United States)

    Adhikari, Atin; Reponen, Tiina; Lee, Shu-An; Grinshpun, Sergey A

    2004-01-01

    Accurate exposure assessment to airborne fungi in agricultural environments is essential for estimating the associated occupational health hazards of workers. The objective of this pilot study was to compare personal and stationary sampling for assessing farmers' exposure to airborne fungi in 3 different agricultural confinements located in Ohio, USA (hog farm, dairy farm, and grain farm), using Button Personal Inhalable Samplers. Personal exposures were measured with samplers worn by 3 subjects (each carrying 2 samplers) during 3 types of activities, including animal feeding in the hog farm, cleaning and animal handling in the dairy farm, and soybean unloading and handling in the grain farm. Simultaneously, the stationary measurements were performed using 5 static Button Samplers and 1 revolving Button Sampler. The study showed that the total concentration of airborne fungi ranged from 1.4 x 10(4)-1.2 x 10(5) spores m(-3) in 3 confinements. Grain unloading and handling activity generated highest concentrations of airborne fungi compared to the other 2 activities. Prevalent airborne fungi belonged to Cladosporium, Aspergillus/Penicillium, Ascospores, smut spores, Epicoccum, Alternaria, and Basidiospores. Lower coefficients of variations were observed for the fungal concentrations measured by personal samplers (7-12%) compared to the concentrations measured by stationary samplers (27-37%). No statistically significant difference was observed between the stationary and personal measurement data for the total concentrations of airborne fungi (p > 0.05). Revolving stationary and static stationary Button Samplers demonstrated similar performance characteristics for the collection of airborne fungi. This reflects the low sensitivity of the sampler's efficiency to the wind speed and direction. The results indicate that personal exposure of agricultural workers in confinements may be adequately assessed by placing several Button Samplers simultaneously operating in a

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

    Directory of Open Access Journals (Sweden)

    Candace L Williams

    2016-05-01

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

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

    Science.gov (United States)

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

    2017-03-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

  12. Optimization of techniques for multiple platform testing in small, precious samples such as human chorionic villus sampling.

    Science.gov (United States)

    Pisarska, Margareta D; Akhlaghpour, Marzieh; Lee, Bora; Barlow, Gillian M; Xu, Ning; Wang, Erica T; Mackey, Aaron J; Farber, Charles R; Rich, Stephen S; Rotter, Jerome I; Chen, Yii-der I; Goodarzi, Mark O; Guller, Seth; Williams, John

    2016-11-01

    Multiple testing to understand global changes in gene expression based on genetic and epigenetic modifications is evolving. Chorionic villi, obtained for prenatal testing, is limited, but can be used to understand ongoing human pregnancies. However, optimal storage, processing and utilization of CVS for multiple platform testing have not been established. Leftover CVS samples were flash-frozen or preserved in RNAlater. Modifications to standard isolation kits were performed to isolate quality DNA and RNA from samples as small as 2-5 mg. RNAlater samples had significantly higher RNA yields and quality and were successfully used in microarray and RNA-sequencing (RNA-seq). RNA-seq libraries generated using 200 versus 800-ng RNA showed similar biological coefficients of variation. RNAlater samples had lower DNA yields and quality, which improved by heating the elution buffer to 70 °C. Purification of DNA was not necessary for bisulfite-conversion and genome-wide methylation profiling. CVS cells were propagated and continue to express genes found in freshly isolated chorionic villi. CVS samples preserved in RNAlater are superior. Our optimized techniques provide specimens for genetic, epigenetic and gene expression studies from a single small sample which can be used to develop diagnostics and treatments using a systems biology approach in the prenatal period. © 2016 John Wiley & Sons, Ltd. © 2016 John Wiley & Sons, Ltd.

  13. Detection and Characterization of Shiga Toxin Producing Escherichia coli, Salmonella spp., and Yersinia Strains from Human, Animal, and Food Samples in San Luis, Argentina.

    Science.gov (United States)

    Favier, Gabriela Isabel; Lucero Estrada, Cecilia; Cortiñas, Teresa Inés; Escudero, María Esther

    2014-01-01

    Shiga toxin producing Escherichia coli (STEC), Salmonella spp., and Yersinia species was investigated in humans, animals, and foods in San Luis, Argentina. A total of 453 samples were analyzed by culture and PCR. The antimicrobial susceptibility of all the strains was studied, the genomic relationships among isolates of the same species were determined by PFGE, and the potencial virulence of Y. enterocolitica strains was analyzed. Yersinia species showed higher prevalence (9/453, 2.0%, 95% CI, 0.7-3.3%) than STEC (4/453, 0.9%, 95% CI, 0-1.8%) and Salmonella spp. (3/453, 0.7%, 95% CI, 0-1.5%). Y. enterocolitica and Y. intermedia were isolated from chicken carcasses (6/80, 7.5%, 95% CI, 1.5-13.5%) and porcine skin and bones (3/10, 30%, 95% CI, 0-65%). One STEC strain was recovered from human feces (1/70, 1.4%, 95% CI, 0-4.2%) and STEC stx1/stx2 genes were detected in bovine stools (3/129, 2.3%, 95% CI, 0-5.0%). S. Typhimurium was isolated from human feces (1/70, 1.4%, 95% CI, 0-4.2%) while one S. Newport and two S. Gaminara strains were recovered from one wild boar (1/3, 33%, 95% CI, 0-99%). The knowledge of prevalence and characteristics of these enteropathogens in our region would allow public health services to take adequate preventive measures.

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

    Science.gov (United States)

    Ledbetter, Laura

    2006-08-01

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

  15. Prevalence of Arcobacter spp. in humans, animals and foods of animal origin including sea food from India.

    Science.gov (United States)

    Patyal, A; Rathore, R S; Mohan, H V; Dhama, K; Kumar, A

    2011-10-01

    The present study reports the prevalence of Arcobacter, an emerging pathogen in human, animals and foods of animal origin in India. A total of 600 samples from various sources, viz. diarrhoeal stools of humans and dogs, faecal swabs of animals (pig, poultry), preputial washings of breeding bulls and food samples (chicken, pork, fish) were examined for presence of Arcobacter spp. Using cultural methods, a total of 63 Arcobacter spp. were isolated of 600 (10.50%) samples with highest isolation rate were from pig faeces (21.33%) followed by sea foods (17.33%), poultry faeces (14.67%), pork (16.00%), chicken meat (12.00%) and human stools (2.67%). The isolates were confirmed as arcobacters by genus-based PCR. PCR screening of all the enriched samples revealed the overall prevalence of Arcobacter spp. to be 12.00% with highest in pig (25.33%), followed by sea food (21.33%), poultry (17.33%), pork (16%), chicken meat (12%) and human stools (4.00%). No Arcobacter spp. was isolated or detected from diarrhoeal faecal samples of dogs and preputial washings. With multiplex PCR, three different species were detected (A. butzleri, A. cryaerophilus and A. skirrowii) with most of the samples showing mixed infections. There are only two recent reports from India; one with cultural isolation and another with PCR detection of Arcobacter spp. in stool samples of humans with clinical diarrhoea. In this context, our present report is the first report of isolation and detection of Arcobacter spp. from various sources of animals and foods including diarrhoeic human stool samples, utilizing both cultural and molecular tools identifying arcobacters at genus and species level. These results support the importance of arcobacters as an emerging food-borne pathogen, possessing zoonotic potential.

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

    Science.gov (United States)

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

    2016-05-01

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

  17. Comparison of 2 chromogenic media for the detection of extended-spectrum β-lactamase producing Enterobacteriaceae stool carriage in nursing home residents.

    Science.gov (United States)

    Blane, Beth; Brodrick, Hayley J; Gouliouris, Theodore; Ambridge, Kirsty E; Kidney, Angela D; Ludden, Catherine M; Limmathurotsakul, Direk; Török, M Estée; Peacock, Sharon J

    2016-03-01

    ChromID ESBL agar and Brilliance ESBL agar were compared for the isolation of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae from 298 stools. These had comparable sensitivity and selectivity for the 116 positive samples. Pre-enrichment with cefpodoxime and extending incubation to 48 hours after direct plating both significantly increased sensitivity but reduced selectivity of both agars.

  18. The correlation between Clostridium-difficile infection and human gut concentrations of Bacteroidetes phylum and clostridial species.

    Science.gov (United States)

    Goldberg, E; Amir, I; Zafran, M; Gophna, U; Samra, Z; Pitlik, S; Bishara, J

    2014-03-01

    We aimed to assess differences in bacterial intensities of Bacteroidetes phylum and different clostridial species in the human intestines with respect to C. difficile infection. Patients with a stool assay for C. difficile toxin were identified via the microbiology laboratory in our institute. Bacterial populations were quantified from stool samples of four groups of patients: Group I-patients with C. difficile associated diarrhea (CDAD); Group II-asymptomatic C. difficile carriers; Group III-patients with non-C. difficile diarrhea; Group IV-patients with no diarrhea and negative stool samples for the C. difficile toxin (control group). Stool was examined for three genes-C. difficile toxin A gene, 16S rRNA gene from Clostridium thermocellum representing other clostridial species, and 16S rRNA gene from Bacteroides fragilis representing the Bacteroidetes phylum. Fifty-nine patients underwent analysis of the stool (CDAD group 14, carriers group 14, non-C. difficile diarrhea group 16, control group 15). C. difficile concentration was highest in the CDAD group, followed by the carriers group. Higher concentrations of both clostridial species and Bacteriodetes were observed in the control and non-C. difficile diarrhea groups compared to the CDAD and carriers groups. We demonstrated an inverse association between infection with C. difficile and the abundance of Bacteroidetes phylum and other clostridial species in human intestines. Studies with larger samples and broader diagnostic procedures are needed in order to better explore and understand this association.

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

    Directory of Open Access Journals (Sweden)

    Arena Christophe

    2012-06-01

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

  20. Human DNA quantification and sample quality assessment: Developmental validation of the PowerQuant(®) system.

    Science.gov (United States)

    Ewing, Margaret M; Thompson, Jonelle M; McLaren, Robert S; Purpero, Vincent M; Thomas, Kelli J; Dobrowski, Patricia A; DeGroot, Gretchen A; Romsos, Erica L; Storts, Douglas R

    2016-07-01

    Quantification of the total amount of human DNA isolated from a forensic evidence item is crucial for DNA normalization prior to short tandem repeat (STR) DNA analysis and a federal quality assurance standard requirement. Previous commercial quantification methods determine the total human DNA and total human male DNA concentrations, but provide limited information about the condition of the DNA sample. The PowerQuant(®) System includes targets for quantification of total human and total human male DNA as well as targets for evaluating whether the human DNA is degraded and/or PCR inhibitors are present in the sample. A developmental validation of the PowerQuant(®) System was completed, following SWGDAM Validation Guidelines, to evaluate the assay's specificity, sensitivity, precision and accuracy, as well as the ability to detect degraded DNA or PCR inhibitors. In addition to the total human DNA and total human male DNA concentrations in a sample, data from the degradation target and internal PCR control (IPC) provide a forensic DNA analyst meaningful information about the quality of the isolated human DNA and the presence of PCR inhibitors in the sample that can be used to determine the most effective workflow and assist downstream interpretation.

  1. Human fascioliasis and anaemia in Dakahlia Governorate, Egypt.

    Science.gov (United States)

    El-Shazly, Atef M; El-Nahas, Hala A; Abdel-Mageed, A A; El Beshbishi, S N; Azab, M S; Abou El Hasan, M; Arafa, Wafaa A S; Morsy, Tosson A

    2005-08-01

    Fasciola infection (fascioliasis) appeared to be endemic in Egypt. Stool samples of fourty eight patients were coprologically diagnosed. According to Fasciola egg counting per gram stool, the severity of infection was divided into light infection in 60.5%, moderate in 27.1% and severe infection in 12.5%. No significant correlation was detected between severity of infection and patients' sex. Complete blood picture, reticylocytic count, serum iron, immunological assays as anti-nuclear, anti-smooth muscle antibody, anti-mitochondrial anti-body, anti-DNA tests and rheumatoid factor and occult blood in stool were investigated. Normocytic normochromic anaemia was detected in 62.5% of the fascioliasis patients, microcytic hypochromic anaemia in 31.3% and macrocytic one in 6.3%. Highly significant negative correlation (R = -0.68) was detected between haemoglobin concentration and egg count per gram faeces. Human fascioliasis was associated with normocytic normochromic anaemia and to a lesser extent microcytic hypochromic anemia.

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

    Directory of Open Access Journals (Sweden)

    Tugba Koca

    2014-02-01

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

  3. Human parechovirus as a minor cause of acute otitis media in children.

    Science.gov (United States)

    Sillanpää, Saara; Oikarinen, Sami; Sipilä, Markku; Seppälä, Elina; Nurminen, Noora; Rautiainen, Markus; Laranne, Jussi; Hyöty, Heikki

    2015-01-01

    Human parechoviruses (HPeVs) cause mild upper respiratory infections, gastrointestinal symptoms, central nervous system infections and some studies have linked them with acute otitis media (AOM). The aim of the present study was to study further the role of HPeV infections in AOM by detecting these viruses directly from middle ear fluid (MEF), respiratory and stool samples collected from children during AOM episodes. A total of 91 MEF samples, 98 nasal swab (NS) samples and 92 stool samples were collected during 100 AOM episodes in a total of 87 children aged between five to 42 months. All specimens were analyzed by real time RT-PCR for the presence of HPeV RNA. HPeV infection was diagnosed in 12 (14%) patients. HPeV RNA was detected in altogether 13 samples, including four MEF samples, three NS samples and six stool samples. One patient was positive in both stool and MEF samples. The results suggest that HPeV may play a role in some AOM cases, but it is not a major cause of AOM in children.

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

    Science.gov (United States)

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

    2017-01-01

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

  5. Bacteria-human somatic cell lateral gene transfer is enriched in cancer samples.

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    David R Riley

    Full Text Available There are 10× more bacterial cells in our bodies from the microbiome than human cells. Viral DNA is known to integrate in the human genome, but the integration of bacterial DNA has not been described. Using publicly available sequence data from the human genome project, the 1000 Genomes Project, and The Cancer Genome Atlas (TCGA, we examined bacterial DNA integration into the human somatic genome. Here we present evidence that bacterial DNA integrates into the human somatic genome through an RNA intermediate, and that such integrations are detected more frequently in (a tumors than normal samples, (b RNA than DNA samples, and (c the mitochondrial genome than the nuclear genome. Hundreds of thousands of paired reads support random integration of Acinetobacter-like DNA in the human mitochondrial genome in acute myeloid leukemia samples. Numerous read pairs across multiple stomach adenocarcinoma samples support specific integration of Pseudomonas-like DNA in the 5'-UTR and 3'-UTR of four proto-oncogenes that are up-regulated in their transcription, consistent with conversion to an oncogene. These data support our hypothesis that bacterial integrations occur in the human somatic genome and may play a role in carcinogenesis. We anticipate that the application of our approach to additional cancer genome projects will lead to the more frequent detection of bacterial DNA integrations in tumors that are in close proximity to the human microbiome.

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

    Science.gov (United States)

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

    2008-09-01

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

  7. Mitochondrial Respiration Chain Enzymatic Activities in the Human Brain: Methodological Implications for Tissue Sampling and Storage.

    Science.gov (United States)

    Ronsoni, Marcelo Fernando; Remor, Aline Pertile; Lopes, Mark William; Hohl, Alexandre; Troncoso, Iris H Z; Leal, Rodrigo Bainy; Boos, Gustavo Luchi; Kondageski, Charles; Nunes, Jean Costa; Linhares, Marcelo Neves; Lin, Kátia; Latini, Alexandra Susana; Walz, Roger

    2016-04-01

    Mitochondrial respiratory chain complexes enzymatic (MRCCE) activities were successfully evaluated in frozen brain samples. Epilepsy surgery offers an ethical opportunity to study human brain tissue surgically removed to treat drug resistant epilepsies. Epilepsy surgeries are done with hemodynamic and laboratory parameters to maintain physiology, but there are no studies analyzing the association among these parameters and MRCCE activities in the human brain tissue. We determined the intra-operative parameters independently associated with MRCCE activities in middle temporal neocortex (Cx), amygdala (AMY) and head of hippocampus (HIP) samples of patients (n = 23) who underwent temporal lobectomy using multiple linear regressions. MRCCE activities in Cx, AMY and HIP are differentially associated to trans-operative mean arterial blood pressure, O2 saturation, hemoglobin, and anesthesia duration to time of tissue sampling. The time-course between the last seizure occurrence and tissue sampling as well as the sample storage to biochemical assessments were also associated with enzyme activities. Linear regression models including these variables explain 13-17 % of MRCCE activities and show a moderate to strong effect (r = 0.37-0.82). Intraoperative hemodynamic and laboratory parameters as well as the time from last seizure to tissue sampling and storage time are associated with MRCCE activities in human samples from the Cx, AMYG and HIP. Careful control of these parameters is required to minimize confounding biases in studies using human brain samples collected from elective neurosurgery.

  8. Molecular identification and characterization of Mycobacterium avium subspecies paratuberculosis in free living non-human primate (Rhesus macaques) from North India.

    Science.gov (United States)

    Singh, S V; Singh, A V; Singh, P K; Kumar, A; Singh, B

    2011-05-01

    In recent years, Mycobacterium avium subspecies paratuberculosis (MAP) has emerged as major animal pathogen with significant zoonotic concerns, worldwide. MAP infection is endemic in domestic and wild ruminant population in India. However, information on MAP infection in free ranging animal species and non human primates is limited. Present study aimed to estimate the status of MAP infection in free living Rhesus macaques suffering with multiple clinical conditions (coughing and loose stool). A total of 25 stool samples were collected from six colonies of Rhesus macaques from Mathura region (North India) and screened for the presence of MAP, using microscopic examination and IS900 PCR, directly from stool samples. PCR positive DNA samples were further genotyped using IS1311 PCR-restriction enzyme analysis. Of the 25 stool samples, 10 (40.0%) and 2 (8.0%) were positive for MAP using microscopic examination and direct IS900 PCR, respectively. IS900 PCR positive DNA samples were genotyped as 'Indian Bison type', which is a major MAP genotype infecting domestic and wild ruminant species and human beings in India. Prevalence of MAP in Rhesus macaques (Indian monkeys) was moderately high and confirmed interspecies sharing of MAP between domestic livestock and non-human primates. Presence of MAP in non-human primates, support the etiological role of MAP in inflammatory bowel disease patients. Indian monkeys may serve as model for understanding the role of non-human primates in sustenance, transmission and pathogenesis of MAP infection.

  9. A duplex PCR for rapid and simultaneous detection of Brucella spp. in human blood samples.

    Science.gov (United States)

    Mirnejad, Reza; Mohamadi, Mozafar; Piranfar, Vahbeh; Mortazavi, Seied Mojtaba; Kachuei, Reza

    2013-06-01

    To design a duplex PCR for rapid and simultaneous detection of Brucella species. in human blood samples. Fifty-two peripheral bloods samples were collected from suspicious patients with brucellosis. Following DNA extraction, PCR assay were performed, using three primers that could simultaneously identify and differentiate three major species of pathogenic Brucella in humans and animals. Of the 52 peripheral bloods samples tested, 25 sample (48%) showed positive reactions in PCR. Twelve samples were positive for Brucella abortus 39 (B. abortus 39) (23%), 13 for Brucella melitensis 39 (B. melitensis 39) (25%) and 0 for Brucella ovis 39 (B. ovis 39) (0%). This work demonstrates that in case where specific primers were utilized, duplex PCR has proved to be a simple, fast, and relatively inexpensive method for simultaneous detection of important species of Brucella in clinical samples. Copyright © 2013 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

  10. A duplex PCR for the rapid and simultaneous detection of Brucella spp. in human blood samples

    Institute of Scientific and Technical Information of China (English)

    Reza Mirnejad; Mozafar mohamadi; Vahbeh Piranfar; Seied Mojtaba Mortazavi; Reza Kachuei

    2013-01-01

    Objective: To design a duplex PCR for rapid and simultaneous detection of Brucella species. in human blood samples. Methods: Fifty-two peripheral bloods samples were collected from suspicious patients with brucellosis. Following DNA extraction, PCR assay were performed, using three primers that could simultaneously identify and differentiate three major species of pathogenic Brucella in humans and animals. Results: Of the 52 peripheral bloods samples tested, 25 sample (48%) showed positive reactions in PCR. Twelve samples were positive for Brucella abortus (B. abortus) (23%), 13 for Brucella melitensis (B. melitensis) (25%) and 0 for Brucella ovis (B. ovis) (0%). Conclusions: This work de=monstrates that in case where specific primers were utilized, duplex PCR has proved to be a simple, fast, and relatively inexpensive method for simultaneous detection of important species of Brucella in clinical samples.

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

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    Lester M Shulman

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

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

    Science.gov (United States)

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

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

    Science.gov (United States)

    Guendel, Lutz; Suehling, Michael; Eckert, Helmut

    2008-03-01

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

  14. Isolation of Bacteroides from fish and human fecal samples for identification of unique molecular markers.

    Science.gov (United States)

    Kabiri, Leila; Alum, Absar; Rock, Channah; McLain, Jean E; Abbaszadegan, Morteza

    2013-12-01

    Bacteroides molecular markers have been used to identify human fecal contamination in natural waters, but recent work in our laboratory confirmed cross-amplification of several human-specific Bacteroides spp. assays with fecal DNA from fish. For identification of unique molecular markers, Bacteroides from human (n = 4) and fish (n = 7) fecal samples were cultured and their identities were further confirmed using Rapid ID 32A API strips. The 16S rDNA from multiple isolates from each sample was PCR amplified, cloned, and sequenced to identify unique markers for development of more stringent human-specific assays. In human feces, Bacteroides vulgatus was the dominant species (75% of isolates), whereas in tilapia feces, Bacteroides eggerthii was dominant (66%). Bacteroides from grass carp, channel catfish, and blue catfish may include Bacteroides uniformis, Bacteroides ovatus, or Bacteroides stercoris. Phylogenic analyses of the 16S rRNA gene sequences showed distinct Bacteroides groupings from each fish species, while human sequences clustered with known B. vulgatus. None of the fish isolates showed significant similarity to Bacteroides sequences currently deposited in NCBI (National Center for Biotechnology Information). This study expands the current sequence database of cultured fish Bacteroides. Such data are essential for identification of unique molecular markers in human Bacteroides that can be utilized in differentiating fish and human fecal contamination in water samples.

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

    Science.gov (United States)

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

    2016-06-01

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

  16. Genotyping of cystic echinococcosis isolates from clinical samples of human and domestic animals

    Directory of Open Access Journals (Sweden)

    S.A. Fadhil

    2016-12-01

    Full Text Available Cystic hydatid disease is a cosmopolitan important disease in both human and animals. Many strains were investigated in this parasite. The aim of study was to characterize genotype variations of Echinococcus granulosus isolates collected from human and domestic animals in Al-Qadisiyah province/ Iraq based on sequencing of nad1 mitochondrial gene. Eighty hydatid cysts of human (12, sheep (15, cattle (36, and camels (17 were collected from hospital and slaughter house of the province, during October 2014 to June 2015; microscopic examination was made for cysts fluid to determine the fertility. DNAs extraction was done for each sample in addition to purify and concentrate of extracted DNA samples was performed to determine nad1 (400bp gene used conventional PCR method. Phylogenetic analysis was performed using NCBI-Blast Alignment identification and Unweighted Pair Group Method with Arithmetic Mean. Twenty five (10 from human and 5 from each studied animals samples were chosen due to their fertility and high DNA purity, in which three strains (genotypes were investigated including sheep strain (G1 40%, buffalo strain (G3 48% and camel strain (G6 12%, where human samples related to G1(20% and G3(80%; sheep samples related to G1(80% and G3(20%; cattle samples related to G1(60%, G3 (20% and G6 (20%; camels samples related to G1(20%, G3(40% and G6(40%. The dominant strain is a buffalo strain (G3; both of buffalo strain (G3 and sheep strain (G1 represented the actual source of human infection. There is no host specificity of detected genotypes.

  17. Human toxocariosis: Seroprevalence in Lima inhabitans by ELISA technique

    OpenAIRE

    ESPINOZA, YRMA; Instituto de Medicina Tropical “Daniel A. Carrión”, Facultad de Medicina, UNMSM; Departamento Académico de Microbiología Médica Facultad de Medicina, UNMSM; Huapaya, Pedro; Instituto de Medicina Tropical “Daniel A. Carrión”, Universidad Nacional Mayor de San Marcos; Sevilla, Carlos; Departamento Académico de Microbiología Médica; Huiza, Alina; Instituto de Medicina Tropical “Daniel A. Carrión”, Universidad Nacional Mayor de San Marcos, Lima, Perú. Bióloga.; Jiménez, Susana; Departamento Académico de Microbiología Médica, Facultad de Medicina, Universidad Nacional Mayor de San Marcos. Lima, Perú.; Náquira, César; Instituto de Medicina Tropical “Daniel A. Carrión”, UNSMM; Instituto Nacional de Salud

    2013-01-01

    Objective: To estimate seroprevalence of human toxocariosis in Lima inhabitants. Design: Cross-sectional study, non aleatory selection. Material and methods: To people living in Lima city urban marginal communities, an interview and clinical examination were done and serum samples obtained to detect antibodies against Toxocara by ELISA technique. Stool samples were also obtained to check parasites causing cross reactions with serology. Results: From 553 persons examined 23,3% were reactive. T...

  18. Is real-time PCR-based diagnosis similar in performance to routine parasitological examination for the identification of Giardia intestinalis, Cryptosporidium parvum/Cryptosporidium hominis and Entamoeba histolytica from stool samples? Evaluation of a new commercial multiplex PCR assay and literature review.

    Science.gov (United States)

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

    2016-02-01

    Microscopy is the reference standard for routine laboratory diagnosis in faecal parasitology but there is growing interest in alternative methods to overcome the limitations of microscopic examination, which is time-consuming and highly dependent on an operator's skills and expertise. Compared with microscopy, DNA detection by PCR is simple and can offer a better turnaround time. However, PCR performances remain difficult to assess as most studies have been conducted on a limited number of positive clinical samples and used in-house PCR methods. Our aim was to evaluate a new multiplex PCR assay (G-DiaParaTrio; Diagenode Diagnostics), targeting Giardia intestinalis, Cryptosporidium parvum/Cryptosporidium hominis and Entamoeba histolytica. To minimize the turnaround time, PCR was coupled with automated DNA extraction (QiaSymphony; Qiagen). The PCR assay was evaluated using a reference panel of 185 samples established by routine microscopic examination using a standardized protocol including Ziehl-Neelsen staining and adhesin detection by ELISA (E. histolytica II; TechLab). This panel, collected from 12 French parasitology laboratories, included 135 positive samples for G. intestinalis (n = 38), C. parvum/C. hominis (n = 26), E. histolytica (n = 5), 21 other gastrointestinal parasites, together with 50 negative samples. In all, the G-DiaParaTrio multiplex PCR assay identified 38 G. intestinalis, 25 C. parvum/C. hominis and five E. histolytica leading to sensitivity/specificity of 92%/100%, 96%/100% and 100%/100% for G. intestinalis, C. parvum/C. hominis and E. histolytica, respectively. This new multiplex PCR assay offers fast and reliable results, similar to microscopy-driven diagnosis for the detection of these gastrointestinal protozoa, allowing its implementation in routine clinical practice.

