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Sample records for qualitative pcr testing

  1. Development of a qualitative real-time PCR for microbiological quality control testing in mammalian cell culture production.

    PubMed

    Kleinschmidt, K; Wilkens, E; Glaeser, S P; Kaempfer, P; Staerk, A; Roesti, D

    2017-04-01

    The aim of this study was to develop and evaluate a real-time PCR technology for microbiological control methods to examine individualized cell therapeutics, an emerging class of pharmaceutical formulations. Oligonucleotide primers and hybridization probe for bacterial detection targeting the 16SrRNA gene were adapted based on Nadkarni et al. [Microbiology148 (2002) 257]. For detection of yeast and moulds, primers and probe were designed from conserved sequences of the 18SrRNA gene in this study. The real-time PCR assays were tested on genomic DNA of Escherichia coli and Candida albicans to assess efficiency and linear dynamic range. After successful establishment of robust real-time PCRs, applicability of the assays was evaluated by extracting microbial target DNA from cell-based preparations. Different commercial DNA extraction methods were compared identifying the MagNA Pure DNA Isolation Kit III as the method of choice. Sensitivity was examined for different strains and a detection limit of 10(2) -10(3) CFU per ml in a sample containing ~10(6) mammalian cells per ml was achieved. This study reports the successful establishment of two qualitative real-time PCR assays, enabling in general the broad-range detection of microbial contaminants in a cell-based sample matrix. Individualized cell therapeutics tend to have a short shelf life. Due to lengthy incubation periods, compendial testing according to current pharmacopoeial guidelines may not be applicable. We report a suitable alternative method upon which future microbiological quality control methods for such products could be based on. However, to implement valid rapid microbiological testing methods using real-time PCR technology, further challenges need to be addressed. © 2016 The Society for Applied Microbiology.

  2. HCG blood test - qualitative

    MedlinePlus

    ... qualitative; Serum HCG - qualitative; HCG in blood serum - qualitative ... Henry's Clinical Diagnosis and Management by Laboratory Methods. 22nd ed. Philadelphia, PA: Elsevier Saunders; 2011:chap ...

  3. Qualitative PCR method for Roundup Ready soybean: interlaboratory study.

    PubMed

    Kodama, Takashi; Kasahara, Masaki; Minegishi, Yasutaka; Futo, Satoshi; Sawada, Chihiro; Watai, Masatoshi; Akiyama, Hiroshi; Teshima, Reiko; Kurosawa, Yasunori; Furui, Satoshi; Hino, Akihiro; Kitta, Kazumi

    2011-01-01

    Quantitative and qualitative methods based on PCR have been developed for genetically modified organisms (GMO). Interlaboratory studies were previously conducted for GMO quantitative methods; in this study, an interlaboratory study was conducted for a qualitative method for a GM soybean, Roundup Ready soy (RR soy), with primer pairs designed for the quantitative method of RR soy studied previously. Fourteen laboratories in Japan participated. Each participant extracted DNA from 1.0 g each of the soy samples containing 0, 0.05, and 0.10% of RR soy, and performed PCR with primer pairs for an internal control gene (Le1) and RR soy followed by agarose gel electrophoresis. The PCR product amplified in this PCR system for Le1 was detected from all samples. The sensitivity, specificity, and false-negative and false-positive rates of the method were obtained from the results of RR soy detection. False-negative rates at the level of 0.05 and 0.10% of the RR soy samples were 6.0 and 2.3%, respectively, revealing that the LOD of the method was somewhat below 0.10%. The current study demonstrated that the qualitative method would be practical for monitoring the labeling system of GM soy in kernel lots.

  4. Testing for Genetically Modified Foods Using PCR

    ERIC Educational Resources Information Center

    Taylor, Ann; Sajan, Samin

    2005-01-01

    The polymerase chain reaction (PCR) is a Nobel Prize-winning technique that amplifies a specific segment of DNA and is commonly used to test for the presence of genetic modifications. Students use PCR to test corn meal and corn-muffin mixes for the presence of a promoter commonly used in genetically modified foods, the cauliflower mosaic virus 35S…

  5. Testing for Genetically Modified Foods Using PCR

    ERIC Educational Resources Information Center

    Taylor, Ann; Sajan, Samin

    2005-01-01

    The polymerase chain reaction (PCR) is a Nobel Prize-winning technique that amplifies a specific segment of DNA and is commonly used to test for the presence of genetic modifications. Students use PCR to test corn meal and corn-muffin mixes for the presence of a promoter commonly used in genetically modified foods, the cauliflower mosaic virus 35S…

  6. PCR-based approach for qualitative molecular analysis of six neurotropic pathogens.

    PubMed

    Ferese, R; Scorzolini, L; Campopiano, R; Albano, V; Griguoli, A M; Giardina, E; Scala, S; Ryskalin, L; D'Alessio, C; Zampatti, S; Fantozzi, R; Storto, M; Fornai, F; Gambardella, S

    2017-01-01

    In the last few years, polymerase chain reaction analysis is frequently required to improve the detection of pathogen infections in central nervous system as a potential cause of neurological disorders and neuropsychiatric symptoms. The goal of this paper is to set up a fast, cheap and reliable molecular approach for qualitative detection of six neurotropic pathogens. A method based on PCR has been designed and implemented to guarantee the qualitative DNA detection of herpes simplex virus types 1 and 2 (HSVI/II), Epstein-Barr virus (EBV), cytomegalovirus (CMV), varicella-zoster virus (VZV), rubella virus (RUBV) and Toxoplasma gondii in the cerebrospinal fluid, where otherwise they are barely detectable. Each PCR assay was tested using dilutions of positive controls, which demonstrated a sensitivity allowing to detect up to 102 copies/ml in PCR and 10 copies/ml in real-time PCR for each pathogen. Once been set up, the protocol was applied to evaluate the cerebrospinal fluid from 100 patients with suspected infectious diseases of the central nervous system and 50 patients without any infection. The method allowed to identify 17 positive cerebrospinal fluid with polymerase chain reaction and 22 with real-time PCR (RT-PCR), respectively. Therefore, application of RT PCR allows a fast and sensitive evaluation of neurotropic DNA pathogens in the course of diagnostic routine within neurological units.

  7. Qualitative and quantitative PCR methods for detection of three lines of genetically modified potatoes.

    PubMed

    Rho, Jae Kyun; Lee, Theresa; Jung, Soon-Il; Kim, Tae-San; Park, Yong-Hwan; Kim, Young-Mi

    2004-06-02

    Qualitative and quantitative polymerase chain reaction (PCR) methods have been developed for the detection of genetically modified (GM) potatoes. The combination of specific primers for amplification of the promoter region of Cry3A gene, potato leafroll virus replicase gene, and potato virus Y coat protein gene allows to identify each line of NewLeaf, NewLeaf Y, and NewLeaf Plus GM potatoes. Multiplex PCR method was also established for the simple and rapid detection of the three lines of GM potato in a mixture sample. For further quantitative detection, the realtime PCR method has been developed. This method features the use of a standard plasmid as a reference molecule. Standard plasmid contains both a specific region of the transgene Cry3A and an endogenous UDP-glucose pyrophosphorylase gene of the potato. The test samples containing 0.5, 1, 3, and 5% GM potatoes were quantified by this method. At the 3.0% level of each line of GM potato, the relative standard deviations ranged from 6.0 to 19.6%. This result shows that the above PCR methods are applicable to detect GM potatoes quantitatively as well as qualitatively.

  8. PCR test for Microsporum canis identification.

    PubMed

    Brillowska-Dabrowska, Anna; Michałek, Ewelina; Saunte, Ditte Marie Lindhardt; Nielsen, Sanne Søgaard; Arendrup, Maiken Cavling

    2013-08-01

    Microsporum canis, for which the natural hosts are cats and dogs, is the most prevalent zoophilic agent causing tinea capitis and tinea corporis in humans. We present here a diagnostic PCR test for M. canis, since its detection and species identification is relevant to the choice of treatment and to the understanding of a probable source of infection. An M. canis-specific PCR was evaluated using 130 clinical isolates of dermatophytes (including M. canis [n = 15] and 13 other species), 10 yeast or mold isolates, 12 hair and skin samples from animals with or without experimental M. canis infection, and 35 patient specimens, including seven specimens positive for M. canis and 15 dermatophyte negative samples. All pure cultures, animal specimens and clinical samples with M. canis were detected by the PCR test, whereas none of the other fungal isolates or samples without M. canis was negative. This study indicates that the PCR test for M. canis identification applied directly to patient specimens or animal hair, as well as to clinical isolates had 100% specificity and sensitivity.

  9. Development of qualitative and quantitative PCR analysis for meat adulteration from RNA samples.

    PubMed

    Cheng, Jai-Hong; Chou, Hsiao-Ting; Lee, Meng-Shiou; Sheu, Shyang-Chwen

    2016-02-01

    Total RNA samples were used to establish qualitative and quantitative PCR-based methods for assessing meat adulteration. The primers were designed based on the mRNA sequences of troponin I (TnI), mitochondrial ribosomal protein (MRP) and tropomodulin genes to distinguish chicken, pork, goat, beef and ostrich. There was no cross reaction between the primers, and the detection limit of the cDNA template was 0.01 and 20 ng in simplex PCR and multiplex PCR, respectively. In the low temperature storage test, the detection limits of cDNA template with 10 and 1 ng were determined at 4 °C and -80 °C. In quantitative assay, the precision of real-time PCR analysis expressed as a coefficient of variation (CV) ranged from 0.25% to 5.24% and the trueness, expressed as an error, ranged from 0.28% to 6.98% for adulteration. Thus, herein, we provided alternative tools for the assessment of meat adulteration using mRNA-based PCR methods.

  10. Evaluation of Altona Diagnostics RealStar Zika Virus RT-PCR Test Kit for Zika virus PCR testing.

    PubMed

    L'Huillier, Arnaud G; Lombos, Ernesto; Tang, Elaine; Perusini, Stephen; Eshaghi, Alireza; Nagra, Sandeep; Frantz, Christine; Olsha, Romy; Kristjanson, Erik; Dimitrova, Kristina; Safronetz, David; Drebot, Mike; Gubbay, Jonathan B

    2017-03-15

    Background: With the emerging ZIKA virus (ZIKV) epidemic, accessible real-time reverse-transcription PCR (rRT-PCR) assays are needed to streamline testing. The commercial Altona Diagnostics RealStar ZIKV rRT-PCR Test Kit has been approved for Emergency Use Authorization by the FDA. Our aim was to verify Altona-PCR, by comparing it to the CDC-designed dual target ZIKV virus rRT-PCR reference assay (Reference-PCR), and describe demographics of patients tested for ZIKV by rRT-PCR in Ontario, Canada.Methods: A large set of clinical specimens were tested for ZIKV by Altona-PCR and Reference-PCR. Positive or equivocal specimens underwent PCR and Sanger sequencing targeting ZIKV NS5 gene.Results: 671 serum specimens were tested by Reference-PCR: 58 (8.6%) were positive, 193 (28.8%) equivocal and 420 (62.6%) negative. Ninety percent of Reference-PCR positive patients were tested in the first 5 days after symptom onset. Altona-PCR was performed on 284/671 tested specimens by Reference-PCR. Altona-PCR was positive in 53/58 (91%) Reference-PCR positive and 16/193 (8%) Reference-PCR equivocal specimens; ZIKV NS5 PCR was positive in all 68 Altona-PCR positive specimens, and negative in all 181 Altona-PCR negative specimens that underwent NS5 PCR.Conclusion: Most ZIKV PCR positive cases are detected in the first five days of illness. Altona-PCR has very good sensitivity (91%) and specificity (97%) compared to Reference-PCR. Altona-PCR can be used for ZIKV diagnostic testing, with less extensive verification requirements compared to a laboratory developed test.

  11. Rapid Qualitative Urinary Tract Infection Pathogen Identification by SeptiFast® Real-Time PCR

    PubMed Central

    Malinka, Thomas; Klaschik, Sven; Weber, Stefan U.; Schewe, Jens-Christian; Stüber, Frank; Book, Malte

    2011-01-01

    Background Urinary tract infections (UTI) are frequent in outpatients. Fast pathogen identification is mandatory for shortening the time of discomfort and preventing serious complications. Urine culture needs up to 48 hours until pathogen identification. Consequently, the initial antibiotic regimen is empirical. Aim To evaluate the feasibility of qualitative urine pathogen identification by a commercially available real-time PCR blood pathogen test (SeptiFast®) and to compare the results with dipslide and microbiological culture. Design of study Pilot study with prospectively collected urine samples. Setting University hospital. Methods 82 prospectively collected urine samples from 81 patients with suspected UTI were included. Dipslide urine culture was followed by microbiological pathogen identification in dipslide positive samples. In parallel, qualitative DNA based pathogen identification (SeptiFast®) was performed in all samples. Results 61 samples were SeptiFast® positive, whereas 67 samples were dipslide culture positive. The inter-methodological concordance of positive and negative findings in the gram+, gram- and fungi sector was 371/410 (90%), 477/492 (97%) and 238/246 (97%), respectively. Sensitivity and specificity of the SeptiFast® test for the detection of an infection was 0.82 and 0.60, respectively. SeptiFast® pathogen identifications were available at least 43 hours prior to culture results. Conclusion The SeptiFast® platform identified bacterial DNA in urine specimens considerably faster compared to conventional culture. For UTI diagnosis sensitivity and specificity is limited by its present qualitative setup which does not allow pathogen quantification. Future quantitative assays may hold promise for PCR based UTI pathogen identification as a supplementation of conventional culture methods. PMID:21359187

  12. Interlaboratory study of DNA extraction from multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for individual kernel detection system of genetically modified maize.

    PubMed

    Akiyama, Hiroshi; Sakata, Kozue; Makiyma, Daiki; Nakamura, Kosuke; Teshima, Reiko; Nakashima, Akie; Ogawa, Asako; Yamagishi, Toru; Futo, Satoshi; Oguchi, Taichi; Mano, Junichi; Kitta, Kazumi

    2011-01-01

    In many countries, the labeling of grains, feed, and foodstuff is mandatory if the genetically modified (GM) organism content exceeds a certain level of approved GM varieties. We previously developed an individual kernel detection system consisting of grinding individual kernels, DNA extraction from the individually ground kernels, GM detection using multiplex real-time PCR, and GM event detection using multiplex qualitative PCR to analyze the precise commingling level and varieties of GM maize in real sample grains. We performed the interlaboratory study of the DNA extraction with multiple ground samples, multiplex real-time PCR detection, and multiplex qualitative PCR detection to evaluate its applicability, practicality, and ruggedness for the individual kernel detection system of GM maize. DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR were evaluated by five laboratories in Japan, and all results from these laboratories were consistent with the expected results in terms of the commingling level and event analysis. Thus, the DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for the individual kernel detection system is applicable and practicable in a laboratory to regulate the commingling level of GM maize grain for GM samples, including stacked GM maize.

  13. Multiplex PCR Tests for Detection of Pathogens Associated with Gastroenteritis

    PubMed Central

    Zhang, Hongwei; Morrison, Scott; Tang, Yi-Wei

    2016-01-01

    Synopsis A wide range of enteric pathogens can cause infectious gastroenteritis. Conventional diagnostic algorithms including culture, biochemical identification, immunoassay and microscopic examination are time consuming and often lack sensitivity and specificity. Advances in molecular technology have as allowed its use as clinical diagnostic tools. Multiplex PCR based testing has made its way to gastroenterology diagnostic arena in recent years. In this article we present a review of recent laboratory developed multiplex PCR tests and current commercial multiplex gastrointestinal pathogen tests. We will focus on two FDA cleared commercial syndromic multiplex tests: Luminex xTAG GPP and Biofire FimArray GI test. These multiplex tests can detect and identify multiple enteric pathogens in one test and provide results within hours. Multiplex PCR tests have shown superior sensitivity to conventional methods for detection of most pathogens. The high negative predictive value of these multiplex tests has led to the suggestion that they be used as screening tools especially in outbreaks. Although the clinical utility and benefit of multiplex PCR test are to be further investigated, implementing these multiplex PCR tests in gastroenterology diagnostic algorithm has the potential to improve diagnosis of infectious gastroenteritis. PMID:26004652

  14. Development of a PCR test to diagnose Haemophilus parasuis infections.

    PubMed

    Oliveira, S; Galina, L; Pijoan, C

    2001-11-01

    A polymerase chain reaction (PCR) test was developed in order to improve the accuracy and speed of diagnosis of Haemophilus parasuis, an economically important respiratory pathogen that affects swine. The gene sequence of the 16S small subunit ribosomal RNA of H. parasuis (GenBank M75065) was compared with 56 16S sequences of related bacteria, including those frequently isolated from pig tissues. Two species-specific primers were designed: HPS forward and HPS reverse. The predicted size of the amplified PCR product was 821 bp. The PCR test could detect a minimum of 102 bacteria and 0.69 pg of DNA. Thirty-one H. parasuis isolates, including 12 different serovars and 19 field isolates, were positive using the PCR test. No amplification was observed when the test was run using DNA from 15 other bacterial species commonly isolated from swine tissues. A weak band was observed when the PCR test was performed using Actinobacillus indolicus DNA as template. Clinical samples tested by PCR included tissues and swabs from 5 animals naturally infected with H. parasuis and 1 experimentally infected animal. The PCR was positive in 26 of 30 clinical samples. Four samples showed weak bands, and these results were not considered positive. Haemophilus parasuis was isolated from 18 of 30 of these samples. Tissues from specific pathogen-free (SPF) pigs and from unrelated species were negative for H. parasuis isolation and PCR. The developed PCR was successfully used in the diagnosis of H. parasuis infection, especially when compared with traditional microbiology techniques.

  15. Validation of qualitative microbiological test methods.

    PubMed

    IJzerman-Boon, Pieta C; van den Heuvel, Edwin R

    2015-01-01

    This paper considers a statistical model for the detection mechanism of qualitative microbiological test methods with a parameter for the detection proportion (the probability to detect a single organism) and a parameter for the false positive rate. It is demonstrated that the detection proportion and the bacterial density cannot be estimated separately, not even in a multiple dilution experiment. Only the product can be estimated, changing the interpretation of the most probable number estimator. The asymptotic power of the likelihood ratio statistic for comparing an alternative method with the compendial method, is optimal for a single dilution experiment. The bacterial density should either be close to two CFUs per test unit or equal to zero, depending on differences in the model parameters between the two test methods. The proposed strategy for method validation is to use these two dilutions and test for differences in the two model parameters, addressing the validation parameters specificity and accuracy. Robustness of these two parameters might still be required, but all other validation parameters can be omitted. A confidence interval-based approach for the ratio of the detection proportions for the two methods is recommended, since it is most informative and close to the power of the likelihood ratio test.

  16. Evaluation of Aspergillus PCR protocols for testing serum specimens.

    PubMed

    White, P Lewis; Mengoli, Carlo; Bretagne, Stéphane; Cuenca-Estrella, Manuel; Finnstrom, Niklas; Klingspor, Lena; Melchers, Willem J G; McCulloch, Elaine; Barnes, Rosemary A; Donnelly, J Peter; Loeffler, Juergen

    2011-11-01

    A panel of human serum samples spiked with various amounts of Aspergillus fumigatus genomic DNA was distributed to 23 centers within the European Aspergillus PCR Initiative to determine analytical performance of PCR. Information regarding specific methodological components and PCR performance was requested. The information provided was made anonymous, and meta-regression analysis was performed to determine any procedural factors that significantly altered PCR performance. Ninety-seven percent of protocols were able to detect a threshold of 10 genomes/ml on at least one occasion, with 83% of protocols reproducibly detecting this concentration. Sensitivity and specificity were 86.1% and 93.6%, respectively. Positive associations between sensitivity and the use of larger sample volumes, an internal control PCR, and PCR targeting the internal transcribed spacer (ITS) region were shown. Negative associations between sensitivity and the use of larger elution volumes (≥100 μl) and PCR targeting the mitochondrial genes were demonstrated. Most Aspergillus PCR protocols used to test serum generate satisfactory analytical performance. Testing serum requires less standardization, and the specific recommendations shown in this article will only improve performance.

  17. PCR test for detecting Taenia solium cysticercosis in pig carcasses.

    PubMed

    Sreedevi, Chennuru; Hafeez, Mohammad; Kumar, Putcha Anand; Rayulu, Vukka Chengalva; Subramanyam, Kothapalli Venkata; Sudhakar, Krovvidi

    2012-01-01

    Polymerase chain reaction (PCR) test was employed to detect Taenia solium DNA in muscle lesions for validation of the meat inspection results of slaughtered pigs. Two sets of oligonucleotide primers, one targeted against the large subunit rRNA gene (TBR primers) and the other targeted against cytochrome c oxidase subunit 1 gene (Cox1 primers) of T. solium were used in this study. On reactivity in PCR test, the TBR primers and the Cox1 primers yielded products of 286 and 984 bp, respectively, in cysticercosis positive cases. Both the sets of primers were found to be highly specific, since they did not yield any PCR product in negative controls. A total of 225 pig carcasses were screened for cysticercosis by meat inspection, out of which 25 carcasses with visible cysts (16 viable and 9 degenerated cysts) were also confirmed to be positive for cysticercosis in PCR test. However, out of the 35 carcasses with suspected lesions on meat inspection, only two were found to be positive for cysticercosis in PCR test. The detection limits for both the primer sets were analyzed. The TBR primer set could detect up to 10 pg of cysticercus DNA, whereas the Cox1 primer set could detect only up to 1 ng. It is evident from the study that PCR test is an efficient tool for validation of meat inspection results and also to rule out ambiguity in carcass judgment of suspected cases of porcine cysticercosis.

  18. Real-time PCR assay for rapid qualitative and quantitative detection of Entamoeba histolytica.

    PubMed

    Orosz, Erika; Perkátai, Katalin; Kapusinszky, Beatrix; Farkas, Agnes; Kucsera, István

    2012-12-01

    Simple real-time PCR assay with one set of primer and probe for rapid, sensitive qualitative and quantitative detection of Entamoeba histolytica has been used. Consensus sequences were used to amplify a species-specific region of the 16S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be a perfect match for the 16S rRNA gene of Entamoeba species, while the acceptor probe sequence was designed for Entamoeba histolytica, which allowed differentiation. The performed characteristics of the real-time PCR assay were compared with ELISA antigen and microscopical detection from 77 samples of individuals with suspected clinical diagnosis of imported E. histolytica infection. Stool and liver abscess pus samples were examined with analytical sensitivity of 5 parasites per PCR reaction. The melting curve means Tms (standard deviation) in clinical isolates were 54°C. The real-time assay was 100% sensitive and specific for differentiation of Entamoeba histolytica, compared with conventional ELISA or microscopy. This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of Entamoeba histolytica. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed.

  19. Validation of a carnation-specific gene, ANS, used as an endogenous reference gene in qualitative and real-time quantitative PCR for carnations.

    PubMed

    Zhu, Hong; Jiang, Lingxi; Tao, Shiru; Lin, Heyan; Wang, Jinbin; Tan, Furong; Zhao, Kai; Wu, Xiao; Li, Peng; Pan, Aihu; Jia, Junwei; Tang, Xueming

    2011-01-01

    The validation of the anthocyanin synthase (ANS) gene as a carnation endogenous reference gene applicable both in classical and real-time PCR methods is a prerequisite for the development of PCR assays for genetically modified (GM) carnation detection. This is important due to the fact that GM carnation lines, developed by Florigene Pty Ltd, have been approved for commercialization. In this study, both methods were tested on 14 different carnation cultivars, and identical amplification products were obtained with all of them. No amplification products were observed with samples from 14 other plant species, which demonstrated that the system was specific to carnation. The results of Southern blot analysis confirmed that the ANS gene had a low copy number in the 10 tested carnation varieties. In qualitative and real-time PCR assays, the LOD values of 0.05 and 0.005 ng carnation DNA, respectively, were validated. Moreover, the real-time PCR system was validated with high PCR efficiency and linearity. Thus, the ANS gene had species specificity, low heterogeneity, and low copy number among the tested cultivars. These results provide evidence that the gene can be used as an endogenous reference gene of carnation, as well as in qualitative and quantitative PCR systems.

  20. Triplet Repeat Primed PCR (TP-PCR) in Molecular Diagnostic Testing for Spinocerebellar Ataxia Type 3 (SCA3).

    PubMed

    Melo, Ana Rosa Vieira; Ramos, Amanda; Kazachkova, Nadiya; Raposo, Mafalda; Bettencourt, Bruno Filipe; Rendeiro, Ana Rita; Kay, Teresa; Vasconcelos, João; Bruges-Armas, Jácome; Lima, Manuela

    2016-12-01

    Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine (polyQ) disorder for which the routine molecular testing is based on PCR and automated capillary electrophoresis. When only a normal allele is detected by standard PCR, the hypothesis of a failed amplification of the expanded allele must be raised. In such cases, complementary techniques such as Southern Blot or triplet repeat primed PCR (TP-PCR) have to be applied. For SCA3, TP-PCR is implemented in some diagnostic laboratories, but a tested protocol has yet to be published. The purpose of this study was to develop and test a TP-PCR protocol for SCA3. Sixty-five blood samples previously genotyped by standard PCR were used in the TP-PCR assay. Fourteen buccal swab samples were also analyzed to confirm the robustness of the technique. The reproducibility of the TP-PCR was evaluated by analyzing all samples in a second laboratory. The results obtained by TP-PCR confirmed the previous PCR results for 64 blood samples; in one sample an expanded allele, previously undetected by PCR, was identified. The results obtained for the buccal swab samples were totally concordant with those obtained for blood. Furthermore, the results obtained in the alternative laboratory were in full agreement with the results obtained in our study. The present TP-PCR protocol developed for SCA3 should constitute a reliable complementary technique to overcome the limitations of standard PCR.

  1. A PCR test for avian malaria in Hawaiian birds.

    PubMed

    Feldman, R A; Freed, L A; Cann, R L

    1995-12-01

    The decline of native Hawaiian forest birds since European contact is attributed to factors ranging from habitat destruction to interactions with introduced species. Remaining populations of Hawaiian honeycreepers (Fringillidae: Drepanidinae) are most abundant and diverse in high elevation refuges above the normal range of disease-carrying mosquitoes. Challenge experiments suggest that honeycreepers are highly susceptible to avian malaria (Plasmodium sp.) but resistance exists in some species. In order to detect low levels of malarial infection and quantify prevalence of Plasmodium in high elevation natural populations of Hawaiian birds, a polymerase chain reaction (PCR) based diagnostic test was developed that identifies rRNA genes of Plasmodium in avian blood samples. Quantitative competitive PCR (QC-PCR) experiments indicate that the detection limit of our test is an order of magnitude greater than that reported for human malaria DNA blot tests. Compared with standard histological methods, the PCR test detected a higher prevalence of diseased birds at mid-elevations. Malaria was detected in three species of native birds living in a high elevation wildlife refuge on the island of Hawaii and in four species from Maui. Our results show that avian malaria is more widespread in Hawaiian forests than previously thought, a finding that has important conservation implications for these threatened species.

  2. Diagnosis of ocular toxoplasmosis by two polymerase chain reaction (PCR) examinations: qualitative multiplex and quantitative real-time.

    PubMed

    Sugita, Sunao; Ogawa, Manabu; Inoue, Shizu; Shimizu, Norio; Mochizuki, Manabu

    2011-09-01

    To establish a two-step polymerase chain reaction (PCR) diagnostic system for ocular toxoplasmosis. A total of 13 ocular fluid samples (11 aqueous humor and 2 vitreous fluid) were collected from 13 patients with clinically suspected ocular toxoplasmosis. Ten ocular samples from other uveitis patients and 20 samples from subjects without ocular inflammation were used as controls. Two polymerase chain reaction (PCR) methods, i.e., qualitative multiplex PCR and quantitative real-time PCR, were used to measure the toxoplasma genome (T. gondii B1 gene). Qualitative multiplex PCR detected T. gondii B1 gene in the ocular fluids of 11 out of 13 patients with clinically suspected ocular toxoplasmosis. In real-time PCR, we detected high copy numbers of T. gondii DNA (5.1 × 10(2)-2.1 × 10(6) copies/mL) in a total of 10 patients (10/13, 77%). Only ocular toxoplasmosis scar lesions were observed in the three real-time PCR-negative patients. PCR assay results for the samples from the two control groups were all negative. The two-step PCR examination to detect toxoplasma DNA is a useful tool for diagnosing ocular toxoplasmosis.

  3. High-Stakes Testing and Curricular Control: A Qualitative Metasynthesis

    ERIC Educational Resources Information Center

    Au, Wayne

    2007-01-01

    Using the method of qualitative metasynthesis, this study analyzes 49 qualitative studies to interrogate how high-stakes testing affects curriculum, defined here as embodying content, knowledge form, and pedagogy. The findings from this study complicate the understanding of the relationship between high-stakes testing and classroom practice by…

  4. Qualitative Examination of Children's Naming Skills through Test Adaptations.

    ERIC Educational Resources Information Center

    Fried-Oken, Melanie

    1987-01-01

    The Double Administration Naming Technique assists clinicians in obtaining qualitative information about a client's visual confrontation naming skills through administration of a standard naming test; readministration of the same test; identification of single and double errors; cuing for double naming errors; and qualitative analysis of naming…

  5. Identification of Candida spp. by phenotypic tests and PCR

    PubMed Central

    Marinho, Sandra Aparecida; Teixeira, Alice Becker; Santos, Otávio Silveira; Cazanova, Ricardo Flores; Ferreira, Carlos Alexandre Sanchez; Cherubini, Karen; de Oliveira, Sílvia Dias

    2010-01-01

    The correct identification of Candida species is of great importance, as it presents prognostic and therapeutical significance, allowing an early and appropriate antifungical therapy. The purpose of this study was to identify isolates of Candida spp. from oral mucosa of 38 patients with oral candidosis evaluated in 2004 by phenotypic methods and PCR, discriminating C. albicans from the other Candida species. The tests used for phenotypic analysis were germ-tube and chlamydoconidia production, culture in CHROMAgar™ Candida, carbohydrate assimilation test, growth at 45ºC and culture in Tween 80 agar. Genotypic confirmation was performed by PCR. Phenotypic tests showed that 63.2% strains formed germ-tubes, 73.7% produced chlamydoconidia, and 63.2% showed green colonies in chromogenic medium, presumptively indicating C. albicans or C. dubliniensis. The carbohydrate assimilation test confirmed these results. A total of 21% strains were identified as C. krusei and 13.2% were indicative of C. tropicalis. Of these later strains, three produced chlamydoconidia. The association of other phenotypic tests with culture in Tween 80 agar identified 95.8% of strains as C. albicans and 4.2% as C. dubliniensis. All 24 strains indicative of C. albicans and C. dubliniensis were confirmed by PCR as C. albicans. PMID:24031493

  6. Multiplex PCR testing for nine different sexually transmitted infections.

    PubMed

    Kriesel, John D; Bhatia, Amiteshwar S; Barrus, Cammie; Vaughn, Mike; Gardner, Jordan; Crisp, Robert J

    2016-12-01

    Current sexually transmitted infection (STI) testing is not optimal due to delays in reporting or missed diagnoses due to a lack of comprehensive testing. The FilmArray® (BioFire Diagnostics, LLC, Salt Lake City, Utah) is a user-friendly, fully automated, multiplex PCR system that is being developed for rapid point-of-care use. A research-use-only STI panel including multiple PCR primer sets for each organism was designed to detect Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, Trichomonas vaginalis, Mycoplasma genitalium, Ureaplasma urealyticum, Haemophilus ducreyi, and herpes simplex virus (HSV) types 1 and 2. Standard clinical testing included Gram stain, nucleic acid amplification, wet mount examination, herpes simplex virus culture, and syphilis IgG. Standard clinical tests were not available for all the organisms tested by the FilmArray STI panel. Two hundred and ninety-five clinical specimens from 190 subjects were directly compared to standard testing. Urine (n = 146), urethral/cervical swabs (31), oral swabs (60), rectal swabs (43), and ulcer swabs (15) were tested. Among the tested samples, FilmArray detected C. trachomatis in 39 (13%), N. gonorrhoeae in 20 (7%), T. vaginalis in nine (3%), HSV 1 in five (2%), HSV 2 in five (2%), U. urealyticum in 36 (12%), M. genitalium in eight (3%), and T. pallidum in 11 (4%). Concordance between the FilmArray STI panel and standard nucleic acid amplification testing for C. trachomatis was 98% and for N. gonorrhoeae was 97%. Multiplex PCR STI testing has the potential to improve public health by providing rapid, sensitive, and reliable results within the clinic or nearby laboratory.

  7. Detection of Genetically Modified Maize in Processed Foods Sold Commercially in Iran by Qualitative PCR

    PubMed Central

    Rabiei, Maryam; Mehdizadeh, Mehrangiz; Rastegar, Hossein; Vahidi, Hossein; Alebouyeh, Mahmoud

    2013-01-01

    Detection of genetically modified organisms (GMOs) in food is an important issue for all the subjects involved in food control and customer’s right. Due to the increasing number of GMOs imported to Iran during the past few years, it has become necessary to screen the products in order to determine the identity of the consumed daily foodstuffs. In this study, following the extraction of genomic DNA from processed foods sold commercially in Iran, qualitative PCR was performed to detect genetically modified maize. The recombinant DNA target sequences were detected with primers highly specific for each investigated transgene such as CaMV35s gene, Bt-11, MON810 and Bt-176 separately. Based on the gel electrophoresis results, Bt- 11 and MON810 events were detected in some maize samples, while, in none of them Bt- 176 modified gene was detected. For the first time, the results demonstrate the presence of genetically modified maize in Iranian food products, reinforcing the need for the development of labeling system and valid quantitative methods in routine analyses. PMID:24250568

  8. Detection of genetically modified maize in processed foods sold commercially in iran by qualitative PCR.

    PubMed

    Rabiei, Maryam; Mehdizadeh, Mehrangiz; Rastegar, Hossein; Vahidi, Hossein; Alebouyeh, Mahmoud

    2013-01-01

    Detection of genetically modified organisms (GMOs) in food is an important issue for all the subjects involved in food control and customer's right. Due to the increasing number of GMOs imported to Iran during the past few years, it has become necessary to screen the products in order to determine the identity of the consumed daily foodstuffs. In this study, following the extraction of genomic DNA from processed foods sold commercially in Iran, qualitative PCR was performed to detect genetically modified maize. The recombinant DNA target sequences were detected with primers highly specific for each investigated transgene such as CaMV35s gene, Bt-11, MON810 and Bt-176 separately. Based on the gel electrophoresis results, Bt- 11 and MON810 events were detected in some maize samples, while, in none of them Bt- 176 modified gene was detected. For the first time, the results demonstrate the presence of genetically modified maize in Iranian food products, reinforcing the need for the development of labeling system and valid quantitative methods in routine analyses.

  9. Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing.

    PubMed

    Li, Jin; Wang, Lilin; Mamon, Harvey; Kulke, Matthew H; Berbeco, Ross; Makrigiorgos, G Mike

    2008-05-01

    PCR is widely employed as the initial DNA amplification step for genetic testing. However, a key limitation of PCR-based methods is the inability to selectively amplify low levels of mutations in a wild-type background. As a result, downstream assays are limited in their ability to identify subtle genetic changes that can have a profound impact in clinical decision-making and outcome. Here we describe co-amplification at lower denaturation temperature PCR (COLD-PCR), a novel form of PCR that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences irrespective of the mutation type or position on the sequence. We replaced regular PCR with COLD-PCR before sequencing or genotyping assays to improve mutation detection sensitivity by up to 100-fold and identified new mutations in the genes encoding p53, KRAS and epidermal growth factor in heterogeneous cancer samples that had been missed by the currently used methods. For clinically relevant microdeletions, COLD-PCR enabled exclusive amplification and isolation of the mutants. COLD-PCR will transform the capabilities of PCR-based genetic testing, including applications in cancer, infectious diseases and prenatal identification of fetal alleles in maternal blood.

  10. Development and validation of qualitative SYBR®Green real-time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes.

    PubMed

    Barbau-Piednoir, Elodie; Botteldoorn, Nadine; Yde, Marc; Mahillon, Jacques; Roosens, Nancy H

    2013-05-01

    A combination of four qualitative SYBR®Green qPCR screening assays targeting two levels of discrimination: Listeria genus (except Listeria grayi) and Listeria monocytogenes, is presented. These assays have been developed to be run simultaneously using the same polymerase chain reaction (PCR) programme. The paper also proposes a new validation procedure to specifically validate qPCR assays applied to food microbiology according to two guidelines: the ISO 22118 norm and the "Definition of minimum performance requirements for analytical methods of GMO testing". The developed assays target the iap, prs and hlyA genes that belong to or neighbour the virulence cluster of Listeria spp. The selected primers were designed to amplify short fragments (60 to 103 bp) in order to obtain optimal PCR efficiency (between 97 and 107 % efficiency). The limit of detection of the SYBR®Green qPCR assays is two to five copies of target genes per qPCR reaction. These assays are highly accurate (98.08 and 100 % accuracy for the Listeria spp. and L. monocytogenes assays, respectively).

  11. Comparison of PCR-RFLP with allele-specific PCR in genetic testing for spinal muscular atrophy.

    PubMed

    Xu, Ruliang; Ogino, Shuji; Lip, Va; Fang, Hong; Wu, Bai-Lin

    2003-01-01

    PCR-based methods for the detection of homozygous deletion of exon 7 of the SMN1 gene have been widely used in genetic testing for spinal muscular atrophy (SMA). We compared the most commonly used PCRrestriction fragment length polymorphism (PCR-RFLP) assay with an allele-specific PCR method, evaluating their potential application in direct testing, prenatal prediction, and preimplantation diagnosis, in terms of a range of DNA amounts used in such testing. We showed that PCR-RFLP could identify the SMN1 exon 7 by amplifying 10 pg of genomic DNA, and could differentiate SMN1 from SMN2 at the 100-pg DNA level (DraIdigested SMN2 fragments served as an internal control for PCR efficiency). In contrast, allele-specific PCR for SMN1, despite some advantages in a rapid preimplantation diagnosis, quickly lost its specificity when 100 pg of genomic DNA was used. In addition, the absence of a SMN1 fragment at the 10-pg DNA level may be due to a PCR amplification failure, and, thus, it is difficult to interpret without a proper internal control. Our data indicate that PCR-RFLP can be used for most diagnostic purposes, whereas the use of allelespecific PCR may be considered with caution under certain circumstances.

  12. Using multiple PCR and CE with chemiluminescence detection for simultaneous qualitative and quantitative analysis of genetically modified organism.

    PubMed

    Guo, Longhua; Qiu, Bin; Chi, Yuwu; Chen, Guonan

    2008-09-01

    In this paper, an ultrasensitive CE-CL detection system coupled with a novel double-on-column coaxial flow detection interface was developed for the detection of PCR products. A reliable procedure based on this system had been demonstrated for qualitative and quantitative analysis of genetically modified organism-the detection of Roundup Ready Soy (RRS) samples was presented as an example. The promoter, terminator, function and two reference genes of RRS were amplified with multiplex PCR simultaneously. After that, the multiplex PCR products were labeled with acridinium ester at the 5'-terminal through an amino modification and then analyzed by the proposed CE-CL system. Reproducibility of analysis times and peak heights for the CE-CL analysis were determined to be better than 0.91 and 3.07% (RSD, n=15), respectively, for three consecutive days. It was shown that this method could accurately and qualitatively detect RRS standards and the simulative samples. The evaluation in terms of quantitative analysis of RRS provided by this new method was confirmed by comparing our assay results with those of the standard real-time quantitative PCR (RT-QPCR) using SYBR Green I dyes. The results showed a good coherence between the two methods. This approach demonstrated the possibility for accurate qualitative and quantitative detection of GM plants in a single run.

  13. Qualitative Aspects of Group-Only Testing

    ERIC Educational Resources Information Center

    Hanshaw, Larry G.

    2012-01-01

    This study sought to determine how students would describe their group-only cooperative testing experiences in terms of key elements of cooperative learning often cited in the literature. Written comments of 159 graduate students were analyzed and 26 related categories of comments were derived from 495 statements of students enrolled in two…

  14. Qualitative Aspects of Group-Only Testing

    ERIC Educational Resources Information Center

    Hanshaw, Larry G.

    2012-01-01

    This study sought to determine how students would describe their group-only cooperative testing experiences in terms of key elements of cooperative learning often cited in the literature. Written comments of 159 graduate students were analyzed and 26 related categories of comments were derived from 495 statements of students enrolled in two…

  15. Triplet repeat primed PCR simplifies testing for Huntington disease.

    PubMed

    Jama, Mohamed; Millson, Alison; Miller, Christine E; Lyon, Elaine

    2013-03-01

    Diagnostic and predictive testing for Huntington disease (HD) requires an accurate determination of the number of CAG repeats in the Huntingtin (HHT) gene. Currently, when a sample appears to be homozygous for a normal allele, additional testing is required to confirm amplification from both alleles. If the sample still appears homozygous, Southern blot analysis is performed to rule out an undetected expanded HTT allele. Southern blot analysis is expensive, time-consuming, and labor intensive and requires high concentrations of DNA. We have developed a chimeric PCR process to help streamline workflow; true homozygous alleles are easily distinguished by this simplified method, and only very large expanded alleles still require Southern blot analysis. Two hundred forty-six HD samples, previously run with a different fragment analysis method, were analyzed with our new method. All samples were correctly genotyped, resulting in 100% concordance between the methods. The chimeric PCR assay was able to identify expanded alleles up to >150 CAG repeats. This method offers a simple strategy to differentiate normal from expanded CAG alleles, thereby reducing the number of samples reflexed to Southern blot analysis. It also provides assurance that expanded alleles are not routinely missed because of allele dropout.

  16. Clinical Performance of Aspergillus PCR for Testing Serum and Plasma: a Study by the European Aspergillus PCR Initiative.

    PubMed

    White, P Lewis; Barnes, Rosemary A; Springer, Jan; Klingspor, Lena; Cuenca-Estrella, Manuel; Morton, C Oliver; Lagrou, Katrien; Bretagne, Stéphane; Melchers, Willem J G; Mengoli, Carlo; Donnelly, J Peter; Heinz, Werner J; Loeffler, Juergen

    2015-09-01

    Aspergillus PCR testing of serum provides technical simplicity but with potentially reduced sensitivity compared to whole-blood testing. With diseases for which screening to exclude disease represents an optimal strategy, sensitivity is paramount. The associated analytical study confirmed that DNA concentrations were greater in plasma than those in serum. The aim of the current investigation was to confirm analytical findings by comparing the performance of Aspergillus PCR testing of plasma and serum in the clinical setting. Standardized Aspergillus PCR was performed on plasma and serum samples concurrently obtained from hematology patients in a multicenter retrospective anonymous case-control study, with cases diagnosed according to European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) consensus definitions (19 proven/probable cases and 42 controls). Clinical performance and clinical utility (time to positivity) were calculated for both kinds of samples. The sensitivity and specificity for Aspergillus PCR when testing serum were 68.4% and 76.2%, respectively, and for plasma, they were 94.7% and 83.3%, respectively. Eighty-five percent of serum and plasma PCR results were concordant. On average, plasma PCR was positive 16.8 days before diagnosis and was the earliest indicator of infection in 13 cases, combined with other biomarkers in five cases. On average, serum PCR was positive 10.8 days before diagnosis and was the earliest indicator of infection in six cases, combined with other biomarkers in three cases. These results confirm the analytical finding that the sensitivity of Aspergillus PCR using plasma is superior to that using serum. PCR positivity occurs earlier when testing plasma and provides sufficient sensitivity for the screening of invasive aspergillosis while maintaining methodological simplicity.

  17. Qualitative and event-specific real-time PCR detection methods for Bt brinjal event EE-1.

    PubMed

    Randhawa, Gurinder Jit; Sharma, Ruchi; Singh, Monika

    2012-01-01

    Bt brinjal event EE-1 with cry1Ac gene, expressing insecticidal protein against fruit and shoot borer, is the first genetically modified food crop in the pipeline for commercialization in India. Qualitative polymerase chain reaction (PCR) along with event-specific conventional as well as real-time PCR methods to characterize the event EE-1 is reported. A multiplex (pentaplex) PCR system simultaneously amplifying cry1Ac transgene, Cauliflower Mosaic Virus (CaMV) 35S promoter, nopaline synthase (nos) terminator, aminoglycoside adenyltransferase (aadA) marker gene, and a taxon-specific beta-fructosidase gene in event EE-1 has been developed. Furthermore, construct-specific PCR, targeting the approximate 1.8 kb region of inserted gene construct comprising the region of CaMV 35S promoter and cry1Ac gene has also been developed. The LOD of developed EE-1 specific conventional PCR assay is 0.01%. The method performance of the reported real-time PCR assay was consistent with the acceptance criteria of Codex Alimentarius Commission ALINORM 10/33/23, with the LOD and LOQ values of 0.05%. The developed detection methods would not only facilitate effective regulatory compliance for identification of genetic traits, risk assessment, management, and postrelease monitoring, but also address consumer concerns and resolution of legal disputes.

  18. Use of Treponema pallidum PCR in Testing of Ulcers for Diagnosis of Primary Syphilis1

    PubMed Central

    Sednaoui, Patrice; Lautenschlager, Stephan; Ferry, Tristan; Toutous-Trellu, Laurence; Cavassini, Matthias; Yassir, Fatima; Martinez de Tejada, Begoña; Emonet, Stéphane; Combescure, Christophe; Schrenzel, Jacques; Perneger, Thomas

    2015-01-01

    Treponema pallidum PCR (Tp-PCR) has been noted as a valid method for diagnosing syphilis. We compared Tp-PCR to a combination of darkfield microscopy (DFM), the reference method, and serologic testing in a cohort of 273 patients from France and Switzerland and found the diagnostic accuracy of Tp-PCR was higher than that for DFM. PMID:25531672

  19. Detection of human herpesvirus-7 by qualitative nested-PCR: comparison between healthy individuals and liver transplant recipients.

    PubMed

    Thomasini, Ronaldo Luis; Martins, Juliana de Moraes; Parola, Daniela Corte; Bonon, Sandra Helena Alves; Boin, Ilka de Fátima Santana Ferreira; Leonardi, Luis Sérgio; Leonardi, Marília; Costa, Sandra Cecília Botelho

    2008-01-01

    Diagnosis of human herpesvirus-7 active infection in transplant patients has proved difficult, because this virus is ubiquitous and can cause persistent infections in the host. The significance of viral DNA detected in leukocytes by PCR is unclear and cross-reaction in serological tests may occur. This study aimed to evaluate nested-PCR to detect human herpesvirus-7 active infection in liver transplant recipients compared to healthy individuals. human herpesvirus-7 nested-PCR was performed on leukocytes and sera of 53 healthy volunteers and sera of 29 liver transplant recipients. In healthy volunteers, human herpesvirus-7 was detected in 28.3% of leukocytes and 0% of serum. human herpesvirus-7 was detected in sera of 48.2% of the liver transplant recipients. Nested-PCR on DNA extracted from leukocytes detected latent infection and the study suggests that nested-PCR performed on serum could be useful to detect human herpesvirus-7 active infection in liver transplant recipients.

  20. Gender, Sexual Orientation, and Adolescent HIV Testing: A Qualitative Analysis

    PubMed Central

    Siegel, Karolynn; Lekas, Helen-Maria; Olson, Kari; VanDevanter, Nancy

    2010-01-01

    Using qualitative data, this article explored the circumstances leading to HIV testing among 59 HIV-infected adolescents recruited from New York City HIV clinics. Results showed differences between the heterosexual women and the gay and bisexual men. Most of the young women were tested during routine health care or self-initiated tests, and most were asymptomatic when they tested positive. Their testing decisions were sometimes based on assessments of their boyfriends’ risk behaviors, rather than their own. Many males were experiencing symptoms of illness when they tested positive, and about half of these recognized their symptoms as related to HIV and sought tests. Some young men expressed fear of learning about positive test results, which delayed their testing, and some providers did not initially recommend HIV testing for males who presented with symptoms. The article concludes that consideration of these gender and sexual orientation-related concerns can facilitate HIV testing among adolescents. PMID:20303793

  1. Interlaboratory Comparison of Quantitative PCR Test Results for Dehalococcoides

    EPA Science Inventory

    Quantitative PCR (qPCR) techniques have been widely used to measure Dehalococcoides (Dhc) DNA in the groundwater at field sites for several years. Interpretation of these data may be complicated when different laboratories using alternate methods conduct the analysis. An...

  2. Interlaboratory Comparison of Quantitative PCR Test Results for Dehalococcoides

    EPA Science Inventory

    Quantitative PCR (qPCR) techniques have been widely used to measure Dehalococcoides (Dhc) DNA in the groundwater at field sites for several years. Interpretation of these data may be complicated when different laboratories using alternate methods conduct the analysis. An...

  3. Added value of PCR-testing for confirmation of invasive meningococcal disease in England.

    PubMed

    Heinsbroek, Ellen; Ladhani, Shamez; Gray, Steve; Guiver, Malcolm; Kaczmarski, Ed; Borrow, Ray; Ramsay, Mary

    2013-11-01

    In England, national guidance recommends that all patients with suspected invasive meningococcal disease (IMD) should be investigated by blood culture and polymerase chain reaction (PCR) testing. The Meningococcal Reference Unit (MRU) provides a national service for meningococcal species confirmation and PCR-testing of clinical samples. We performed a population-level assessment of the added value of PCR-testing for IMD to augment traditional culture confirmation. We analysed all PCR-samples and invasive meningococcal isolates received by MRU in 2009 and 2010. We assumed that all patients with PCR-samples submitted to MRU also had blood cultures performed and that positive blood cultures were referred to MRU. We confirmed this assertion by case ascertainment. In total, 25,379 specimens from 22,039 patients were submitted for PCR-testing and 1492 (6.8%) tested PCR-positive. MRU received 825 invasive meningococcal isolates; 393 confirmed by PCR and culture, 405 without a PCR-specimen submitted and 27 with a PCR-negative result. Thus, of 1924 reported IMD cases, 1099 (57.1%) were confirmed by PCR only, 432 (22.5%) by culture only and 393 (20.4%) by both tests. More than half of all confirmed IMD cases were confirmed by PCR only, indicating this service ensures high case ascertainment for national surveillance. Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

  4. Analyses quantitatives et qualitatives des tests cloze: syntaxe et semantique (Quantitative and Qualitative Analyses of Cloze Tests: Syntax and Semantics).

    ERIC Educational Resources Information Center

    Connors, Kathleen; Toker, Mia Beer

    1984-01-01

    Linguistic and statistical analyses of cloze tests as a measure of second language competence revealed qualitative and quantitative differences between the performances of Anglophone immersion and French control groups. The diagnostic limitations of the procedure and the usefulness of the linguistic information derived from it are discussed. (MSE)

  5. Predicting Gene Structures from Multiple RT-PCR Tests

    NASA Astrophysics Data System (ADS)

    Kováč, Jakub; Vinař, Tomáš; Brejová, Broňa

    It has been demonstrated that the use of additional information such as ESTs and protein homology can significantly improve accuracy of gene prediction. However, many sources of external information are still being omitted from consideration. Here, we investigate the use of product lengths from RT-PCR experiments in gene finding. We present hardness results and practical algorithms for several variants of the problem and apply our methods to a real RT-PCR data set in the Drosophila genome. We conclude that the use of RT-PCR data can improve the sensitivity of gene prediction and locate novel splicing variants.

  6. [PCR "real time" to analyze the quantitative and qualitative relations microbiota of periodontal pockets].

    PubMed

    Zorina, O A; Kulakov, A A; Boriskina, O A; Rebrikov, D V

    2011-01-01

    The introduction of a broad medical practice PCR "real time" is just beginning and dentistry is no exception. Modern molecular genetic methods provide numerous opportunities for diagnosis, assessment and prediction in patients with inflammatory periodontal diseases. Early and accurate diagnosis can allow in the future reduce the incidence of periodontitis and the progression of its course.

  7. Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing

    PubMed Central

    Espy, M. J.; Uhl, J. R.; Sloan, L. M.; Buckwalter, S. P.; Jones, M. F.; Vetter, E. A.; Yao, J. D. C.; Wengenack, N. L.; Rosenblatt, J. E.; Cockerill, F. R.; Smith, T. F.

    2006-01-01

    Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory. PMID:16418529

  8. Real-time qualitative PCR for 57 human adenovirus types from multiple specimen sources.

    PubMed

    Buckwalter, Seanne P; Teo, Rose; Espy, Mark J; Sloan, Lynne M; Smith, Thomas F; Pritt, Bobbi S

    2012-03-01

    Human adenoviruses (HAdVs) are ubiquitous double-stranded DNA viruses that cause a wide array of diseases in humans including pharyngitis, pneumonia, gastroenteritis, hemorrhagic cystitis, and keratoconjunctivitis. They also cause life-threatening opportunistic infections in immunocompromised individuals and are responsible for outbreaks in certain populations. Diagnosis is traditionally by cell culture or antigen detection methods. However, some HAdVs can take up to 4 weeks to isolate, and diarrheagenic types 40 and 41 will not grow in routine cell culture. Therefore, the goal of this study was to design a rapid, real-time PCR assay to detect all known 57 HAdV types from multiple different specimen sources. Primers and fluorescence resonance energy transfer hybridization probes were designed to target a 185-bp region of the penton base gene of HAdV. The analytical sensitivity was determined to be 10 copies/μl for HAdV types showing exact primer/probe homology in specimen matrix. Using whole-virus strains, the analytical sensitivity for representative HAdV types ranged from 10(-1) to 10(3) 50% tissue culture infective dose (TCID(50))/ml. The assay demonstrated 100% sensitivity and 99% specificity. This real-time PCR assay provides a rapid method for the detection of all 57 known HAdV types from respiratory specimens, blood, stool, urine, and ocular swabs.

  9. Intrapartum detection of Group B streptococci colonization by rapid PCR-test on labor ward.

    PubMed

    Mueller, Martin; Henle, Ariane; Droz, Sara; Kind, Andre B; Rohner, Susanne; Baumann, Marc; Surbek, Daniel

    2014-05-01

    Group B streptococci (GBS) may lead to early onset neonatal sepsis with severe morbidity and mortality of newborns. Intrapartum detection of GBS is needed. The objective was to compare a PCR-based test performed in the laboratory versus labor ward. 300 patients were included prospectively. In phase I, swabs were analyzed by selective culture and rapid PCR in the laboratory. In phase II, swabs were analyzed accordingly, but the PCR test was conducted in labor ward. Test performances were analyzed and compared. In phase I the rapid PCR test had a sensitivity of 85.71% and a specificity of 95.9%. The GBS colonization rate was 18.67%. Overall 8.5% of the PCR results were invalid. In phase II the PCR test showed a sensitivity of 85.71% and a specificity of 95.65%. The GBS colonization rate was 23.3%. Overall 23.5% of swabs tested with PCR were invalid. Initiation of specific, short 2-hour training for operating personnel in the labor ward reduced the invalid test rate to 13.4%. The rapid PCR-based test yields adequate results to identify GBS colonization when performed in labor ward. In order to reduce the number of invalid tests a short training period is needed. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  10. [The development of a test-system for the quantitative and qualitative evaluation of DNA content in criminalistic objects by the real-time polymerase chain reaction].

    PubMed

    Lapenkov, M I; Plakhina, N V; Alekseev, Ia I; Varlamov, D A

    2011-01-01

    An original test-system for the preliminary quantitative and qualitative evaluation of isolated DNA is proposed by the polymerase chain reaction in real time (PCR-RT) based on the TaqMan technology. This test-system permits to simultaneously measure the amount of DNA in the sample, identify the genetic gender, and detect PCR inhibitors. The method has been approbated in the practical work of forensic medical experts.

  11. [Application of droplet digital PCR technology for genetic testing and prenatal diagnosis of spinal muscular atrophy].

    PubMed

    Zou, Yang; Xu, Peiwen; Li, Jie; Huang, Sexin; Gao, Ming; Kang, Ranran; Gao, Xuan; Gao, Yuan

    2016-10-01

    To explore the clinical application of droplet digital PCR (ddPCR) for genetic testing and prenatal diagnosis of spinal muscular atrophy (SMA) with deletion of SMN1 gene exon 7. A total of 138 clinical samples, including 121 peripheral blood, 13 amniotic fluid, 2 umbilical cord blood and 2 chorionic villi from 56 SMA families, were tested by both ddPCR and multiplex ligation-dependent probe amplification (MLPA). Results of the two approaches were analyzed with commercial software QuantaSoft (ddPCR) and Coffalyser (MLPA), respectively. Among the 138 cases, 25 had two copies, 84 had one copy, and 29 had null copy of exon 7 of the SMN1 gene. The results of ddPCR and MLPA were completely consistent. As a rapid, precise and economically efficient method, ddPCR will provide a new choice for genetic testing of SMA.

  12. Why test for tuberculosis? A qualitative study from South Africa

    PubMed Central

    Skinner, D.

    2016-01-01

    Setting: Early testing and treatment initiation are crucial for controlling the tuberculosis (TB) epidemic, especially in high-burden countries such as South Africa. Objective: To explore reasons why patients opted to test for TB and the context in which they were tested. Design: This qualitative study was nested in a larger study evaluating patients who did not initiate anti-tuberculosis treatment after diagnosis. In-depth interviews were conducted with 41 patients across five provinces of South Africa. Results: While most patients presented for testing because of their symptoms, unfortunately many waited until their symptoms were severe and thus remained infectious for longer. Outreach campaigns and TB screening at primary health care facilities were perceived favourably, although some respondents were unclear as to the nature of the tests being performed and had concerns about the implications. Positive health care worker attitudes towards presumptive TB patients contributed towards prompt testing and treatment initiation. Conclusion: As patients often delayed presenting for testing, strategies to engage early with presumptive TB patients so that testing and treatment can commence without delay should be a priority for TB programmes. PMID:28123955

  13. Why test for tuberculosis? A qualitative study from South Africa.

    PubMed

    Skinner, D; Claassens, M

    2016-12-21

    Setting: Early testing and treatment initiation are crucial for controlling the tuberculosis (TB) epidemic, especially in high-burden countries such as South Africa. Objective: To explore reasons why patients opted to test for TB and the context in which they were tested. Design: This qualitative study was nested in a larger study evaluating patients who did not initiate anti-tuberculosis treatment after diagnosis. In-depth interviews were conducted with 41 patients across five provinces of South Africa. Results: While most patients presented for testing because of their symptoms, unfortunately many waited until their symptoms were severe and thus remained infectious for longer. Outreach campaigns and TB screening at primary health care facilities were perceived favourably, although some respondents were unclear as to the nature of the tests being performed and had concerns about the implications. Positive health care worker attitudes towards presumptive TB patients contributed towards prompt testing and treatment initiation. Conclusion: As patients often delayed presenting for testing, strategies to engage early with presumptive TB patients so that testing and treatment can commence without delay should be a priority for TB programmes.