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2016-10-01

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

  1. Scientific Opinion on the substantiation of a health claim related to beta-palmitate and contribution to softening of stools pursuant to Article 14 of Regulation (EC No 1924/2006

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    EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA

    2014-02-01

    Full Text Available Following an application from Specialised Nutrition Europe (formerly IDACE, submitted for authorisation of a health claim pursuant to Article 14 of Regulation (EC No 1924/2006 via the Competent Authority of France, the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA was asked to deliver an opinion on the scientific substantiation of a health claim related to beta-palmitate and contribution to softening of stools. The food constituent, beta-palmitate, that is the subject of the health claim, is sufficiently characterised. Contribution to softening of stools is a beneficial physiological effect for infants. In weighing the evidence the Panel took into account that, out of two human intervention studies with important methodological limitations, one suggested a stool-softening effect of beta-palmitate whereas the second did not, that one animal study did not support a stool-softening effect of beta-palmitate, and that the evidence provided for a mechanism by which beta-palmitate could contribute to the softening of stools is weak. The Panel concludes that a cause and effect relationship has not been established between the consumption of beta-palmitate and softening of stools.

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

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    Rajesh Kumar

    2016-06-01

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

  3. Simultaneous detection and differentiation of Entamoeba histolytica, E. dispar, E. moshkovskii, Giardia lamblia and Cryptosporidium spp. in human fecal samples using multiplex PCR and qPCR-MCA.

    Science.gov (United States)

    Zebardast, Nozhat; Yeganeh, Farshid; Gharavi, Mohammad Javad; Abadi, Alireza; Seyyed Tabaei, Seyyed Javad; Haghighi, Ali

    2016-10-01

    Entamoeba histolytica, Giardia lamblia and Cryptosporidium spp. are common causes of diarrheal and intestinal diseases all over the world. Microscopic methods are useful in the diagnosis of intestinal parasites (IPs), but their sensitivity was assessed approximately 60 percent. Recently, molecular techniques have been used increasingly for the identification and characterization of the parasites. Among those, in this study we have used multiplex PCR and Real-time PCR with melting curve analysis (qPCR-MCA) for simultaneous detection and differentiation of E. histolytica, E. dispar, E. moshkovskii, G. lamblia and Cryptosporidium spp. in human fecal samples. Twenty DNA samples from 12 E. histolytica and 8 E. dispar samples and twenty stool samples confirmed positive for G. lamblia and Cryptosporidium spp. were analyzed. After DNA extraction from the samples, multiplex PCR was done for detection and differentiation of above mentioned parasites. QPCR-MCA was also performed for the detection and differentiation of 11 isolates of above mentioned parasite in a cycle with a time and temperature. Multiplex PCR was able to simultaneous detect and differentiate of above mentioned parasite in a single reaction. QPCR-MCA was able to differentiate genus and species those five protozoa using melting temperature simultaneously at the same time and temperature programs. In total, qPCR-MCA diagnosed 7/11 isolation of E. histolytica, 6/8 isolation of E. dispar, 1/1 E. moshkovskii Laredo, 10/11 G. Lamblia and 6/11 Cryptosporidium spp. Application of multiplex PCR for detection of more than one species in a test in developing countries, at least in reference laboratories has accurate diagnosis and plays a critical role in differentiation of protozoan species. Multiplex PCR assay with a template and multi template had different results and it seems that using a set of primers with one template has higher diagnostic capability in compare with multi template. The results of this study

  4. ELISA y técnica de sedimentación espontánea para el diagnóstico de infección por Giardia lamblia en muestras fecales de niños de Perú ELISA and spontaneous sedimentation technique for the diagnosis of Giardia lamblia infection in stool samples of Peruvian children

    Directory of Open Access Journals (Sweden)

    Claudia Rodríguez-Ulloa

    2011-12-01

    Full Text Available OBJETIVO: Comparar un kit ELISA comercial para coproantígenos y la técnica de sedimentación espontánea en tubo (TSET para el diagnóstico de Giardia lamblia en muestras fecales de niños de una zona endémica peruana. MATERIAL Y MÉTODOS: Fueron analizadas 174 muestras mediante la TSET y el kit Giardia 2nd Generation ELISA. RESULTADOS: Fueron positivas 51 muestras por ELISA y 49 por TSET. CONCLUSIONES: El ELISA resultó ser altamente sensible y específico, sencillo y rápido; sin embargo, la muy buena concordancia, alta precisión, bajo costo y capacidad para detectar otros enteroparásitos hace que la TSET sea recomendable para el diagnóstico en zonas endémicas del Perú.OBJETIVE: To compare a commercial coproantigen ELISA kit and the technique of spontaneous sedimentation in tube (TSET for the diagnosis of Giardia lamblia in fecal specimens from children in a Peruvian endemic area. MATERIAL AND METHODS: 174 fecal samples were analyzed by TSET and 2nd Generation Giardia ELISA kit. RESULTS: 51 samples were positive by ELISA and 49 by TSET. CONCLUSIONS: The ELISA was highly sensitive and specific, simple and fast. However, the very good agreement, high precision, low cost and ability to detect other intestinal parasites makes use of TSET recommended for laboratory diagnosis in endemic areas of Peru.

  5. Determination of cadmium and lead in human biological samples by spectrometric techniques: a review.

    Science.gov (United States)

    Lemos, Valfredo Azevedo; de Carvalho, Anaildes Lago

    2010-12-01

    The analysis of human biological samples, such as blood, urine, nails, and hair, is generally used for the verification of human exposure to toxic metals. In this review, various spectrometric methods for the determination of cadmium and lead in biological samples are discussed and compared. Several spectrometric techniques are presented and discussed with respect to various characteristics such as sensitivity, selectivity, and cost. Special attention is drawn to the procedures for digestion prior to the determination of cadmium and lead in hair, nails, blood, and urine.

  6. Detection of Campylobacter in human and animal field samples in Cambodia.

    Science.gov (United States)

    Osbjer, Kristina; Tano, Eva; Chhayheng, Leang; Mac-Kwashie, Akofa Olivia; Fernström, Lise-Lotte; Ellström, Patrik; Sokerya, Seng; Sokheng, Choup; Mom, Veng; Chheng, Kannarath; San, Sorn; Davun, Holl; Boqvist, Sofia; Rautelin, Hilpi; Magnusson, Ulf

    2016-06-01

    Campylobacter are zoonotic bacteria and a leading cause of human gastroenteritis worldwide with Campylobacter jejuni and C. coli being the most commonly detected species. The aim of this study was to detect Campylobacter in humans and livestock (chickens, ducks, pigs, cattle, water buffalo, quail, pigeons and geese) in rural households by routine culturing and multiplex PCR in faecal samples frozen before analysis. Of 681 human samples, 82 (12%) tested positive by PCR (C. jejuni in 66 samples and C. coli in 16), but none by routine culture. Children were more commonly Campylobacter positive (19%) than adult males (8%) and females (7%). Of 853 livestock samples, 106 (12%) tested positive by routine culture and 352 (41%) by PCR. Campylobacter jejuni was more frequent in chickens and ducks and C. coli in pigs. In conclusion, Campylobacter proved to be highly prevalent by PCR in children (19%), ducks (24%), chickens (56%) and pigs (72%). Routine culturing was insufficiently sensitive in detecting Campylobacter in field samples frozen before analysis. These findings suggest that PCR should be the preferred diagnostic method for detection of Campylobacter in humans and livestock where timely culture is not feasible. © 2016 The Authors. APMIS published by John Wiley & Sons Ltd on behalf of Scandinavian Societies for Medical Microbiology and Pathology.

  7. Discovery of a novel polyomavirus in acute diarrheal samples from children.

    Directory of Open Access Journals (Sweden)

    Guixia Yu

    Full Text Available Polyomaviruses are small circular DNA viruses associated with chronic infections and tumors in both human and animal hosts. Using an unbiased deep sequencing approach, we identified a novel, highly divergent polyomavirus, provisionally named MX polyomavirus (MXPyV, in stool samples from children. The ∼5.0 kB viral genome exhibits little overall homology (<46% amino acid identity to known polyomaviruses, and, due to phylogenetic variation among its individual proteins, cannot be placed in any existing taxonomic group. PCR-based screening detected MXPyV in 28 of 834 (3.4% fecal samples collected from California, Mexico, and Chile, and 1 of 136 (0.74% of respiratory samples from Mexico, but not in blood or urine samples from immunocompromised patients. By quantitative PCR, the measured titers of MXPyV in human stool at 10% (weight/volume were as high as 15,075 copies. No association was found between the presence of MXPyV and diarrhea, although girls were more likely to shed MXPyV in the stool than boys (p=0.012. In one child, viral shedding was observed in two stools obtained 91 days apart, raising the possibility of chronic infection by MXPyV. A multiple sequence alignment revealed that MXPyV is a closely related variant of the recently reported MWPyV and HPyV10 polyomaviruses. Further studies will be important to determine the association, if any, of MXPyV with disease in humans.

  8. Proteomic biomarker discovery in 1000 human plasma samples with mass spectrometry

    DEFF Research Database (Denmark)

    Cominetti, Ornella; Núñez Galindo, Antonio; Corthésy, John

    2016-01-01

    the quality of the MS data and provided descriptive statistics. The data set was interrogated for proteins with most stable expression levels in that set of plasma samples. We evaluated standard clinical variables that typically impact forthcoming results and assessed body mass index-associated and gender......-specific proteins at two time points. We demonstrate that analyzing a large number of human plasma samples for biomarker discovery with MS using isobaric tagging is feasible, providing robust and consistent biological results....

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

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    Khosro Hazrati Tappeh

    2014-12-01

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

  10. Diagnostic efficacy of monoclonal antibody based sandwich enzyme linked immunosorbent assay (ELISA for detection of Fasciola gigantica excretory/secretory antigens in both serum and stool

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    Zoheiry Mona K

    2011-09-01

    Full Text Available Abstract Background This research was carried out to develop a reliable monoclonal antibody (MoAb-based sandwich enzyme linked immunosorbent assay (ELISA for the diagnosis of active Fasciola gigantica infection in both serum and stool for comparative purposes. Methods From a panel of MoAbs raised against F. gigantica excretory/secretory antigens (ES Ags, a pair (12B/11D/3F and 10A/9D/10G was chosen due to its high reactivity and strict specificity to F. gigantica antigen by indirect ELISA. Results The two MoAbs were of the IgG1 and IgG2a subclasses, respectively. Using SDS-PAGE and EITB, the selected MoAbs recognized 83, 64, 45 and 26 kDa bands of ES Ags. The lower detection limit of ELISA assay was 3 ng/ml. In stool, the sensitivity, specificity and diagnostic efficacy of ELISA was 96%, 98.2 and 97.1%; while in serum they were 94%, 94.6% and 94.3%, respectively. Moreover, a positive correlation was found between ova count in stool of F. gigantica infected patients and the OD readings of ELISA in both stool and serum samples (r = 0.730, p Conclusions These data showed that the use of MoAb-based sandwich ELISA for the detection of F. gigantica coproantigens in stool specimens was superior to serum samples; it provides a highly efficient, non-invasive technique for the diagnosis of active F. gigantica infection.

  11. Comparison of three stool antigen assays with the 13C- urea breath test for the primary diagnosis of Helicobacter pylori infection and monitoring treatment outcome.

    LENUS (Irish Health Repository)

    Hooton, Carmel

    2012-02-03

    BACKGROUND: The urea breath test (UBT) is the gold-standard non-invasive test for the detection of Helicobacter pylori infection, however, the lack of availability of the UBT due to the high cost of the test, and in particular the need for expensive analytical instrumentation, limits the usefulness of this method. Stool antigen assays may offer an alternative non-invasive method for the diagnosis of infection. OBJECTIVE: To compare the accuracy of three stool antigen assays (HpSA, IDEIA HpStAR, and ImmunoCard STAT) against the UBT for the primary diagnosis of H. pylori infection and for monitoring treatment outcome. METHODS: A total of 102 patients attending two gastroenterology day-case clinics for the investigation of dyspepsia were included. Each patient provided breath and stool samples for analysis. Patients who tested positive for H. pylori by the validated UBT were prescribed triple therapy and invited to return for repeat breath and stool sample analysis 6 weeks post-treatment. RESULTS: Of the 102 patients tested, 48 were diagnosed with H. pylori infection by the UBT. The HpSA assay interpreted 38 of these as positive (79% sensitive). Of the 54 UBT-negative patients the HpSA assay interpreted all 54 as negative (100% specific). The IDEIA HpStAR assay correctly identified 44 patients as positive (92% sensitive) and 50 as negative (92.5% specific). The ImmunoCard STAT assay interpreted 38 patients as positive (79% sensitive) and 52 as negative (96.3% specific). CONCLUSION: The findings indicate that the IDEIA HpStAR stool antigen kit is the most accurate assay of the three assays evaluated, and possibly represents a viable alternative to the UBT for the primary diagnosis of H. pylori infection and for monitoring treatment outcome.

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

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

    Science.gov (United States)

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

    2007-01-01

    AIM: To investigate the clinical performances of rapid stool test (ImmunoCard STAT HpSA, Meridian Diagnostic Inc.) in the evaluation of eradication therapy of H pylori and to compare it with a well-known and validated laboratory stool test (Amplified IDEA Hp StAR, Dako). METHODS: Stool samples of 122 patients were evaluated after eradication therapy of H pylori. H pylori status was assessed by 13C-urea breath test (UBT). Stool specimens were tested using either the rapid immunoassay kit or the laboratory immunoassay kit. RESULTS: Forty-three patients were infected and 79 non-infected. Sensitivity and specificity of ImmunoCard STAT and Hp StAR were 58.14% and 76.4%, and 97.47% and 98.73%, respectively (P > 0.05). Overall agreement between the two tests was 92.6% (113 of 122 cases). CONCLUSION: ImmunoCard STAT seems to have rather low performances, and it cannot be regarded as a reliable tool in the post-treatment setting. Also Hp StAR cannot be recommended to confirm H pylori eradication after treatment. PMID:17724805

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

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    Zohreh Torabi

    2014-09-01

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

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

    Science.gov (United States)

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

    2017-07-01

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

  16. China's human resources for maternal and child health: a national sampling survey.

    Science.gov (United States)

    Ren, Zhenghong; Song, Peige; Theodoratou, Evropi; Guo, Sufang; An, Lin

    2015-12-16

    In order to achieve the Millennium Development Goals (MDG) 4 and 5, the Chinese Government has invested greatly in improving maternal and child health (MCH) with impressive results. However, one of the most important barriers for further improvement is the uneven distribution of MCH human resources. There is little information about the distribution, quantity and capacity of the Chinese MCH human resources and we sought to investigate this. Cities at prefectural level were selected by random cluster sampling. All medical and health institutions providing MCH-related services in the sampled areas were investigated using a structured questionnaire. The data were weighted based on the proportion of the sampled districts/cities. Amount, proportions and numbers per 10,000 population of MCH human resources were estimated in order to reveal the quantity of the Chinese MCH human resources. The capacity of MCH human resources was evaluated by analyzing data on the education level and professional skills of the staff. There were 77,248 MCH workers in China in 2010. In general, 67.6% and 71.9% of the women's and children's health care professionals had an associate degree or higher, whereas around 30% had only high-school or lower degrees. More than 40% of the women's health workers were capable of providing skilled birth attendance, but these proportions varied between different institutions and locations. Evidence from this study highlights that Chinese MCH human resources are not in shortage in the national level. However, the quantity and capacity of MCH human resources are not evenly distributed among different institutions and locations. Finally there is a need in the improvement of the MCH services by improving the quality of MCH human resources.

  17. Efficient discrimination and removal of phospholipids during electromembrane extraction from human plasma samples

    DEFF Research Database (Denmark)

    Vårdal, Linda; Gjelstad, Astrid; Huang, Chuixiu

    2017-01-01

    AIM: For the first time, extracts obtained from human plasma samples by electromembrane extraction (EME) were investigated comprehensively with particular respect to phospholipids using ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Thhe purpose was to invest...

  18. Imitation of Tongue Protrusion in Human Neonates: Specificity of the Response in a Large Sample

    Science.gov (United States)

    Nagy, Emese; Pilling, Karen; Orvos, Hajnalka; Molnar, Peter

    2013-01-01

    Although a large body of evidence has accumulated on the young human infant's ability to imitate, the phenomenon has failed to gain unanimous acceptance. Imitation of tongue protrusion, the most tested gesture to date, was examined in a sample of 115 newborns in the first 5 days of life in 3 seating positions. An ethologically based…

  19. Analyses of human colonic mucus obtained by an in vivo sampling technique

    NARCIS (Netherlands)

    Hamer, H.M.; Jonkers, D.M.A.E.; Loof, A.; Houtvin, S.A.L.W. van; Troost, F.J.; Venema, K.; Kodde, A.; Koek, G.H.; Schipper, R.G.; Heerde, W.L. van; Brummer, R.J.

    2009-01-01

    Background: The mucus layer is an important dynamic component of the epithelial barrier. It contains mucin glycoproteins and other compounds secreted by the intestinal epithelium, such as secretory IgA. However, a standardized in vivo sampling technique of mucus in humans is not yet available. Aim:

  20. Groundwater sampling methods using glass wool filtration to trace human enteric viruses in Madison, Wisconsin

    Science.gov (United States)

    Human enteric viruses have been detected in the Madison, Wisconsin deep municipal well system. Earlier projects by the Wisconsin Geological and Natural History Survey (WGNHS) have used glass wool filters to sample groundwater for these viruses directly from the deep municipal wells. Polymerase chain...

  1. Elimination of bioweapons agents from forensic samples during extraction of human DNA.

    Science.gov (United States)

    Timbers, Jason; Wilkinson, Della; Hause, Christine C; Smith, Myron L; Zaidi, Mohsin A; Laframboise, Denis; Wright, Kathryn E

    2014-11-01

    Collection of DNA for genetic profiling is a powerful means for the identification of individuals responsible for crimes and terrorist acts. Biologic hazards, such as bacteria, endospores, toxins, and viruses, could contaminate sites of terrorist activities and thus could be present in samples collected for profiling. The fate of these hazards during DNA isolation has not been thoroughly examined. Our goals were to determine whether the DNA extraction process used by the Royal Canadian Mounted Police eliminates or neutralizes these agents and if not, to establish methods that render samples safe without compromising the human DNA. Our results show that bacteria, viruses, and toxins were reduced to undetectable levels during DNA extraction, but endospores remained viable. Filtration of samples after DNA isolation eliminated viable spores from the samples but left DNA intact. We also demonstrated that contamination of samples with some bacteria, endospores, and toxins for longer than 1 h compromised the ability to complete genetic profiling.

  2. Long-term performance and stability of molecular shotgun lipidomic analysis of human plasma samples.

    Science.gov (United States)

    Heiskanen, Laura A; Suoniemi, Matti; Ta, Hung Xuan; Tarasov, Kirill; Ekroos, Kim

    2013-09-17

    The stability of the lipid concentration levels in shotgun lipidomics analysis was tracked over a period of 3.5 years. Concentration levels in several lipid classes, such as phospholipids, were determined in human plasma lipid extracts. Impact of the following factors on the analysis was investigated: sample amount, internal standard amount, and sample dilution factor. Moreover, the reproducibility of lipid profiles obtained in both polarity modes was evaluated. Total number of samples analyzed was approximately 6800 and 7300 samples in negative and positive ion modes, respectively, out of which 610 and 639 instrument control samples were used in stability calculations. The assessed shotgun lipidomics approach showed to be remarkably robust and reproducible, requiring no batch corrections. Coefficients of variation (CVs) of lipid mean concentration measured with optimized analytical parameters were typically less than 15%. The high reproducibility indicated that no lipid degradation occurred during the monitored time period.

  3. Easily-handled method to isolate mesenchymal stem cells from coagulated human bone marrow samples

    Institute of Scientific and Technical Information of China (English)

    Heng-Xiang; Wang; Zhi-Yong; Li; Zhi-Kun; Guo; Zi-Kuan; Guo

    2015-01-01

    AIM:To establish an easily-handled method to isolate mesenchymal stem cells(MSCs) from coagulated human bone marrow samples. METHODS: Thrombin was added to aliquots of seven heparinized human bone marrow samples to mimic marrow coagulation. The clots were untreated,treated with urokinase or mechanically cut into pieces before culture for MSCs. The un-coagulated samples and the clots were also stored at 4 ℃ for 8 or 16 h before the treatment. The numbers of colony-forming unit-fibroblast(CFU-F) in the different samples were determined. The adherent cells from different groups were passaged and their surface profile was analyzed with flow cytometry. Their capacities of in vitro osteogenesis and adipogenesis were observed after the cells were exposed to specific inductive agents.RESULTS: The average CFU-F number of urokinasetreated samples(16.85 ± 11.77/106) was comparable to that of un-coagulated control samples(20.22 ± 10.65/106,P = 0.293),which was significantly higher than those of mechanically-cut clots(6.5 ± 5.32/106,P < 0.01) and untreated clots(1.95 ± 1.86/106,P < 0.01). The CFU-F numbers decreased after samples were stored,but those of control and urokinase-treated clots remained higher than the other two groups. Consistently,the numbers of the attached cells at passage 0 were higher in control and urokinase-treated clots than those of mechanically-cut clots and untreated clots.The attached cells were fibroblast-like in morphology and homogenously positive for CD44,CD73 and CD90,and negative for CD31 and CD45. Also,they could be induced to differentiate into osteoblasts and adipocytes in vitro. CONCLUSION: Urokinase pretreatment is an optimal strategy to isolate MSCs from human bone marrow samples that are poorly aspirated and clotted.

  4. Rigorous Training of Dogs Leads to High Accuracy in Human Scent Matching-To-Sample Performance.

    Directory of Open Access Journals (Sweden)

    Sophie Marchal

    Full Text Available Human scent identification is based on a matching-to-sample task in which trained dogs are required to compare a scent sample collected from an object found at a crime scene to that of a suspect. Based on dogs' greater olfactory ability to detect and process odours, this method has been used in forensic investigations to identify the odour of a suspect at a crime scene. The excellent reliability and reproducibility of the method largely depend on rigor in dog training. The present study describes the various steps of training that lead to high sensitivity scores, with dogs matching samples with 90% efficiency when the complexity of the scents presented during the task in the sample is similar to that presented in the in lineups, and specificity reaching a ceiling, with no false alarms in human scent matching-to-sample tasks. This high level of accuracy ensures reliable results in judicial human scent identification tests. Also, our data should convince law enforcement authorities to use these results as official forensic evidence when dogs are trained appropriately.

  5. Sequence space coverage, entropy of genomes and the potential to detect non-human DNA in human samples

    Directory of Open Access Journals (Sweden)

    Maley Carlo C

    2008-10-01

    Full Text Available Abstract Background Genomes store information for building and maintaining organisms. Complete sequencing of many genomes provides the opportunity to study and compare global information properties of those genomes. Results We have analyzed aspects of the information content of Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Saccharomyces cerevisiae, and Escherichia coli (K-12 genomes. Virtually all possible (> 98% 12 bp oligomers appear in vertebrate genomes while 98% to D. melanogaster (12–17 bp, C. elegans (11–17 bp, A. thaliana (11–17 bp, S. cerevisiae (10–16 bp and E. coli (9–15 bp. Frequencies of unique oligomers in the genomes follow similar patterns. We identified a set of 2.6 M 15-mers that are more than 1 nucleotide different from all 15-mers in the human genome and so could be used as probes to detect microbes in human samples. In a human sample, these probes would detect 100% of the 433 currently fully sequenced prokaryotes and 75% of the 3065 fully sequenced viruses. The human genome is significantly more compact in sequence space than a random genome. We identified the most frequent 5- to 20-mers in the human genome, which may prove useful as PCR primers. We also identified a bacterium, Anaeromyxobacter dehalogenans, which has an exceptionally low diversity of oligomers given the size of its genome and its GC content. The entropy of coding regions in the human genome is significantly higher than non-coding regions and chromosomes. However chromosomes 1, 2, 9, 12 and 14 have a relatively high proportion of coding DNA without high entropy, and chromosome 20 is the opposite with a low frequency of coding regions but relatively high entropy. Conclusion Measures of the frequency of oligomers are useful for designing PCR assays and for identifying chromosomes and organisms with hidden structure that had not been previously recognized. This information may be used to detect

  6. SAMPLING INTENSITY WITH FIXED PRECISION WHEN ESTIMATING VOLUME OF HUMAN BRAIN COMPARTMENTS

    Directory of Open Access Journals (Sweden)

    Rhiannon Maudsley

    2011-05-01

    Full Text Available Cavalieri sampling and point counting are frequently applied in combination with magnetic resonance (MR imaging to estimate the volume of human brain compartments. Current practice involves arbitrarily choosing the number of sections and sampling intensity within each section, and subsequently applying error prediction formulae to estimate the precision. The aim of this study is to derive a reference table for researchers who are interested in estimating the volume of brain regions, namely grey matter, white matter, and their union, to a given precision. In particular, this table, which is based on subsampling of a large brain data set obtained from coronal MR images, offers a recommendation for the minimum number of sections and mean number of points per section that are required to achieve a pre-defined coefficient of error of the volume estimator. Further analysis onMR brain data from a second human brain shows that the sampling intensity recommended is appropriate.

  7. Determination of cholesterol concentration in human milk samples using attenuated total reflectance Fourier transform infrared spectroscopy

    Science.gov (United States)

    Kamelska, A. M.; Pietrzak-Fiećko, R.; Bryl, K.

    2013-03-01

    Results of an inexpensive and rapid evaluation of the cholesterol concentration in human milk using ATR-FTIR techniques are presented. The FTIR spectrum of pure cholesterol was characterized and quantitatively estimated in the region between 2800 and 3200 cm-1. 125 samples at different stages of lactation were analyzed. There were no differences between the cholesterol concentrations in the samples of early (1-3 months), medium (4-6 months), and late (> 6 months) lactation stages ( p = 0.096968). The cholesterol concentration ranged from 4.30 to 21.77 mg/100 cm3. Such a broad range was due to the differences between the samples from different women ( p = 0.000184). The results indicate that ATR-FTIR has potential for rapid estimation of cholesterol concentration in human milk.

  8. Effect of Mass Stool Examination and Mass Treatment For Decreasing Intestinal Helminth and Protozoan Infection Rates in Bolivian Children: A Cross-Sectional Study

    Science.gov (United States)

    Còrdova Vidal, Claudia; Strauss, Wilma; Ikoma, Toshikazu; Endoh, Kazuo; Yamamoto, Masaharu

    2016-01-01

    Bolivia is one of the countries with a high intestinal helminth and protozoan infection rate. Despite the high prevalence of the parasitic infection, nationwide preventive measures for Bolivian children have not yet been implemented. We evaluated the effect of mass stool examination and treatment as a strategy for decreasing the infection rate. This study was conducted between 2013 and 2015 in children aged 2–18 years. A total of 2,033 stool samples (575 in 2013, 815 in 2014 and 642 in 2015) were collected and examined using the formalin-ether medical sedimentation method. As an anthelminthic medicine, nitazoxanide was given to all infected children within 2 months post-examination, each year. The effect of mass stool examination and treatment was evaluated based on the changes in the overall or individual parasitic infection rates during the study period. The overall parasitic infection rate decreased significantly from 65.2% in 2013 to 43.0% in 2015; a 22.2 percentage point decrease (PHymenolepis nana, decreased significantly from 9.0% in 2013 to 6.4% in 2014 to 3.4% in 2015 (P<0.001). Prevalence of the most common pathogenic protozoan infection, Entamoeba histolytica, decreased significantly from 19.0% in 2013 to 3.0% in 2015 (P<0.001). Conversely, the rate of Giardia intestinalis increased significantly from 16.5% in 2013 to 21.2% in 2015 (P<0.01). Mass stool examination and treatment for intestinal helminth and protozoan infections was effective for decreasing the overall parasitic infection rate in the study population, excluding Giardia intestinalis. Further studies on the long-term effect of mass stool examination and treatment for decreasing all intestinal parasitic infection rates in Bolivian children are needed. PMID:27923058

  9. Total reflection X-ray fluorescence as a fast multielemental technique for human placenta sample analysis

    Science.gov (United States)

    Marguí, E.; Ricketts, P.; Fletcher, H.; Karydas, A. G.; Migliori, A.; Leani, J. J.; Hidalgo, M.; Queralt, I.; Voutchkov, M.