  14. Validation of qualitative test for phosphine gas in human tissues.

    PubMed

    Raina, Anupuma; Shrivastava, H C; Mathur, Nitin; Dogra, T D

    2003-08-01

    Phosphine has been known to science since the birth of modern chemistry. WHO reports that the technical product usually has a foul odour, like "fishy" or "garlicky" because of the presence of substituted phosphines and diphosphine (P2H4). Many medico-legal autopsy cases have been reported positive for aluminium phosphide even though there was neither any suspicion of consuming aluminium phosphide nor any clinical findings, postmortem findings or circumstantial evidences. The present study was carried out to validate the qualitative test, presently applied in many laboratories for testing phosphine. It was observed that 65% of human tissues in saturated solution of common salt show positivity for phosphine gas on the first day of autopsy.

  15. DNA methylation testing and marker validation using PCR: diagnostic applications.

    PubMed

    Egger, Gerda; Wielscher, Matthias; Pulverer, Walter; Kriegner, Albert; Weinhäusel, Andreas

    2012-01-01

    DNA methylation provides a fundamental epigenetic mechanism to establish and promote cell-specific gene-expression patterns, which are inherited by subsequent cell generations. Thus, the epigenome determines the differentiation into a cell lineage but can also program cells to become abnormal or malignant. In humans, different germline and somatic diseases have been linked to faulty DNA methylation. In this article, we will discuss the available PCR-based technologies to assess differences in DNA methylation levels mainly affecting 5-methylcytosine in the CpG dinucleotide context in hereditary syndromal and somatic pathological conditions. We will discuss some of the current diagnostic applications and provide an outlook on how DNA methylation-based biomarkers might provide novel tools for diagnosis, prognosis or patient stratification for diseases such as cancer.

  16. Development and in-house validation of the event-specific qualitative and quantitative PCR detection methods for genetically modified cotton MON15985.

    PubMed

    Jiang, Lingxi; Yang, Litao; Rao, Jun; Guo, Jinchao; Wang, Shu; Liu, Jia; Lee, Seonghun; Zhang, Dabing

    2010-02-01

    To implement genetically modified organism (GMO) labeling regulations, an event-specific analysis method based on the junction sequence between exogenous integration and host genomic DNA has become the preferential approach for GMO identification and quantification. In this study, specific primers and TaqMan probes based on the revealed 5'-end junction sequence of GM cotton MON15985 were designed, and qualitative and quantitative polymerase chain reaction (PCR) assays were established employing the designed primers and probes. In the qualitative PCR assay, the limit of detection (LOD) was 0.5 g kg(-1) in 100 ng total cotton genomic DNA, corresponding to about 17 copies of haploid cotton genomic DNA, and the LOD and limit of quantification (LOQ) for quantitative PCR assay were 10 and 17 copies of haploid cotton genomic DNA, respectively. Furthermore, the developed quantitative PCR assays were validated in-house by five different researchers. Also, five practical samples with known GM contents were quantified using the developed PCR assay in in-house validation, and the bias between the true and quantification values ranged from 2.06% to 12.59%. This study shows that the developed qualitative and quantitative PCR methods are applicable for the identification and quantification of GM cotton MON15985 and its derivates.

  17. Comparison of PCR, Wright agglutination test and blood culture for diagnosis of brucellosis in suspected patients.

    PubMed

    Hekmatimoghaddam, Seyedhosssein; Sadeh, Maryam; Khalili, Mohammad Bagher; Mollaabedin, Mansour; Sazmand, Alireza

    2013-11-15

    Brucellosis has long been prevalent in Iran, with considerable medical and economic importance. Timely diagnosis is needed for early management and effective prevention of its consequences in human beings and animals. Current diagnostic methods impose peculiar challenges in terms of analytical method performance. This study compares diagnostic sensitivity, specificity, predictive Value of Positive (PVP) and Predictive Value of Negative (PVN) for Polymerase Chain Reaction (PCR), Wright agglutination test and blood culture used for patients suspected of brucellosis. In 120 patients clinically suspected of brucellosis and referred by physicians to the Yazd central Medical Laboratory, some relevant demographic, occupational, nutritional and clinical data were collected. Also, venous blood samples were drawn for diagnosis of brucellosis using PCR, Wright agglutination test and blood culture techniques. The most frequent symptom of patients was arthralgia (82 cases, 68.3%). PCR was positive in 25 cases (20.8%), wright test in 21 patients (17.5%) and blood culture in 6 cases (5%). In 20 out of 21 wright-positive cases, PCR was positive and all of the culture-positive patients had positive PCR. Sensitivity, specificity, PVP and PVN of blood culture compared to PCR (as the gold standard test) were 24, 100, 100 and 86%, respectively, but the above parameters when PCR is compared with blood culture (as gold standard) were 100, 83, 24 and 95%, respectively. PCR has better analytical performances than blood culture for diagnosis of brucellosis and is suitable for confirmation of Wright-positive cases.

  18. PCR as a screening test for invasive aspergillosis in haematological patients: a pilot study.

    PubMed

    da Silva, Thomas Victor Maciel; Carneiro, Lilian Carla; Ramos, Francine dos Santos; Baethgen, Ludmila Fiorenzano; Paz, Alessandra Aparecida; Larentis, Daniela Zilio; Daudt, Liane Esteves; Tusset, Cintia; Lopes-Bezerra, Leila Maria; Pasqualotto, Alessandro C

    2014-02-01

    Invasive aspergillosis is a leading cause of morbidity and mortality in immunocompromised patients, particularly in individuals with haematological malignancy and in haematopoietic stem cell transplant recipients. Nowadays, the galactomannan (GM) assay has been widely used as an indication of invasive aspergillosis, even though the test is known to generate false-positive results. The aim of this study was to compare the performance of GM and real-time PCR (qPCR) to detected Aspergillus in blood samples obtained from high-risk haematological patients. Haematological patients were screened twice weekly with GM testing, which was performed by the Platelia ELISA kit. An additional sample of whole blood (4 ml) was obtained for the purpose of qPCR testing. Sixty-four samples from 12 patients with haematopoietic stem cell transplant or haematological malignancy were studied. The overall accordance between GM and qPCR tests was 96.9 % (62 samples). Only two samples showed contradictory results, with positive GM test and negative real-time PCR results. Based on the high concordance between GM and qPCR in terms of negative results, the main utility of qPCR could be in the confirmation of positive results seen with GM testing.

  19. Overnight Enrichment is Essential for Reliable Salmonella PCR Testing from Fecal Samples.

    PubMed

    Wolff, Dietmar; Gerritzen, Andreas

    2017-03-01

    A rapid molecular test for identifying cases of infectious diarrhea might be beneficial for patients. We established a LightMix modular multiplex PCR assay for rapid detection of Campylobacter, Salmonella, Yersinia, and Shigella species from fecal samples. Unlike Campylobacter sp., direct detection of Salmonella from feces by multiplex PCR was significantly less sensitive than culture. Only 11 out of 21 (52%) Salmonella culture positive specimens were positive for Salmonella in the direct multiplex PCR assay (Campylobacter: 52/53 samples [98%]). In contrast, an overnight selenite enrichment step prior to multiplex PCR increased the Salmonella assay sensitivity significantly: 18 out of 18 Salmonella culture positive samples were then also detected by PCR. An overnight enrichment step is necessary for reliable PCR detection of Salmonella from fecal samples.

  20. [Retrospective study of the implementation of the qualitative PCR technique in biological samples for monitoring toxoplasmosis in pediatric patients receiving hematopoietic stem cell transplantation].

    PubMed

    Nigro, Mónica G; Figueroa, Carlos; Ledesma, Bibiana A

    2014-01-01

    Toxoplasmosis is an opportunistic infection caused by the parasite Toxoplasma gondii. The infection is severe and difficult to diagnose in patients receiving allogeneic hematopoietic stem cell transplantation (HSCT). Twelve patients receiving HSCT were monitored post-transplant, by qualitative PCR at the Children's Hospital S.A.M.I.C. "Prof. Dr. Juan P. Garrahan". The monitoring of these patients was defined by a history of positive serology for toxoplasmosis in the donor or recipient and because their hematologic condition did not allow the use of trimethoprim-sulfamethoxazole for prophylaxis. During the patients' monitoring, two of them with positive PCR results showed signs of illness by T. gondii and were treated with pyrimethamine-clindamycin. In two other patients, toxoplasmosis was the cause of death and an autopsy finding, showing negative PCR results. Four patients without clinical manifestations received treatment for toxoplasmosis because of positive PCR detection. In four patients there were no signs of toxoplasmosis disease and negative PCR results during follow-up. The qualitative PCR technique proved useful for the detection of toxoplasmosis reactivation in HSCT recipients, but has limitations in monitoring and making clinical decisions due to the persistence of positive PCR over time and manifestations of toxicity caused by the treatment. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España. All rights reserved.

  1. COLD-PCR: a new platform for highly improved mutation detection in cancer and genetic testing.

    PubMed

    Li, Jin; Makrigiorgos, G Mike

    2009-04-01

    PCR is widely employed as the initial DNA amplification step for genetic testing and cancer biomarker detection. However, a key limitation of PCR-based methods, including real-time PCR, is the inability to selectively amplify low levels of variant alleles in a wild-type allele background. As a result, downstream assays are limited in their ability to identify subtle genetic changes that can have a profound impact on clinical decision-making and outcome or that can serve as cancer biomarkers. We developed COLD-PCR (co-amplification at lower denaturation temperature-PCR) [Li, Wang, Mamon, Kulke, Berbeco and Makrigiorgos (2008) Nat. Med. 14, 579-584], a novel form of PCR that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences irrespective of the mutation type or position on the sequence. Consequently, COLD-PCR amplification from genomic DNA yields PCR products containing high-prevalence variant alleles that can be detected. Since PCR constitutes a ubiquitous initial step for almost all genetic analysis, COLD-PCR provides a general platform to improve the sensitivity of essentially all DNA-variation detection technologies including Sanger sequencing, pyrosequencing, single molecule sequencing, mutation scanning, mutation genotyping or methylation assays. COLD-PCR combined with real-time PCR provides a new approach to boost the capabilities of existing real-time mutation detection methods. We replaced regular PCR with COLD-PCR before sequencing or real-time mutation detection assays to improve mutation detection-sensitivity by up to 100-fold and identified novel p53/Kras/EGFR (epidermal growth factor receptor) mutations in heterogeneous cancer samples that were missed by all existing methods. For clinically relevant micro-deletions, COLD-PCR enabled exclusive amplification and isolation of the mutants. COLD-PCR is expected to have diverse applications in the fields of biomarker identification and tracing, genomic

  2. Identification of Streptococcus pyogenes – Phenotypic Tests vs Molecular Assay (spy1258PCR): A Comparative Study

    PubMed Central

    Abraham, Tintu

    2016-01-01

    Introduction Traditionally Group A Streptococcus pyogenes (GAS) is differentiated from other beta haemolytic streptococci (BHS) by certain presumptive tests such as bacitracin sensitivity and production of Pyrollidonyl Aryl Sulfatase (PYR). The phenotypic and genotypic confirmatory tests are Lancefield grouping for cell wall carbohydrate antigen and PCR for spy1258 gene respectively. Reliance on presumptive tests alone may lead to misidentification of isolates. Aim To compare the predictive values of routine phenotypic tests with spy1258 PCR for the identification of Streptococcus pyogenes. Materials and Methods This comparative analytical study was carried out in the Department of Microbiology, JIPMER, Puducherry, over a period of 18 months (1st November 2013 to 30th April 2015). Two hundred and six consecutive BHS isolates from various clinical samples were subjected to phenotypic tests such as bacitracin sensitivity, PYR test and Lancefield grouping. The results were compared with spy1258 PCR which was considered 95 the confirmatory test for identification. Results The sensitivity and specificity of phenotypic tests were as follows; Susceptibility to bacitracin – 95.42%, 70.96%, PYR test – 95.42%, 77.41%, Lancefield grouping- 97.71%, 80.64%. Conclusion Clinical laboratories should not depend on bacitracin sensitivity as a single presumptive test for the routine identification of GAS but should use supplemental tests such as PYR test or latex agglutination test and for best results use spy1258 PCR. PMID:27630838

  3. Identification of Streptococcus pyogenes - Phenotypic Tests vs Molecular Assay (spy1258PCR): A Comparative Study.

    PubMed

    Abraham, Tintu; Sistla, Sujatha

    2016-07-01

    Traditionally Group A Streptococcus pyogenes (GAS) is differentiated from other beta haemolytic streptococci (BHS) by certain presumptive tests such as bacitracin sensitivity and production of Pyrollidonyl Aryl Sulfatase (PYR). The phenotypic and genotypic confirmatory tests are Lancefield grouping for cell wall carbohydrate antigen and PCR for spy1258 gene respectively. Reliance on presumptive tests alone may lead to misidentification of isolates. To compare the predictive values of routine phenotypic tests with spy1258 PCR for the identification of Streptococcus pyogenes. This comparative analytical study was carried out in the Department of Microbiology, JIPMER, Puducherry, over a period of 18 months (1(st) November 2013 to 30(th) April 2015). Two hundred and six consecutive BHS isolates from various clinical samples were subjected to phenotypic tests such as bacitracin sensitivity, PYR test and Lancefield grouping. The results were compared with spy1258 PCR which was considered 95 the confirmatory test for identification. The sensitivity and specificity of phenotypic tests were as follows; Susceptibility to bacitracin - 95.42%, 70.96%, PYR test - 95.42%, 77.41%, Lancefield grouping- 97.71%, 80.64%. Clinical laboratories should not depend on bacitracin sensitivity as a single presumptive test for the routine identification of GAS but should use supplemental tests such as PYR test or latex agglutination test and for best results use spy1258 PCR.

  4. Species-specific identification of ruminant components contaminating industrial crude porcine heparin using real-time fluorescent qualitative and quantitative PCR.

    PubMed

    Huang, Qing; Xu, Ting; Wang, Gui-Yu; Huang, Jun-Fu; Xia, Han; Yin, Richard; Tang, Angie; Fu, Wei-Ling

    2012-02-01

    Ever since the emergence of bovine spongiform encephalopathy, the source of pharmaceutical heparin has been restricted to porcine intestinal mucosa. In this project, two real-time fluorescent PCR methods were developed to assist with quality control analysis. The first is a qualitative method which relies on SYBR Green I chemistry to confirm the porcine origin of industrial crude porcine heparin (ICPH), identify any ruminant contaminants, and generally control purity. The second is based on TaqMan chemistry and is able to quantitatively identify porcine, bovine, caprine, and ovine components and contaminants in ICPH. By targeting mitochondrial DNA, both PCR systems showed a detection limit of 1 pg DNA and amplification efficiencies ranging between 96% and 102%. Moreover, quantitative PCR showed a detection limit of 0.02 ppm in samples comprising porcine, bovine, caprine, and ovine DNA. The results of qualitative PCR over 27 ICPH samples showed that all samples were porcine in origin and that 17 had ruminant contaminants. The results of quantitative PCR further showed that out of all 17 samples with ruminant contaminants, seven samples had bovine, ovine, and caprine contaminants, two samples had bovine and ovine contaminants, and eight samples had only ovine contaminants. In conclusion, the qualitative PCR system was found to be a relatively inexpensive, rapid, and flexible method of identifying the porcine origin of and ruminant contaminants in ICPH, while the quantitative PCR was found suitable to accurately analyze the components and contaminants in detail. Both methods are suitable for routine control assays for the evaluation of ICPH purity and origins of contaminants.

  5. Rapid identification of HPV 16 and 18 by multiplex nested PCR-immunochromatographic test.

    PubMed

    Kuo, Yung-Bin; Li, Yi-Shuan; Chan, Err-Cheng

    2015-02-01

    Human papillomavirus (HPV) types 16 and 18 are known to be high-risk viruses that cause cervical cancer. An HPV rapid testing kit that could help physicians to make early and more informed decisions regarding patient care is needed urgently but not yet available. This study aimed to develop a multiplex nested polymerase chain reaction-immunochromatographic test (PCR-ICT) for the rapid identification of HPV 16 and 18. A multiplex nested PCR was constructed to amplify the HPV 16 and 18 genotype-specific L1 gene fragments and followed by ICT which coated with antibodies to identify rapidly the different PCR products. The type-specific gene regions of high-risk HPV 16 and 18 could be amplified successfully by multiplex nested PCR at molecular sizes of approximately 99 and 101bp, respectively. The capture antibodies raised specifically against the moleculars labeled on the PCR products could be detected simultaneously both HPV 16 and 18 in one strip. Under optimal conditions, this PCR-ICT assay had the capability to detect HPV in a sample with as low as 100 copies of HPV viral DNA. The PCR-ICT system has the advantage of direct and simultaneous detection of two high-risk HPV 16 and 18 DNA targets in one sample, which suggested a significant potential of this assay for clinical application.

  6. PCR artifact in testing for homologous recombination in genomic editing in zebrafish

    PubMed Central

    Won, Minho

    2017-01-01

    We report a PCR-induced artifact in testing for homologous recombination in zebrafish. We attempted to replace the lnx2a gene with a donor cassette, mediated by a TALEN induced double stranded cut. The donor construct was flanked with homology arms of about 1 kb at the 5’ and 3’ ends. Injected embryos (G0) were raised and outcrossed to wild type fish. A fraction of the progeny appeared to have undergone the desired homologous recombination, as tested by PCR using primer pairs extending from genomic DNA outside the homology region to a site within the donor cassette. However, Southern blots revealed that no recombination had taken place. We conclude that recombination happened during PCR in vitro between the donor integrated elsewhere in the genome and the lnx2a locus. We conclude that PCR alone may be insufficient to verify homologous recombination in genome editing experiments in zebrafish. PMID:28362803

  7. Monitoring Babesia bovis infections in cattle by using PCR-based tests.

    PubMed Central

    Calder, J A; Reddy, G R; Chieves, L; Courtney, C H; Littell, R; Livengood, J R; Norval, R A; Smith, C; Dame, J B

    1996-01-01

    The sensitivity and specificity of PCR tests based on the small-subunit rRNA gene sequence of Babesia bovis were compared in a blind study of experimentally infected cattle with the corresponding parameters of the complement fixation (CF) test currently used in the United States to screen for bovine babesiosis. Cattle were experimentally infected with a single inoculum of a cloned laboratory strain of B. bovis. Blood samples were collected and tested over a period covering from the day of infection to 10 months postinfection. The level of parasitemia (percent infected erythrocytes) present in each sample was estimated from test results and was plotted as a function of time postinfection. These data are the first describing the course of infection by methods capable of detecting parasitemias in the range of 10(-7)%, which frequently occur in the carrier state. Parasitemias in the samples tested strongly influenced the sensitivity and negative predictive value of the PCR-based tests which varied with time postinfection. The average sensitivities of the three PCR-based tests for B. bovis ranged from 58 to 70% for a single determination, while the sensitivity of the CF test was only 6%. Both PCR-based and CF tests for B. bovis had high specificity values ranging from 96 to 100%. PMID:8897177

  8. Specific PCR detection of tiger, leopard, and lion ingredients from test samples.

    PubMed

    Cao, Jijuan; Xu, Junyi; Liu, Ran; Yu, Ke; Wang, Changwen

    2011-01-01

    A PCR method was developed for specific detection of tiger, leopard, and lion DNA from test specimens for inspection and quarantine or for law-enforced animal protection. Three pairs of specific primers were designed based on the mitochondrial cytochrome b gene of tiger, leopard, and lion and used in the PCR testing. To mimic the effect of food processing on the sensitivity of the test, the tiger muscle and bovine bonemeal powder samples were treated at 133 degrees C for 30 min. At this processing condition, the method was sensitive enough to detect as low as 0.05% of tiger-derived ingredients from the mixed bonemeal powders. The data demonstrate that our PCR method is convenient and economic, with high sensitivity and repeatability, and can be used to detect and identify tiger, leopard, and lion ingredients from various test samples.

  9. Comparison of PCR and common clinical tests for the diagnosis of H. pylori in dyspeptic patients.

    PubMed

    Pacheco, N; Mago, V; Gómez, I; Gueneau, P; Guelrud, M; Reyes, N; Pericchi, L R; Domínguez-Bello, M G

    2001-04-01

    Helicobacter pylori has been recognized as a major gastric pathogen. The objective of this study was to assess the diagnostic value of common clinical tests to detect H. pylori infection, by comparison with PCR. Serum and gastric biopsy specimens from 106 dyspeptic patients were examined. Serology was performed with Pyloriset Dry test, and biopsies were examined histologically, for rapid urease activity and PCR amplification of an ureA gene segment of H. pylori. PCR primers were specific for H. pylori and required at least 1.47 pg of H. pylori DNA, corresponding to about 800 bacterial cells. According to serology, histology, rapid urease, and PCR, positive results were respectively found in 56%, 86%, 64%, and 85% of dyspeptic patients, primarily with gastritis. Relative to PCR, the sensitivity (and specificity) was 55% (38%) for serology, 86% (13%) for histology, 70% (69%) for urease. When combining histology and urease, Bayesian analysis of data indicated no advantage of using combined methods over rapid urease test alone. Histology should not any longer be considered a gold standard test for Helicobacter pylori. Urea breath test still seems the first option for non invasive diagnostic. If an invasive diagnostic is justified, highly specific and sensitive molecular methods should be used to examine specimens.

  10. Development of a qualitative real-time PCR method to detect 19 targets for identification of genetically modified organisms.

    PubMed

    Peng, Cheng; Wang, Pengfei; Xu, Xiaoli; Wang, Xiaofu; Wei, Wei; Chen, Xiaoyun; Xu, Junfeng

    2016-01-01

    As the amount of commercially available genetically modified organisms (GMOs) grows recent years, the diversity of target sequences for molecular detection techniques are eagerly needed. Considered as the gold standard for GMO analysis, the real-time PCR technology was optimized to produce a high-throughput GMO screening method. With this method we can detect 19 transgenic targets. The specificity of the assays was demonstrated to be 100 % by the specific amplification of DNA derived from reference material from 20 genetically modified crops and 4 non modified crops. Furthermore, most assays showed a very sensitive detection, reaching the limit of ten copies. The 19 assays are the most frequently used genetic elements present in GM crops and theoretically enable the screening of the known GMO described in Chinese markets. Easy to use, fast and cost efficient, this method approach fits the purpose of GMO testing laboratories.

  11. Clinical characteristics of patients who test positive for Clostridium difficile by repeat PCR.

    PubMed

    Green, Daniel A; Stotler, Brie; Jackman, Dana; Whittier, Susan; Della-Latta, Phyllis

    2014-11-01

    The high sensitivity of PCR assays for diagnosing Clostridium difficile infection (CDI) has greatly reduced the need for repeat testing after a negative result. Nevertheless, a small subset of patients do test positive within 7 days of a negative test. The aim of this study was to evaluate the clinical characteristics of these patients to determine when repeat testing may be appropriate. The results of all Xpert C. difficile PCR (Cepheid, Sunnyvale CA) tests performed in the clinical microbiology laboratory at New York-Presbyterian Hospital, Columbia University Medical Center (NYPH/CUMC) from 1 May 2011 through 6 September 2013, were reviewed. A retrospective case-control study was performed, comparing patients who tested positive within 7 days of a negative test result to a random selection of 50 controls who tested negative within 7 days of a negative test result. During the study period, a total of 14,875 tests were performed, of which 1,066 were repeat tests (7.2%). Eleven of these repeat tests results were positive (1.0%). The only risk factor independently associated with repeat testing positive was history of a prior CDI (odds ratio [OR], 19.6 [95% confidence interval {CI}, 4.0 to 19.5], P < 0.001). We found that patients who test positive for C. difficile by PCR within 7 days of a negative test are more likely to have a history of CDI than are patients who test negative with repeat PCR. This finding may be due to the high rate of disease relapse or the increased likelihood of empirical therapy leading to false-negative results in these patients.

  12. [Selection of suitable polypropylene tubes for DNA testing using real-time PCR].

    PubMed

    Shimizu, Eri; Futo, Satoshi; Masubuchi, Tomoko; Minegishi, Yasutaka; Kasahara, Masaki; Akiyama, Hiroshi; Teshima, Reiko; Hino, Akihiro; Mano, Jyunichi; Furui, Satoshi; Kitta, Kazumi

    2010-01-01

    Polypropylene microtubes (tubes) are generally used for bio-material tests in addition to PCR tests such as genetically modified organism (GMO) testings. However, the choice of suitable tubes is quite important, because it might influence the results: DNA binding and/or elution of chemical substances sometimes occurs. In this study, we established methods to select tubes with the most suitable characteristics for DNA testing.

  13. Development of a qualitative, multiplex real-time PCR kit for screening of genetically modified organisms (GMOs).

    PubMed

    Dörries, Hans-Henno; Remus, Ivonne; Grönewald, Astrid; Grönewald, Cordt; Berghof-Jäger, Kornelia

    2010-03-01

    The number of commercially available genetically modified organisms (GMOs) and therefore the diversity of possible target sequences for molecular detection techniques are constantly increasing. As a result, GMO laboratories and the food production industry currently are forced to apply many different methods to reliably test raw material and complex processed food products. Screening methods have become more and more relevant to minimize the analytical effort and to make a preselection for further analysis (e.g., specific identification or quantification of the GMO). A multiplex real-time PCR kit was developed to detect the 35S promoter of the cauliflower mosaic virus, the terminator of the nopaline synthase gene of Agrobacterium tumefaciens, the 35S promoter from the figwort mosaic virus, and the bar gene of the soil bacterium Streptomyces hygroscopicus as the most widely used sequences in GMOs. The kit contains a second assay for the detection of plant-derived DNA to control the quality of the often processed and refined sample material. Additionally, the plant-specific assay comprises a homologous internal amplification control for inhibition control. The determined limits of detection for the five assays were 10 target copies/reaction. No amplification products were observed with DNAs of 26 bacterial species, 25 yeasts, 13 molds, and 41 not genetically modified plants. The specificity of the assays was further demonstrated to be 100% by the specific amplification of DNA derived from reference material from 22 genetically modified crops. The applicability of the kit in routine laboratory use was verified by testing of 50 spiked and unspiked food products. The herein described kit represents a simple and sensitive GMO screening method for the reliable detection of multiple GMO-specific target sequences in a multiplex real-time PCR reaction.

  14. A point-of-care PCR test for HIV-1 detection in resource-limited settings.

    PubMed

    Jangam, Sujit R; Agarwal, Abhishek K; Sur, Kunal; Kelso, David M

    2013-04-15

    A low-cost, fully integrated sample-to-answer, quantitative PCR (qPCR) system that can be used for detection of HIV-1 proviral DNA in infants at the point-of-care in resource-limited settings has been developed and tested. The system is based on a novel DNA extraction method, which uses a glass fiber membrane, a disposable assay card that includes on-board reagent storage, provisions for thermal cycling and fluorescence detection, and a battery-operated portable analyzer. The system is capable of automated PCR mix assembly using a novel reagent delivery system and performing qPCR. HIV-1 and internal control targets are detected using two spectrally separated fluorophores, FAM and Quasar 670. In this report, a proof-of-concept of the platform is demonstrated. Initial results with whole blood demonstrate that the test is capable of detecting HIV-1 in blood samples containing greater than 5000 copies of HIV-1. In resource-limited settings, a point-of-care HIV-1 qPCR test would greatly increase the number of test results that reach the infants caregivers, allowing them to pursue anti-retroviral therapy.

  15. Diagnosis of periprosthetic joint infection using alpha-defensin test or multiplex-PCR: ideal diagnostic test still not found.

    PubMed

    Suda, Arnold J; Tinelli, Marco; Beisemann, Nils D; Weil, Yoram; Khoury, Amal; Bischel, Oliver E

    2017-07-01

    Diagnosing periprosthetic infection remains a challenge. Multiplex-PCR and biomarkers such as alpha-defensin are potentially useful and fast methods for detecting periprosthetic infection. This study compared these new methods with clinical assessment, conventional microbiological methods and histo-pathological examination. Twenty-eight consecutive patients with 30 joints and a mean age of 67.7 years (range 39 to 88) with removal of total hip arthroplasty (THA) or total knee replacement (TKR) were included in this study. Patients were classified according to the modified Musculoskeletal Infection Society score (MSIS) for infected joints. Punction fluid and tissue specimens were taken for conventional microbiological examination, alphadefensin test was performed, a synovial membrane specimen was used for multiplex-PCR and histopathological examination was carried out. The alpha-defensin test and multiplex-PCR showed a sensitivity of 76.9 vs. 30.8% and a specificity of 82.4 vs. 100%, respectively. We found a significant difference between the positive and negative results (p = 0.0023). The conventional microbiological methods were not significantly different from the alpha-defensin test (p = 0.244) with a sensitivity of 84.6% and a specificity of 100% but did differ significantly from the multiplex PCR (p = 0.0030). There was a significant difference between modified MSIS classification and multiplex PCR (p = 0.0007). Neither alpha-defensin test nor multiplex-PCR could detect periprosthetic infection immediately and reliably. Multiplex-PCR was suitable for detecting the non-infected but not the truly infected. Alpha-defensin test was helpful but showed no satisfactory results. Conventional microbiological methods remain the most reliable for periprosthetic infection diagnosis.

  16. False-Positive Results after Environmental Pinworm PCR Testing due to Rhabditid Nematodes in Corncob Bedding

    PubMed Central

    Leblanc, Mathias; Berry, Kristina; Graciano, Sandy; Becker, Brandon; Reuter, Jon D

    2014-01-01

    Modern rodent colonies are housed in individually ventilated cages to protect the animals from contamination with adventitious pathogens. Standard health monitoring through soiled-bedding sentinels does not always detect infections, especially in the context of low pathogen prevalence. Recently proposed alternatives include analyzing environmental samples from the cages or rack exhaust by PCR to improve the detection of rodent pathogens but optimal sampling strategies have not yet been established for different microorganisms. Although generally very sensitive and specific, these molecular assays are not foolproof and subject to false-positive and –negative results and should always be interpreted cautiously with an overall understanding of the intrinsic controls and all the variables that may affect the results. Here, we report a limited Aspiculuris tetraptera outbreak in a mouse barrier facility that was detected by fecal PCR in sentinels and confirmed by fecal flotation and direct cecal examination of both sentinels and colony animals. The outbreak led to a widespread survey of all facilities for pinworms by using environmental PCR from ventilated rack exhaust plenums. Environmental PCR suggested an unexpected widespread contamination of all ventilated racks holding nonautoclaved cages, but results could not be confirmed in sentinel or colony animals by fecal flotation, cecal and colonic examination, or cage PCR testing. After additional investigation, the unexpected environmental PCR results were confirmed as false-positive findings due to the nonspecificity of the assay, leading to the amplification of rhabditid nematodes, which are not infectious in rodents but which contaminated the corncob bedding. PMID:25650980

  17. False-positive results after environmental pinworm PCR testing due to Rhabditid nematodes in Corncob bedding.

    PubMed

    Leblanc, Mathias; Berry, Kristina; Graciano, Sandy; Becker, Brandon; Reuter, Jon D

    2014-11-01

    Modern rodent colonies are housed in individually ventilated cages to protect the animals from contamination with adventitious pathogens. Standard health monitoring through soiled-bedding sentinels does not always detect infections, especially in the context of low pathogen prevalence. Recently proposed alternatives include analyzing environmental samples from the cages or rack exhaust by PCR to improve the detection of rodent pathogens but optimal sampling strategies have not yet been established for different microorganisms. Although generally very sensitive and specific, these molecular assays are not foolproof and subject to false-positive and -negative results and should always be interpreted cautiously with an overall understanding of the intrinsic controls and all the variables that may affect the results. Here, we report a limited Aspiculuris tetraptera outbreak in a mouse barrier facility that was detected by fecal PCR in sentinels and confirmed by fecal flotation and direct cecal examination of both sentinels and colony animals. The outbreak led to a widespread survey of all facilities for pinworms by using environmental PCR from ventilated rack exhaust plenums. Environmental PCR suggested an unexpected widespread contamination of all ventilated racks holding nonautoclaved cages, but results could not be confirmed in sentinel or colony animals by fecal flotation, cecal and colonic examination, or cage PCR testing. After additional investigation, the unexpected environmental PCR results were confirmed as false-positive findings due to the nonspecificity of the assay, leading to the amplification of rhabditid nematodes, which are not infectious in rodents but which contaminated the corncob bedding.

  18. Event-specific qualitative and quantitative PCR detection of the GMO carnation (Dianthus caryophyllus) variety Moonlite based upon the 5'-transgene integration sequence.

    PubMed

    Li, P; Jia, J W; Jiang, L X; Zhu, H; Bai, L; Wang, J B; Tang, X M; Pan, A H

    2012-04-27

    To ensure the implementation of genetically modified organism (GMO)-labeling regulations, an event-specific detection method was developed based on the junction sequence of an exogenous integrant in the transgenic carnation variety Moonlite. The 5'-transgene integration sequence was isolated by thermal asymmetric interlaced PCR. Based upon the 5'-transgene integration sequence, the event-specific primers and TaqMan probe were designed to amplify the fragments, which spanned the exogenous DNA and carnation genomic DNA. Qualitative and quantitative PCR assays were developed employing the designed primers and probe. The detection limit of the qualitative PCR assay was 0.05% for Moonlite in 100 ng total carnation genomic DNA, corresponding to about 79 copies of the carnation haploid genome; the limit of detection and quantification of the quantitative PCR assay were estimated to be 38 and 190 copies of haploid carnation genomic DNA, respectively. Carnation samples with different contents of genetically modified components were quantified and the bias between the observed and true values of three samples were lower than the acceptance criterion (<25%) of the GMO detection method. These results indicated that these event-specific methods would be useful for the identification and quantification of the GMO carnation Moonlite.

  19. Comparison of PCR with other tests for early diagnosis of canine ehrlichiosis.

    PubMed Central

    Iqbal, Z; Chaichanasiriwithaya, W; Rikihisa, Y

    1994-01-01

    The purpose of the study was to compare the sensitivity of PCR with those of cell culture reisolation of Ehrlichia canis, the indirect fluorescent antibody test (IFA), and Western immunoblotting (WI) in the early diagnosis of canine ehrlichiosis. Five German shepherd dogs were intravenously inoculated with 10(7) E. canis-infected DH82 cells. Blood was collected on alternate days during a 2-week postinoculation period. Mononuclear cell fractions were harvested and used for E. canis reisolation and DNA extraction for PCR. The plasma was used for assaying antibodies against E. canis. By PCR, the 16S rRNA gene of E. canis was detected in the mononuclear cell specimens collected as early as day 4 to 10 postexposure (PE). E. canis was reisolated from the blood starting on day 2 PE from all five dogs. The indirect fluorescent antibody test and Western immunoblotting could detect E. canis antibodies as early as 2 to 8 days PE. Cell culture reisolation proved to be the most sensitive and definitive for early diagnosis of ehrlichiosis, but it is not very convenient, since it takes a long time (14 to 34 days) to show up positive. The sensitivity of PCR is comparable to or slightly less than that of other established methods; however, the convenience, quickness, and direct nature of detecting E. canis DNA is expected to make PCR more useful for clinical diagnosis. Images PMID:7929754

  20. Qualitative research on point-of-care testing strategies and programs for HIV.

    PubMed

    Engel, Nora; Pant Pai, Nitika

    2015-01-01

    Point-of-care (POC) testing in communities, home settings and primary healthcare centers plays an important role in cutting delays in HIV diagnosis and in the uptake of voluntary testing and counseling. Qualitative research methods have important potential to overcome the current challenges in expanding HIV POC testing programs and strategies, by examining the diagnostic processes, complex inter-relationships and patterns involved in making POC diagnostics work in real-world settings. This article reviews existing qualitative studies on POC testing strategies and programs for HIV. Qualitative research on POC diagnostics around the uptake of POC tests, the actual diagnostic and testing processes involved, the influence of POC tests on clinical decision-making, communication of decisions and decisions exercised by patients are limited. Equally limited are studies that explore adaptation of POC programs to various socio-cultural contexts. More qualitative research is needed to inform test developers, funders and policymakers.

  1. A proficiency testing method for detecting antibodies against Brucella abortus in quantitative and qualitative serological tests.

    PubMed

    Gall, D; Nielsen, K; Nicola, A; Renteria, T

    2008-12-01

    A proficiency testing panel for detecting antibodies against Brucella abortus was developed and evaluated by both primary binding and conventional serological tests, using the guidelines of the World Organisation for Animal Health and the International Organization for Standardization Guide 43-1. All serological tests were judged satisfactory. Among the primary binding tests, the competitive enzyme-linked immunosorbent assay (ELISA 2) and the indirect enzyme-linked immunosorbent assay (ELISA 1), with standard deviation indices (z-scores) of -0.06 and 0.10, respectively, performed best. Similarly, E(n) numbers (i.e. a way of comparing different measurements of performance) of 0 for both the competitive ELISA 2 and the indirect ELISA 1 indicated that these tests performed best in the initial round of proficiency testing. The conventional serological tests all passed the panel. Comparing data from both the quantitative and qualitative tests demonstrated that this proficiency testing scheme was fit for the purpose for which it was designed.

  2. Generic RT-PCR tests for detection and identification of tospoviruses.

    PubMed

    Hassani-Mehraban, A; Westenberg, M; Verhoeven, J T J; van de Vossenberg, B T L H; Kormelink, R; Roenhorst, J W

    2016-07-01

    A set of tests for generic detection and identification of tospoviruses has been developed. Based on a multiple sequence alignment of the nucleocapsid gene and its 5' upstream untranslated region sequence from 28 different species, primers were designed for RT-PCR detection of tospoviruses from all recognized clades, i.e. the American, Asian and Eurasian clades, and from the small group of distinct and floating species. Pilot experiments on isolates from twenty different species showed that the designed primer sets successfully detected all species by RT-PCR, as confirmed by nucleotide sequence analysis of the amplicons. In a final optimized design, the primers were applied in a setting of five RT-PCR tests. Seven different tospoviruses were successfully identified from diagnostic samples and in addition a non-described tospovirus species from alstroemeria plants. The results demonstrate that the newly developed generic RT-PCR tests provide a relevant tool for broad detection and identification of tospoviruses in plant quarantine and diagnostic laboratories.

  3. Comparison of QIAGEN automated nucleic acid extraction methods for CMV quantitative PCR testing.

    PubMed

    Miller, Steve; Seet, Henrietta; Khan, Yasmeen; Wright, Carolyn; Nadarajah, Rohan

    2010-04-01

    We examined the effect of nucleic acid extraction methods on the analytic characteristics of a quantitative polymerase chain reaction (PCR) assay for cytomegalovirus (CMV). Human serum samples were extracted with 2 automated instruments (BioRobot EZ1 and QIAsymphony SP, QIAGEN, Valencia, CA) and CMV PCR results compared with those of pp65 antigenemia testing. Both extraction methods yielded results that were comparably linear and precise, whereas the QIAsymphony SP had a slightly lower limit of detection (1.92 log(10) copies/mL vs 2.26 log(10) copies/mL). In both cases, PCR was more sensitive than CMV antigen detection, detecting CMV viremia in 12% (EZ1) and 21% (QIAsymphony) of antigen-negative specimens. This study demonstrates the feasibility of using 2 different extraction techniques to yield results within 0.5 log(10) copies/mL of the mean value, a level that would allow for clinical comparison between different laboratory assays.

  4. Evaluation of a PCR Test for Detection of Treponema pallidum in Swabs and Blood

    PubMed Central

    Grange, P. A.; Gressier, L.; Dion, P. L.; Farhi, D.; Benhaddou, N.; Gerhardt, P.; Morini, J. P.; Deleuze, J.; Pantoja, C.; Bianchi, A.; Lassau, F.; Avril, M. F.; Janier, M.

    2012-01-01

    Syphilis diagnosis is based on clinical observation, serological analysis, and dark-field microscopy (DFM) detection of Treponema pallidum subsp. pallidum, the etiological agent of syphilis, in skin ulcers. We performed a nested PCR (nPCR) assay specifically amplifying the tpp47 gene of T. pallidum from swab and blood specimens. We studied a cohort of 294 patients with suspected syphilis and 35 healthy volunteers. Eighty-seven of the 294 patients had primary syphilis, 103 had secondary syphilis, 40 had latent syphilis, and 64 were found not to have syphilis. The T. pallidum nPCR results for swab specimens were highly concordant with syphilis diagnosis, with a sensitivity of 82% and a specificity of 95%. Reasonable agreement was observed between the results obtained with the nPCR and DFM methods (kappa = 0.53). No agreement was found between the nPCR detection of T. pallidum in blood and the diagnosis of syphilis, with sensitivities of 29, 18, 14.7, and 24% and specificities of 96, 92, 93, and 97% for peripheral blood mononuclear cell (PBMC), plasma, serum, and whole-blood fractions, respectively. HIV status did not affect the frequency of T. pallidum detection in any of the specimens tested. Swab specimens from mucosal or skin lesions seemed to be more useful than blood for the efficient detection of the T. pallidum genome and, thus, for the diagnosis of syphilis. PMID:22219306

  5. [Usefulness of Anti-HCV ELISA Test and HCV Reverse Transcriptase-PCR for the Diagnosis of Hepatits C Viral Infection.].

    PubMed

    Kim, Myeong Hee; Lee, Hee Joo; Park, Su Yon; Lee, Youn Sik; Suh, Jin Tae

    2006-12-01

    The diagnosis of hepatitis C virus (HCV) infection is screened by anti-HCV enzymelinked immunosorbant assay (ELISA) and confirmed by recombinant immunoblotting assay (RIBA) or HCV RT-PCR. We attempted to evaluate the results between anti-HCV ELISA and a qualitative HCV RT-PCR. Four hundred and twenty patients who were tested with anti-HCV ELISA and HCV RTPCR, simultaneously, from January 2002 to June 2005 were enrolled in this study. Anti-HCV ELISA was performed by AxSYM HCV version 3.0 (Abbott Laboratories, USA). HCV RT-PCR was performed using in-house RT-nested PCR methods from January 2002 to October 2004 and HCV Genotype Amplification Kit (LiPA) (Bayer Healthcare, USA) from November 2004 to June 2005. Of the 420 patients tested, 321 were positive for anti-HCV ELISA, and 204 were positive for RT-PCR. The positive predictability of anti-HCV ELISA was 63.6%. Among anti-HCV positive patients, RT-PCR was positive in 7.3% of the patients with sample/cut-off (S/CO)<6, compared with 82.8% of the patients with S/CO>/=6. Among the 117 patients with positive anti-HCV, but with negative HCV RT-PCR, 64 had liver diseases such as chronic hepatitis C, chronic hepatitis B, or hepatocellular carcinoma. Twelve patients showed positive HCV RT-PCR, but negative anti-HCV results; of these 9 had hepatic dysfunction. In the patients who were positive for anti-HCV ELISA with a low S/CO, HCV RT-PCR positivity was shown in a low proportion. Therefore, in such cases, the results should be confirmed by RIBA or HCV RT-PCR. The liver function test showed increased levels of hepatic enzymes in patients with positive HCV RT-PCR, but negative anti-HCV. Such findings correlate to an early phase of chronic hepatitis C, suggesting the necessity of continuous follow up.

  6. An Improved Flame Test for Qualitative Analysis Using a Multichannel UV-Visible Spectrophotometer

    ERIC Educational Resources Information Center

    Blitz, Jonathan P.; Sheeran, Daniel J.; Becker, Thomas L.

    2006-01-01

    Qualitative analysis schemes are used in undergraduate laboratory settings as a way to introduce equilibrium concepts and logical thinking. The main component of all qualitative analysis schemes is a flame test, as the color of light emitted from certain elements is distinctive and a flame photometer or spectrophotometer in each laboratory is…

  7. An Improved Flame Test for Qualitative Analysis Using a Multichannel UV-Visible Spectrophotometer

    ERIC Educational Resources Information Center

    Blitz, Jonathan P.; Sheeran, Daniel J.; Becker, Thomas L.

    2006-01-01

    Qualitative analysis schemes are used in undergraduate laboratory settings as a way to introduce equilibrium concepts and logical thinking. The main component of all qualitative analysis schemes is a flame test, as the color of light emitted from certain elements is distinctive and a flame photometer or spectrophotometer in each laboratory is…

  8. Statistical tests for comparison of quantitative and qualitative models developed with near infrared spectral data

    NASA Astrophysics Data System (ADS)

    Roggo, Y.; Duponchel, L.; Ruckebusch, C.; Huvenne, J.-P.

    2003-06-01

    Near-infrared spectroscopy (NIRS) has been applied for both qualitative and quantitative evaluation of sugar beet. However, chemometrics methods are numerous and a choice criterion is sometime difficult to determine. In order to select the most accurate chemometrics method, statistical tests are developed. In the first part, quantitative models, which predict sucrose content of sugar beet, are compared. To realize a systematic study, 54 models are developed with different spectral pre-treatments (Standard Normal Variate (SNV), Detrending (D), first and second Derivative), different spectral ranges and different regression methods (Principal Component Regression (PCR), Partial Least Squares (PLS), Modified PLS (MPLS)). Analyze of variance and Fisher's tests are computed to compare respectively bias and Standard Error of Prediction Corrected for bias (SEP(C)). The model developed with full spectra pre-treated by SNV, second derivative and MPLS methods gives accurate results: bias is 0.008 and SEP(C) is 0.097 g of sucrose per 100 g of sample on a concentration range between 14 and 21 g/100 g. In the second part, McNemar's test is applied to compare the classification methods. The classifications are used with two data sets: the first data set concerns the disease resistance of sugar beet and the second deals with spectral differences between four spectrometers. The performances of four well-known classification methods are compared on the NIRS data: Linear Discriminant Analysis (LDA), K Nearest Neighbors method (KNN), Simple Modeling of Class Analogy (SIMCA) and Learning Vector Quantization neural network (LVQ) are computed. In this study, the most accurate method (SIMCA) has a prediction rate of 81.9% of good classification on the disease resistance determination and has 99.4% of good classification on the instrument data set.

  9. Sturgeon nucleo-cytoplasmic large DNA virus phylogeny and PCR tests.

    PubMed

    Clouthier, Sharon C; VanWalleghem, Elissa; Anderson, Eric D

    2015-12-09

    Sturgeon epitheliotropic nucleo-cytoplasmic large DNA viruses (NCLDVs) can cause a lethal disease of the integumentary system. These viruses have not been assigned to any currently recognized family or genus. In this study, phylogenetic analyses using the major capsid protein (MCP) showed that the sturgeon NCLDVs formed a cohesive taxonomic group, could be identified to the species or possibly sub-species level and formed a distinct evolutionary lineage within the Megavirales. The genetic relatedness of the sturgeon virus MCP allowed design of 3 PCR diagnostic tests with analytical specificity (ASp) inclusive of this group of viruses. The conventional PCR test, C1, had broader ASp than the 2 quantitative PCR tests, Q1 and Q2, and was inclusive of the sturgeon viruses as well as some viruses belonging to the families Mimi-, Phycodna-, or Iridoviridae. Q2 had broader specificity than Q1 but both tests recognized the sturgeon NCLDVs and did not cross-react with co-localizing sturgeon herpesviruses. Analytical test performance characteristics evaluated for Q1 and Q2 revealed sensitive assays with observed 50% limits of detection between 3 and 6.25 plasmid copies and high intra- and inter-assay repeatability. Q1 was used to test for sturgeon viruses in endangered populations of lake sturgeon Acipenser fulvescens within the Winnipeg River or Nelson River drainage systems of Manitoba, Canada. Test results indicated that namao virus is endemic in the Nelson River water basin. These tests meet the analytical requirements for diagnostic testing in Canada and are useful tools for disease management in sturgeon conservation stocking programs in North America.

  10. Optimisation of forensic genetics procedures used in disputed paternity testing: adjustment of the PCR reaction volume.

    PubMed

    Marjanović, Damir; Bakal, Narcisa; Kovacević, Lejla; Hodzić, Melisa; Haverić, Anja; Haverić, Sanin; Ibrulj, Slavica; Durmić, Adaleta

    2006-05-01

    Standard molecular techniques, with only a slight modification, are very useful in obtaining and interpreting the final results in the field of forensic genetic. Data obtained through such analysis are highly reliable and can be used as a very powerful tool that produces valuable results. However, success and swiftness of DNA typing of biological evidence either that found at a crime scene or used in disputed paternity testing, depends on the optimization of numerous factors. One of the most important and critical phases that ensures reliability of the whole procedure is the choice of the most suitable volume for the amplification protocol. Buccal swabs were collected from volunteers. DNA was extracted by Qiagen Dnaeasy Tissue Kit. PowerPlex 16 kit was used to simultaneously amplify 15 STR loci by PCR. Amplification was carried out as described previously. The tested total working reaction volumes were 5, 10 and 25 microl. The PCR amplification was carried out in PE Gene Amp PCR System Thermal Cycler (ABI, Foster City, CA). Amplification products were analyzed on an ABI PRISM 377 instrument (ABI, Foster City, CA) in 5% bis-acrilamide gel. Amplification was generally successful for all the tested reaction volumes. Lower partial to complete DNA profiles ratio, the quality of obtained STR profiles, significantly reduced amount of reaction's components give advantage to 5 microl reaction volume over other two tested volumes in this case.

  11. Detection limits of the strip test and PCR for genetically modified corn in Brazil.

    PubMed

    Nascimento, V E; Von Pinho, É V R; Von Pinho, R G; do Nascimento, A D

    2012-08-16

    Brazilian legislation establishes a labeling limit for products that contain more than 1% material from genetically modified organisms (GMOs). We assessed the sensitivity of the lateral flow strip test in detection of the GMO corn varieties Bt11 and MON810 and the specificity and sensitivity of PCR techniques for their detection. For the strip test, the GMO seeds were mixed with conventional seeds at levels of 0.2, 0.4 and 0.8% for Bt11, and 0.4, 0.8 and 1.6% for MON810. Three different methodologies were assessed and whole seeds, their endosperm and embryonic axis were used. For the PCR technique, the GMO seeds of each of the two varieties were mixed with conventional seeds at levels of 20, 10, 5, 2, 1, and 0.5%. The seeds were ground and the DNA extracted. For detection of the GMO material, specific primers were used for MON810 and Bt11 and maize zein as an endogenous control. The sensitivity of the strip test varied for both maize varieties and methodologies. The test was positive for Bt11 only at 0.8%, in contrast with the detection limit of 0.4% indicated by the manufacturer. In the multiplex PCR, the primers proved to be specific for the different varieties. These varieties were detected in samples with one GMO seed in 100. Thus, this technique proved to be efficient in detecting contaminations equal to or greater than 1%.

  12. Accuracy of malaria diagnosis by microscopy, rapid diagnostic test, and PCR methods and evidence of antimalarial overprescription in non-severe febrile patients in two Tanzanian hospitals.

    PubMed

    Nicastri, Emanuele; Bevilacqua, Nazario; Sañé Schepisi, Monica; Paglia, Maria G; Meschi, Silvia; Ame, Shaali M; Mohamed, Jape A; Mangi, Sabina; Fumakule, Robert; Di Caro, Antonino; Capobianchi, Maria R; Kitua, Andrew; Molteni, Fabrizio; Racalbuto, Vincenzo; Ippolito, Giuseppe

    2009-05-01

    The study was aimed to evaluate the malaria over/underdiagnosis and over/underprescription of antimalarial drugs. Between February and March 2007 blood samples were collected from 336 non-severe febrile outpatients attended in two peripheral Tanzanian hospitals. Microscopy and a rapid diagnostic test (RDT) were done locally and the accuracy evaluated by qualitative polymerase chain reaction (PCR) for Plasmodium spp. The testing was performed at National Institute for Infectious Diseases Lazzaro Spallanzani (INMI), Rome, Italy. As a result of PCR, we identified 26 malaria cases out of 336 (7.7%) patients. Microscopy and RDT accuracies were 93.5% and 97.6%, respectively. Overprescription and underdiagnosis rates were 29.3% and 30.8%, respectively. On-field training, clinical management of febrile illness, and malaria microscopy in remote settings should be considered.

  13. Testing the feasibility of DNA typing for human identification by PCR and an oligonucleotide ligation assay

    SciTech Connect

    Delahunty, C.; Ankener, W.; Deng, Qiang

    1996-06-01

    The use of DNA typing in human genome analysis is increasing and finding widespread application in the area of forensic and paternity testing. In this report, we explore the feasibility of typing single nucleotide polymorphisms (SNPs) by using a semiautomated method for analyzing human DNA samples. In this approach, PCR is used to amplify segments of human DNA containing a common SNP. Allelic nucleotides in the amplified product are then typed by a calorimetric implementation of the oligonucleotide ligation assay (OLA). The results of the combined assay, PCR/OLA, are read directly by a spectrophotometer; the absorbances are compiled and the genotypes are automatically determined. A panel of 20 markers has been developed for DNA typing and has been tested using a sample panel from the CEPH pedigrees (CEPH parents). The results of this typing, as well as the potential to apply this method to larger populations, are discussed. 62 refs., 2 figs., 4 tabs.

  14. Testing the feasibility of DNA typing for human identification by PCR and an oligonucleotide ligation assay.

    PubMed Central

    Delahunty, C.; Ankener, W.; Deng, Q.; Eng, J.; Nickerson, D. A.

    1996-01-01

    The use of DNA typing in human genome analysis is increasing and finding widespread application in the area of forensic and paternity testing. In this report, we explore the feasibility of typing single nucleotide polymorphisms (SNPs) by using a semiautomated method for analyzing human DNA samples. In this approach, PCR is used to amplify segments of human DNA containing a common SNP. Allelic nucleotides in the amplified product are then typed by a colorimetric implementation of the oligonucleotide ligation assay (OLA). The results of the combined assay, PCR/OLA, are read directly by a spectrophotometer; the absorbances are compiled; and the genotypes are automatically determined. A panel of 20 markers has been developed for DNA typing and has been tested using a sample panel from the CEPH pedigrees (CEPH parents). The results of this typing, as well as the potential to apply this method to larger populations, are discussed. Images Figure 2 PMID:8651301

  15. Trypanosoma evansi: A comparison of PCR and parasitological diagnostic tests in experimentally infected mice.

    PubMed

    Fernández, D; González-Baradat, B; Eleizalde, M; González-Marcano, E; Perrone, T; Mendoza, M

    2009-01-01

    Trypanosoma evansi is the causative agent of equine trypanosomosis, disease that affects horse's productivity and health. Parasitological and molecular methods are mostly used to detect the infection. The aim of this work was evaluate PCR sensitivity to detect T. evansi using the primers 21/22-mer, ITS1, ESAG 6/7 and TBR 1/2 designed from repetitive (multicopies) genomic sequences. The results were compare with two parasitological tests in mice, micro-haematocrite centrifugation technique and direct microscopic examination. The results shows (a) that the minimum amount of DNA from blood of highly parasitaemic mice that was detectable by PCR was 0.001 ng, using the ESAG6/7 and TBR1/2 primer, (b) using TBR1/2 primer for parasites purified could detect 0.000001 ng and (c) in the prepatent period PCR detect the presence of parasites earlier than parasitological techniques. Nevertheless, the percentage of detection for PCR varies depending on primer employed with 60% and 66% for ITS1 and 21/22-mer, and 80% for ESAG6/7 and TBR1/2. Consequently, TBR1/2 and ESAG6/7 were the best primers to monitor T. evansi infections in mice. For epidemiological application, such comparative evaluation should be made for detection of T. evansi in livestock such as horses.

  16. A Targeted Q-PCR-Based Method for Point Mutation Testing by Analyzing Circulating DNA for Cancer Management Care.

    PubMed

    Thierry, Alain R

    2016-01-01

    Circulating cell-free DNA (cfDNA) is a valuable source of tumor material available with a simple blood sampling enabling a noninvasive quantitative and qualitative analysis of the tumor genome. cfDNA is released by tumor cells and exhibits the genetic and epigenetic alterations of the tumor of origin. Circulating cell-free DNA (cfDNA) analysis constitutes a hopeful approach to provide a noninvasive tumor molecular test for cancer patients. Based upon basic research on the origin and structure of cfDNA, new information on circulating cell-free DNA (cfDNA) structure, and specific determination of cfDNA fragmentation and size, we revisited Q-PCR-based method and recently developed a the allele-specific-Q-PCR-based method with blocker (termed as Intplex) which is the first multiplexed test for cfDNA. This technique, named Intplex(®) and based on a refined Q-PCR method, derived from critical observations made on the specific structure and size of cfDNA. It enables the simultaneous determination of five parameters: the cfDNA total concentration, the presence of a previously known point mutation, the mutant (tumor) cfDNA concentration (ctDNA), the proportion of mutant cfDNA, and the cfDNA fragmentation index. Intplex(®) has enabled the first clinical validation of ctDNA analysis in oncology by detecting KRAS and BRAF point mutations in mCRC patients and has demonstrated that a blood test could replace tumor section analysis for the detection of KRAS and BRAF mutations. The Intplex(®) test can be adapted to all mutations, genes, or cancers and enables rapid, highly sensitive, cost-effective, and repetitive analysis. As regards to the determination of mutations on cfDNA Intplex(®) is limited to the mutational status of known hotspot mutation; it is a "targeted approach." However, it offers the opportunity in detecting quantitatively and dynamically mutation and could constitute a noninvasive attractive tool potentially allowing diagnosis, prognosis, theranostics

  17. 9 CFR 147.30 - Laboratory procedure recommended for the polymerase chain reaction (PCR) test for Mycoplasma...

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... the polymerase chain reaction (PCR) test for Mycoplasma gallisepticum and M. synoviae. 147.30 Section... Examination Procedures § 147.30 Laboratory procedure recommended for the polymerase chain reaction (PCR) test... should consist of the following sequences: ER12JA07.005 (c) Polymerase chain reaction. (1) Treat...

  18. 9 CFR 147.30 - Laboratory procedure recommended for the polymerase chain reaction (PCR) test for Mycoplasma...