    2017-04-01

    In the present contribution, benchtop total reflection X-ray fluorescence spectrometry (TXRF) has been evaluated as a cost-effective multielemental analytical technique for human placenta analysis. An easy and rapid sample preparation consisting of suspending 50 mg of sample in 1 mL of a Triton 1% solution in deionized water showed to be the most suitable for this kind of samples. However, for comparison purposes, an acidic microwave acidic digestion procedure was also applied. For both sample treatment methodologies, limits of detection for most elements were in the low mg/kg level. Accurate and precise results were obtained using internal standardization as quantification approach and applying a correction factor to compensate for absorption effects. The correction factor was based on the proportional ratio between the slurry preparation results and those obtained for the analysis of a set of human placenta samples analysed by microwave acidic digestion and ICP-AES analysis. As a study case, the developed TXRF methodology was applied for multielemental analysis (K, Ca, Fe, Cu, Zn, As, Se, Br, Rb and Sr) of several healthy women's placenta samples from two regions in Jamaica.

  10. Residues of PCDDs and PCDFs in human milk samples in Ahmedabad, India

    Energy Technology Data Exchange (ETDEWEB)

    Kashyap, R.; Bhatnagar, V.; Sadhu, H.; Jhamb, N.; Karanjkar, R.; Saiyed, H. [National Inst. of Occupational Health, Ahmedabad (India)

    2004-09-15

    Polychlorinated dibenzo-p-dioxins (PCDDs) and Polychlorinated dibenzo furans (PCDFs) represent a class of organic environmental pollutants. They are unwanted byproduct of incineration, uncontrolled burning and certain industrial processes. They are persistent in nature and bioaccumulates through food chain. These are hazardous to human health and environment. The residues of these toxicants have been detected in human adipose tissue, blood and milk. WHO has coordinated two rounds of follow up studies on levels of PCDD/Fs and PCBs in human milk and the data shows a decreasing trend during the last 30 years. However, in India there is no data available on the exposure and residues of these contaminants. This study presents first time the levels of dioxin and furans in human milk samples collected from the Ahmedabad city in India.

  11. Evaluation of an immunochromatographic dip strip test for simultaneous detection of Cryptosporidium spp, Giardia duodenalis, and Entamoeba histolytica antigens in human faecal samples.

    Science.gov (United States)

    Goñi, P; Martín, B; Villacampa, M; García, A; Seral, C; Castillo, F J; Clavel, A

    2012-08-01

    Immunochromatographic (IC) tests may play an important role in the future diagnosis of parasitic diseases because of their speed and simplicity of use. A recently developed test to detect Cryptosporidium spp, Giardia duodenalis and Entamoeba histolytica was evaluated. Microscopy and PCR were the "gold standard" reference techniques and the results of this IC test were compared with those obtained with ELISA and IC single test for the three parasites. One hundred sixty stool samples were assayed. Using microscopy, 22 samples were diagnosed as positive for Cryptosporidium spp., 31 for Giardia duodenalis, 41 for Entamoeba histolytica/dispar, and 68 had a negative diagnosis for the three parasites. Results of IC tests show sensitivities of 70-72% for Cryptosporidium, 90-97% for Giardia and 62.5% for Entamoeba histolytica. Specificities were of 93.6-94.9%, >99% and 96.1%, respectively. In all diagnoses, agreement with microscopy and PCR was over 90%, except in the triple test and microscopy in E. histolytica detection that was 76.3%, due to the inability of microscopy to differentiate E. histolytica from nonpathogenic species such as E. dispar or E. moshkovskii. The triple stool immunoassays provide adequate sensitivities and specificities for use in outbreak situations, for screening proposals and for massive assays in endemic areas where a large number of samples must be analysed or as complementary test for individual diagnosis.

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

    Science.gov (United States)

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

    2014-02-25

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

  13. Sensitive Procedure for Rapid Detection of Human Brucellosis, Based on PCR Method in Contaminated Serum Samples

    Directory of Open Access Journals (Sweden)

    Eslam Ghezelsofla

    2013-07-01

    Full Text Available AbstractBackground and objective: Brucellosis is a zoonosis transmittable to humans poses a significant public health problem in many developing countries and requires rapid and accurate diagnostic methods. Here, our aim was to develop a diagnostic polymerase chain reaction (PCR assay in artificially contaminated serum samples as a model for rapid and accurate laboratory confirmation of human brucellosis. Material and methods: In this study, initially the standard Brucella abortus strain (2308 were cultured on Brucella agar medium and then colonies were inactivated by formalin 10 %. Genomic DNA was extracted from inactivated bacterial colonies. Serial dilutions of bacterial-DNA were prepared in fetal bovine serum (FBS and water and subsequently DNA extraction were repeated on these artificially contaminated samples. The two pairs of primers amplified two different fragments included in: a gene encoding an outer membrane protein (omp-2 (primers JPF/JPR and a sequence 16S rRNA of B. abortus (primers F4/R2. Results: The two primers assayed showed a difference in sensitivity for detecting Brucella DNA, ranging between 5 pg and 50 pg for artificially contaminated serum samples and 50Fg and 5 pg for contaminated control samples. Therefore, the sensitivity of PCR using F4/R2 primers was greater than the PCR using JPF/JPR primers.Conclusion: Although the sensitivity of PCR using these primers was affected by serum inhibitors, they are still the most sensitive and they could provide a useful tool for the diagnosis of human brucellosis.

  14. A percutaneous needle biopsy technique for sampling the supraclavicular brown adipose tissue depot of humans.

    Science.gov (United States)

    Chondronikola, M; Annamalai, P; Chao, T; Porter, C; Saraf, M K; Cesani, F; Sidossis, L S

    2015-10-01

    Brown adipose tissue (BAT) has been proposed as a potential target tissue against obesity and its related metabolic complications. Although the molecular and functional characteristics of BAT have been intensively studied in rodents, only a few studies have used human BAT specimens due to the difficulty of sampling human BAT deposits. We established a novel positron emission tomography and computed tomography-guided Bergström needle biopsy technique to acquire human BAT specimens from the supraclavicular area in human subjects. Forty-three biopsies were performed on 23 participants. The procedure was tolerated well by the majority of participants. No major complications were noted. Numbness (9.6%) and hematoma (2.3%) were the two minor complications noted, which fully resolved. Thus, the proposed biopsy technique can be considered safe with only minimal risk of adverse events. Adoption of the proposed method is expected to increase the sampling of the supraclavicular BAT depot for research purposes so as to augment the scientific knowledge of the biology of human BAT.

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

  16. [Detection of human parvovirus B19, human bocavirus and human parvovirus 4 infections in blood samples among 95 patients with liver disease in Nanjing by nested PCR].

    Science.gov (United States)

    Tong, Rui; Zhou, Wei-Min; Liu, Xi-Jun; Wang, Yue; Lou, Yong-Liang; Tan, Wen-Jie

    2013-04-01

    To analyze the infection of human parvovirus B19, human bocavirus (HBoV) and human parvovirus 4 (PARV4) in blood samples among patients with liver disease in Nanjing by molecular detection. Nested PCR assays were designed and validated to detect B19, HBoV and PARV4, respectively. The assays were used to screen three parvoviruses in blood samples from 95 patients with different liver disease in Nanjing. The parvovirus infection was analyzed statistically. The detection limits were 10 copies of genomic DNA equivalents per reaction for each assays and the good specificity were observed. The frequency of B19 and HBoV were 2/95 (2.1%) and 9/95 (9.5%) in blood samples respectively. No PARV4 was detected. HBoV was detected in 3/5 patients with drug-induced hepatitis. Both B19 and HBoV infection were detected in blood from patients with liver disease.

  17. Comparison of 2 chromogenic media for the detection of extended-spectrum β-lactamase producing Enterobacteriaceae stool carriage in nursing home residents

    Science.gov (United States)

    Blane, Beth; Brodrick, Hayley J.; Gouliouris, Theodore; Ambridge, Kirsty E.; Kidney, Angela D.; Ludden, Catherine M.; Limmathurotsakul, Direk; Török, M. Estée; Peacock, Sharon J.

    2016-01-01

    ChromID ESBL agar and Brilliance ESBL agar were compared for the isolation of extended-spectrum β-lactamase (ESBL)–producing Enterobacteriaceae from 298 stools. These had comparable sensitivity and selectivity for the 116 positive samples. Pre-enrichment with cefpodoxime and extending incubation to 48 hours after direct plating both significantly increased sensitivity but reduced selectivity of both agars. PMID:26712266

  18. Antigenic typing of brazilian rabies virus samples isolated from animals and humans, 1989-2000

    Directory of Open Access Journals (Sweden)

    FAVORETTO Silvana Regina

    2002-01-01

    Full Text Available Animal and human rabies samples isolated between 1989 and 2000 were typified by means of a monoclonal antibody panel against the viral nucleoprotein. The panel had been previously established to study the molecular epidemiology of rabies virus in the Americas. Samples were isolated in the Diagnostic Laboratory of the Pasteur Institute and in other rabies diagnostic centers in Brazil. In addition to the fixed virus samples CVS-31/96-IP, preserved in mouse brain, and PV-BHK/97, preserved in cell culture, a total of 330 rabies virus samples were isolated from dogs, cats, cattle, horses, bats, sheep, goat, swine, foxes, marmosets, coati and humans. Six antigenic variants that were compatible with the pre-established monoclonal antibodies panel were defined: numbers 2 (dog, 3 (Desmodus rotundus, 4 (Tadarida brasiliensis, 5 (vampire bat from Venezuela, 6 (Lasiurus cinereus and Lab (reacted to all used antibodies. Six unknown profiles, not compatible with the panel, were also found. Samples isolated from insectivore bats showed the greatest variability and the most commonly isolated variant was variant-3 (Desmodus rotundus. These findings may be related to the existence of multiple independent transmission cycles, involving different bat species.

  19. Comparison of chlorzoxazone one-sample methods to estimate CYP2E1 activity in humans

    DEFF Research Database (Denmark)

    Kramer, Iza; Dalhoff, Kim; Clemmesen, Jens O

    2003-01-01

    OBJECTIVE: Comparison of a one-sample with a multi-sample method (the metabolic fractional clearance) to estimate CYP2E1 activity in humans. METHODS: Healthy, male Caucasians ( n=19) were included. The multi-sample fractional clearance (Cl(fe)) of chlorzoxazone was compared with one...... estimates, Cl(est) at 3 h or 6 h, and MR at 3 h, can serve as reliable markers of CYP2E1 activity. The one-sample clearance method is an accurate, renal function-independent measure of the intrinsic activity; it is simple to use and easily applicable to humans.......-time-point clearance estimation (Cl(est)) at 3, 4, 5 and 6 h. Furthermore, the metabolite/drug ratios (MRs) estimated from one-time-point samples at 1, 2, 3, 4, 5 and 6 h were compared with Cl(fe). RESULTS: The concordance between Cl(est) and Cl(fe) was highest at 6 h. The minimal mean prediction error (MPE) of Cl...

  20. Selection of Candidate Housekeeping Genes for Normalization in Human Postmortem Brain Samples

    Directory of Open Access Journals (Sweden)

    Aldo Pagano

    2011-08-01

    Full Text Available The most frequently used technique to study the expression profile of genes involved in common neurological disorders is quantitative real-time RT-PCR, which allows the indirect detection of very low amounts of selected mRNAs in tissue samples. Expression analysis by RT-qPCR requires an appropriate normalization to the expression level of genes characterized by a stable, constitutive transcription. However, the identification of a gene transcribed at a very stable level is difficult if not impossible, since significant fluctuations of the level of mRNA synthesis often accompanies changes of cell behavior. The aim of this study is to identify the most stable genes in postmortem human brain samples of patients affected by Alzheimer’s disease (AD suitable as reference genes. The experiments analyzed 12 commonly used reference genes in brain samples from eight individuals with AD and seven controls. After a careful analysis of the results calculated by geNorm and NormFinder algorithms, we found that CYC1 and EIF4A2 are the best reference genes. We remark on the importance of the determination of the best reference genes for each sample to be analyzed and suggest a practical combination of reference genes to be used in the analysis of human postmortem samples.

  1. Quality assesment for the analysis of PCDDs/PCDFs in individual human serum samples

    Energy Technology Data Exchange (ETDEWEB)

    Perez, F. [IIQAB-CSIC, Barcelona (Spain). Dept. of Ecotechnologies, Lab. of Dioxins; Abad, E.; Llerena, J.J.; Caixach, J.; Rivera, J.

    2004-09-15

    The aim of this work was to optimise a relevant methodology for the ultratrace analysis of PCDDs/PCDFs in individual human serum samples. In order to carry out the study, different strategies including the elaboration of quality control samples, parallel sample analysis, control blanks and a number of quality assurance measures were implemented as analytical current practices. Some of the main drawbacks in the analysis of PCDDs/PCDFs in these kind of samples come from two conflicting aspects: the small sample size and the low levels expected to be found. Taking this into account, an unavoidable compromise between the sample amount and the minimum analytical requirements, mainly the detection limit (LOD), is mandatory. To reach this goal C{sub 18} solid phase extraction was used to remove the analytes from the matrix. Clean up was performed by solid-liquid adsorption chromatography using a variety of adsorbents. Instrumental analysis was achieved by high-resolution gas chromatography coupled to high-resolution mass spectrometry (HRGC/HRMS). Finally, the optimised methodology was applied to evaluate the potential impact in general population living in the surroundings of an obsolete municipal waste incinerator plant (MWI). Thus, more than 400 individuals serum samples potentially exposed to the emission of the incinerator and people not exposed were considered in this study.

  2. Microbiome Analysis of Stool Samples from African Americans with Colon Polyps

    NARCIS (Netherlands)

    Brim, H.; Yooseph, S.; Zoetendal, E.G.; Lee, E.; Torralbo, M.; Laiyemo, A.O.; Shokrani, B.; Nelson, K.; Ashktorab, H.

    2013-01-01

    Background: Colonic polyps are common tumors occurring in similar to 50% of Western populations with similar to 10% risk of malignant progression. Dietary agents have been considered the primary environmental exposure to promote colorectal cancer (CRC) development. However, the colonic mucosa is per

  3. Microbiome Analysis of Stool Samples from African Americans with Colon Polyps

    NARCIS (Netherlands)

    Brim, H.; Yooseph, S.; Zoetendal, E.G.; Lee, E.; Torralbo, M.; Laiyemo, A.O.; Shokrani, B.; Nelson, K.; Ashktorab, H.

    2013-01-01

    Background: Colonic polyps are common tumors occurring in similar to 50% of Western populations with similar to 10% risk of malignant progression. Dietary agents have been considered the primary environmental exposure to promote colorectal cancer (CRC) development. However, the colonic mucosa is

  4. Laboratory Testing of Donors and Stool Samples for Fecal Microbiota Transplantation for Recurrent Clostridium difficile Infection.

    Science.gov (United States)

    Woodworth, Michael H; Neish, Emma M; Miller, Nancy S; Dhere, Tanvi; Burd, Eileen M; Carpentieri, Cynthia; Sitchenko, Kaitlin L; Kraft, Colleen S

    2017-04-01

    Fecal microbiota transplantation is an efficacious and inexpensive therapy for recurrent Clostridium difficile infection, yet its safety is thought to depend on appropriate fecal donor screening. FDA guidance for regulation of this procedure is in flux, but screening and manufacture of fecal material from asymptomatic donors present many challenges to clinical laboratories. This minireview summarizes FDA regulatory changes, principles of donor selection, and recommended laboratory screening practices for fecal microbiota transplantation. Copyright © 2017 American Society for Microbiology.

  5. Barriers to acceptance of self-sampling for human papillomavirus across ethnolinguistic groups of women.

    Science.gov (United States)

    Howard, Michelle; Lytwyn, Alice; Lohfeld, Lynne; Redwood-Campbell, Lynda; Fowler, Nancy; Karwalajtys, Tina

    2009-01-01

    Immigrant and low socio-economic (SES) women in North America underutilize Papanicolaou screening. Vaginal swab self-sampling for oncogenic human papillomavirus (HPV) has the potential to increase cervical cancer screening participation. The purpose of this qualitative study was to understand the perceptions of lower SES and immigrant women regarding self-sampling for HPV. Eleven focus-group interviews were conducted: one with Canadian-born English-speaking lower SES women, and two groups each with Arabic, Cantonese, Dari (Afghani), Somali and Spanish (Latino)-speaking women (one group conducted in English, the other in the native language) recently immigrated to Canada. Five to nine women aged 35 to 65 years and married with children participated in each group. Themes included 1) who might use self-sampling and why; 2) aversion to self-sampling and reasons to prefer physician; 3) ways to improve the appeal of self-sampling. Women generally perceived benefits of self-sampling and a small number felt they might use the method, but all groups had some reservations. Reasons included: uncertainty over performing the sampling correctly; fear of hurting themselves; concern about obtaining appropriate material; and concerns about test accuracy. Women preferred testing by a health care professional because they were accustomed to pelvic examinations, it was more convenient, or they trusted the results. Perceptions of self-sampling for HPV were similar across cultures and pertained to issues of confidence in self-sampling and need for physician involvement in care. These findings can inform programs and studies planning to employ self-sampling as a screening modality for cervical cancer.

  6. Pepper mild mottle virus, a plant virus associated with specific immune responses, Fever, abdominal pains, and pruritus in humans.

    Directory of Open Access Journals (Sweden)

    Philippe Colson

    Full Text Available BACKGROUND: Recently, metagenomic studies have identified viable Pepper mild mottle virus (PMMoV, a plant virus, in the stool of healthy subjects. However, its source and role as pathogen have not been determined. METHODS AND FINDINGS: 21 commercialized food products containing peppers, 357 stool samples from 304 adults and 208 stool samples from 137 children were tested for PMMoV using real-time PCR, sequencing, and electron microscopy. Anti-PMMoV IgM antibody testing was concurrently performed. A case-control study tested the association of biological and clinical symptoms with the presence of PMMoV in the stool. Twelve (57% food products were positive for PMMoV RNA sequencing. Stool samples from twenty-two (7.2% adults and one child (0.7% were positive for PMMoV by real-time PCR. Positive cases were significantly more likely to have been sampled in Dermatology Units (p<10(-6, to be seropositive for anti-PMMoV IgM antibodies (p = 0.026 and to be patients who exhibited fever, abdominal pains, and pruritus (p = 0.045, 0.038 and 0.046, respectively. CONCLUSIONS: Our study identified a local source of PMMoV and linked the presence of PMMoV RNA in stool with a specific immune response and clinical symptoms. Although clinical symptoms may be imputable to another cofactor, including spicy food, our data suggest the possibility of a direct or indirect pathogenic role of plant viruses in humans.

  7. Genomic studies of envelope gene sequences from mosquito and human samples from Bangkok, Thailand.

    Science.gov (United States)

    Pitaksajjakul, Pannamthip; Benjathummarak, Surachet; Son, Hyun Ngoc; Thongrungkiat, Supatra; Ramasoota, Pongrama

    2016-01-01

    Dengue virus (DENV) is an RNA virus showing a high degree of genetic variation as a consequence of its proofreading inability. This variation plays an important role in virus evolution and pathogenesis. Although levels of within-host genetic variation are similar following equilibrium, variation among different hosts is frequently different. To identify dengue quasispecies present among two hosts, we collected patient samples from six acute DENV cases and two pools of Aedes aegypti mosquitoes and analyzed the genetic variation of regions of the viral envelope gene. Among human and mosquito samples, we found three major clusters originating from two subpopulations. Although several shared lineages were observed in the two hosts, only one lineage showing evidence of neutral selection was observed among two hosts. Taken together, our data provide evidence for the existence of a DENV quasispecies, with less genetic variation observed in mosquitoes than humans and with circulating lineages found in both host types.

  8. Load and failure behavior of human muscle samples in the context of proximal femur replacement

    OpenAIRE

    Schleifenbaum, Stefan; Schmidt, Michael; Möbius, Robert; Wolfskämpf, Thomas; Schröder, Christian; Grunert, Ronny; Hammer, Niels; Prietzel, Torsten

    2016-01-01

    Background: To ensure adequate function after orthopedic tumor reconstruction, it is important to reattach the remaining soft tissue to the implant. This study aimed at obtaining mechanical properties of textile muscle-implant and muscle-bone connections in a preliminary test. Methods: Two groups of soft-tissue attachment were mechanically tested and compared: Native bone-muscle samples obtained from human femora and muscles attached to a prosthetic implant by means of Trevira® attachment tu...

  9. Dioxin-like activity of environmental compounds in human blood and environmental samples

    DEFF Research Database (Denmark)

    Long, Manhai; Bonefeld-Jørgensen, Eva Cecilie

    2012-01-01

    R transactivation bioassay is utilized in an array of projects to study the AhR-mediated activities of individual chemicals and mixtures and for epidemiological purposes. This review summarizes a series of studies regarding the DL-activity of single compounds and complex compound mixtures in the environment...... a cost-effective and integrated screening tool for measurement of the DL-activity in human, environmental and commercial samples....

  10. Separation of proteins from human plasma by sample displacement chromatography in hydrophobic interaction mode

    OpenAIRE

    Josic, Djuro; Breen, Lucas; Clifton, James; Gajdosik, Martina Srajer; Gaso-Sokac, Dajana; Rucevic, Marijana; Müller, Egbert

    2012-01-01

    Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately twenty years ago. This method was first used for the preparative purification of peptides and proteins. Recently, SDC in ion-exchange mode was also successfully used for enrichment of low abundance proteins from human plasma. In this paper, the use of SDC for the separation of plasma proteins in hydrophobic interaction mode is demonstrated. By use of two or more columns coupled in se...

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

    Science.gov (United States)

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

    2015-08-01

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

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

    Science.gov (United States)

    2016-01-01

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

  13. Dioxin-like activity of environmental compounds in human blood and environmental samples

    DEFF Research Database (Denmark)

    Long, Manhai; Bonefeld-Jørgensen, Eva Cecilie

    2012-01-01

    and humans. We found that some pesticides, plasticizers and phytoestrogens can activate the AhR, and the combined effect of compounds with no or weak AhR potency cannot be ignored. The significant DL-activity in the wastewater effluent indicates the treatment is not sufficient to prevent contamination...... of surface waters with dioxins. Our results from human studies suggest that the serum DL-activity reflect the complex mixture of persistent organic pollutants (POPs). Greenlandic Inuit had lower serum DL-activity level compared to Europeans, probably due to long distance from the dioxin sources and UV...... a cost-effective and integrated screening tool for measurement of the DL-activity in human, environmental and commercial samples....

  14. Dioxin-like activity in environmental and human samples from Greenland and Denmark

    DEFF Research Database (Denmark)

    Long, Manhai; Bonefeld-Jørgensen, Eva Cecilie

    2012-01-01

    and humans. We found that some pesticides, plasticizers and phytoestrogens can activate the AhR, and the combined effect of compounds with no or weak AhR potency cannot be ignored. The significant DL-activity in the wastewater effluent indicates the treatment is not sufficient to prevent contamination...... of surface waters with dioxins. Our results from human studies suggest that the serum DL-activity reflect the complex mixture of persistent organic pollutants (POPs). Greenlandic Inuit had lower serum DL-activity level compared to Europeans, probably due to long distance from the dioxin sources and UV...... a cost-effective and integrated screening tool for measurement of the DL-activity in human, environmental and commercial samples....

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

    Science.gov (United States)

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

    2017-04-01

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

  16. Least destructive sampling of human remains using laser drilling for Sr isotope analysis by TIMS

    Science.gov (United States)

    Willmes, Malte; Moffat, Ian; Grün, Rainer; Armstrong, Richard; Kinsley, Les; McMorrow, Linda

    2013-04-01

    Strontium isotope ratios (87Sr/86Sr) measured in ancient human remains can be used to reconstruct migration patterns of ancient human populations. This application is based on the fact that different geologic regions have distinct Sr isotope signatures that are cycled through the soils, plants and rivers, and eventually enter the food cycle. Sr isotope ratios measured in skeletal remains (bones and teeth) reflect the average of dietary Sr that was consumed when the tissue was formed, allowing the investigation of human migration between geologically distinct terrains. The analysis of human remains is always a sensitive topic requiring minimal damage to the sample, while at the same time providing highly precise and accurate results. Samples can be analysed either by solution methods like thermal ionisation mass spectrometry (TIMS), or by in-situ laser ablation MC-ICP-MS. For TIMS a drill is used to extract a small amount of sample, which is then digested in acid and Sr is separated out using ion exchange chromatography. This technique provides highly precise and accurate results, because any isobaric interferences are removed during chemical separation. The drawback is that drilling may cause visible damage to the sample, restricting access to precious human remains. LA-MC-ICP-MS analysis is very fast and nearly destruction free. However, the accuracy and precision of LA-MC-ICP-MS is limited by a number of factors including large instrumental mass discrimination, laser-induced isotopic and elemental fractionations and molecular interferences on 87Sr. Its application thus requires rigorous data reduction, which can introduce significant uncertainties into the analysis. This is especially true for samples with relatively low Sr concentrations such as human teeth (e.g., Woodhead et al., 2005; Horstwood et al., 2008; Vroon et al., 2008). In addition, LA-MC-ICP-MS has traditionally required a flat sample surface, thus an unbroken tooth needs to be cut, which is rather

  17. Laser induced breakdown spectroscopy of human liver samples with Wilson's disease

    Science.gov (United States)

    Grolmusová, Zuzana; Horňáčková, Michaela; Plavčan, Jozef; Kopáni, Martin; Babál, Pavel; Veis, Pavel

    2013-08-01

    Laser induced breakdown spectroscopy (LIBS) is an elemental analytical technique with various applications. The paper demonstrates the first LIBS measurements of human liver samples for the purpose of detecting the higher copper content related with the advanced stage of Wilson's disease. These measurements were implemented using a Nd:YAG laser working at the wavelength of 532 nm and an echelle type spectrometer equipped with an intensified CCD camera allowing for a wide spectral range coverage (200-950 nm) and rapid camera gating (minimum gating time of 5 ns). Seven liver samples with suspected Wilson's disease and five reference samples were investigated. The main parameter of interest was the Cu/C ratio obtained at first from spectra and secondly directly from an iCCD image. Our experiment is a pilot study, which shows LIBS analysis of human liver samples for the purpose of detecting the normal and higher copper content for the first time. The method proved to be a quick and a low-cost approach for the detection of pathological accumulation of copper in the affected tissue.

  18. DNA purification from crude samples for human identification using gradient elution isotachophoresis.

    Science.gov (United States)

    Strychalski, Elizabeth A; Konek, Christopher; Butts, Erica L R; Vallone, Peter M; Henry, Alyssa C; Ross, David

    2013-09-01

    Gradient elution isotachophoresis (GEITP) was demonstrated for DNA purification, concentration, and quantification from crude samples, represented here by soiled buccal swabs, with minimal sample preparation prior to human identification using STR analysis. During GEITP, an electric field applied across leading and trailing electrolyte solutions resulted in isotachophoretic focusing of DNA at the interface between these solutions, while a pressure-driven counterflow controlled the movement of the interface from the sample reservoir into a microfluidic capillary. This counterflow also prevented particulates from fouling or clogging the capillary and reduced or eliminated contamination of the delivered DNA by PCR inhibitors. On-line DNA quantification using laser-induced fluorescence compared favorably with quantitative PCR measurements and potentially eliminates the need for quantitative PCR prior to STR analysis. GEITP promises to address the need for a rapid and robust method to deliver DNA from crude samples to aid the forensic community in human identification. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Detection of Streptococcus mutans Genomic DNA in Human DNA Samples Extracted from Saliva and Blood

    Science.gov (United States)

    Vieira, Alexandre R.; Deeley, Kathleen B.; Callahan, Nicholas F.; Noel, Jacqueline B.; Anjomshoaa, Ida; Carricato, Wendy M.; Schulhof, Louise P.; DeSensi, Rebecca S.; Gandhi, Pooja; Resick, Judith M.; Brandon, Carla A.; Rozhon, Christopher; Patir, Asli; Yildirim, Mine; Poletta, Fernando A.; Mereb, Juan C.; Letra, Ariadne; Menezes, Renato; Wendell, Steven; Lopez-Camelo, Jorge S.; Castilla, Eduardo E.; Orioli, Iêda M.; Seymen, Figen; Weyant, Robert J.; Crout, Richard; McNeil, Daniel W.; Modesto, Adriana; Marazita, Mary L.

    2011-01-01

    Caries is a multifactorial disease, and studies aiming to unravel the factors modulating its etiology must consider all known predisposing factors. One major factor is bacterial colonization, and Streptococcus mutans is the main microorganism associated with the initiation of the disease. In our studies, we have access to DNA samples extracted from human saliva and blood. In this report, we tested a real-time PCR assay developed to detect copies of genomic DNA from Streptococcus mutans in 1,424 DNA samples from humans. Our results suggest that we can determine the presence of genomic DNA copies of Streptococcus mutans in both DNA samples from caries-free and caries-affected individuals. However, we were not able to detect the presence of genomic DNA copies of Streptococcus mutans in any DNA samples extracted from peripheral blood, which suggests the assay may not be sensitive enough for this goal. Values of the threshold cycle of the real-time PCR reaction correlate with higher levels of caries experience in children, but this correlation could not be detected for adults. PMID:21731912

  20. Novel genotype of Ehrlichia canis detected in samples of human blood bank donors in Costa Rica.

    Science.gov (United States)

    Bouza-Mora, Laura; Dolz, Gaby; Solórzano-Morales, Antony; Romero-Zuñiga, Juan José; Salazar-Sánchez, Lizbeth; Labruna, Marcelo B; Aguiar, Daniel M

    2017-01-01

    This study focuses on the detection and identification of DNA and antibodies to Ehrlichia spp. in samples of blood bank donors in Costa Rica using molecular and serological techniques. Presence of Ehrlichia canis was determined in 10 (3.6%) out of 280 blood samples using polymerase chain reaction (PCR) targeting the ehrlichial dsb conserved gene. Analysis of the ehrlichial trp36 polymorphic gene in these 10 samples revealed substantial polymorphism among the E. canis genotypes, including divergent tandem repeat sequences. Nucleotide sequences of dsb and trp36 amplicons revealed a novel genotype of E. canis in blood bank donors from Costa Rica. Indirect immunofluorescence assay (IFA) detected antibodies in 35 (35%) of 100 serum samples evaluated. Thirty samples showed low endpoint titers (64-256) to E. canis, whereas five sera yielded high endpoint titers (1024-8192); these five samples were also E. canis-PCR positive. These findings represent the first report of the presence of E. canis in humans in Central America.

  1. Isolation and clinical sample typing of human leptospirosis cases in Argentina.

    Science.gov (United States)

    Chiani, Yosena; Jacob, Paulina; Varni, Vanina; Landolt, Noelia; Schmeling, María Fernanda; Pujato, Nazarena; Caimi, Karina; Vanasco, Bibiana

    2016-01-01

    Leptospira typing is carried out using isolated strains. Because of difficulties in obtaining them, direct identification of infective Leptospira in clinical samples is a high priority. Multilocus sequence typing (MLST) proved highly discriminatory for seven pathogenic species of Leptospira, allowing isolate characterization and robust assignment to species, in addition to phylogenetic evidence for the relatedness between species. In this study we characterized Leptospira strains circulating in Argentina, using typing methods applied to human clinical samples and isolates. Phylogenetic studies based on 16S ribosomal RNA gene sequences enabled typing of 8 isolates (6 Leptospira interrogans, one Leptospira wolffii and one Leptospira broomii) and 58 out of 85 (68.2%) clinical samples (55 L. interrogans, 2 Leptospira meyeri, and one Leptospira kirschneri). MLST results for the L. interrogans isolates indicated that five were probably Canicola serogroup (ST37) and one was probably Icterohaemorrhagiae serogroup (ST17). Eleven clinical samples (21.6%), provided MLST interpretable data: five were probably Pyrogenes serogroup (ST13), four Sejroe (ST20), one Autumnalis (ST22) and one Canicola (ST37). To the best of our knowledge this study is the first report of the use of an MLST typing scheme with seven loci to identify Leptospira directly from clinical samples in Argentina. The use of clinical samples presents the advantage of the possibility of knowing the infecting strain without resorting to isolates. This study also allowed, for the first time, the characterization of isolates of intermediate pathogenicity species (L. wolffii and L. broomii) from symptomatic patients.

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

    Directory of Open Access Journals (Sweden)

    Maria Antonietta Distasi

    2011-06-01

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

  3. Dioxin and PCB levels in human samples from the Greek population

    Energy Technology Data Exchange (ETDEWEB)

    Leondiadis, L.; Vassiliadou, I.; Costopoulou, D.; Papadopoulos, A. [Mass Spectrometry and Dioxin Analysis Lab. - NCSR Demokritos, Athens (Greece)

    2004-09-15

    Polychlorinated biphenyls (PCBs) are commercial chemical substances produced in a large scale since 1930, with a wide range of applications in industry, such as for coolant fluids in transformers and dielectric fluids in capacitors. After their health effects became apparent, PCB production was banned in the late 1970s. However, humans are still exposed through PCB leakage of old capacitors and transformers and disposal of contaminated materials. Dioxins (polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzo-furans (PCDFs)), are formed as undesirable by-products mainly during the production of chlorinated chemicals and during the combustion of municipal and hazardous waste. Due to potential health hazard (dermal toxicity, immunotoxicity, reproductive effects, teratogenicity, endocrine disruption and carcinogenicity), their monitoring in humans is of high general concern. Enough information on POP presence in human tissues from industrialized countries is available to suggest that the concentration of these compounds has decreased during the last 10 years. Monitoring of human exposure to PCBs and dioxins, contaminants that accumulate in lipid tissue, is most conveniently performed by analysis of blood plasma or blood serum. Monitoring of dioxins in human milk is of also great importance, since it is especially feared that lactational exposure to dioxins and related compounds may adversely affect brain development and the immune system of infants and children. The present study includes the analyses of non-ortho, mono-ortho, indicator PCBs, and PCDD/Fs in human blood and human milk samples collected between November 2002 and February 2004 and is the first study of this kind to be undertaken in Greece.

  4. Genetic identification of missing persons: DNA analysis of human remains and compromised samples.

    Science.gov (United States)

    Alvarez-Cubero, M J; Saiz, M; Martinez-Gonzalez, L J; Alvarez, J C; Eisenberg, A J; Budowle, B; Lorente, J A

    2012-01-01

    Human identification has made great strides over the past 2 decades due to the advent of DNA typing. Forensic DNA typing provides genetic data from a variety of materials and individuals, and is applied to many important issues that confront society. Part of the success of DNA typing is the generation of DNA databases to help identify missing persons and to develop investigative leads to assist law enforcement. DNA databases house DNA profiles from convicted felons (and in some jurisdictions arrestees), forensic evidence, human remains, and direct and family reference samples of missing persons. These databases are essential tools, which are becoming quite large (for example the US Database contains 10 million profiles). The scientific, governmental and private communities continue to work together to standardize genetic markers for more effective worldwide data sharing, to develop and validate robust DNA typing kits that contain the reagents necessary to type core identity genetic markers, to develop technologies that facilitate a number of analytical processes and to develop policies to make human identity testing more effective. Indeed, DNA typing is integral to resolving a number of serious criminal and civil concerns, such as solving missing person cases and identifying victims of mass disasters and children who may have been victims of human trafficking, and provides information for historical studies. As more refined capabilities are still required, novel approaches are being sought, such as genetic testing by next-generation sequencing, mass spectrometry, chip arrays and pyrosequencing. Single nucleotide polymorphisms offer the potential to analyze severely compromised biological samples, to determine the facial phenotype of decomposed human remains and to predict the bioancestry of individuals, a new focus in analyzing this type of markers.

  5. A simple sample preparation method for measuring amoxicillin in human plasma by hollow fiber centrifugal ultrafiltration.

    Science.gov (United States)

    Dong, Wei-Chong; Hou, Zi-Li; Jiang, Xin-Hui; Jiang, Ye

    2013-02-01

    A simple sample preparation method has been developed for the determination of amoxicillin in human plasma by hollow fiber centrifugal ultrafiltration (HF-CF-UF). A 400-μL plasma sample was placed directly into the HF-CF-UF device, which consisited of a slim glass tube and a U-shaped hollow fiber. After centrifugation at 1.25 × 10(3) g for 10 min, the filtrate was withdrawn from the hollow fiber and 20 µL was directly injected into the high-performance liquid chromatography (HPLC) for analysis. The calibration curve was linear over the range of 0.1-20 µg/mL (r = 0.9996) and the limit of detection was as low as 0.025 µg/mL. The average recovery and absolute recovery were 99.9% and 84.5%, respectively. Both the intra-day and inter-day precisions (relative standard deviation) were less than 3.1% for three concentrations (0.25, 2.5 and 10 µg/mL). The sample preparation process was simplified. Only after a single centrifugal ultrafiltration can the filtrate be injected directly into HPLC. The present method is simple, sensitive and accurate. It could be effective for the analysis of biological samples with high protein contents, especially for the biopharmaceutical analysis of drugs that use traditional isolation techniques for sample preparation such as the protein precipitation method.

  6. Small Sample Kernel Association Tests for Human Genetic and Microbiome Association Studies.

    Science.gov (United States)

    Chen, Jun; Chen, Wenan; Zhao, Ni; Wu, Michael C; Schaid, Daniel J

    2016-01-01

    Kernel machine based association tests (KAT) have been increasingly used in testing the association between an outcome and a set of biological measurements due to its power to combine multiple weak signals of complex relationship with the outcome through the specification of a relevant kernel. Human genetic and microbiome association studies are two important applications of KAT. However, the classic KAT framework relies on large sample theory, and conservativeness has been observed for small sample studies, especially for microbiome association studies. The common approach for addressing the small sample problem relies on computationally intensive resampling methods. Here, we derive an exact test for KAT with continuous traits, which resolve the small sample conservatism of KAT without the need for resampling. The exact test has significantly improved power to detect association for microbiome studies. For binary traits, we propose a similar approximate test, and we show that the approximate test is very powerful for a wide range of kernels including common variant- and microbiome-based kernels, and the approximate test controls the type I error well for these kernels. In contrast, the sequence kernel association tests have slightly inflated genomic inflation factors after small sample adjustment. Extensive simulations and application to a real microbiome association study are used to demonstrate the utility of our method. © 2015 WILEY PERIODICALS, INC.

  7. A salting out and resin procedure for extracting Schistosoma mansoni DNA from human urine samples

    Directory of Open Access Journals (Sweden)

    Rodrigues Nilton B

    2010-04-01

    Full Text Available Abstract Background In this paper a simple and cheap salting out and resin (InstaGene matrix® resin - BioRad DNA extraction method from urine for PCR assays is introduced. The DNA of the fluke Schistosoma mansoni was chosen as the target since schistosomiasis lacks a suitable diagnostic tool which is sensitive enough to detect low worm burden. It is well known that the PCR technique provides high sensitivity and specificity in detecting parasite DNA. Therefore it is of paramount importance to take advantage of its excellent performance by providing a simple to handle and reliable DNA extraction procedure, which permits the diagnosis of the disease in easily obtainable urine samples. Findings The description of the extraction procedure is given. This extraction procedure was tested for reproducibility and efficiency in artificially contaminated human urine samples. The reproducibility reached 100%, showing positive results in 5 assay repetitions of 5 tested samples each containing 20 ng DNA/5 ml. The efficiency of the extraction procedure was also evaluated in a serial dilution of the original 20 ng DNA/5 ml sample. Detectable DNA was extracted when it was at a concentration of 1.28 pg DNA/mL, revealing the high efficiency of this procedure. Conclusions This methodology represents a promising tool for schistosomiasis diagnosis utilizing a bio-molecular technique in urine samples which is now ready to be tested under field conditions and may be applicable to the diagnosis of other parasitic diseases.

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

    Directory of Open Access Journals (Sweden)

    Hanifi Körkoca

    2015-03-01

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

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

    Directory of Open Access Journals (Sweden)

    Alvarez Marina M

    2011-05-01

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

  10. Recognition of serous ovarian tumors in human samples by multimodal nonlinear optical microscopy

    Science.gov (United States)

    Adur, Javier; Pelegati, Vitor B.; Costa, Leverson F. L.; Pietro, Luciana; de Thomaz, Andre A.; Almeida, Diogo B.; Bottcher-Luiz, Fatima; Andrade, Liliana A. L. A.; Cesar, Carlos L.

    2011-09-01

    We used a multimodal nonlinear optics microscopy, specifically two-photon excited fluorescence (TPEF), second and third harmonic generation (SHG/THG) microscopies, to observe pathological conditions of ovarian tissues obtained from human samples. We show that strong TPEF + SHG + THG signals can be obtained in fixed samples stained with hematoxylin and eosin (H&E) stored for a very long time, and that H&E staining enhanced the THG signal. We then used the multimodal TPEF-SHG-THG microscopies in a stored file of H&E stained samples of human ovarian cancer to obtain complementary information about the epithelium/stromal interface, such as the transformation of epithelium surface (THG) and the overall fibrillary tissue architecture (SHG). This multicontrast nonlinear optics microscopy is able to not only differentiate between cancerous and healthy tissue, but can also distinguish between normal, benign, borderline, and malignant specimens according to their collagen disposition and compression levels within the extracellular matrix. The dimensions of the layers of epithelia can also be measured precisely and automatically. Our data demonstrate that optical techniques can detect pathological changes associated with ovarian cancer.

  11. Exploring the acceptability of human papillomavirus self-sampling among Muslim immigrant women.

    Science.gov (United States)

    Lofters, Aisha K; Vahabi, Mandana; Fardad, Mitra; Raza, Afrah

    2017-01-01

    With appropriate screening (ie, the Papanicolaou [Pap] test), cervical cancer is highly preventable, and high-income countries, including Canada, have observed significant decreases in cervical cancer mortality. However, certain subgroups, including immigrants from countries with large Muslim populations, experience disparities in cervical cancer screening. Little is known about the acceptability of human papillomavirus (HPV) self-sampling as a screening strategy among Muslim immigrant women in Canada. This study assessed cervical cancer screening practices, knowledge and attitudes, and acceptability of HPV self-sampling among Muslim immigrant women. A convenience sample of 30 women was recruited over a 3-month period (June-August 2015) in the Greater Toronto Area. All women were between 21 and 69 years old, foreign-born, and self-identified as Muslim, and had good knowledge of English. Data were collected through a self-completed questionnaire. More than half of the participants falsely indicated that Pap tests may cause cervical infection, and 46.7% indicated that the test is an intrusion on privacy. The majority of women reported that they would be willing to try HPV self-sampling, and more than half would prefer this method to provider-administered sampling methods. Barriers to self-sampling included confidence in the ability to perform the test and perceived cost, and facilitators included convenience and privacy being preserved. The results demonstrate that HPV self-sampling may provide a favorable alternative model of care to the traditional provider-administered Pap testing. These findings add important information to the literature related to promoting cancer screening among women who are under or never screened for cervical cancer.

  12. Human-parathormone assay for use in dogs: validation, sample handling studies, and parathyroid function testing.

    Science.gov (United States)

    Torrance, A G; Nachreiner, R

    1989-07-01

    Ten commercially available parathormone (PTH) assays were competitively validated, using dilutional parallelism, intra-assay and interassay coefficients of variation, and sensitivity and measured responses of 2 dogs to calcium and EDTA infusions. A 2-site immunoradiometric assay for intact human-PTH was superior to the others for estimating canine-PTH, met the criteria for validity, and was further investigated. A series of sample-handling studies was performed. Serum and plasma samples stored at 24 C lost 15% (n = 5; P less than 0.05) of PTH between 2 and 24 hours. This did not occur at 6 C. The mean PTH concentration of sera from blood samples clotted at 24 C was 6% (P less than 0.05) higher than equivalent EDTA samples. Serum samples stored at 6 and 37 C deteriorated 35% and 100% (n = 5; P less than 0.05), respectively, after 1 week, whereas samples stored at -20 and -70 C for 4 weeks did not deteriorate. There was no significant deterioration of PTH in samples frozen (-40 C) and thawed up to 7 times (n = 5). Parathyroid function testing was investigated by use of 2-hour infusions of disodium EDTA (25 mg/kg/h), 10-minute infusions of calcium gluconate (3 mg of elemental calcium/kg/10 min), and physiologic saline controls (n = 8). Renal function was monitored before and after EDTA infusion by exogenous creatinine clearance. Infusion of disodium EDTA increased mean PTH concentration from 67 (time 0) to 317 and 235 pg/ml at 90 and 180 minutes, respectively (P less than 0.001). Infusion of calcium gluconate decreased mean PTH concentration from 84 (time 0) to 14 and 12 pg/ml at 15 and 60 minutes, respectively (P less than 0.005). There were no observable side effects of the infusions in normal conscious dogs and no differences in exogenous creatinine clearance after EDTA infusion.

  13. Freezing adversely affects measurement of vascular endothelial growth factor levels in human aqueous samples

    Directory of Open Access Journals (Sweden)

    Sankarathi Balaiya

    2011-01-01

    Full Text Available Sankarathi Balaiya Sandeep Grover Ravi K Murthy Kakarla V ChalamDepartment of Ophthalmology, University of Florida College of Medicine, Jacksonville, FL, USAPurpose: Aqueous levels of vascular endothelial growth factor (VEGF can be a surrogate marker of intraocular VEGF activity and a measure of efficacy of anti-VEGF treatment in a variety of vasoproliferative retinal disorders, including diabetic retinopathy, age-related macular degeneration, and central retinal vein occlusion. Measurement of the VEGF level may be adversely affected by premeasurement variables, such as freezing and delay, in sample analysis. We aim to evaluate the effect of storage and delayed measurement of human aqueous VEGF levels in these conditions.Methods: Aqueous samples collected from patients receiving intravitreal injection of bevacizumab for various retinal diseases were divided into two groups. In Group 1, the VEGF levels were analyzed on the same day; in Group 2, the VEGF levels were analyzed after 21 days of freezer storage (-80°C using immunobead assay. Statistical comparison using a paired t-test was performed between the two groups.Results: Thirty-one aqueous humor samples were collected, and the VEGF concentration for fresh samples was 7.8 ± 5.9 pg/mL (mean ± SD compared to 6.5 ± 6.0 pg/mL in frozen samples, resulting in a statistically significant difference (P = 0.03.Conclusions: Accurate measurement of the VEGF level is a vital component of clinical decision-making. Delayed analysis of VEGF levels in aqueous samples may result in significant sample degradation and lower levels of measured VEGF.Keywords: VEGF level, aqueous humor, immunobead assay, VEGF storage

  14. Study of microtip-based extraction and purification of DNA from human samples for portable devices

    Science.gov (United States)

    Fotouhi, Gareth

    DNA sample preparation is essential for genetic analysis. However, rapid and easy-to-use methods are a major challenge to obtaining genetic information. Furthermore, DNA sample preparation technology must follow the growing need for point-of-care (POC) diagnostics. The current use of centrifuges, large robots, and laboratory-intensive protocols has to be minimized to meet the global challenge of limited access healthcare by bringing the lab to patients through POC devices. To address these challenges, a novel extraction method of genomic DNA from human samples is presented by using heat-cured polyethyleneimine-coated microtips generating a high electric field. The microtip extraction method is based on recent work using an electric field and capillary action integrated into an automated device. The main challenges to the method are: (1) to obtain a stable microtip surface for the controlled capture and release of DNA and (2) to improve the recovery of DNA from samples with a high concentration of inhibitors, such as human samples. The present study addresses these challenges by investigating the heat curing of polyethyleneimine (PEI) coated on the surface of the microtip. Heat-cured PEI-coated microtips are shown to control the capture and release of DNA. Protocols are developed for the extraction and purification of DNA from human samples. Heat-cured PEI-coated microtip methods of DNA sample preparation are used to extract genomic DNA from human samples. It is discovered through experiment that heat curing of a PEI layer on a gold-coated surface below 150°C could inhibit the signal of polymerase chain reaction (PCR). Below 150°C, the PEI layer is not completely cured and dissolved off the gold-coated surface. Dissolved PEI binds with DNA to inhibit PCR. Heat curing of a PEI layer above 150°C on a gold-coated surface prevents inhibition to PCR and gel electrophoresis. In comparison to gold-coated microtips, the 225°C-cured PEI-coated microtips improve the

  15. Paenibacillus spp. isolated from human and environmental samples in Spain: detection of 11 new species

    Directory of Open Access Journals (Sweden)

    J.A. Sáez-Nieto

    2017-09-01

    Full Text Available One hundred thirty-six isolates, 88 human and 48 environmental, that met the requirements to belong to the genus Paenibacillus were identified using a polyphasic taxonomic approach known as 16S rRNA plus phenotypic traits. Thirty-seven Paenibacillus species were identified; some had not been previously reported from clinical samples. The main species were P. pabuli (13 isolates, P. provencensis (11, P. phoenicis (9 and P. lautus (8. P. pabuli (11/13 and P. provencensis (8/11 were mainly environmental isolates, while P. phoenicis (9/9 and P. lautus (6/8 were mainly human isolates. Despite the difficulties in assigning to human Paenibacillus isolates a role as a pathogen or contaminant, here 25% of the isolates were involved in true infections, especially in those cases that affected abscesses, wound exudates, ocular infections and diverse fluids. In addition, 15 isolates were identified as 11 ‘Candidatus’ to a new species, all of them from human specimens except one that was obtained from laboratory air. The antimicrobial susceptibility testing showed 95.6% of isolates were resistant to ampicillin, 44% were resistant to cotrimoxazole, 20 to 30% were resistant to cefotaxime and vancomycin and 13% were resistant to rifampicin and erythromycin.

  16. Applicability of the CALUX bioassay for screening of dioxin levels in human milk samples

    DEFF Research Database (Denmark)

    Laier, P.; Cederberg, Tommy Licht; Larsen, John Christian;

    2003-01-01

    . The results obtained with the bioassay when testing milk extracts fractionated into dioxins/furans, non-ortho PCB and mono/di-ortho PCB fractions indicated that the correlation between the bioassay and the chemical analyses depends primarily on the A receptor activity observed in the mono/di-ortho PCB......The CALUX (chemically activated luciferase expression) bioassay based on rat hepatoma (H4IIE) cells is a sensitive assay for the detection of Ah receptor agonists like 2,3,7,8-substituted chlorinated dibenzo-p-dioxins and dibenzofurans and related PCBs. In this paper, the assay was optimized...... and applied for monitoring levels of dioxins in human milk samples. Combination effects of dioxin-like compounds were evaluated by testing potential mechanisms of interaction between seven of the major dioxin-like compounds in human milk using the isobole method. Results showed that the compounds acted...

  17. Dioxin-like activity in environmental and human samples from Greenland and Denmark

    DEFF Research Database (Denmark)

    Long, Manhai; Bonefeld-Jørgensen, Eva Cecilie

    2012-01-01

    of surface waters with dioxins. Our results from human studies suggest that the serum DL-activity reflect the complex mixture of persistent organic pollutants (POPs). Greenlandic Inuit had lower serum DL-activity level compared to Europeans, probably due to long distance from the dioxin sources and UV...... degradation of the high potent dioxin and/or the inhibitory effect of the high level of non-DL POPs. Selective bioaccumulation of PCBs in the food chain may contribute to the negative correlation between serum POPs and DL-activity observed in Greenlandic Inuit. Hence the AhR transactivation bioassay provides...... a cost-effective and integrated screening tool for measurement of the DL-activity in human, environmental and commercial samples....

  18. Nondestructive sampling of human skeletal remains yields ancient nuclear and mitochondrial DNA.

    Science.gov (United States)

    Bolnick, Deborah A; Bonine, Holly M; Mata-Míguez, Jaime; Kemp, Brian M; Snow, Meradeth H; LeBlanc, Steven A

    2012-02-01

    Museum curators and living communities are sometimes reluctant to permit ancient DNA (aDNA) studies of human skeletal remains because the extraction of aDNA usually requires the destruction of at least some skeletal material. Whether these views stem from a desire to conserve precious materials or an objection to destroying ancestral remains, they limit the potential of aDNA research. To help address concerns about destructive analysis and to minimize damage to valuable specimens, we describe a nondestructive method for extracting DNA from ancient human remains. This method can be used with both teeth and bone, but it preserves the structural integrity of teeth much more effectively than that of bone. Using this method, we demonstrate that it is possible to extract both mitochondrial and nuclear DNA from human remains dating between 300 BC and 1600 AD. Importantly, the method does not expose the remains to hazardous chemicals, allowing them to be safely returned to curators, custodians, and/or owners of the samples. We successfully amplified mitochondrial DNA from 90% of the individuals tested, and we were able to analyze 1-9 nuclear loci in 70% of individuals. We also show that repeated nondestructive extractions from the same tooth can yield amplifiable mitochondrial and nuclear DNA. The high success rate of this method and its ability to yield DNA from samples spanning a wide geographic and temporal range without destroying the structural integrity of the sampled material may make possible the genetic study of skeletal collections that are not available for destructive analysis. Copyright © 2011 Wiley Periodicals, Inc.

  19. An Improved Method for High Quality Metagenomics DNA Extraction from Human and Environmental Samples

    DEFF Research Database (Denmark)

    Bag, Satyabrata; Saha, Bipasa; Mehta, Ojasvi

    2016-01-01

    and human origin samples. We introduced a combination of physical, chemical and mechanical lysis methods for proper lysis of microbial inhabitants. The community microbial DNA was precipitated by using salt and organic solvent. Both the quality and quantity of isolated DNA was compared with the existing...... methodologies and the supremacy of our method was confirmed. Maximum recovery of genomic DNA in the absence of substantial amount of impurities made the method convenient for nucleic acid extraction. The nucleic acids obtained using this method are suitable for different downstream applications. This improved...