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... the polymerase chain reaction (PCR) test for Mycoplasma gallisepticum and M. synoviae. 147.30 Section... Examination Procedures § 147.30 Laboratory procedure recommended for the polymerase chain reaction (PCR) test... should consist of the following sequences: ER12JA07.005 (c) Polymerase chain reaction. (1) Treat...

  19. 9 CFR 147.30 - Laboratory procedure recommended for the polymerase chain reaction (PCR) test for Mycoplasma...

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... the polymerase chain reaction (PCR) test for Mycoplasma gallisepticum and M. synoviae. 147.30 Section... Examination Procedures § 147.30 Laboratory procedure recommended for the polymerase chain reaction (PCR) test... should consist of the following sequences: ER12JA07.005 (c) Polymerase chain reaction. (1) Treat...

  20. 9 CFR 147.30 - Laboratory procedure recommended for the polymerase chain reaction (PCR) test for Mycoplasma...

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... the polymerase chain reaction (PCR) test for Mycoplasma gallisepticum and M. synoviae. 147.30 Section... Examination Procedures § 147.30 Laboratory procedure recommended for the polymerase chain reaction (PCR) test... should consist of the following sequences: ER12JA07.005 (c) Polymerase chain reaction. (1) Treat...

  1. 9 CFR 147.30 - Laboratory procedure recommended for the polymerase chain reaction (PCR) test for Mycoplasma...

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... the polymerase chain reaction (PCR) test for Mycoplasma gallisepticum and M. synoviae. 147.30 Section... Examination Procedures § 147.30 Laboratory procedure recommended for the polymerase chain reaction (PCR) test... should consist of the following sequences: ER12JA07.005 (c) Polymerase chain reaction. (1) Treat...

  2. A PCR detection method for testing Mycoplasma contamination of veterinary vaccines and biological products.

    PubMed

    Ingebritson, A L; Gibbs, C P; Tong, C; Srinivas, G B

    2015-02-01

    A rapid test method was developed for detecting mycoplasma contamination in veterinary biological products. The method reduces testing time by 2 weeks and shows comparable sensitivity to the current agar-based detection model. The primary goals for the development of the test were to reduce the testing time, incorporate a method that was easily adaptable across the veterinary biologics industry and reduce the subjective interpretation of results. We found that biological enrichment is necessary to maintain sensitivity of the detection method when compared to the standard culture-based test and that periodic sampling of enrichment cultures is essential to detect a wide variety of mycoplasma species that may be present as contaminants. The PCR detection method is comparable to the agar-based model and can reduce the overall testing time by up to 14 days.

  3. TQAP for Verification of Qualitative Lead Test Kits

    EPA Science Inventory

    There are lead-based paint test kits available to help home owners and contractors identify lead-based paint hazards before any Renovation, Repair, and Painting (RRP) activities take place so that proper health and safety meaures can be enacted. However, many of these test kits ...

  4. TQAP for Verification of Qualitative Lead Test Kits

    EPA Science Inventory

    There are lead-based paint test kits available to help home owners and contractors identify lead-based paint hazards before any Renovation, Repair, and Painting (RRP) activities take place so that proper health and safety meaures can be enacted. However, many of these test kits ...

  5. An Improved Method for Students' Flame Tests in Qualitative Analysis.

    ERIC Educational Resources Information Center

    Bare, William D.; Bradley, Tom; Pulliam, Elizabeth

    1998-01-01

    Describes a revised laboratory set-up for performing flame tests that enables students to pay more attention to the results of the tests and less attention to the manipulation of equipment. Employs a watch glass to add the analytical solutions to the flame. (DDR)

  6. Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection.

    PubMed

    Buh Gasparic, Meti; Tengs, Torstein; La Paz, Jose Luis; Holst-Jensen, Arne; Pla, Maria; Esteve, Teresa; Zel, Jana; Gruden, Kristina

    2010-03-01

    Several techniques have been developed for detection and quantification of genetically modified organisms, but quantitative real-time PCR is by far the most popular approach. Among the most commonly used real-time PCR chemistries are TaqMan probes and SYBR green, but many other detection chemistries have also been developed. Because their performance has never been compared systematically, here we present an extensive evaluation of some promising chemistries: sequence-unspecific DNA labeling dyes (SYBR green), primer-based technologies (AmpliFluor, Plexor, Lux primers), and techniques involving double-labeled probes, comprising hybridization (molecular beacon) and hydrolysis (TaqMan, CPT, LNA, and MGB) probes, based on recently published experimental data. For each of the detection chemistries assays were included targeting selected loci. Real-time PCR chemistries were subsequently compared for their efficiency in PCR amplification and limits of detection and quantification. The overall applicability of the chemistries was evaluated, adding practicability and cost issues to the performance characteristics. None of the chemistries seemed to be significantly better than any other, but certain features favor LNA and MGB technology as good alternatives to TaqMan in quantification assays. SYBR green and molecular beacon assays can perform equally well but may need more optimization prior to use.

  7. PCR-based verification of positive rapid diagnostic tests for intestinal protozoa infections with variable test band intensity.

    PubMed

    Becker, Sören L; Müller, Ivan; Mertens, Pascal; Herrmann, Mathias; Zondie, Leyli; Beyleveld, Lindsey; Gerber, Markus; du Randt, Rosa; Pühse, Uwe; Walter, Cheryl; Utzinger, Jürg

    2017-10-01

    Stool-based rapid diagnostic tests (RDTs) for pathogenic intestinal protozoa (e.g. Cryptosporidium spp. and Giardia intestinalis) allow for prompt diagnosis and treatment in resource-constrained settings. Such RDTs can improve individual patient management and facilitate population-based screening programmes in areas without microbiological laboratories for confirmatory testing. However, RDTs are difficult to interpret in case of 'trace' results with faint test band intensities and little is known about whether such ambiguous results might indicate 'true' infections. In a longitudinal study conducted in poor neighbourhoods of Port Elizabeth, South Africa, a total of 1428 stool samples from two cohorts of schoolchildren were examined on the spot for Cryptosporidium spp. and G. intestinalis using an RDT (Crypto/Giardia DuoStrip; Coris BioConcept). Overall, 121 samples were positive for G. intestinalis and the RDT suggested presence of cryptosporidiosis in 22 samples. After a storage period of 9-10 months in cohort 1 and 2-3 months in cohort 2, samples were subjected to multiplex PCR (BD Max™ Enteric Parasite Panel, Becton Dickinson). Ninety-three percent (112/121) of RDT-positive samples for G. intestinalis were confirmed by PCR, with a correlation between RDT test band intensity and quantitative pathogen load present in the sample. For Cryptosporidium spp., all positive RDTs had faintly visible lines and these were negative on PCR. The performance of the BD Max™ PCR was nearly identical in both cohorts, despite the prolonged storage at disrupted cold chain conditions in cohort 1. The Crypto/Giardia DuoStrip warrants further validation in communities with a high incidence of diarrhoea. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Introduction of a novel parechovirus RT-PCR clinical test in a regional medical center.

    PubMed

    Renaud, Christian; Kuypers, Jane; Ficken, Elle; Cent, Anne; Corey, Lawrence; Englund, Janet A

    2011-05-01

    Although data documenting the severity and frequency of human parechovirus (HPeV) infections have been published, detection of HPeV is not routinely performed in most clinical virology laboratories. To describe diagnostic yield, epidemiology and clinical characteristics of patients infected with HPeV during the first year using a new HPeV reverse transcription (RT)-PCR. We introduced an HPeV RT-PCR for the routine testing of cerebrospinal fluid (CSF) and blood samples submitted to our clinical laboratory for detection of human enteroviruses (HEV). Prospective testing of samples with retrospective analysis of medical charts was performed. Of the 499 clinical samples received between May, 2009 and May, 2010, 9.6% (46 patients) had HEV detected and 3.4% (15 patients) had HPeV detected. All patients infected by HPeV were <3 months old, hospitalized between June and October 2009, and all typed viruses were HPeV3. Clinical characteristics of HPeV and HEV infected infants were similar. However, patients infected with HPeV were more likely to have a normal leukocyte count in their CSF (p<0.001). One HPeV3-infected infant developed encephalitis and another developed hepatitis. In our institution, the HPeV RT-PCR was useful to diagnose a novel pathogen in infants with sepsis-like disease. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. A Quantitative PCR-Electrochemical Genosensor Test for the Screening of Biotech Crops.

    PubMed

    Moura-Melo, Suely; Miranda-Castro, Rebeca; de-Los-Santos-Álvarez, Noemí; Miranda-Ordieres, Arturo J; Dos Santos Junior, José Ribeiro; da Silva Fonseca, Rosana A; Lobo-Castañón, María Jesús

    2017-04-18

    The design of screening methods for the detection of genetically modified organisms (GMOs) in food would improve the efficiency in their control. We report here a PCR amplification method combined with a sequence-specific electrochemical genosensor for the quantification of a DNA sequence characteristic of the 35S promoter derived from the cauliflower mosaic virus (CaMV). Specifically, we employ a genosensor constructed by chemisorption of a thiolated capture probe and p-aminothiophenol gold surfaces to entrap on the sensing layer the unpurified PCR amplicons, together with a signaling probe labeled with fluorescein. The proposed test allows for the determination of a transgene copy number in both hemizygous (maize MON810 trait) and homozygous (soybean GTS40-3-2) transformed plants, and exhibits a limit of quantification of at least 0.25% for both kinds of GMO lines.

  10. A Quantitative PCR-Electrochemical Genosensor Test for the Screening of Biotech Crops

    PubMed Central

    Moura-Melo, Suely; Miranda-Castro, Rebeca; de-los-Santos-Álvarez, Noemí; Miranda-Ordieres, Arturo J.; dos Santos Junior, José Ribeiro; da Silva Fonseca, Rosana A.; Lobo-Castañón, María Jesús

    2017-01-01

    The design of screening methods for the detection of genetically modified organisms (GMOs) in food would improve the efficiency in their control. We report here a PCR amplification method combined with a sequence-specific electrochemical genosensor for the quantification of a DNA sequence characteristic of the 35S promoter derived from the cauliflower mosaic virus (CaMV). Specifically, we employ a genosensor constructed by chemisorption of a thiolated capture probe and p-aminothiophenol gold surfaces to entrap on the sensing layer the unpurified PCR amplicons, together with a signaling probe labeled with fluorescein. The proposed test allows for the determination of a transgene copy number in both hemizygous (maize MON810 trait) and homozygous (soybean GTS40-3-2) transformed plants, and exhibits a limit of quantification of at least 0.25% for both kinds of GMO lines. PMID:28420193

  11. Further Validation of the Qualitative Scoring System for the Modified Bender-Gestalt Test.

    ERIC Educational Resources Information Center

    Brannigan, Gary G.; And Others

    1995-01-01

    Compares the Qualitative Scoring System and the Developmental Scoring Systems, both Bender-Gestalt tests, in predicting achievement on the Metropolitan Achievement Test (MAT). In this study, first through fourth graders (n=409) from regular elementary schools were subjected to both tests; both systems correlated significantly with school…

  12. Further Validation of the Qualitative Scoring System for the Modified Bender-Gestalt Test.

    ERIC Educational Resources Information Center

    Brannigan, Gary G.; And Others

    1995-01-01

    Compares the Qualitative Scoring System and the Developmental Scoring Systems, both Bender-Gestalt tests, in predicting achievement on the Metropolitan Achievement Test (MAT). In this study, first through fourth graders (n=409) from regular elementary schools were subjected to both tests; both systems correlated significantly with school…

  13. The prevalence of canine Leishmania infantum infection in western China detected by PCR and serological tests.

    PubMed

    Wang, Jun-Yun; Ha, Yu; Gao, Chun-Hua; Wang, Yong; Yang, Yue-Tao; Chen, Hai-Tang

    2011-05-09

    Canine leishmaniasis (CanL) is endemic in western China, resulting in important public health problem. It is essential to evaluate the prevalence of canine Leishmania infantum infection for designing control policy. In the present study we report for the first time prevalence of Leishmania infection in dogs living in Jiuzhaigou County (Sichuan Provence, China), which is not only an important endemic area of CanL but also a tourism scenic spot, detected by PCR, ELISA and dipstick test. The results could provide key information for designing control programs against canine and human leishmaniasis. In addition, the complete sequence of the Leishmania isolate from Sichuan Province has not been reported to date and we present the sequences of 116 base-pair (bp) fragment of the conserved region in the minicircle kinetoplast DNA (kDNA) and the results of phylogenetic analyses based on the sequence of the amplified fragment. The proportion of dogs infected with Leishmania in Jiuzhaigou County was 36.79%, 9.43%, and 51.88% detected by ELISA, dipstick test, and PCR, respectively. The ELISA and PCR tests were more sensitive than dipstick test. The PCR method is the most sensitive way to detect dogs infected with Leishmania parasites. The total positive rate for infected dogs in the area was 59.43% by the three methods. The PCR products of 116-bp fragment amplified from the kDNA conserved region of dog blood samples and laboratory maintained L. infantum were DNA sequenced and the variation of the sequences was observed. The phylogenetic tree based on the sequences of 116-bp fragment reveals that L. infantum is more genetically related to visceralizing species L. donovani than to the Leishmania species associated with cutaneous disease. More than half of dogs living in the endemic Jiuzhaigou County were infected by L. infantum. Control measures, such as treatment or eradication of infected dogs, or prohibition of maintaining dogs, must be taken against these infected dogs due

  14. Quantitative and qualitative validations of a sonication-based DNA extraction approach for PCR-based molecular biological analyses.

    PubMed

    Dai, Xiaohu; Chen, Sisi; Li, Ning; Yan, Han

    2016-05-15

    The aim of this study was to comprehensively validate the sonication-based DNA extraction method, in hope of the replacement of the so-called 'standard DNA extraction method' - the commercial kit method. Microbial cells in the digested sludge sample, containing relatively high amount of PCR-inhibitory substances, such as humic acid and protein, were applied as the experimental alternatives. The procedure involving solid/liquid separation of sludge sample and dilution of both DNA templates and inhibitors, the minimum templates for PCR-based analyses, and the in-depth understanding from the bias analysis by pyrosequencing technology were obtained and confirmed the availability of the sonication-based DNA extraction method. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Event-specific qualitative and quantitative PCR detection methods for transgenic rapeseed hybrids MS1xRF1 and MS1xRF2.

    PubMed

    Wu, Yuhua; Wu, Gang; Xiao, Ling; Lu, Changming

    2007-10-17

    Except for the events RT73, MS8, RF3, and T45, event-specific detection methods for most commercialized genetically modified (GM) rapeseed varieties have not been established, and as a result, the enforcement of genetically modified organism labeling policies has been hindered. The genetically modified rapeseeds, MS1xRF1 and MS1xRF2, are 2 of 11 approved GM-rapeseed varieties for commercialization. In this study, the right border junction fragments between the gene construct and the rapeseed genome of events RF1, RF2, and MS1 were isolated using the commercially available GenomeWalker technology. Homology analysis indicated that the gene construct of RF1 integrated upstream of the nuclease gene, and that of the RF2 and MS1 inserted into the exon region of a gene encoding for an unknown protein. The event-specific primer pairs and corresponding probes were designed on the basis of the revealed right border junction fragments. Then, we successfully developed the identification and quantification methods for the gene-stacked hybrids MS1xRF1 and MS1xRF2 using those primers and probes. The relative limit of detection in the qualitative polymerase chain reaction (PCR) was 0.013% for the RF2 and MS1 assays using 100 ng of rapeseed DNA per reaction and 0.13% for the RF1 assay. The absolute limit of detection in the quantitative PCR was approximately one to two initial copies for each of the three event-specific assays. The evaluation of the real-time PCR assays revealed that the qualitative and quantitative methods developed by focusing on the gene-stacked hybrids MS1xRF1 and MS1xRF2 were highly specific, sensitive, and suitable for samples with a low quantity of DNA.

  16. Fluorescent detection of Southern blots and PCR-based genetic typing tests

    SciTech Connect

    Mansfield, E.S.; Worley, J.M.; Zimmerman, P.A.

    1994-09-01

    The Southern blot is used to study gene organization, to identify disease-causing genomic rearrangements, or for typing RFLP markers in forensic, paternity, or prenatal diagnostic testing. Fluorescence offers a much greater dynamic range and a more linear response than film used in radioactive or chemiluminescent detection of RFLPs. We therefore investigated using the Fluorimager{trademark} 575 (Molecular Dynamics, Inc.) for analyzing Southern blots. Using a single-locus probe to D2S44 (YNH24) (Promega Corp.), we detect as little as 100 ng (0.05 attomole) genomic DNA. The alkaline phosphatase-labeled probe is detected using AttoPhos (JBL Scientific), and the developed membrane is scanned with the Fluorimager. Biotinylated hybridization probes can also be developed using a streptavidin-alkaline phosphatase conjugate and AttoPhos. The instrument scan parameters can be adjusted to prevent overexposure and accompanying loss of resolution in images of blots, gels, or 96-well microplates. We have used these other sample formats in PCR-based genetic typing assays. We use FluorKit DQS (Molecular Dynamics) to accurately quantify PCR template DNA (1-500 ng) in 96-well microplates scanned using the same instrument. Mutation detection assays run include heteroduplex gels (5% polyacrylamide, 2.7 M urea), short tandem repeat (STR) markers, amplified fragment length polymorphisms (AmpFLP), competitive priming PCR, and allele-specific oligotyping. These assays are run using either 1- or 2-color labeling. We detect unlabeled PCR products, such as the AmpFLP marker D1S80 (Perkin-Elmer) by post-staining gels for 10 minutes with SYBR Green 1 (Molecular Probes) and scanning the wet gel. The Fluorimager scans a 20 x 25 cm sample within three minutes, allowing rapid optimization of fluorescent protocols and high sample throughput.

  17. PCR testing of a ventilated caging system to detect murine fur mites.

    PubMed

    Jensen, Eric S; Allen, Kenneth P; Henderson, Kenneth S; Szabo, Aniko; Thulin, Joseph D

    2013-01-01

    Rodents housed in microisolation caging are commonly monitored for infectious agents by the use of soiled bedding sentinels. This strategy relies on the successful transmission of rodent pathogens from the index rodents via soiled bedding to sentinel cages and the subsequent infection or colonization of sentinel rodents. When the prevalence of a pathogen is low or the target agent is not readily transmitted by soiled bedding, alternative testing methodologies should be used. Given the continued prevalence of institutions self-reporting murine fur mites and with the advent of a new sensitive and specific PCR assay for mites, we sought to determine whether the exhaust system of an individual ventilated caging (IVC) system could be used for monitoring the rack's rodent population for mites rather than relying on the responses of sentinels. We deployed single cages of mice (Mus musculus) that were known to be infested with either Radfordia affinis or Myobia musculi on a 70-cage rack, sampled the horizontal exhaust manifolds weekly, and used the new PCR assay to test these samples for mite DNA. We detected the presence of fur mites at a 94.1% probability of detection within 4 wk of placement. Therefore, we recommend swabbing and testing the shelf exhaust manifolds of IVC racks rather than relying on soiled-bedding sentinels as an indicator of the mite status of the rodents on that rack.

  18. PCR Testing of a Ventilated Caging System to Detect Murine Fur Mites

    PubMed Central

    Jensen, Eric S; Allen, Kenneth P; Henderson, Kenneth S; Szabo, Aniko; Thulin, Joseph D

    2013-01-01

    Rodents housed in microisolation caging are commonly monitored for infectious agents by the use of soiled bedding sentinels. This strategy relies on the successful transmission of rodent pathogens from the index rodents via soiled bedding to sentinel cages and the subsequent infection or colonization of sentinel rodents. When the prevalence of a pathogen is low or the target agent is not readily transmitted by soiled bedding, alternative testing methodologies should be used. Given the continued prevalence of institutions self-reporting murine fur mites and with the advent of a new sensitive and specific PCR assay for mites, we sought to determine whether the exhaust system of an individual ventilated caging (IVC) system could be used for monitoring the rack's rodent population for mites rather than relying on the responses of sentinels. We deployed single cages of mice (Mus musculus) that were known to be infested with either Radfordia affinis or Myobia musculi on a 70-cage rack, sampled the horizontal exhaust manifolds weekly, and used the new PCR assay to test these samples for mite DNA. We detected the presence of fur mites at a 94.1% probability of detection within 4 wk of placement. Therefore, we recommend swabbing and testing the shelf exhaust manifolds of IVC racks rather than relying on soiled-bedding sentinels as an indicator of the mite status of the rodents on that rack. PMID:23562030

  19. A specific test for transthyretin 122 (Val yields Ile), based on PCR-primer-introduced restriction analysis (PCR-PIRA): Confirmation of the gene frequency in blacks

    SciTech Connect

    Jacobson, D.R. )

    1992-01-01

    The variant transthyretin (TTR) allele, TTR (122 Val {yields} Ile), associated with cardiac amyloidosis in blacks, is caused by a G {yields} A transition which destroys a MaeIII site. This variant has previously been detected by PCR around codon 122, followed by MaeIII digestion, but this test is not specific: any of 12 mutations in the MaeIII recognition site, each of which yields a different amino acid change, would also destroy this site. A modification of PCR, termed PCR-primer-introduced restriction analysis, was used to introduce a new FokI site into the PCR products derived from the variant (122 Ile) but not wild-type (122 Val) allele. This test demonstrated that each of six previously identified MaeIII ({minus}) alleles had lost its MaeIII site because of a G {yields} A transition encoding TTR (122 Val {yields} Ile), confirming that the same TTR variant was present both in 4/177 healthy black individuals and as a homozygous variant in an individual with cardiac amyloidosis.

  20. Applications of abduction: hypothesis testing of neuroendocrinological qualitative compartmental models.

    PubMed

    Menzies, T; Compton, P

    1997-06-01

    It is difficult to assess hypothetical models in poorly measured domains such as neuroendocrinology. Without a large library of observations to constrain inference, the execution of such incomplete models implies making assumptions. Mutually exclusive assumptions must be kept in separate worlds. We define a general abductive multiple-worlds engine that assesses such models by (i) generating the worlds and (ii) tests if these worlds contain known behaviour. World generation is constrained via the use of relevant envisionment. We describe QCM, a modeling language for compartmental models that can be processed by this inference engine. This tool has been used to find faults in theories published in international refereed journals; i.e. QCM can detect faults which are invisible to other methods. The generality and computational limits of this approach are discussed. In short, this approach is applicable to any representation that can be compiled into an and-or graph, provided the graphs are not too big or too intricate (fanout < 7).

  1. Detection and differentiation of human parvovirus variants by commercial quantitative real-time PCR tests.

    PubMed

    Hokynar, Kati; Norja, Päivi; Laitinen, Harri; Palomäki, Pekka; Garbarg-Chenon, Antoine; Ranki, Annamari; Hedman, Klaus; Söderlund-Venermo, Maria

    2004-05-01

    Parvovirus B19 causes a variety of diseases in humans, with outcomes ranging from asymptomatic to severe, such as chronic anemia in immunocompromised patients or fetal hydrops and death after maternal infection during pregnancy. The virus may be transmitted via plasma-derived products. According to the results of solvent-detergent safety studies, an upper limit of B19 DNA in plasma pools was recently defined. To restrict the input of B19 virus into production pools, a quantitative nucleic acid test is a prerequisite. We examined the suitability of the two commercial quantitative B19 PCR tests, LightCycler-Parvovirus B19 quantification kit (Roche Diagnostics) and RealArt Parvo B19 LC PCR (Artus) for detection, quantification, and differentiation of the three known B19 genotypes, including the newly described erythrovirus variants (genotypes 2 and 3). The former kit was highly sensitive for genotype 1 but was not suitable for detection of genotype 2 or one of two genotype 3 strains. The latter kit detected and differentiated all three genotypes, albeit with lower sensitivity for one of the genotype-3 strains. We furthermore assessed the prevalence of the three B19 virus genotypes in blood donors, by screening pooled plasma samples derived from 140,160 Finnish blood-donor units. None of the pools contained detectable levels of B19 virus genotypes 2 or 3. The origin, mode of transmission, and clinical significance of these genotypes are unknown and deserve further study. The RealArt Parvo B19 LC PCR is suitable for detection, quantification, and differentiation of all three B19 virus genotypes in molecular and clinical research.

  2. [Canine visceral leishmaniasis diagnosis by immunohistochemistry and PCR in skin tissues in association with IFAT and ELISA-test].

    PubMed

    de Queiroz, Nina M G P; de Assis, Juliana; Oliveira, Trícia M F S; Machado, Rosângela Z; Nunes, Cáris M; Starke-Buzetti, Wilma A

    2010-01-01

    The purpose of the present study was to evaluate the immunohistochemistry (IMHC) and PCR (Polymerase Chain Reaction) tests for Canine Visceral Leishmaniasis (CVL) diagnosis and compare the results with serological tests such as the indirect fluorescence antibody test (IFAT), ELISA and a parasitological test (microscopic direct examination of the parasite stained with haematoxylin and eosin--HE). For this study, samples of healthy or lesion skin tissues were obtained from 34 CVL naturally infected dogs classified in three groups: asymptomatic, oligosymptomatic and polisymptomatic. Not only lesion (56.5%) but also healthy skins (31.8%) were positives by IMHC and confirmed by PCR in 97.8% of skin samples. In asymptomatic group, 87.5% dogs were negatives by serological tests, but positives by IMHC in 50% and by PCR in 100%. In oligosymptomatic group, 100%, 85.7% and 28.6% of dogs were positives, respectively by PCR, serological and IMHC tests. In addition, 91.7% of polisymptomatic dogs were serum positive and had intact parasites in the skin. In general, PCR showed higher positivity (100%). The efficiency of each test varied with the evolution of the disease. IMHC may be used to confirm the results of the serology and PCR in inconclusive cases after HE and IMHC. The association of techniques proposed in this study may increase the positivity and contributed to the control of this canine disease.

  3. Establishment of Multiplex Solid-Phase Strip PCR Test for Detection of 24 Ocular Infectious Disease Pathogens.

    PubMed

    Nakano, Satoko; Sugita, Sunao; Tomaru, Yasuhiro; Hono, Ayumi; Nakamuro, Takako; Kubota, Toshiaki; Takase, Hiroshi; Mochizuki, Manabu; Takahashi, Masayo; Shimizu, Norio

    2017-03-01

    To establish and evaluate a new multiplex solid-phase strip polymerase chain reaction (strip PCR) for concurrent detection of common ocular infectious disease pathogens. A new multiplex strip PCR was established to detect 24 common ocular infectious disease pathogens: herpes simplex virus (HSV) 1, HSV2, varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpes virus (HHV) 6, HHV7, HHV8, human T-cell lymphotropic virus (HTLV)-1, adenovirus, Mycobacterium tuberculosis, Treponema pallidum, Propionibacterium acnes (P. acnes), bacterial 16S ribosomal RNA (rRNA), Candida species (Candida sp.), C. glabrata, C. krusei, Aspergillus, Fusarium, fungal 28S rRNA, Toxoplasma (T. gondii), Toxocara, Chlamydia trachomatis (C. trachomatis), and Acanthamoeba. Strip PCR was tested with a negative control (distilled water) and standard positive control DNA. Cutoffs of quantification cycle (Cq) values were determined with noninfectious ocular samples to avoid false-positives caused by contamination with P. acnes, bacterial 16S, and fungal 28S from reagents and ocular surfaces. A pilot study to evaluate the strip PCR was performed using infectious ocular samples (aqueous humor, vitreous, cornea, and tears) by strip PCR and previously developed capillary-type multiplex PCR and quantitative real-time PCR (qPCR). Strip PCR was verified with negative and positive controls. Strip PCR rapidly detected HSV1, HSV2, VZV, EBV, CMV, HHV6, HHV7, HTLV-1, adenovirus, P. acnes, bacterial 16S, Candida sp., C. glabrata, Aspergillus, fungal 28S, T. gondii, C. trachomatis, and Acanthamoeba in patient samples. The sensitivity was comparable to that of qPCR. Our novel strip PCR assay is a simple, rapid, and high-sensitivity method for detecting ocular infectious disease pathogens.

  4. Evaluation of Legionella real-time PCR against traditional culture for routine and public health testing of water samples.

    PubMed

    Collins, S; Stevenson, D; Walker, J; Bennett, A

    2017-06-01

    To evaluate the usefulness of Legionella qPCR alongside traditional culture for enumeration of Legionella from water samples as part of both routine and public health investigation testing. Routine water samples (n = 2002) and samples from public health investigations (n = 215) were analysed by culture and qPCR for Legionella spp., Legionella pneumophila and L. pneumophila sg-1. A negative qPCR result was highly predictive of a negative culture result for all water systems (negative predictive values, NPV from 97·4 to 100%). Positive predictive values (PPV) were lower (0-50%). Results for qPCR were generally larger than culture with average log10 differences of 1·1 for Legionella spp. and 1·2 for L. pneumophila. Alert and action levels of 1000 and 10 000 GU per litre, respectively, are proposed for Legionella qPCR for hot and cold water systems (HCWS). The use of qPCR significantly reduced the time to results for public health investigations by rapidly identifying potential sources and ruling out others, thus enabling a more rapid and efficient response. The high NPV of qPCR supports its use to rapidly screen out negative samples without culture. Alert and action levels for Legionella qPCR for HCWS are proposed. Quantitative PCR will be a valuable tool for both routine and public health testing. This study generated comparative data of >2000 water samples by qPCR and culture. Action and alert levels have been recommended that could enable duty holders to interpret qPCR results to facilitate timely Legionella control and public health protection. © 2017 Crown copyright. Journal of Applied Microbiology © 2017 The Society for Applied Microbiology.

  5. Adaptive and qualitative changes in encoding strategy with experience: evidence from the test-expectancy paradigm.

    PubMed

    Finley, Jason R; Benjamin, Aaron S

    2012-05-01

    Three experiments demonstrated learners' abilities to adaptively and qualitatively accommodate their encoding strategies to the demands of an upcoming test. Stimuli were word pairs. In Experiment 1, test expectancy was induced for either cued recall (of targets given cues) or free recall (of targets only) across 4 study-test cycles of the same test format, followed by a final critical cycle featuring either the expected or the unexpected test format. For final tests of both cued and free recall, participants who had expected that test format outperformed those who had not. This disordinal interaction, supported by recognition and self-report data, demonstrated not mere differences in effort based on anticipated test difficulty, but rather qualitative and appropriate differences in encoding strategies based on expected task demands. Participants also came to appropriately modulate metacognitive monitoring (Experiment 2) and study-time allocation (Experiment 3) across study-test cycles. Item and associative recognition performance, as well as self-report data, revealed shifts in encoding strategies across trials; these results were used to characterize and evaluate the different strategies that participants employed for cued versus free recall and to assess the optimality of participants' metacognitive control of encoding strategies. Taken together, these data illustrate a sophisticated form of metacognitive control, in which learners qualitatively shift encoding strategies to match the demands of anticipated tests.

  6. Adaptive and Qualitative Changes in Encoding Strategy With Experience: Evidence From the Test-Expectancy Paradigm

    PubMed Central

    Finley, Jason R.; Benjamin, Aaron S.

    2012-01-01

    Three experiments demonstrated learners’ abilities to adaptively and qualitatively accommodate their encoding strategies to the demands of an upcoming test. Stimuli were word pairs. In Experiment 1, test expectancy was induced for either cued recall (of targets given cues) or free recall (of targets only) across 4 study–test cycles of the same test format, followed by a final critical cycle featuring either the expected or the unexpected test format. For final tests of both cued and free recall, participants who had expected that test format outperformed those who had not. This disordinal interaction, supported by recognition and self-report data, demonstrated not mere differences in effort based on anticipated test difficulty, but rather qualitative and appropriate differences in encoding strategies based on expected task demands. Participants also came to appropriately modulate metacognitive monitoring (Experiment 2) and study-time allocation (Experiment 3) across study–test cycles. Item and associative recognition performance, as well as self-report data, revealed shifts in encoding strategies across trials; these results were used to characterize and evaluate the different strategies that participants employed for cued versus free recall and to assess the optimality of participants’ metacognitive control of encoding strategies. Taken together, these data illustrate a sophisticated form of metacognitive control, in which learners qualitatively shift encoding strategies to match the demands of anticipated tests. PMID:22103783

  7. Will droplet digital PCR become the test of choice for detecting and quantifying ocular Chlamydia trachomatis infection? Maybe not.

    PubMed

    Schachter, Julius

    2013-11-01

    Evaluation of: Roberts CH, Last A, Molina-Gonzalez S et al. Development and evaluation of a next-generation digital PCR diagnostic assay for ocular chlamydia trachomatis infections. J. Clin. Microbiol. 51(7), 2195-2203 (2013). Trachoma is the leading infectious cause of blindness in developing countries. Currently, there is no program to eliminate blinding trachoma as a public health problem. We need better diagnostic tests for research and to assess progress in control programs. Roberts et al. adapted droplet digital PCR (ddPCR), an emulsion PCR process that performs absolute quantitation of nucleic acids, to detect and quantify Chlamydia trachomatis infections. They compared the results with ddPCR on conjunctival swab specimens collected in trachoma-endemic area to results using Roche's Amplicor® C. trachomatis/Neisseria gonorrhoeae (CT/NG) PCR and found that ddPCR sensitivity was 73.3%. The authors concluded that 'ddPCR is an effective diagnostic technology suitable for both research and clinical use in diagnosing ocular C. trachomatis infections'. This reviewer disagrees, feeling that if the stated sensitivity is accurate, it is too low, and suggests there may be good reasons to adapt commercially available tests for this purpose.

  8. A novel real-time RT-PCR assay for influenza C tested in Peruvian children.

    PubMed

    Howard, Leigh M; Johnson, Monika; Gil, Ana I; Pekosz, Andrew; Griffin, Marie R; Edwards, Kathryn M; Lanata, Claudio F; Grijalva, Carlos G; Williams, John V

    2017-09-01

    Influenza C virus (ICV) is associated with acute respiratory illness. Yet ICV remains under recognized, with most previous studies using only culture to identify cases. To develop a sensitive and specific real-time RT-PCR assay for ICV that allows for rapid and accurate detection in a clinical or research setting. Multiple ICV sequences obtained from GenBank were analyzed, including 141 hemagglutinin-esterase (HE), 106 matrix (M), and 97 nucleoprotein (NP) sequences. Primers and probes were designed based on conserved regions. Multiple primer-probe sets were tested against multiple ICV strains. The ICV M and NP genes offered the most conserved sequence regions. Primers and probes based on newer sequence data offered enhanced detection of ICV, especially for low titer specimens. An NP-targeted assay yielded the best performance and was capable of detecting 10-100 RNA copies per reaction. The NP assay detected multiple clinical isolates of ICV collected in a field epidemiology study conducted in Peru. We report a new real-time RT-PCR assay for ICV with high sensitivity and specificity. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Prevalence of PCR detectable malaria infection among febrile patients with a negative Plasmodium falciparum specific rapid diagnostic test in Zanzibar.

    PubMed

    Baltzell, Kimberly A; Shakely, Deler; Hsiang, Michelle; Kemere, Jordan; Ali, Abdullah Suleiman; Björkman, Anders; Mårtensson, Andreas; Omar, Rahila; Elfving, Kristina; Msellem, Mwinyi; Aydin-Schmidt, Berit; Rosenthal, Philip J; Greenhouse, Bryan

    2013-02-01

    We screened for malaria in 594 blood samples from febrile patients who tested negative by a Plasmodium falciparum-specific histidine-rich protein-2-based rapid diagnostic test at 12 health facilities in Zanzibar districts North A and Micheweni, from May to August 2010. Screening was with microscopy, polymerase chain reaction (PCR) targeting the cytochrome b gene (cytbPCR) of the four major human malaria species, and quantitative PCR (qPCR). The prevalence of cytbPCR-detectable malaria infection was 2% (12 of 594), including 8 P. falciparum, 3 Plasmodium malariae, and 1 Plasmodium vivax infections. Microscopy identified 4 of 8 P. falciparum infections. Parasite density as estimated by microscopy or qPCR was > 4,000 parasites/μL in 5 of 8 cytbPCR-detectable P. falciparum infections. The infections that were missed by the rapid diagnostic test represent a particular challenge in malaria elimination settings and highlight the need for more sensitive point-of-care diagnostic tools to improve case detection of all human malaria species in febrile patients.

  10. Comparative Evaluation of Norovirus Infection in Children with Acute Gastroenteritis by Rapid Immunochromatographic Test, RT-PCR and Real-time RT-PCR.

    PubMed

    Kumthip, Kattareeya; Khamrin, Pattara; Saikruang, Wilaiporn; Supadej, Kanittapon; Ushijima, Hiroshi; Maneekarn, Niwat

    2017-03-02

    Immunochromatographic (IC) test for norovirus detection is a rapid and simple detection method. This study evaluated the sensitivity and specificity of a recent version of R-Biopharm RIDA®QUICK Norovirus IC assay for norovirus detection in fecal specimens from children hospitalized with acute gastroenteritis. Fecal specimens were tested by IC kit in comparison with gold standard reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR. The IC kit showed high sensitivity and specificity comparable with PCR-based methods. None of false positive and false negative was found and the assay did not cross-react with other gastroenteritis viruses. The IC assay could detect genogroup I.5 (GI.5) and a wide range of genotypes in the GII noroviruses including GII.3, GII.4, GII.6, GII.7, GII.14, GII.15, GII.21, and also newly emerging GII.17 norovirus. In conclusion, this norovirus IC kit could be an alternative choice for rapid screening or a quick diagnostic tool for norovirus detection in fecal specimens of acute gastroenteritis patients.

  11. Somatic Mutation Allelic Ratio Test Using ddPCR (SMART-ddPCR): An Accurate Method for Assessment of Preferential Allelic Imbalance in Tumor DNA

    PubMed Central

    de Smith, Adam J.; Walsh, Kyle M.; Hansen, Helen M.; Endicott, Alyson A.; Wiencke, John K.; Metayer, Catherine; Wiemels, Joseph L.

    2015-01-01

    The extent to which heritable genetic variants can affect tumor development has yet to be fully elucidated. Tumor selection of single nucleotide polymorphism (SNP) risk alleles, a phenomenon called preferential allelic imbalance (PAI), has been demonstrated in some cancer types. We developed a novel application of digital PCR termed Somatic Mutation Allelic Ratio Test using Droplet Digital PCR (SMART-ddPCR) for accurate assessment of tumor PAI, and have applied this method to test the hypothesis that heritable SNPs associated with childhood acute lymphoblastic leukemia (ALL) may demonstrate tumor PAI. These SNPs are located at CDKN2A (rs3731217) and IKZF1 (rs4132601), genes frequently lost in ALL, and at CEBPE (rs2239633), ARID5B (rs7089424), PIP4K2A (rs10764338), and GATA3 (rs3824662), genes located on chromosomes gained in high-hyperdiploid ALL. We established thresholds of AI using constitutional DNA from SNP heterozygotes, and subsequently measured allelic copy number in tumor DNA from 19–142 heterozygote samples per SNP locus. We did not find significant tumor PAI at these loci, though CDKN2A and IKZF1 SNPs showed a trend towards preferential selection of the risk allele (p = 0.17 and p = 0.23, respectively). Using a genomic copy number control ddPCR assay, we investigated somatic copy number alterations (SCNA) underlying AI at CDKN2A and IKZF1, revealing a complex range of alterations including homozygous and hemizygous deletions and copy-neutral loss of heterozygosity, with varying degrees of clonality. Copy number estimates from ddPCR showed high agreement with those from multiplex ligation-dependent probe amplification (MLPA) assays. We demonstrate that SMART-ddPCR is a highly accurate method for investigation of tumor PAI and for assessment of the somatic alterations underlying AI. Furthermore, analysis of publicly available data from The Cancer Genome Atlas identified 16 recurrent SCNA loci that contain heritable cancer risk SNPs associated with a

  12. Improving early identification of HIV-infected neonates with birth PCR testing in a large urban hospital in Johannesburg, South Africa: successes and challenges.

    PubMed

    Technau, Karl-Günter; Kuhn, Louise; Coovadia, Ashraf; Carmona, Sergio; Sherman, Gayle

    2017-04-10

    Timely diagnosis is necessary to avert early death in HIV-infected neonates. Birth PCR testing may improve early identification and facilitate access to care. We implemented a birth HIV diagnosis programme in Johannesburg, South Africa and present successes and challenges of the first two and a half years of operation. Between June 2014 and December 2016, we sought to identify all HIV-exposed births and offer newborn HIV PCR testing before discharge after delivery. The programme identified newly delivered women who had tested positive during pregnancy and provided post-partum HIV antibody testing for women without recent negative results. HIV-positive women were required to consent for neonatal birth testing and asked to return a week later to obtain their results. Neonatal venous blood was sampled and tested at the national laboratory using Roche COBAS® TaqMan® HIV-1 Qualitative Test (Version 2.0). Non-negative results triggered active follow-up for confirmatory testing and appropriate treatment. Of 30,591 women with live births, 6864 (22.4%) were known to be HIV positive and an additional 221 women (1.4% of those tested) were identified during maternal postnatal testing. Of 7085 HIV-positive women, 6372 (89.9%) were interviewed and agreed to data collection, 6358 (99.8%) consented to birth testing for 6467 neonates and a blood sample was collected for 6377 (98.6%). If tested, 6210 (97.4%) tested negative, 91 (1.4%) positive, 57 (0.9%) revealed errors and 19 (0.3%) were indeterminate . Seven of the 19 neonates with indeterminate results and one with initial error result were found to be infected on subsequent testing yielding an intrauterine transmission rate of 1.6% (95% CI: 1.3-1.9). Sixteen (16%) of 99 infected infants were born to women (n = 221) identified during postnatal testing. With active outreach, 95/99 (96%) infected infants were initiated on antiretroviral therapy. Of 6261 neonates with negative results, 3251 (52%) returned to receive their test

  13. HIV Testing among Men Who Have Sex with Men (MSM): Systematic Review of Qualitative Evidence

    ERIC Educational Resources Information Center

    Lorenc, Theo; Marrero-Guillamon, Isaac; Llewellyn, Alexis; Aggleton, Peter; Cooper, Chris; Lehmann, Angela; Lindsay, Catriona

    2011-01-01

    We conducted a systematic review of qualitative evidence relating to the views and attitudes of men who have sex with men (MSM) concerning testing for HIV. Studies conducted in high-income countries (Organisation for Economic Co-operation and Development members) since 1996 were included. Seventeen studies were identified, most of gay or bisexual…

  14. Employing Online S-P Diagnostic Table for Qualitative Comments on Test Results

    ERIC Educational Resources Information Center

    Wang, Chien-hwa; Chen, Cheng-ping

    2013-01-01

    The major concerns of adaptive testing studies have concentrated on effectiveness and efficiency of the system built for the research experiments. It has been criticised that such general information has fallen short of providing qualitative descriptions regarding learning performance. Takahiro Sato of Japan proposed an analytical diagram called…

  15. Laboratory Tests of Multiplex Detection of PCR Amplicons Using the Luminex 100 Flow Analyzer

    SciTech Connect

    Venkateswaran, K.S.; Nasarabadi, S.; Langlois, R.G.

    2000-05-05

    Lawrence Livermore National Laboratory (LLNL) demonstrated the power of flow cytometry in detecting the biological agents simulants at JFT III. LLNL pioneered in the development of advanced nucleic acid analyzer (ANM) for portable real time identification. Recent advances in flow cytometry provide a means for multiplexed nucleic acid detection and immunoassay of pathogenic microorganisms. We are presently developing multiplexed immunoassays for the simultaneous detection of different simulants. Our goal is to build an integrated instrument for both nucleic acid analysis and immuno detection. In this study we evaluated the Luminex LX 100 for concurrent identification of more than one PCR amplified product. ANAA has real-time Taqman fluorescent detection capability for rapid identification of field samples. However, its multiplexing ability is limited by the combination of available fluorescent labels. Hence integration of ANAA with flow cytometry can give the rapidity of ANAA amplification and the multiplex capability of flow cytometry. Multiplexed flow cytometric analysis is made possible using a set of fluorescent latex microsphere that are individually identified by their red and infrared fluorescence. A green fluorochrome is used as the assay signal. Methods were developed for the identification of specific nucleic acid sequences from Bacillus globigii (Bg), Bacillus thuringensis (Bt) and Erwinia herbicola (Eh). Detection sensitivity using different reporter fluorochromes was tested with the LX 100, and also different assay formats were evaluated for their suitability for rapid testing. A blind laboratory trial was carried out December 22-27, 1999 to evaluate bead assays for multiplex identification of Bg and Bt PCR products. This report summarizes the assay development, fluorochrome comparisons, and the results of the blind trial conducted at LLNL for the laboratory evaluation of the LX 100 flow analyzer.

  16. Comparison of a fur mite PCR assay and the tape test for initial and posttreatment diagnosis during a natural infection.

    PubMed

    Weiss, Erica E; Evans, Kristin D; Griffey, Stephen M

    2012-01-01

    Fur mites were diagnosed in a colony of mice at our research institution. In the current study, we compared the effectiveness of PCR and tape test in a small population of mice at the onset of diagnosis and throughout treatment. Samples were collected 1 d prior to treatment with permethrin impregnated cotton balls and 6 and 12 wk after treatment. PCR confirmed the presence of Myocoptes musculinus and Radfordia affinis or Myobia musculi, but tape test confirmed only the presence of Myocoptes spp. The results of the PCR and tape test agreed 97.2% of the time during active infection on day 1, but only 59.5% and 48.4% of results coincided at 6 and 12 wk after treatment, respectively. At 6 wk, 11 of the 37 samples were PCR-negative but tape-test-positive, compared with 9 of the 31 samples at 12 wk. Our results show that PCR is a reliable diagnostic method during active fur mite infection but that false-negative results are possible after treatment. Negative PCR results after treatment should be interpreted carefully, and a secondary diagnostic method should be considered.

  17. Effects of recent Leptospira vaccination on whole blood real-time PCR testing in healthy client-owned dogs.

    PubMed

    Midence, J N; Leutenegger, C M; Chandler, A M; Goldstein, R E

    2012-01-01

    Bacterin-based canine Leptospira vaccines could present a challenge for the use of whole blood real-time polymerase chain reaction (PCR) as a diagnostic tool. Recent vaccination could induce positive results if the targeted DNA fragment is present within the vaccine and in the blood of the recently vaccinated dog. The objective of this study was to assess whether 2 available 4-serovar vaccines induce a positive real-time PCR reaction in the blood of healthy recently vaccinated dogs. Twenty healthy dogs. This was a prospective study. Dogs were assigned to 1 of 2 vaccine groups. Both vaccines were culture-based and include Leptospira interrogans serovars Pomona, Canicola, and Icterohaemorrhagiae and Leptospira kirschneri serovar Grippotyphosa. Whole blood for real-time PCR and serum for the microscopic agglutination test (MAT) were collected prior to and 3 and 7 days after vaccination and weekly thereafter for 8 weeks. Two real-time PCR tests targeting 2 different genes were performed independently in a blinded fashion. Both Leptospira vaccines produced positive real-time PCR reactions when assayed undiluted or diluted 1 : 100 in canine blood. However, blood samples drawn from all dogs at all time points after vaccination were negative on PCR. All dogs developed MAT titers. Recent vaccination with 2 commercially available vaccines does not interfere with the use of real-time PCR for the identification of acute Leptospira infection in dogs. Copyright © 2011 by the American College of Veterinary Internal Medicine.

  18. [Laboratory diagnosis of Treponema pallidum infection in patients with early syphilis and neurosyphilis through a PCR-based test].

    PubMed

    García, Patricia; Grassi, Bruno; Fich, Félix; Salvo, Aurelio; Araya, Luis; Abarzúa, Fernando; Soto, Julia; Poggi, Helena; Lagos, Marcela; Vásquez, Patricia; León, Eugenia P; Pérez, Carlos; Wozniak, Aniela

    2011-08-01

    Syphilis is a sexually transmitted disease caused by Treponema pallidum. The diagnosis is based mainly in clinical presentation and non-specific assays. PCR-based diagnosis has been suggested as an attractive alternative method. The aim of this study was the validation of a PCR-based test for the diagnosis of early syphilis (ES) and neurosyphilis (NS). Clinical samples of mucocutaneous lesions and cerebrospinal fluid (CSF) specimens from patients previously diagnosed for ES and NS respectively using an enlarged gold standard, were tested by PCR. The reaction was done using primers targeting the tpN47 gene. Twenty out of 21 mucocutaneous samples from patients diagnosed with ES were positive by PCR, with a clinical sensitivity of 95%. Four out of 8 CSF samples from patients previously diagnosed with NS were positive by PCR, with a clinical sensitivity of 50%. The clinical specificity for both ES and NS was 100%. The PCR sensitivity and specificity for mucocutaneous samples allowed us to implement this assay in our laboratory for routine diagnosis. Although the sensitivity of the PCR in CSF was low, it may be useful to support clinical diagnosis.

  19. Screening biological stains with qPCR versus lateral flow immunochromatographic test strips: a quantitative comparison using analytical figures of merit.

    PubMed

    Oechsle, Crystal Simson; Haddad, Sandra; Sgueglia, Joanne B; Grgicak, Catherine M

    2014-01-01

    Biological fluid identification is an important facet of evidence examination in forensic laboratories worldwide. While identifying bodily fluids may provide insight into which downstream DNA methods to employ, these screening techniques consume a vital portion of the available evidence, are usually qualitative, and rely on visual interpretation. In contrast, qPCR yields information regarding the amount and proportion of amplifiable genetic material. In this study, dilution series of either semen or male saliva were prepared in either buffer or female blood. The samples were subjected to both lateral flow immunochromatographic test strips and qPCR analysis. Analytical figures of merit-including sensitivity, minimum distinguishable signal (MDS) and limit of detection (LOD)-were calculated and compared between methods. By applying the theory of the propagation of random errors, LODs were determined to be 0.05 μL of saliva for the RSID™ Saliva cards, 0.03 μL of saliva for Quantifiler(®) Duo, and 0.001 μL of semen for Quantifiler(®) Duo. In conclusion, quantitative PCR was deemed a viable and effective screening method for subsequent DNA profiling due to its stability in different matrices, sensitivity, and low limits of detection.

  20. [Sensitivity and specificity of nested PCR pyrosequencing in hepatitis B virus drug resistance gene testing].

    PubMed

    Sun, Shumei; Zhou, Hao; Zhou, Bin; Hu, Ziyou; Hou, Jinlin; Sun, Jian

    2012-05-01

    To evaluate the sensitivity and specificity of nested PCR combined with pyrosequencing in the detection of HBV drug-resistance gene. RtM204I (ATT) mutant and rtM204 (ATG) nonmutant plasmids mixed at different ratios were detected for mutations using nested-PCR combined with pyrosequencing, and the results were compared with those by conventional PCR pyrosequencing to analyze the linearity and consistency of the two methods. Clinical specimens with different viral loads were examined for drug-resistant mutations using nested PCR pyrosequencing and nested PCR combined with dideoxy sequencing (Sanger) for comparison of the detection sensitivity and specificity. The fitting curves demonstrated good linearity of both conventional PCR pyrosequencing and nested PCR pyrosequencing (R(2)>0.99, P<0.05). Nested PCR showed a better consistency with the predicted value than conventional PCR, and was superior to conventional PCR for detection of samples containing 90% mutant plasmid. In the detection of clinical specimens, Sanger sequencing had a significantly lower sensitivity than nested PCR pyrosequencing (92% vs 100%, P<0.01). The detection sensitivity of Sanger sequencing varied with the viral loads, especially in samples with low viral copies (HBV DNA ≤3log10 copies/ml), where the sensitivity was 78%, significantly lower than that of pyrosequencing (100%, P<0.01). Neither of the two methods yielded positive results for the negative control samples, suggesting their good specificity. Compared with nested PCR and Sanger sequencing method, nested PCR pyrosequencing has a higher sensitivity especially in clinical specimens with low viral copies, which can be important for early detection of HBV mutant strains and hence more effective clinical management.

  1. Comparison of a Fur Mite PCR Assay and the Tape Test for Initial and Posttreatment Diagnosis during a Natural Infection

    PubMed Central

    Weiss, Erica E; Evans, Kristin D; Griffey, Stephen M

    2012-01-01

    Fur mites were diagnosed in a colony of mice at our research institution. In the current study, we compared the effectiveness of PCR and tape test in a small population of mice at the onset of diagnosis and throughout treatment. Samples were collected 1 d prior to treatment with permethrin impregnated cotton balls and 6 and 12 wk after treatment. PCR confirmed the presence of Myocoptes musculinus and Radfordia affinis or Myobia musculi, but tape test confirmed only the presence of Myocoptes spp. The results of the PCR and tape test agreed 97.2% of the time during active infection on day 1, but only 59.5% and 48.4% of results coincided at 6 and 12 wk after treatment, respectively. At 6 wk, 11 of the 37 samples were PCR-negative but tape-test–positive, compared with 9 of the 31 samples at 12 wk. Our results show that PCR is a reliable diagnostic method during active fur mite infection but that false-negative results are possible after treatment. Negative PCR results after treatment should be interpreted carefully, and a secondary diagnostic method should be considered. PMID:23312085

  2. Novel methylation specific real-time PCR test for the diagnosis of Klinefelter syndrome

    PubMed Central

    Mehta, Akanksha; Mielnik, Anna; Schlegel, Peter N; Paduch, Darius A

    2014-01-01

    The aim of this study was to design a molecular assay for the diagnosis of Klinefelter syndrome (KS), based on the detection of supernumerary X-chromosomes (X-chs). DNA was extracted from peripheral blood samples of twenty-six 47,XXY males; two 46,XY/47,XXY males; twenty-two 46,XY males; and 15 females; and deaminated. Methylation-specific quantitative polymerase chain reaction (MS-qPCR) was performed using primers for unmethylated and methylated copies of the X-ch inactive-specific transcript (XIST-U and XIST-M) gene. X-ch disomy was determined on the basis of XIST methylation status. Degree of mosaicism in the 46,XY/47,XXY males was compared with karyotype and fluorescent in situ hybridization (FISH) results. Data analysis was performed using the Roche® LightCycler software V. 3.5.3., including determination of crossing points (CPs) by fit-point analysis and melting curve analysis. X-ch disomy was detected in all female controls and KS patients; male controls expressed XIST-M only. CPs ranged from 29.5 to 32.5 (standard deviation (s.d.) 0.8) for XIST-U and from 29 to 31 (s.d. 0.6) for XIST-M. Limit of detection of mosaicism was 1%. Based on XIST-U/XIST-M ratios for the two 47,XXY/46,XY patients, the calculated degree of mosaicism (1.8% and 17.8%) was comparable to FISH results (2.3% and 15%, respectively). Turnaround time from DNA deamination to final data analysis was under 9 h. We conclude that MS-qPCR is a sensitive, specific and rapid test for the detection of X-ch disomy, with applicability for the screening and diagnosis of KS, even in the setting of low grade 47,XXY/46,XY mosaicism. PMID:24923458

  3. Comparison of N95 disposable filtering facepiece fits using bitrex qualitative and TSI Portacount quantitative fit testing.

    PubMed

    Clapham, S J

    2000-01-01

    As a means of evaluating the use of denatonium benzoate (bitrex) as a qualitative fit test agent with filtering facepiece respirators, the bitrex qualitative and TSI Protacount(R) quantitative fit-test methods were compared using N95 filtering facepieces. Seventy-nine paired tests (trial) were performed. Detection of bitrex during a qualitative fit test or measurement of a fit factor of <100 during a quantitative fit test constituted a failure. Qualitative and quantitative methods were performed using identical test protocols. Data were analyzed using pass/fail criteria, and matched-pair analysis methods were applied. The results of this study indicate that the use of bitrex during qualitative fit testing of N95 disposable filtering facepieces results in an increase in failure and/or rejection in cases where a TSI Portacount (plus N95 companion accessory) quantitatively establishes an acceptable fit.

  4. [PCR testing for Bordetella pertussis in household contacts as a diagnostic tool for atypical whooping cough in unvaccinated young infants].

    PubMed

    Cosnes-Lambe, Cecile; Raymond, Josette; Vallet, Christelle; Armengaud, Jean-Baptiste; Bosdure, Emmanuelle; Catalano-Pons, Charlotte; Chalumeau, Martin; El Hajje, Marie-Joelle; Moulin, Florence; de Suremain, Nathalie; Reglier-Poupet, Hélène; Poyart, Claire; Gendrel, Dominique

    2008-10-01

    False-negative findings of polymerase chain reaction (PCR) for genuine pertussis as well as the numerous atypical forms of whooping cough make it difficult to diagnose this disease in young babies. For two years, real-time PCR was performed to test for Bordetella pertussis in 86 infants younger than 6 months hospitalized for apnea or paroxysmal and/or vomiting cough and in 205 of their household contacts, whether or not they coughed. Group 1 included 30 infants for whom PCR detected B. pertussis (25 of whom were also RSV+). PCR was also positive for at least one household contact in 25/30 families. This group included 16 babies with apnea and 12 who developed a whooping cough during follow-up. Group 2 comprised 12 infants whose PCR was negative while at least one household contact had positive results. Five of these infants had severe apnea and 6 developed a whooping cough. Group 3 included 44 infants (28 RSV +) for whom PCR was negative in the index case and in the household contacts: none developed a whooping cough during follow-up. Only 3 of the 54 positive household contacts had a paroxysmal cough or a typical whooping cough and 12 had no cough at all. Positive PCR in a household contact, symptomatic or not, is helpful for the diagnosis of atypical whooping cough in young infants.

  5. False negative PCR despite high levels of JC virus DNA in spinal fluid: Implications for diagnostic testing.

    PubMed

    Landry, Marie L; Eid, Tore; Bannykh, Serguei; Major, Eugene

    2008-10-01

    Genome amplification methods such as polymerase chain reaction (PCR) have revolutionized our ability to detect viruses in spinal fluids of patients with neurologic diseases. It is not as well appreciated among clinicians that PCR protocols, quality assurance, and technical expertise vary significantly among laboratories. In a multi-laboratory blinded study of herpes simplex virus PCR, the most widely used and best validated CSF PCR assay, low-level positives were often missed and false positives were not uncommon [Schloss L, van Loon AM, Cinque P, Cleator G, Echevarria JM, Falk KI, et al. An international external quality assessment of nucleic acid amplification of herpes simplex virus. J Clin Virol 2003;28(2):175-85]. In addition, genome variability and mutations, which are increasingly recognized for a number of different viruses, can lead to falsely low or negative results. Both clinicians and laboratories must recognize the limitations of PCR, since misleading results may have serious consequences. We present here a case of a rapidly progressive, fatal neurologic illness in a young mother, whose CSF JCV DNA PCR at a reference laboratory was falsely negative. Ultimately, brain biopsy established the diagnosis of progressive multifocal leukoencephalopathy (PML). Repeat PCR testing of the same CSF targeting a different region of the genome yielded a high positive result.