  20. Modular Sampling and Analysis Techniques for the Real-Time Analysis of Human Breath

    Energy Technology Data Exchange (ETDEWEB)

    Frank, M; Farquar, G; Adams, K; Bogan, M; Martin, A; Benner, H; Spadaccini, C; Steele, P; Davis, C; Loyola, B; Morgan, J; Sankaran, S

    2007-07-09

    At LLNL and UC Davis, we are developing several techniques for the real-time sampling and analysis of trace gases, aerosols and exhaled breath that could be useful for a modular, integrated system for breath analysis. Those techniques include single-particle bioaerosol mass spectrometry (BAMS) for the analysis of exhaled aerosol particles or droplets as well as breath samplers integrated with gas chromatography mass spectrometry (GC-MS) or MEMS-based differential mobility spectrometry (DMS). We describe these techniques and present recent data obtained from human breath or breath condensate, in particular, addressing the question of how environmental exposure influences the composition of breath.

  1. Genetic Characterization of Atypical Mansonella (Mansonella) ozzardi Microfilariae in Human Blood Samples from Northeastern Peru

    Science.gov (United States)

    Marcos, Luis A.; Arrospide, Nancy; Recuenco, Sergio; Cabezas, Cesar; Weil, Gary J.; Fischer, Peter U.

    2012-01-01

    DNA sequence comparisons are useful for characterizing proposed new parasite species or strains. Microfilariae with an atypical arrangement of nuclei behind the cephalic space have been recently described in human blood samples from the Amazon region of Peru. Three blood specimens containing atypical microfilariae were genetically characterized using three DNA markers (5S ribosomal DNA, 12S ribosomal DNA, and cytochrome oxidase I). All atypical microfilariae were clustered into the Mansonella group and indistinguishable from M. ozzardi based on these DNA markers. PMID:22826497

  2. [The quality control of preanalytical variations for the determination of lead in samples of human origin].

    Science.gov (United States)

    Zhong, Kun; Wang, Wei; He, Falin; Wang, Zhiguo

    2015-02-01

    The aims of this article was to provide the quality control requirements of preanalytical variation for the determination of lead in samples of human origin, reduce the influence of preanalytical variation on the test results. According to the Clinical and Laboratory Standards Institute documents, control of preanalytical variation in trace element determinations, analytical procedures for the determination of lead in blood and urine and other references and guidelines, the methods of quality control of lead determination had been made, including: the factors needed to be considered before collection, preservation, transportation and other preanalytical factors, the abilities and considerations of laboratory staff, etc.

  3. Antibiotic resistance genes in the bacteriophage DNA fraction of human fecal samples.

    Science.gov (United States)

    Quirós, Pablo; Colomer-Lluch, Marta; Martínez-Castillo, Alexandre; Miró, Elisenda; Argente, Marc; Jofre, Juan; Navarro, Ferran; Muniesa, Maite

    2014-01-01

    A group of antibiotic resistance genes (ARGs) (blaTEM, blaCTX-M-1, mecA, armA, qnrA, and qnrS) were analyzed by real-time quantitative PCR (qPCR) in bacteriophage DNA isolated from feces from 80 healthy humans. Seventy-seven percent of the samples were positive in phage DNA for one or more ARGs. blaTEM, qnrA, and, blaCTX-M-1 were the most abundant, and armA, qnrS, and mecA were less prevalent. Free bacteriophages carrying ARGs may contribute to the mobilization of ARGs in intra- and extraintestinal environments.

  4. [Human fasciolosis in Mara municipality, Zulia state. Venezuela: prevalence and asociated factors].

    Science.gov (United States)

    Freites, Azael; Colmenares, Cecilia; Alarcón-Noya, Belkisyolé; García, María Eugenia; Díaz-Suárez, Odelis

    2009-12-01

    In Venezuela, human Fasciolosis shows a low frequency. However, Mara Municipality is a highly endemic region for bovine fasciolosis and there are no reports of this parasite infection in humans. To determine the prevalence and associated factors to human fasciolosis in Mara municipality - Zulia state, a total of 51 blood and stool samples were collected. Serums were tested by ELISA and Western Blot (WB) assays, with excretion-secretion antigens of Fasciola hepatica (AFhES). The serum samples that resulted positive by these assays were tested by ELISA IgG anti Toxocara sp, Toxoplasma gondii and cysticerosis. Stool samples were concentrated by the Ritchie and rapid sedimentation techniques. Two serum samples were reactive to ELISA AFhES (3.9%) and these did not recognize the specific molecules of WB-AFhES detected by serum from patients with an absolutely demonstrated infection. Both participants were not positive to IgG anti Toxocara sp, Toxoplasma gondii, cysticerosis, and stool samples of these were negative to intestinal parasites. The general prevalence of intestinal parasites was 52.9% (27/51), being protozoa more frequent than helminthes. No Fasciola eggs were found. The two positives participants had in common that both had worked as fresh pasture cutters. These results suggest that the population had been in contact with F. hepatica, with no active infection because of the lack of specific molecules recognition and the absence of eggs in stool samples. Human fasciolosis has a low frequency in Venezuela and is underestimated and underrecognized by health workers and the general population.

  5. Calcium isolation from large-volume human urine samples for 41Ca analysis by accelerator mass spectrometry.

    Science.gov (United States)

    Miller, James J; Hui, Susanta K; Jackson, George S; Clark, Sara P; Einstein, Jane; Weaver, Connie M; Bhattacharyya, Maryka H

    2013-08-01

    Calcium oxalate precipitation is the first step in preparation of biological samples for (41)Ca analysis by accelerator mass spectrometry. A simplified protocol for large-volume human urine samples was characterized, with statistically significant increases in ion current and decreases in interference. This large-volume assay minimizes cost and effort and maximizes time after (41)Ca administration during which human samples, collected over a lifetime, provide (41)Ca:Ca ratios that are significantly above background.

  6. Calcium Isolation from Large-Volume Human Urine Samples for 41Ca Analysis by Accelerator Mass Spectrometry

    Science.gov (United States)

    Miller, James J; Hui, Susanta K; Jackson, George S; Clark, Sara P; Einstein, Jane; Weaver, Connie M; Bhattacharyya, Maryka H

    2013-01-01

    Calcium oxalate precipitation is the first step in preparation of biological samples for 41Ca analysis by accelerator mass spectrometry. A simplified protocol for large-volume human urine samples was characterized, with statistically significant increases in ion current and decreases in interference. This large-volume assay minimizes cost and effort and maximizes time after 41Ca administration during which human samples, collected over a lifetime, provide 41Ca:Ca ratios that are significantly above background. PMID:23672965

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

    Science.gov (United States)

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

    2007-11-01

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

  8. Human Cytomegalovirus and Human Umbilical Vein Endothelial Cells: Restriction of Primary Isolation to Blood Samples and Susceptibilities of Clinical Isolates from Other Sources to Adaptation

    OpenAIRE

    2002-01-01

    In immunocompromised patients with disseminated infection, human cytomegalovirus (HCMV) is widespread in the microvascular endothelium of multiple organs. Human umbilical vein endothelial cells (HUVEC) were used in parallel to human embryonic lung fibroblasts (HELF) to recover HCMV from blood samples of immunocompromised patients. Using the shell vial technique, comparable median numbers of p72-positive HUVEC and HELF cells were found with the 26 HCMV-positive buffy coat samples out of 150 ex...

  9. Optimization of a Pre-MEKC Separation SPE Procedure for Steroid Molecules in Human Urine Samples

    Directory of Open Access Journals (Sweden)

    Ilona Olędzka

    2013-11-01

    Full Text Available Many steroid hormones can be considered as potential biomarkers and their determination in body fluids can create opportunities for the rapid diagnosis of many diseases and disorders of the human body. Most existing methods for the determination of steroids are usually time- and labor-consuming and quite costly. Therefore, the aim of analytical laboratories is to develop a new, relatively low-cost and rapid implementation methodology for their determination in biological samples. Due to the fact that there is little literature data on concentrations of steroid hormones in urine samples, we have made attempts at the electrophoretic determination of these compounds. For this purpose, an extraction procedure for the optimized separation and simultaneous determination of seven steroid hormones in urine samples has been investigated. The isolation of analytes from biological samples was performed by liquid-liquid extraction (LLE with dichloromethane and compared to solid phase extraction (SPE with C18 and hydrophilic-lipophilic balance (HLB columns. To separate all the analytes a micellar electrokinetic capillary chromatography (MECK technique was employed. For full separation of all the analytes a running buffer (pH 9.2, composed of 10 mM sodium tetraborate decahydrate (borax, 50 mM sodium dodecyl sulfate (SDS, and 10% methanol was selected. The methodology developed in this work for the determination of steroid hormones meets all the requirements of analytical methods. The applicability of the method has been confirmed for the analysis of urine samples collected from volunteers—both men and women (students, amateur bodybuilders, using and not applying steroid doping. The data obtained during this work can be successfully used for further research on the determination of steroid hormones in urine samples.

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

    Directory of Open Access Journals (Sweden)

    Anna E Scott

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

  11. High-throughput miRNA profiling of human melanoma blood samples

    Directory of Open Access Journals (Sweden)

    Rass Knuth

    2010-06-01

    Full Text Available Abstract Background MicroRNA (miRNA signatures are not only found in cancer tissue but also in blood of cancer patients. Specifically, miRNA detection in blood offers the prospect of a non-invasive analysis tool. Methods Using a microarray based approach we screened almost 900 human miRNAs to detect miRNAs that are deregulated in their expression in blood cells of melanoma patients. We analyzed 55 blood samples, including 20 samples of healthy individuals, 24 samples of melanoma patients as test set, and 11 samples of melanoma patients as independent validation set. Results A hypothesis test based approch detected 51 differentially regulated miRNAs, including 21 miRNAs that were downregulated in blood cells of melanoma patients and 30 miRNAs that were upregulated in blood cells of melanoma patients as compared to blood cells of healthy controls. The tets set and the independent validation set of the melanoma samples showed a high correlation of fold changes (0.81. Applying hierarchical clustering and principal component analysis we found that blood samples of melanoma patients and healthy individuals can be well differentiated from each other based on miRNA expression analysis. Using a subset of 16 significant deregulated miRNAs, we were able to reach a classification accuracy of 97.4%, a specificity of 95% and a sensitivity of 98.9% by supervised analysis. MiRNA microarray data were validated by qRT-PCR. Conclusions Our study provides strong evidence for miRNA expression signatures of blood cells as useful biomarkers for melanoma.

  12. Detection of Human Papillomavirus 18 in Cervical Cancer Samples Using PCR-ELISA (DIAPOPS

    Directory of Open Access Journals (Sweden)

    KN Tafreshi

    2011-12-01

    Full Text Available Background and Objectives: Human Papillomavirus (HPV infection is a major risk factor for adenocarcinoma of the cervix. The high-risk types of the virus such as HPV16 and HPV18, which possess the E6 and E7 oncogenes, are responsible for approximately 50% of all cervical cancers. A rapid, sensitive and specific test has been proposed for detection of HPV to improve cervical cancer screening programs.Objectives: The aim of this study was to develop a fast PCR-ELISA assay designated as DIAPOPS (Detection of Immobilized Amplified Products in a One Phase Systemfor detection of HPV16 and HPV18 types in SCC samples and Pap smears. The type specific primers and probes were designed for PCR and PCR-ELISA. The amplified products were hybridized with a specific biotin-labeled probe for HPV18 inner amplicons. The hybrids were detected with peroxidase conjugated avidin. The test was performed on the paraffin block and Pap smear samples from the cervical cancer patients, and the results of DIAPOPS were compared with conventional PCR assay.Results: The 70 samples (SCC and Pap smear samples were collected from Imam Khomeini and Mirzakoochak Khan Hospitals in Tehran. The PCR-based method detected six HPV16 positive, three HPV18 positive and Two HPV33 positive samples. DIAPOPS results were compared with the conventional PCR results and they showed an increase in sensitivity of the DIAPOPS test. Not only all of them were confirmed by PCR-ELISA but also three samples that conventional PCR showed negative for HPV18, were demonstrated positive by the PCR-ELISA method.Conclusion: The results of the study show that modified PCR-ELISA assay is more sensitive to detect HPV types and can be used for diagnostic purposes.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2016-05-01

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

  15. Multi-elemental imaging of paraffin-embedded human samples by laser-induced breakdown spectroscopy

    Science.gov (United States)

    Moncayo, S.; Trichard, F.; Busser, B.; Sabatier-Vincent, M.; Pelascini, F.; Pinel, N.; Templier, I.; Charles, J.; Sancey, L.; Motto-Ros, V.

    2017-07-01

    Chemical elements play central roles for physiological homeostasis in human cells, and their dysregulation might lead to a certain number of pathologies. Novel imaging techniques that improve the work of pathologists for tissue analysis and diagnostics are continuously sought. We report the use of Laser-Induced Breakdown Spectroscopy (LIBS) to perform multi-elemental images of human paraffin-embedded skin samples on the entire biopsy scale in a complementary and compatible way with microscope histopathological examination. A specific instrumental configuration is proposed in order to detect most of the elements of medical interest (i.e. P, Al, Mg, Na, Zn, Si, Fe, and Cu). As an example of medical application, we selected and analysed skin biopsies, including healthy skin tissue, cutaneous metastasis of melanoma, Merkel-cell carcinoma and squamous cell carcinoma. Clear distinctions in the distribution of chemical elements are observed from the different samples investigated. This study demonstrates the high complementarity of LIBS elemental imaging with conventional histopathology, opening new opportunities for any medical application involving metals.

  16. Quantitative second-harmonic generation imaging to detect osteogenesis imperfecta in human skin samples

    Science.gov (United States)

    Adur, J.; Ferreira, A. E.; D'Souza-Li, L.; Pelegati, V. B.; de Thomaz, A. A.; Almeida, D. B.; Baratti, M. O.; Carvalho, H. F.; Cesar, C. L.

    2012-03-01

    Osteogenesis Imperfecta (OI) is a genetic disorder that leads to bone fractures due to mutations in the Col1A1 or Col1A2 genes that affect the primary structure of the collagen I chain with the ultimate outcome in collagen I fibrils that are either reduced in quantity or abnormally organized in the whole body. A quick test screening of the patients would largely reduce the sample number to be studied by the time consuming molecular genetics techniques. For this reason an assessment of the human skin collagen structure by Second Harmonic Generation (SHG) can be used as a screening technique to speed up the correlation of genetics/phenotype/OI types understanding. In the present work we have used quantitative second harmonic generation (SHG) imaging microscopy to investigate the collagen matrix organization of the OI human skin samples comparing with normal control patients. By comparing fibril collagen distribution and spatial organization, we calculated the anisotropy and texture patterns of this structural protein. The analysis of the anisotropy was performed by means of the two-dimensional Discrete Fourier Transform and image pattern analysis with Gray-Level Co-occurrence Matrix (GLCM). From these results, we show that statistically different results are obtained for the normal and disease states of OI.

  17. Goblet cells of the normal human bulbar conjunctiva and their assessment by impression cytology sampling.

    Science.gov (United States)

    Doughty, Michael J

    2012-07-01

    Goblet cells of the conjunctiva are the main source of mucus for the ocular surface. The objectives of this review are to consider the goblet cells as assessed by various histological, cytological and electron microscopy methods, and to assess the consistency of published reports (over more than 25 years) of goblet cell density (GCD) from impression cytology specimens from nominally healthy human subjects. Reported GCD values have been notably variable, with a range from 24 to 2226 cells/mm² for average values. Data analysis suggests that a high density of goblet cells should be expected for the healthy human conjunctiva, with a tendency toward higher values in samples taken from normally covered locations (inferior and superior bulbar conjunctiva) of the open eye (at 973 +/- 789 cells/ mm²) than in samples taken from exposed (interpalpebral) locations (at 427 +/- 376 cells/mm²). No obvious change in GCD was found with respect to age, perhaps because the variability of the data did not allow detection of any age-related decline in GCD. Analyses of published data from 33 other sources indicated a trend for GCD to be lower than normal across a spectrum of ocular surface diseases.

  18. Deep RNA sequencing analysis of readthrough gene fusions in human prostate adenocarcinoma and reference samples

    Directory of Open Access Journals (Sweden)

    Nacu Serban

    2011-01-01

    Full Text Available Abstract Background Readthrough fusions across adjacent genes in the genome, or transcription-induced chimeras (TICs, have been estimated using expressed sequence tag (EST libraries to involve 4-6% of all genes. Deep transcriptional sequencing (RNA-Seq now makes it possible to study the occurrence and expression levels of TICs in individual samples across the genome. Methods We performed single-end RNA-Seq on three human prostate adenocarcinoma samples and their corresponding normal tissues, as well as brain and universal reference samples. We developed two bioinformatics methods to specifically identify TIC events: a targeted alignment method using artificial exon-exon junctions within 200,000 bp from adjacent genes, and genomic alignment allowing splicing within individual reads. We performed further experimental verification and characterization of selected TIC and fusion events using quantitative RT-PCR and comparative genomic hybridization microarrays. Results Targeted alignment against artificial exon-exon junctions yielded 339 distinct TIC events, including 32 gene pairs with multiple isoforms. The false discovery rate was estimated to be 1.5%. Spliced alignment to the genome was less sensitive, finding only 18% of those found by targeted alignment in 33-nt reads and 59% of those in 50-nt reads. However, spliced alignment revealed 30 cases of TICs with intervening exons, in addition to distant inversions, scrambled genes, and translocations. Our findings increase the catalog of observed TIC gene pairs by 66%. We verified 6 of 6 predicted TICs in all prostate samples, and 2 of 5 predicted novel distant gene fusions, both private events among 54 prostate tumor samples tested. Expression of TICs correlates with that of the upstream gene, which can explain the prostate-specific pattern of some TIC events and the restriction of the SLC45A3-ELK4 e4-e2 TIC to ERG-negative prostate samples, as confirmed in 20 matched prostate tumor and normal

  19. Exploring the acceptability of human papillomavirus self-sampling among Muslim immigrant women

    Directory of Open Access Journals (Sweden)

    Lofters AK

    2017-07-01

    Full Text Available Aisha K Lofters,1–4 Mandana Vahabi,5,6 Mitra Fardad,7 Afrah Raza8 1Centre for Urban Health Solutions, Li Ka Shing Knowledge Institute, St. Michael’s Hospital, 2Department of Family and Community Medicine, University of Toronto, 3Department of Family and Community Medicine, St. Michael’s Hospital, 4Institute for Clinical Evaluative Sciences, 5Faculty of Community Services, Daphne Cockwell School of Nursing, 6Graduate Program in Immigration and Settlement Studies, Ryerson University, 7Faculty of Community Service, Daphne Cockwell School of Nursing, Ryerson University, Toronto, ON, Canada; 8University of Michigan Medical School, Ann Arbor, MI, USA Background: With appropriate screening (ie, the Papanicolaou [Pap] test, cervical cancer is highly preventable, and high-income countries, including Canada, have observed significant decreases in cervical cancer mortality. However, certain subgroups, including immigrants from countries with large Muslim populations, experience disparities in cervical cancer screening. Little is known about the acceptability of human papillomavirus (HPV self-sampling as a screening strategy among Muslim immigrant women in Canada. This study assessed cervical cancer screening practices, knowledge and attitudes, and acceptability of HPV self-sampling among Muslim immigrant women. Methods: A convenience sample of 30 women was recruited over a 3-month period (June–August 2015 in the Greater Toronto Area. All women were between 21 and 69 years old, foreign-born, and self-identified as Muslim, and had good knowledge of English. Data were collected through a self-completed questionnaire. Results: More than half of the participants falsely indicated that Pap tests may cause cervical infection, and 46.7% indicated that the test is an intrusion on privacy. The majority of women reported that they would be willing to try HPV self-sampling, and more than half would prefer this method to provider-administered sampling methods

  20. Prevalence of human papillomavirus infection in a clinic sample of transsexuals in Italy.

    Science.gov (United States)

    Loverro, Giuseppe; Di Naro, Edoardo; Caringella, Anna Maria; De Robertis, Anna Lisa; Loconsole, Daniela; Chironna, Maria

    2016-02-01

    Detectable human papillomavirus (HPV) DNA is the most common sexually transmitted infection. Reports on the prevalence of detectable HPV DNA among transsexuals (not sex workers) are scarce. The objective of the study was to determine the prevalence of detectable HPV DNA in a clinic sample of transsexuals and to assess the relationship between detectable HPV DNA and cytological outcomes. Clinical samples (oral, anal, vaginal, cervicovaginal and penile scraped cells) from 35 transsexuals (surgically treated and surgically untreated) who attended the outpatient Clinic of Gender Identity Dysphoria of the Department of Obstetrics and Gynecology of Policlinico Hospital (Bari, Italy) were collected for cytological analysis and HPV DNA detection and typing. All enrolled subjects answered an anonymous structured questionnaire about their sexual habits. Serological status for other sexually transmitted diseases (hepatitis B virus (HBV), hepatitis C virus (HCV), HIV and syphilis) was also evaluated. HPV DNA was detected in 14 of 35 patients (40.0%). The prevalence of detectable HPV DNA was 38.2% (13/34) in tested anal samples, 9.1% (2/22) in vaginal samples and 8.3% (1/12) in penile samples. Oncogenic HPV genotypes have been detected in 93% of HPV-positive transsexuals. More than one-third (35.7%) of HPV-positive transsexuals were infected with at least one of the four vaccine-preventable genotypes, 6, 11, 16 and 18. The high rate of detectable HPV DNA by oncogenic types suggests that periodic cytological screening and clinical evaluation may be necessary since transsexuals are at high risk of anogenital cancer. Also promoting HPV vaccination in younger subjects may be advisable. 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/

  1. Comparison of sequencing platforms for single nucleotide variant calls in a human sample.

    Science.gov (United States)

    Ratan, Aakrosh; Miller, Webb; Guillory, Joseph; Stinson, Jeremy; Seshagiri, Somasekar; Schuster, Stephan C

    2013-01-01

    Next-generation sequencings platforms coupled with advanced bioinformatic tools enable re-sequencing of the human genome at high-speed and large cost savings. We compare sequencing platforms from Roche/454(GS FLX), Illumina/HiSeq (HiSeq 2000), and Life Technologies/SOLiD (SOLiD 3 ECC) for their ability to identify single nucleotide substitutions in whole genome sequences from the same human sample. We report on significant GC-related bias observed in the data sequenced on Illumina and SOLiD platforms. The differences in the variant calls were investigated with regards to coverage, and sequencing error. Some of the variants called by only one or two of the platforms were experimentally tested using mass spectrometry; a method that is independent of DNA sequencing. We establish several causes why variants remained unreported, specific to each platform. We report the indel called using the three sequencing technologies and from the obtained results we conclude that sequencing human genomes with more than a single platform and multiple libraries is beneficial when high level of accuracy is required.

  2. Accumulation levels and characteristics of some pesticides in human adipose tissue samples from Southeast China.

    Science.gov (United States)

    Wang, Na; Shi, Lili; Kong, Deyang; Cai, Daoji; Cao, Yanzhong; Liu, Yongming; Pang, Guofang; Yu, Rongbin

    2011-08-01

    This paper presents a comprehensive study of pesticide levels and bio-accumulation characteristics in human adipose tissues among residents of Southeast China. A large number of adipose samples (n=633) were selected for 58 pesticides and were analyzed by high sensitive Gas Chromatography-Tandem Mass Spectrometry (GC-MS/MS). The results showed that POPs pesticides were frequently detected, including 2,4'-DDD, 2,4'-DDE, 2,4'-DDT, 4,4'-DDD, 4,4'-DDE, 4,4'-DDT, α-HCH, β-HCH, γ-HCH, δ-HCH, hexachlorobenzene (HCB), and mirex. Other detected pesticide species were dicofol, methamidophos and chlordimeform, which have rarely been reported. Comparing to different countries, the concentrations of total DDT and HCH in these three Chinese southeastern sites were in the middle range, whereas the HCB and mirex were in the lower end. A significant correlation was observed between region as well as age and POPs pesticide levels. Some pesticide residue levels were also found significantly correlated to occupation. However, there was no significant correlation between gender and pesticides. Meanwhile, it is interesting to find that mortality of malignant tumors tends to associate with the pesticides levels in human adipose tissue. More importantly, the measured data presented in this study provide realistic information which is useful for assessing human exposure to pesticides in the general population of Southeast China. Copyright © 2011 Elsevier Ltd. All rights reserved.

  3. Tracking human footprints in Antarctica through passive sampling of polycyclic aromatic hydrocarbons in inland lakes.

    Science.gov (United States)

    Yao, Yao; Meng, Xiang-Zhou; Wu, Chen-Chou; Bao, Lian-Jun; Wang, Feng; Wu, Feng-Chang; Zeng, Eddy Y

    2016-06-01

    Freely dissolved polycyclic aromatic hydrocarbons (PAHs) were monitored in seven inland lakes of Antarctica by a polyethylene (PE)-based passive sampling technique, with the objective of tracking human footprints. The measured concentrations of PAHs were in the range of 14-360 ng L(-1) with the highest values concentrated around the Russian Progress II Station, indicating the significance of human activities to the loading of PAHs in Antarctica. The concentrations of PAHs in the inland lakes were in the upper part of the PAHs levels in aquatic environments from remote and background regions across the globe. The composition profiles of PAHs indicated that PAHs in the inland lakes were derived mainly from local oil spills, which was corroborated by a large number of fuel spillage reports from ship and plane crash incidents in Antarctica during recent years. Clearly, local human activities, rather than long-range transport, are the dominant sources of PAH contamination to the inland lakes. Finally, the present study demonstrates the efficacy of PE-based passive samplers for investigating PAHs in the aquatic environment of Antarctica under complex field conditions.

  4. Detection of Mycobacterium tuberculosis and Mycobacterium bovis from human sputum samples through multiplex PCR.

    Science.gov (United States)

    Jabbar, Abdul; Khan, Jafar; Ullah, Aman; Rehman, Hazir; Ali, Ijaz

    2015-07-01

    Tuberculosis (TB) has a long history and being present even before the start of recording history. It has left detrimental effects on all aspect of the life and geared the developments in the science of health. TB is caused by Mycobacterium tuberculosis complex (MTBC) including five species M. tuberculosis, M. bovis, M. africanum, M. canetti, and M. microti. M. tuberculosis and M. bovis infect both animals and humans. Therefore, differentiation of these two closely related species is very important for epidemiological and management purpose. We undertook the present study to characterize mycobacteria isolated from sputum of known TB patients by conventional methods and further, by multiplex PCR (mPCR) to detect the prevalence of Zoonotic TB (TB caused by M. bovis). Sputum samples from TB patient were collected from two tertiary care hospitals in Peshawar i.e. Lady Reading Hospital and Hayatabad Medical Complex. All the samples were subjected to Ziehl Neelsen (ZN) stain, culture on Lowenstein Jensen (LJ) and Stone Brink medium, Nitrate reduction test and multiplex PCR. A total of hundred mycobacterial strains were isolated from these samples on the basis of ZN staining, cultural and biochemical methods. Later on, these isolates were subjected to multiplex PCR by using pncATB-1.2 and pncAMT-2 primers specific to M. tuberculosis and JB21, JB22 primers specific to M. bovis. By means of conventional method, these hundred cultures isolates were differentiated into M. tuberculosis (ninety six) and M. bovis (four). Furthermore, by mPCR, it was determined that out of hundred isolates, ninety-eight were identified as M. tuberculosis and two isolates as M. bovis. This molecular method enables to differentiate M. bovis from M. tuberculosis in human sputum.

  5. Human genomic DNA analysis using a semi-automated sample preparation, amplification, and electrophoresis separation platform.

    Science.gov (United States)

    Raisi, Fariba; Blizard, Benjamin A; Raissi Shabari, Akbar; Ching, Jesus; Kintz, Gregory J; Mitchell, Jim; Lemoff, Asuncion; Taylor, Mike T; Weir, Fred; Western, Linda; Wong, Wendy; Joshi, Rekha; Howland, Pamela; Chauhan, Avinash; Nguyen, Peter; Petersen, Kurt E

    2004-03-01

    The growing importance of analyzing the human genome to detect hereditary and infectious diseases associated with specific DNA sequences has motivated us to develop automated devices to integrate sample preparation, real-time PCR, and microchannel electrophoresis (MCE). In this report, we present results from an optimized compact system capable of processing a raw sample of blood, extracting the DNA, and performing a multiplexed PCR reaction. Finally, an innovative electrophoretic separation was performed on the post-PCR products using a unique MCE system. The sample preparation system extracted and lysed white blood cells (WBC) from whole blood, producing DNA of sufficient quantity and quality for a polymerase chain reaction (PCR). Separation of multiple amplicons was achieved in a microfabricated channel 30 microm x 100 microm in cross section and 85 mm in length filled with a replaceable methyl cellulose matrix operated under denaturing conditions at 50 degrees C. By incorporating fluorescent-labeled primers in the PCR, the amplicons were identified by a two-color (multiplexed) fluorescence detection system. Two base-pair resolution of single-stranded DNA (PCR products) was achieved. We believe that this integrated system provides a unique solution for DNA analysis.

  6. [Detection and typing by molecular biology of human papillomavirus in genital samples].

    Science.gov (United States)

    Suárez Moya, A; Esquivias Gómez, J I; Vidart Aragón, J A; Picazo de la Garza, J J

    2006-06-01

    Recently, there has been a marked increase in human papillomavirus (HPV) infection, and the etiological relationship between some HPV genotypes and genital cancer has been confirmed. Therefore, we used current molecular biology techniques to evaluate the prevalence of these viruses and their genotype in genital samples. We processed 401 genital samples from 281 women and 120 men, all with a diagnosis compatible with HPV infection. Virus was detected using PCR, and positive samples were typed using an array technique which enabled us to detect the 35 most common types of mucous-associated HPV. Of the 401 patients studied, 185 (46.1%) were positive, and only one type of HPV was detected in 133 cases. We found that 41.6% of the women and 56.7% of the men were positive. A total of 260 HPVs were typed; 154 were high oncogenic risk. They infected 16 men (23.5%) and 88 women (75.2%). The difference was statistically significant (pHVP 16 in 52 cases. We found a 46% prevalence of HPV infection. More than half of these patients were infected by high-risk HPV. The presence of high-risk HPV was significantly higher in women.

  7. Polychlorinated biphenyls and organochlorine pesticides in human milk samples from two regions in Croatia.

    Science.gov (United States)

    Klinčić, D; Herceg Romanić, S; Matek Sarić, M; Grzunov, J; Dukić, B

    2014-03-01

    We analyzed 20 polychlorinated biphenyls (PCBs) and seven organochlorine pesticides (OCPs) in milk samples collected during 2009-2011 from primiparae living in two different regions in Croatia. p,p'-DDE is the dominant organochlorine pesticide. α-HCH/γ-HCH and p,p'-DDE/p,p'-DDT ratios indicate that there is fresh input of γ-HCH in investigated population on both locations, while this is not applicable to p,p'-DDT. The PCB profile was dominated by higher chlorinated congeners. Non-ortho PCB congeners which have the highest TEF values were not detected in any of individual samples. Toxic equivalents for mono-ortho substituted PCB congeners indicated higher exposure to toxic PCBs in Zadar, but estimated daily intakes for both locations indicate that infants consuming mother's milk are not at risk of adverse effects caused by PCBs and OCPs. Our study builds on the previous research of human milk samples collected in Zagreb and reveals that over 10-year period, levels of investigated organochlorine compounds decreased significantly. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Analysis of inflammatory response in human plasma samples by an automated multicapillary electrophoresis system.

    Science.gov (United States)

    Larsson, Anders; Hansson, Lars-Olof

    2004-01-01

    A new automated multicapillary zone electrophoresis instrument with a new high-resolution (HR) buffer (Capillarys with HR buffer) for analysis of human plasma proteins was evaluated. Albumin, alpha(1)-antitrypsin, alpha(1)-acid glycoprotein, haptoglobin, fibrinogen, immunoglobulin (Ig)A, IgG and IgM were determined nephelometrically in 200 patient plasma samples. The same samples were then analyzed on the Capillarys system (Sebia, Paris, France). The albumin concentration from the nephelometric determination was used for quantification of the individual peaks in the capillary electrophoresis (CE) electropherogram. There was strong linear correlation between the nephelometric and electrophoretic determination of alpha(1)-antitrypsin (R(2) = 0.906), alpha(1)-acid glycoprotein (R(2) =0.894) and haptoglobin (R(2) = 0.913). There was also good correlation between the two determinations of gamma-globulins (R(2) = 0.883), while the correlation was weaker for fibrinogen (R(2) = 0.377). The Capillarys instrument is a reliable system for plasma protein analysis, combining the advantages of full automation, good analytical performance and high throughput. The HR buffer in combination with albumin quantification allows the simultaneous quantification of inflammatory markers in plasma samples without the need for nephelometric determination of these proteins.

  9. PERT: a method for expression deconvolution of human blood samples from varied microenvironmental and developmental conditions.

    Directory of Open Access Journals (Sweden)

    Wenlian Qiao

    Full Text Available The cellular composition of heterogeneous samples can be predicted using an expression deconvolution algorithm to decompose their gene expression profiles based on pre-defined, reference gene expression profiles of the constituent populations in these samples. However, the expression profiles of the actual constituent populations are often perturbed from those of the reference profiles due to gene expression changes in cells associated with microenvironmental or developmental effects. Existing deconvolution algorithms do not account for these changes and give incorrect results when benchmarked against those measured by well-established flow cytometry, even after batch correction was applied. We introduce PERT, a new probabilistic expression deconvolution method that detects and accounts for a shared, multiplicative perturbation in the reference profiles when performing expression deconvolution. We applied PERT and three other state-of-the-art expression deconvolution methods to predict cell frequencies within heterogeneous human blood samples that were collected under several conditions (uncultured mono-nucleated and lineage-depleted cells, and culture-derived lineage-depleted cells. Only PERT's predicted proportions of the constituent populations matched those assigned by flow cytometry. Genes associated with cell cycle processes were highly enriched among those with the largest predicted expression changes between the cultured and uncultured conditions. We anticipate that PERT will be widely applicable to expression deconvolution strategies that use profiles from reference populations that vary from the corresponding constituent populations in cellular state but not cellular phenotypic identity.

  10. PERT: a method for expression deconvolution of human blood samples from varied microenvironmental and developmental conditions.

    Science.gov (United States)

    Qiao, Wenlian; Quon, Gerald; Csaszar, Elizabeth; Yu, Mei; Morris, Quaid; Zandstra, Peter W

    2012-01-01

    The cellular composition of heterogeneous samples can be predicted using an expression deconvolution algorithm to decompose their gene expression profiles based on pre-defined, reference gene expression profiles of the constituent populations in these samples. However, the expression profiles of the actual constituent populations are often perturbed from those of the reference profiles due to gene expression changes in cells associated with microenvironmental or developmental effects. Existing deconvolution algorithms do not account for these changes and give incorrect results when benchmarked against those measured by well-established flow cytometry, even after batch correction was applied. We introduce PERT, a new probabilistic expression deconvolution method that detects and accounts for a shared, multiplicative perturbation in the reference profiles when performing expression deconvolution. We applied PERT and three other state-of-the-art expression deconvolution methods to predict cell frequencies within heterogeneous human blood samples that were collected under several conditions (uncultured mono-nucleated and lineage-depleted cells, and culture-derived lineage-depleted cells). Only PERT's predicted proportions of the constituent populations matched those assigned by flow cytometry. Genes associated with cell cycle processes were highly enriched among those with the largest predicted expression changes between the cultured and uncultured conditions. We anticipate that PERT will be widely applicable to expression deconvolution strategies that use profiles from reference populations that vary from the corresponding constituent populations in cellular state but not cellular phenotypic identity.

  11. Analysis of persistence of human papillomavirus infection in men evaluated by sampling multiple genital sites.

    Science.gov (United States)

    Capra, G; Nyitray, A G; Lu, B; Perino, A; Marci, R; Schillaci, R; Matranga, D; Firenze, A; Caleca, M; Bellavia, C; Guarneri, F; Giuliano, A; Giovannelli, L

    2015-11-01

    Although human papillomavirus (HPV) infection has been studied extensively in women, data on male infection are limited. The purpose of this study was to investigate persistence of HPV infection at multiple genital sites in men and to define potential associations with socio-behavioural characteristics. Penile, urethral and seminal specimens were tested by the INNO-LiPA HPV system (Innogenetics) and a PCR assay. Persistence was defined as the detection of same HPV type at ≥ 2 consecutive visits. The Kaplan-Meier method and the log-rank test were applied to estimate the likelihood of persistence. A total of 50 men (median age: 33 years) were followed for a median of 14.7 months. Altogether, 49%, 36%, 26% and 11% of baseline HPV-positive men had 6-, 12-, 18- and 24-month persistent infection with any HPV type, respectively. The 6-, 12- and 18- month persistence was more common for oncogenic HPV infections; 24-month persistence was similar. The median duration of persistence was 21.7 months for any HPV. The median duration of persistence for any HPV type was significantly longer in the penile sample (22.5 months, 95% CI: 18.3-26.7) than the semen sample (15.3 months, 95% CI: 14.5-16.1). Over a third of type-specific HPV infections in men remained persistent over a 24-month period. The median duration of HPV infection was longer in penile samples compared to seminal samples. As being increasing the attention of HPV vaccination as a potential preventive approach also for men, it is imperative to obtain additional insight on natural history of HPV infection in men, particularly as far as incidence and duration are concerned.

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

    Science.gov (United States)

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

    2014-01-01

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

  13. First reported case of Campylobacter lanienae enteritis in a human

    Science.gov (United States)

    Lemay, Frédéric; Bekal, Sadjia; Frost, Eric H.; Michaud, Sophie

    2016-01-01

    Introduction: Campylobacters are the most frequently identified bacteria causing diarrhoea in humans worldwide. Campylobacter lanienae was isolated for the first time in 2000 from faecal samples of two asymptomatic abattoir workers in Switzerland during a routine hygiene screen, but has never been associated with human disease. Case presentation: At hospital admission, the patient reported diarrhoea, lower abdominal cramps, nausea, one episode of bilious vomiting and low-grade fever of 38 °C. The patient was having 10 or more diarrheic stools per day as well as during the night, and had noticed blood mixed with the stools on several occasions. Stool cultures were negative for species of Salmonella and Shigella, Escherichia coli O157:H7 and Yersinia enterocolitica, but were positive for C. lanienae. Identification was made by classical biochemical testing, as well as 16S rRNA gene and cpn60 sequencing. The patient slowly improved without antibiotic treatment and was discharged nine days after admission with complete resolution of symptoms. Conclusion: On the whole it seems very likely that C. lanienae was the causative agent. Clinical microbiologists should be aware of this micro-organism which can be identified by phenotypic and molecular methods. The real burden of C. lanienae infection in humans might be underestimated and should be further investigated as a potential cause of human diarrhoea disease.

  14. Comparison of two adult mosquito sampling methods with human landing catches in south-central Ethiopia.

    Science.gov (United States)

    Kenea, Oljira; Balkew, Meshesha; Tekie, Habte; Gebre-Michael, Teshome; Deressa, Wakgari; Loha, Eskindir; Lindtjørn, Bernt; Overgaard, Hans J

    2017-01-13

    The human landing catch (HLC) is the standard reference method for measuring human exposure to mosquito bites. However, HLC is labour-intensive, exposes collectors to infectious mosquito bites and is subjected to collector bias. These necessitate local calibration and application of alternative methods. This study was undertaken to determine the relative sampling efficiency (RSE) of light traps with or without yeast-produced carbon dioxide bait vs. HLC in south-central Ethiopia. The experiment was conducted for 39 nights in a 3 × 3 Latin square randomized design with Anopheles arabiensis as the target species in the period between July and November 2014 in Edo Kontola village, south-central Ethiopia. Center for Disease Control and Prevention light trap catches (LTC) and yeast-generated carbon dioxide-baited light trap catches (CB-LTC) were each evaluated against HLC. The total nightly mosquito catches for each Anopheles species in either method was compared with HLC by Pearson correlation and simple linear regression analysis on log-transformed [log10(x + 1)] values. To test if the RSE of each alternative method was affected by mosquito density, the ratio of the number of mosquitoes in each method to the number of mosquitoes in HLC was plotted against the average mosquito abundance. Overall, 7606 Anopheles females were collected by the three sampling methods. Among these 5228 (68.7%) were Anopheles ziemanni, 1153 (15.2%) An. arabiensis, 883 (11.6%) Anopheles funestus s.l., and 342 (4.5%) Anopheles pharoensis. HLC yielded 3392 (44.6%), CB-LTC 2150 (28.3%), and LTC 2064 (27.1%) Anopheles females. The RSEs of LTC and HLC for An. arabiensis were significantly correlated (p method for sampling An. arabiensis. LTC can be used for large-scale indoor An. arabiensis surveillance and monitoring when it is difficult to use HLC. CB-LTC does not substantially improve sampling of this major vector compared to LTC in this setting. Trial registration PACTR201411000882128

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

    Directory of Open Access Journals (Sweden)

    Floch Pauline

    2012-12-01

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

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

    Science.gov (United States)

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

    2014-09-11

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

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

    Science.gov (United States)

    Chrzanowski, Rose-Ann C.

    2010-01-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  19. In Vitro Efficient Expansion of Tumor Cells Deriving from Different Types of Human Tumor Samples

    Directory of Open Access Journals (Sweden)

    Ilaria Turin

    2014-03-01

    Full Text Available Obtaining human tumor cell lines from fresh tumors is essential to advance our understanding of antitumor immune surveillance mechanisms and to develop new ex vivo strategies to generate an efficient anti-tumor response. The present study delineates a simple and rapid method for efficiently establishing primary cultures starting from tumor samples of different types, while maintaining the immuno-histochemical characteristics of the original tumor. We compared two different strategies to disaggregate tumor specimens. After short or long term in vitro expansion, cells analyzed for the presence of malignant cells demonstrated their neoplastic origin. Considering that tumor cells may be isolated in a closed system with high efficiency, we propose this methodology for the ex vivo expansion of tumor cells to be used to evaluate suitable new drugs or to generate tumor-specific cytotoxic T lymphocytes or vaccines.

  20. Determination of Human Chorionic Gonadotropin in Postmortem Samples in Ectopic Pregnancies.

    Science.gov (United States)

    Palmiere, Cristian; Lesta, Maria del Mar; Fanton, Laurent; Ventura, Francesco; Bonsignore, Alessandro; Reggiani Bonetti, Luca

    2016-01-01

    Increased human chorionic gonadotropin levels (HCG) can be detected in femoral blood, bile, and vitreous humor collected during autopsy of pregnant women using a standard kit designed for living patients. In the study herein, the concentrations of HCG were measured in postmortem serum, vitreous, bile, cerebrospinal, and pericardial fluids in 4 cases of fatal ectopic pregnancy and 40 controls using a quantitative electrochemiluminescence immunoassay designed for living patients. No false-negative cases were identified in any of the analyzed samples in any of the ectopic pregnancy cases. No correlations were found between total HCG levels in postmortem serum and the other tested specimens. The results of this study would suggest that higher HCG in bile, vitreous, pericardial, and cerebrospinal fluids may confirm the existence of ectopic pregnancy and therefore identify other situations in which this hormone is increased, although gestational age cannot be reliably estimated using these values. © 2015 American Academy of Forensic Sciences.

  1. Ultrasensitive PCR and real-time detection from human genomic samples using a bidirectional flow microreactor.

    Science.gov (United States)

    Chen, Lin; West, Jonathan; Auroux, Pierre-Alain; Manz, Andreas; Day, Philip J R

    2007-12-01

    In this paper we present a reliable bidirectional flow DNA amplification microreactor for processing real-world genomic samples. This system shares the low-power thermal responsiveness of a continuous flow reactor with the low surface area to volume ratio character of stationary reactors for reducing surface inhibitory effects. Silanization with dimethyldichlorosilane in combination with dynamic surface passivation was used to enhance PCR compatibility and enable efficient amplification. For real-time fragment amplification monitoring we have implemented an epimodal fluorescent detection capability. The passivated bidirectional flow system was ultrasensitive, achieving an RNase P gene detection limit of 24 human genome copies with a reaction efficiency of 77%. This starts to rival the performance of a conventional real-time PCR instrument with a reaction efficiency of 93% and revitalizes flow-through PCR as a viable component of lab on a chip DNA analysis formats.

  2. Analyses of robotic traverses and sample sites in the Schrödinger basin for the HERACLES human-assisted sample return mission concept

    Science.gov (United States)

    Steenstra, Edgar S.; Martin, Dayl J. P.; McDonald, Francesca E.; Paisarnsombat, Sarinya; Venturino, Christian; O'Hara, Sean; Calzada-Diaz, Abigail; Bottoms, Shelby; Leader, Mark K.; Klaus, Kurt K.; van Westrenen, Wim; Needham, Debra H.; Kring, David A.

    2016-09-01

    The International Space Exploration Coordination Group (ISECG) developed an integrated Global Exploration Roadmap (GER) that outlines plans for human-assisted sample return from the lunar surface in ∼2024 and for human presence on the lunar surface in ∼2028. Previous studies have identified the Schrödinger basin, situated on the far side of the Moon, as a prime target for lunar science and exploration where a significant number of the scientific concepts reviewed by the National Research Council (NRC, 2007) can be addressed. In this study, two robotic mission traverses within the Schrödinger basin are proposed based on a 3 year mission plan in support of the HERACLES human-assisted sample return mission concept. A comprehensive set of modern remote sensing data (LROC imagery, LOLA topography, M3 and Clementine spectral data) has been integrated to provide high-resolution coverage of the traverses and to facilitate identification of specific sample localities. We also present a preliminary Concept of Operations (ConOps) study based on a set of notional rover capabilities and instrumental payload. An extended robotic mission to the Schrödinger basin will allow for significant sample return opportunities from multiple distinct geologic terrains and will address multiple high-priority NRC (2007) scientific objectives. Both traverses will offer the first opportunity to (i) sample pyroclastic material from the lunar farside, (ii) sample Schrödinger impact melt and test the lunar cataclysm hypothesis, (iii) sample deep crustal lithologies in an uplifted peak ring and test the lunar magma ocean hypothesis and (iv) explore the top of an impact melt sheet, enhancing our ability to interpret Apollo samples. The shorter traverse will provide the first opportunity to sample farside mare deposits, whereas the longer traverse has significant potential to collect SPA impact melt, which can be used to constrain the basin-forming epoch. These robotic missions will revalidate

  3. Trace samples of human blood in mosquitoes as a forensic investigation tool.

    Science.gov (United States)

    Rabêlo, K C N; Albuquerque, C M R; Tavares, V B; Santos, S M; Souza, C A; Oliveira, T C; Oliveira, N C L; Crovella, S

    2015-11-23

    Investigations of any type of crime invariably starts at the crime scene by collecting evidence. Thus, the purpose of this research was to collect and analyze an entomological trace from an environment that is similar to those of indoor crime scenes. Hematophagous mosquitoes were collected from two residential units; saliva of volunteers that were residents in the units was also collected for genetic analysis as reference samples. We examined the allele frequencies of 15 short tandem repeat loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA) and amelogenin. A total of 26 female hematophagous mosquitoes were identified as Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus; we were able to obtain 11 forensically valid genetic profiles, with a minimum of 0.028203 ng/μL of human DNA. Thus, the results of this study showed that it was possible to correlate human genetic information from mosquitoes with the volunteer reference samples, which validates the use of this information as forensic evidence. Furthermore, we observed mixed genetic profiles from one mosquito. Therefore, it is clearly important to collect these insects indoors where crimes were committed, because it may be possible to find intact genetic profiles of suspects in the blood found in the digestive tract of hematophagous mosquitoes for later comparison to identify an offender and/or exclude suspects.

  4. Characterization of quinolone resistance in Salmonella spp. isolates from food products and human samples in Brazil

    Science.gov (United States)

    Pribul, Bruno Rocha; Festivo, Marcia Lima; de Souza, Miliane Moreira Soares; dos Prazeres Rodrigues, Dalia

    2016-01-01

    Non-typhoidal salmonellosis is an important zoonotic disease caused by Salmonella enterica. The aim of this study was to investigate the prevalence of plasmid-mediated quinolone resistance in Salmonella spp. and its association with fluoroquinolone susceptibility in Brazil. A total of 129 NTS isolates (samples from human origin, food from animal origin, environmental, and animal) grouped as from animal (n = 62) and human (n = 67) food were evaluated between 2009 and 2013. These isolates were investigated through serotyping, antimicrobial susceptibility testing, and the presence of plasmid-mediated quinolone resistance (PMQR) genes (qnr, aac(6′)-Ib) and associated integron genes (integrase, and conserved integron region). Resistance to quinolones and/or fluoroquinolones, from first to third generations, was observed. Fifteen isolates were positive for the presence of qnr genes (8 qnrS, 6 qnrB, and 1 qnrD) and twenty three of aac(6′)-Ib. The conserved integron region was detected in 67 isolates as variable regions, from ±600 to >1000 pb. The spread of NTS involving PMQR carriers is of serious concern and should be carefully monitored. PMID:26887245

  5. Assessment of malathion and its effects on leukocytes in human blood samples

    Science.gov (United States)

    Sharma, Amit Kumar; Tiwari, Udita; Gaur, Mulayam Singh; Tiwari, Rajeev Kumar

    2016-01-01

    Abstract In the present paper, we report a reproducible, cost effective, fast response method for detection of malathion and its effects on leukocytes in different human blood groups. Spectroscopic methods (UV-Vis spectrometry) and Fourier transform infrared coupled with solid phase extraction were applied for analyzing malathion content in human blood plasma. The spiking levels of malathion in the range of 0.1-1.7 µg/mL were extracted from blood plasma samples using SPE. The present active functional groups (C = O; P-O-C; -OH; P = S) were also characterized. The recovery rate of malathion was 80%±4.5%. The calculated correlation coefficient was 0.9799, indicating the linearity of the results. The limit of detection (LOD) and limit of quantification (LOQ) were (0.1-1.7) µg/mL and (0.3-1.5) µg/mL, respectively. Malathion <1.0 µg/mL showed no significant change while higher levels of malathion exposure (1.5 µg/mL and 3.0 µg/mL) reduced the number of white blood cells. In conclusion, the spectroscopic results may be useful to understand the mechanism of other pesticides such as methyl parathion and parathion.

  6. Characterization of quinolone resistance in Salmonella spp. isolates from food products and human samples in Brazil.

    Science.gov (United States)

    Pribul, Bruno Rocha; Festivo, Marcia Lima; de Souza, Miliane Moreira Soares; Rodrigues, Dalia dos Prazeres

    2016-01-01

    Non-typhoidal salmonellosis is an important zoonotic disease caused by Salmonella enterica. The aim of this study was to investigate the prevalence of plasmid-mediated quinolone resistance in Salmonella spp. and its association with fluoroquinolone susceptibility in Brazil. A total of 129 NTS isolates (samples from human origin, food from animal origin, environmental, and animal) grouped as from animal (n=62) and human (n=67) food were evaluated between 2009 and 2013. These isolates were investigated through serotyping, antimicrobial susceptibility testing, and the presence of plasmid-mediated quinolone resistance (PMQR) genes (qnr, aac(6')-Ib) and associated integron genes (integrase, and conserved integron region). Resistance to quinolones and/or fluoroquinolones, from first to third generations, was observed. Fifteen isolates were positive for the presence of qnr genes (8 qnrS, 6 qnrB, and 1 qnrD) and twenty three of aac(6')-Ib. The conserved integron region was detected in 67 isolates as variable regions, from ±600 to >1000pb. The spread of NTS involving PMQR carriers is of serious concern and should be carefully monitored.

  7. Determination of human albumin in serum and urine samples by constant-energy synchronous fluorescence method.

    Science.gov (United States)

    Madrakian, Tayyebeh; Bagheri, Habibollah; Afkhami, Abbas

    2015-08-01

    A sensitive spectrofluorimetric method using constant-energy synchronous fluorescence technique is proposed for the determination of human albumin without separation. In this method, no reagent was used for enhancement of the fluorescence signal of albumin in the solution. Effects of some parameters, such as energy difference between excitation and emission monochromators (ΔE), emission and excitation slit widths and scan rate of wavelength were studied and the optimum conditions were established. For this purpose factorial design and response surface method were employed for optimization of the effective parameters on the fluorescence signal. The results showed that the scan rate of the wavelength has no significant effect on the analytical signal. The calibration curve was linear in the range 0.1-220.0 µg mL(-1) of albumin with a detection limit of 7.0 × 10(-3)  µg mL(-1). The relative standard deviations (RSD) for six replicate measurements of albumin were calculated as 2.2%, 1.7% and 1.3% for 0.5, 10.0 and 100.0 µg mL(-1) albumin, respectively. Furthermore the proposed method has been employed for the determination of albumin in human serum and urine samples.

  8. Characterization of a Hemoglobin Adduct from Ethyl Vinyl Ketone Detected in Human Blood Samples.

    Science.gov (United States)

    Carlsson, Henrik; Motwani, Hitesh V; Osterman Golkar, Siv; Törnqvist, Margareta

    2015-11-16

    Electrophiles have the ability to form adducts to nucleophilic sites in proteins and DNA. Internal exposure to such compounds thus constitutes a risk for toxic effects. Screening of adducts using mass spectrometric methods by adductomic approaches offers possibilities to detect unknown electrophiles present in tissues. Previously, we employed untargeted adductomics to detect 19 unknown adducts to N-terminal valine in hemoglobin (Hb) in human blood. This article describes the characterization of one of these adducts, which was identified as the adduct from ethyl vinyl ketone (EVK). The mean adduct level was 40 ± 12 pmol/g Hb in 12 human blood samples; adduct levels from acrylamide (AA) and methyl vinyl ketone (MVK) were quantified for comparison. Using l-valine p-nitroanilide (Val-pNA), introduced as a model of the N-terminal valine, the rate of formation of the EVK adduct was studied, and the rate constant determined to 200 M(-1)h(-1) at 37 °C. In blood, the reaction rate was too fast to be feasibly measured, EVK showing a half-life adduct was found to be unstable, with a half-life of 7.6 h. From the mean adduct level measured in human blood, a daily dose (area under the concentration-time-curve, AUC) of 7 nMh EVK was estimated. The AUC of AA from intake via food is about 20 times higher. EVK is naturally present in a wide range of foods and is also used as a food additive. Most probably, naturally formed EVK is a major source to observed adducts. Evaluation of available toxicological data and information on occurrence of EVK indicate that further studies of EVK are motivated. This study illustrates a quantitative strategy in the initial evaluation of the significance of an adduct detected through adduct screening.