  6. Using probit regression to disclose the analytical performance of qualitative and semi-quantitative tests.

    PubMed

    Åsberg, Arne; Johnsen, Harald; Mikkelsen, Gustav; Hov, Gunhild Garmo

    2016-11-01

    The analytical performance of qualitative and semi-quantitative tests is usually studied by calculating the fraction of positive results after replicate testing of a few specimens with known concentrations of the analyte. We propose using probit regression to model the probability of positive results as a function of the analyte concentration, based on testing many specimens once with a qualitative and a quantitative test. We collected laboratory data where urine specimens had been analyzed by both a urine albumin ('protein') dipstick test (Combur-Test strips) and a quantitative test (BN ProSpec System). For each dipstick cut-off level probit regression was used to estimate the probability of positive results as a function of urine albumin concentration. We also used probit regression to estimate the standard deviation of the continuous measurement signal that lies behind the binary test response. Finally, we used probit regression to estimate the probability of reading a specific semi-quantitative dipstick result as a function of urine albumin concentration. Based on analyses of 3259 specimens, the concentration of urine albumin with a 0.5 (50%) probability of positive result was 57 mg/L at the lowest possible cut-off limit, and 246 and 750 mg/L at the next (higher) levels. The corresponding standard deviations were 29, 83, and 217 mg/L, respectively. Semi-quantitatively, the maximum probability of these three readings occurred at a u-albumin of 117, 420, and 1200 mg/L, respectively. Probit regression is a useful tool to study the analytical performance of qualitative and semi-quantitative tests.

  7. Comparison of quantitative and qualitative tests for glucose-6-phosphate dehydrogenase deficiency in the neonatal period.

    PubMed

    Keihanian, F; Basirjafari, S; Darbandi, B; Saeidinia, A; Jafroodi, M; Sharafi, R; Shakiba, M

    2017-06-01

    Considering the high prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency among newborns, different screening methods have been established in various countries. In this study, we aimed to assess the prevalence of G6PD deficiency among newborns in Rasht, Iran, and compare G6PD activity in cord blood samples, using quantitative and qualitative tests. This cross-sectional, prospective study was performed at five largest hospitals in Rasht, Guilan Province, Iran. The screening tests were performed for all the newborns, referred to these hospitals. Specimens were characterized in terms of G6PD activity under ultraviolet light, using the kinetic method and the qualitative fluorescent spot test (FST). We also determined the sensitivity, specificity, negative predictive value, and positive predictive value of the qualitative assay. Blood samples were collected from 1474 newborns. Overall, 757 (51.4%) subjects were male. As the findings revealed, 1376 (93.4%) newborns showed normal G6PD activity, while 98 (6.6%) had G6PD deficiency. There was a significant difference in the mean G6PD level between males and females (P = 0.0001). Also, a significant relationship was detected between FST results and the mean values obtained in the quantitative test (P < 0.0001). According to the present study, FST showed acceptable sensitivity and specificity for G6PD activity, although it appeared inefficient for diagnostic purposes in some cases. © 2017 John Wiley & Sons Ltd.

  8. PCR detection of cytomegalovirus DNA in serum as a diagnostic test for congenital cytomegalovirus infection.

    PubMed Central

    Nelson, C T; Istas, A S; Wilkerson, M K; Demmler, G J

    1995-01-01

    PCR detected cytomegalovirus (CMV) DNA in the serum of 18 of 18 infants with symptomatic congenital CMV infection, 1 of 2 infants with asymptomatic congenital CMV infection, and 0 of 32 controls. Serum CMV PCR provided a rapid, sensitive, and specific method for diagnosis of congenital CMV infection in infants who were symptomatic at birth. PMID:8586726

  9. Production, Extraction, and Qualitative Testing of Penicillin: A Biochemistry Experiment for Health Science Chemistry Courses

    NASA Astrophysics Data System (ADS)

    Stevens, Richard E.; Billingsley, Kara C.

    1998-10-01

    This laboratory procedure guides students through the growth of a submerged Penicillium chrysogenum culture. Subsequent steps include extraction of the penicillin by adsorption onto activated charcoal, extraction with acetone, and qualitative testing of the drug on a bacterial culture. The laboratory procedure is designed for freshman-level health science chemistry courses. This procedure produces minimal waste, which can be disposed of by the appropriate use of an autoclave.

  10. Evaluation of a rapid fecal PCR test for detection of Mycobacterium avium subsp. paratuberculosis in dairy cattle.

    PubMed

    Wells, Scott J; Collins, Michael T; Faaberg, Kay S; Wees, Carrie; Tavornpanich, Saraya; Petrini, Kristine R; Collins, James E; Cernicchiaro, Natalia; Whitlock, Robert H

    2006-10-01

    A high-throughput TaqMan PCR assay for detection of bovine paratuberculosis was evaluated by using fecal samples from 1,808 dairy cattle in seven naturally infected herds and 347 dairy cattle in seven herds considered free of paratuberculosis. Fecal, blood, and milk samples were submitted to laboratories where the PCR-based assay, three different fecal culture procedures for Mycobacterium avium subsp. paratuberculosis (centrifugation, sedimentation, and the BACTEC filter concentration method), two serologic enzyme-linked immunosorbent assays (ELISAs), and one milk ELISA were performed. Results from testing of dairy cattle in herds free of M. avium subsp. paratuberculosis showed that the PCR assay's specificity was 99.7%. Twenty-three percent of the dairy cows that were fecal culture positive by at least one of the three methods were positive by the PCR assay. By Bayesian non-"gold standard" analysis methods, the TaqMan PCR assay had a higher specificity than the serum ELISAs (99.3%; 95% confidence interval [CI]=98.6 to 99.7%) and a test sensitivity similar to that of the serum ELISAs (29%; 95% CI=24 to 35%). By classical methods, the estimated relative sensitivity of the fecal PCR assay was 4% for light and moderate fecal shedders (compared to 12 to 13% for the ELISAs) and 76% for heavy fecal shedders (compared to 67% for the milk ELISA). The PCR assay has higher sensitivity for detection of heavy fecal shedders than the evaluated milk ELISA but lower sensitivity than a serum or milk ELISA for detection of light and moderate fecal shedders. This assay can be used as a quick test for detection of cattle with heavy fecal shedding, those cattle with the highest risk of transmitting infection to susceptible cattle.

  11. Development of novel noninvasive prenatal testing protocol for whole autosomal recessive disease using picodroplet digital PCR

    PubMed Central

    Chang, Mun Young; Kim, Ah Reum; Kim, Min Young; Kim, Soyoung; Yoon, Jinsun; Han, Jae Joon; Ahn, Soyeon; Kang, Changsoo; Choi, Byung Yoon

    2016-01-01

    We developed a protocol of noninvasive prenatal testing (NIPT), employing a higher-resolution picodroplet digital PCR, to detect genetic imbalance in maternal plasma DNA (mpDNA) caused by cell-free fetal DNA (cffDNA). In the present study, this approach was applied to four families with autosomal recessive (AR) congenital sensorineural hearing loss. First, a fraction of the fetal DNA in mpDNA was calculated. Then, we made artificial DNA mixtures (positive and negative controls) to simulate mpDNA containing the fraction of cffDNA with or without mutations. Next, a fraction of mutant cluster signals over the total signals was measured from mpDNA, positive controls, and negative controls. We determined whether fetal DNA carried any paternal or maternal mutations by calculating and comparing the sum of the log-likelihood of the study samples. Of the four families, we made a successful prediction of the complete fetal genotype in two cases where a distinct cluster was identified for each genotype and the fraction of cffDNA in mpDNA was at least 6.4%. Genotyping of only paternal mutation was possible in one of the other two families. This is the first NIPT protocol potentially applicable to any AR monogenic disease with various genotypes, including point mutations. PMID:27924908

  12. ‘I’m fishing really’ — inflammatory marker testing in primary care: a qualitative study

    PubMed Central

    Watson, Jessica; de Salis, Isabel; Hamilton, Willie; Salisbury, Chris

    2016-01-01

    Background Inflammatory markers can be helpful as part of the diagnostic workup for specific diseases or for monitoring disease activity. A third use is as a screening and/or triage tool to differentiate between the presence or absence of disease. Most research into inflammatory markers looks at diagnosis of specific diseases and comes from secondary care. Qualitative studies to explore when and why clinicians use these tests in primary care are lacking. Aim To identify clinicians’ approaches to inflammatory marker testing in primary care. Design and setting Qualitative study with 26 GPs and nurse practitioners. Method Interviews were conducted using a semi-structured topic guide. Clinicians reviewed recent cases of inflammatory marker testing in their pathology inbox. Interviews were audiorecorded and transcribed. Qualitative analysis was conducted by two of the authors. Results Clinicians are uncertain about the appropriate use of inflammatory markers and differ in their approach to testing patients with undifferentiated symptoms. Normal or significantly elevated inflammatory markers are seen as helpful, but mildly raised inflammatory markers in the context of non-specific symptoms are difficult to interpret. Clinicians describe a tension between not wanting to ‘miss anything’ and, on the other hand, being wary of picking up borderline abnormalities that can lead to cascades of further tests. Diagnostic uncertainty is a common reason for inflammatory marker testing, with the aim to reassure; however, paradoxically, inconclusive results can generate a cycle of uncertainty and anxiety. Conclusion Further research is needed to define when inflammatory marker testing is useful in primary care and how to interpret results. PMID:26852797

  13. 'I'm fishing really'--inflammatory marker testing in primary care: a qualitative study.

    PubMed

    Watson, Jessica; de Salis, Isabel; Hamilton, Willie; Salisbury, Chris

    2016-03-01

    Inflammatory markers can be helpful as part of the diagnostic workup for specific diseases or for monitoring disease activity. A third use is as a screening and/or triage tool to differentiate between the presence or absence of disease. Most research into inflammatory markers looks at diagnosis of specific diseases and comes from secondary care. Qualitative studies to explore when and why clinicians use these tests in primary care are lacking. To identify clinicians' approaches to inflammatory marker testing in primary care. Qualitative study with 26 GPs and nurse practitioners. Interviews were conducted using a semi-structured topic guide. Clinicians reviewed recent cases of inflammatory marker testing in their pathology inbox. Interviews were audiorecorded and transcribed. Qualitative analysis was conducted by two of the authors. Clinicians are uncertain about the appropriate use of inflammatory markers and differ in their approach to testing patients with undifferentiated symptoms. Normal or significantly elevated inflammatory markers are seen as helpful, but mildly raised inflammatory markers in the context of non-specific symptoms are difficult to interpret. Clinicians describe a tension between not wanting to 'miss anything' and, on the other hand, being wary of picking up borderline abnormalities that can lead to cascades of further tests. Diagnostic uncertainty is a common reason for inflammatory marker testing, with the aim to reassure; however, paradoxically, inconclusive results can generate a cycle of uncertainty and anxiety. Further research is needed to define when inflammatory marker testing is useful in primary care and how to interpret results. © British Journal of General Practice 2016.

  14. An extremely sensitive species-specific ARMS PCR test for the presence of tiger bone DNA.

    PubMed

    Wetton, Jon H; Tsang, Carol S F; Roney, Chris A; Spriggs, Adrian C

    2002-04-18

    The survival of the tiger (Panthera tigris) is seriously threatened by poaching to provide raw materials for traditional Chinese medicines (TCMs). Most highly prized are the tiger's bones, which are used in combination with other animal and plant derivatives in pills and plasters for the treatment of rheumatism and other ailments. Hundreds of patent remedies have been produced which claim to contain tiger bone, but proof of its presence is needed if legislation prohibiting the trade in endangered species is to be enforced.A highly sensitive tiger-specific real-time PCR assay has been developed to address this problem. Using primers specific to the tiger mitochondrial cytochrome b gene, successful amplification has been reliably achieved from blood, hair and bone as well as from a range of TCMs spiked with 0.5% tiger bone. Although capable of detecting fewer than 10 substrate molecules, the seven varieties of TCM pills and plasters tested showed no detectable trace of tiger DNA before spiking. Furthermore, sequencing several "tiger bone" fragments seized from TCM shops has shown that they actually originated from cattle and pigs. The potential effects of traditional bone preparation methods, evidence that much lower concentrations are used than alleged on TCM packaging, and substitution of bones from other species all suggest a low likelihood of detecting tiger DNA in patent medicines. Despite this, the basic methods have been thoroughly proven and can be readily applied to derivatives from other Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) protected species, providing a rapid and highly sensitive forensic test for species of origin. Potential applications to the monitoring of wild populations are demonstrated by the successful identification of shed hairs and faecal samples.

  15. Fast test for assessing the susceptibility of Mycobacterium tuberculosis to isoniazid and rifampin by real-time PCR

    PubMed Central

    Gonçalves, Maria Gisele; Fukasawa, Lucila Okuyama; Oliveira, Rosangela Siqueira; Salgado, Maristela Marques; Harrison, Lee H; Shutt, Kathleen A; Sacchi, Claudio Tavares

    2015-01-01

    Mycobacterium tuberculosis is the bacterium that causes tuberculosis (TB), a leading cause of death from infectious disease worldwide. Rapid diagnosis of resistant strains is important for the control of TB. Real-time polymerase chain reaction (RT-PCR) assays may detect all of the mutations that occur in the M. tuberculosis 81-bp core region of the rpoB gene, which is responsible for resistance to rifampin (RIF) and codon 315 of the katG gene and the inhA ribosomal binding site, which are responsible for isoniazid (INH). The goal of this study was to assess the performance of RT-PCR compared to traditional culture-based methods for determining the drug susceptibility of M. tuberculosis. BACTEC™ MGIT™ 960 was used as the gold standard method for phenotypic drug susceptibility testing. Susceptibilities to INH and RIF were also determined by genotyping of katG, inhA and rpoB genes. RT-PCR based on molecular beacons probes was used to detect specific point mutations associated with resistance. The sensitivities of RT-PCR in detecting INH resistance using katG and inhA targets individually were 55% and 25%, respectively and 73% when combined. The sensitivity of the RT-PCR assay in detecting RIF resistance was 99%. The median time to complete the RT-PCR assay was three-four hours. The specificities for tests were both 100%. Our results confirm that RT-PCR can detect INH and RIF resistance in less than four hours with high sensitivity. PMID:23147147

  16. Non-Invasive Cytology Brush PCR Diagnostic Testing in Mucosal Leishmaniasis: Superior Performance to Conventional Biopsy with Histopathology

    PubMed Central

    Veland, Nicolas; Pilar Ramos, Ana; Calderon, Flor; Arevalo, Jorge; Low, Donald E.; Llanos-Cuentas, Alejandro

    2011-01-01

    Background Traditional methods of diagnosing mucosal leishmaniasis (ML), such as biopsy with histopathology, are insensitive and require collection of an invasive diagnostic specimen. Methods We compared standard invasive procedures including biopsy histopathology, biopsy PCR, and leishmanin skin test (LST) to a novel, non-invasive, cytology-brush based PCR for the diagnosis of ML in Lima, Peru. Consensus reference standard was 2/4 tests positive, and outcome measures were sensitivity and specificity. Leishmania species identification was performed by PCR-based assays of positive specimens. Results Twenty-eight patients were enrolled, 23 of whom fulfilled criteria for a diagnosis of ML. Sensitivity and specificity of biopsy with histopathology were 21.7% [95% CI 4.9–38.5%] and 100%; 69.6% [95% CI 50.8–88.4%] and 100% for LST; 95.7% [95% CI 87.4–100%] and 100% for biopsy PCR; and 95.7% [95% CI 87.4–100%] and 90% [95% CI 71.4–100%] for cytology brush PCR using both Cervisoft® and Histobrush® cervical cytology brushes. Represented species identified by PCR-RFLP included: L. (V). braziliensis (n = 4), and L. (V). peruviana (n = 3). Conclusions Use of commercial grade cytology brush PCR for diagnosis of ML is sensitive, rapid, well tolerated, and carries none of the risks of invasive diagnostic procedures such as biopsy. Further optimization is required for adequate species identification. Further evaluation of this method in field and other settings is warranted. PMID:22046280

  17. Comparison of pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus PCR and biochemical tests to characterize Lactococcus garvieae.

    PubMed

    Ture, M; Altinok, I; Capkin, E

    2015-01-01

    Biochemical test, pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) were used to compare 42 strains of Lactococcus garvieae isolated from different regions of Turkey, Italy, France and Spain. Twenty biotypes of L. garvieae were formed based on 54 biochemical tests. ERIC-PCR of genomic DNA from different L. garvieae strains resulted in amplification of multiple fragments of DNA in sizes ranging between 200 and 5000 bp with various band intensities. After cutting DNA with ApaI restriction enzyme and running on the PFGE, 11–22 resolvable bands ranging from 2 to 194 kb were observed. Turkish isolates were grouped into two clusters, and only A58 (Italy) strain was connected with Turkish isolates. Similarities between Turkish, Spanish, Italian and French isolates were <50% except 216-6 Rize strain. In Turkey, first lactococcosis occurred in Mugla, and then, it has been spread all over the country. Based on ERIC-PCR, Spanish and Italian strains of L. garvieae were related to Mugla strains. Therefore, after comparing PFGE profiles, ERIC-PCR profiles and phenotypic characteristics of 42 strains of L. garvieae, there were no relationships found between these three typing methods. PFGE method was more discriminative than the other methods. © 2014 John Wiley & Sons Ltd.

  18. Evaluation of Quantitative Real-Time PCR as a Hepatitis C Virus Supplementary Test After RIBA Discontinuation.

    PubMed

    Gong, Shunyou; Schmotzer, Christine L; Zhou, Lan

    2016-09-01

    Laboratory testing plays a major role in hepatitis C virus (HCV) diagnosis and patient follow-up. The high false positive rates of HCV screening tests require confirmation through a supplementary test. According to the 2003 CDC guidelines, recombinant immunoblot assay (RIBA) is indispensible to confirm positive screening results and differentiate biologic false positivity from true HCV exposure. However, RIBA has been permanently discontinued since 2011. In the 2013 update of its guidelines, CDC called for further studies on HCV laboratory testing without RIBA. In this study, we analyzed the applicability of quantitative real-time PCR (qPCR) as a supplementary HCV diagnostic test. By comparing our HCV testing performances before and after RIBA discontinuation, we found that omitting RIBA has no significant effect on the accurate and efficient identification of HCV infection, provided that HCV antibody signal-to-cutoff ratio is considered. Furthermore, we proposed a new HCV testing algorithm that incorporates semiquantitative assessment of HCV antibody positivity and HCV viral load measurement by qPCR. By following the algorithm, we were able to address confirmation of positive HCV screening results and to provide useful information generally required by clinicians, including the needs of further laboratory testing or clinical follow-up, as well as HCV viral titers.

  19. Systematic review and meta-analysis: rapid diagnostic tests versus placental histology, microscopy and PCR for malaria in pregnant women

    PubMed Central

    2011-01-01

    Background During pregnancy, malaria infection with Plasmodium falciparum or Plasmodium vivax is related to adverse maternal health and poor birth outcomes. Diagnosis of malaria, during pregnancy, is complicated by the absence or low parasite densities in peripheral blood. Diagnostic methods, other than microscopy, are needed for detection of placental malaria. Therefore, the diagnostic accuracy of rapid diagnostic tests (RDTs), detecting antigen, and molecular techniques (PCR), detecting DNA, for the diagnosis of Plasmodium infections in pregnancy was systematically reviewed. Methods MEDLINE, EMBASE and Web of Science were searched for studies assessing the diagnostic accuracy of RDTs, PCR, microscopy of peripheral and placental blood and placental histology for the detection of malaria infection (all species) in pregnant women. Results The results of 49 studies were analysed in metandi (Stata), of which the majority described P. falciparum infections. Although both placental and peripheral blood microscopy cannot reliably replace histology as a reference standard for placental P. falciparum infection, many studies compared RDTs and PCR to these tests. The proportion of microscopy positives in placental blood (sensitivity) detected by peripheral blood microscopy, RDTs and PCR are respectively 72% [95% CI 62-80], 81% [95% CI 55-93] and 94% [95% CI 86-98]. The proportion of placental blood microscopy negative women that were negative in peripheral blood microscopy, RDTs and PCR (specificity) are 98% [95% CI 95-99], 94% [95% CI 76-99] and 77% [95% CI 71-82]. Based on the current data, it was not possible to determine if the false positives in RDTs and PCR are caused by sequestered parasites in the placenta that are not detected by placental microscopy. Conclusion The findings suggest that RDTs and PCR may have good performance characteristics to serve as alternatives for the diagnosis of malaria in pregnancy, besides any other limitations and practical considerations

  20. Comparison of quantitative and qualitative tests for glucose-6-phosphate dehydrogenase deficiency.

    PubMed

    LaRue, Nicole; Kahn, Maria; Murray, Marjorie; Leader, Brandon T; Bansil, Pooja; McGray, Sarah; Kalnoky, Michael; Zhang, Hao; Huang, Huiqiang; Jiang, Hui; Domingo, Gonzalo J

    2014-10-01

    A barrier to eliminating Plasmodium vivax malaria is inadequate treatment of infected patients. 8-Aminoquinoline-based drugs clear the parasite; however, people with glucose-6-phosphate dehydrogenase (G6PD) deficiency are at risk for hemolysis from these drugs. Understanding the performance of G6PD deficiency tests is critical for patient safety. Two quantitative assays and two qualitative tests were evaluated. The comparison of quantitative assays gave a Pearson correlation coefficient of 0.7585 with significant difference in mean G6PD activity, highlighting the need to adhere to a single reference assay. Both qualitative tests had high sensitivity and negative predictive value at a cutoff G6PD value of 40% of normal activity if interpreted conservatively and performed under laboratory conditions. The performance of both tests dropped at a cutoff level of 45%. Cytochemical staining of specimens confirmed that heterozygous females with > 50% G6PD-deficient cells can seem normal by phenotypic tests. © The American Society of Tropical Medicine and Hygiene.

  1. Evaluation of primer and probe mismatches in sensitivity of select RRT-PCR tests for avian influenza

    USDA-ARS?s Scientific Manuscript database

    The recent outbreak of pH1N1 in animals highlighted an imperfection of the matrix real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) that has become the primary screening test for avian and swine influenza viruses. Four mismatches in one primer resulted in an important loss of sens...

  2. [Comparison of the CMV antigenemia test and CMV-DNA PCR results in solid organ transplant recipients].

    PubMed

    Özkarata, Emre; Özkarataş, Emre; Özbek, Ö Alpay; Avkan Oğuz, Vildan; Sayıner, A Arzu

    2016-01-01

    Cytomegalovirus (CMV) infection is among the most common important viral infections in solid organ transplant (SOT) recipients. Diagnostic tests for detecting CMV replication are widely used for this group of patients, however there is no clear agreement on the cut-off levels for interpretation of clinical decisions especially when the low level of viral load is detected. In this study, CMV pp65 antigenemia test results were compared with plasma CMV-DNA levels detected by quantitative real-time polymerase chain reaction (qPCR) in samples of kidney and liver transplant recipients in the Central Laboratory of Dokuz Eylul University Hospital between 2011 and 2013, and the correlation between these two tests and viral load equivalent to antigenemia positivity were determined. In the study, pp65 antigenemia and CMV-DNA qPCR results were evaluated retrospectively. The samples from the same patients were included if the time between antigenemia and CMV-DNA qPCR tests were less than 48 hours. SPSS v15.0 was used for correlation, regression and ROC curve analysis. The results of the 217 samples collected from 100 patients (59 male, 41 female; age range: 16-71, mean age: 46 ± 13 years), 36 liver and 64 kidney recipients were evaluated in the study. Of the patients 80% were CMV IgM negative, IgG positive; 1% was CMV IgG and IgM positive; 2% were CMV IgM and IgG negative, while for 17 patients serological results could not be reached. CMV pp65 antigenemia and CMV-DNA were both negative in 102 (47%) samples, while both were positive in 37 (17%) samples. The single sample from a case with CMV IgM and IgG positivity yielded negative results for both antigenemia and CMV-DNA tests. In 78 samples antigenemia were negative and CMV-DNA qPCR were positive, while there were no samples with antigenemia positive and qPCR negative. Mean values of antigenemia and qPCR tests were 23 positive cells/200.000 leukocytes (range: 1 to 230 positive cells) and 12.595 copies/ml (range: 180 to 106

  3. Diagnostic Accuracy of PCR Alone and Compared to Urinary Antigen Testing for Detection of Legionella spp.: a Systematic Review

    PubMed Central

    Green, Hefziba; Steinmetz, Tali; Leibovici, Leonard; Paul, Mical

    2015-01-01

    The diagnosis of Legionnaires' disease (LD) is based on the isolation of Legionella spp., a 4-fold rise in antibodies, a positive urinary antigen (UA), or direct immunofluorescence tests. PCR is not accepted as a diagnostic tool for LD. This systematic review assesses the diagnostic accuracy of PCR in various clinical samples with a direct comparison versus UA. We included prospective or retrospective cohort and case-control studies. Studies were included if they used the Centers for Disease Control and Prevention consensus definition criteria of LD or a similar one, assessed only patients with clinical pneumonia, and reported data for all true-positive, false-positive, true-negative, and false-negative results. Two reviewers abstracted data independently. Risk of bias was assessed using Quadas-2. Summary sensitivity and specificity values were estimated using a bivariate model and reported with a 95% confidence interval (CI). Thirty-eight studies were included. A total of 653 patients had confirmed LD, and 3,593 patients had pneumonia due to other pathogens. The methodological quality of the studies as assessed by the Quadas-2 tool was poor to fair. The summary sensitivity and specificity values for diagnosis of LD in respiratory samples were 97.4% (95% CI, 91.1% to 99.2%) and 98.6% (95% CI, 97.4% to 99.3%), respectively. These results were mainly unchanged by any covariates tested and subgroup analysis. The diagnostic performance of PCR in respiratory samples was much better than that of UA. Compared to UA, PCR in respiratory samples (especially in sputum samples or swabs) revealed a significant advantage in sensitivity and an additional diagnosis of 18% to 30% of LD cases. The diagnostic performance of PCR in respiratory samples was excellent and preferable to that of the UA. Results were independent on the covariate tested. PCR in respiratory samples should be regarded as a valid tool for the diagnosis of LD. PMID:26659202

  4. Diagnostic Accuracy of PCR Alone and Compared to Urinary Antigen Testing for Detection of Legionella spp.: a Systematic Review.

    PubMed

    Avni, Tomer; Bieber, Amir; Green, Hefziba; Steinmetz, Tali; Leibovici, Leonard; Paul, Mical

    2016-02-01

    The diagnosis of Legionnaires' disease (LD) is based on the isolation of Legionella spp., a 4-fold rise in antibodies, a positive urinary antigen (UA), or direct immunofluorescence tests. PCR is not accepted as a diagnostic tool for LD. This systematic review assesses the diagnostic accuracy of PCR in various clinical samples with a direct comparison versus UA. We included prospective or retrospective cohort and case-control studies. Studies were included if they used the Centers for Disease Control and Prevention consensus definition criteria of LD or a similar one, assessed only patients with clinical pneumonia, and reported data for all true-positive, false-positive, true-negative, and false-negative results. Two reviewers abstracted data independently. Risk of bias was assessed using Quadas-2. Summary sensitivity and specificity values were estimated using a bivariate model and reported with a 95% confidence interval (CI). Thirty-eight studies were included. A total of 653 patients had confirmed LD, and 3,593 patients had pneumonia due to other pathogens. The methodological quality of the studies as assessed by the Quadas-2 tool was poor to fair. The summary sensitivity and specificity values for diagnosis of LD in respiratory samples were 97.4% (95% CI, 91.1% to 99.2%) and 98.6% (95% CI, 97.4% to 99.3%), respectively. These results were mainly unchanged by any covariates tested and subgroup analysis. The diagnostic performance of PCR in respiratory samples was much better than that of UA. Compared to UA, PCR in respiratory samples (especially in sputum samples or swabs) revealed a significant advantage in sensitivity and an additional diagnosis of 18% to 30% of LD cases. The diagnostic performance of PCR in respiratory samples was excellent and preferable to that of the UA. Results were independent on the covariate tested. PCR in respiratory samples should be regarded as a valid tool for the diagnosis of LD.

  5. A comparison of EGFR mutation testing methods in lung carcinoma: direct sequencing, real-time PCR and immunohistochemistry.

    PubMed

    Angulo, Bárbara; Conde, Esther; Suárez-Gauthier, Ana; Plaza, Carlos; Martínez, Rebeca; Redondo, Pilar; Izquierdo, Elisa; Rubio-Viqueira, Belén; Paz-Ares, Luis; Hidalgo, Manuel; López-Ríos, Fernando

    2012-01-01

    The objective of this study is to compare two EGFR testing methodologies (a commercial real-time PCR kit and a specific EGFR mutant immunohistochemistry), with direct sequencing and to investigate the limit of detection (LOD) of both PCR-based methods. We identified EGFR mutations in 21 (16%) of the 136 tumours analyzed by direct sequencing. Interestingly, the Therascreen EGFR Mutation Test kit was able to characterize as wild-type one tumour that could not be analyzed by direct sequencing of the PCR product. We then compared the LOD of the kit and that of direct sequencing using the available mutant tumours. The kit was able to detect the presence of a mutation in a 1% dilution of the total DNA in nine of the 18 tumours (50%), which tested positive with the real-time quantitative PCR method. In all cases, EGFR mutation was identified at a dilution of 5%. Where the mutant DNA represented 30% of the total DNA, sequencing was able to detect mutations in 12 out of 19 cases (63%). Additional experiments with genetically defined standards (EGFR ΔE746-A750/+ and EGFR L858R/+) yielded similar results. Immunohistochemistry (IHC) staining with exon 19-specific antibody was seen in eight out of nine cases with E746-A750del detected by direct sequencing. Neither of the two tumours with complex deletions were positive. Of the five L858R-mutated tumours detected by the PCR methods, only two were positive for the exon 21-specific antibody. The specificity was 100% for both antibodies. The LOD of the real-time PCR method was lower than that of direct sequencing. The mutation specific IHC produced excellent specificity.

  6. A Comparison of EGFR Mutation Testing Methods in Lung Carcinoma: Direct Sequencing, Real-time PCR and Immunohistochemistry

    PubMed Central

    Angulo, Bárbara; Conde, Esther; Suárez-Gauthier, Ana; Plaza, Carlos; Martínez, Rebeca; Redondo, Pilar; Izquierdo, Elisa; Rubio-Viqueira, Belén; Paz-Ares, Luis; Hidalgo, Manuel; López-Ríos, Fernando

    2012-01-01

    The objective of this study is to compare two EGFR testing methodologies (a commercial real-time PCR kit and a specific EGFR mutant immunohistochemistry), with direct sequencing and to investigate the limit of detection (LOD) of both PCR-based methods. We identified EGFR mutations in 21 (16%) of the 136 tumours analyzed by direct sequencing. Interestingly, the Therascreen EGFR Mutation Test kit was able to characterize as wild-type one tumour that could not be analyzed by direct sequencing of the PCR product. We then compared the LOD of the kit and that of direct sequencing using the available mutant tumours. The kit was able to detect the presence of a mutation in a 1% dilution of the total DNA in nine of the 18 tumours (50%), which tested positive with the real-time quantitative PCR method. In all cases, EGFR mutation was identified at a dilution of 5%. Where the mutant DNA represented 30% of the total DNA, sequencing was able to detect mutations in 12 out of 19 cases (63%). Additional experiments with genetically defined standards (EGFR ΔE746-A750/+ and EGFR L858R/+) yielded similar results. Immunohistochemistry (IHC) staining with exon 19-specific antibody was seen in eight out of nine cases with E746-A750del detected by direct sequencing. Neither of the two tumours with complex deletions were positive. Of the five L858R-mutated tumours detected by the PCR methods, only two were positive for the exon 21-specific antibody. The specificity was 100% for both antibodies. The LOD of the real-time PCR method was lower than that of direct sequencing. The mutation specific IHC produced excellent specificity. PMID:22952784

  7. Evaluation and Comparison of Multiple Test Methods, Including Real-time PCR, for Legionella Detection in Clinical Specimens

    PubMed Central

    Peci, Adriana; Winter, Anne-Luise; Gubbay, Jonathan B.

    2016-01-01

    Legionella is a Gram-negative bacterium that can cause Pontiac fever, a mild upper respiratory infection and Legionnaire’s disease, a more severe illness. We aimed to compare the performance of urine antigen, culture, and polymerase chain reaction (PCR) test methods and to determine if sputum is an acceptable alternative to the use of more invasive bronchoalveolar lavage (BAL). Data for this study included specimens tested for Legionella at Public Health Ontario Laboratories from 1st January, 2010 to 30th April, 2014, as part of routine clinical testing. We found sensitivity of urinary antigen test (UAT) compared to culture to be 87%, specificity 94.7%, positive predictive value (PPV) 63.8%, and negative predictive value (NPV) 98.5%. Sensitivity of UAT compared to PCR was 74.7%, specificity 98.3%, PPV 77.7%, and NPV 98.1%. Out of 146 patients who had a Legionella-positive result by PCR, only 66 (45.2%) also had a positive result by culture. Sensitivity for culture was the same using either sputum or BAL (13.6%); sensitivity for PCR was 10.3% for sputum and 12.8% for BAL. Both sputum and BAL yield similar results regardless testing methods (Fisher Exact p-values = 1.0, for each test). In summary, all test methods have inherent weaknesses in identifying Legionella; therefore, more than one testing method should be used. Obtaining a single specimen type from patients with pneumonia limits the ability to diagnose Legionella, particularly when urine is the specimen type submitted. Given ease of collection and similar sensitivity to BAL, clinicians are encouraged to submit sputum in addition to urine when BAL submission is not practical from patients being tested for Legionella. PMID:27630979

  8. Detection of feline upper respiratory tract disease pathogens using a commercially available real-time PCR test.

    PubMed

    Litster, A; Wu, C C; Leutenegger, C M

    2015-11-01

    Feline herpesvirus (FHV-1), feline calicivirus (FCV), Bordetella bronchiseptica (Bb), Chlamydia felis (Cf) and Mycoplasma felis (Mf) are common infectious agents identified in cats with upper respiratory tract disease (URTD). Each of these agents can either act as primary pathogens or cause subclinical infections, and pathogen identification can be used to prevent disease transmission in shelters, or to manage individual cats with recurrent URTD. The aim of this study was to compare pathogen detection rates using real-time PCR testing and virus isolation (VI) or bacterial culture in conjunctival, nasal and oropharyngeal swabs from 18 shelter-housed cats with clinical URTD. Co-infections were common; FHV-1 was most prevalent and Cf and FCV were least prevalent. Agents detected by PCR were FCV 2/18 (11%), FHV-1 17/18 (94%), Bb 8/18 (44%) and Mf 15/18 (83%). Agents detected by VI and bacterial culture were FCV 1/18 (6%), FHV-1 12/18 (67%), Bb 8/18 (44%) and Mf 12/18 (67%). Agreement between PCR results and the other two methods was: FHV-1, 57.4%; FCV, 98.1%; Bb, 75.0%; Mf, 60.0%. Discordancies included PCR-positive, VI-negative (FCV, n = 1/54, 1.9%; FHV-1, n = 23/54, 42.6%), PCR-positive, culture-negative (Bb, n = 6/36, 16.7%; Mf, n = 13/36, 36.1%) or PCR-negative, culture-positive (Bb, n = 3/36, 8.3%; Mf, n = 2/36, 5.6%) results. A combination of an oropharyngeal swab and either a conjunctival or a nasal swab submitted for PCR testing was able to detect all infectious agents tested for in each cat. PCR testing was a sensitive and convenient method of detection of infectious agents in cats with clinical signs of URTD.

  9. Direct Comparison of Flow-FISH and qPCR as Diagnostic Tests for Telomere Length Measurement in Humans

    PubMed Central

    Gutierrez-Rodrigues, Fernanda; Santana-Lemos, Bárbara A.; Scheucher, Priscila S.; Alves-Paiva, Raquel M.; Calado, Rodrigo T.

    2014-01-01

    Telomere length measurement is an essential test for the diagnosis of telomeropathies, which are caused by excessive telomere erosion. Commonly used methods are terminal restriction fragment (TRF) analysis by Southern blot, fluorescence in situ hybridization coupled with flow cytometry (flow-FISH), and quantitative PCR (qPCR). Although these methods have been used in the clinic, they have not been comprehensively compared. Here, we directly compared the performance of flow-FISH and qPCR to measure leukocytes' telomere length of healthy individuals and patients evaluated for telomeropathies, using TRF as standard. TRF and flow-FISH showed good agreement and correlation in the analysis of healthy subjects (R2 = 0.60; p<0.0001) and patients (R2 = 0.51; p<0.0001). In contrast, the comparison between TRF and qPCR yielded modest correlation for the analysis of samples of healthy individuals (R2 = 0.35; p<0.0001) and low correlation for patients (R2 = 0.20; p = 0.001); Bland-Altman analysis showed poor agreement between the two methods for both patients and controls. Quantitative PCR and flow-FISH modestly correlated in the analysis of healthy individuals (R2 = 0.33; p<0.0001) and did not correlate in the comparison of patients' samples (R2 = 0.1, p = 0.08). Intra-assay coefficient of variation (CV) was similar for flow-FISH (10.8±7.1%) and qPCR (9.5±7.4%; p = 0.35), but the inter-assay CV was lower for flow-FISH (9.6±7.6% vs. 16±19.5%; p = 0.02). Bland-Altman analysis indicated that flow-FISH was more precise and reproducible than qPCR. Flow-FISH and qPCR were sensitive (both 100%) and specific (93% and 89%, respectively) to distinguish very short telomeres. However, qPCR sensitivity (40%) and specificity (63%) to detect telomeres below the tenth percentile were lower compared to flow-FISH (80% sensitivity and 85% specificity). In the clinical setting, flow-FISH was more accurate, reproducible, sensitive, and specific in the

  10. Direct comparison of flow-FISH and qPCR as diagnostic tests for telomere length measurement in humans.

    PubMed

    Gutierrez-Rodrigues, Fernanda; Santana-Lemos, Bárbara A; Scheucher, Priscila S; Alves-Paiva, Raquel M; Calado, Rodrigo T

    2014-01-01

    Telomere length measurement is an essential test for the diagnosis of telomeropathies, which are caused by excessive telomere erosion. Commonly used methods are terminal restriction fragment (TRF) analysis by Southern blot, fluorescence in situ hybridization coupled with flow cytometry (flow-FISH), and quantitative PCR (qPCR). Although these methods have been used in the clinic, they have not been comprehensively compared. Here, we directly compared the performance of flow-FISH and qPCR to measure leukocytes' telomere length of healthy individuals and patients evaluated for telomeropathies, using TRF as standard. TRF and flow-FISH showed good agreement and correlation in the analysis of healthy subjects (R(2) = 0.60; p<0.0001) and patients (R(2) = 0.51; p<0.0001). In contrast, the comparison between TRF and qPCR yielded modest correlation for the analysis of samples of healthy individuals (R(2) = 0.35; p<0.0001) and low correlation for patients (R(2) = 0.20; p = 0.001); Bland-Altman analysis showed poor agreement between the two methods for both patients and controls. Quantitative PCR and flow-FISH modestly correlated in the analysis of healthy individuals (R(2) = 0.33; p<0.0001) and did not correlate in the comparison of patients' samples (R(2) = 0.1, p = 0.08). Intra-assay coefficient of variation (CV) was similar for flow-FISH (10.8 ± 7.1%) and qPCR (9.5 ± 7.4%; p = 0.35), but the inter-assay CV was lower for flow-FISH (9.6 ± 7.6% vs. 16 ± 19.5%; p = 0.02). Bland-Altman analysis indicated that flow-FISH was more precise and reproducible than qPCR. Flow-FISH and qPCR were sensitive (both 100%) and specific (93% and 89%, respectively) to distinguish very short telomeres. However, qPCR sensitivity (40%) and specificity (63%) to detect telomeres below the tenth percentile were lower compared to flow-FISH (80% sensitivity and 85% specificity). In the clinical setting, flow-FISH was more accurate, reproducible, sensitive, and specific in the measurement of human

  11. Compounding diagnostic delays: a qualitative study of point-of-care testing in South Africa.

    PubMed

    Engel, Nora; Davids, Malika; Blankvoort, Nadine; Pai, Nitika Pant; Dheda, Keertan; Pai, Madhukar

    2015-04-01

    Successful point-of-care (POC) testing (completion of test-and-treat cycle in one patient encounter) has immense potential to reduce diagnostic and treatment delays, and improve patient and public health outcomes. We explored what tests are done and how in public/private, rural/urban hospitals and clinics in South Africa and whether they can ensure successful POC testing. This qualitative research study examined POC testing across major diseases in Cape Town, Durban and Eastern Cape. We conducted 101 semi-structured interviews and seven focus group discussions with doctors, nurses, community health workers, patients, laboratory technicians, policymakers, hospital managers and diagnostic manufacturers. In South Africa, diagnostics are characterised by a centralised system. Most tests conducted on the spot can be made to work successfully as POC tests. The majority of public/private clinics and smaller hospitals send samples via couriers to centralised laboratories and retrieve results the same way, via internet, fax or phone. The main challenge to POC testing lies in transporting samples and results, while delays risk patient loss from diagnostic/treatment pathways. Strategies to deal with associated delays create new problems, such as artificially prolonged turnaround times, strains on human resources and quality of testing, compounding additional diagnostic and treatment delays. For POC testing to succeed, particular characteristics of diagnostic ecosystems and adaptations of professional practices to overcome associated challenges must be taken into account. © 2014 John Wiley & Sons Ltd.

  12. Prophylactic strategies for acute hemolysis secondary to plasma-incompatible platelet transfusions: correlation between qualitative hemolysin test and isohemagglutinin titration

    PubMed Central

    Landim, Cinthia Silvestre; Gomes, Francisco Carlos Almeida; Zeza, Bernardete Martin; Mendrone-Júnior, Alfredo; Dinardo, Carla Luana

    2015-01-01

    Objective Brazilian legislation has recently suggested the use of the qualitative hemolysin test instead of isohemagglutinin titers as prophylaxis for acute hemolysis related to plasma-incompatible platelet transfusions. The efficacy of this test in preventing hemolytic reactions has never been evaluated while isohemagglutinin titers have been extensively studied. The main objective of this study was to evaluate the correlation between the results of these two tests. The impact of each type of prophylaxis on the platelet inventory management and the ability of the qualitative hemolysin test to prevent red cell sensitization after the transfusion of incompatible units were also studied. Methods A total of 246 donor blood samples were evaluated using both isohemagglutinin titers and the qualitative hemolysin test, and the results were statistically compared. Subsequently, 600 platelet units were tested using the hemolysin assay and the percentage of units unsuitable for transfusion was compared to historical data using isohemagglutinin titers (cut-off: 100). Moreover, ten patients who received units with minor ABO incompatibilities that were negative for hemolysis according to the qualitative hemolysin test were evaluated regarding the development of hemolysis and red cell sensitization (anti-A or anti-B). Results Isohemagglutinin titration and the results of qualitative hemolysin test did not correlate. The routine implementation of the qualitative hemolysin test significantly increased the percentage of platelet units found unsuitable for transfusions (15–65%; p-value <0.001). Furthermore the qualitative hemolysin test did not prevent red blood cell sensitization in a small exploratory analysis. Conclusion Qualitative hemolysin test results do not correlate to those of isohemagglutinin titers and its implementation as the prophylaxis of choice for hemolysis associated with plasma-incompatible platelet transfusions lacks clinical support of safety and

  13. Knowing is not enough: a qualitative report on HIV testing among heterosexual African-American men

    PubMed Central

    Bond, Keosha T.; Frye, Victoria; Taylor, Raekiela; Williams, Kim; Bonner, Sebastian; Lucy, Debbie; Cupid, Malik; Weiss, Linda; Koblin, Beryl A.

    2015-01-01

    Despite having higher rates of HIV testing than all other racial groups, African-Americans continue to be disproportionately affected by the HIV epidemic in the United States. Knowing one’s status is the key step to maintaining behavioral changes that could stop the spread of the virus, yet little is known about the individual- and socio-structural-level barriers associated with HIV testing and communication among heterosexual African-American men. To address this and inform the development of an HIV prevention behavioral intervention for heterosexual African-American men, we conducted computerized, structured interviews with 61 men, focus group interviews with 25 men in 5 different groups, and in-depth qualitative interviews with 30 men living in high HIV prevalence neighborhoods in New York City. Results revealed that HIV testing was frequent among the participants. Even with high rates of testing, the men in the study had low levels of HIV knowledge; perceived little risk of HIV; and misused HIV testing as a prevention method. Factors affecting HIV testing, included stigma, relationship dynamics and communication, and societal influences, suggesting that fear, low perception of risk, and HIV stigma may be the biggest barriers to HIV testing. These results also suggest that interventions directed toward African-American heterosexual men must address the use of “testing as prevention” as well as correct misunderstandings of the window period and the meaning of HIV test results, and interventions should focus on communicating about HIV. PMID:25298014

  14. HIV Testing and Risk Perceptions: A Qualitative Analysis of Secondary School Students in Kampala, Uganda

    PubMed Central

    Aluzimbi, George; Lubwama, George; Muyonga, Michael; Hladik, Wolfgang

    2017-01-01

    The purpose of this paper is to explore the perceptions of self-reported HIV testing and risk behavior among sexually active adolescents and youth in secondary schools in Kampala Uganda. This was a cross-sectional survey conducted between June and October 2010 among secondary school students in Kampala, Uganda. Forty eight (48) students across the 54 schools were purposively selected for the qualitative sub-study based on their responses to particular questions. We thematically analyzed 28 interviews for our qualitative study using Nvivo software. Drug and alcohol use coupled with peers pressure impaired students’ perceptions towards HIV risk and therefore increased their susceptibility to HIV risk behaviors. Of the 28 scripts analyzed, 82% (23/28) had ever had sexual partners, 79% (22/28) were currently sexually active, and 57% (16/28) had ever been tested for HIV. In conclusion, most adolescents interviewed did not perceive HIV testing to be important to HIV prevention and reported low perception of susceptibility to HIV infection. Development of an adolescent HIV prevention model is important in improving uptake of HIV services. PMID:28878868

  15. Human cytomegalovirus detection by real-time PCR and pp65-antigen test in hematopoietic stem cell transplant recipients: a challenge in low and middle-income countries

    PubMed Central

    Breda, Giovanni; Almeida, Bernado; Carstensen, Suzana; Bonfim, Carmem M; Nogueira, Meri B; Vidal, Luine R; Almeida, Sergio M; Raboni, Sonia M

    2013-01-01

    Aim: Cytomegalovirus (CMV) infection is one of the most common complications in patients submitted to hematopoietic stem cell transplant (HSCT). Pre-emptive therapy has been indicated in patients with laboratory evidence of CMV replication. The aims of this study were to compare real-time PCR or pp65 antigen assay methodologies to detect CMV replication in HSCT patients, define a viral load threshold for initiation of pre-emptive therapy, and assess the feasibility of its implementation in hospitals of countries with low and middle income. Material and methods: Human CMV detection by real-time PCR and pp65 antigen assay was carried out in blood and plasma samples of HSCT patients collected weekly during 3 months. Pre-emptive therapy was based on CMV antigenemia results. Results: Twenty-one patients were monitored with a total of 227 samples collected; 13 (62%) patients were children. A poor correlation was observed between qualitative results, though quantitative results showed statistically significant difference, with higher viral loads detected in patients with positive antigenemia. Compared to a positive antigenemia, a cutoff value of 1067.5 copies/ml, 3.03 log10/ml, for viral load was obtained with 100% sensitivity and 71% specificity. Conclusion: CMV real-time PCR in whole blood was suitable for monitoring HSCT patients. However, its high cost is a limiting factor, and it could be used to monitor selected patients, those with prolonged leukopenia and underweight children, and subsequently switched to pp65 antigen test. Further studies involving larger numbers of patients should be performed to confirm this statement. PMID:24188241

  16. Social and psychological impact of HPV testing in cervical screening: a qualitative study

    PubMed Central

    McCaffery, K; Waller, J; Nazroo, J; Wardle, J

    2006-01-01

    Objective: Human papillomavirus (HPV) testing has been proposed for inclusion in the UK cervical screening programme. While testing may bring some benefits to the screening programme, testing positive for HPV, a sexually transmitted virus, may have adverse social and psychological consequences for women. The aim of this study was to examine the social and psychological impact of HPV testing in the context of cervical cancer screening. Method: In-depth interviews generating qualitative data were carried out with 74 women participating in HPV testing in England between June 2001 and December 2003. Purposive sampling was used to ensure heterogeneity in age, ethnic group, marital status, socioeconomic background, cytology, and HPV results among participants. Results: Testing positive for HPV was associated with adverse social and psychological consequences, relating primarily to the sexually transmitted nature of the virus and its link to cervical cancer. Women described feeling stigmatised, anxious and stressed, concerned about their sexual relationships, and were worried about disclosing their result to others. Anxiety about the infection was widespread, but the impact of testing positive varied. The psychological burden of the infection related to women’s relationship status and history, their social and cultural norms and practices around sex and relationships, and their understanding of key features of HPV. Conclusion: HPV testing should be accompanied by extensive health education to inform women and to de-stigmatise infection with the virus to ensure that any adverse impact of the infection on women’s wellbeing is minimised. PMID:16581749

  17. An alternative and sensitive method based on LCM and Q-PCR for HER2 testing in breast cancer.

    PubMed

    Fetica, Bogdan; Balacescu, Ovidiu; Balacescu, Loredana; Rus, Meda; Berindan-Neagoe, Ioana

    2014-01-01

    Nowadays, HER2 testing in breast cancer represents a necessity for both prognostic and therapy. Despite widespread use of immunohistochemistry (IHC) for assessing HER2 status, there are some limitations to identify truly negative or positive HER2 cases. Fluorescence in situ hybridization (FISH) or chromogenic in situ hybridization (CISH) could solve the equivocal HER2 IHC cases but there is no consensus on which is the best method. Consequently, finding a sensitive method for HER2 testing is critical for the management of the disease. In addition, tumor heterogeneity is an important factor which could affect accuracy of molecular diagnostics. Laser capture micro-dissection (LCM) is used to isolate pure cell populations from heterogeneous tumor tissue. The combination between LCM and quantitative polymerase chain reaction (Q-PCR), the gold standard in molecular biology for quantifying gene amplification levels, could define an important tool to improve the molecular diagnostics of HER2 status.In our pilot study we used LCM and Q-PCR to evaluate HER2 gene amplification for invasive breast carcinoma samples. The samples were selected based on HER2 status assessed by IHC and CISH. Our results demonstrated high sensitivity of Q-PCR for assessing HER2 DNA amplification as well as a good concordance between Q-PCR and IHC/ CISH assay.

  18. Accurate, Fast and Cost-Effective Diagnostic Test for Monosomy 1p36 Using Real-Time Quantitative PCR

    PubMed Central

    Cunha, Pricila da Silva; Pena, Heloisa B.; D'Angelo, Carla Sustek; Koiffmann, Celia P.; Rosenfeld, Jill A.; Shaffer, Lisa G.; Stofanko, Martin; Gonçalves-Dornelas, Higgor; Pena, Sérgio Danilo Junho

    2014-01-01

    Monosomy 1p36 is considered the most common subtelomeric deletion syndrome in humans and it accounts for 0.5–0.7% of all the cases of idiopathic intellectual disability. The molecular diagnosis is often made by microarray-based comparative genomic hybridization (aCGH), which has the drawback of being a high-cost technique. However, patients with classic monosomy 1p36 share some typical clinical characteristics that, together with its common prevalence, justify the development of a less expensive, targeted diagnostic method. In this study, we developed a simple, rapid, and inexpensive real-time quantitative PCR (qPCR) assay for targeted diagnosis of monosomy 1p36, easily accessible for low-budget laboratories in developing countries. For this, we have chosen two target genes which are deleted in the majority of patients with monosomy 1p36: PRKCZ and SKI. In total, 39 patients previously diagnosed with monosomy 1p36 by aCGH, fluorescent in situ hybridization (FISH), and/or multiplex ligation-dependent probe amplification (MLPA) all tested positive on our qPCR assay. By simultaneously using these two genes we have been able to detect 1p36 deletions with 100% sensitivity and 100% specificity. We conclude that qPCR of PRKCZ and SKI is a fast and accurate diagnostic test for monosomy 1p36, costing less than 10 US dollars in reagent costs. PMID:24839341

  19. Accurate, fast and cost-effective diagnostic test for monosomy 1p36 using real-time quantitative PCR.

    PubMed

    Cunha, Pricila da Silva; Pena, Heloisa B; D'Angelo, Carla Sustek; Koiffmann, Celia P; Rosenfeld, Jill A; Shaffer, Lisa G; Stofanko, Martin; Gonçalves-Dornelas, Higgor; Pena, Sérgio Danilo Junho

    2014-01-01

    Monosomy 1p36 is considered the most common subtelomeric deletion syndrome in humans and it accounts for 0.5-0.7% of all the cases of idiopathic intellectual disability. The molecular diagnosis is often made by microarray-based comparative genomic hybridization (aCGH), which has the drawback of being a high-cost technique. However, patients with classic monosomy 1p36 share some typical clinical characteristics that, together with its common prevalence, justify the development of a less expensive, targeted diagnostic method. In this study, we developed a simple, rapid, and inexpensive real-time quantitative PCR (qPCR) assay for targeted diagnosis of monosomy 1p36, easily accessible for low-budget laboratories in developing countries. For this, we have chosen two target genes which are deleted in the majority of patients with monosomy 1p36: PRKCZ and SKI. In total, 39 patients previously diagnosed with monosomy 1p36 by aCGH, fluorescent in situ hybridization (FISH), and/or multiplex ligation-dependent probe amplification (MLPA) all tested positive on our qPCR assay. By simultaneously using these two genes we have been able to detect 1p36 deletions with 100% sensitivity and 100% specificity. We conclude that qPCR of PRKCZ and SKI is a fast and accurate diagnostic test for monosomy 1p36, costing less than 10 US dollars in reagent costs.

  20. PCR and Mammalian Cell Selection Assays for Short-Term Genotoxicity Testing

    DTIC Science & Technology

    1993-11-05

    quantitative PCR, the specific DNA fragments (-330- and, when observed, 830-bp products) were excised from the gel and their Cerenkov radiation (i.e., light ...amplification bands so that the proced..re could be used to detect newly arising t(14;18) amplification producto wnose exact size could not be predicted. We

  1. Evaluation of a novel real-time PCR test based on the ssrA gene for the identification of group B streptococci in vaginal swabs

    PubMed Central

    2009-01-01

    Background Despite the implementation of prevention guidelines, early-onset group B streptococci (GBS) disease remains a cause of neonatal morbidity and mortality worldwide. Strategies to identify women who are at risk of transmitting GBS to their infant and the administration of intrapartum antibiotics have greatly reduced the incidence of neonatal GBS disease. However, there is a requirement for a rapid diagnostic test for GBS that can be carried out in a labour ward setting especially for women whose GBS colonisation status is unknown at the time of delivery. We report the design and evaluation of a real-time PCR test (RiboSEQ GBS test) for the identification of GBS in vaginal swabs from pregnant women. Methods The qualitative real-time PCR RiboSEQ GBS test was designed based on the bacterial ssrA gene and incorporates a competitive internal standard control. The analytical sensitivity of the test was established using crude lysate extracted from serial dilutions of overnight GBS culture using the IDI Lysis kit. Specificity studies were performed using DNA prepared from a panel of GBS strains, related streptococci and other species found in the genital tract environment. The RiboSEQ GBS test was evaluated on 159 vaginal swabs from pregnant women and compared with the GeneOhm™ StrepB Assay and culture for the identification of GBS. Results The RiboSEQ GBS test is specific and has an analytical sensitivity of 1-10 cell equivalents. The RiboSEQ GBS test was 96.4% sensitive and 95.8% specific compared to "gold standard" culture for the identification of GBS in vaginal swabs from pregnant women. In this study, the RiboSEQ GBS test performed slightly better than the commercial BD GeneOhm™ StrepB Assay which gave a sensitivity of 94.6% and a specificity of 89.6% compared to culture. Conclusion The RiboSEQ GBS test is a valuable method for the rapid, sensitive and specific detection of GBS in pregnant women. This study also validates the ssrA gene as a suitable and

  2. Usefulness of PCR and Antigen Latex Agglutination Test with Samples Obtained by Transthoracic Needle Aspiration for Diagnosis of Pneumococcal Pneumonia

    PubMed Central

    García, Amparo; Rosón, Beatriz; Pérez, José Luis; Verdaguer, Ricard; Dorca, Jordi; Carratalà, Jordi; Casanova, Aurora; Manresa, Frederic; Gudiol, Francesc

    1999-01-01

    In a large number of cases, the etiology of community-acquired pneumonia (CAP) is not established. Some cases are probably caused by Streptococcus pneumoniae. Transthoracic needle aspiration (TNA) culture has a limited sensitivity which might be improved by antigen detection or gene amplification techniques. We evaluated the capacity of a PCR assay and a latex agglutination test to detect S. pneumoniae in samples obtained by TNA from 95 patients with moderate-to-severe CAP. Latex agglutination and PCR had sensitivities of 52.2 and 91.3%, specificities of 88.7 and 83.3%, positive predictive values of 62.3 and 65.6%, and negative predictive values of 83.3 and 96.5%, respectively, when culture techniques were used as the “gold standard.” When we considered expanded criteria for the diagnosis of pneumococcal pneumonia as a standard for our calculations, latex agglutination and PCR had sensitivities of 53.6 and 89.7%, specificities of 93.0 and 90.0%, positive predictive values of 78.9 and 81.3%, and negative predictive values of 80.3 and 94.7%, respectively. The additional diagnosis provided by the PCR assay compared to latex agglutination was 12.2% (95% confidence interval of the difference from 0.4 to 20.1%). PCR was more sensitive than TNA culture, particularly in patients who had received prior antibiotic therapy (83.3 versus 33.3%). Although PCR is a very sensitive and specific technique, it has not proved to be cost-effective in clinical practice. Conversely, latex agglutination is a fast and simple method whose results might have significant implications for initial antibiotic therapy. PMID:9986837

  3. Using a field quantitative real-time PCR test to rapidly identify highly viremic rift valley fever cases.

    PubMed

    Njenga, M Kariuki; Paweska, Janusz; Wanjala, Rose; Rao, Carol Y; Weiner, Matthew; Omballa, Victor; Luman, Elizabeth T; Mutonga, David; Sharif, Shanaaz; Panning, Marcus; Drosten, Christian; Feikin, Daniel R; Breiman, Robert F

    2009-04-01

    Approximately 8% of Rift Valley fever (RVF) cases develop severe disease, leading to hemorrhage, hepatitis, and/or encephalitis and resulting in up to 50% of deaths. A major obstacle in the management of RVF and other viral hemorrhagic fever cases in outbreaks that occur in rural settings is the inability to rapidly identify such cases, with poor prognosis early enough to allow for more-aggressive therapies. During an RVF outbreak in Kenya in 2006 to 2007, we evaluated whether quantitative real-time reverse transcription-PCR (qRT-PCR) could be used in the field to rapidly identify viremic RVF cases with risk of death. In 52 of 430 RVF cases analyzed by qRT-PCR and virus culture, 18 died (case fatality rate [CFR] = 34.6%). Levels of viremia in fatal cases were significantly higher than those in nonfatal cases (mean of 10(5.2) versus 10(2.9) per ml; P < 0.005). A negative correlation between the levels of infectious virus particles and the qRT-PCR crossover threshold (C(T)) values allowed the use of qRT-PCR to assess prognosis. The CFR was 50.0% among cases with C(T) values of <27.0 (corresponding to 2.1 x 10(4) viral RNA particles/ml of serum) and 4.5% among cases with C(T) values of >or=27.0. This cutoff yielded 93.8% sensitivity and a 95.5% negative predictive value; the specificity and positive predictive value were 58% and 50%, respectively. This study shows a correlation between high viremia and fatality and indicates that qRT-PCR testing can identify nearly all fatal RVF cases.

  4. Sensitivity testing of trypanosome detection by PCR from whole blood samples using manual and automated DNA extraction methods.

    PubMed

    Dunlop, J; Thompson, C K; Godfrey, S S; Thompson, R C A

    2014-11-01

    Automated extraction of DNA for testing of laboratory samples is an attractive alternative to labour-intensive manual methods when higher throughput is required. However, it is important to maintain the maximum detection sensitivity possible to reduce the occurrence of type II errors (false negatives; failure to detect the target when it is present), especially in the biomedical field, where PCR is used for diagnosis. We used blood infected with known concentrations of Trypanosoma copemani to test the impact of analysis techniques on trypanosome detection sensitivity by PCR. We compared combinations of a manual and an automated DNA extraction method and two different PCR primer sets to investigate the impact of each on detection levels. Both extraction techniques and specificity of primer sets had a significant impact on detection sensitivity. Samples extracted using the same DNA extraction technique performed substantially differently for each of the separate primer sets. Type I errors (false positives; detection of the target when it is not present), produced by contaminants, were avoided with both extraction methods. This study highlights the importance of testing laboratory techniques with known samples to optimise accuracy of test results.

  5. Women’s perspectives of the fetal fibronectin testing process: a qualitative descriptive study

    PubMed Central

    2014-01-01

    Background In 2009 the Ontario Ministry of Health and Long Term Care funded the implementation of province-wide fetal fibronectin testing in Ontario hospitals. This paper reports results from the provincial evaluation that sought to describe the experience of fetal fibronectin testing from the perspective of women with symptoms of preterm labour. Methods A descriptive qualitative design was used, employing semi-structured telephone and face-to-face interviews with women who had fetal fibronectin testing. Results Five hospitals participated in recruiting women for the study and 17 women were interviewed. Women described their experiences of fetal fibronectin testing as an emotional process that moves from expecting, to feeling, to hoping for reassurance; and then to re-defining what is required to feel reassured. Women described feeling anxious while waiting for fetal fibronectin results. When test results were negative, women described feeling a sense of relief that their symptoms would not likely lead to an imminent preterm birth. Women with positive results expressed feeling reassured by the care decisions and quick action taken by the health care team. Conclusion Fetal fibronectin testing was acceptable and beneficial to these women with symptoms of preterm labour. Implications for practice and future research are suggested. PMID:24894630

  6. Women's perspectives of the fetal fibronectin testing process: a qualitative descriptive study.

    PubMed

    Peterson, Wendy E; Sprague, Ann E; Reszel, Jessica; Walker, Mark; Fell, Deshayne B; Perkins, Sherry L; Dunn, Sandra I; Johnson, Moya

    2014-06-04

    In 2009 the Ontario Ministry of Health and Long Term Care funded the implementation of province-wide fetal fibronectin testing in Ontario hospitals. This paper reports results from the provincial evaluation that sought to describe the experience of fetal fibronectin testing from the perspective of women with symptoms of preterm labour. A descriptive qualitative design was used, employing semi-structured telephone and face-to-face interviews with women who had fetal fibronectin testing. Five hospitals participated in recruiting women for the study and 17 women were interviewed. Women described their experiences of fetal fibronectin testing as an emotional process that moves from expecting, to feeling, to hoping for reassurance; and then to re-defining what is required to feel reassured. Women described feeling anxious while waiting for fetal fibronectin results. When test results were negative, women described feeling a sense of relief that their symptoms would not likely lead to an imminent preterm birth. Women with positive results expressed feeling reassured by the care decisions and quick action taken by the health care team. Fetal fibronectin testing was acceptable and beneficial to these women with symptoms of preterm labour. Implications for practice and future research are suggested.

  7. Simple PCR Assays Improve the Sensitivity of HIV-1 Subtype B Drug Resistance Testing and Allow Linking of Resistance Mutations

    PubMed Central

    Johnson, Jeffrey A.; Li, Jin-Fen; Wei, Xierong; Lipscomb, Jonathan; Bennett, Diane; Brant, Ashley; Cong, Mian-er; Spira, Thomas; Shafer, Robert W.; Heneine, Walid

    2007-01-01

    Background The success of antiretroviral therapy is known to be compromised by drug-resistant HIV-1 at frequencies detectable by conventional bulk sequencing. Currently, there is a need to assess the clinical consequences of low-frequency drug resistant variants occurring below the detection limit of conventional genotyping. Sensitive detection of drug-resistant subpopulations, however, requires simple and practical methods for routine testing. Methodology We developed highly-sensitive and simple real-time PCR assays for nine key drug resistance mutations and show that these tests overcome substantial sequence heterogeneity in HIV-1 clinical specimens. We specifically used early wildtype virus samples from the pre-antiretroviral drug era to measure background reactivity and were able to define highly-specific screening cut-offs that are up to 67-fold more sensitive than conventional genotyping. We also demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants. Resistance mutation associations revealed in mutation-specific amplicon sequences were verified by clonal sequencing. Significance Combined, sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations. PMID:17653265

  8. Qualitative risk assessment during polymer mortar test specimens preparation - methods comparison

    NASA Astrophysics Data System (ADS)

    Silva, F.; Sousa, S. P. B.; Arezes, P.; Swuste, P.; Ribeiro, M. C. S.; Baptista, J. S.

    2015-05-01

    Polymer binder modification with inorganic nanomaterials (NM) could be a potential and efficient solution to control matrix flammability of polymer concrete (PC) materials without sacrificing other important properties. Occupational exposures can occur all along the life cycle of a NM and “nanoproducts” from research through scale-up, product development, manufacturing, and end of life. The main objective of the present study is to analyse and compare different qualitative risk assessment methods during the production of polymer mortars (PM) with NM. The laboratory scale production process was divided in 3 main phases (pre-production, production and post-production), which allow testing the assessment methods in different situations. The risk assessment involved in the manufacturing process of PM was made by using the qualitative analyses based on: French Agency for Food, Environmental and Occupational Health & Safety method (ANSES); Control Banding Nanotool (CB Nanotool); Ecole Polytechnique Fédérale de Lausanne method (EPFL); Guidance working safely with nanomaterials and nanoproducts (GWSNN); Istituto Superiore per la Prevenzione e la Sicurezza del Lavoro, Italy method (ISPESL); Precautionary Matrix for Synthetic Nanomaterials (PMSN); and Stoffenmanager Nano. It was verified that the different methods applied also produce different final results. In phases 1 and 3 the risk assessment tends to be classified as medium-high risk, while for phase 2 the more common result is medium level. It is necessary to improve the use of qualitative methods by defining narrow criteria for the methods selection for each assessed situation, bearing in mind that the uncertainties are also a relevant factor when dealing with the risk related to nanotechnologies field.

  9. Comparison of the Qualitative and Developmental Scoring Systems for the Modified Version of the Bender-Gestalt Test.

    ERIC Educational Resources Information Center

    Brannigan, Gary G.; Brunner, Nancy A.

    1993-01-01

    Examined two scoring systems for Modified Version of the Bender-Gestalt Test. Administered Bender-Gestalt and Otis-Lennon School Ability Test to 75 first-grade and 84 second-grade students. Both systems were significantly correlated with school ability. Results of tests for differences between correlations indicated that Qualitative Scoring System…

  10. Comparison of the Qualitative and Developmental Scoring Systems for the Modified Version of the Bender-Gestalt Test.

    ERIC Educational Resources Information Center

    Brannigan, Gary G.; Brunner, Nancy A.

    1993-01-01

    Examined two scoring systems for Modified Version of the Bender-Gestalt Test. Administered Bender-Gestalt and Otis-Lennon School Ability Test to 75 first-grade and 84 second-grade students. Both systems were significantly correlated with school ability. Results of tests for differences between correlations indicated that Qualitative Scoring System…

  11. Validation of a qualitative immunochromatographic test for the noninvasive assessment of stress in dogs.

    PubMed

    Di Nardo, F; Anfossi, L; Ozella, L; Saccani, A; Giovannoli, C; Spano, G; Baggiani, C

    2016-08-15

    Salivary cortisol is regarded as a reliable parameter for the noninvasive assessment of the welfare of animals, because it is strictly related to stress levels. Several methods are available for salivary cortisol measurement in mammals, however rapid diagnostic test for detecting salivary cortisol are confined to humans. The availability of such non invasive diagnostic tools operable in situ would facilitate monitoring of animal welfare. The Cortisol stress™ test provides a simple and rapid tool to discriminate cortisol levels in canine saliva above or below 4ng/ml, which has been suggested as the cut-off value for distinguishing unstressed dogs from those experiencing stress. The test is based on a competitive immunochromatographic assay (ICT) using gold nanoparticles as probes, in which the color intensity of the Test line is inversely correlated to the salivary cortisol level. The qualitative result is obtained by the visual observation of the color formed on the Test line compared to that of the Control line We evaluated the accuracy of the test by determining salivary cortisol in 85 samples of canine saliva belonging to dogs with very variable age, sex, breed, and life history, and comparing the qualitative results to those obtained by a reference ELISA kit. Agreeing results were obtained through the two methods, and the ICT showed high diagnostic sensitivity, specificity and efficiency (100%, 98.4%, and 98.8%, respectively). Furthermore, we evaluated the precision of the test by an experimental design approach, which combines errors due to within-day and between-day variation with the biological variability, and demonstrated that the test could be reliably applied for correctly classifying canine samples, according to their salivary cortisol level. Moreover, we studied the shelf-life of the device in three experimental conditions. We confirmed the stability of the ICT at 4°C and 25°C for at least six months and observed similar results for an accelerated

  12. Decision making about Pap test use among Korean immigrant women: A qualitative study.

    PubMed

    Kim, Kyounghae; Kim, Soohyun; Gallo, Joseph J; Nolan, Marie T; Han, Hae-Ra

    2017-08-01

    Understanding how individuals make decisions about Pap tests concerning their personal values helps health-care providers offer tailored approaches to guide patients' decision making. Yet research has largely ignored decision making about Pap tests among immigrant women who experience increased risk of cervical cancer. To explore decision making about Pap tests among Korean immigrant women. We conducted a qualitative descriptive study using 32 semi-structured, in-depth interviews with Korean immigrant women residing in a north-eastern metropolitan area. Data were audio-recorded, transcribed verbatim and analysed using inductive coding. Although most women with positive decisions made their own decisions, some women deferred to their providers, and others made decisions in collaboration with their providers and significant others. While women making positive decisions tended to consider both barriers to and facilitators of having Pap tests, women making negative decisions predominantly discussed the barriers to having Pap tests, such as modesty and differences between the South Korean and US health-care systems. The women's reflections on their decisions differed regarding their Pap test decisions. Women's desired roles in the decision-making process and reflection on their decision outcome appeared to vary, although most participants with positive decisions made their own decisions and were satisfied with their decisions. Future research should conduct longitudinal, quantitative studies to test our findings regarding decision-making processes and outcomes about Pap tests. The findings should be incorporated into cervical cancer screening practices to fulfil the unmet needs of immigrant women in patient-provider communication and to facilitate women's decision making about Pap tests. © 2016 The Authors. Health Expectations published by John Wiley & Sons Ltd.

  13. Estimation of Microbial Concentration in Food Products from Qualitative, Microbiological Test Data with the MPN Technique.

    PubMed

    Fujikawa, Hiroshi

    2017-01-01

    Microbial concentration in samples of a food product lot has been generally assumed to follow the log-normal distribution in food sampling, but this distribution cannot accommodate the concentration of zero. In the present study, first, a probabilistic study with the most probable number (MPN) technique was done for a target microbe present at a low (or zero) concentration in food products. Namely, based on the number of target pathogen-positive samples in the total samples of a product found by a qualitative, microbiological examination, the concentration of the pathogen in the product was estimated by means of the MPN technique. The effects of the sample size and the total sample number of a product were then examined. Second, operating characteristic (OC) curves for the concentration of a target microbe in a product lot were generated on the assumption that the concentration of a target microbe could be expressed with the Poisson distribution. OC curves for Salmonella and Cronobacter sakazakii in powdered formulae for infants and young children were successfully generated. The present study suggested that the MPN technique and the Poisson distribution would be useful for qualitative microbiological test data analysis for a target microbe whose concentration in a lot is expected to be low.

  14. [Staphylococcus aureus enterotoxin A detection using the polymerase chain reaction (PCR) and its correlation with coagulase and thermonuclease tests].

    PubMed

    Suarez, María José; Arias, María Laura; del Mar Gamboa, María

    2008-03-01

    Staphylococcus aureus is a pathogenic bacterium, widely distributed on nature and associated to general infection and food borne outbreaks. The relationship between this bacterium and food borne outbreaks has been done, historically, using several tests, including coagulase, thermonuclease and actually, PCR for the genes codifying for the enterotoxin responsible of clinical symptoms. The objective of this work is to detect enterotoxin A codifying gene through PCR in a group of S. aureus strains isolated from food samples, and also to correlate the presence of this gene with the production of coagulase and thermonuclease enzymes. A total of 69 staphylococcal strains were analyzed, 58 obtained from non pasteurized milk samples from the Estación Experimental Alfredo Volio Mata and 11 from the Food and Water Microbiology Laboratory collection, Universidad de Costa Rica. Coagulase, thermonuclease and enterotoxin A were analyzed in all the strains, and a statistical correlation was performed in order to verify possible associations. Results show that there is no correlation between the three variables, nevertheless, all coagulase positive strains were thermonuclease positive, and all enterotoxin positive strains were coagulase and thermonuclease positive, but not inversely. These results show that the use of presumptive or indirect tests for establishing entorotoxigenity of S. aureus strains is not truthful, more sensible and specific analysis, as PCR, shall be performed.

  15. Comparison of Performance Characteristics of Aspergillus PCR in Testing a Range of Blood-Based Samples in Accordance with International Methodological Recommendations.

    PubMed

    Springer, Jan; White, P Lewis; Hamilton, Shanna; Michel, Denise; Barnes, Rosemary A; Einsele, Hermann; Löffler, Juergen

    2016-03-01

    Standardized methodologies for the molecular detection of invasive aspergillosis (IA) have been established by the European Aspergillus PCR Initiative for the testing of whole blood, serum, and plasma. While some comparison of the performance of Aspergillus PCR when testing these different sample types has been performed, no single study has evaluated all three using the recommended protocols. Standardized Aspergillus PCR was performed on 423 whole-blood pellets (WBP), 583 plasma samples, and 419 serum samples obtained from hematology patients according to the recommendations. This analysis formed a bicenter retrospective anonymous case-control study, with diagnosis according to the revised European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) consensus definitions (11 probable cases and 36 controls). Values for clinical performance using individual and combined samples were calculated. For all samples, PCR positivity was significantly associated with cases of IA (for plasma, P = 0.0019; for serum, P = 0.0049; and for WBP, P = 0.0089). Plasma PCR generated the highest sensitivity (91%); the sensitivities for serum and WBP PCR were 80% and 55%, respectively. The highest specificity was achieved when testing WBP (96%), which was significantly superior to the specificities achieved when testing serum (69%, P = 0.0238) and plasma (53%, P = 0.0002). No cases were PCR negative in all specimen types, and no controls were PCR positive in all specimens. This study confirms that Aspergillus PCR testing of plasma provides robust performance while utilizing commercial automated DNA extraction processes. Combining PCR testing of different blood fractions allows IA to be both confidently diagnosed and excluded. A requirement for multiple PCR-positive plasma samples provides similar diagnostic utility and is technically less demanding. Time

  16. Comparison of Performance Characteristics of Aspergillus PCR in Testing a Range of Blood-Based Samples in Accordance with International Methodological Recommendations

    PubMed Central

    Springer, Jan; Hamilton, Shanna; Michel, Denise; Barnes, Rosemary A.; Einsele, Hermann; Löffler, Juergen

    2016-01-01

    Standardized methodologies for the molecular detection of invasive aspergillosis (IA) have been established by the European Aspergillus PCR Initiative for the testing of whole blood, serum, and plasma. While some comparison of the performance of Aspergillus PCR when testing these different sample types has been performed, no single study has evaluated all three using the recommended protocols. Standardized Aspergillus PCR was performed on 423 whole-blood pellets (WBP), 583 plasma samples, and 419 serum samples obtained from hematology patients according to the recommendations. This analysis formed a bicenter retrospective anonymous case-control study, with diagnosis according to the revised European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) consensus definitions (11 probable cases and 36 controls). Values for clinical performance using individual and combined samples were calculated. For all samples, PCR positivity was significantly associated with cases of IA (for plasma, P = 0.0019; for serum, P = 0.0049; and for WBP, P = 0.0089). Plasma PCR generated the highest sensitivity (91%); the sensitivities for serum and WBP PCR were 80% and 55%, respectively. The highest specificity was achieved when testing WBP (96%), which was significantly superior to the specificities achieved when testing serum (69%, P = 0.0238) and plasma (53%, P = 0.0002). No cases were PCR negative in all specimen types, and no controls were PCR positive in all specimens. This study confirms that Aspergillus PCR testing of plasma provides robust performance while utilizing commercial automated DNA extraction processes. Combining PCR testing of different blood fractions allows IA to be both confidently diagnosed and excluded. A requirement for multiple PCR-positive plasma samples provides similar diagnostic utility and is technically less demanding. Time

  17. [Introduction of rapid syphilis and HIV testing in prenatal care in Colombia: qualitative analysis].

    PubMed

    Ochoa-Manjarrés, María Teresa; Gaitán-Duarte, Hernando Guillermo; Caicedo, Sidia; Gómez, Berta; Pérez, Freddy

    2016-12-01

    Interpret perceptions of Colombian health professionals concerning factors that obstruct and facilitate the introduction of rapid syphilis and HIV testing in prenatal care services. A qualitative study based on semi-structured interviews was carried out. A convenience sample was selected with 37 participants, who included health professionals involved in prenatal care services, programs for pregnant women, clinical laboratories, and directors of health care units or centers, as well as representatives from regional departments and the Ministry of Health. Colombia does not do widespread screening with rapid syphilis and HIV tests in prenatal care. The professionals interviewed stated they did not have prior experience in the use of rapid tests-except for laboratory staff-or in the course of action in response to a positive result. The insurance system hinders access to timely diagnosis and treatment. Health authorities perceive a need to review existing standards, strengthen the first level of care, and promote comprehensive prenatal care starting with contracts between insurers and health service institutional providers. Participants recommended staff training and integration between health-policymaking and academic entities for updating training programs. The market approach and the characteristics of the Colombian health system constitute the main barriers to implementation of rapid testing as a strategy for elimination of mother-to-child transmission of syphilis and HIV. Measures identified include making changes in contracts between insurers and health service institutional providers, adapting the timing and duration of prenatal care procedures, and training physicians and nurses involved in prenatal care.

  18. A PCR blood test outperforms chromogranin A in carcinoid detection and is unaffected by proton pump inhibitors.

    PubMed

    Modlin, Irvin M; Aslanian, Harry; Bodei, Lisa; Drozdov, Ignat; Kidd, Mark

    2014-12-01

    A critical requirement in neuroendocrine tumor (NET) management is a blood biomarker test that is sensitive, specific and reproducible. We evaluated a PCR-based 51-transcript signature to detect tumors, compared it with chromogranin A (CgA) and examined the confounding effect of proton pump inhibitors (PPIs), which cause falsely elevated CgA levels. The multigene signature was evaluated in two groups. Group 1: 125 prospectively collected NETs: gastroenteropancreatic NETs (n=91, including 42 pancreatic and 40 small intestinal), carcinoids of unknown primary (n=18) and other sites (n=16). Group 2: prospectively collected non-NET patients receiving PPIs (>1 month; dyspepsia, n=19; GERD, n=6; and pancreatitis, n=4) and 50 controls. All samples were analyzed by PCR (marker genes) and ELISA (DAKO-CgA). Sensitivity comparisons included χ(2), non-parametric measurements, and receiver operating characteristic (ROC) curves. Group 1: 123 NETs were PCR-positive (98.4%) compared with 50 (40%) CgA-positive (χ(2)=97.3, P<10(-26)). Significant differences (P<0.001) were noted between pancreas: PCR 95% vs CgA 29.2% (P<10(-9)) and small intestine: 100 vs 58% (P<10(-4)). The multigene test was elevated in all grades (G1-G3), in both local and disseminated disease, and was not normalized by somatostatin analog therapy. It was also elevated in 97% of CgA normal NETs. Group 2: PPI administration increased CgA in 83% and CgA was elevated in 26% of controls. PCR values were not elevated in either group. PCR performance metrics were as follows: sensitivity 98.4%, specificity 100%, positive predictive value 100%, negative predictive value 97.8%, and the ROC-derived area under the curve (AUC) was 0.997. These were significantly better than CgA (all metrics <60%; AUC, 0.54; Z-statistic, 10.44, P<0.0001). A 51-panel multigene blood transcript analysis is significantly more sensitive than plasma CgA for NET detection and is unaffected by acid suppression therapy.

  19. HPV is detectable in virtually all abnormal cervical cytology samples after reinvestigation of HPV negatives with multiple alternative PCR tests.

    PubMed

    Evans, Mark Francis; Adamson, Christine Stewart-Crawford; Schned, Laura Meredith; St John, Timothy Louis; Leiman, Gladwyn; Ashikaga, Takamaru; Cooper, Kumarasen

    2010-09-01

    The demonstration of human papillomavirus (HPV) in 99.7% of cervical carcinoma surgical specimens from around the world required investigations by multiple alternative polymerase chain reaction (PCR) assays. A similar approach may therefore be necessary to best characterize HPV prevalence and genotype distribution among cervical cytology samples. In an earlier study, 752 of 799 (94.1%) abnormal and 82 of 300 (27.3%) normal cytology specimens tested HPV positive after PCR using GP5+/6+primers. This study has reinvestigated the "HPV negative" abnormal samples (20 atypical squamous cells of undetermined significance, 5 low-grade squamous intraepithelial lesion, 14 atypical squamous cells, cannot exclude HSIL, 6 high-grade squamous intraepithelial lesion) and an age-matched cohort of "HPV negative" normal (negative for an intraepithelial lesion or malignancy) samples by PCR using PGMY09/11, FAP59/64, and LCR-E7 primers. PGMY09/11-GP5+/6+ nested PCR was performed on samples that were HPV negative by PGMY09/11 PCR. After the first 3 assays, HPV was detected in 41 of 45 (91.1%) abnormal and in 10 of 47 (21.3%) normal samples (P<0.0001). Eighteen HPV genotypes were detected and in some samples the genotype that was identified differed between the tests. The nondetection of common HPV genotypes (eg, HPVs 6, 11, 16, and 18) was notable. High-grade histopathology was found for 2 patients with HPV52-positive cytopathology. Combined with our earlier study, HPV (40 different genotypes) is shown in 99.5% of abnormal samples (99.8% inclusive of the nested PCR data). These findings show that HPV genotype and prevalence estimates are dependent on the method(s) of detection and indicate that suboptimal analytical sensitivity for one or more of the less common high-risk HPV genotypes could lead to impaired clinical sensitivity. HPV may be causal in almost every instance of abnormal cervical cytology; however, passenger HPV that is incidental to an abnormality may also have been

  20. Isolation and identification of bovine Brucella isolates from Pakistan by biochemical tests and PCR.

    PubMed

    Ali, Shahzad; Ali, Qurban; Melzer, Falk; Khan, Iahtasham; Akhter, Shamim; Neubauer, Heinrich; Jamal, Syed M

    2014-01-01

    Brucellosis is endemic in bovines in Pakistan. The Brucella species and biovars involved, however, are unknown. The objectives of the present study were to isolate and characterize brucellae from seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes which had recently aborted. The seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes were collected from the Potohar Plateau, Pakistan. Isolation of brucellae was done on modified Farrell's serum dextrose agar. Isolates were characterized by conventional biotyping methods, while molecular typing was done by genus (B4/B5) and species-specific (Brucella abortus, Brucella melitensis, Brucella ovis, and Brucella suis) polymerase chain reaction (PCR). A total of 30 isolates were recovered from milk (n = 5), aborted fetuses (n = 13), and vaginal swabs (n = 12). Most isolates were from cattle (56.7 %). All of them were identified as B. abortus biovar 1 based on conventional biotyping methods and genus and species-specific PCR. This preliminary study provides the first report on the prevalence of B. abortus biovar 1 in cattle and buffaloes in Pakistan.

  1. Pregnant women's experiences of routine counselling and testing for HIV in Eastern Uganda: a qualitative study.

    PubMed

    Rujumba, Joseph; Neema, Stella; Tumwine, James K; Tylleskär, Thorkild; Heggenhougen, Harald K

    2013-05-24

    Routine HIV counselling and testing as part of antenatal care has been institutionalized in Uganda as an entry point for pregnant women into the prevention of mother-to-child transmission of HIV (PMTCT) programme. Understanding how women experience this mode of HIV testing is important to generate ideas on how to strengthen the PMTCT programme. We explored pregnant HIV positive and negative women's experiences of routine counselling and testing in Mbale District, Eastern Uganda and formulated suggestions for improving service delivery. This was a qualitative study conducted at Mbale Regional Referral Hospital in Eastern Uganda between January and May 2010. Data were collected using in-depth interviews with 30 pregnant women (15 HIV positive and 15 HIV negative) attending an antenatal clinic, six key informant interviews with health workers providing antenatal care and observations. Data were analyzed using a content thematic approach. Prior to attending their current ANC visit, most women knew that the hospital provided HIV counselling and testing services as part of antenatal care (ANC). HIV testing was perceived as compulsory for all women attending ANC at the hospital but beneficial, for mothers, especially those who test HIV positive and their unborn babies. Most HIV positive women were satisfied with the immediate counselling they received from health workers, but identified the need to provide follow up counselling and support after the test, as areas for improvement. However, most HIV negative women mentioned that they were given inadequate attention during post-test counselling. This left them with unanswered questions and, for some, doubts about the negative test results. In this setting, routine HIV counselling and testing services are known and acceptable to mothers. There is need to strengthen post-test and follow up counselling for both HIV positive and negative women in order to maximize opportunities for primary and post exposure HIV prevention

  2. Patients' understanding of genetic susceptibility testing in mainstream medicine: qualitative study on thrombophilia

    PubMed Central

    Saukko, Paula M; Ellard, Sian; Richards, Suzanne H; Shepherd, Maggie H; Campbell, John L

    2007-01-01

    Background UK and US policy initiatives have suggested that, in the future, patients and clinicians in mainstream medicine could use genetic information to prevent common illnesses. There are no studies on patients' experience and understanding of the process of testing for common genetic susceptibilities in mainstream medicine. Methods Qualitative interviews with 42 individuals who had undergone testing for a genetic susceptibility for deep vein thrombosis in primary and secondary care in the UK. Results Some participants, often from higher social classes, had a good understanding of the test and its implications. They had often sought additional information on thrombophilia from relatives and from the Internet. Others, often from less privileged backgrounds, had a poorer understanding of the test – seven individuals were unaware of having had the genetic test. Features of genetic information led to misunderstandings: (i) at referral, (ii) when communicating results, and (iii) when making sense of the implications of testing. Participants' accounts indicated that non-specialist doctors may feel obliged to refer a patient for a genetic test they know little about, because a patient requests it after a relative had tested positive. Sometimes a referral for a genetic test was lost under information overload when multiple tests and issues were considered. The inconsistent and informal ways of communicating test results – for example by phone – in mainstream medicine also led to confusion. Participants did not generally overestimate their risk, but some were uncertain about whether they were taking the right preventive actions and/or whether their children were at risk. Information about genetic susceptibilities was difficult to make sense of, as it related to ambiguous risks for participants and family members, complicated and unfamiliar terminology and multiple genes and preventive strategies. Conclusion Policy visions of clinicians and patients in mainstream

  3. Point-Counterpoint: Large Multiplex PCR Panels Should Be First-Line Tests for Detection of Respiratory and Intestinal Pathogens.

    PubMed

    Schreckenberger, Paul C; McAdam, Alexander J

    2015-10-01

    The first FDA-approved multiplex PCR panel for a large number of respiratory pathogens was introduced in 2008. Since then, other PCR panels for detection of several respiratory and gastrointestinal pathogens have been approved by the FDA and are commercially available, and more such panels are likely to become available. These assays detect 12 to 20 pathogens, and some include pathogens that typically cause different manifestations of infection, although they infect the same organ system. Some of these tests are labor-intensive, while others require little labor, and all of them are expensive, both for the laboratory and for the patient or insurer. They include a bundle of tests with limited or no options for selecting which tests will be performed. Laboratories and hospitals have adopted different strategies for offering these assays. Some have implemented strategies to limit the use of the tests, such as limiting the frequency with which patients can be tested, restricting testing to specific groups of patients (e.g., immunocompromised patients), or providing education to encourage the use of less expensive tests before using large multiplex panels. Others have offered these assays without limiting their use, either relying on the ordering provider to exercise good judgment or because such assays are thought to be appropriate for first-line diagnostic testing. In this Point-Counterpoint, Paul Schreckenberger of Loyola University Medical Center explains why his laboratory offers these assays without restriction. Alex McAdam of Boston's Children Hospital explains the concerns about the use of these assays as first-line tests and why some limitations on their use might be appropriate. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  4. Point-Counterpoint: Large Multiplex PCR Panels Should Be First-Line Tests for Detection of Respiratory and Intestinal Pathogens

    PubMed Central

    2015-01-01

    The first FDA-approved multiplex PCR panel for a large number of respiratory pathogens was introduced in 2008. Since then, other PCR panels for detection of several respiratory and gastrointestinal pathogens have been approved by the FDA and are commercially available, and more such panels are likely to become available. These assays detect 12 to 20 pathogens, and some include pathogens that typically cause different manifestations of infection, although they infect the same organ system. Some of these tests are labor-intensive, while others require little labor, and all of them are expensive, both for the laboratory and for the patient or insurer. They include a bundle of tests with limited or no options for selecting which tests will be performed. Laboratories and hospitals have adopted different strategies for offering these assays. Some have implemented strategies to limit the use of the tests, such as limiting the frequency with which patients can be tested, restricting testing to specific groups of patients (e.g., immunocompromised patients), or providing education to encourage the use of less expensive tests before using large multiplex panels. Others have offered these assays without limiting their use, either relying on the ordering provider to exercise good judgment or because such assays are thought to be appropriate for first-line diagnostic testing. In this Point-Counterpoint, Paul Schreckenberger of Loyola University Medical Center explains why his laboratory offers these assays without restriction. Alex McAdam of Boston's Children Hospital explains the concerns about the use of these assays as first-line tests and why some limitations on their use might be appropriate. PMID:25762770

  5. Adult Patients' Perspectives on the Benefits and Harms of Overused Screening Tests: a Qualitative Study.

    PubMed

    Sutkowi-Hemstreet, Anne; Vu, Maihan; Harris, Russell; Brewer, Noel T; Dolor, Rowena J; Sheridan, Stacey L

    2015-11-01

    In recent years, there has been a growing interest in reducing the overuse of healthcare services. However, little is known about how patients conceptualize the benefits and harms of overused screening tests or how patients make decisions regarding these tests. To determine how patients think about the harms and benefits of overused screening tests and how they consider these and other factors when making decisions. Semi-structured, qualitative interviews. The study comprised 50 patients, ages 50-84, who had previously received or not received any of four overused screening services: 1) prostate cancer screening (men ages 50-69), 2) colon cancer screening (men and women ages 76-85), 3) osteoporosis screening (low-risk women ages 50-64), or 4) cardiovascular disease screening (low-risk men and women ages 50-85). We conducted a thematic analysis, using a hybrid inductive-deductive approach. Two independent coders analyzed interview transcriptions to identify themes and exemplifying quotes. Many patients could not name a harm of screening. When they did name harms, patients often focused on only the harms of the screening test itself and rarely mentioned harms further along the screening cascade (e.g., from follow-up testing and treatment). In contrast, patients could easily name benefits of screening, although many seemed to misunderstand or overestimate the magnitude of the benefits. Furthermore, patients described many additional factors they considered when making screening decisions, including their clinicians' recommendations, their age, family or friends' experiences with disease, and insurance coverage. This study highlights the need to help adults recognize and understand the benefits and harms of screening and make appropriate decisions about overused screening tests.

  6. Patient perspectives on test result communication in primary care: a qualitative study

    PubMed Central

    Litchfield, Ian J; Bentham, Louise M; Lilford, Richard J; McManus, Richard J; Greenfield, Sheila M

    2015-01-01

    Background Although the number of blood tests ordered in primary care continues to increase, efficient systems for the communication of blood test results to patients are lacking. This is a concern in terms of both patient safety and patient satisfaction. Aim To gain an understanding of patient perspectives on organisational and technological aspects of current and prospective systems for communicating laboratory test results in primary care, and the influences that impact patients’ preferred methods for receiving results. Design and setting Qualitative study using patient focus groups in four primary care practices in Birmingham, UK. Method The primary care practices were purposively selected to ensure they varied in size, socioeconomic environment, and the default pathways they used to communicate test results. A total of 26 patients from the four practices who had had a recent blood test were recruited. Over a 6 month period in 2011, six, 1-hour focus groups were conducted at the four practices involved in the study. Results Patients expressed a preference for receiving results from the ordering GP or a clinically qualified member of staff. Suggestions for refining current systems included improved access to phlebotomy appointments, better management of patient telephone calls, and a clear, accessible protocol for the communication of results. Conclusion Despite the testing and result communication process being a core activity in primary care, it was found that practices could improve their service in a number of areas. Patients described frequent delays and inconsistency in both the level of information and the method of communication, as well as dissatisfaction with non-clinical staff relaying results. Patient preferences for result communication based on their experience of current systems have produced practical suggestions to improve processes. PMID:25733434

  7. Agreement between quantitative and qualitative sensory testing of changes in oro-facial somatosensory sensitivity.

    PubMed

    Agbaje, J; De Laat, A; Constantinus, P; Svensson, P; Baad-Hansen, L

    2017-01-01

    Qualitative somatosensory testing (QualST) is a simple chairside test. It can be used to roughly assess the presence or absence of altered somatosensory function. To use QualST clinically, it is important to assess its agreement with quantitative sensory testing (QST). The aims of this study were to assess the agreement between QST and QualST when testing the modulation of facial sensitivity by capsaicin in healthy participants and to explore the agreement between QST and QualST in assessing the intraoral sensory function in clinical atypical odontalgia (AO) patients. Eighteen healthy pain-free adults and data from 27 AO patients were included in the study. Thirteen QST and three QualST parameters were evaluated at each site. Z-scores were computed for healthy participants, and Loss-Gain scores were created. The agreement observed between QST and QualST in participants with no alterations in facial sensation (placebo) was good, that is ranging from 89% to 94%. A poorer agreement was seen after capsaicin application in all test modalities with agreement ranging from 50% to 72%. The commonest misclassification observed was participants classified as normal according to QST, but hyper- or hyposensitive according to QualST after capsaicin application, especially for cold and pinprick. A similar trend was observed in AO patients where patients classified as normal using QST were misclassified as hypersensitive and in few patients as hyposensitive by QualST. In conclusion, the study showed that QualST may be used as a screening tool in the clinical setting, especially to show that subjects have normal sensory function.

  8. Efficacy of species-specific recA PCR tests in the identification of Burkholderia cepacia complex environmental isolates.

    PubMed

    Dalmastri, Claudia; Pirone, Luisa; Tabacchioni, Silvia; Bevivino, Annamaria; Chiarini, Luigi

    2005-05-01

    In this study, we evaluated if recA species-specific PCR assays could be successfully applied to identify environmental isolates of the widespread Burkholderia cepacia complex (Bcc) species. A total of 729 Bcc rhizosphere isolates collected in different samplings were assigned to the species B. cepacia genomovar I (61), B. cenocepacia recA lineage IIIB (514), B. ambifaria (124) and B. pyrrocinia (30), by means of recA (RFLP) analysis, and PCR tests were performed to assess sensitivity and specificity of recA species-specific primers pairs. B. cepacia genomovar I specific primers produced the expected amplicon with all isolates of the corresponding species (sensitivity, 100%), and cross-reacted with all B. pyrrocinia isolates. On the contrary, B. cenocepacia IIIB primers did not give the expected amplicon in 164 B. cenocepacia IIIB isolates (sensitivity, 68.1%), and isolates of distinct populations showed different sensitivity. B. ambifaria primers failed to amplify a recA-specific fragment only in a few isolates of this species (sensitivity, 93.5%). The absence of specific amplification in a high number of B. cenocepacia rhizosphere isolates indicates that recA specific PCR assays can lead to an underestimation of environmental microorganisms belonging to this bacterial species.

  9. Virtual PCR

    SciTech Connect

    Gardner, S N; Clague, D S; Vandersall, J A; Hon, G; Williams, P L

    2006-02-23

    The polymerase chain reaction (PCR) stands among the keystone technologies for analysis of biological sequence data. PCR is used to amplify DNA, to generate many copies from as little as a single template. This is essential, for example, in processing forensic DNA samples, pathogen detection in clinical or biothreat surveillance applications, and medical genotyping for diagnosis and treatment of disease. It is used in virtually every laboratory doing molecular, cellular, genetic, ecologic, forensic, or medical research. Despite its ubiquity, we lack the precise predictive capability that would enable detailed optimization of PCR reaction dynamics. In this LDRD, we proposed to develop Virtual PCR (VPCR) software, a computational method to model the kinetic, thermodynamic, and biological processes of PCR reactions. Given a successful completion, these tools will allow us to predict both the sequences and concentrations of all species that are amplified during PCR. The ability to answer the following questions will allow us both to optimize the PCR process and interpret the PCR results: What products are amplified when sequence mixtures are present, containing multiple, closely related targets and multiplexed primers, which may hybridize with sequence mismatches? What are the effects of time, temperature, and DNA concentrations on the concentrations of products? A better understanding of these issues will improve the design and interpretation of PCR reactions. The status of the VPCR project after 1.5 years of funding is consistent with the goals of the overall project which was scoped for 3 years of funding. At half way through the projected timeline of the project we have an early beta version of the VPCR code. We have begun investigating means to improve the robustness of the code, performed preliminary experiments to test the code and begun drafting manuscripts for publication. Although an experimental protocol for testing the code was developed, the preliminary

  10. Exploring Operational Test and Evaluation of Unmanned Aircraft Systems: A Qualitative Case Study

    NASA Astrophysics Data System (ADS)

    Saliceti, Jose A.

    The purpose of this qualitative case study was to explore and identify strategies that may potentially remedy operational test and evaluation procedures used to evaluate Unmanned Aircraft Systems (UAS) technology. The sample for analysis consisted of organizations testing and evaluating UASs (e.g., U.S. Air Force, U.S. Navy, U.S. Army, U.S. Marine Corps, U.S. Coast Guard, and Customs Border Protection). A purposeful sampling technique was used to select 15 subject matter experts in the field of operational test and evaluation of UASs. A questionnaire was provided to participants to construct a descriptive and robust research. Analysis of responses revealed themes related to each research question. Findings revealed operational testers utilized requirements documents to extrapolate measures for testing UAS technology and develop critical operational issues. The requirements documents were (a) developed without the contribution of stakeholders and operational testers, (b) developed with vague or unrealistic measures, and (c) developed without a systematic method to derive requirements from mission tasks. Four approaches are recommended to develop testable operational requirements and assist operational testers: (a) use a mission task analysis tool to derive requirements for mission essential tasks for the system, (b) exercise collaboration among stakeholders and testers to ensure testable operational requirements based on mission tasks, (c) ensure testable measures are used in requirements documents, and (d) create a repository list of critical operational issues by mission areas. The preparation of operational test and evaluation processes for UAS technology is not uniform across testers. The processes in place are not standardized, thus test plan preparation and reporting are different among participants. A standard method to prepare and report UAS technology should be used when preparing and reporting on UAS technology. Using a systematic process, such as mission

  11. [THE COMPARATIVE ANALYSIS OF RESULTS OF DETECTION OF CARCINOGENIC TYPES OF HUMAN PAPILLOMA VIRUS BY QUALITATIVE AND QUANTITATIVE TESTS].

    PubMed

    Kuzmenko, E T; Labigina, A V; Leshenko, O Ya; Rusanov, D N; Kuzmenko, V V; Fedko, L P; Pak, I P

    2015-05-01

    The analysis of results of screening (n = 3208; sexually active citizen aged from 18 to 59 years) was carried out to detect oncogene types of human papilloma virus in using qualitative (1150 females and 720 males) and quantitative (polymerase chain reaction in real-time (843 females and 115 males) techniques. The human papilloma virus of high oncogene type was detected in 65% and 68.4% of females and in 48.6% and 53% of males correspondingly. Among 12 types of human papilloma virus the most frequently diagnosed was human papilloma virus 16 independently of gender of examined and technique of analysis. In females, under application of qualitative tests rate of human papilloma virus 16 made up to 18.3% (n = 280) and under application of quantitative tests Rte of human papilloma virus made up to 14.9% (n = 126; p ≤ 0.05). Under examination of males using qualitative tests rate of human papilloma virus 16 made up to 8.3% (n = 60) and under application of qualitative tests made up to 12.2% (n = 14; p ≥ 0.05). Under application of qualitative tests rate of detection on the rest ofoncogene types of human papilloma virus varied in females from 3.4% to 8.4% and in males from 1.8% to 5.9%. Under application of qualitative tests to females rate of human papilloma virus with high viral load made up to 68.4%, with medium viral load - 2.85% (n = 24) and with low viral load -0.24% (n = 2). Under application of quantitative tests in males rate of detection of types of human papilloma virus made up to 53% and at that in all high viral load was established. In females, the most of oncogene types of human papilloma virus (except for 31, 39, 59) are detected significantly more often than in males.

  12. Comparison of Binax NOW urine antigen test and pneumococcal DNA assay using qPCR before and after nasopharyngeal swabbing in healthy Malawian children.

    PubMed

    Lees, E A; Ho, D K K; Guiver, M; Mankhambo, L A; French, N; Carrol, E D

    2015-11-01

    Diagnosis of invasive pneumococcal disease is challenging. We compared Binax NOW pneumococcal urinary antigen test with blood pneumococcal PCR in healthy Malawian children with and without pneumococcal carriage, and we found a high false-positive rate with Binax NOW. Blood pneumococcal PCR positivity was 66/88 (75%) compared to 5/27 (18%) when nasopharyngeal swabbing was performed first compared to after blood sampling for pneumococcal blood PCR. We speculate that nasopharyngeal swabbing may be causing a breach of mucosal integrity, leading to invasion into the bloodstream. These findings need to be confirmed with autolysin-based PCR assays.

  13. Test Takers' Experiences with Computer-Administered Listening Comprehension Tests: Interviewing for Qualitative Explorations of Test Validity

    ERIC Educational Resources Information Center

    Gorsuch, Greta

    2004-01-01

    In this study, retrospective interviews were used to investigate reliability (and thus validity) threats to a computerized ESL listening comprehension test administered at a university in the US. The participants in the investigation, six international graduate students, were asked to respond to semi- and open-ended questions during individual…

  14. A comparison of qualitative and quantitative fecal immunochemical tests in the Korean national colorectal cancer screening program.

    PubMed

    Park, Mi Jin; Choi, Kui Son; Lee, You Kyoung; Jun, Jae Kwan; Lee, Hoo-Yeon

    2012-04-01

    The National Cancer Screening Program (NCSP) has since 2004 provided annual colorectal cancer screening using the fecal immunochemical test (FIT) for individuals aged 50 years or older. The aim of this study was to examine the positivity and detection rates of the FIT and to compare the detection rates of the qualitative and quantitative FITs in participants in the 2009 NCSP. We analyzed positivity and detection rates according to FIT type (qualitative and quantitative). We used a multinomial logistic regression to analyze the odds ratio of "benign" or "suspicious cancer and cancer" compared to "normal," adjusted for gender, age, health insurance type, region of residence, hospital type, and FIT type. Of the 1,181,904 participants, 72.8% received a qualitative and 27.2% a quantitative FIT. The positivity rates were 8.1% for the qualitative and 2.5% for the quantitative FIT. The detection rate was 5.2% for the qualitative and 14.4% for the quantitative FIT. The odds ratio of a "suspicious cancer and cancer" versus a "normal" result was 2.73 (95% CI = 2.22-3.35) for the quantitative compared to qualitative FIT, after adjustment. The positivity rate of the qualitative FIT was around three times higher than that of the quantitative FIT. However, the odds ratio for detection of "suspicious cancer and cancer" versus "normal" of the quantitative FIT was about three times higher than that of the qualitative FIT. These findings suggest that quality control may be important, particularly for the qualitative FIT.

  15. SEROLOGIC SURVEY AND RESULTS OF URINARY PCR TESTING FOR LEPTOSPIROSIS IN CAPTIVE BLACK-TAILED PRAIRIE DOGS (CYNOMYS LUDOVICIANUS).

    PubMed

    Olds, June E; Sun, Yaxuan; Baum, David H; Gauger, Phillip

    2015-12-01

    Leptospirosis is an important zoonotic disease occurring clinically and subclinically in humans and a wide variety of mammal species worldwide. Often, rodents and wild animals are identified as important reservoirs for the disease. Twenty-two captive black-tailed prairie dogs (Cynomys ludovicianus) housed within a zoo were examined as part of a routine census and preventive medicine program. During examinations, blood and urine were collected to screen for exposure to, or infection with, leptospirosis. All animals were apparently healthy at the time of examination. Leptospira microscopic agglutination test identified 12 of 22 (54.5%) prairie dogs with antibody titers ≥1 : 100 against Leptospira interrogans serovar bratislava on initial serologic examination. All prairie dogs within this collection were serologically negative for L. interrogans serovars canicola, hardjo, icterohaemorrhagiae, and pomona and Leptospira kirschneri serovar grippotyphosa. Leptospira polymerase chain reaction (PCR) testing of urine was negative in all animals tested. This report describes evidence that captive prairie dogs may be exposed to leptospirosis, most likely from wild rodent reservoirs; however, serum titers are low, and lack of leptospiral DNA detected by PCR indicates that these captive animals are unlikely to be important reservoirs for the disease.

  16. New Tests for Syphilis: Rational Design of a PCR Method for Detection of Treponema pallidum in Clinical Specimens Using Unique Regions of the DNA Polymerase I Gene

    PubMed Central

    Liu, Hsi; Rodes, Berta; Chen, C.-Y.; Steiner, Bret

    2001-01-01

    A sensitive and specific PCR method to detect Treponema pallidum in clinical specimens was developed. PCR primers were designed based on two unique features of the DNA polymerase I gene (polA). The first distinctive characteristic is that the region codes for a high cysteine content and has low homology with similar regions of DNA polymerase I gene from known microorganisms. The second unique feature is the presence of four insertions in the gene. PCR tests using primers designed on the basis these regions reacted with various pathogenic T. pallidum subspecies but did not react with nonpathogenic treponemal species or other spirochetes. An additional 59 species of bacteria and viruses, including those that cause genital ulcers, tested negative. This PCR method is extremely robust and sensitive. The detection limit is about 10 to 25 organisms when analyzed on gel. However, the analytic sensitivity can be increased by at least 1 log, to a detection limit of a single organism, when the ABI 310 Prism Genetic Analyzer is used to detect fluorescence-labeled amplicons. We further used this test in a clinical setting and compared the results with results from a previously reported multiplex-PCR test (for T. pallidum, Haemophilus ducreyi, and herpes simplex virus). We tested 112 genital ulcer specimens by the polA PCR, obtaining a sensitivity of 95.8% and a specificity of 95.7%. These results suggest that the polA PCR is applicable as a routine clinical diagnostic test for syphilis. PMID:11326018

  17. Comparative Application of PLS and PCR Methods to Simultaneous Quantitative Estimation and Simultaneous Dissolution Test of Zidovudine - Lamivudine Tablets.

    PubMed

    Üstündağ, Özgür; Dinç, Erdal; Özdemir, Nurten; Tilkan, M Günseli

    2015-01-01

    In the development strategies of new drug products and generic drug products, the simultaneous in-vitro dissolution behavior of oral dosage formulations is the most important indication for the quantitative estimation of efficiency and biopharmaceutical characteristics of drug substances. This is to force the related field's scientists to improve very powerful analytical methods to get more reliable, precise and accurate results in the quantitative analysis and dissolution testing of drug formulations. In this context, two new chemometric tools, partial least squares (PLS) and principal component regression (PCR) were improved for the simultaneous quantitative estimation and dissolution testing of zidovudine (ZID) and lamivudine (LAM) in a tablet dosage form. The results obtained in this study strongly encourage us to use them for the quality control, the routine analysis and the dissolution test of the marketing tablets containing ZID and LAM drugs.

  18. Clinical Validation of a PCR Assay for the Detection of EGFR Mutations in Non–Small-Cell Lung Cancer: Retrospective Testing of Specimens from the EURTAC Trial

    PubMed Central

    Benlloch, Susana; Botero, Maria Luisa; Beltran-Alamillo, Jordi; Mayo, Clara; Gimenez-Capitán, Ana; de Aguirre, Itziar; Queralt, Cristina; Ramirez, Jose Luis; Cajal, Santiago Ramón y.; Klughammer, Barbara; Schlegel, Mariette; Bordogna, Walter; Chen, David; Zhang, Guili; Kovach, Barbara; Shieh, Felice; Palma, John F.; Wu, Lin; Lawrence, H. Jeffrey; Taron, Miquel

    2014-01-01

    The EURTAC trial demonstrated that the tyrosine kinase inhibitor (TKI) erlotinib was superior to chemotherapy as first-line therapy for advanced non-small cell lung cancers (NSCLC) that harbor EGFR activating mutations in a predominantly Caucasian population. Based on EURTAC and several Asian trials, anti-EGFR TKIs are standard of care for EGFR mutation-positive NSCLC. We sought to validate a rapid multiplex EGFR mutation assay as a companion diagnostic assay to select patients for this therapy. Samples from the EURTAC trial were prospectively screened for EGFR mutations using a combination of laboratory-developed tests (LDTs), and tested retrospectively with the cobas EGFR mutation test (EGFR PCR test). The EGFR PCR test results were compared to the original LDT results and to Sanger sequencing, using a subset of specimens from patients screened for the trial. Residual tissue was available from 487 (47%) of the 1044 patients screened for the trial. The EGFR PCR test showed high concordance with LDT results with a 96.3% overall agreement. The clinical outcome of patients who were EGFR-mutation detected by the EGFR PCR test was very similar to the entire EURTAC cohort. The concordance between the EGFR PCR test and Sanger sequencing was 90.6%. In 78.9% of the discordant samples, the EGFR PCR test result was confirmed by a sensitive deep sequencing assay. This retrospective study demonstrates the clinical utility of the EGFR PCR test in the accurate selection of patients for anti-EGFR TKI therapy. The EGFR PCR test demonstrated improved performance relative to Sanger sequencing. PMID:24586842

  19. Comparison of the clinical performances of the AdvanSure HPV Screening Real-Time PCR, the Abbott Real-Time High-Risk HPV Test, and the Hybrid Capture High-Risk HPV DNA Test for Cervical Cancer Screening.

    PubMed

    Chung, Hae-Sun; Hahm, Chorong; Lee, Miae

    2014-09-01

    The clinical performance of three human papillomavirus (HPV) DNA commercial assays for cervical cancer screening was evaluated; the AdvanSure HPV Screening Real-Time PCR (AdvanSure PCR; LG Life Sciences) that was developed recently for the detection of both high-risk and low-risk genotypes, the Abbott RealTime High-Risk HPV Test (Abbott PCR; Abbott Molecular) and the Hybrid Capture High-Risk HPV DNA test (HC2; Qiagen). The three different HPV DNA tests were compared using cytology samples obtained from 619 women who underwent routine cervical cancer screening. The gold-standard assay was histopathological confirmation of cervical intraepithelial neoplasia of grade 2 or worse. The clinical sensitivities of the AdvanSure PCR, the Abbott PCR and the HC2 for the detection of cervical intraepithelial neoplasia of grade 2 or worse were 95.5%, 95.5% and 100%, respectively, while the clinical specificities were 61.6%, 86.4% and 83.3%, respectively. There were no significant differences in the clinical sensitivities of the Abbott PCR and the AdvanSure PCR compared to the HC2. The clinical specificities of the Abbott PCR and the AdvanSure PCR for the detection of HPV types 16/18 were 97.8% and 98.5%, respectively. For cervical cancer screening, all three tests showed relatively good clinical sensitivities, but the AdvanSure PCR had lower clinical specificity than the Abbott PCR and the HC2. The AdvanSure PCR and the Abbott PCR assays have the advantage of being automated and the ability to distinguish between HPV types 16/18 and other HPV types. The two real-time PCR assays could be useful tools in HPV testing for cervical cancer screening.

  20. Reference genes for QRT-PCR tested under various stress conditions in Folsomia candida and Orchesella cincta (Insecta, Collembola)

    PubMed Central

    de Boer, Muriel E; de Boer, Tjalf E; Mariën, Janine; Timmermans, Martijn JTN; Nota, Benjamin; van Straalen, Nico M; Ellers, Jacintha; Roelofs, Dick

    2009-01-01

    Background Genomic studies measuring transcriptional responses to changing environments and stress currently make their way into the field of evolutionary ecology and ecotoxicology. To investigate a small to medium number of genes or to confirm large scale microarray studies, Quantitative Reverse Transcriptase PCR (QRT-PCR) can achieve high accuracy of quantification when key standards, such as normalization, are carefully set. In this study, we validated potential reference genes for their use as endogenous controls under different chemical and physical stresses in two species of soil-living Collembola, Folsomia candida and Orchesella cincta. Treatments for F. candida were cadmium exposure, phenanthrene exposure, desiccation, heat shock and pH stress, and for O. cincta cadmium, desiccation, heat shock and starvation. Results Eight potential reference genes for F. candida and seven for O. cincta were ranked by their stability per stress factor using the programs geNorm and Normfinder. For F. candida the succinate dehydrogenase (SDHA) and eukaryotic transcription initiation factor 1A (ETIF) genes were found the most stable over the different treatments, while for O. cincta, the beta actin (ACTb) and tyrosine 3-monooxygenase (YWHAZ) genes were the most stable. Conclusion We present a panel of reference genes for two emerging ecological genomic model species tested under a variety of treatments. Within each species, different treatments resulted in differences in the top stable reference genes. Moreover, the two species differed in suitable reference genes even when exposed to similar stresses. This might be attributed to dissimilarity of physiology. It is vital to rigorously test a panel of reference genes for each species and treatment, in advance of relative quantification of QRT-PCR gene expression measurements. PMID:19486513

  1. Qualitative and quantitative agar invasion test based on bacterial colony/biofilm.

    PubMed

    Corcuera, María Teresa; Gómez-Aguado, Fernando; Gómez-Lus, María Luisa; Ramos, Carmen; de la Parte, María Antonia; Alonso, María José; Prieto, José

    2013-09-01

    Invasion of the culture medium is a feature frequently studied in yeasts, in which it has been related to a greater virulence, but it is practically unknown in bacteria. Recently, it has been demonstrated that several clinically relevant bacterial species were also able of invading agar media, so it was necessary to design a microbiological assay to study the expression of this character in bacteria. Accordingly, a bacterial agar invasion test based on colony/biofilm development was designed, which allows qualitative and quantitative characterization of bacterial growth into the agar culture medium. Once the culture conditions were optimized, the test was applied to 90 strains from nine bacterial species, validating its usefulness for differentiating invasive strains (positive) from those non invasive (negative). The test also allows sorting invasive strains according to agar invasion intensity (low, moderate, high) and topographic invasion pattern (peripheral, homogeneous, mixed). Moreover, an image analysis routine to quantify the invasion was developed. Implemented method enables direct measuring of two invasion parameters (invasion area and number of invasion dots), automated calculation of three relative variables (invasion relative area, invasion dots relative density, and invasion dot average area), and the establishment of strain specific frequency histograms. This new methodology is simple, fast, reproducible, objective, inexpensive and can be used to study a great number of specimens simultaneously, all of which make it suitable for incorporation to the routine of any microbiology laboratory. It could also be a useful tool for additional studies related to clinical aspects of bacterial isolates such as virulence and antimicrobial response. © 2013.