  9. Inactivation of human immunodeficiency virus type 1 in blood samples stored as high-salt lysates.

    Science.gov (United States)

    Zolg, J W; Lanciotti, R S; Wendlinger, M; Meyer, W A

    1990-09-01

    Blood samples to be tested for the presence of parasite DNA by using specific DNA probes are routinely stored in our laboratory as high-salt lysates (HSL). To safeguard against the risk of accidental infection with etiological agents such as the human immunodeficiency virus type 1 (HIV-1) while manipulating large numbers of blood samples in preparation for DNA probing, we determined the residual infectivity of HIV-1 after exposure to HSL components. Both high-titer virus stocks or provirus-carrying cells, suspended either in tissue culture medium or freshly drawn blood, were completely inactivated upon contact with the HSL components. This was verified by the absence of any detectable HIV-1-specific antigen in the supernatants of long-term cultures and the absence of virus-specific DNA fragments after amplification by polymerase chain reaction with DNA from such cultures as target DNA. These results support the conclusion that the virus is in fact completely inactivated by contact with the HSL components, rendering blood specimens stored as HSL noninfectious in regard to HIV-1.

  10. Aqueous two phase system based on ionic liquid for isolation of quinine from human plasma sample.

    Science.gov (United States)

    Flieger, J; Czajkowska-Żelazko, A

    2015-01-01

    Aqueous two phase system was applied for selective extraction of quinine from human plasma. Bi-phase was constructed from ionic liquid: butyl-methyl-imidazolium chloride after addition kosmotropic salts K₃PO₄ or KH₂PO₄. Quinine was determined in plasma samples after drinking of tonic containing quinine. Determination was performed by HPLC on 5-μm Zorbax SB-CN column and eluent containing 40% acetonitrile (v/v), 20 mM phosphate buffer at pH 3 and 40 mM NaPF₆ using external standard method. The spectrophotometric detection was set λ=214 nm. Selective fluorescence detection was performed at excitation of 325 nm and emission of 375 nm. Proposed strategy provides suitable sample purification and gives extraction yields in the range of 89-106%. The determination coefficient (R(2)) has a value ≥0.997 in the range of 50-800 ng/ml quinine concentration. The limit of quantification was set at 27.9 ng/ml and the detection limit was found to be 8.4 ng/ml under fluorescence detection.

  11. Analysis of Fluconazole in Human Urine Sample by High Performance Liquid Chromatography Method

    Science.gov (United States)

    Hermawan, D.; Ali, N. A. Md; Ibrahim, W. A. Wan; Sanagi, M. M.

    2013-04-01

    A method for determination of fluconazole, antifungal drug in human urine by using reversed-phased high performance liquid chromatography (RP-HPLC) with ultraviolet (UV) detector was developed. Optimization HPLC conditions were carried out by changing the flow rate and composition of mobile phase. The optimum separation conditions at a flow rate 0.85 mL/min with a composition of mobile phase containing methanol:water (70:30, v/v) with UV detection at a wavelength 254 nm was able to analyze fluconazole within 3 min. The excellent linearity was obtained in the range of concentration 1 to 10 μg/mL with r2 = 0.998. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.39 μg/mL and 1.28 μg/mL, respectively. Solid phase extraction (SPE) method using octadecylsilane (C18) as a sorbent was used to clean-up and pre-concentrated of the urine sample prior to HPLC analysis. The average recoveries of fluconazole in spiked urine sample was 72.4% with RSD of 3.21% (n=3).

  12. Proteomic and oxidative stress analysis in human brain samples of Huntington disease.

    Science.gov (United States)

    Sorolla, Ma Alba; Reverter-Branchat, Gemma; Tamarit, Jordi; Ferrer, Isidre; Ros, Joaquim; Cabiscol, Elisa

    2008-09-01

    Huntington disease (HD) is a neurodegenerative disorder caused by expansion of CAG repeats in exon 1 of the huntingtin gene, affecting initially the striatum and progressively the cortex. This work reports a proteomic analysis of human brain postmortem samples obtained from striatum and cortex of patients with HD compared to samples of age- and sex-matched controls. Antioxidant defense proteins that were strongly induced in striatum, but also detectable in cortex, were identified as peroxiredoxins 1, 2, and 6, as well as glutathione peroxidases 1 and 6. The activities of other antioxidant enzymes such as mitochondrial superoxide dismutase and catalase were also increased in HD. Aconitase, a protein involved in energy metabolism, showed decreased activities in striatum of HD patients. Protein carbonyls, used as markers of oxidative stress, were increased in HD, and glial fibrillary acidic protein, aconitase, gamma-enolase, and creatine kinase B were identified as the main targets. Taken together, these results indicate that oxidative stress and damage to specific macromolecules would participate in the disease progression. Also, these data support the rationale for therapeutic strategies that either potentiate antioxidant defenses or avoid oxidative stress generation to delay disease progression.

  13. Performance of the Fecal Immunochemical Test for Colorectal Cancer Screening Using Different Stool-Collection Devices: Preliminary Results from a Randomized Controlled Trial.

    Science.gov (United States)

    Shin, Hye Young; Suh, Mina; Baik, Hyung Won; Choi, Kui Son; Park, Boyoung; Jun, Jae Kwan; Hwang, Sang-Hyun; Kim, Byung Chang; Lee, Chan Wha; Oh, Jae Hwan; Lee, You Kyoung; Han, Dong Soo; Lee, Do-Hoon

    2016-11-15

    We are in the process of conducting a randomized trial to determine whether compliance with the fecal immunochemical test (FIT) for colorectal cancer screening differs according to the stool-collection method. This study was an interim analysis of the performance of two stool-collection devices (sampling bottle vs conventional container). In total, 1,701 individuals (age range, 50 to 74 years) were randomized into the sampling bottle group (intervention arm) or the conventional container group (control arm). In both groups, we evaluated the FIT positivity rate, the positive predictive value for advanced neoplasia, and the detection rate for advanced neoplasia. The FIT positivity rates were 4.1% for the sampling bottles and 2.0% for the conventional containers; these values were significantly different. The positive predictive values for advanced neoplasia in the sampling bottles and conventional containers were 11.1% (95% confidence interval [CI], -3.4 to 25.6) and 12.0% (95% CI, -0.7 to 24.7), respectively. The detection rates for advanced neoplasia in the sampling bottles and conventional containers were 4.5 per 1,000 persons (95% CI, 2.0 to 11.0) and 2.4 per 1,000 persons (95% CI, 0.0 to 5.0), respectively. The impact of these findings on FIT screening performance was unclear in this interim analysis. This impact should therefore be evaluated in the final analysis following the final enrollment period.

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

    DEFF Research Database (Denmark)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Science.gov (United States)

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

    2012-03-01

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

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

    Directory of Open Access Journals (Sweden)

    Cecilia Stålsby Lundborg

    2012-03-01

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

  18. Prevalence and genotyping of high risk human papillomavirus in cervical cancer samples from Punjab, Pakistan.

    Science.gov (United States)

    Siddiqa, Abida; Zainab, Maidah; Qadri, Ishtiaq; Bhatti, Muhammad Faraz; Parish, Joanna L

    2014-07-17

    Cervical cancer is the third most common cause of cancer-related death in women worldwide. Infection with high-risk human papillomavirus (HPV) is established as the cause of cervical carcinoma, therefore, high risk HPV detection may have prognostic significance for the women who are at increased risk of disease progression. The paucity of data on the incidence of cervical cancer in Pakistan makes it difficult to determine disease burden. Even less information is available regarding the prevalent HPV strains in cervical specimens collected from this region. Cervical cancer is a neglected disease in Pakistan in terms of screening, prevention, and vaccination. Identification and accurate genotyping of the virus burden in cancer specimens is important to inform intervention policies for future management of HPV associated disease and to potentially stratify patients dependent on HPV status. In this study, detection and genotyping of HPV types 16 and 18 from 77 cervical specimens were carried out. Consensus primers GP5+/GP6+, which detect 44 genital HPV types, and type specific primers (TS16 and TS18) were used in conjunction with newly designed type specific primers. Using a combination of these methods of detection, a total of 94.81% (95% CI ±4.95) of cervical lesions were positive for HPV. Single infections of HPV16 were detected in 24.68% (95% CI ±9.63) of total samples and HPV18 was found in 25.97% (95% CI ±9.79) samples. Interestingly, a high proportion of samples (40.26%, 95% CI ±10.95) was positive for both HPV16 and 18, indicating a higher incidence of co-infection than previously reported for similar ethnic regions. The HPV genotype of 3.90% of HPV positive samples remained undetected, although these samples were positive with the GP5+/GP6+ primer set indicating infection with an HPV type other than 16 or 18. These data indicate that the overall incidence of high risk HPV infection in cervical cancer and intraepithelial neoplasia specimens in Punjab

  19. SEROTYPING AND ANTIMICROBIAL DRUG RESISTANCE OF SALMONELLA ISOLATED FROM LETTUCE AND HUMAN DIARRHEA SAMPLES IN BURKINA FASO.

    Science.gov (United States)

    Siourimè, Somda Namwin; Isidore, Bonkoungou Ouindgueta Juste; Oumar, Traoré; Nestor, Bassolé Ismael Henri; Yves, Traoré; Nicolas, Barro; Aly, Savadogo

    2017-01-01

    Background: In Burkina Faso dirty water in particular those of the stoppings and the gutter ones are used for vegetables irrigation in the gardens. The aim of this study was to determine the prevalence and antibiotic susceptibility of Salmonella serotypes from humans and lettuce samples inBurkina Faso. Materials and Methods:Salmonella strains isolated from patients in 2009 to 2015 and lettuce samples in 2014 in Burkina Faso were serotyped using specific antisera. All strains were subjected to a set of 14 antibiotics to study their antibiogram by using Baeur–Kirby disk diffusion method. Results: Out of 154 Salmonella isolated, 60 were from human and 94 from lettuce samples. Serotyping revealed four different serotypes and 39% (60) untypeable strains from human and lettuce (14 and 46 strains). Salmonella serotypes from human and lettuce samples were: Paratyphi A (10% and 22%), Paratyphi B (34% and 8%), Paratyphi C (14% and 18%) and Typhi (21% and 1%). A high resistance of Salmonella Paratyphi B and Salmonella spp to tetracycline were 70% from human and 35 % from lettuce samples. Multiresistance was observed to tetracycline, chloramphenicol and amoxicillin/clavulanic-acid or ampicillin with Salmonella ParatyphiB 35% and Salmonella Typhi 33% from human samples and Salmonella spp 4% from lettuce samples. Conclusion: This study showed the diversity of Salmonella serotypes from both clinical and environmental samples and emergence of multiresistant Salmonella to antibiotics in Burkina Faso. A lettuce is a potential source of transmission of Salmonella causing diarrhea among human in Burkina Faso. List of non-standard Abbreviations : HDB: Hôpital du District de Bogodogo, LNSP: Laboratoire National de Santé Publique, DSG : District Sanitaire de Gourcy, DSB : District Sanitaire de Boromo PMID:28670637

  20. Development of the human infant intestinal microbiota.

    Directory of Open Access Journals (Sweden)

    Chana Palmer

    2007-07-01

    Full Text Available Almost immediately after a human being is born, so too is a new microbial ecosystem, one that resides in that person's gastrointestinal tract. Although it is a universal and integral part of human biology, the temporal progression of this process, the sources of the microbes that make up the ecosystem, how and why it varies from one infant to another, and how the composition of this ecosystem influences human physiology, development, and disease are still poorly understood. As a step toward systematically investigating these questions, we designed a microarray to detect and quantitate the small subunit ribosomal RNA (SSU rRNA gene sequences of most currently recognized species and taxonomic groups of bacteria. We used this microarray, along with sequencing of cloned libraries of PCR-amplified SSU rDNA, to profile the microbial communities in an average of 26 stool samples each from 14 healthy, full-term human infants, including a pair of dizygotic twins, beginning with the first stool after birth and continuing at defined intervals throughout the first year of life. To investigate possible origins of the infant microbiota, we also profiled vaginal and milk samples from most of the mothers, and stool samples from all of the mothers, most of the fathers, and two siblings. The composition and temporal patterns of the microbial communities varied widely from baby to baby. Despite considerable temporal variation, the distinct features of each baby's microbial community were recognizable for intervals of weeks to months. The strikingly parallel temporal patterns of the twins suggested that incidental environmental exposures play a major role in determining the distinctive characteristics of the microbial community in each baby. By the end of the first year of life, the idiosyncratic microbial ecosystems in each baby, although still distinct, had converged toward a profile characteristic of the adult gastrointestinal tract.

  1. Tracking human activity and well-being in natural environments using wearable sensors and experience sampling.

    Science.gov (United States)

    Doherty, Sean T; Lemieux, Christopher J; Canally, Culum

    2014-04-01

    A growing range of studies have begun to document the health and well-being benefits associated with contact with nature. Most studies rely on generalized self-reports following engagement in the natural environment. The actual in-situ experience during contact with nature, and the environmental features and factors that evoke health benefits have remained relatively unexplored. Smartphones offer a new opportunity to monitor and interact with human subjects during everyday life using techniques such as Experience Sampling Methods (ESM) that involve repeated self-reports of experiences as they occur in-situ. Additionally, embedded sensors in smartphones such as Global Positioning Systems (GPS) and accelerometers can accurately trace human activities. This paper explores how these techniques can be combined to comprehensively explore the perceived health and well-being impacts of contact with nature. Custom software was developed to passively track GPS and accelerometer data, and actively prompt subjects to complete an ESM survey at regular intervals throughout their visit to a provincial park in Ontario, Canada. The ESM survey includes nine scale questions concerning moods and emotions, followed by a series of open-ended experiential questions that subjects provide recorded audio responses to. Pilot test results are used to illustrate the nature, quantity and quality of data obtained. Participant activities were clearly evident from GPS maps, including especially walking, cycling and sedate activities. From the ESM surveys, participants reported an average of 25 words per question, taking an average of 15 s to record them. Further qualitative analysis revealed that participants were willing to provide considerable insights into their experiences and perceived health impacts. The combination of passive and interactive techniques is sure to make larger studies of this type more affordable and less burdensome in the future, further enhancing the ability to understand

  2. Molecular epidemiology of human papillomavirus infections in cervical samples from cuban women older than 30 years.

    Science.gov (United States)

    Soto, Yudira; Torres, Griselda; Kourí, Vivian; Limia, Celia María; Goicolea, Adibel; Capó, Virginia; Pérez, Lissette; de la Torre, Ana Isabel; López, Ledy Xiomara; Govín, Anamays; Correa, Consuelo Beatriz; Alemán, Yoan; Alvarez, Alina Ana; Manzano, Blanca Rosa

    2014-07-01

    This study aimed to provide information about the molecular epidemiology of human papillomavirus (HPV) in a group of Cuban women. DNA from cervical samples was analyzed using a quantitative real-time polymerase chain reaction (PCR), which detects 6 of the clinically most relevant high-risk HPV types. Furthermore, end point PCR and sequencing were performed. Three hundred twenty-two women (211 with positive and 111 with negative cytologic results) aged between 30 and 69 years were enrolled. Risk factors associated with HPV infections and premalignant lesions were also investigated. HPV DNA was detected in 76.1% (245/322) of the studied population, and 34 different genotypes were found. There was an association between HPV infection and low educational level, history of oral contraceptives, menopausal stage, as well as cigarette and/or alcohol consumption. Besides, in a multivariate analysis, previous positive Pap test result and positive colposcopy finding were both predictor variables for HPV infections and for premalignant lesions. Human papillomavirus infection was found in 94.3% of women (199/211) with positive cytologic result and in 41.4% (46/111) of those with negative results, being more likely that the first group was infected with any HPV (odds ratio = 23.43; 95% CI = 11.70-46.92; p = .000). The most common genotypes were HPV types 16, 18, 31, 58, 33, and 45. All the cases with HPV positive findings had at least 1 high-risk HPV genotype. This is the first report of the molecular epidemiology of HPV in Cuban women, based on results from a DNA sequence and quantitative PCR. Most individuals were infected with high-risk HPV types. These findings support the inclusion of HPV vaccine in Cuba.

  3. Comparison of the Compositions of the Stool Microbiotas of Infants Fed Goat Milk Formula, Cow Milk-Based Formula, or Breast Milk

    OpenAIRE

    Tannock, Gerald W.; Lawley, Blair; Munro, Karen; Gowri Pathmanathan, Siva; Zhou, Shao J.; Makrides, Maria; Robert A Gibson; Sullivan, Thomas; Prosser, Colin G.; Lowry, Dianne; Alison J. Hodgkinson

    2013-01-01

    The aim of the study was to compare the compositions of the fecal microbiotas of infants fed goat milk formula to those of infants fed cow milk formula or breast milk as the gold standard. Pyrosequencing of 16S rRNA gene sequences was used in the analysis of the microbiotas in stool samples collected from 90 Australian babies (30 in each group) at 2 months of age. Beta-diversity analysis of total microbiota sequences and Lachnospiraceae sequences revealed that they were more similar in breast...

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

    Science.gov (United States)

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

    2007-01-01

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

  5. Levels and complexity of IgA antibody against oral bacteria in samples of human colostrum.

    Science.gov (United States)

    Petrechen, L N; Zago, F H; Sesso, M L T; Bertoldo, B B; Silva, C B; Azevedo, K P; de Lima Pereira, S A; Geraldo-Martins, V R; Ferriani, V P L; Nogueira, R D

    2015-01-01

    Streptococcus mutans (SM) have three main virulence antigens: glucan binding protein B (gbpB), glucosyltransferase (Gtf) and antigens I/II (Ag I/II) envolved in the capacity of those bacteria to adhere and accumulate in the dental biofilm. Also, the glycosyltransferases 153 kDa of Streptococcus gordonii (SGO) and 170kDa of Streptococcus sanguinis (SSA) were important antigens associated with the accumulation of those bacterias. Streptococcus mitis (SMI) present IgA1 protease of 202 kDa. We investigated the specificity and levels IgA against those antigens of virulence in samples of human colostrum. This study involved 77 samples of colostrum that were analyzed for levels of immunoglobulian A, M and G by Elisa. The specificity of IgA against extracts of SM and initials colonizators (SSA, SMI, SGO) were analyzed by the Western blot. The mean concentration of IgA was 2850.2 (±2567.2) mg/100 mL followed by IgM and IgG (respectively 321.8±90.3 and 88.3±51.5), statistically different (pbacteria antigens and theirs virulence antigens. To SM, the GbpB was significantly lower detected than others antigens of SM (p0.4). So, the breast milk from first hours after birth presented significant levels of IgA specific against important virulence of antigens those oral streptococci, which can disrupt the installation and accumulation process of these microorganisms in the oral cavity. Copyright © 2014 Elsevier GmbH. All rights reserved.

  6. Novel encapsulation improves recovery of probiotic strains in fecal samples of human volunteers.

    Science.gov (United States)

    Mai, Volker; Waugh, Sheldon; Byrd, Doratha; Simpson, Damion; Ukhanova, Maria

    2017-02-01

    Probiotic supplements can contribute to maintaining health and ameliorating various disease symptoms. Probiotics can be delivered in many forms with crucial differences in their survival during gastrointestinal (GI) passage. Previously, a novel encapsulation, Probiotic Pearls™ Acidophilus, Integrative Therapeutics, LLC, USA (Pearls), was shown to increase survival in vitro after exposure to gastric conditions. Here, we compare fecal recovery in human volunteers consuming Pearls or a conventional hard-shelled gelatin capsule. We performed a randomized double-blinded, two-armed trial, with six healthy subjects in each 12-day study arm. In fecal samples collected at baseline, twice during the intervention period, and after washout, we compared colony counts between the two encapsulation methods. The identity of the colonies was confirmed by colony morphology, strain-specific PCR, and 16S rRNA gene sequencing. We further performed a comprehensive 16S rRNA gene sequencing-based analysis to identify differential effects on overall microbiota composition. We detected an average log increase in bifidobacteria of 0.152 cfu/g with gelatin and 0.651 cfu/g with Pearls capsules (p > 0.05). Total lactobacilli counts increased in both groups with no difference between the groups. However, the supplemented Lactobacillus acidophilus NCFM decreased to baseline levels within 7 days after end of supplementation with gelatin capsules while 3.11 log cfu/g higher counts compared to baseline (p = 0.05) remained for Pearls. Targeted qPCR largely confirmed the trends observed by viable plate counts. Protecting the probiotic strains by Pearls encapsulation results in higher recovery rates of the supplemented lactobacilli and bifidobacteria in fecal samples and increased persistence, suggesting an improved survival and viability that might increase efficacy towards achieving desired health benefits.

  7. Detection of nandrolone, testosterone, and their esters in rat and human hair samples.

    Science.gov (United States)

    Höld, K M; Borges, C R; Wilkins, D G; Rollins, D E; Joseph, R E

    1999-10-01

    Nandrolone and testosterone are anabolic androgenic steroids occasionally abused by athletes. A sensitive, specific, and reproducible gas chromatography-mass spectrometry method for the quantitative determination of nandrolone, testosterone, and their esters in hair has been developed. The limits of quantitation of this method, based on 20 mg of hair, were 50 pg/mg for nandrolone and testosterone, 100 pg/mg for testosterone acetate, and 200 pg/mg for nandrolone-decanoate. Nandrolone-d3 and testosterone-d3 were used as internal standards. This method has been applied to the analysis of these compounds incorporated into rat and human hair. Male Long-Evans rats were given nandrolone decanoate 60 mg/kg intraperitoneally (i.p.) once daily for 10 days over a time period of 14 days. Two of the three rats contained nandrolone in the pigmented hair collected at day 21 at a concentration of 63 and 76 pg/mg, respectively. No drug was found in the corresponding nonpigmented hair. The rat hair samples that tested positive for nandrolone contained also nandrolone decanoate in concentrations of 0.9 and 1.2 ng/mg, respectively. In a separate experiment rats were given testosterone acetate 10 mg/kg i.p. once daily for five days. No testosterone or testosterone acetate was detected in the rat hair samples. Hair specimens were also obtained from four self-reported steroid users. The hair of two subjects were determined to be positive for testosterone in concentrations of 54 and 81 pg/mg. These data demonstrate that it is possible to detect the steroids nandrolone, testosterone, and nandrolone decanoate in hair after systemic administration.

  8. The human gut microbiome impacts health and disease.

    Science.gov (United States)

    Ehrlich, Stanislav Dusko

    2016-01-01

    The human gut microbiome can now be characterized in unprecedented detail by an approach based on high-throughput sequencing of total stool DNA, that we name quantitative metagenomics. Central to the approach is a catalog that lists all the genes of intestinal microbes that are known - 9.9 millions, identified by the analysis of 1267 stool samples. Beyond the gene list, genetic units that carry them begun to be known; many of these correspond to bacterial species that were never isolated and cultured yet. Quantitative metagenomics allows developing powerful algorithms to diagnose a disease, monitor patients and identify individuals at risk to progress towards a disease. This lays ground for developing new approaches to better restore and even preserve the health by modulation of the altered microbiome, which contributes to promote or aggravate a disease.

  9. Prevalence of Human Papillomavirus Infection in Unselected SurePath Samples Using the APTIMA HPV mRNA Assay

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Preisler, Sarah; Ejegod, Ditte M;

    2013-01-01

    The APTIMA Human Papillomavirus (HPV) Assay detects E6/E7 mRNA from 14 human papillomavirus genotypes. Horizon was a population-based split-sample study among well-screened women, with an aim to compare APTIMA, Hybrid Capture 2 (HC2), and liquid-based cytology (LBC) using SurePath samples. APTIMA...... agreement between APTIMA and HC2. This is the first APTIMA study using SurePath samples on the PANTHER platform. The trends in positivity rates on SurePath samples for APTIMA, HC2, and LBC were consistent with studies based on PreservCyt samples, and the agreement between the two HPV assays was substantial....... The high proportions of women testing positive suggest that in countries with a high HPV prevalence, caution will be needed if HPV tests, including mRNA-based tests, are to replace LBC....

  10. Sensitivity of PCR assays for murine gammaretroviruses and mouse contamination in human blood samples.

    Directory of Open Access Journals (Sweden)

    Li Ling Lee

    Full Text Available Gammaretroviruses related to murine leukemia virus (MLV have variously been reported to be present or absent in blood from chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME patients and healthy controls. Using subjects from New York State, we have investigated by PCR methods whether MLV-related sequences can be identified in nucleic acids isolated from whole blood or from peripheral blood mononuclear cells (PBMCs or following PBMC culture. We have also passaged the prostate cancer cell line LNCaP following incubation with plasma from patients and controls and assayed nucleic acids for viral sequences. We have used 15 sets of primers that can effectively amplify conserved regions of murine endogenous and exogenous retrovirus sequences. We demonstrate that our PCR assays for MLV-related gag sequences and for mouse DNA contamination are extremely sensitive. While we have identified MLV-like gag sequences following PCR on human DNA preparations, we are unable to conclude that these sequences originated in the blood samples.

  11. Gene expression profiling of human breast tissue samples using SAGE-Seq.

    Science.gov (United States)

    Wu, Zhenhua Jeremy; Meyer, Clifford A; Choudhury, Sibgat; Shipitsin, Michail; Maruyama, Reo; Bessarabova, Marina; Nikolskaya, Tatiana; Sukumar, Saraswati; Schwartzman, Armin; Liu, Jun S; Polyak, Kornelia; Liu, X Shirley

    2010-12-01

    We present a powerful application of ultra high-throughput sequencing, SAGE-Seq, for the accurate quantification of normal and neoplastic mammary epithelial cell transcriptomes. We develop data analysis pipelines that allow the mapping of sense and antisense strands of mitochondrial and RefSeq genes, the normalization between libraries, and the identification of differentially expressed genes. We find that the diversity of cancer transcriptomes is significantly higher than that of normal cells. Our analysis indicates that transcript discovery plateaus at 10 million reads/sample, and suggests a minimum desired sequencing depth around five million reads. Comparison of SAGE-Seq and traditional SAGE on normal and cancerous breast tissues reveals higher sensitivity of SAGE-Seq to detect less-abundant genes, including those encoding for known breast cancer-related transcription factors and G protein-coupled receptors (GPCRs). SAGE-Seq is able to identify genes and pathways abnormally activated in breast cancer that traditional SAGE failed to call. SAGE-Seq is a powerful method for the identification of biomarkers and therapeutic targets in human disease.

  12. Haematospirillum jordaniae gen. nov., sp. nov., isolated from human blood samples.

    Science.gov (United States)

    Humrighouse, B W; Emery, B D; Kelly, A J; Metcalfe, M G; Mbizo, J; McQuiston, J R

    2016-04-01

    A Gram-negative, aerobic, motile, spiral-shaped bacterium, strain H5569(T), was isolated from a human blood sample. Phenotypic and molecular characteristics of the isolate were investigated. Optimal growth was found to occur at 35 °C under aerobic conditions on Heart Infusion Agar supplemented with 5 % rabbit blood. The major fatty acids present in the cells were identified as C16:0, C16:1ω7c and C18:1ω7c. The predominant respiratory quinone was found to be ubiquinone-Q10. The G+C content of genomic DNA for strain H5569(T) was found to be 49.9 %. Based on 16S rRNA gene sequence analysis results, 13 additional isolates were also analysed in this study. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the organism, represented by strain H5569(T), forms a distinct lineage within the family Rhodospirillaceae, closely related to two Novispirillum itersonii subspecies (93.9-94.1 %) and two Caenispirillum sp. (91.2-91.6 %). Based on these results, the isolate H5569(T) is concluded to represent a new genus and species for which the name Haematospirillum jordaniae gen. nov., sp. nov. is proposed. The type strain is H5569(T) (=DSM(T) 28903 = CCUG 66838(T)).