  2. The Quantitative-Qualitative Distinction and the Null Hypothesis Significance Testing Procedure

    ERIC Educational Resources Information Center

    Ratnesar, Nimal; Mackenzie, Jim

    2006-01-01

    Conventional discussion of research methodology contrast two approaches, the quantitative and the qualitative, presented as collectively exhaustive. But if qualitative is taken as the understanding of lifeworlds, the two approaches between them cover only a tiny fraction of research methodologies; and the quantitative, taken as the routine…

  3. Multiplex PCR tests sentinel the appearance of pandemic influenza viruses including H1N1 swine influenza.

    PubMed

    Mahony, James B; Hatchette, Todd; Ojkic, Davor; Drews, Steven J; Gubbay, Jonathan; Low, Donald E; Petric, Martin; Tang, Patrick; Chong, Sylvia; Luinstra, Kathy; Petrich, Astrid; Smieja, Marek

    2009-07-01

    Since the turn of the century seven new respiratory viruses have infected man and two of these have resulted in worldwide epidemics. Both SARS Coronavirus which quickly spread to 29 countries in February 2003 and H1N1 swine influenza that recently spread from Mexico to 30 countries in three weeks represent major pandemic threats for mankind. Diagnostic assays are required to detect novel influenza strains with pandemic potential. In this report we evaluate the ability of a multiplex PCR test (xTAG RVP) to detect new, "non-seasonal" influenza viruses including the H1N1 swine influenza A/swine/California/04/2009. Laboratory based study using retrospective and prospective specimens. This multiplex PCR test detected the present of non-seasonal (non-H1, non-H3) influenza in 20 of 20 patients infected with H1N1 swine flu virus. In addition to detecting the current swine flu the xTAG RVP test detected the H5N1 A/Vietnam/1203/2004 high pathogenicity avian influenza virus that circulated in South East Asia in 2003 as well as 17 out of 17 influenza A viruses representing 11 HA subtypes isolated from birds, swine and horses not yet seen in the human population. Based on these results we believe that this molecular test can perform an important role as a sentinel test to detect novel non-seasonal influenza A viruses in patients presenting with influenza-like illness (ILI) and therefore act as an early warning system for the detection of future pandemic influenza threats.

  4. Identification of Eastern United States Reticulitermes Termite Species via PCR-RFLP, Assessed Using Training and Test Data

    PubMed Central

    Garrick, Ryan C.; Collins, Benjamin D.; Yi, Rachel N.; Dyer, Rodney J.; Hyseni, Chaz

    2015-01-01

    Reticulitermes termites play key roles in dead wood decomposition and nutrient cycling in forests. They also damage man-made structures, resulting in considerable economic loss. In the eastern United States, five species (R. flavipes, R. virginicus, R. nelsonae, R. hageni and R. malletei) have overlapping ranges and are difficult to distinguish morphologically. Here we present a molecular tool for species identification. It is based on polymerase chain reaction (PCR) amplification of a section of the mitochondrial cytochrome oxidase subunit II gene, followed by a three-enzyme restriction fragment length polymorphism (RFLP) assay, with banding patterns resolved via agarose gel electrophoresis. The assay was designed using a large set of training data obtained from a public DNA sequence database, then evaluated using an independent test panel of Reticulitermes from the Southern Appalachian Mountains, for which species assignments were determined via phylogenetic comparison to reference sequences. After refining the interpretive framework, the PCR-RFLP assay was shown to provide accurate identification of four co-occurring species (the fifth species, R. hageni, was absent from the test panel, so accuracy cannot yet be extended to training data). The assay is cost- and time-efficient, and will help improve knowledge of Reticulitermes species distributions. PMID:26463202

  5. Comparison of a qualitative canine C-reactive protein test to a quantitative test and traditional markers of inflammation - Short communication.

    PubMed

    Smuts, Celia M; Mills, Jennifer N; Gaál, Tibor

    2015-09-01

    Qualitative tests for C-reactive protein (CRP) are available for use in dogs, and provide a rapid in-house method of detecting acute inflammation. The aim of this study was to compare results from a qualitative CRP lateral flow test (Teco CRP FASTest) to those obtained from a quantitative CRP ELISA and to traditional methods of detecting inflammation, including total leukocyte and neutrophil numbers, presence of immature neutrophils and a left shift, presence or absence of toxic changes in neutrophils and plasma fibrinogen concentration in whole blood and serum samples collected from 113 client-owned dogs. More dogs had CRP FASTest positive results than had quantitatively increased CRP (ELISA) or increases in traditional methods used for measuring inflammation. Few dogs had increases in markers of inflammation but no elevated CRP. The qualitative CRP FASTest was found to be a sensitive test for detecting increased CRP concentration and was positive more frequently than were traditional markers of inflammation.

  6. Usefulness of two Aspergillus PCR assays and Aspergillus galactomannan and β-D-glucan testing of bronchoalveolar lavage fluid for diagnosis of chronic pulmonary aspergillosis.

    PubMed

    Urabe, Naohisa; Sakamoto, Susumu; Sano, Go; Suzuki, Junko; Hebisawa, Akira; Nakamura, Yasuhiko; Koyama, Kazuya; Ishii, Yoshikazu; Tateda, Kazuhiro; Homma, Sakae

    2017-03-22

    We evaluated the usefulness of an Aspergillus galactomannan (GM) test, a β-D-glucan (βDG) test, and two different Aspergillus polymerase chain reaction (PCR) assays of bronchoalveolar lavage fluid (BALF) samples for diagnosis of chronic pulmonary aspergillosis (CPA). BALF samples from 28 patients with and 120 patients without CPA were collected. We calculated the sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio for each test individually and in combination with other tests. The optical density index values, as determined by receiver operating characteristic analysis, for diagnosis of CPA were 0.5 and 105 for GM and βDG testing of BALF, respectively. The sensitivity and specificity of the GM test, βDG test, and PCR assays 1 and 2 were 77.8% and 90.0%, 77.8% and 72.5%, 86.7% and 84.2%, and 66.7% and 94.2%, respectively. Comparison of PCR assays showed that PCR assay 1 had better sensitivity, negative predictive value, and negative likelihood ratio and PCR assay 2 had better specificity, positive predictive value, and positive likelihood ratio. The combination of the GM and βDG tests had the highest diagnostic odds ratio. The combination of the GM and βDG tests of BALF was more useful than any single test for diagnosing CPA.

  7. False-negative post-18-month confirmatory HIV tests in HIV DNA PCR-positive children: a retrospective analysis.

    PubMed

    Garcia-Prats, Anthony J; Draper, Heather R; Sanders, Jill E; Agrawal, Anurag K; Mohapi, Edith Q; Schutze, Gordon E

    2012-09-24

    The WHO guidelines for children less than 18 months old diagnosed with HIV based on presumptive clinical diagnosis or one virologic test recommend confirmatory HIV antibody testing after 18 months of age. This study describes post-18-month HIV test results following this WHO-recommended confirmatory testing strategy. Case series and retrospective review of routine program data. Children enrolled at the Baylor Children's Clinical Center of Excellence, a pediatric and family HIV clinic in Maseru, Lesotho from December 2005 through January 2009 with a positive HIV DNA PCR at less than 18 months of age and HIV rapid test results after 18 months of age were included. Post-18-month confirmatory HIV test results are described. Factors associated with non-positive confirmatory rapid tests were determined using binary logistic regression. Of the 109 children meeting inclusion criteria, 22 (20.2%) had negative and 27 (24.8%) discordant confirmatory rapid tests. Forty-six of these 49 were on antiretroviral therapy (ART). Among these 49, 11 of 24 post-18-month HIV DNA PCRs were negative, whereas nine of 10 post-18-month HIV ELISAs were positive; 29 were definitively and 17 probably HIV-infected, two were uninfected, and one had undetermined status. Only age less than 9 months at ART initiation (odds ratio 4.25, P = 0.002) was associated with non-positive rapid tests. False-negative post-18-month confirmatory rapid tests and HIV DNA PCRs in children on ART are common, associated with early ART initiation, and may lead to inappropriate ART discontinuation and discharge from care of truly HIV-infected children.

  8. Prospective clinical evaluation of Amplicor Mycobacterium tuberculosis PCR test as a screening method in a low-prevalence population.

    PubMed Central

    Cartuyvels, R; De Ridder, C; Jonckheere, S; Verbist, L; Van Eldere, J

    1996-01-01

    Of 656 respiratory samples analyzed for Mycobacterium tuberculosis by microscopy, culture, and the Amplicor PCR method, 25 were positive by culture, 12 were positive by microscopy, and 17 were positive by the Amplicor PCR method; 16 samples were Amplicor PCR positive and culture negative. No patient except one with culture-negative, Amplicor PCR-positive samples had clinical indications of tuberculosis. The sensitivity and specificity of the Amplicor PCR compared with those of culture were 68 and 97.4%, respectively. For culture-positive, smear-negative samples, the sensitivity of the Amplicor PCR was 46%. PMID:8818898

  9. Sensitive immunochemical approaches for quantitative (FPIA) and qualitative (lateral flow tests) determination of gentamicin in milk.

    PubMed

    Beloglazova, N V; Shmelin, P S; Eremin, S A

    2016-01-01

    Three kinds of immunoassays for the determination of gentamicin in milk samples were developed and validated. First, a fast and easily-performed fluorescence polarization immunoassay was used for characterization of the employed polyclonal antibody. The calculated Kaff were (1.9±0.4)×10(9)М(-1) and (6.0±0.2)×10(6)М(-1) for the high- and low-affinity fractions respectively. The assay was characterized with a good sensitivity, the limit of detection being 5μgkg(-1). Two different kinds of detection labels, i.e. colloidal gold (CG) and quantum dots (QDs), were evaluated for use in lateral-flow format with respect to rapid visual on-site testing. The cut-off levels for both qualitative formats were selected based on the maximum level for gentamicin in milk established by the European Commission, 100μgkg(-1), resulting in a 10μgkg(-1) cut-off considering sample dilution. The intra-laboratory validation was performed with sterilized milk samples artificially spiked with gentamicin at concentrations less than, equal to, and greater than the cut-off level. It was shown that milk products could be analyzed without any sample preparation, except for dilution with the buffer solution. The rates of false-positive and false-negative results were below 5% for both labels. The different developed immunoassays were tested towards gentamicin determination in artificially-spiked and naturally contaminated milk samples. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Evaluation of two singleplex reverse transcription-Insulated isothermal PCR tests and a duplex real-time RT-PCR test for the detection of porcine epidemic diarrhea virus and porcine deltacoronavirus.

    PubMed

    Zhang, Jianqiang; Tsai, Yun-Long; Lee, Pei-Yu Alison; Chen, Qi; Zhang, Yan; Chiang, Cheng-Jen; Shen, Yu-Han; Li, Fu-Chun; Chang, Hsiao-Fen Grace; Gauger, Phillip C; Harmon, Karen M; Wang, Hwa-Tang Thomas

    2016-08-01

    Recent outbreaks of porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) in multiple countries have caused significant economic losses and remain a serious challenge to the swine industry. Rapid diagnosis is critical for the implementation of efficient control strategies before and during PEDV and PDCoV outbreaks. Insulated isothermal PCR (iiPCR) on the portable POCKIT™ device is user friendly for on-site pathogen detection. In the present study, a singleplex PEDV RT-iiPCR, a singleplex PDCoV RT-iiPCR, and a duplex PEDV/PDCoV real-time RT-PCR (rRT-PCR) commercial reagents targeting the M gene were compared to an N gene-based PEDV rRT-PCR and an M gene-based PDCoV rRT-PCR that were previously published and used as reference PCRs. All PCR assays were highly specific and did not cross react with other porcine enteric pathogens. Analytical sensitivities of the PEDV RT-iiPCR, PDCoV RT-iiPCR and duplex PEDV/PDCoV rRT-PCR were determined using in vitro transcribed RNA as well as viral RNA extracted from ten-fold serial dilutions of PEDV and PDCoV cell culture isolates. Performance of each PCR assay was further evaluated using 170 clinical samples (86 fecal swabs, 24 feces, 19 intestines, and 41 oral fluids). Compared to the reference PEDV rRT-PCR, the sensitivity, specificity and accuracy of the PEDV RT-iiPCR were 97.73%, 98.78%, and 98.24%, respectively, and those of the duplex PEDV/PDCoV rRT-PCR were 98.86%, 96.34%, and 97.65%, respectively. Compared to the reference PDCoV rRT-PCR, the sensitivity, specificity and accuracy of the PDCoV RT-iiPCR were 100%, 100%, and 100%, respectively, and those of the PEDV/PDCoV duplex rRT-PCR were 96.34%, 100%, and 98.24%, respectively. Overall, all three new PCR assays were comparable to the reference rRT-PCRs for detection of PEDV and/or PDCoV. The PEDV and PDCoV RT-iiPCRs are potentially useful tools for on-site detection and the duplex PEDV/PDCoV rRT-PCR provides a convenient method to simultaneously detect

  11. Correlations Between the Porteus Maze Test Qualitative Score and Age and Recidivism Rates of Female Correctional Inmates.

    ERIC Educational Resources Information Center

    Pearson, Virginia L.

    This study investigated correlations between the Qualitative score of the Porteus Maze Test and age and rates of recidivism of correctional institution inmates. In addition, the study was structured to provide answers to the following questions: (1) Is there a relationship between age and rates of recidivism and the Conformity-Variability score of…

  12. Tests of the Dynamic Field Theory and The Spatial Precision Hypothesis: Capturing a Qualitative Developmental Transition in Spatial Working Memory

    ERIC Educational Resources Information Center

    Schutte, Anne R.; Spencer, John P.

    2009-01-01

    This study tested a dynamic field theory (DFT) of spatial working memory and an associated spatial precision hypothesis (SPH). Between 3 and 6 years of age, there is a qualitative shift in how children use reference axes to remember locations: 3-year-olds' spatial recall responses are biased toward reference axes after short memory delays, whereas…

  13. qPCR-High resolution melt analysis for drug susceptibility testing of Mycobacterium leprae directly from clinical specimens of leprosy patients.

    PubMed

    Araujo, Sergio; Goulart, Luiz Ricardo; Truman, Richard W; Goulart, Isabela Maria B; Vissa, Varalakshmi; Li, Wei; Matsuoka, Masanori; Suffys, Philip; Fontes, Amanda B; Rosa, Patricia S; Scollard, David M; Williams, Diana L

    2017-06-01

    Real-Time PCR-High Resolution Melting (qPCR-HRM) analysis has been recently described for rapid drug susceptibility testing (DST) of Mycobacterium leprae. The purpose of the current study was to further evaluate the validity, reliability, and accuracy of this assay for M. leprae DST in clinical specimens. The specificity and sensitivity for determining the presence and susceptibility of M. leprae to dapsone based on the folP1 drug resistance determining region (DRDR), rifampin (rpoB DRDR) and ofloxacin (gyrA DRDR) was evaluated using 211 clinical specimens from leprosy patients, including 156 multibacillary (MB) and 55 paucibacillary (PB) cases. When comparing the results of qPCR-HRM DST and PCR/direct DNA sequencing, 100% concordance was obtained. The effects of in-house phenol/chloroform extraction versus column-based DNA purification protocols, and that of storage and fixation protocols of specimens for qPCR-HRM DST, were also evaluated. qPCR-HRM results for all DRDR gene assays (folP1, rpoB, and gyrA) were obtained from both MB (154/156; 98.7%) and PB (35/55; 63.3%) patients. All PCR negative specimens were from patients with low numbers of bacilli enumerated by an M. leprae-specific qPCR. We observed that frozen and formalin-fixed paraffin embedded (FFPE) tissues or archival Fite's stained slides were suitable for HRM analysis. Among 20 mycobacterial and other skin bacterial species tested, only M. lepromatosis, highly related to M. leprae, generated amplicons in the qPCR-HRM DST assay for folP1 and rpoB DRDR targets. Both DNA purification protocols tested were efficient in recovering DNA suitable for HRM analysis. However, 3% of clinical specimens purified using the phenol/chloroform DNA purification protocol gave false drug resistant data. DNA obtained from freshly frozen (n = 172), formalin-fixed paraffin embedded (FFPE) tissues (n = 36) or archival Fite's stained slides (n = 3) were suitable for qPCR-HRM DST analysis. The HRM-based assay was also able to

  14. Pre-Clinical Testing of a Real-Time PCR Assay for Diahhreal Disease Agent Cryptosporidium

    DTIC Science & Technology

    2014-05-16

    tests on freeze-dried Shigella ipaH, enterotoxigenic E . coli (ETEC) ST1b, ETEC LT, ETEC ST1a, Cryptosporidium, and Leptospira (Idaho Technologies, Salt...Citrobacter freundii 1 Negative EHEC 1 Negative Enterobacter aerogenes 1 Negative Enterobacter cloacae 1 Negative Enterotoxigenic E . coli (ETEC...2 Negative Enteroinvasive E . coli (EIEC) 1 Negative Escherichia coli 1 Negative K. pneumoniae 1 Negative P. aeruginosa 1 Negative Proteus

  15. Predictive power of quantitative and qualitative fecal immunochemical tests for hemoglobin in population screening for colorectal neoplasm.

    PubMed

    Huang, Yanqin; Li, Qilong; Ge, Weiting; Cai, Shanrong; Zhang, Suzhan; Zheng, Shu

    2014-01-01

    The aim of this study was to evaluate the performance of qualitative and quantitative fecal immunochemical tests (FITs) in population screening for colorectal neoplasm. A total of 9000 participants aged between 40 and 74 years were enrolled in this study. Each participant received two stool sampling tubes and was asked to simultaneously submit two stool samples from the same bowel movement. The stool samples of each participant were tested using an immunogold labeling FIT dipstick (qualitative FIT) and an automated fecal blood analyzer (quantitative FIT). Colonoscopy was performed for those who test positive in either FIT. The positive predictive values and population detection rates of the FITs for predicting colorectal neoplasm were compared. A total of 6494 (72.16%) participants simultaneously submitted two stool samples. The diagnostic consistency for a positive result between quantitative and qualitative FITs was poor (κ=0.278, 95% confidence interval=0.223-0.333). The positive predictive values of the quantitative FIT were significantly higher than those of the qualitative FIT for predicting large (≥1 cm) adenomas (23 cases, 14.29% and 16 cases, 6.72%, P=0.013) and colorectal cancer (10 cases, 6.21% and 5 cases, 2.10%, P=0.034); however, the population detection rate for advanced neoplasm of the quantitative FIT was not significantly different from that of the qualitative FIT. Quantitative FIT is superior to qualitative FIT in predicting advanced colorectal neoplasm during colorectal cancer screening. Further studies are needed to elucidate the causes of the predictive superiority.

  16. [Evaluation of immunochromatography test for rapid detection of influenza A and B viruses using real-time PCR].

    PubMed

    Hara, Michimaru; Sadamasu, Kenji; Takao, Shinichi; Shinkai, Takayuki; Kai, Akemi; Fukuda, Shinji; Shimazu, Yukie; Kuwayama, Masaru; Miyazaki, Kazuo

    2006-09-01

    The sensitivity of rapid diagnostic kits to influenza B is lower than to influenza A. The cause-poor performance of the kit or the scarcity of viruses in type B specimens-has yet to be clarified. Using real-time PCR, we measured the amount of influenza viruses with nasopharyngeal aspirate fluid previously identified by virus isolation culture and passing the rapid diagnosis test by four types of kits, including the ESPLINE Influenza A&B-N (Fujirebio Corp., Japan). We classified the results of virus isolation and rapid diagnosis tests into three groups and examined them: group 1 (12 specimens, influenza B, all negative in tests using four types of kits); group 2 (57 specimens, influenza B, all positive in tests); and group 3 (36 specimens, AH3, all positive in tests). The average amount of viruses in group 1 (6.60 +/- 0.81 log10copies/mL) was significantly lower (p<0.0001) than that in group 2 (8.51 +/- 0.57 log10copies/mL) or group 3 (8.72 +/- 0.63 log10copies/mL). No significant difference was seen in the amount of viruses between groups 2 and 3. We concluded that the cause of low sensitivity in rapid diagnostic kits to influenza B are attributable to the scarcity of viruses in the specimen.

  17. Development and performance assessment of a qualitative SYBR® green real-time PCR assay for the detection of Aspergillus versicolor in indoor air.

    PubMed

    Libert, X; Chasseur, C; Bladt, S; Packeu, A; Bureau, F; Roosens, N H; De Keersmaecker, S C J

    2015-09-01

    Currently, contamination of indoor environment by fungi and molds is considered as a public health problem. The monitoring of indoor airborne fungal contamination is a common tool to help understanding the link between fungi in houses and respiratory problems. Classical analytical monitoring methods, based on cultivation and microscopic identification, depend on the growth of the fungi. Consequently, they are biased by difficulties to grow some species on certain culture media and under certain conditions or by noncultivable or dead fungi that can consequently not be identified. However, they could have an impact on human health as they might be allergenic. Since molecular methods do not require a culture step, they seem an excellent alternative for the monitoring of indoor fungal contaminations. As a case study, we developed a SYBR® green real-time PCR-based assay for the specific detection and identification of Aspergillus versicolor, which is frequently observed in indoor environment and known to be allergenic. The developed primers amplify a short region of the internal transcribed spacer 1 from the 18S ribosomal DNA complex. Subsequently, the performance of this quantitative polymerase chain reaction (qPCR) method was assessed using specific criteria, including an evaluation of the selectivity, PCR efficiency, dynamic range, and repeatability. The limit of detection was determined to be 1 or 2 copies of genomic DNA of A. versicolor. In order to demonstrate that this SYBR® green qPCR assay is a valuable alternative for monitoring indoor fungal contamination with A. versicolor, environmental samples collected in contaminated houses were analyzed and the results were compared to the ones obtained with the traditional methods.

  18. Rapid molecular haemagglutinin subtyping of avian influenza isolates by specific real-time RT-PCR tests.

    PubMed

    Elizalde, Maia; Agüero, Montserrat; Buitrago, Dolores; Yuste, María; Arias, María Luisa; Muñoz, María Jesús; Lelli, Davide; Pérez-Ramírez, Elisa; Moreno-Martin, Ana María; Fernández-Pinero, Jovita

    2014-02-01

    Sixteen haemagglutinin (HA) subtypes of avian influenza viruses (AIV) have been described to date. Rapid subtype identification of any AIV is of major interest because of the possible serious consequences for the poultry industry and even public health. Molecular techniques currently allow immediate accurate subtype characterisation prior to virus isolation. In this study, a set of fourteen specific real-time RT-PCR methods were developed and evaluated for AIV HA subtyping (H1-H4, H6-H8, H10-H16), H5 and H9 being excluded on the basis of the current validity of the European Union (EU) recommended specific assays. Specific primers and probes sets for each HA-subtype were designed to hybridise the largest isolates range within each single subtype, considering the Eurasian lineage as a major target. The robustness and general application of the 14 HA-subtype methods were verified by the analysis of 110 AIV isolates belonging to all 16 HA-subtypes, performed in different laboratories. The developed real-time RT-PCR assays proved to be highly specific and revealed suitable sensitivity, allowing direct HA-subtyping of clinical material. In summary, this study provides for the first time a panel of molecular tests using specific hydrolysis probes for rapid and complete AIV HA-subtype identification.

  19. Counselors' Clinical Use of Definitive Drug Testing Results in Their Work with Substance-Use Clients: a Qualitative Study.

    PubMed

    Rzetelny, Adam; Zeller, Barbara; Miller, Nicholas; City, Kathy Egan; Kirsh, Kenneth L; Passik, Steven D

    We conducted a psychotherapeutic examination of the use of definitive drug testing (liquid chromatography with tandem mass spectrometry) in the treatment of substance use disorders (SUD). Employing a generic qualitative method (Caelli et al. in International Journal of Qualitative Methods, 2(2), 2003; Merriam, 2009) we asked SUD counselors to provide narratives about cases where drug testing had revealed new or unexpected information about clients' drug-taking behaviors. Semi-structured interviews with 12 SUD counselors were conducted by phone and analyzed for themes derived from the literature. These counselors reported many new positive drug tests in clients previously believed to be adherent with treatment. Key themes assessed in counselors' narratives included initial client denial that was often followed by later acknowledgement of relapse and increased motivation, at times presenting new opportunities for clients to engage in treatment and enhance the therapeutic alliance. These results suggest that definitive drug testing can be used in a non-stigmatizing and therapeutic manner.

  20. Current State of PCR-Based Epstein-Barr Virus DNA Testing for Nasopharyngeal Cancer.

    PubMed

    Kim, Kelly Y; Le, Quynh-Thu; Yom, Sue S; Pinsky, Benjamin A; Bratman, Scott V; Ng, Raymond H W; El Mubarak, Haja S; Chan, K C Allen; Sander, Miriam; Conley, Barbara A

    2017-04-01

    Clinical studies have shown plasma Epstein-Barr virus (EBV) DNA level to be an independent prognostic biomarker for nasopharyngeal carcinoma (NPC). However, the proportion of NPC patients whose tumors are associated with EBV vary with geographic location, and there are a variety of assays for plasma EBV. To develop the level of evidence needed to demonstrate the clinical utility of plasma EBV DNA detection for NPC patients and encourage widespread adoption of this biomarker test in clinical laboratories, validated harmonized assays are needed. In 2015, the National Cancer Institute (NCI) convened a Workshop on Harmonization of EBV Testing for Nasopharyngeal Cancer, where experts in head and neck oncology and laboratory medicine addressed the limitations of currently available polymerase chain reaction-based EBV DNA quantitation assays and discussed strategies for advancing the development of harmonized EBV DNA assays and their appropriate clinical use. This article presents the key recommendations to direct future efforts in assay harmonization and validation. Published by Oxford University Press 2017. This work is written by US Government employees and is in the public domain in the US.

  1. Analysis of Helicobacter pylori Prevalence in Chittagong, Bangladesh, Based on PCR and CLO Test

    PubMed Central

    Habib, Abdul Musaweer; Alam, Md. Jibran; Rudra, Bashudev; Quader, Md. Abdul; Al-Forkan, Mohammad

    2016-01-01

    The pathogenic bacterium Helicobacter pylori is a causative agent of gastric diseases in Bangladesh as well as throughout the world. This study aimed at analyzing the prevalence of H. pylori infection among dyspeptic patients in Chittagong, the second most populous city of Bangladesh, using 16S rRNA-based H. pylori-specific Polymerase Chain Reaction and Campylobacter-like organism test. We found that 67% of the population under study was positive for H. pylori infection. Gastric ulcer and duodenal ulcer disease showed statistically significant association with H. pylori infection; however, no association of H. pylori infection was observed in terms of age and gender. This study would play a crucial role in managing H. pylori-induced gastric diseases by understanding the current trend of H. pylori infection in the Chittagong region of Bangladesh. PMID:27891051

  2. Could Digital PCR Be an Alternative as a Non-Invasive Prenatal Test for Trisomy 21: A Proof of Concept Study

    PubMed Central

    El Khattabi, Laïla Allach; Rouillac-Le Sciellour, Christelle; Le Tessier, Dominique; Luscan, Armelle; Coustier, Audrey; Porcher, Raphael; Bhouri, Rakia; Nectoux, Juliette; Sérazin, Valérie; Quibel, Thibaut; Mandelbrot, Laurent; Tsatsaris, Vassilis

    2016-01-01

    Objective NIPT for fetal aneuploidy by digital PCR has been hampered by the large number of PCR reactions needed to meet statistical requirements, preventing clinical application. Here, we designed an octoplex droplet digital PCR (ddPCR) assay which allows increasing the number of available targets and thus overcomes statistical obstacles. Method After technical optimization of the multiplex PCR on mixtures of trisomic and euploid DNA, we performed a validation study on samples of plasma DNA from 213 pregnant women. Molecular counting of circulating cell-free DNA was performed using a mix of hydrolysis probes targeting chromosome 21 and a reference chromosome. Results The results of our validation experiments showed that ddPCR detected trisomy 21 even when the sample’s trisomic DNA content is as low as 5%. In a validation study of plasma samples from 213 pregnant women, ddPCR discriminated clearly between the trisomy 21 and the euploidy groups. Conclusion Our results demonstrate that digital PCR can meet the requirements for non-invasive prenatal testing of trisomy 21. This approach is technically simple, relatively cheap, easy to implement in a diagnostic setting and compatible with ethical concerns regarding access to nucleotide sequence information. These advantages make it a potential technique of choice for population-wide screening for trisomy 21 in pregnant women. PMID:27167625

  3. Could Digital PCR Be an Alternative as a Non-Invasive Prenatal Test for Trisomy 21: A Proof of Concept Study.

    PubMed

    El Khattabi, Laïla Allach; Rouillac-Le Sciellour, Christelle; Le Tessier, Dominique; Luscan, Armelle; Coustier, Audrey; Porcher, Raphael; Bhouri, Rakia; Nectoux, Juliette; Sérazin, Valérie; Quibel, Thibaut; Mandelbrot, Laurent; Tsatsaris, Vassilis; Vialard, François; Dupont, Jean-Michel

    2016-01-01

    NIPT for fetal aneuploidy by digital PCR has been hampered by the large number of PCR reactions needed to meet statistical requirements, preventing clinical application. Here, we designed an octoplex droplet digital PCR (ddPCR) assay which allows increasing the number of available targets and thus overcomes statistical obstacles. After technical optimization of the multiplex PCR on mixtures of trisomic and euploid DNA, we performed a validation study on samples of plasma DNA from 213 pregnant women. Molecular counting of circulating cell-free DNA was performed using a mix of hydrolysis probes targeting chromosome 21 and a reference chromosome. The results of our validation experiments showed that ddPCR detected trisomy 21 even when the sample's trisomic DNA content is as low as 5%. In a validation study of plasma samples from 213 pregnant women, ddPCR discriminated clearly between the trisomy 21 and the euploidy groups. Our results demonstrate that digital PCR can meet the requirements for non-invasive prenatal testing of trisomy 21. This approach is technically simple, relatively cheap, easy to implement in a diagnostic setting and compatible with ethical concerns regarding access to nucleotide sequence information. These advantages make it a potential technique of choice for population-wide screening for trisomy 21 in pregnant women.

  4. The Fecal Virome of Children with Hand, Foot, and Mouth Disease that Tested PCR Negative for Pathogenic Enteroviruses

    PubMed Central

    Linsuwanon, Piyada; Poovorawan, Yong; Li, Linlin; Deng, Xutao; Vongpunsawad, Sompong; Delwart, Eric

    2015-01-01

    Hand, foot, and mouth disease (HFMD) affects infant and young children. A viral metagenomic approach was used to identify the eukaryotic viruses in fecal samples from 29 Thai children with clinical diagnosis of HFMD collected during the 2012 outbreak. These children had previously tested negative by PCR for enterovirus 71 and coxsackievirus A16 and A6. Deep sequencing revealed nine virus families: Picornaviridae, Astroviridae, Parvoviridae, Caliciviridae, Paramyxoviridae, Adenoviridae, Reoviridae, Picobirnaviridae, and Polyomaviridae. The highest number of viral sequences belonged to human rhinovirus C, astrovirus-MLB2, and coxsackievirus A21. Our study provides an overview of virus community and highlights a broad diversity of viruses found in feces from children with HFMD. PMID:26288145

  5. Concordance between RT-PCR-based detection of respiratory viruses from nasal swabs collected for viral testing and nasopharyngeal swabs collected for bacterial testing

    PubMed Central

    Grijalva, Carlos G.; Griffin, Marie R.; Edwards, Kathryn M.; Johnson, Monika; Gil, Ana I.; Verastegui, Héctor; Lanata, Claudio F.; Williams, John V.

    2014-01-01

    Background Epidemiologic studies of respiratory infections frequently rely on separate sample collections for the detection of bacteria and viruses. The requirement for two specimens presents cost, logistical, and acceptability challenges. Objectives To determine the agreement in detection of respiratory viruses using RT-PCR between two different types of samples collected on the same day: nasal swabs preserved in viral transport medium (NS) and nasopharyngeal swabs preserved in skim milk-tryptone-glucose-glycerol [STGG] medium (NP), the current standard for pneumococcal colonization studies. Study design Paired NS and NP samples were collected between May 2009 and September 2011 as part of the RESPIRA-PERU study, a large prospective cohort of Andean children <3 years of age. NS samples used polyester swabs and viral transport medium whereas NP samples used rayon wire-handled swabs and STGG medium. Samples were tested for influenza, human metapneumovirus (MPV), respiratory syncytial virus (RSV), human rhinovirus (HRV), parainfluenza virus 3 (PIV3) and adenovirus (ADV) using real-time RT-PCR. We calculated the agreement, and compared cycle thresholds (CT) between NP and NS samples. Results Among 226 paired NP-NS samples, we observed very high agreement with a Kappa statistic ranging from 0.71 for ADV to 0.97 for MPV. CT values were similar for both strategies. Conclusions NP samples preserved in STGG provide a simple and reliable strategy for identification of both pneumococcus and respiratory viruses. This single specimen collection strategy could be used for epidemiologic studies, especially in resource-limited settings. Furthermore, archived NP-STGG specimens from previous studies could be reliably tested by RT-PCR for viruses. PMID:24875136

  6. Concordance between RT-PCR-based detection of respiratory viruses from nasal swabs collected for viral testing and nasopharyngeal swabs collected for bacterial testing.

    PubMed

    Grijalva, Carlos G; Griffin, Marie R; Edwards, Kathryn M; Johnson, Monika; Gil, Ana I; Verástegui, Héctor; Lanata, Claudio F; Williams, John V

    2014-07-01

    Epidemiologic studies of respiratory infections frequently rely on separate sample collections for the detection of bacteria and viruses. The requirement for two specimens presents cost, logistical, and acceptability challenges. To determine the agreement in detection of respiratory viruses using RT-PCR between two different types of samples collected on the same day: nasal swabs preserved in viral transport medium (NS) and nasopharyngeal swabs preserved in skim milk-tryptone-glucose-glycerol [STGG] medium (NP), the current standard for pneumococcal colonization studies. Paired NS and NP samples were collected between May 2009 and September 2011 as part of the RESPIRA-PERU study, a large prospective cohort of Andean children <3 years of age. NS samples used polyester swabs and viral transport medium whereas NP samples used rayon wire-handled swabs and STGG medium. Samples were tested for influenza, human metapneumovirus (MPV), respiratory syncytial virus (RSV), human rhinovirus (HRV), parainfluenza virus 3 (PIV3) and adenovirus (ADV) using real-time RT-PCR. We calculated the agreement, and compared cycle thresholds (CT) between NP and NS samples. Among 226 paired NP-NS samples, we observed very high agreement with a Kappa statistic ranging from 0.71 for ADV to 0.97 for MPV. CT values were similar for both strategies. NP samples preserved in STGG provide a simple and reliable strategy for identification of both pneumococcus and respiratory viruses. This single specimen collection strategy could be used for epidemiologic studies, especially in resource-limited settings. Furthermore, archived NP-STGG specimens from previous studies could be reliably tested by RT-PCR for viruses. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. DNA testing of Klinefelter's syndrome in a criminal case using XY chromosomal STR multiplex-PCR.

    PubMed

    Honda, K; Tun, Z; Matoba, R

    2001-09-01

    We report genetic typing of Klinefelter's syndrome applied to casework in forensic DNA testing. In this case, by using extracted DNA from body samples (muscle and bones), we could identify two distinct X alleles in two out of three X-STR loci (HPRTB and ARA), in addition to Y alleles (DYS390, DYS393). The extra X was found to have originated from father, and the victim turned out to have 47XXY Klinefelter's syndrome. The victim was a 30-year-old male, born from relatively elderly parents as a second child. His father was a severe alcoholic and had been malnourished for more than 20 years at the moment of his birth. He exhibited slight mental retardation as a child, and belonged to a criminal group as an adult. The method presented here was useful to accurately diagnose sex chromosomal abnormality instead of conventional chromosomal analysis and Xg blood group typing. A subtype of this syndrome, 48 XXXY or mosaic, for example, could be identified if the intensity of the overlapped X bands were calculated.

  8. The development and testing of a qualitative instrument designed to assess critical thinking

    NASA Astrophysics Data System (ADS)

    Clauson, Cynthia Louisa

    This study examined a qualitative approach to assess critical thinking. An instrument was developed that incorporates an assessment process based on Dewey's (1933) concepts of self-reflection and critical thinking as problem solving. The study was designed to pilot test the critical thinking assessment process with writing samples collected from a heterogeneous group of students. The pilot test included two phases. Phase 1 was designed to determine the validity and inter-rater reliability of the instrument using two experts in critical thinking, problem solving, and literacy development. Validity of the instrument was addressed by requesting both experts to respond to ten questions in an interview. The inter-rater reliability was assessed by analyzing the consistency of the two experts' scorings of the 20 writing samples to each other, as well as to my scoring of the same 20 writing samples. Statistical analyses included the Spearman Rho and the Kuder-Richardson (Formula 20). Phase 2 was designed to determine the validity and reliability of the critical thinking assessment process with seven science teachers. Validity was addressed by requesting the teachers to respond to ten questions in a survey and interview. Inter-rater reliability was addressed by comparing the seven teachers' scoring of five writing samples with my scoring of the same five writing samples. Again, the Spearman Rho and the Kuder-Richardson (Formula 20) were used to determine the inter-rater reliability. The validity results suggest that the instrument is helpful as a guide for instruction and provides a systematic method to teach and assess critical thinking while problem solving with students in the classroom. The reliability results show the critical thinking assessment instrument to possess fairly high reliability when used by the experts, but weak reliability when used by classroom teachers. A major conclusion was drawn that teachers, as well as students, would need to receive instruction

  9. Comparison of two quantitative real-time CMV-PCR tests calibrated against the 1st WHO international standard for viral load monitoring of renal transplant patients.

    PubMed

    Mannonen, Laura; Loginov, Raisa; Helanterä, Ilkka; Dumoulin, Alexis; Vilchez, Regis A; Cobb, Bryan; Hirsch, Hans H; Lautenschlager, Irmeli

    2014-04-01

    Cytomegalovirus (CMV) replication in organ transplant recipients is commonly diagnosed by quantitative PCR methods. However, there has been a poor inter-laboratory correlation of viral load values due to the lack of an international reference standard. In a recent study, the COBAS® AmpliPrep/COBAS® TaqMan® (CAP/CTM) CMV test calibrated to the 1st WHO CMV standard, showed good reproducibility in CMV load values across multiple laboratories. Fifty-seven follow-up plasma specimens from 10 kidney transplant recipients with CMV replication were examined using the new quantitative CAP/CTM CMV test and the "in-house" quantitative CMV real-time PCR method, also calibrated against the 1st WHO CMV standard for their clinical applicability for monitoring CMV load in renal transplant patients. By CAP/CTM CMV test 49/57 specimens were CMV-DNA positive compared to 44/57 by the "in-house" PCR test. The "in-house" PCR and CAP/CTM CMV test correlated well in monitoring individual kidney transplant patients. Conversion of the CMV-DNA copies to IUs made the results of the "in-house" PCR and CAP/CTM CMV test less uniform in analysis of the patient samples. In specimens of one patient, significant underquantification of CMV load with "in-house" PCR emerged during follow-up due to a point mutation in the "in-house" PCR primer sequence. The CAP/CTM CMV test was found suitable for diagnosing and monitoring CMV replication in renal transplant patients. Multicenter studies are needed to provide more information of the commutability of the 1st WHO CMV standard and to define the clinical thresholds. © 2013 Wiley Periodicals, Inc.

  10. Chlamydia infection in patients with and without cervical intra-epithelial lesions tested by real-time PCR vs. direct immunofluorescence.

    PubMed

    Oliveira, Micheline de Lucena; Amorim, Melania Maria Ramos de; Souza, Paulo Roberto Eleutério de; Albuquerque, Lúcia Cristina Bezerra de; Brandão, Lucas André Cavalcanti; Guimarães, Rafael Lima

    2008-08-01

    This study compares the detection rates of Chlamydia trachomatis by two techniques, direct immunofluorescence (IMF) and real time polymerase chain reaction (PCR), in patients with and without intra-epithelial cervical lesions (SIL) in Recife. We conducted a transversal study involving 35 women with SIL and 35 without SIL attended at Ambulatório Especializado da Mulher, Recife, Brazil. They were tested for Chlamydia trachomatis using two techniques, direct IMF or real time PCR. The rates of Chlamydia trachomatis detection were compared and the association with intra-epithelial cervical lesions was determined using the chi-square test at a 5% level of significance. Concordance between the tests was evaluated using kappa. The global prevalence of Chlamydia infection was 47.1% by direct IMF and 58.6% by real time PCR. A significant association was observed between Chlamydia diagnosis and presence of intra-epithelial cervical lesions, with about 80% positive results by direct IMF and 77.1% by real time PCR. However, the detected rate of infection with Chlamydia trachomatis was significantly greater in patients without intra-epithelial cervical lesions tested by real time PCR (40%) when compared to direct IMF (14.3%). The concordance between the tests was weak, with a kappa coefficient of 0.4. Both real time PCR and direct IMF detected elevated rates of Chlamydia infection in patients with intra-epithelial cervical lesions (80%) but the tests were discordant when patients without cervical lesions were tested, possibly because sensitivity of real time PCR is greater.

  11. Nasal Methicillin-Resistant Staphylococcus aureus (MRSA) PCR Testing Reduces the Duration of MRSA-Targeted Therapy in Patients with Suspected MRSA Pneumonia.

    PubMed

    Baby, Nidhu; Faust, Andrew C; Smith, Terri; Sheperd, Lyndsay A; Knoll, Laura; Goodman, Edward L

    2017-04-01

    The objective of this study was to evaluate the impact of pharmacist-ordered methicillin-resistant Staphylococcus aureus (MRSA) PCR testing on the duration of empirical MRSA-targeted antibiotic therapy in patients with suspected pneumonia. This is a retrospective analysis of patients who received vancomycin or linezolid for suspected pneumonia before and after the implementation of a pharmacist-driven protocol for nasal MRSA PCR testing. Patients were included if they were adults of >18 years of age and initiated on vancomycin or linezolid for suspected MRSA pneumonia. The primary endpoint was the duration of vancomycin or linezolid therapy. After screening 368 patients, 57 patients met inclusion criteria (27 pre-PCR and 30 post-PCR). Baseline characteristics were similar between the two groups, with the majority of patients classified as having health care-associated pneumonia (68.4%). The use of the nasal MRSA PCR test reduced the mean duration of MRSA-targeted therapy by 46.6 h (74.0 ± 48.9 h versus 27.4 ± 18.7 h; 95% confidence interval [CI], 27.3 to 65.8 h; P < 0.0001). Fewer patients in the post-PCR group required vancomycin serum levels and dose adjustment (48.1% versus 16.7%; P = 0.02). There were no significant differences between the pre- and post-PCR groups regarding days to clinical improvement (1.78 ± 2.52 versus 2.27 ± 3.34; P = 0.54), length of hospital stay (11.04 ± 9.5 versus 8.2 ± 7.8; P = 0.22), or hospital mortality (14.8% versus 6.7%; P = 0.41). The use of nasal MRSA PCR testing in patients with suspected MRSA pneumonia reduced the duration of empirical MRSA-targeted therapy by approximately 2 days without increasing adverse clinical outcomes.

  12. A human papilloma virus testing algorithm comprising a combination of the L1 broad-spectrum SPF10 PCR assay and a novel E6 high-risk multiplex type-specific genotyping PCR assay.

    PubMed

    van Alewijk, Dirk; Kleter, Bernhard; Vent, Maarten; Delroisse, Jean-Marc; de Koning, Maurits; van Doorn, Leen-Jan; Quint, Wim; Colau, Brigitte

    2013-04-01

    Human papillomavirus (HPV) epidemiological and vaccine studies require highly sensitive HPV detection and genotyping systems. To improve HPV detection by PCR, the broad-spectrum L1-based SPF10 PCR DNA enzyme immunoassay (DEIA) LiPA system and a novel E6-based multiplex type-specific system (MPTS123) that uses Luminex xMAP technology were combined into a new testing algorithm. To evaluate this algorithm, cervical swabs (n = 860) and cervical biopsy specimens (n = 355) were tested, with a focus on HPV types detected by the MPTS123 assay (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 6, and 11). Among the HPV-positive samples, identifications of individual HPV genotypes were compared. When all MPTS123 targeted genotypes were considered together, good overall agreement was found (κ = 0.801, 95% confidence interval [CI], 0.784 to 0.818) with identification by SPF10 LiPA, but significantly more genotypes (P < 0.0001) were identified by the MPTS123 PCR Luminex assay, especially for HPV types 16, 35, 39, 45, 58, and 59. An alternative type-specific assay was evaluated that is based on detection of a limited number of HPV genotypes by type-specific PCR and a reverse hybridization assay (MPTS12 RHA). This assay showed results similar to those of the expanded MPTS123 Luminex assay. These results confirm the fact that broad-spectrum PCRs are hampered by type competition when multiple HPV genotypes are present in the same sample. Therefore, a testing algorithm combining the broad-spectrum PCR and a range of type-specific PCRs can offer a highly accurate method for the analysis of HPV infections and diminish the rate of false-negative results and may be particularly useful for epidemiological and vaccine studies.

  13. A Human Papilloma Virus Testing Algorithm Comprising a Combination of the L1 Broad-Spectrum SPF10 PCR Assay and a Novel E6 High-Risk Multiplex Type-Specific Genotyping PCR Assay

    PubMed Central

    Kleter, Bernhard; Vent, Maarten; Delroisse, Jean-Marc; de Koning, Maurits; van Doorn, Leen-Jan; Quint, Wim; Colau, Brigitte

    2013-01-01

    Human papillomavirus (HPV) epidemiological and vaccine studies require highly sensitive HPV detection and genotyping systems. To improve HPV detection by PCR, the broad-spectrum L1-based SPF10 PCR DNA enzyme immunoassay (DEIA) LiPA system and a novel E6-based multiplex type-specific system (MPTS123) that uses Luminex xMAP technology were combined into a new testing algorithm. To evaluate this algorithm, cervical swabs (n = 860) and cervical biopsy specimens (n = 355) were tested, with a focus on HPV types detected by the MPTS123 assay (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 6, and 11). Among the HPV-positive samples, identifications of individual HPV genotypes were compared. When all MPTS123 targeted genotypes were considered together, good overall agreement was found (κ = 0.801, 95% confidence interval [CI], 0.784 to 0.818) with identification by SPF10 LiPA, but significantly more genotypes (P < 0.0001) were identified by the MPTS123 PCR Luminex assay, especially for HPV types 16, 35, 39, 45, 58, and 59. An alternative type-specific assay was evaluated that is based on detection of a limited number of HPV genotypes by type-specific PCR and a reverse hybridization assay (MPTS12 RHA). This assay showed results similar to those of the expanded MPTS123 Luminex assay. These results confirm the fact that broad-spectrum PCRs are hampered by type competition when multiple HPV genotypes are present in the same sample. Therefore, a testing algorithm combining the broad-spectrum PCR and a range of type-specific PCRs can offer a highly accurate method for the analysis of HPV infections and diminish the rate of false-negative results and may be particularly useful for epidemiological and vaccine studies. PMID:23363835

  14. Reverse-Transcriptase PCR Detection of Leptospira: Absence of Agreement with Single-Specimen Microscopic Agglutination Testing

    PubMed Central

    Waggoner, Jesse J.; Balassiano, Ilana; Mohamed-Hadley, Alisha; Vital-Brazil, Juliana Magalhães; Sahoo, Malaya K.; Pinsky, Benjamin A.

    2015-01-01

    Background Reference diagnostic tests for leptospirosis include nucleic acid amplification tests, bacterial culture, and microscopic agglutination testing (MAT) of acute and convalescent serum. However, clinical laboratories often do not receive paired specimens. In the current study, we tested serum samples using a highly sensitive real-time nucleic acid amplification test for Leptospira and compared results to MAT performed on the same specimens. Methods/Principal Findings 478 serum samples from suspected leptospirosis cases in Rio de Janeiro were tested using a real-time RT-PCR for the diagnosis of leptospirosis, malaria and dengue (the Lepto-MD assay). The Lepto-MD assay detects all species of Leptospira (saprophytic, intermediate, and pathogenic), and in the current study, we demonstrate that this assay amplifies both Leptospira RNA and DNA. Dengue virus RNA was identified in 10 patients, and no cases of malaria were detected. A total of 65 samples (13.6%) were positive for Leptospira: 35 samples (7.3%) in the Lepto-MD assay, 33 samples (6.9%) by MAT, and 3 samples tested positive by both (kappa statistic 0.02). Poor agreement between methods was consistent regardless of the titer used to define positive MAT results or the day of disease at sample collection. Leptospira nucleic acids were detected in the Lepto-MD assay as late as day 22, and cycle threshold values did not differ based on the day of disease. When Lepto-MD assay results were added to the MAT results for all patients in 2008 (n=818), the number of detected leptospirosis cases increased by 30.4%, from 102 (12.5%) to 133 (16.3%). Conclusions/Significance This study demonstrates a lack of agreement between nucleic acid detection of Leptospira and single-specimen MAT, which may result from the clearance of bacteremia coinciding with the appearance of agglutinating antibodies. A combined testing strategy for acute leptospirosis, including molecular and serologic testing, appears necessary to maximize

  15. Qualitative Organic Analysis: An Efficient, Safer, and Economical Approach to Preliminary Tests and Functional Group Analysis

    ERIC Educational Resources Information Center

    Dhingra, Sunita; Angrish, Chetna

    2011-01-01

    Qualitative organic analysis of an unknown compound is an integral part of the university chemistry laboratory curriculum. This type of training is essential as students learn to approach a problem systematically and to interpret the results logically. However, considerable quantities of waste are generated by using conventional methods of…

  16. Qualitative Organic Analysis: An Efficient, Safer, and Economical Approach to Preliminary Tests and Functional Group Analysis

    ERIC Educational Resources Information Center

    Dhingra, Sunita; Angrish, Chetna

    2011-01-01

    Qualitative organic analysis of an unknown compound is an integral part of the university chemistry laboratory curriculum. This type of training is essential as students learn to approach a problem systematically and to interpret the results logically. However, considerable quantities of waste are generated by using conventional methods of…

  17. Primary care clinicians’ attitudes towards point-of-care blood testing: a systematic review of qualitative studies

    PubMed Central

    2013-01-01

    Background Point-of-care blood tests are becoming increasingly available and could replace current venipuncture and laboratory testing for many commonly used tests. However, at present very few have been implemented in most primary care settings. Understanding the attitudes of primary care clinicians towards these tests may help to identify the barriers and facilitators to their wider adoption. We aimed to systematically review qualitative studies of primary care clinicians’ attitudes to point-of-care blood tests. Methods We systematically searched Medline, Embase, ISI Web of Knowledge, PsycINFO and CINAHL for qualitative studies of primary care clinicians’ attitudes towards point-of-care blood tests in high income countries. We conducted a thematic synthesis of included studies. Results Our search identified seven studies, including around two hundred participants from Europe and Australia. The synthesis generated three main themes: the impact of point-of-care testing on decision-making, diagnosis and treatment; impact on clinical practice more broadly; and impact on patient-clinician relationships and perceived patient experience. Primary care clinicians believed point-of-care testing improved diagnostic certainty, targeting of treatment, self-management of chronic conditions, and clinician-patient communication and relationships. There were concerns about test accuracy, over-reliance on tests, undermining of clinical skills, cost, and limited usefulness. Conclusions We identified several perceived benefits and barriers regarding point-of-care tests in primary care. These imply that if point-of-care tests are to become more widely adopted, primary care clinicians require evidence of their accuracy, rigorous testing of the impact of introduction on patient pathways and clinical practice, and consideration of test funding. PMID:23945264

  18. A multianalyte PCR blood test outperforms single analyte ELISAs (chromogranin A, pancreastatin, neurokinin A) for neuroendocrine tumor detection.

    PubMed

    Modlin, Irvin M; Drozdov, Ignat; Alaimo, Daniele; Callahan, Stephen; Teixiera, Nancy; Bodei, Lisa; Kidd, Mark

    2014-08-01

    A critical requirement in neuroendocrine tumor (NET) management is a sensitive, specific and reproducible blood biomarker test. We evaluated a PCR-based 51 transcript signature (NETest) and compared it to chromogranin A (CgA), pancreastatin (PST) and neurokinin A (NKA). The multigene signature was evaluated in two groups: i) a validation set of 40 NETs and controls and ii) a prospectively collected group of NETs (n=41, 61% small intestinal, 50% metastatic, 44% currently treated and 41 age-sex matched controls). Samples were analyzed by a two-step PCR (51 marker genes) protocol and ELISAs for CgA, PST and NKA. Sensitivity comparisons included χ(2), non-parametric measurements, ROC curves and predictive feature importance (PFAI) analyses. NETest identified 38 of 41 NETs. Performance metrics were: sensitivity 92.8%, specificity 92.8%, positive predictive value 92.8% and negative predictive value 92.8%. Single analyte ELISA metrics were: CgA 76, 59, 65, and 71%; PST 63, 56, 59, and 61% and NKA 39, 93, 84, and 60%. The AUCs (ROC analysis) were: NETest: 0.96±0.025, CgA: 0.67±0.06, PST 0.56±0.06, NKA: 0.66±0.06. NETest significantly outperformed single analyte tests (area differences: 0.284-0.403, Z-statistic 4.85-5.9, P<0.0001). PFAI analysis determined NETest had most value (69%) in diagnosis (CgA (13%), PST (9%), and NKA (9%)). Test data were consistent with the validation set (NETest >95% sensitivity and specificity, AUC =0.98 vs single analytes: 59-67% sensitivity, AUCs: 0.58-0.63). The NETest is significantly more sensitive and efficient (>93%) than single analyte assays (CgA, PST or NKA) in NET diagnosis. Blood-based multigene analytic measurement will facilitate early detection of disease recurrence and can predict therapeutic efficacy. ©  2014 Society for Endocrinology.

  19. Cross-Institute Evaluations of Inhibitor-Resistant PCR Reagents for Direct Testing of Aerosol and Blood Samples Containing Biological Warfare Agent DNA

    PubMed Central

    Minogue, Timothy D.; Rachwal, Phillip A.; Trombley Hall, Adrienne; Koehler, Jeffery W.

    2014-01-01

    Rapid pathogen detection is crucial for the timely introduction of therapeutics. Two groups (one in the United Kingdom and one in the United States) independently evaluated inhibitor-resistant PCR reagents for the direct testing of substrates. In the United Kingdom, a multiplexed Bacillus anthracis (target) and Bacillus subtilis (internal-control) PCR was used to evaluate 4 reagents against 5 PCR inhibitors and down-selected the TaqMan Fast Virus 1-Step master mix (Life Technologies Inc.). In the United States, four real-time PCR assays (targeting B. anthracis, Brucella melitensis, Venezuelan equine encephalitis virus [VEEV], and Orthopoxvirus spp.) were used to evaluate 5 reagents (plus the Fast Virus master mix) against buffer, blood, and soil samples and down-selected the KAPA Blood Direct master mix (KAPA Biosystems Inc.) with added Platinum Taq (Life Technologies). The down-selected reagents underwent further testing. In the United Kingdom experiments, both reagents were tested against seven contrived aerosol collector samples containing B. anthracis Ames DNA and B. subtilis spores from a commercial formulation (BioBall). In PCR assays with reaction mixtures containing 40% crude sample, an airfield-collected sample induced inhibition of the B. subtilis PCR with the KAPA reagent and complete failure of both PCRs with the Fast Virus reagent. However, both reagents allowed successful PCR for all other samples—which inhibited PCRs with a non-inhibitor-resistant reagent. In the United States, a cross-assay limit-of-detection (LoD) study in blood was conducted. The KAPA Blood Direct reagent allowed the detection of agent DNA (by four PCRs) at higher concentrations of blood in the reaction mixture (2.5%) than the Fast Virus reagent (0.5%), although LoDs differed between assays and reagent combinations. Across both groups, the KAPA Blood Direct reagent was determined to be the optimal reagent for inhibition relief in PCR. PMID:24334660

  20. Cross-institute evaluations of inhibitor-resistant PCR reagents for direct testing of aerosol and blood samples containing biological warfare agent DNA.

    PubMed

    Minogue, Timothy D; Rachwal, Phillip A; Trombley Hall, Adrienne; Koehler, Jeffery W; Weller, Simon A

    2014-02-01

    Rapid pathogen detection is crucial for the timely introduction of therapeutics. Two groups (one in the United Kingdom and one in the United States) independently evaluated inhibitor-resistant PCR reagents for the direct testing of substrates. In the United Kingdom, a multiplexed Bacillus anthracis (target) and Bacillus subtilis (internal-control) PCR was used to evaluate 4 reagents against 5 PCR inhibitors and down-selected the TaqMan Fast Virus 1-Step master mix (Life Technologies Inc.). In the United States, four real-time PCR assays (targeting B. anthracis, Brucella melitensis, Venezuelan equine encephalitis virus [VEEV], and Orthopoxvirus spp.) were used to evaluate 5 reagents (plus the Fast Virus master mix) against buffer, blood, and soil samples and down-selected the KAPA Blood Direct master mix (KAPA Biosystems Inc.) with added Platinum Taq (Life Technologies). The down-selected reagents underwent further testing. In the United Kingdom experiments, both reagents were tested against seven contrived aerosol collector samples containing B. anthracis Ames DNA and B. subtilis spores from a commercial formulation (BioBall). In PCR assays with reaction mixtures containing 40% crude sample, an airfield-collected sample induced inhibition of the B. subtilis PCR with the KAPA reagent and complete failure of both PCRs with the Fast Virus reagent. However, both reagents allowed successful PCR for all other samples-which inhibited PCRs with a non-inhibitor-resistant reagent. In the United States, a cross-assay limit-of-detection (LoD) study in blood was conducted. The KAPA Blood Direct reagent allowed the detection of agent DNA (by four PCRs) at higher concentrations of blood in the reaction mixture (2.5%) than the Fast Virus reagent (0.5%), although LoDs differed between assays and reagent combinations. Across both groups, the KAPA Blood Direct reagent was determined to be the optimal reagent for inhibition relief in PCR.

  1. Qualitative point-of-care D-dimer testing compared with quantitative D-dimer testing in excluding pulmonary embolism in primary care.

    PubMed

    Lucassen, W A M; Erkens, P M G; Geersing, G J; Büller, H R; Moons, K G M; Stoffers, H E J H; van Weert, H C P M

    2015-06-01

    General practitioners can safely exclude pulmonary embolism (PE) by using the Wells PE rule combined with D-dimer testing. To compare the accuracy of a strategy using the Wells rule combined with either a qualitative point-of-care (POC) D-dimer test performed in primary care or a quantitative laboratory-based D-dimer test. We used data from a prospective cohort study including 598 adults suspected of PE in primary care in the Netherlands. General practitioners scored the Wells rule and carried out a qualitative POC test. All patients were referred to hospital for reference testing. We obtained quantitative D-dimer test results as performed in hospital laboratories. The primary outcome was the prevalence of venous thromboembolism in low-risk patients. Prevalence of PE was 12.2%. POC D-dimer test results were available in 582 patients (97%). Quantitative test results were available in 401 patients (67%). We imputed results in 197 patients. The quantitative test and POC test missed one (0.4%) and four patients (1.5%), respectively, with a negative strategy (Wells ≤ 4 points and D-dimer test negative) (P = 0.20). The POC test could exclude 23 more patients (4%) (P = 0.05). The sensitivity and specificity of the Wells rule combined with a POC test were 94.5% and 51.0% and, combined with a quantitative test, 98.6% and 47.2%, respectively. Combined with the Wells PE rule, both tests are safe to use in excluding PE. The quantitative test seemed to be safer than the POC test, albeit not statistically significant. The specificity of the POC test was higher, resulting in more patients in whom PE could be excluded. © 2015 International Society on Thrombosis and Haemostasis.

  2. Bayesian techniques for comparison of the test performance of PCR and culture for the identification of Campylobacter in enriched comminuted chicken samples.

    PubMed

    Ebel, E D; Williams, M S; Golden, N J; Tankson, J

    2016-05-01

    Using Bayesian methods that do not require the definition of a gold standard, the diagnostic sensitivity and specificity of a real-time polymerase chain reaction (PCR) assay are compared to those of an enriched culture assay for detection of Campylobacter in enriched comminuted chicken samples. Food Safety and Inspection Service comminuted chicken samples were collected from production facilities across the United States. Enriched samples were examined using a commercial real-time PCR kit and plated for culture. Allowing for conditional dependence between these approaches and defining relatively uninformed prior distributions, the 'no gold standard' Bayesian methods generated estimates of the means (95% credible interval) of the posterior distributions for sensitivity and specificity of the PCR as 93% (79, 100%) and 95% (87, 100%) respectively. The estimated sensitivity implies a mean false negative frequency of 7%. The estimated means of the posterior distributions for sensitivity and specificity of the culture assay were 91% (76, 100%) and 96% (88, 100%) respectively. In this case, the mean false negative frequency is 9%. Graphical comparisons of the posterior distributions with their corresponding prior distributions suggested only subtle differences in the sensitivities of both tests, but the posterior distributions for specificities are substantially more certain than the prior distributions. The study suggests that the commercial real-time PCR assay is a more sensitive screening test that would provide timelier negative test results. The modest 1% reduction in specificity of this PCR assay, as compared to an enriched culture assay, is less of a concern for regulatory testing programs if a culture-based confirmatory assay is applied to all presumptive positive samples. The sensitivity and specificity of a PCR assay and a culture assay for Campylobacter in comminuted poultry produced in the United States were estimated. The PCR assay was shown to be an

  3. Rapid colorimetric testing for pyrazinamide susceptibility of M. tuberculosis by a PCR-based in-vitro synthesized pyrazinamidase method.

    PubMed

    Zhou, Man; Geng, Xuelei; Chen, Jun; Wang, Xude; Wang, Dianbing; Deng, Jiaoyu; Zhang, Zhiping; Wang, Weihua; Zhang, Xian-En; Wei, Hongping

    2011-01-01

    Pyrazinamide (PZA) is an important first-line anti-tuberculosis drug. But PZA susceptibility test is challenging because PZA activity is optimal only in an acid environment that inhibits the growth of M. tuberculosis. For current phenotypic methods, inconsistent results between different labs have been reported. Direct sequencing of pncA gene is being considered as an accurate predictor for PZA susceptibility, but this approach needs expensive sequencers and a mutation database to report the results. An in-vitro synthesized Pyrazinamidase (PZase) assay was developed based on PCR amplification of pncA gene and an in vitro wheat germ system to express the pncA gene into PZase. The activity of the synthesized PZase was used as an indicator for PZA susceptibility. Fifty-one clinical isolates were tested along with pncA sequencing and the BACTEC MGIT 960 methods. The in-vitro synthesized PZase assay was able to detect PZA susceptibility of M. tuberculosis within 24 h through observing the color difference either by a spectrometer or naked eyes. This method showed agreements of 100% (33/33) and 88% (14/16) with the pncA sequencing method, and agreements of 96% (27/28) and 65% (15/23) with the BACTEC MGIT 960 method, for susceptible and resistant strains, respectively. The novel in-vitro synthesized PZase assay has significant advantages over current methods, such as its fast speed, simplicity, no need for expensive equipment, and the potentials of being a direct test, predicting resistance level and easy reading results by naked eyes. After confirmation by more clinical tests, this method may provide a radical change to the current PZA susceptibility assays.

  4. Barriers to Point-of-Care Testing in India: Results from Qualitative Research across Different Settings, Users and Major Diseases

    PubMed Central

    Engel, Nora; Ganesh, Gayatri; Patil, Mamata; Yellappa, Vijayashree; Pant Pai, Nitika; Vadnais, Caroline; Pai, Madhukar

    2015-01-01

    Background Successful point-of-care testing, namely ensuring the completion of the test and treat cycle in the same encounter, has immense potential to reduce diagnostic and treatment delays, and impact patient outcomes. However, having rapid tests is not enough, as many barriers may prevent their successful implementation in point-of-care testing programs. Qualitative research on diagnostic practices may help identify such barriers across different points of care in health systems. Methods In this exploratory qualitative study, we conducted 78 semi-structured interviews and 13 focus group discussions in an urban and rural area of Karnataka, India, with healthcare providers (doctors, nurses, specialists, traditional healers, and informal providers), patients, community health workers, test manufacturers, laboratory technicians, program managers and policy-makers. Participants were purposively sampled to represent settings of hospitals, peripheral labs, clinics, communities and homes, in both the public and private sectors. Results In the Indian context, the onus is on the patient to ensure successful point-of-care testing across homes, clinics, labs and hospitals, amidst uncoordinated providers with divergent and often competing practices, in settings lacking material, money and human resources. We identified three overarching themes affecting point-of-care testing: the main theme is ‘relationships’ among providers and between providers and patients, influenced by the cross-cutting theme of ‘infrastructure’. Challenges with both result in ‘modified practices’ often favouring empirical (symptomatic) treatment over treatment guided by testing. Conclusions Even if tests can be conducted on the spot and infrastructure challenges have been resolved, relationships among providers and between patients and providers are crucial for successful point-of-care testing. Furthermore, these barriers do not act in isolation, but are interlinked and need to be examined

  5. Real-time PCR Tests in Dutch Exotic Mosquito Surveys; Implementation of Aedes aegypti and Aedes albopictus Identification Tests, and the Development of Tests for the Identification of Aedes atropalpus and Aedes japonicus japonicus (Diptera: Culicidae).

    PubMed

    van de Vossenberg, B T L H; Ibáñez-Justicia, A; Metz-Verschure, E; van Veen, E J; Bruil-Dieters, M L; Scholte, E J

    2015-05-01

    Since 2009, The Netherlands Food and Consumer Product Safety Authority carries out surveys focusing on, amongst others, the presence of invasive mosquito species (IMS). Special attention is given to exotic container-breeding Aedes species Aedes aegypti (L.), Aedes albopictus (Skuse), Aedes atropalpus (Coquillett), and Aedes japonicus japonicus (Theobald). This study describes the implementation of real-time PCR tests described by Hill et al. (2008) for the identification of Ae. aegypti and Ae. albopictus, and the development of two novel real-time PCR tests for the identification of Ae. atropalpus and Ae. j. japonicus. Initial test showed that optimization of elements of the Ae. aegypti and Ae. albopictus tests was needed. Method validation tests were performed to determine if the implemented and newly developed tests are fit for routine diagnostics. Performance criteria of analytical sensitivity, analytical specificity, selectivity, repeatability, and reproducibility were determined. In addition, experiments were performed to determine the influence of environmental conditions on the usability of DNA extracted from mosquito specimens trapped in BG-Sentinel traps. The real-time PCR tests were demonstrated to be sensitive, specific, repeatable, reproducible, and are less prone to false negative results compared to partial cytochrome c oxidase I gene sequencing owing to the DNA fragmentation caused by environmental influences.

  6. Mitochondrial D-loop {open_quotes}signatures{close_quotes} produced by low-stringency single specific primer PCR constitute a simple comparative human identity test

    SciTech Connect

    Barreto, G.; Vago, A.R.; Pena, S.D.J.

    1996-03-01

    We have developed a technique called {open_quotes}LSSP-PCR{close_quotes} (low-stringency single specific primer PCR) that detects single or multiple mutations in DNA. A purified DNA fragment is submitted to PCR by using a single primer specific for one of the extremities of the fragment, under conditions of very low stringency. The primer hybridizes specifically to its complementary extremity and nonspecifically to multiple sites within the fragment, in a sequence-dependent manner. A complex set of reaction products is thus created that, when separated by electrophoresis, constitutes a unique {open_quotes}gene signature.{close_quotes} We here report the application of LSSP-PCR to the detection of sequence variation in the control (D-loop) region of human mtDNA, which is known to differ significantly between unrelated individuals. We prepared human DNA samples from blood and amplified a 1,024-bp portion of the mtDNA control region, using primers L15996 and H408. The amplified mtDNA fragments were then reamplified under LSSP-PCR conditions by using L15996 or H408 as drivers to produce complex signatures that always differed between unrelated individuals and yet were highly reproducible. In contrast, all mother-child pairs tested were identical, as expected from the matrilineal inheritance of mtDNA. Thus, the use of LSSP-PCR to produce D-loop signatures constitutes a powerful new technique for mtDNA-based comparative identity testing. 18 refs., 7 figs.

  7. Comparison of a real-time PCR method with serology and blood smear analysis for diagnosis of human anaplasmosis: importance of infection time course for optimal test utilization.

    PubMed

    Schotthoefer, A M; Meece, J K; Ivacic, L C; Bertz, P D; Zhang, K; Weiler, T; Uphoff, T S; Fritsche, T R

    2013-07-01

    Anaplasmosis and ehrlichiosis are emerging tick-borne diseases with clinically similar presentations caused by closely related pathogens. Currently, laboratories rely predominantly on blood smear analysis (for the detection of intracellular morulae) and on serologic tests, both of which have recognized limitations, for diagnostic purposes. We compared the performance of a published real-time PCR assay that incorporates melt curve analysis to differentiate Anaplasma and Ehrlichia species with blood smear and serologic methods in an upper Midwest population. Overall, 38.5% of the specimens selected for evaluation had one or more tests that were positive for anaplasmosis. The PCR positivity for all specimens was maximal (21.2%; 29/137) during the early acute phase of illness (0 to 4 days since illness onset) and significantly less frequent (11.5%; 20/174) during later phases (>4 days since illness onset). All positive specimens were Anaplasma phagocytophilum; no Ehrlichia species were identified. The real-time PCR detected 100% of infections that were detected by blood smear analysis (14/14) and broadened the detection window from a maximum of 14 days for smear positivity to 30 days for PCR. Additional infections were detected by real-time PCR in 12.9% (11/85) of smear-negative patients. There was poor agreement between the real-time PCR assay and serologic test results: 19.8% (19/96) and 13.7% (29/212) of seropositive and -negative patients, respectively, were PCR positive. Seropositivity increased with increasing days of illness, demonstrating that serologic detection methods are best utilized during presumed convalescence. Our results indicate that the optimal performance and utilization of laboratory tests for the diagnosis of anaplasmosis require knowledge regarding time of symptom onset or days of illness.

  8. Development of PCR-based tests for the identification of North American isolates of epizootic haemorrhagic disease virus.

    PubMed Central

    Harding, M J; Prud'homme, I; Rola, J; Dulac, G C

    1996-01-01

    A serogroup-specific polymerase chain reaction (PCR) assay and isolate identification strategies (restriction endonuclease analysis (REA) and nucleotide sequencing) were developed for the detection of North American isolates of epizootic haemorrhagic disease virus (EHDV). PCR primers (EHDV-pr4, EHDV-pr5) were designed to hybridize to the L3 gene of a North American isolate of EHDV serotype 1. Total nucleic acid was extracted from preparations of infected tissue culture and PCR was performed using a cDNA-PCR kit, according to the manufacturer's specifications. The PCR assay generated a 459 base pair product from North American isolates of EHDV serotypes 1 and 2, while bluetongue virus (BTV) serotypes 10, 11, 13, and 17, and cell controls, failed to demonstrate PCR products. Slight modifications allowed for the PCR detection of EHDV-1 and -2 in white-tailed deer blood (Odocoileus virginiatus); PCR fragments were not amplified from uninfected deer blood. A number of restriction endonucleases and sequencing primers were evaluated for their utility in isolate identification experiments. Specifically, REA employing HincII and cycle sequencing with an internal primer (EHDV-1-pr3) proved most successful for identifying isolate-specific genome markers. The techniques presented are expected to prove valuable for rapid and specific detection of possible future EHDV incursions in wild and domestic animal species. Images Figure 1. Figure 2. Figure 3. PMID:8825996

  9. Everyday uses of standardized test information in a geriatric setting: a qualitative study exploring occupational therapist and physiotherapist test administrators’ justifications

    PubMed Central

    2014-01-01

    Background Health professionals are required to collect data from standardized tests when assessing older patients’ functional ability. Such data provide quantifiable documentation on health outcomes. Little is known, however, about how physiotherapists and occupational therapists who administer standardized tests use test information in their daily clinical work. This article aims to investigate how test administrators in a geriatric setting justify the everyday use of standardized test information. Methods Qualitative study of physiotherapists and occupational therapists on two geriatric hospital wards in Norway that routinely tested their patients with standardized tests. Data draw on seven months of fieldwork, semi-structured interviews with eight physiotherapists and six occupational therapists (12 female, two male), as well as observations of 26 test situations. Data were analyzed using Systematic Text Condensation. Results We identified two test information components in everyday use among physiotherapist and occupational therapist test administrators. While the primary component drew on the test administrators’ subjective observations during testing, the secondary component encompassed the communication of objective test results and test performance. Conclusions The results of this study illustrate the overlap between objective and subjective data in everyday practice. In clinical practice, by way of the clinicians’ gaze on how the patient functions, the subjective and objective components of test information are merged, allowing individual characteristics to be noticed and made relevant as test performance justifications and as rationales in the overall communication of patient needs. PMID:24528609

  10. Development and evaluation of a novel RT-qPCR based test for the quantification of HER2 gene expression in breast cancer.

    PubMed

    El Hadi, Hicham; Abdellaoui-Maane, Imane; Kottwitz, Denise; El Amrani, Manal; Bouchoutrouch, Nadia; Qmichou, Zineb; Karkouri, Mehdi; ElAttar, Hicham; Errihani, Hassan; Fernandez, Pedro L; Bakri, Youssef; Sefrioui, Hassan; Moumen, Abdeladim

    2017-03-20

    Accurate measurement of Human epidermal growth factor receptor (HER2) gene expression is central for breast or stomach cancer therapy orientation and prognosis. The current standards testing methods for HER2 expression are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). In the current study, we explored the use of quantitative real time reverse transcription-PCR (RT-qPCR) as a potential method for the accurate relative quantification of the HER2 gene using formalin fixed paraffin embedded (FFPE) breast cancer biopsy samples. The main aim of the current study is to measure the level of concordance of RT-qPCR based quantification of HER2 overexpression with both IHC and FISH. Accordingly, an endogenous control gene (ECG) is required for this relative quantification and should ideally be expressed equivalently across tested samples. Stably expressed ECGs have been selected from a panel of seven genes using GenEx V6 software which is based on geNorm and NormFinder and statistical methods. Quantification of HER2 gene expression was performed by our RT-qPCR-based test and compared to the results obtained by both IHC and FISH methods. HER2 gene quantification using RT-qPCR test was normalized using the two ECGs (RPL30 and RPL37A) that were successfully identified and selected from a panel of seven genes as the most stable and reliable ECGs. We evaluated a total of 216 FFPE tissue samples from breast cancer patients. The results obtained with RT-qPCR in the current study were compared to both IHC and FISH data collected for the same patients. In addition to an internal evaluation, an external evaluation of this assay was also performed in a recognized pathology center in Europe (Clinic Barcelona Hospital Universitari, Spain) using 116 FFPE breast cancer tissue samples. The results demonstrated a high concordance between RT-qPCR and either IHC (98%) or FISH (72%) methods. Accordantly, the overall concordance was 85%. To our knowledge, this is the

  11. Amplification of Mycoplasma haemofelis DNA by a PCR for point-of-care use.

    PubMed

    Hawley, Jennifer; Yaaran, Tal; Maurice, Sarah; Lappin, Michael R

    2017-09-01

    We compared a qualitative in-clinic (IC)-PCR for the detection of Mycoplasma haemofelis DNA with the results of a commercial qualitative laboratory-based, conventional (c)PCR. In order to determine the specificity of both tests, Bartonella spp. samples were included. Forty-three previously tested blood samples with known PCR results for hemoplasmas and Bartonella spp. were selected. The samples were split between 2 laboratories. At the first laboratory, DNA was purified and run on 2 cPCR assays for the detection of hemoplasmas and Bartonella spp. At the second laboratory, DNA was purified using 2 purification protocols and both run in the IC-PCR assay. The cPCR results confirmed that 18 samples were positive for M. haemofelis, 5 for ' Candidatus M. haemominutum', 8 for Bartonella henselae, 2 for Bartonella clarridgeiae, and 10 were negative for both genera. No mixed infections were observed. The IC-PCR assay for the detection of M. haemofelis had a sensitivity of 94.4% and specificity of 96%, when using the same DNA purification method as the first laboratory. Using the second purification method, the sensitivity of the IC-PCR assay was 77.8% and specificity was 96%. Bartonella species were not detected by the IC-PCR M. haemofelis assay. The IC-PCR assay decreased the amount of time to final result compared to a cPCR assay.

  12. Narrative-based educational nursing intervention for managing hospitalized older adults at risk for delirium: field testing and qualitative evaluation.

    PubMed

    Bélanger, Louise; Ducharme, Francine

    2015-01-01

    Though delirium is a common complication among hospitalized older adults and the nursing care required in these situations is complex, the subject has received little attention in the literature on continuing nursing education. A study was undertaken to field test and qualitatively evaluate a narrative-based educational intervention for nurses in hospital units with a high incidence of delirium. Triangulated data collection allowed carrying out a qualitative evaluation of the intervention process and outcomes. Process evaluation showed that the intervention was facilitated by the participants' attitudes and diversity of experience, as well as by the use of real care situations, which allowed integrating theory and practice. Outcome evaluation brought to light numerous elements of empirical, ethical and esthetic knowledge expressed by the participants. Study results evidence the applicability of such interventions as part of continuing nursing education and their contribution to knowledge development. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. A systematic review of qualitative findings on factors enabling and deterring uptake of HIV testing in Sub-Saharan Africa

    PubMed Central

    2013-01-01

    Background Despite Sub-Saharan Africa (SSA) being the epicenter of the HIV epidemic, uptake of HIV testing is not optimal. While qualitative studies have been undertaken to investigate factors influencing uptake of HIV testing, systematic reviews to provide a more comprehensive understanding are lacking. Methods Using Noblit and Hare’s meta-ethnography method, we synthesised published qualitative research to understand factors enabling and deterring uptake of HIV testing in SSA. We identified 5,686 citations out of which 56 were selected for full text review and synthesised 42 papers from 13 countries using Malpass’ notion of first-, second-, and third-order constructs. Results The predominant factors enabling uptake of HIV testing are deterioration of physical health and/or death of sexual partner or child. The roll-out of various HIV testing initiatives such as ‘opt-out’ provider-initiated HIV testing and mobile HIV testing has improved uptake of HIV testing by being conveniently available and attenuating fear of HIV-related stigma and financial costs. Other enabling factors are availability of treatment and social network influence and support. Major barriers to uptake of HIV testing comprise perceived low risk of HIV infection, perceived health workers’ inability to maintain confidentiality and fear of HIV-related stigma. While the increasingly wider availability of life-saving treatment in SSA is an incentive to test, the perceived psychological burden of living with HIV inhibits uptake of HIV testing. Other barriers are direct and indirect financial costs of accessing HIV testing, and gender inequality which undermines women’s decision making autonomy about HIV testing. Despite differences across SSA, the findings suggest comparable factors influencing HIV testing. Conclusions Improving uptake of HIV testing requires addressing perception of low risk of HIV infection and perceived inability to live with HIV. There is also a need to continue

  14. Wet-lab tested microRNA assays for qPCR studies with SYBR® Green and DNA primers in pig tissues.

    PubMed

    Mentzel, Caroline M J; Skovgaard, Kerstin; Córdoba, Sarai; Herrera Uribe, Juber; Busk, Peter K; Cirera, Susanna

    2014-01-01

    MicroRNAs are key post-transcriptional regulators of gene expression that are involved in several biological processes including those that mediate disease pathophysiology. Hence, quantifying microRNA expression levels can provide important and novel insights into disease biology. In recent years, the pig has emerged as an excellent large animal model for studying human diseases and conditions (e.g. obesity) due to similarities in organ size, gastro-intestinal tract, metabolism, immune response, genetics and the availability of relevant tissues that are not normally easily available in humans. We have previously developed two useful tools in the field of microRNA quantitative real time PCR (qPCR): 1) a very specific, sensitive and simple qPCR method based on DNA primers, MiR-specific qPCR; and 2) the free primer-design software miRprimer. The present study integrates in a publicly accessible database all available information on validated porcine microRNA qPCR assays that have utilized these tools. Due to the high phylogenetic conservation in microRNA sequence between pig, humans and other domestic species this database is a very valuable resource for the broader scientist community who are working on microRNAs and want to use readily tested qPCR assays in a simple and cost-effective manner.

  15. Performance of Roche CAP/CTM HIV-1 qualitative test version 2.0 using dried blood spots for early infant diagnosis.

    PubMed

    Gueye, Sokhna Bousso; Diop-Ndiaye, Halimatou; Diallo, Mamadou Malick; Ly, Omar; Sow-Ndoye, Aissatou; Diagne-Gueye, Ndèye Diabou; Kébé-Fall, Khady; Diop, Fatou; Gaye-Diallo, Aïssatou; Belec, Laurent; Mboup, Souleymane; Touré-Kane, Coumba

    2016-03-01

    In the context of early infant diagnosis (EID) decentralization in sub-Saharan Africa, dried blood spot (DBS) is now widely used for HIV proviral DNA detection in resource-limited settings. A new version of CAP/CTM (version 2) has been introduced, recently by Roche Diagnosis as a new real-time PCR assay to replace previous technologies on qualitative detection of HIV-1 DNA using whole blood and DBS samples. The objective of this study was to evaluate CAP/CTM version 2 compared to CAP/CTM version 1 and Amplicor on DBS. A total of 261 DBS were collected from children aged 4 weeks to 17 months born from HIV-seropositive mothers and tested by the three techniques. CAP/CTM version 2 showed 100% of agreement with Amplicor including 74 positive results and 187 negative results. CAP/CTM version 2 versus CAP/CTM version 1 as well as CAP/CTM version 1 versus Amplicor showed two discordant results giving a sensitivity of 98.6%, specificity of 99.5%, positive predictive value of 98.6% and negative predictive value of 99.5%. The concordance was 99.12% (95% of confidence interval) giving a Kappa coefficient of 0.97 (p<0.001). These findings confirmed the expected good performance of CAP/CTM version 2 for HIV-1 EID.

  16. Aspergillus-Specific Lateral-Flow Device and Real-Time PCR Testing of Bronchoalveolar Lavage Fluid: a Combination Biomarker Approach for Clinical Diagnosis of Invasive Pulmonary Aspergillosis.

    PubMed

    Johnson, Gemma L; Sarker, Shah-Jalal; Nannini, Francesco; Ferrini, Arianna; Taylor, Emma; Lass-Flörl, Cornelia; Mutschlechner, Wolfgang; Bustin, Stephen A; Agrawal, Samir G

    2015-07-01

    Clinical experience with the impact of serum biomarkers for invasive fungal disease (IFD) varies markedly in hemato-oncology. Invasive pulmonary aspergillosis (IPA) is the most common manifestation, so we evaluated biomarkers in bronchoalveolar lavage (BAL) fluid. An Aspergillus-specific lateral-flow device (LFD), quantitative real-time PCR (qPCR), and the galactomannan (GM) test were used with 32 BAL fluid samples from 32 patients at risk of IPA. Eight patients had proven IPA, 3 had probable IPA, 6 had possible IPA, and 15 patients had no IPA by European Organization for Research and Treatment of Cancer Invasive Fungal Infections Cooperative Group/Mycoses Study Group of the National Institute of Allergy and Infectious Diseases (EORTC/MSG) criteria. The diagnostic accuracies of the tests were evaluated, and pairwise agreement between biomarkers was calculated. The diagnostic performance of the EORTC/MSG criteria was evaluated against the test(s) identified to be the most useful for IPA diagnosis. Using the EORTC/MSG criteria, the sensitivities of qPCR and LFD were 100% and the sensitivity of the GM test was 87.5% (GM test index cutoff, >0.8), with the tests having specificities of between 66.7 and 86.7%. The agreement between the results of qPCR and LFD was almost perfect (Cohen's kappa coefficient = 0.93, 95% confidence interval, 0.81 to 1.00). LFD and qPCR combined had a sensitivity of 100% and a specificity of 85.7%. Calcofluor staining and culture of all BAL fluid samples were negative for fungal infection. The median time from the start of mold-active antifungal therapy to the time of collection of BAL fluid was 6 days. Reversing roles and using dual testing by LFD and qPCR to classify cases, the EORTC/MSG criteria had a sensitivity of 83.3%. All three tests are useful for the diagnosis of IPA in BAL fluid samples. Despite the significant delays between the start of antifungal therapy and bronchoscopy, unlike microscopy and culture, the biomarkers remained

  17. A Qualitative Study of Barriers to the Utilization of HIV Testing Services Among Rural African American Cocaine Users

    PubMed Central

    Wright, Patricia B.; Stewart, Katharine E.; Curran, Geoffrey M.; Booth, Brenda M.

    2013-01-01

    This qualitative study is about barriers to the utilization of HIV testing as perceived by African Americans who have recently used cocaine and who live in the rural Delta region of Arkansas. Affordability, physical accessibility, and geographic availability were not perceived as barriers to HIV testing in this sample, yet acceptability was still perceived as poor. Acceptability due to social mores and norms was a major barrier. Many said testing was unacceptable because of fear of social costs. Many were confident of being HIV-negative based on risky assumptions about testing and the notification process. Small-town social and sexual networks added to concerns about reputation and risk. System approaches may fail if they focus solely on improving access to HIV services but do not take into consideration deeply internalized experiences of rural African Americans as well as involvement of the community in developing programs and services. PMID:24039279

  18. Correlation between Clostridium difficile bacterial load, commercial real-time PCR cycle thresholds, and results of diagnostic tests based on enzyme immunoassay and cell culture cytotoxicity assay.

    PubMed

    Dionne, Léa-Laurence; Raymond, Frédéric; Corbeil, Jacques; Longtin, Jean; Gervais, Philippe; Longtin, Yves

    2013-11-01

    The impact of Clostridium difficile fecal loads on diagnostic test results is poorly understood, but it may have clinical importance. In this study, we investigated the relationship between C. difficile fecal load and the results of four assays: a glutamate dehydrogenase (GDH) enzyme immunoassay (EIA), a toxin A/B antigen EIA (ToxAB), a cell culture cytotoxicity assay (CCA), and PCR targeting the tcdB gene. We also compared the PCR cycle threshold (CT) with the results of quantitative culture using Spearman's rank correlation coefficient. Finally, we sequenced the genomes of 24 strains with different detection profiles. A total of 203 clinical samples harboring toxigenic C. difficile were analyzed and sorted into one of four groups: 17 PCR(+) (group 1), 37 PCR(+) GDH(+) (group 2), 24 PCR(+) GDH(+) CCA(+) (group 3), and 125 PCR(+) GDH(+) ToxAB(+) (group 4). The overall median fecal load in log10 CFU/g was 6.67 (interquartile range [IQR], 5.57 to 7.54). The median fecal bacterial load of groups 1, 2, 3, and 4 were 4.15 (IQR, 3.00 to 4.98), 5.74 (IQR, 4.75 to 6.16), 6.20 (IQR, 5.23 to 6.80), and 7.08 (IQR, 6.35 to 7.83), respectively. Group 1 samples had lower fecal loads than those from each of the other groups (P < 0.001). Group 2 samples had lower fecal loads than those from groups 3 and 4 (P < 0.001). There was a significant correlation between PCR CT and fecal loads (ρ = -0.697; P < 0.001). NAP1 strains were associated with the detection of toxins by EIA or CCA (P = 0.041). This study demonstrates an association between C. difficile fecal load and the results of routinely used diagnostic tests.

  19. When Are Qualitative Testing Results Sufficient To Predict a Reduction in Illnesses in a Microbiological Food Safety Risk Assessment?

    PubMed

    Ebel, Eric D; Williams, Michael S

    2015-08-01

    Process models that include the myriad pathways that pathogen-contaminated food may traverse before consumption and the dose-response function to relate exposure to likelihood of illness may represent a "gold standard" for quantitative microbial risk assessment. Nevertheless, simplifications that rely on measuring the change in contamination occurrence of a raw food at the end of production may provide reasonable approximations of the effects measured by a process model. In this study, we parameterized three process models representing different product-pathogen pairs (i.e., chicken-Salmonella, chicken-Campylobacter, and beef-E. coli O157:H7) to compare with predictions based on qualitative testing of the raw product before consideration of mixing, partitioning, growth, attenuation, or dose-response processes. The results reveal that reductions in prevalence generated from qualitative testing of raw finished product usually underestimate the reduction in likelihood of illness for a population of consumers. Qualitative microbial testing results depend on the test's limit of detection. The negative bias is greater for limits of detection that are closer to the center of the contamination distribution and becomes less as the limit of detection is moved further into the right tail of the distribution. Nevertheless, a positive bias can result when the limit of detection refers to very high contamination levels. Changes in these high levels translate to larger consumed doses for which the slope of the dose-response function is smaller compared with the larger slope associated with smaller doses. Consequently, in these cases, a proportional reduction in prevalence of contamination results in a less than proportional reduction in probability of illness. The magnitudes of the biases are generally less for nonscalar (versus scalar) adjustments to the distribution.

  20. qPCR-High resolution melt analysis for drug susceptibility testing of Mycobacterium leprae directly from clinical specimens of leprosy patients

    PubMed Central

    Goulart, Luiz Ricardo; Truman, Richard W.; Goulart, Isabela Maria B.; Vissa, Varalakshmi; Li, Wei; Matsuoka, Masanori; Suffys, Philip; Fontes, Amanda B.; Rosa, Patricia S.; Scollard, David M.; Williams, Diana L.

    2017-01-01

    Background Real-Time PCR-High Resolution Melting (qPCR-HRM) analysis has been recently described for rapid drug susceptibility testing (DST) of Mycobacterium leprae. The purpose of the current study was to further evaluate the validity, reliability, and accuracy of this assay for M. leprae DST in clinical specimens. Methodology/Principal findings The specificity and sensitivity for determining the presence and susceptibility of M. leprae to dapsone based on the folP1 drug resistance determining region (DRDR), rifampin (rpoB DRDR) and ofloxacin (gyrA DRDR) was evaluated using 211 clinical specimens from leprosy patients, including 156 multibacillary (MB) and 55 paucibacillary (PB) cases. When comparing the results of qPCR-HRM DST and PCR/direct DNA sequencing, 100% concordance was obtained. The effects of in-house phenol/chloroform extraction versus column-based DNA purification protocols, and that of storage and fixation protocols of specimens for qPCR-HRM DST, were also evaluated. qPCR-HRM results for all DRDR gene assays (folP1, rpoB, and gyrA) were obtained from both MB (154/156; 98.7%) and PB (35/55; 63.3%) patients. All PCR negative specimens were from patients with low numbers of bacilli enumerated by an M. leprae-specific qPCR. We observed that frozen and formalin-fixed paraffin embedded (FFPE) tissues or archival Fite’s stained slides were suitable for HRM analysis. Among 20 mycobacterial and other skin bacterial species tested, only M. lepromatosis, highly related to M. leprae, generated amplicons in the qPCR-HRM DST assay for folP1 and rpoB DRDR targets. Both DNA purification protocols tested were efficient in recovering DNA suitable for HRM analysis. However, 3% of clinical specimens purified using the phenol/chloroform DNA purification protocol gave false drug resistant data. DNA obtained from freshly frozen (n = 172), formalin-fixed paraffin embedded (FFPE) tissues (n = 36) or archival Fite’s stained slides (n = 3) were suitable for qPCR-HRM DST

  1. Anticipated motivation for genetic testing among smokers, nonsmokers, and former smokers: an exploratory qualitative study of decision making.

    PubMed

    Giordimaina, Alicia M; Sheldon, Jane P; Petty, Elizabeth M

    2014-01-01

    This qualitative study explores the public's interest in genetic testing related to cigarette smoking, comparing the public's motivations with researchers' intentions for this technology. Adult nonsmokers (n=463), former smokers (n=163), and current smokers (n=129) completed an online survey. Within a hypothetical scenario, respondents decided whether they desired genetic testing related to smoking and explained their decision making. A non-parametric Kruskal-Wallis test was used to compare the interest in genetic testing by smoking history group. Inductive content analysis was used to investigate respondents' explanations for their testing decisions. Most nonsmokers (64%) and former smokers (58%) did not want genetic testing. While most current daily smokers were interested in testing (56%), most current occasional smokers were not (52%). Respondents' decision-making explanations were categorized into 3 major themes: Causality, Relevancy and Utility (e.g. personal benefits or harms). The use of causality, relevancy and utility explanations varied by smoking history. Notable perceived benefits of testing included recreation and altruism. Notable perceived harms included fear of fatalistic thoughts and concern about genetic discrimination. Interest in genetic testing was highest among current daily smokers, despite potential utility in other groups. Although respondents' motivations for testing paralleled researchers' intentions of tailoring smoking cessation therapies and increasing motivation to quit or abstain, respondents also raised alternative motivations and fears that healthcare providers would need to address. © 2014 S. Karger AG, Basel.

  2. A qualitative study of secondary distribution of HIV self-test kits by female sex workers in Kenya

    PubMed Central

    Maman, Suzanne; Murray, Katherine R.; Napierala Mavedzenge, Sue; Oluoch, Lennah; Sijenje, Florence; Agot, Kawango; Thirumurthy, Harsha

    2017-01-01

    Promoting awareness of serostatus and frequent HIV testing is especially important among high risk populations such as female sex workers (FSW) and their sexual partners. HIV self-testing is an approach that is gaining ground in sub-Saharan Africa as a strategy to increase knowledge of HIV status and promote safer sexual decisions. However, little is known about self-test distribution strategies that are optimal for increasing testing access among hard-to-reach and high risk individuals. We conducted a qualitative study with 18 FSW who participated in a larger study that provided them with five oral fluid-based self-tests, training on how to use the tests, and encouragement to offer the self-tests to their sexual partners using their discretion. Women demonstrated agency in the strategies they used to introduce self-tests to their partners and to avoid conflict with partners. They carefully considered with whom to share self-tests, often assessing the possibility for negative reactions from partners as part of their decision making process. When women faced negative reactions from partners, they drew on strategies they had used before to avoid conflict and physical harm from partners, such as not responding to angry partners and forgoing payment to leave angry partners quickly. Some women also used self-tests to make more informed sexual decisions with their partners. PMID:28346527

  3. A qualitative study of secondary distribution of HIV self-test kits by female sex workers in Kenya.

    PubMed

    Maman, Suzanne; Murray, Katherine R; Napierala Mavedzenge, Sue; Oluoch, Lennah; Sijenje, Florence; Agot, Kawango; Thirumurthy, Harsha

    2017-01-01

    Promoting awareness of serostatus and frequent HIV testing is especially important among high risk populations such as female sex workers (FSW) and their sexual partners. HIV self-testing is an approach that is gaining ground in sub-Saharan Africa as a strategy to increase knowledge of HIV status and promote safer sexual decisions. However, little is known about self-test distribution strategies that are optimal for increasing testing access among hard-to-reach and high risk individuals. We conducted a qualitative study with 18 FSW who participated in a larger study that provided them with five oral fluid-based self-tests, training on how to use the tests, and encouragement to offer the self-tests to their sexual partners using their discretion. Women demonstrated agency in the strategies they used to introduce self-tests to their partners and to avoid conflict with partners. They carefully considered with whom to share self-tests, often assessing the possibility for negative reactions from partners as part of their decision making process. When women faced negative reactions from partners, they drew on strategies they had used before to avoid conflict and physical harm from partners, such as not responding to angry partners and forgoing payment to leave angry partners quickly. Some women also used self-tests to make more informed sexual decisions with their partners.

  4. Perceptions and experiences of pregnant women about routine HIV testing and counselling in Ghimbi town, Ethiopia: a qualitative study.

    PubMed

    Mitiku, Israel; Addissie, Adamu; Molla, Mitike

    2017-02-16

    Ethiopia has implemented routine HIV testing and counselling using a provider initiated HIV testing ('opt-out' approach) to achieve high coverage of testing and prevention of mother-to-child transmission of HIV. However, women's perceptions and experiences with this approach have not been well studied. We conducted a qualitative study to explore pregnant women's perceptions and experiences of routine HIV testing and counselling in Ghimbi town, Ethiopia, in May 2013. In-depth interviews were held with 28 women tested for HIV at antenatal clinics (ANC), as well as four health workers involved in routine HIV testing and counselling. Data were analyzed using the content analysis approach. We found that most women perceived routine HIV testing and counselling beneficial for women as well as unborn babies. Some women perceived HIV testing as compulsory and a prerequisite to receive delivery care services. On the other hand, health workers reported that they try to emphasise the importance HIV testing during pre-test counselling in order to gain women's acceptance. However, both health workers and ANC clients perceived that the pre-test counselling was limited. Routine HIV testing and counselling during pregnancy is well acceptable among pregnant women in the study setting. However, there is a sense of obligation as women felt the HIV testing is a pre-requisite for delivery services. This may be related to the limited pre-test counselling. There is a need to strengthen pre-test counselling to ensure that HIV testing is implemented in a way that ensures pregnant women's autonomy and maximize opportunities for primary prevention of HIV.

  5. A lottery incentive system to facilitate dialogue and social support for workplace HIV counselling and testing: A qualitative inquiry

    PubMed Central

    Weihs, Martin; Meyer-Weitz, Anna

    2014-01-01

    Abstract Despite South African mid-sized companies' efforts to offer HIV counselling and testing (HCT) in the workplace, companies report relatively poor uptake rates. An urgent need for a range of different interventions aimed at increasing participation in workplace HCT has been identified. The aim of this study was to explore qualitatively the influence of a lottery incentive system (LIS) as an intervention to influence shop-floor workers' workplace HIV testing behaviour. A qualitative study was conducted among 17 shop-floor workers via convenience sampling in two mid-sized South African automotive manufacturing companies in which an LIS for HCT was implemented. The in-depth interviews employed a semi-structured interview schedule and thematic analysis was used to analyse the data. The interviews revealed that the LIS created excitement in the companies and renewed employees' personal interest in HCT. The excitement facilitated social interactions that resulted in a strong group cohesion pertaining to HCT that mitigated the burden of HIV stigma in the workplace. Open discussions allowed for the development of supportive social group pressure to seek HCT as a collective in anticipation of a reward. Lotteries were perceived as a supportive and innovative company approach to workplace HCT. The study identified important aspects for consideration by companies when using an LIS to enhance workplace HIV testing. The significance of inter- and intra-player dialogue in activating supportive social norms for HIV testing in collectivist African contexts was highlighted. PMID:25023208

  6. A lottery incentive system to facilitate dialogue and social support for workplace HIV counselling and testing: a qualitative inquiry.

    PubMed

    Weihs, Martin; Meyer-Weitz, Anna

    2014-01-01

    Despite South African mid-sized companies' efforts to offer HIV counselling and testing (HCT) in the workplace, companies report relatively poor uptake rates. An urgent need for a range of different interventions aimed at increasing participation in workplace HCT has been identified. The aim of this study was to explore qualitatively the influence of a lottery incentive system (LIS) as an intervention to influence shop-floor workers' workplace HIV testing behaviour. A qualitative study was conducted among 17 shop-floor workers via convenience sampling in two mid-sized South African automotive manufacturing companies in which an LIS for HCT was implemented. The in-depth interviews employed a semi-structured interview schedule and thematic analysis was used to analyse the data. The interviews revealed that the LIS created excitement in the companies and renewed employees' personal interest in HCT. The excitement facilitated social interactions that resulted in a strong group cohesion pertaining to HCT that mitigated the burden of HIV stigma in the workplace. Open discussions allowed for the development of supportive social group pressure to seek HCT as a collective in anticipation of a reward. Lotteries were perceived as a supportive and innovative company approach to workplace HCT. The study identified important aspects for consideration by companies when using an LIS to enhance workplace HIV testing. The significance of inter- and intra-player dialogue in activating supportive social norms for HIV testing in collectivist African contexts was highlighted.

  7. Development of an immunochromatographic test strip for simultaneous qualitative and quantitative detection of ochratoxin A and zearalenone in cereal.

    PubMed

    Sun, Yaning; Xing, Guangxu; Yang, Jifei; Wang, Fangyu; Deng, Ruiguang; Zhang, Gaiping; Hu, Xiaofei; Zhang, Yong

    2016-08-01

    Ochratoxin A (OTA) and zearalenone (ZEN) are natural products of filamentous fungi that are harmful to humans and animals exposed to them even in extremely low concentration. The immunochromatographic test strip has become a popular diagnostic tool for detecting analytes. Its major advantages are that results can be obtained within 5-10 min, all needed reagents are included in the strip and it can be used to detect OTA and ZEN contamination in spots. In this study a colloidal gold-based immunochromatographic test strip of competitive format was developed for the rapid simultaneous qualitative and quantitative detection of OTA and ZEN in corn and other cereals. The test strip results with the naked eye showed that the sensitivities were 6 µg kg(-1) OTA and 20 µg kg(-1) ZEN in cereal, while those with a TSR3000 membrane strip reader showed that the IC50 values of OTA and ZEN were 1.7905 and 4.3514 ng mL(-1) and the lower detection limit (LDL) values were 0.7697 and 1.2000 µg kg(-1) respectively. These results were confirmed by high-performance liquid chromatography. The immunochromatographic test strip developed in this study could be used for the rapid simultaneous, qualitative and quantitative screening of OTA and ZEN in corn samples. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  8. Optimization of rapid detection of Escherichia coli O157:H7 and Listeria monocytogenes by PCR and application to field test.

    PubMed

    Moon, Gi-Seong; Kim, Wang June; Shin, Weon-Sun

    2004-08-01

    For rapid detection of Escherichia coli O157:H7 and Listeria monocytogenes, simple methods for sample preparation and PCR were established and applied to a field test. To improve specificity, primer sets LP43-LP44 and C(+)-D(-) were selected for E. coli O157:H7 and L. monocytogenes, respectively. Through centrifugation and partial heat treatment after enrichment, E. coli O157:H7 and L. monocytogenes were detected at 1 initial CFU without genomic DNA extraction in the culture and with artificially inoculated food samples including milk, chicken, ham, and pork. Based on the optimized PCR method, a feasibility test was carried out using randomly collected field samples. To remove false positives and false negatives, a PCR method using several primer sets, including the optimized primer set, and a standard culture method were used. With the PCR detection and standard culture methods, two pork samples were positive for L. monocytogenes after enrichment, indications that the PCR assay could be effectively used for rapid, sensitive, and species-specific detection of foodborne pathogens.

  9. [PCR-based analysis of the D1S80 locus and its application in paternity test].

    PubMed

    Que, T; Lin, Y; Li, L

    1998-01-01

    Allelic data for the D1S80 locus was obtained by using the PCR and subsequent analysis with high-resolution horizontal PAGE technique. In a sample of 184 unrelated Chinese, the D1S80 locus had 75 phenotypes and 20 allelic. The distribution of genotype was in agreement with expected values according to the Hardy-Weinberg equilibrium. Analysis of the D1S80 locus by the PCR has been successfully applied in 150 cases of questioned paternity.

  10. The physiologic state of Escherichia coli O157:H7 does not affect its detection in two commercial real-time PCR-based tests

    USDA-ARS?s Scientific Manuscript database

    Multiplex real-time PCR detection of Escherichia coli O157:H7 is an efficient molecular tool with high sensitivity and specificity for meat safety and quality assurance in the beef industry. The Biocontrol GDS and the DuPont Qualicon BAX®-RT rapid detection systems are two commercial tests based on...

  11. Evaluation of commercial universal rRNA gene PCR plus sequencing tests for identification of bacteria and fungi associated with infectious endocarditis.

    PubMed

    Kühn, Christian; Disqué, Claudia; Mühl, Helge; Orszag, Peter; Stiesch, Meike; Haverich, Axel

    2011-08-01

    Two new commercially available universal rRNA gene PCR plus sequencing tests, SepsiTest and universal microbe detection (UMD; Molzym, Bremen, Germany), were evaluated using blood specimens and heart valves from 30 patients with suspected infectious endocarditis (IE). The sensitivity of PCR (85%) was nearly twice as high as that of culture (45%), which in 10/20 IE cases presumably stayed negative as a consequence of growth inhibition of the pathogens by antibiotics. Further, PCR provided the basis for reclassification of 5/10 non-IE cases into IE cases. Culture-negative infections were identified by PCR, including single infections due to streptococci and Gram-negative bacteria (Escherichia coli, Haemophilus parainfluenzae) and mixed infections involving two Gram-positive bacteria or Candida spp. with Gram-positive bacteria. The new commercial tests proved to be of value for the rapid diagnosis of IE, particularly in cases of culture-negative infections. Issues regarding the feasibility of these tests for routine use are discussed.

  12. Analytical validation of a real-time RT-PCR test for Pan-American lineage H7 subtype avian influenza viruses

    USDA-ARS?s Scientific Manuscript database

    Rapid detection of avian influenza virus and identification of the H5 and H7 hemagglutinin subtypes some of which are associated with high pathogenicity in poultry is critical for clinical diagnosis and wild bird monitoring programs. A real-time RT-PCR test for identification of the H7 subtype in N...

  13. Analysis of the Reasons for Non-Uptake of Predictive Testing for Huntington's Disease in Spain: A Qualitative Study.

    PubMed

    Rivera-Navarro, Jesús; Cubo, Esther; Mariscal, Natividad

    2015-12-01

    Children of persons affected by Huntington's disease (HD) have a 50% chance of inheriting the disease. Genetic testing in Spain is offered to individuals (presymptomatic test) or mothers of fetuses (prenatal) who run the risk of suffering from HD. The objective of this study is to analyze the factors that influence the decisions of adult children of persons affected with HD regarding predictive testing. A qualitative research methodology was used involving 4 focus groups (FGs) made up of adult children of persons with HD in different cities in Spain. The results of the study showed that over half of the focus group participants were inclined to decline genetic testing. The main explanatory determinants for taking or not taking the predictive test are: Maturity of the individual at risk, which was directly related to age; Ability to cope with a positive test result; Experience of living with HD sufferers; Information about testing and psychological support; Attitude of the family; Social visibility of genetic testing; Personality and temperament of each subject at risk of HD. These results imply that these factors should be analyzed in more detail in quantitative studies in order to help the Spanish Department of Health understand why some children of parents with HD decline genetic testing, so that they may and apply these data when creating specific policy regarding this issue.

  14. Acceptability of Using Electronic Vending Machines to Deliver Oral Rapid HIV Self-Testing Kits: A Qualitative Study

    PubMed Central

    Young, Sean D.; Daniels, Joseph; Chiu, ChingChe J.; Bolan, Robert K.; Flynn, Risa P.; Kwok, Justin; Klausner, Jeffrey D.

    2014-01-01

    Introduction Rates of unrecognized HIV infection are significantly higher among Latino and Black men who have sex with men (MSM). Policy makers have proposed that HIV self-testing kits and new methods for delivering self-testing could improve testing uptake among minority MSM. This study sought to conduct qualitative assessments with MSM of color to determine the acceptability of using electronic vending machines to dispense HIV self-testing kits. Materials and Methods African American and Latino MSM were recruited using a participant pool from an existing HIV prevention trial on Facebook. If participants expressed interest in using a vending machine to receive an HIV self-testing kit, they were emailed a 4-digit personal identification number (PIN) code to retrieve the test from the machine. We followed up with those who had tested to assess their willingness to participate in an interview about their experience. Results Twelve kits were dispensed and 8 interviews were conducted. In general, participants expressed that the vending machine was an acceptable HIV test delivery method due to its novelty and convenience. Discussion Acceptability of this delivery model for HIV testing kits was closely associated with three main factors: credibility, confidentiality, and convenience. Future research is needed to address issues, such as user-induced errors and costs, before scaling up the dispensing method. PMID:25076208

  15. Using Rapid Diagnostic Tests as a Source of Viral RNA for Dengue Serotyping by RT-PCR - A Novel Epidemiological Tool

    PubMed Central

    Vongsouvath, Manivanh; Phommasone, Koukeo; Sengvilaipaseuth, Onanong; Kosoltanapiwat, Nathamon; Chantratita, Narisara; Blacksell, Stuart D.; Lee, Sue J.; de Lamballerie, Xavier; Mayxay, Mayfong; Keomany, Sommay; Newton, Paul N.; Dubot-Pérès, Audrey

    2016-01-01

    Background Dengue virus infection causes major public health problems in tropical and subtropical areas. In many endemic areas, including the Lao PDR, inadequate access to laboratory facilities is a major obstacle to surveillance and study of dengue epidemiology. Filter paper is widely used for blood collection for subsequent laboratory testing for antibody and nucleic acid detection. For the first time, we demonstrate that dengue viral RNA can be extracted from dengue rapid diagnostic tests (RDT) and then submitted to real-time RT-PCR for serotyping. Methodology/Principal Findings We evaluated the Standard Diagnostics (SD) Bioline Dengue Duo RDT, a commonly used test in dengue endemic areas. First, using the QIAamp RNA kit, dengue RNA was purified from the sample pad of the NS1 RDT loaded with virus isolates of the four serotypes, then quantified by RT-PCR. We observed greater recovery of virus, with a mean of 27 times more RNA recovered from RDT, than from filter paper. Second, we evaluated dengue NS1 RDTs from patients at Mahosot Hospital, Vientiane, (99 patients) and from rural Salavan Provincial Hospital (362 patients). There was good agreement between dengue RT-PCR from NS1 RDT with RT-PCR performed on RNA extracted from patient sera, either using RDT loaded with blood (82.8% and 91.4%, in Vientiane and Salavan, respectively) or serum (91.9% and 93.9%). There was 100% concordance between RDT and serum RT-PCR of infecting dengue serotype. Conclusions/Significance Therefore, the collection of NS1 positive RDTs, which do not require cold storage, may be a novel approach for dengue serotyping by RT-PCR and offers promising prospects for the collection of epidemiological data from previously inaccessible tropical areas to aid surveillance and public health interventions. PMID:27159058

  16. Performance of the COBAS AMPLICOR HCV MONITOR Test, Version 2.0, an Automated Reverse Transcription-PCR Quantitative System for Hepatitis C Virus Load Determination

    PubMed Central

    Gerken, G.; Rothaar, T.; Rumi, M. G.; Soffredini, R.; Trippler, M.; Blunk, M. J.; Butcher, A.; Soviero, S.; Colucci, G.

    2000-01-01

    A clinical evaluation of an automated quantitative PCR assay, the COBAS AMPLICOR HCV MONITOR test, version 2.0 (v2.0), was carried out to assess the performance of this test in comparison with that of the previous, manual version, the AMPLICOR HCV MONITOR test, and with that of nested PCR. Serial dilutions of serum samples infected with genotype 1b, 2a, or 3, as well as synthetic RNA transcripts and serum samples derived from 87 patients with chronic hepatitis C and infected with genotype 1a, 1b, 2a, 2b, 3a, 3b, 4, or 5, were analyzed to determine the ability of the system to efficiently quantify various hepatitis C virus (HCV) genotypes. These experiments showed that the COBAS AMPLICOR HCV MONITOR test, v2.0, has mean intra-assay, interassay, and interoperator coefficients of variation that range from 22 to 34.5% and a 3-logarithm dynamic range, which spans from 103 to 106 copies/ml. Compared to the previous, manual version of the test, the COBAS AMPLICOR HCV MONITOR test, v2.0, showed an improved efficacy for all genotypes, especially genotypes 2, 3, and 4, whose estimated concentrations were on average 1 logarithm higher. When used to monitor patients under treatment, however, both versions showed the same patterns of viremia, indicating that the COBAS AMPLICOR HCV MONITOR test, v2.0, and the AMPLICOR HCV MONITOR test were equally effective at detecting relative viremia changes in serial samples. As expected, the automated test was less sensitive than nested PCR; among specimens from a cohort of patients treated with interferon, nested PCR identified three more viremic specimens, which probably contained very low concentrations of HCV RNA. PMID:10834978

  17. Comparison of real-time PCR with disk diffusion, agar screen and E-test methods for detection of methicillin-resistant Staphylococcus aureus.

    PubMed

    Shariati, Laleh; Validi, Majid; Tabatabaiefar, Mohammad Amin; Karimi, Ali; Nafisi, Mohammad Reza

    2010-12-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a nosocomial pathogen. Our main objective was to compare oxacillin disk test, oxacillin E-test, and oxacillin agar screen for detection of methicillin resistance in S. aureus, using real-time PCR for mecA as the "gold standard" comparison assay. 196 S. aureus isolates were identified out of 284 Staphylococcus isolates. These isolates were screened for MRSA with several methods: disk diffusion, agar screen (6.0 μg/ml), oxacillin E-test, and real-time PCR for detection of mecA gene. Of the 196 S. aureus isolates tested, 96 isolates (49%) were mecA-positive and 100 isolates (51%) mecA-negative. All methods tested had a statistically significant agreement with real-time PCR. E-test was 100% sensitive and specific for mecA presence. The sensitivity and specificity of oxacillin agar screen method were 98 and 99%, respectively and sensitivity and specificity of oxacillin disk diffusion method were 95 and 93%, respectively. In the present study, oxacillin E-test is proposed as the best phenotypic method. For economic reasons, the oxacillin agar screen method (6.0 μg/ml), which is suitable for the detection of MRSA, is recommended due to its accuracy and low cost.

  18. Standard Setting in Specific-Purpose Language Testing: What Can a Qualitative Study Add?

    ERIC Educational Resources Information Center

    Manias, Elizabeth; McNamara, Tim

    2016-01-01

    This paper explores the views of nursing and medical domain experts in considering the standards for a specific-purpose English language screening test, the Occupational English Test (OET), for professional registration for immigrant health professionals. Since individuals who score performances in the test setting are often language experts…

  19. Standard Setting in Specific-Purpose Language Testing: What Can a Qualitative Study Add?

    ERIC Educational Resources Information Center

    Manias, Elizabeth; McNamara, Tim

    2016-01-01

    This paper explores the views of nursing and medical domain experts in considering the standards for a specific-purpose English language screening test, the Occupational English Test (OET), for professional registration for immigrant health professionals. Since individuals who score performances in the test setting are often language experts…

  20. Bayesian estimation of test characteristics of real-time PCR, bacteriological culture and California mastitis test for diagnosis of intramammary infections with Staphylococcus aureus in dairy cattle at routine milk recordings.

    PubMed

    Mahmmod, Yasser S; Toft, Nils; Katholm, Jørgen; Grønbæk, Carsten; Klaas, Ilka C

    2013-11-01

    Danish farmers can order a real-time PCR mastitis diagnostic test on routinely taken cow-level samples from milk recordings. Validation of its performance in comparison to conventional mastitis diagnostics under field conditions is essential for efficient control of intramammary infections (IMI) with Staphylococcus aureus (S. aureus). Therefore, the objective of this study was to estimate the sensitivity (Se) and specificity (Sp) of real-time PCR, bacterial culture (BC) and California mastitis test (CMT) for the diagnosis of the naturally occurring IMI with S. aureus in routinely collected milk samples using latent class analysis (LCA) to avoid the assumption of a perfect reference test. Using systematic random sampling, a total of 609 lactating dairy cows were selected from 6 dairy herds with bulk tank milk PCR cycle threshold (Ct) value ≤39 for S. aureus. At routine milk recordings, automatically obtained cow-level (composite) milk samples were analyzed by PCR and at the same milking, 2436 quarter milk samples were collected aseptically for BC and CMT. Results showed that 140 cows (23%) were positive for S. aureus IMI by BC while 170 cows (28%) were positive by PCR. Estimates of Se and Sp for PCR were higher than test estimates of BC and CMT. SeCMT was higher than SeBC however, SpBC was higher than SpCMT. SePCR was 91%, while SeBC was 53%, and SeCMT was 61%. SpPCR was 99%, while SpBC was 89%, and SpCMT was 65%. In conclusion, PCR has a higher performance than the conventional diagnostic tests (BC and CMT) suggesting its usefulness as a routine test for accurate diagnosis of S. aureus IMI from dairy cows at routine milk recordings. The use of LCA provided estimates of the test characteristics for two currently diagnostic tests (BC, CMT) and a novel technique (real-time PCR) for diagnosing S. aureus IMI under field conditions at routine milk recordings in Denmark. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Attitudes toward genetic testing for cancer risk after genetic counseling and decision support: a qualitative comparison between hereditary cancer types.

    PubMed

    Wakefield, Claire E; Kasparian, Nadine A; Meiser, Bettina; Homewood, Judi; Kirk, Judy; Tucker, Kathy

    2007-01-01

    This study aimed to qualitatively assess individuals' attitudes toward genetic testing for cancer risk after genetic counseling and decision support. As part of a larger study, 78 women considering genetic testing for hereditary breast/ovarian cancer (HBOC) risk and 22 individuals considering genetic testing for hereditary nonpolyposis colorectal cancer (HNPCC) completed an open-ended table of their perceived pros and cons of genetic testing. The most frequently reported pros were "to help manage my risk of developing cancer," "to help my family," and "to know my cancer risk." With regards to risk management, the HBOC group perceived genetic testing as most helpful in informing their general risk management practices, while the HN-PCC group focused on the potential to clarify their need for bowel cancer screening, suggesting that patients' perceptions of the benefits of genetic testing may differ across cancer syndromes. Individuals in both groups expressed concern about the potential psychological impact of genetic testing. We also found that some affected individuals may not fully comprehend the meaning of their potential test results. Eliciting patients' perceived pros and cons during genetic counseling is likely to be a valuable tool for improving patient care. This data also provides an improved evidence base for the development of patient education tools.

  2. Detection of multiple Mycoplasma species in bulk tank milk samples using real-time PCR and conventional culture and comparison of test sensitivities.