  13. Sources of technical variability in quantitative LC-MS proteomics: human brain tissue sample analysis.

    Science.gov (United States)

    Piehowski, Paul D; Petyuk, Vladislav A; Orton, Daniel J; Xie, Fang; Moore, Ronald J; Ramirez-Restrepo, Manuel; Engel, Anzhelika; Lieberman, Andrew P; Albin, Roger L; Camp, David G; Smith, Richard D; Myers, Amanda J

    2013-05-03

    To design a robust quantitative proteomics study, an understanding of both the inherent heterogeneity of the biological samples being studied as well as the technical variability of the proteomics methods and platform is needed. Additionally, accurately identifying the technical steps associated with the largest variability would provide valuable information for the improvement and design of future processing pipelines. We present an experimental strategy that allows for a detailed examination of the variability of the quantitative LC-MS proteomics measurements. By replicating analyses at different stages of processing, various technical components can be estimated and their individual contribution to technical variability can be dissected. This design can be easily adapted to other quantitative proteomics pipelines. Herein, we applied this methodology to our label-free workflow for the processing of human brain tissue. For this application, the pipeline was divided into four critical components: Tissue dissection and homogenization (extraction), protein denaturation followed by trypsin digestion and SPE cleanup (digestion), short-term run-to-run instrumental response fluctuation (instrumental variance), and long-term drift of the quantitative response of the LC-MS/MS platform over the 2 week period of continuous analysis (instrumental stability). From this analysis, we found the following contributions to variability: extraction (72%) > instrumental variance (16%) > instrumental stability (8.4%) > digestion (3.1%). Furthermore, the stability of the platform and its suitability for discovery proteomics studies is demonstrated.

  14. Sources of Technical Variability in Quantitative LC-MS Proteomics: Human Brain Tissue Sample Analysis.

    Energy Technology Data Exchange (ETDEWEB)

    Piehowski, Paul D.; Petyuk, Vladislav A.; Orton, Daniel J.; Xie, Fang; Moore, Ronald J.; Ramirez Restrepo, Manuel; Engel, Anzhelika; Lieberman, Andrew P.; Albin, Roger L.; Camp, David G.; Smith, Richard D.; Myers, Amanda J.

    2013-05-03

    To design a robust quantitative proteomics study, an understanding of both the inherent heterogeneity of the biological samples being studied as well as the technical variability of the proteomics methods and platform is needed. Additionally, accurately identifying the technical steps associated with the largest variability would provide valuable information for the improvement and design of future processing pipelines. We present an experimental strategy that allows for a detailed examination of the variability of the quantitative LC-MS proteomics measurements. By replicating analyses at different stages of processing, various technical components can be estimated and their individual contribution to technical variability can be dissected. This design can be easily adapted to other quantitative proteomics pipelines. Herein, we applied this methodology to our label-free workflow for the processing of human brain tissue. For this application, the pipeline was divided into four critical components: Tissue dissection and homogenization (extraction), protein denaturation followed by trypsin digestion and SPE clean-up (digestion), short-term run-to-run instrumental response fluctuation (instrumental variance), and long-term drift of the quantitative response of the LC-MS/MS platform over the 2 week period of continuous analysis (instrumental stability). From this analysis, we found the following contributions to variability: extraction (72%) >> instrumental variance (16%) > instrumental stability (8.4%) > digestion (3.1%). Furthermore, the stability of the platform and its’ suitability for discovery proteomics studies is demonstrated.

  15. Association of human papillomavirus and Chlamydia trachomatis with intraepithelial alterations in cervix samples

    Directory of Open Access Journals (Sweden)

    Denise Wohlmeister

    2016-02-01

    Full Text Available The influence of different infectious agents and their association with human papillomavirus (HPV in cervical carcinogenesis have not been completely elucidated. This study describes the association between cytological changes in cervical epithelium and the detection of the most relevant aetiological agents of sexually transmitted diseases. Samples collected from 169 patients were evaluated by conventional cytology followed by molecular analysis to detect HPV DNA, Chlamydia trachomatis, herpes simplex virus 1 and 2,Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, andTreponema pallidum, besides genotyping for most common high-risk HPV. An association between cytological lesions and different behavioural habits such as smoking and sedentariness was observed. Intraepithelial lesions were also associated with HPV and C. trachomatis detection. An association was also found between both simple and multiple genotype infection and cytological changes. The investigation of HPV and C. trachomatisproved its importance and may be considered in the future for including in screening programs, since these factors are linked to the early diagnosis of patients with precursor lesions of cervical cancer.

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

    Science.gov (United States)

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

    2016-12-01

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

  17. Human sexuality education in the middle grades classroom: A review of curricula in a sample of Florida school districts

    Science.gov (United States)

    Myrick, Melinda D.

    2007-12-01

    This study examined the extent to which human sexuality topics are covered in Florida middle school science classrooms and the process by which curricular decisions are made regarding human sexuality education on a county-wide basis. Primary data included interviews with county-level administrators who oversee curricular decisions related to the middle-grades science curriculum or health curriculum in twelve school districts within the state. These districts represented four geographic locations and districts of various sizes. Administrators from four of the twelve studies in the sample chose to provide information regarding their human sexuality education curriculum. In two cases, teacher leads were identified and were interviewed to understand the implementation of the curriculum within the classroom. Additional data were collected from the district curriculum guides for human sexuality education and the adopted middle-grades science textbook for each county. The interview and documentary data were analyzed by comparison to established criteria for a comprehensive human sexuality education curriculum. The analysis revealed that the scope of human sexuality education varied considerably within the sample and that much of the curricula in place failed to include topics and activities that have been identified as important in a successful human sexuality education program. These findings are limited because few counties chose to fully participate. Additional research is clearly needed to examine the effectiveness of existing human sexuality education curricula in Florida. In addition, research is needed to understand the characteristics, values, and beliefs of successful human sexuality education instructors across the state.

  18. Comparison of Nested Polymerase Chain Reaction and Real-Time Polymerase Chain Reaction with Parasitological Methods for Detection of Strongyloides stercoralis in Human Fecal Samples.

    Science.gov (United States)

    Sharifdini, Meysam; Mirhendi, Hossein; Ashrafi, Keyhan; Hosseini, Mostafa; Mohebali, Mehdi; Khodadadi, Hossein; Kia, Eshrat Beigom

    2015-12-01

    This study was performed to evaluate nested polymerase chain reaction (PCR) and real-time PCR methods for detection of Strongyloides stercoralis in fecal samples compared with parasitological methods. A total of 466 stool samples were examined by conventional parasitological methods (formalin ether concentration [FEC] and agar plate culture [APC]). DNA was extracted using an in-house method, and mitochondrial cytochrome c oxidase subunit 1 and 18S ribosomal genes were amplified by nested PCR and real-time PCR, respectively. Among 466 samples, 12.7% and 18.2% were found infected with S. stercoralis by FEC and APC, respectively. DNA of S. stercoralis was detected in 18.9% and 25.1% of samples by real-time PCR and nested PCR, respectively. Considering parasitological methods as the diagnostic gold standard, the sensitivity and specificity of nested PCR were 100% and 91.6%, respectively, and that of real-time PCR were 84.7% and 95.8%, respectively. However, considering sequence analyzes of the selected nested PCR products, the specificity of nested PCR is increased. In general, molecular methods were superior to parasitological methods. They were more sensitive and more reliable in detection of S. stercoralis in comparison with parasitological methods. Between the two molecular methods, the sensitivity of nested PCR was higher than real-time PCR.

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

    Science.gov (United States)

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

    2004-06-01

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

  20. Accurate measurement of circulating mitochondrial DNA content from human blood samples using real-time quantitative PCR.

    Science.gov (United States)

    Ajaz, Saima; Czajka, Anna; Malik, Afshan

    2015-01-01

    We describe a protocol to accurately measure the amount of human mitochondrial DNA (MtDNA) in peripheral blood samples which can be modified to quantify MtDNA from other body fluids, human cells, and tissues. This protocol is based on the use of real-time quantitative PCR (qPCR) to quantify the amount of MtDNA relative to nuclear DNA (designated the Mt/N ratio). In the last decade, there have been increasing numbers of studies describing altered MtDNA or Mt/N in circulation in common nongenetic diseases where mitochondrial dysfunction may play a role (for review see Malik and Czajka, Mitochondrion 13:481-492, 2013). These studies are distinct from those looking at genetic mitochondrial disease and are attempting to identify acquired changes in circulating MtDNA content as an indicator of mitochondrial function. However, the methodology being used is not always specific and reproducible. As more than 95 % of the human mitochondrial genome is duplicated in the human nuclear genome, it is important to avoid co-amplification of nuclear pseudogenes. Furthermore, template preparation protocols can also affect the results because of the size and structural differences between the mitochondrial and nuclear genomes. Here we describe how to (1) prepare DNA from blood samples; (2) pretreat the DNA to prevent dilution bias; (3) prepare dilution standards for absolute quantification using the unique primers human mitochondrial genome forward primer (hMitoF3) and human mitochondrial genome reverse primer(hMitoR3) for the mitochondrial genome, and human nuclear genome forward primer (hB2MF1) and human nuclear genome reverse primer (hB2MR1) primers for the human nuclear genome; (4) carry out qPCR for either relative or absolute quantification from test samples; (5) analyze qPCR data; and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high-throughput use.

  1. Performance of chromID Clostridium difficile agar compared with BBL C. difficile selective agar for detection of C. difficile in stool specimens.

    Science.gov (United States)

    Han, Sang Bong; Chang, Jiyoung; Shin, Sang Hyun; Park, Kang Gyun; Lee, Gun Dong; Park, Yong Gyu; Park, Yeon-Joon

    2014-09-01

    We evaluated the performance of a new chromogenic medium for detection of Clostridium difficile, chromID C. difficile agar (CDIF; bioMérieux, France), by comparison with BBL C. difficile Selective Agar (CDSA; Becton Dickinson and Company, USA). After heat pre-treatment (80℃, 5 min), 185 diarrheal stool samples were inoculated onto the two media types and incubated anaerobically for 24 hr and 48 hr for CDIF and for 48 hr and 72 hr for CDSA. All typical colonies on each medium were examined by Gram staining, and the gram-positive rods confirmed to contain the tpi gene by PCR were identified as C. difficile. C. difficile was recovered from 36 samples by using a combination of the two media. The sensitivity with CDIF 48 hr was highest (100%) and was significantly higher than that with CDIF 24 hr (58.3%; P<0.001), because samples with a low burden of C. difficile tended to require prolonged incubation up to 48 hr (P<0.001). The specificity of CDIF 24 hr and CDIF 48 hr (99.3% and 90.6%, respectively) was significantly higher than that of CDSA 48 hr and CDSA 72 hr (72.5% and 67.1%, respectively; P<0.001). CDIF was effective for detecting C. difficile in heat-pretreated stool specimens, thus reducing unnecessary testing for toxin production in non-C. difficile isolates and turnaround time.

  2. Multicenter clinical evaluation of the portrait toxigenic C. difficile assay for detection of toxigenic Clostridium difficile strains in clinical stool specimens.

    Science.gov (United States)

    Buchan, Blake W; Mackey, Tami-Lea A; Daly, Judy A; Alger, Garrison; Denys, Gerald A; Peterson, Lance R; Kehl, Sue C; Ledeboer, Nathan A

    2012-12-01

    We compared the Portrait Toxigenic C. difficile Assay, a new semiautomated sample-to-result molecular test, to a toxigenic bacterial culture/cell cytotoxin neutralization assay (TBC/CCNA) for the detection of toxigenic Clostridium difficile in 549 stool specimens. Stool specimens were also tested by one of three alternative FDA-cleared molecular tests for toxigenic C. difficile (Xpert C. difficile, Illumigene C. difficile, or GeneOhm Cdiff). The sensitivities and specificities of the molecular tests compared to TBC/CCNA were as follows: 98.2% and 92.8% for the Portrait assay, 100% and 91.7% for the Xpert assay, 93.3% and 95.1% for the Illumigene assay, and 97.4% and 98.5% for the GeneOhm assay, respectively. The majority of Portrait false-positive results (20/31; 64.5%) were also positive for C. difficile by an alternative molecular test, suggesting an increased sensitivity compared to the culture-based "gold standard" method. The Portrait test detected an assay input of 30 CFU in 100% of spiked samples and detected an input of 10 CFU in 96.7% of samples tested.

  3. Stool Tests

    Science.gov (United States)

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

  4. Stool Culture

    Science.gov (United States)

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

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

    Science.gov (United States)

    Kazerouni, Abbas; Wein, Lawrence M

    2017-01-01

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

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

    Science.gov (United States)

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

    2017-05-01

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

  7. Interferon-λ4 (IFNL4 transcript expression in human liver tissue samples.

    Directory of Open Access Journals (Sweden)

    Ahmad Amanzada

    Full Text Available Eradication of hepatitis C virus (HCV infection, both spontaneous and treatment-induced, is marked by the wildtype allele C of a single nucleotide polymorphism upstream of the IL28B gene, rs12979860. This favorable allele was recently described to be in linkage disequilibrium with the wildtype allele TT of a dinucleotide polymorphism, ss469415590, located within a new protein-coding gene. While the TT allele introduces a frame-shift and disrupts the open reading frame, only the variant allele, ΔG, creates a novel type III interferon (IFN protein, IFN-λ4/IFNL4. Absence of IFNL4 is thus supposed to favor resolution of HCV infection. As to date IFNL4 mRNA transcription has only been investigated in polyI:C-stimulated primary human hepatocytes and not yet in HCV infection in vivo, this study analyzed IFNL4 mRNA expression in human liver biopsy specimens. Samples were obtained from patients with a broad panel of disorders including no liver disease, liver diseases of non-viral etiology, chronic hepatitis B and chronic hepatitis C. Hepatic IFNL4 transcripts were detectable exclusively in a subgroup of chronic hepatitis C patients (24/45. Their amounts were positively related to liver HCV RNA copy numbers (p = 0.0023, r = 0.56 suggesting that the hepatic viral load influences IFNL4 transcription irrespective of IFNL4 governing genotype. Both, the IFNL4 creating allele ΔG (p<0.0001 and actual IFNL4 transcription (p = 0.0015 were found to be correlated to the activation of IFN stimulatory genes (ISGs. By contrast, IFNL4 ss469415590 genotypes were not found to be related to IFN-λ2/3/IL28 or IFN-λ1/IL29 gene expression. In conclusion, this study is the first report on intrahepatic transcript levels of the recently discovered IFNL4 gene. Data indicate that HCV infection in particular might activate IFNL4 transcription in the liver. It provides a possible explanation as to why hepatitis C patients show ISG stimulation in their livers in the

  8. A study on the levels of a polybrominated biphenyl in Chinese human milk samples collected in 2007 and 2011.

    Science.gov (United States)

    Liu, Xiao; Wen, Sheng; Li, Jingguang; Zhang, Lei; Zhao, Yunfeng; Wu, Yongning

    2016-09-01

    The levels of a 2,2',4,4',5,5'-hexabromobiphenyl (BB-153) were measured in human milk samples collected in 2007 and 2011 from residents in China by high-resolution gas chromatography-high-resolution mass chromatography (HRGC-HRMS) with isotope dilution. The median concentrations of BB-153 from the samples collected in 2007 and 2011 were 8.3 and 7.2 pg/g lipid weight, respectively. The levels of BB-153 in the human milk collected from rural areas were not significantly different to those collected from the urban areas in China. Meanwhile, significant positive correlations were found between the levels of BB-153 in human milk and the consumption of animal-origin foods. In the present study, the mean levels of BB-153 in human milk from Chinese mothers were found to be lower than those from European and American mothers.

  9. Multiple types of human papillomavirus in cervical samples in women in Campo Grande, MS, Brazil

    Directory of Open Access Journals (Sweden)

    Inês Aparecida Tozetti

    2006-10-01

    Full Text Available We analyzed 87 cervical samples from Campo Grande, Mato Grosso do Sul, with a PGMY/GP+ nested PCR system. Positive samples were typed using E7 type-specific primer pairs for HPV 6/11, 16, 18, 45 and 66. Eighteen samples (22% were infected with HPV6/11, 18 samples (22% with HPV66, 13 samples (15.9% with HPV45, 8 samples (9.8% with HPV18 and 7 samples (8.5% with HPV16. Seventeen samples (20.7% were infected by two HPV types, and five samples (6.1% by three HPV types. We conclude that infection with multiple types is present at a high frequency in our population and that there is a relation between some types and cytological finds.

  10. High risk human papillomavirus genotyping in clinical samples: evaluation of different commercial tests.

    Science.gov (United States)

    Paolini, F; Rollo, F; Brandi, R; Benevolo, M; Mariani, L; Cercato, M C; Vocaturo, A; Venuti, A

    2011-01-01

    The aim of the present study is to compare the performance of several commercial human papillomavirus (HPV) tests in a cohort of 281 women. The hybrid capture II, the PreTect-HPV-Proofer, the linear array, and DR.HPVTMIVD were utilized to detect and type HPV in parallel with in-house PCR tests followed by direct automated sequencing or by sub-cloning and sequencing. The concordance levels along with other tests were evaluated with a Cohen's K value varying between 0.60 to 0.88, indicating good correlation with nearly perfect agreement between hybrid capture II, (HCII) and the linear array test. High sensitivity was recorded by the linear array and HCII with 100% (95% CI, 0.8021 to 1.0000) detection of cervical intraepithelial neoplasia (CIN) III by both methods. Conversely, the PreTect-HPV-Proofer showed high specificity with 12% (95% CI, 0.7966 to 0.9163) positivity on normal samples. The genotyping analysis showed that agreement among tests was only low to moderate with great differences between different HPV types. Multiple infections were detected with poor concordance and sub-cloning assays revealed the presence of a lower number of HPV in comparison to the other methods. In summary, the use of different HPV tests applied to the same group of cervical smears may possibly lead to incongruent results, suggesting the need to standardize type-specific sensitivity of genotyping methods and the need to evaluate their accuracy in detecting multiple HPV infections. This would be a prerequisite for the use of genotyping assays in cervical cancer screening programs.

  11. A multiclass method for the analysis of endocrine disrupting chemicals in human urine samples. Sample treatment by dispersive liquid-liquid microextraction.

    Science.gov (United States)

    Vela-Soria, F; Ballesteros, O; Zafra-Gómez, A; Ballesteros, L; Navalón, A

    2014-11-01

    The population is continuously exposed to endocrine disrupting chemicals (EDCs). This has influenced an increase in diseases and syndromes that are more frequent nowadays. Therefore, it is necessary to develop new analytical procedures to evaluate the exposure with the ultimate objective of establishing, in an accurate way, relationships between EDCs and harmful health effects. In the present work, a new method based on a sample treatment by dispersive liquid-liquid microextraction (DLLME) for the extraction of six parabens (methyl-, ethyl-, isopropyl-, propyl-, isobutyl and butylparaben), six benzophenones (benzophenone-1, benzophenone-2, benzophenone-3, benzophenone-6, benzophenone-8 and 4-hydroxybenzophenone) and two bisphenols (bisphenol A and bisphenol S) in human urine samples, followed by gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis is proposed. An enzymatic treatment allows determining the total content of the target EDCs. The extraction parameters were accurately optimized using multivariate optimization strategies. Ethylparaben ring-(13)C6 and bisphenol A-d16 were used as surrogates. Found limits of quantification ranging from 0.2 to 0.5 ng mL(-1) and inter-day variability (evaluated as relative standard deviation) ranging from 2.0% to 14.9%. The method was validated using matrix-matched standard calibration followed by a recovery assay with spiked samples. Recovery rates ranged from 94% to 105%. A good linearity, for concentrations up to 300 ng mL(-1) for parabens and 40 ng mL(-1) for benzophenones and bisphenols, respectively, was obtained. The method was satisfactorily applied for the determination of target compounds in human urine samples from 20 randomly selected individuals.

  12. Chromatofocusing profile of purified human alpha-fetoprotein and albumin differs from those of crude samples: effect of protein concentration of the elution of the sample.

    Science.gov (United States)

    Leal, J A; Eddy, K B; Keel, B A

    1991-02-01

    Chromatofocusing was utilized to characterize charge microheterogeneity of purified human alpha-fetoprotein (AFP) and human serum albumin (HSA). Crude cord blood samples yielded three isoforms: AFP-IA, IB, and II, with pIs 4.57 (52%), 4.27 (43%), and less than 4.00 (5%), respectively. In contrast, 10 micrograms of purified AFP or 250,000 cpm of 125I-AFP eluted entirely as isoform AFP-II. 125I-AFP focused in the presence of crude cord blood, amniotic fluid, adult male serum, or 25 mg purified HSA resulted in elution profiles similar to those of crude cord blood. Pure AFP focused along with 0.1, 1.0, 5.0, or 10 mg HSA showed a gradual shift from AFP-II to AFP-I. With greater than or equal to 5 mg HSA, isoform I was further resolve into AFP-IA and IB. Similarly, 250,000 cpm of 125I-HSA, which also eluted entirely as isoform II, showed a gradual shift to isoform I when increasing concentrations of unlabeled HSA were added. The resolution of isoform HSA-I in HSA-IA, IB, and IC was again improved with greater than or equal to 5 mg unlabeled HSA. When carrier proteins of varying pI values were chromatofocused along with purified AFP, it was observed that only those proteins with pIs in the range of AFP caused significant alteration in the relative distribution of AFP. We conclude that sample protein concentration and composition must be carefully considered when chromatofocusing is being used for purified samples and when the elution profiles of samples from different origins and varying protein concentrations are being compared.

  13. Microfluidic biosensor for cholera toxin detection in fecal samples.

    Science.gov (United States)

    Bunyakul, Natinan; Promptmas, Chamras; Baeumner, Antje J

    2015-01-01

    Sample preparation and processing steps are the most critical assay aspects that require our attention in the development of diagnostic devices for analytes present in complex matrices. In the best scenarios, diagnostic devices should use only simple sample processing. We have therefore investigated minimal preparation of stool samples and their effect on our sensitive microfluidic immunosensor for the detection of cholera toxin. This biosensor was previously developed and tested in buffer solutions only, using either fluorescence or electrochemical detection strategies. The microfluidic devices were made from polydimethylsiloxane using soft lithography and silicon templates. Cholera toxin subunit B (CTB)-specific antibodies immobilized onto superparamagnetic beads and ganglioside GM1-containing liposomes were used for CTB recognition in the detection system. Quantification of CTB was tested by spiking it in human stool samples. Here, optimal minimal sample processing steps, including filtration and centrifugation, were optimized using a microtiter plate assay owing to its high-throughput capabilities. Subsequently, it was transferred to the microfluidic systems, enhancing the diagnostic characteristic of the biosensor. It was found that the debris removal obtained through simple centrifugation resulted in an acceptable removal of matrix effects for the fluorescence format, reaching a limit of detection of only 9.0 ng/mL. However, the electron transfer in the electrochemical format was slightly negatively affected (limit of detection of 31.7 ng/mL). Subsequently, cross-reactivity using the heat-labile Escherichia coli toxin was investigated using the electrochemical microfluidic immunosensors and was determined to be negligible. With minimal sample preparation required, these microfluidic liposome-based systems have demonstrated excellent analytical performance in a complex matrix and will thus be applicable to other sample matrices.

  14. pH adjustment of human blood plasma prior to bioanalytical sample preparation

    NARCIS (Netherlands)

    Hendriks, G.; Uges, D. R. A.; Franke, J. P.

    2008-01-01

    pH adjustment in bioanalytical sample preparation concerning ionisable compounds is one of the most common sample treatments. This is often done by mixing an aliquot of the sample with a proper buffer adjusted to the proposed pH. The pH of the resulting mixture however, does not necessarily have to

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

    Science.gov (United States)

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

    2015-02-01

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

  16. Novel method for simultaneous aqueous in situ derivatization of THC and THC-COOH in human urine samples: validation and application to real samples.

    Science.gov (United States)

    Chericoni, S; Battistini, I; Dugheri, S; Pacenti, M; Giusiani, M

    2011-05-01

    The present work describes the validation of a novel aqueous in situ derivatization procedure with trimethyloxonium tetrafluoroborate (TMO) as methylating agent for the simultaneous, quantitative analysis of Δ(9)-tetrahydrocannabinol (THC) and 11-nor-Δ(9)-tetrahydrocannabinol carboxylic acid (THC-COOH) in human urine. The derivatizing agent is directly added to the urine sample and the methyl-derivatives are then recovered by liquid-liquid extraction procedure. Gas chromatography-mass spectrometry was used to detect the derivatives in selected ion monitoring mode. The limits of detection were 0.7 ng/mL for THC and 0.5 ng/mL for THC-COOH, whereas the limits of quantification were 1.9 and 0.9 ng/mL, respectively. The method has been applied to 60 real samples both positive and negative to immunochemical screening test resulting to be very useful and reliable in routine analysis of THC-COOH in human urine for toxicological and forensic purposes.

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

    Science.gov (United States)

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

    2016-08-01

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

  18. A Comparison of Microscopy and Enzyme Linked Immunosorbent Assay for Diagnosis of Giardia lamblia in Human Faecal Specimens.

    Science.gov (United States)

    Jahan, Noor; Khatoon, Razia; Ahmad, Siraj

    2014-11-01

    Giardia lamblia, a flagellate protozoa, is a common causative agent of parasitic diarrhoeal diseases of humans. Laboratory diagnosis mainly consists of direct microscopic examination of stool specimen for trophozoite and cysts of Giardia. However, due to intermittent faecal excretion of parasite, the case may be miss diagnosed and the patient may continue excreting the parasite and infecting others. Therefore, other mode of diagnosis should be looked for, which overcome the above drawbacks of microscopy used alone for diagnosis. The present study was done to evaluate the efficacy of RIDASCREEN Giardia (ELISA) test in comparison to direct microscopy in the diagnosis of Giardia lamblia in stool specimens from patients with diarrhea and other gastrointestinal symptoms. A total of 1680 patients were included in the study and three faecal specimens were taken from each patient which was divided into two parts. One part was used for direct wet mount examination and second part was used to put ELISA by using RIDASCREEN Giardia test. Out of 1680 stool samples, 380 specimens (22.6%) were found to be positive for Giardia lamblia. Maximum cases were detected by RIDASCREEN Giardia (ELISA) test with sensitivity of 100% and specificity of 91.5%. Maximum cases of giardiasis were detected in children less than 10 y of age (12.8%). RIDASCREEN Giardia test is a rapid and effective method with high sensitivity and specificity and detects Giardia antigens in stool specimens even when the count of parasite is low, thus reducing the chances of missing even the asymptomatic cases.

  19. Sample size calculations in human electrophysiology (EEG and ERP) studies: A systematic review and recommendations for increased rigor.

    Science.gov (United States)

    Larson, Michael J; Carbine, Kaylie A

    2017-01-01

    There is increasing focus across scientific fields on adequate sample sizes to ensure non-biased and reproducible effects. Very few studies, however, report sample size calculations or even the information needed to accurately calculate sample sizes for grants and future research. We systematically reviewed 100 randomly selected clinical human ele