    PubMed

    Justice-Allen, A; Trujillo, J; Goodell, G; Wilson, D

    2011-07-01

    The objective of this study was to further validate a SYBR PCR protocol for Mycoplasma spp. by comparing it with standard microbial culture in the detection of Mycoplasma spp. in bulk tank milk samples. Additionally, we identified Mycoplasma spp. present by analysis of PCR-generated amplicons [dissociation (melt) temperature (T(m)), length, and DNA sequence]. The research presented herein tests the hypothesis that the SYBR PCR protocol is as sensitive as conventional culture for the detection of Mycoplasma spp. in bulk tank milk samples. Mycoplasmas cause several important disease syndromes in cattle, including mastitis in dairy cows. The standard diagnostic method at the herd level has been microbial isolation of mycoplasmas on 1 of several specialized media and speciation through biochemical or immunological techniques; repeated sampling schemes are recommended. The development of a real-time SYBR PCR protocol offers advantages in decrease of time to detection, cost, and complexity. The T(m) of the double-stranded DNA generated from the PCR reaction was used to detect the presence of and tentatively identify the species of mycoplasmas other than Mycoplasma bovis. In the SYBR PCR protocol, the presence of multiple species of mycoplasmas is indicated by an atypical dissociation curve. Gel electrophoresis and sequencing of the amplicons was used to confirm the mycoplasma species present when a non-M. bovis organism was detected (T(m) not equal to M. bovis) and used to identify all the mycoplasma species present for the samples with atypical dissociation curves. Mycoplasma bovis was identified in 83% of SYBR PCR mycoplasma-positive bulk tank samples. Another mycoplasma was identified either alone or in addition to M. bovis in 25% of SYBR PCR mycoplasma-positive bulk tank milk samples. Four species of mycoplasma other than M. bovis (Mycoplasma alkalescens, Mycoplasma arginini, Mycoplasma bovigenitalium, and Mycoplasma gateae) were identified in bulk tank milk samples

  3. Comparison of qualitative and quantitative fit-testing results for three commonly used respirators in the healthcare sector.

    PubMed

    Hon, Chun-Yip; Danyluk, Quinn; Bryce, Elizabeth; Janssen, Bob; Neudorf, Mike; Yassi, Annalee; Shen, Hui; Astrakianakis, George

    2017-03-01

    N95 filtering facepiece respirators are used by healthcare workers when there is a risk of exposure to airborne hazards during aerosol-generating procedures. Respirator fit-testing is required prior to use to ensure that the selected respirator provides an adequate face seal. Two common fit-test methods can be employed: qualitative fit-test (QLFT) or quantitative fit-test (QNFT). Respiratory protection standards deem both fit-tests to be acceptable. However, previous studies have indicated that fit-test results may differ between QLFT and QNFT and that the outcomes may also be influenced by the type of respirator model. The aim of this study was to determine if there is a difference in fit-test outcomes with our suite of respirators, 3M - 1860S, 1860, AND 1870, and whether the model impacts the fit-test results. Subjects were recruited from residential care facilities. Each participant was assigned a respirator and underwent sequential QLFT and QNFT fit-tests and the results (either pass or fail) were recorded. To ascertain the degree of agreement between the two fit-tests, a Kappa (Κ) statistic was conducted as per the American National Standards Institute (ANSI) respiratory protection standard. The pass-fail rates were stratified by respirator model and a Kappa statistic was calculated for each to determine effect of model on fit-test outcomes. We had 619 participants and the aggregate Κ statistic for all respirators was 0.63 which is below the suggested ANSI threshold of 0.70. There was no statistically significant difference in results when stratified by respirator model. QNFT and QLFT produced different fit-test outcomes for the three respirator models examined. The disagreement in outcomes between the two fit-test methods with our suite of N95 filtering facepiece respirators was approximately 12%. Our findings may benefit other healthcare organizations that use these three respirators.

  4. Fetal Tissues Tested for Microbial Sterility by Culture- and PCR-Based Methods Can be Safely Used in Clinics.

    PubMed

    Vitrenko, Yakov; Kostenko, Iryna; Kulebyakina, Kateryna; Duda, Alla; Klunnyk, Mariya; Sorochynska, Khrystyna

    2017-02-16

    Cell preparations to be used in clinical practice must be free of infectious agents. Safety concerns are especially elevated upon the use of human fetal tissues, which are otherwise highly advantageous in cell therapy. We demonstrate that treating fetal samples with antibiotic, extensive washing, and homogenization prior to cryoconservation efficiently removes microbes in general. Screening a large collection by an automatic culture system showed that 89.2% fetal tissue samples were sterile, while contamination was detected in 10.8% samples. Liver and chorion were contaminated more than the brain, kidney, lung, and soft tissues. Broad-range PCR from the bacterial 16s rRNA gene was adopted as a confirmatory assay; however, the concordance between the culture-based and PCR assays was weak. Taxonomic identification was done for contaminated samples by bacteriological methods and sequencing 16s rRNA PCR products. The two approaches revealed different spectra of taxonomic groups sharing only Lactobacillus, the most frequently found genus. In addition, other representatives of vaginal microbiota were detected by culture-based identification, while PCR product sequencing has also revealed a subset of nosocomial microorganisms. Importantly, species known to cause sepsis were identified by both techniques, arguing for their indispensability and mutual complementarity. We suggest that most contaminations are taken up during collection of fetal material rather than originating from an in utero infection. In conclusion, a rigorous microbiological control by culture and PCR is a prerequisite for safe clinical use of fetal tissue suspensions.

  5. A novel poisson distribution-based approach for testing boundaries of real-time PCR assays for food pathogen quantification.

    PubMed

    Rossmanith, Peter; Wagner, Martin

    2011-09-01

    The validation of quantitative real-time PCR systems and above all, proof of the detection limit of this method, is a frequently and intensively discussed topic in food pathogen detection. Among proper sample collection, assay design, careful experimental design, execution of real-time PCR, and data analysis, the validation of the method per se ensuring reliable quantification data is of prime importance. The purpose of this study was to evaluate a novel validation tool for real-time PCR assays, based on the theoretical possibility of the amplification of a single DNA target. The underlying mathematical basis for the work is Poisson distribution, which describes patterns of low particle numbers in a volume. In this context, we focused on the quantitative aspect of real-time PCR for the first time. This allowed for demonstration of the reliable amplification of a lone target DNA molecule and the demonstration of the distinct discrimination between integer molecular numbers when using low initial copy numbers. A real-time PCR assay amplifying a 274-bp fragment of the positive regulatory protein A locus of Listeria monocytogenes was used for this work. Evidence for a linear range of quantification from a single target copy to 10 ng of target DNA was experimentally demonstrated, and evidence for the significance of this novel validation approach is presented here.

  6. Understanding laboratory testing in diagnostic uncertainty: a qualitative study in general practice.

    PubMed Central

    van der Weijden, Trudy; van Bokhoven, Marloes A; Dinant, Geert-Jan; van Hasselt, Cathelijne M; Grol, Richard P T M

    2002-01-01

    BACKGROUND: Better knowledge of the professional's motives for ordering laboratory tests in the case of diagnostic uncertainty may lead to interventions directed at reducing unnecessary testing. AIM: To gain insight into the general practitioner's (GP's) motives for ordering laboratory tests for patients presenting with unexplained complaints. DESIGN OF STUDY: Semi-structured interviews based on surgery observations. SETTING: Twenty-one general practices in rural and urban areas of The Netherlands. METHOD: Investigation of the GP's perception of determinants of test-ordering behaviour in the situation of diagnostic uncertainty. The interviews were structured by evaluating the consultations and test-ordering performance of that day. RESULTS: Dutch GPs vary considerably in their motives for ordering tests. Numerous motives emerged from the data. Some examples of important themes include: personal routines; tolerance of diagnostic uncertainty; time pressure; and tactical motives for test ordering. Complying with the perceived needs of the patient for reassurance through testing is seen as an easy, cost- and time-effective strategy. A clear hierarchy in the determinants was not found. CONCLUSION: The decision to request laboratory testing is the result of a complex interaction of considerations that are often conflicting. Designers of interventions meant to improve the ordering of tests should be aware of the numerous determinants, and take contextual variables into account. PMID:12528582

  7. Men "missing" from population-based HIV testing: insights from qualitative research.

    PubMed

    Camlin, Carol S; Ssemmondo, Emmanuel; Chamie, Gabriel; El Ayadi, Alison M; Kwarisiima, Dalsone; Sang, Norton; Kabami, Jane; Charlebois, Edwin; Petersen, Maya; Clark, Tamara D; Bukusi, Elizabeth A; Cohen, Craig R; R Kamya, Moses; Havlir, Diane

    2016-01-01

    Men's uptake of HIV testing is critical to the success of "test and treat" strategies in generalized epidemics. This study sought to identify cultural factors and community processes that influence men's HIV testing uptake in the baseline year of an ongoing test-and-treat trial among 334,479 persons in eastern Africa (SEARCH, NCT#01864603). Data were collected using participant observation at mobile community health campaigns (CHCs) (n = 28); focus group discussions (n = 8 groups) with CHC participants; and in-depth interviews with care providers (n = 50), leaders (n = 32), and members (n = 112) of eight communities in Kenya and Uganda. An 8-person research team defined analytical codes and iteratively refined them during data collection using grounded theoretical approaches, and textual data were coded using Atlas.ti software. Structural and cultural barriers, including men's mobility and gender norms valorizing risk-taking and discouraging health-seeking behavior, were observed, and contributed to men's lower participation in HIV testing relative to women. Men's labor opportunities often require extended absences from households: during planting season, men guarded fields from monkeys from dawn until nightfall; lake fishermen traveled long distances and circulated between beaches. Men often tested "by proxy", believing their wives' HIV test results to be their status. Debates about HIV risks were vigorous, with many men questioning "traditional" masculine gender norms that enhanced risks. The promise of antiretroviral therapy (ART) to prolong health was a motivating factor for many men to participate in testing. Flexibility in operating hours of HIV testing including late evening and weekend times along with multiple convenient locations that moved were cited as facilitating factors enhancing male participating in HIV testing. Mobile testing reduced but did not eliminate barriers to men's participation in a large-scale "test & treat" effort

  8. Communication with patients during the prenatal testing procedure: an explorative qualitative study.

    PubMed

    van Zwieten, Myra; Willems, Dick; Knegt, Lia; Leschot, Nico

    2006-10-01

    While generally two phases of prenatal genetic counseling are distinguished, i.e. pre- and post-test counseling, we revealed a third form of communication during the testing procedure. The content of this intermediate communication was explored. A secondary analysis was performed on data obtained in another observational study, which was focussed on how indefinite testing results are clarified. Thirteen testing trajectories in which communication with parents took place during the testing procedure were further analysed. In the majority of cases the content of intermediate communication was similar to the content of pre-test counseling. In four cases the content was different, because the communication involved the parents in decision-making about a testing result, which was still being processed. Communication in (prenatal) genetic testing is not always restricted to separate phases, but can be an ongoing process occurring parallel to, and sometimes even intertwined with, the testing process. The advocated model of shared decision-making might work better once it is determined if the decision concerns the area wherein the provider is the expert, or the patient. Further research into the process of continuing decision-making could clarify how providers' and patients' responsibilities regarding the diagnostic process are distributed. Meanwhile, the possible occurrence of continuous decision-making should be mentioned in (prenatal) genetic counseling.

  9. Restriction-Site-Specific PCR as a Rapid Test To Detect Enterohemorrhagic Escherichia coli O157:H7 Strains in Environmental Samples

    PubMed Central

    Kimura, Richard; Mandrell, Robert E.; Galland, John C.; Hyatt, Doreene; Riley, Lee W.

    2000-01-01

    Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important food-borne pathogen in industrialized countries. We developed a rapid and simple test for detecting E. coli O157:H7 using a method based on restriction site polymorphisms. Restriction-site-specific PCR (RSS-PCR) involves the amplification of DNA fragments using primers based on specific restriction enzyme recognition sequences, without the use of endonucleases, to generate a set of amplicons that yield “fingerprint” patterns when resolved electrophoretically on an agarose gel. The method was evaluated in a blinded study of E. coli isolates obtained from environmental samples collected at beef cattle feedyards. The 54 isolates were all initially identified by a commonly used polyclonal antibody test as belonging to O157:H7 serotype. They were retested by anti-O157 and anti-H7 monoclonal antibody enzyme-linked immunosorbent assay (ELISA). The RSS-PCR method identified all 28 isolates that were shown to be E. coli O157:H7 by the monoclonal antibody ELISA as belonging to the O157:H7 serotype. Of the remaining 26 ELISA-confirmed non-O157:H7 strains, the method classified 25 strains as non-O157:H7. The specificity of the RSS-PCR results correlated better with the monoclonal antibody ELISA than with the polyclonal antibody latex agglutination tests. The RSS-PCR method may be a useful test to distinguish E. coli O157:H7 from a large number of E. coli isolates from environmental samples. PMID:10831431

  10. Impediments to DNA testing and cascade screening for hypertrophic cardiomyopathy and Long QT syndrome: a qualitative study of patient experiences.

    PubMed

    Smart, Andrew

    2010-12-01

    This paper reports data from a qualitative study of patient experiences of DNA testing and cascade screening for hypertrophic cardiomyopathy and long QT syndrome, cardiac conditions that place sufferers at risk of sudden death. The paper particularly focuses on potential impediments to testing and screening. Semi-structured interviews were undertaken with a purposive sample of 27 people in the UK who had undergone testing. In the context of the uncertainties that can characterize experiences of these disorders, the majority of participants in this sample embraced testing and screening as a way of providing health information for themselves or their relatives (particularly children). There was nevertheless evidence of ambivalence about the value and impact of the DNA test information which could influence participants' dispositions toward testing, and play into dilemmas about family communication. Other concerns arose in relation to communicating about these disorders, decisions to involve elderly relatives and pressures relating to family responsibility. The evidence of ambivalence provides insight into why some people may be resistant to testing, screening and sharing information. The findings about communication processes indicate potential areas of concern for the cascading process.

  11. General practitioners' barriers and facilitators towards new provider-initiated HIV testing strategies: a qualitative study.

    PubMed

    Joore, Ivo K; van Roosmalen, Sanne Lc; van Bergen, Jan Eam; van Dijk, Nynke

    2017-04-01

    European guidelines recommend offering an HIV test to individuals who display HIV indicator conditions (ICs). UK guidelines recommend performing a 'routine offer of HIV testing' in primary care where HIV prevalence exceeds 2 in 1000. Implementation of new provider-initiated HIV testing strategies in general practice is limited, while the numbers of undiagnosed and late for care HIV patients remain high. We have explored Dutch general practitioners' barriers to and facilitators of both strategies. We combined semi-structured in-depth interviews with focus groups. Nine general practitioners - key informants of sexually transmitted infection/HIV prevention and control - were selected for the interviews. Additionally, we organised focus groups with a broad sample of general practitioners (n = 81). Framework analysis was used to analyse the data. Various barriers were found, related to (1) the content of the guidelines (testing the right group and competing priorities in general practice), (2) their organisational implementation (lack of time, unclear when to repeat the HIV test and overlong list of ICs) and (3) the patient population (creating fear among patients, stigmatising them and fear regarding financial costs). Multiple general practitioners stated that performing a sexual risk assessment of patients is important before applying either strategy. Also, they recommended implementing the IC-guided approach only in high-prevalence areas and combining HIV tests with other laboratory blood tests. General practitioners tend to cling to old patterns of risk-based testing. Promoting awareness of HIV testing and educating general practitioners about the benefits of new provider-initiated HIV testing strategies is important for the actual uptake of HIV testing.

  12. Women's understanding of abnormal cervical smear test results: a qualitative interview study.

    PubMed Central

    Kavanagh, A. M.; Broom, D. H.

    1997-01-01

    OBJECTIVE: To describe how women interpret their experiences of diagnosis and treatment of a cervical abnormality and how healthcare services for such women can be improved. DESIGN: Qualitative study using detailed individual interviews. SETTING: Australian gynaecology clinics. SUBJECTS: 29 Women who had a cervical cytological abnormality and who attended a gynaecologist. MAIN OUTCOME MEASURES: Women's views on their diagnosis and their information needs. RESULTS: Most women wanted to participate in decisions about their care but found it difficult to get the information they required from doctors because they were confused by what their doctors told them and felt unable to ask questions in the consultation. Medical terms such as wart virus and precancer were difficult to understand. Not being able to see their cervix also made it hard for women to understand what their abnormality meant and what treatment entailed. Most women tried to make sense of their abnormality in the context of their everyday lives. For some women their gynaecological care was not consistent with the way they understood their abnormality. CONCLUSIONS: The inherent power structure of medical practice combined with time pressures often make it difficult for doctors to give the detailed information and reassurance patients need when a diagnosis is distressing or when investigation and treatment are strange and upsetting. PMID:9161314

  13. Detection of Helicobacter pylori in adenotonsillar tissue of children with chronic adenotonsillitis using rapid urease test, PCR and blood serology: a prospective study.

    PubMed

    Abdel-Monem, Mohamed H; Magdy, Emad A; Nour, Yasser A; Harfoush, Reem A; Ibreak, Alnagy

    2011-04-01

    Contradictory results have been reported regarding Helicobacter pylori (H. pylori) detection in adenotonsillar tissue. The aims of this study were to investigate whether adenotonsillar tissue of symptomatic children with chronic adenotonsillitis harbors the H. pylori organism, using two biopsy-based invasive methods namely; rapid urease test (RUT) and polymerase chain reaction (PCR) as well as blood serology and to compare the results obtained from each of these methods to the "gold standard". This prospective clinical study was carried out on 20 children aged between 2 and 10 years scheduled for tonsillectomy +/- adenoidectomy in a tertiary referral center. Exclusion criteria included: use of antacids, H(2) blockers or antibiotics during the previous month before surgery and adenotonsillectomy for obstructive sleep apnea. Core biopsy samples from resected adenotonsillar tissue was tested for H. pylori detection using both RUT and PCR assay for the ureC gene. Preoperative patient venous blood samples were also tested for H. pylori IgG antibodies. As a "gold standard", examined tissue was considered to be H. pylori infected if the two biopsy specimen-based methods (RUT and PCR) yielded positive results. Thirty adenotonsillectomy specimens were tested (20 tonsils and 10 adenoids). RUT was positive in 16 (53.3%) specimens (12 tonsils and 4 adenoids). According to the "gold standard", 11/16 were considered false-positive, yielding this test sensitivity 100% and specificity 56%. The ureC gene sequence was detected by PCR in 5 (16.6%) specimens (3 tonsils and 2 adenoids), all of which were also positive by RUT, thus were considered H. pylori infected. Accordingly, PCR had a 100% sensitivity and specificity. Serology testing was positive for H. pylori IgG antibodies in 4/20 patients (20%), only two of them were found to have H. pylori infected adenotonsillar tissue. Based on our findings it seems that adenotonsillar tissue may constitute an extra-gastric reservoir for H

  14. General practitioners’ barriers and facilitators towards new provider-initiated HIV testing strategies: a qualitative study

    PubMed Central

    van Roosmalen, Sanne LC; van Bergen, Jan EAM; van Dijk, Nynke

    2016-01-01

    European guidelines recommend offering an HIV test to individuals who display HIV indicator conditions (ICs). UK guidelines recommend performing a ‘routine offer of HIV testing’ in primary care where HIV prevalence exceeds 2 in 1000. Implementation of new provider-initiated HIV testing strategies in general practice is limited, while the numbers of undiagnosed and late for care HIV patients remain high. We have explored Dutch general practitioners’ barriers to and facilitators of both strategies. We combined semi-structured in-depth interviews with focus groups. Nine general practitioners – key informants of sexually transmitted infection/HIV prevention and control – were selected for the interviews. Additionally, we organised focus groups with a broad sample of general practitioners (n = 81). Framework analysis was used to analyse the data. Various barriers were found, related to (1) the content of the guidelines (testing the right group and competing priorities in general practice), (2) their organisational implementation (lack of time, unclear when to repeat the HIV test and overlong list of ICs) and (3) the patient population (creating fear among patients, stigmatising them and fear regarding financial costs). Multiple general practitioners stated that performing a sexual risk assessment of patients is important before applying either strategy. Also, they recommended implementing the IC-guided approach only in high-prevalence areas and combining HIV tests with other laboratory blood tests. General practitioners tend to cling to old patterns of risk-based testing. Promoting awareness of HIV testing and educating general practitioners about the benefits of new provider-initiated HIV testing strategies is important for the actual uptake of HIV testing. PMID:27207253

  15. Assessment of the Usefulness of Multiplex Real-Time PCR Tests in the Diagnostic and Therapeutic Process of Pneumonia in Hospitalized Children: A Single-Center Experience

    PubMed Central

    Bartkowska-Śniatkowska, Alicja; Jończyk-Potoczna, Katarzyna; Wysocka-Leszczyńska, Joanna; Bobkowski, Waldemar; Fichna, Piotr; Sobkowiak, Paulina; Mazur-Melewska, Katarzyna; Bręborowicz, Anna; Wysocki, Jacek; Januszkiewicz-Lewandowska, Danuta

    2017-01-01

    The aim of the study was assessment of the usefulness of multiplex real-time PCR tests in the diagnostic and therapeutic process in children hospitalized due to pneumonia and burdened with comorbidities. Methods. The study group included 97 children hospitalized due to pneumonia at the Karol Jonscher Teaching Hospital in Poznań, in whom multiplex real-time PCR tests (FTD respiratory pathogens 33; fast-track diagnostics) were used. Results. Positive test results of the test were achieved in 74 patients (76.3%). The average age in the group was 56 months. Viruses were detected in 61 samples (82% of all positive results); bacterial factors were found in 29 samples (39% of all positive results). The presence of comorbidities was established in 90 children (92.78%). On the basis of the obtained results, 5 groups of patients were established: viral etiology of infection, 34 patients; bacterial etiology, 7 patients; mixed etiology, 23 patients; pneumocystis, 9 patients; and no etiology diagnosed, 24 patients. Conclusions. Our analysis demonstrated that the participation of viruses in causing severe lung infections is significant in children with comorbidities. Multiplex real-time PCR tests proved to be more useful in establishing the etiology of pneumonia in hospitalized children than the traditional microbiological examinations. PMID:28182108

  16. Assessment of the Usefulness of Multiplex Real-Time PCR Tests in the Diagnostic and Therapeutic Process of Pneumonia in Hospitalized Children: A Single-Center Experience.

    PubMed

    Gowin, Ewelina; Bartkowska-Śniatkowska, Alicja; Jończyk-Potoczna, Katarzyna; Wysocka-Leszczyńska, Joanna; Bobkowski, Waldemar; Fichna, Piotr; Sobkowiak, Paulina; Mazur-Melewska, Katarzyna; Bręborowicz, Anna; Wysocki, Jacek; Januszkiewicz-Lewandowska, Danuta

    2017-01-01

    The aim of the study was assessment of the usefulness of multiplex real-time PCR tests in the diagnostic and therapeutic process in children hospitalized due to pneumonia and burdened with comorbidities. Methods. The study group included 97 children hospitalized due to pneumonia at the Karol Jonscher Teaching Hospital in Poznań, in whom multiplex real-time PCR tests (FTD respiratory pathogens 33; fast-track diagnostics) were used. Results. Positive test results of the test were achieved in 74 patients (76.3%). The average age in the group was 56 months. Viruses were detected in 61 samples (82% of all positive results); bacterial factors were found in 29 samples (39% of all positive results). The presence of comorbidities was established in 90 children (92.78%). On the basis of the obtained results, 5 groups of patients were established: viral etiology of infection, 34 patients; bacterial etiology, 7 patients; mixed etiology, 23 patients; pneumocystis, 9 patients; and no etiology diagnosed, 24 patients. Conclusions. Our analysis demonstrated that the participation of viruses in causing severe lung infections is significant in children with comorbidities. Multiplex real-time PCR tests proved to be more useful in establishing the etiology of pneumonia in hospitalized children than the traditional microbiological examinations.

  17. Comparison of the Eragen Multi-Code Respiratory Virus Panel with Conventional Viral Testing and Real-Time Multiplex PCR Assays for Detection of Respiratory Viruses▿ †

    PubMed Central

    Arens, Max Q.; Buller, Richard S.; Rankin, Anne; Mason, Sheila; Whetsell, Amy; Agapov, Eugene; Lee, Wai-Ming; Storch, Gregory A.

    2010-01-01

    High-throughput multiplex assays for respiratory viruses are an important step forward in diagnostic virology. We compared one such assay, the PLx Multi-Code Respiratory Virus Panel (PLx-RVP), manufactured by Eragen Biosciences, Inc. (Madison, WI), with conventional virologic testing, consisting of fluorescent-antibody staining plus testing with the R-mix system and fibroblast tube cultures. The test set consisted of 410 archived respiratory specimens, mostly nasopharyngeal swabs, including 210 that had been positive by conventional testing for a balanced selection of common respiratory viruses. Specimens yielding discrepant results were evaluated using a panel of respiratory virus PCR assays developed, characterized, and validated with clinical specimens. PLx-RVP increased the total rate of detection of viruses by 35.8%, and there was a 25.7% increase in the rate of detection of positive specimens. Reference PCR assay results corroborated the PLx-RVP result for 54 (82%) of 66 discrepancies with conventional testing. Of the 12 specimens with discrepancies between PLx-RVp and the reference PCRs, 6 were positive for rhinovirus by PLx-RVP and the presence of rhinovirus was confirmed by nucleotide sequencing. The remaining six specimens included five in which the PLx-RVP failed to detect parainfluenza virus and one in which the detection of influenza A virus by PLx-RVP could not be confirmed by the reference PCR. Taking the results of the reference PCR assay results into account, the sensitivities of the PLx-RVP for individual viruses ranged from 94 to 100% and the specificities ranged from 99 to 100%. We conclude that PLx-RVP is a highly accurate system for the detection of respiratory viruses and significantly improves the rate of detection of these viruses compared to that by conventional virologic testing. PMID:20484608

  18. Differentiation between Staphylococcus aureus and coagulase-negative Staphylococcus species by real-time PCR including detection of methicillin resistants in comparison to conventional microbiology testing.

    PubMed

    Klaschik, Sven; Lehmann, Lutz E; Steinhagen, Folkert; Book, Malte; Molitor, Ernst; Hoeft, Andreas; Stueber, Frank

    2015-03-01

    Staphylococcus aureus has long been recognized as a major pathogen. Methicillin-resistant strains of S. aureus (MRSA) and methicillin-resistant strains of S. epidermidis (MRSE) are among the most prevalent multiresistant pathogens worldwide, frequently causing nosocomial and community-acquired infections. In the present pilot study, we tested a polymerase chain reaction (PCR) method to quickly differentiate Staphylococci and identify the mecA gene in a clinical setting. Compared to the conventional microbiology testing the real-time PCR assay had a higher detection rate for both S. aureus and coagulase-negative Staphylococci (CoNS; 55 vs. 32 for S. aureus and 63 vs. 24 for CoNS). Hands-on time preparing DNA, carrying out the PCR, and evaluating results was less than 5 h. The assay is largely automated, easy to adapt, and has been shown to be rapid and reliable. Fast detection and differentiation of S. aureus, CoNS, and the mecA gene by means of this real-time PCR protocol may help expedite therapeutic decision-making and enable earlier adequate antibiotic treatment. © 2014 Wiley Periodicals, Inc.

  19. Autism genetic testing: a qualitative study of awareness, attitudes, and experiences among parents of children with autism spectrum disorders.

    PubMed

    Chen, Lei-Shih; Xu, Lei; Huang, Tse-Yang; Dhar, Shweta U

    2013-04-01

    The goal of this first-of-its-kind qualitative study was to examine the awareness, attitudes, and experiences among parents of autistic children regarding autism genetic testing. We conducted in-depth, individual, and semistructured interviews with 42 parents of autistic children with diverse racial/ethnic backgrounds. All interviews were audio-taped, transcribed, and coded into major themes and subthemes. Approximately one-quarter of participants had two or more autistic children, and about half of them were ethnic/racial minorities. The majority of participants postulated favorable attitudes toward autism genetic testing for three main reasons: early intervention and treatment, identifying the etiology of autism, and informed family planning. Nevertheless, among parents who had taken their children for genetic testing, some expressed frustration and questioned the competency of their providers in interpreting test results. Asian parents and those with a low socioeconomic status expressed lower awareness and tended to have more limited access to autism genetic testing. As health-care providers play a vital role in providing genetic services and education, these professionals should be educated and be sensitive to the needs of parents with autistic children. Further quantitative research is required to examine the effects of socio-demographic factors on parents' awareness, attitudes, and experiences regarding autism genetic testing.

  20. Trauma-Informed Decision-Making Among Providers and Victims of Intimate Partner Violence During HIV Testing: A Qualitative Study.

    PubMed

    Williams, Jessica R; Gonzalez-Guarda, Rosa M; Ilias, Vanessa

    We explored the process of implementing an HIV testing program at an intimate partner violence (IPV) service agency from the client and provider perspectives. A qualitative descriptive approach was used wherein semi-structured interviews were conducted with 19 key informants (i.e., women with a history of IPV, HIV service providers, IPV service providers). Interviews focused on facilitators and barriers to HIV testing implementation, the decision-making process during HIV testing, and support needs. All interviews were recorded and transcribed. The text of the interviews was analyzed using directed content analysis. Unique factors were found to influence HIV testing in victims of IPV including potential for re-traumatization, readiness for testing, competing priorities, and the influence of children. The results provided important information that can be used to improve the implementation of HIV testing, tailoring processes so they are more trauma-informed; and better support individuals with a history of IPV. Copyright © 2017 Association of Nurses in AIDS Care. Published by Elsevier Inc. All rights reserved.

  1. Quantitative immunochemical fecal occult blood testing for colorectal adenoma detection: evaluation in the target population of screening and comparison with qualitative tests.

    PubMed

    Haug, Ulrike; Hundt, Sabrina; Brenner, Hermann

    2010-03-01

    Quantitative and qualitative immunochemical fecal occult blood tests (FOBTs) have been proposed for noninvasive colorectal cancer screening, but comparative evaluation is lacking. The aim of this study was to determine the diagnostic accuracy of two (quantitative) enzyme-linked immunosorbent assay (ELISA)-based immunochemical FOBTs for identifying colorectal adenomas in the target population of screening and to compare the results with six (qualitative) immunochromatographic FOBTs, previously evaluated in the same study participants using the same stool samples. A total of 1,319 participants of screening colonoscopy at average risk for colorectal neoplasia (mean age 63 years; age range 31-86 years; 50% men) were recruited prospectively from January 2006 to December 2007 in collaboration with 20 gastroenterological practices in Germany. Fecal hemoglobin and hemoglobin-haptoglobin levels were measured using an automated ELISA (RIDASCREEN). Test performance characteristics at different cutoff values were derived by comparing the results of stool testing with the results of colonoscopy in a blinded manner. A total of 130 participants (10%) had an advanced adenoma. The area under the receiver-operating characteristic curve with regard to advanced adenomas was 0.68 (0.65-0.71) for hemoglobin and 0.64 (0.61-0.67) for hemoglobin-haptoglobin (P=0.034). At a specificity of approximately 95%, the sensitivity (95% confidence interval) for advanced adenomas was 33% (25-42%) for hemoglobin and 24% (17-32%) for hemoglobin-haptoglobin, respectively. The sensitivity for hemoglobin was very close to sensitivities of the six qualitative FOBTs at (strongly divergent) levels of specificity observed for the latter. ELISA-based measurement of hemoglobin was superior to hemoglobin-haptoglobin, but showed a similar sensitivity for advanced adenomas compared with (qualitative) immunochromatographic FOBTs at defined levels of specificity. Compared with the latter, its quantitative nature

  2. Qualitative study of how women define and use information about breast symptoms and diagnostic tests.

    PubMed

    Davey, Heather M; Butow, Phyllis N

    2006-10-01

    Unstructured interviews were conducted with 14 women with recent experience of diagnostic testing to explore the definition and role of information in women's experiences of undergoing diagnostic breast tests. Analysis showed that women see information as more than what they are told by healthcare professionals. Information also comes from significant others, administration and reception staff, media, comparison with previous breast symptoms, silences, gestures and behavior of healthcare professionals and significant others, and the number of tests they undergo. Information is about more than understanding. It can provide support and control, reduce anxiety, facilitate question asking and promote women's trust and confidence in themselves and their healthcare professionals. Healthcare professionals need to be aware that women define information more broadly than what they are told, and of the importance women attach to this other information. They also need to be aware that information serves more purposes than helping women understand the diagnostic testing process and their result.

  3. Toward a Model for Field-Testing Patient Decision-Support Technologies: A Qualitative Field-Testing Study

    PubMed Central

    Elwyn, Glyn; Edwards, Adrian; Watson, Eila; Austoker, Joan; Grol, Richard

    2007-01-01

    Background Field-testing is a quality assurance criterion in the development of patient decision-support technologies (PDSTs), as identified in the consensus statement of the International Patient Decision Aids Standards Collaboration. We incorporated field-testing into the development of a Web-based, prostate-specific antigen PDST called Prosdex, which was commissioned as part of the UK Prostate Cancer Risk Management Programme. Objectives The aim of this study was to develop a model for the future field-testing of PDSTs, based on the field-testing of Prosdex. Our objectives were (1) to explore the reactions of men to evolving prototypes of Prosdex, (2) to assess the effect of these responses on the development process, and (3) to develop a model for field-testing PDSTs based on the responses and their effect on the development process. Methods Semistructured interviews were conducted with the men after they had viewed evolving prototypes of Prosdex in their homes. The men were grouped according to the prototype viewed. Men between 40 and 75 years of age were recruited from two family practices in different parts of Wales, United Kingdom. In the interviews, the men were asked for their views on Prosdex, both as a whole and in relation to specific sections such as the introduction and video clips. Comments and technical issues that arose during the viewings were noted and fed back to the developers in order to produce subsequent prototypes. Results A total of 27 men were interviewed, in five groups, according to the five prototypes of Prosdex that were developed. The two main themes from the interviews were the responses to the information provided in Prosdex and the responses to specific features of Prosdex. Within these themes, two of the most frequently encountered categories were detail of the information provided and balance between contrasting viewpoints. Criticisms were encountered, particularly with respect to navigation of the site. In addition, we found

  4. Utility of routine urine CMV PCR and total serum IgM testing of small for gestational age infants: a single center review.

    PubMed

    Espiritu, Michael M; Bailey, Sean; Wachtel, Elena V; Mally, Pradeep V

    2017-08-14

    Due to the extremely low incidence of TORCH (toxoplasmosis, rubella, CMV, herpes simplex virus) infections, diagnostic testing of all small for gestational age (SGA) infants aimed at TORCH etiologies may incur unnecessary tests and cost. To determine the frequency of urine CMV PCR and total IgM testing among infants with birth weight <10% and the rate of test positivity. To evaluate the frequency of alternative etiologies of SGA in tested infants. Retrospective chart review of SGA infants admitted to the neonatal intensive care unit (NICU) at NYU Langone Medical Center between 2007 and 2012. Subjects were classified as being SGA with or without intrauterine growth restriction (IUGR). The IUGR subjects were then further categorized as having either symmetric or asymmetric IUGR utilizing the Fenton growth chart at birth. Initial testing for TORCH infections, which included serum total IgM, CMV PCR and head ultrasound, were reviewed and analyzed. Three hundred and eighty-six (13%) infants from a total of 2953 NICU admissions had a birth weight ≤10th percentile. Of these, 44% were IUGR; 34% being symmetric IUGR and 10% asymmetric. A total of 32% of SGA infants had urine CMV PCR and total IgM tested with no positive results. As expected, significantly higher percentage of symmetric IUGR infants were tested compared to asymmetric IUGR and non-IUGR SGA infants, (64% vs. 47% vs. 19%) P≤0.01. However, 63% of infants with a known cause for IUGR had same testing done aimed at TORCH infections. We calculated additional charges of $64,065 that were incurred by such testing. The majority of infants in our study who received testing for urine CMV PCR and total IgM aimed at TORCH infections had one or more other known non-infectious etiologies for IUGR. Because the overall yield of such testing is extremely low, we suggest tests for possible TORCH infections may be limited to symmetric IUGR infants without other known etiologies. Improved guidelines testing for TORCH

  5. Identification and testing of reference genes for Sesame gene expression analysis by quantitative real-time PCR.

    PubMed

    Wei, Libin; Miao, Hongmei; Zhao, Ruihong; Han, Xiuhua; Zhang, Tide; Zhang, Haiyang

    2013-03-01

    Sesame (Sesamum indicum L.) is an ancient and important oilseed crop. However, few sesame reference genes have been selected for quantitative real-time PCR until now. Screening and validating reference genes is a requisite for gene expression normalization in sesame functional genomics research. In this study, ten candidate reference genes, i.e., SiACT, SiUBQ6, SiTUB, Si18S rRNA, SiEF1α, SiCYP, SiHistone, SiDNAJ, SiAPT and SiGAPDH, were chosen and examined systematically in 32 sesame samples. Three qRT-PCR analysis methods, i.e., geNorm, NormFinder and BestKeeper, were evaluated systematically. Results indicated that all ten candidate reference genes could be used as reference genes in sesame. SiUBQ6 and SiAPT were the optimal reference genes for sesame plant development; SiTUB was suitable for sesame vegetative tissue development, SiDNAJ for pathogen treatment, SiHistone for abiotic stress, SiUBQ6 for bud development and SiACT for seed germination. As for hormone treatment and seed development, SiHistone, SiCYP, SiDNAJ or SiUBQ6, as well as SiACT, SiDNAJ, SiTUB or SiAPT, could be used as reference gene, respectively. To illustrate the suitability of these reference genes, we analyzed the expression variation of three functional sesame genes of SiSS, SiLEA and SiGH in different organs using the optimal qRT-PCR system for the first time. The stability levels of optimal and worst reference genes screened for seed development, anther sterility and plant development were validated in the qRT-PCR normalization. Our results provided a reference gene application guideline for sesame gene expression characterization using qRT-PCR system.

  6. Exploring informed choice in the context of prenatal testing: findings from a qualitative study

    PubMed Central

    Potter, Beth K.; O’Reilly, Natasha; Etchegary, Holly; Howley, Heather; Graham, Ian D.; Walker, Mark; Coyle, Doug; Chorny, Yelena; Cappelli, Mario; Boland, Isabelle; Wilson, Brenda J.

    2008-01-01

    Abstract Purpose  This study explored whether and how a sample of women made informed choices about prenatal testing for foetal anomalies; its aim was to provide insights for future health policy and service provision. Methods  We conducted semi‐structured interviews with 38 mothers in Ottawa, Ontario, all of whom had been offered prenatal tests in at least one pregnancy. Using the Multi‐dimensional Measure of Informed Choice as a general guide to analysis, we explored themes relevant to informed choice, including values and knowledge, and interactions with health professionals. Results  Many, but not all, participants seemed to have made informed decisions about prenatal testing. Values and knowledge were interrelated and important components of informed choice, but the way they were discussed differed from the way they have been presented in scientific literature. In particular, ‘values’ related to expressions of women’s moral views or ideas about ‘how life should be lived’ and ‘knowledge’ related to the ways in which women prioritized and interpreted factual information, through their own and others’ experiences and in ‘thinking through’ the personal implications of testing. While some women described non‐directive discussions with health professionals, others perceived testing as routine or felt pressured to accept it. Conclusions  Our findings suggest a need for maternity care providers to be vigilant in promoting active decision making about prenatal testing, particularly around the consideration of personal implications. Further development of measures of informed choice may be necessary to fully evaluate decision support tools and to determine whether prenatal testing programmes are meeting their objectives. PMID:18798759

  7. Probe-based quantitative PCR assay for detecting constitutional and somatic deletions in the NF1 gene: application to genetic testing and tumor analysis.

    PubMed

    Terribas, Ernest; Garcia-Linares, Carles; Lázaro, Conxi; Serra, Eduard

    2013-06-01

    About 5% of patients with neurofibromatosis type 1 (NF1) bear constitutional microdeletions that encompass NF1 (neurofibromin 1) and neighboring genes. These patients are characterized by the development of a high number of dermal neurofibromas (dNFs), mental retardation, and an increased risk of developing a malignant peripheral nerve sheath tumor (MPNST). Additionally, 10% of somatic second hits identified in dNFs are caused by deletions involving the NF1 gene. To detect constitutional and somatic deletions, we developed a probe-based quantitative PCR (qPCR) assay for interrogating the copy number status of 11 loci distributed along a 2.8-Mb region around the NF1 gene. We developed the qPCR assay with Universal ProbeLibrary technology (Roche) and designed a Microsoft Excel spreadsheet to analyze qPCR data for copy number calculations. The assay fulfilled the essential aspects of the MIQE (minimum information for publication of quantitative real-time PCR experiments) guidelines and used the qBase relative quantification framework for calculations. The assay was validated with a set of DNA samples with known constitutional or somatic NF1 deletions. The assay showed high diagnostic sensitivity and specificity and distinguished between Type-1, Type-2, and atypical constitutional microdeletions in 14 different samples. It also identified 16 different somatic deletions in dNFs. These results were confirmed by multiplex ligation-dependent probe amplification. The qPCR assay provides a methodology for detecting constitutional NF1 microdeletions that could be incorporated as an additional technique in a genetic-testing setting. It also permits the identification of somatic NF1 deletions in tissues with a high percentage of cells bearing 2 copies of the NF1 gene. © 2013 American Association for Clinical Chemistry.

  8. Y-chromosomal testing of brown bears (Ursus arctos): Validation of a multiplex PCR-approach for nine STRs suitable for fecal and hair samples.

    PubMed

    Aarnes, Siv Grethe; Hagen, Snorre B; Andreassen, Rune; Schregel, Julia; Knappskog, Per M; Hailer, Frank; Stenhouse, Gordon; Janke, Axel; Eiken, Hans Geir

    2015-11-01

    High-resolution Y-chromosomal markers have been applied to humans and other primates to study population genetics, migration, social structures and reproduction. Y-linked markers allow the direct assessment of the genetic structure and gene flow of uniquely male inherited lineages and may also be useful for wildlife conservation and forensics, but have so far been available only for few wild species. Thus, we have developed two multiplex PCR reactions encompassing nine Y-STR markers identified from the brown bear (Ursus arctos) and tested them on hair, fecal and tissue samples. The multiplex PCR approach was optimized and analyzed for species specificity, sensitivity and stutter-peak ratios. The nine Y-STRs also showed specific STR-fragments for male black bears and male polar bears, while none of the nine markers produced any PCR products when using DNA from female bears or males from 12 other mammals. The multiplex PCR approach in two PCR reactions could be amplified with as low as 0.2 ng template input. Precision was high in DNA templates from hairs, fecal scats and tissues, with standard deviations less than 0.14 and median stutter ratios from 0.04 to 0.63. Among the eight di- and one tetra-nucleotide repeat markers, we detected simple repeat structures in seven of the nine markers with 9-25 repeat units. Allelic variation was found for eight of the nine Y-STRs, with 2-9 alleles for each marker and a total of 36 alleles among 453 male brown bears sampled mainly from Northern Europe. We conclude that the multiplex PCR approach with these nine Y-STRs would provide male bear Y-chromosomal specificity and evidence suited for samples from conservation and wildlife forensics.

  9. Making HIV testing work at the point of care in South Africa: a qualitative study of diagnostic practices.

    PubMed

    Engel, Nora; Davids, Malika; Blankvoort, Nadine; Dheda, Keertan; Pant Pai, Nitika; Pai, Madhukar

    2017-06-17

    Point of care testing promises to reduce delays in diagnosing and initiating treatment for infectious diseases such as Human Immuno-deficiency Virus (HIV). In South Africa, decentralized HIV testing with rapid tests offers important lessons for point of care testing programs. Yet, little is known about the strategies of providers and clients to make HIV testing successful in settings short of equipment, human resources and space. We aimed at examining these strategies. This paper is based on a larger qualitative study of diagnostic practices across major diseases and actors in homes, clinics, communities, hospitals and laboratories in South Africa. We conducted 101 semi-structured interviews and 7 focus group discussions with doctors, nurses, community health workers, patients, laboratory technicians, policymakers, hospital managers and manufacturers between September 2012 and June 2013 in Durban, Cape Town and Eastern Cape. The topics explored included diagnostic processes and challenges, understanding of diagnosis, and visions of ideal tests. For this paper, the data on HIV testing processes in clinics, communities and hospitals was used. Strategies to make HIV testing work at point of care involve overcoming constraints in equipment, spaces, human resources and workload and actively managing diagnostic processes. We grouped these strategies into subthemes: maintaining relationships, adapting testing guidelines and practices to stock-outs, to physical space, and to different clients, turning the test into a tool to reach another aim and turning the testing process into a tool to enhance adherence. These adaptive strategies are locally negotiated solutions, often ad-hoc, depending on personal commitment, relationships, human resources, physical space and referral systems. In the process, testing is redefined and repurposed. Not all of these repurposing acts are successful in ensuring a timely diagnosis. Some lead to disruptions, unnecessary testing or delays with

  10. The application of uniplex, duplex, and multiplex PCR for the absence of specified microorganism testing of pharmaceutical excipients and drug products.

    PubMed

    Ragheb, Suzan M; Yassin, Aymen S; Amin, Magdy A

    2012-01-01

    Notable progress has been made in methods that encourage the use of polymerase chain reaction (PCR) as a rapid and accurate tool in microbiological testing of pharmaceuticals. In this study, the detection of the four main specified microorganisms according to the pharmacopeial recommendations, Salmonella spp, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, was optimized in different pharmaceutical dosage forms and raw materials. Uniplex PCR was performed for the detection of each microorganism individually targeting the conserved region in each bacterial genome. Further optimizations were done to perform duplex and multiplex PCR assays considering relative concentrations of competitor primers used in the reaction. The uniplex PCR amplicons were successfully sequenced, confirming the conservation of used primers. Other validation parameters such as specificity, sensitivity, and robustness were examined closely. The method provides a high-throughput screening method to test different pharmaceutical preparations for specified microorganisms for the detection of microbiological contamination. Strict regulations govern the production of pharmaceutical products whether they are sterile or nonsterile. Certain official tests are carried out in microbiology testing laboratory in any pharmaceutical production facility to ensure the pharmaceuticals microbiological quality according to the standard pharmacopeial recommendations. Nonsterile products must be free of specified microorganisms that are used as a check for their quality. Topical preparations must be free of Pseudomonas aeruginosa and Staphylococcus aureus, and oral preparations must be free of Salmonella spp and Escherichia coli. Conventional microbiological methods are time-consuming, labor-intensive, and require long incubation times, resulting in delaying the release of the products. In this study, we tested and validated a polymerase chain reaction identification approach to detect indicator

  11. Accessing HIV Testing and Treatment among Men Who Have Sex with Men in China: A Qualitative Study

    PubMed Central

    Wei, Chongyi; Yan, Hongjing; Yang, Chuankun; Raymond, H. Fisher; Li, Jianjun; Yang, Haitao; Zhao, Jinkou; Huan, Xiping; Stall, Ron

    2013-01-01

    Barriers to HIV testing and HIV care and treatment pose significant challenges to HIV prevention among men who have sex with men (MSM) in China. We carried out a qualitative study to identify barriers and facilitators to HIV testing and treatment among Chinese MSM. In 2012, 7 focus group discussions were conducted with 49 MSM participants in Nanjing, China. Purposive sampling was used to recruit a diverse group of MSM participants. Semi-structured interviews were conducted to collect FG data. Major barriers to testing included gay- and HIV-related stigma and discrimination, relationship type and partner characteristics, low perception of risk or threat, HIV is incurable or equals death, concerns of confidentiality, unaware that testing is offered for free, and name-based testing. Key facilitators of testing included engaging in high-risk sex, sense of responsibility for partner, collectivism, testing as a part of standard/routine medical care, MSM-friendly medical personnel, increased acceptance of gay/bisexual men by the general public, legal recognition and protection of homosexuals, and home self-testing. Barriers to treatment included negative coping, non-disclosure to families, misconceptions of domestically produced antiretroviral drugs and the benefits of treatment, and costs associated with long-term treatment. Facilitators of treatment included sense of hopefulness that a cure would be found, the cultural value of longevity, peer social support and professional psychological counseling, affordable and specialized treatment and care, and reduced HIV-related stigma and discrimination. Finally, for both testing and treatment, more educational and promotional activities within MSM communities and among the general public are needed. PMID:23909807

  12. Accessing HIV testing and treatment among men who have sex with men in China: a qualitative study.

    PubMed

    Wei, Chongyi; Yan, Hongjing; Yang, Chuankun; Raymond, H Fisher; Li, Jianjun; Yang, Haitao; Zhao, Jinkou; Huan, Xiping; Stall, Ron

    2014-01-01

    Barriers to HIV testing and HIV care and treatment pose significant challenges to HIV prevention among men who have sex with men (MSM) in China. We carried out a qualitative study to identify barriers and facilitators to HIV testing and treatment among Chinese MSM. In 2012, seven focus group (FG) discussions were conducted with 49 MSM participants in Nanjing, China. Purposive sampling was used to recruit a diverse group of MSM participants. Semi-structured interviews were conducted to collect FG data. Major barriers to testing included gay- and HIV-related stigma and discrimination, relationship type and partner characteristics, low perception of risk or threat, HIV is incurable or equals death, concerns of confidentiality, unaware that testing is offered for free, and name-based testing. Key facilitators of testing included engaging in high-risk sex, sense of responsibility for partner, collectivism, testing as a part of standard/routine medical care, MSM-friendly medical personnel, increased acceptance of gay/bisexual men by the general public, legal recognition and protection of homosexuals, and home self-testing. Barriers to treatment included negative coping, nondisclosure to families, misconceptions of domestically produced antiretroviral drugs (ARVs) and the benefits of treatment, and costs associated with long-term treatment. Facilitators of treatment included sense of hopefulness that a cure would be found, the cultural value of longevity, peer social support and professional psychological counseling, affordable and specialized treatment and care, and reduced HIV-related stigma and discrimination. Finally, for both testing and treatment, more educational and promotional activities within MSM communities and among the general public are needed.

  13. Evaluating functional clothing in climatic chamber tests versus field tests: a comparison of quantitative and qualitative methods in product development.

    PubMed

    Karlsson, I C; Rosenblad, E F

    1998-10-01

    For the evaluation of clothing by human beings, two different approaches can be identified--laboratory tests (i.e. climatic chamber tests) or field trials/wear trials. It has been suggested that the performance of clothing can be adequately predicted on the basis of the data obtained from climatic chamber tests and that field trials may be expendable altogether. From a product development perspective, this paper discusses the information provided by standard data collection methods and tools for the assessment of thermal comfort used in laboratory settings and compares this information with that acquired in field trials. When assessing the information provided in the laboratory test against those questions posed in a development process, the results highlight the insufficiency of objectively measurable criteria. Therefore, objective measurements alone cannot verify the adequacy of stated requirements for thermal comfort. The use of appropriate and sensitive tools for collecting subjective votes should also be noted, since small differences in thermal sensation affected the individual's preference for clothing. Exposed differences between subjects, in terms of thermophysiological as well as subjective responses, illustrate the importance of studying individual values and deviations as opposed to strict mean values for large populations in order to satisfy a potential user group. It is argued that complementary interviews form a basis for further understanding of ratings and preferences and that they should also be included in the climatic chamber evaluation 'tool box'. User satisfaction is based on a simultaneous assessment of partly opposing properties into a satisfying use value. Although laboratory test procedures can be developed and improved in relation to design issues, field evaluations must be regarded as an integrated part of an iterative development process. Only the actual use situation can provide the total spectrum of conditions on which basis

  14. Comparison of bacterial culture and qPCR testing of rectal and pen floor samples as diagnostic approaches to detect enterotoxic Escherichia coli in nursery pigs.

    PubMed

    Weber, N R; Nielsen, J P; Hjulsager, C K; Jorsal, S E; Haugegaard, S; Hansen, C F; Pedersen, K S

    2017-08-01

    Enterotoxigenic E. coli (ETEC) are a major cause of diarrhoea in weaned pigs. The objective of this study was to evaluate the agreement at pen level among three different diagnostic approaches for the detection of ETEC in groups of nursery pigs with diarrhoea. The diagnostic approaches used were: bacterial culturing of faecal samples from three pigs (per pen) with clinical diarrhoea and subsequent testing for virulence genes in E. coli isolates; bacterial culturing of pen floor samples and subsequent testing for virulence genes in E. coli isolates; qPCR testing of pen floor samples in order to determine the quantity of F18 and F4 genes. The study was carried out in three Danish pig herds and included 31 pens with a pen-level diarrhoea prevalence of > 25%, as well as samples from 93 diarrhoeic nursery pigs from these pens. All E. coli isolates were analysed by PCR and classified as ETEC when genes for one or more adhesin factors and one or more enterotoxins were detected. A total of 208 E. coli colonies from pig samples and 172 E. coli colonies from pen floor samples were isolated. Haemolytic activity was detected on blood agar plates in 111 (29.2%) of the 380 colonies that were isolated. The only adhesin factor detected in this study was F18. When comparing bacterial culture or qPCR testing of pen floor samples with detection of ETEC-positive diarrhoeic pigs by culture, agreement was found in 26 (83.9%, Kappa = 0.665) and 23 (74.2%, Kappa = 0.488) of the pens, respectively. Agreement was observed between the detection of ETEC by bacterial culture and qPCR in the same pen floor sample in 26 (83.9%, Kappa = 0.679) pens. We observed an acceptable agreement for the detection of ETEC-positive diarrhoeic nursery pigs in pen samples for both bacterial culture of pen floor samples and qPCR. This study showed that both bacterial culture and qPCR testing of pen floor samples can be used as a diagnostic approach for detecting groups of ETEC-positive diarrhoeic nursery pigs

  15. A qualitative study looking at informed choice in the context of non‐invasive prenatal testing for aneuploidy

    PubMed Central

    Hill, Melissa; Chitty, Lyn S.

    2016-01-01

    Abstract Objective To explore women's attitudes towards non‐invasive prenatal testing (NIPT) and determine factors influencing their decisions around uptake of NIPT. Method We conducted qualitative interviews to assess knowledge, attitude and deliberation amongst women offered NIPT in a public health service. In total, 45 women took part in telephone interviews (79% participation rate). Results Most women could recount the key aspects of NIPT discussed during pre‐test counselling but had variable knowledge about Down syndrome. Analysis of women's attitudes towards undergoing NIPT revealed three dominant factors they considered when reflecting on the test: (1) how NIPT compared with alternative testing options, (2) reflections on coping and (3) moral or religious values. Exploring the deliberative process revealed the different paths women take when making decisions. For some, it was an extension of the decision to have Down syndrome screening; some considered it early on following the booking‐in appointment; others made step‐wise decisions about NIPT when it became relevant to them. Conclusion Our findings support the importance of personalised counselling, whereby women and their partners have the opportunity to reflect on the implications of the test results in the context of their own lives and values. Our data highlight the influence of personal circumstances on decision‐making. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd. PMID:27477537

  16. A qualitative assessment of shoulder girdle elevation during the upper limb tension test 1.

    PubMed

    Coppieters, M W; Stappaerts, K H; Everaert, D G; Staes, F F

    1999-02-01

    The 'feel through range' and the 'end-feel' of the elbow extension, and the elevation of the shoulder girdle during the upper limb tension test for the median nerve are considered important in clinical decision making. As no objective measurements have been published regarding these parameters, a special device was designed in order to obtain objective information on the force by which the shoulder girdle is elevated during the test. The purpose of this technical note is to describe the technical aspects of the device and its practical application. In a pilot study, five asymptomatic subjects have been assessed. The relationship between the elevation of the shoulder girdle and the range of elbow extension has been analysed. The data of five subjects and representative curves of one subject are presented. All five subjects demonstrate a considerable increase in force elevating the shoulder girdle. The results show that the device can be used to measure the force by which the shoulder girdle is elevated throughout the test. Although the number of subjects and the number of repetitions is limited, the data suggest that the method is accurate and that the phenomenon of shoulder girdle elevation during the upper limb tension test in asymptomatic subjects is consistent.

  17. Implementation of repeat HIV testing during pregnancy in Kenya: a qualitative study.

    PubMed

    Rogers, Anna Joy; Weke, Elly; Kwena, Zachary; Bukusi, Elizabeth A; Oyaro, Patrick; Cohen, Craig R; Turan, Janet M

    2016-07-11

    Repeat HIV testing in late pregnancy has the potential to decrease rates of mother-to-child transmission of HIV by identifying mothers who seroconvert after having tested negative for HIV in early pregnancy. Despite being national policy in Kenya, the available data suggest that implementation rates are low. We conducted 20 in-depth semi-structured interviews with healthcare providers and managers to explore barriers and enablers to implementation of repeat HIV testing guidelines for pregnant women. Participants were from the Nyanza region of Kenya and were purposively selected to provide variation in socio-demographics and job characteristics. Interview transcripts were coded and analyzed in Dedoose software using a thematic analysis approach. Four themes were identified a priori using Ferlie and Shortell's Framework for Change and additional themes were allowed to emerge from the data. Participants identified barriers and enablers at the client, provider, facility, and health system levels. Key barriers at the client level from the perspective of providers included late initial presentation to antenatal care and low proportions of women completing the recommended four antenatal visits. Barriers to offering repeat HIV testing for providers included heavy workloads, time limitations, and failing to remember to check for retest eligibility. At the facility level, inconsistent volume of clients and lack of space required for confidential HIV retesting were cited as barriers. Finally, at the health system level, there were challenges relating to the HIV test kit supply chain and the design of nationally standardized antenatal patient registers. Enablers to improving the implementation of repeat HIV testing included client dissemination of the benefits of antenatal care through word-of-mouth, provider cooperation and task shifting, and it was suggested that use of an electronic health record system could provide automatic reminders for retest eligibility. This study

  18. Application of real time RT-PCR for the genetic homogeneity and stability tests of the seed candidates for live attenuated influenza vaccine production.

    PubMed

    Shcherbik, Svetlana; Sergent, Sheila B; Davis, William G; Shu, Bo; Barnes, John; Kiseleva, Irina; Larionova, Natalie; Klimov, Alexander; Bousse, Tatiana

    2014-01-01

    Development and improvement of quality control tests for live attenuated vaccines are a high priority because of safety concerns. Live attenuated influenza vaccine (LAIV) viruses are 6:2 reassortants containing the hemagglutinin (HA) and neuraminidase (NA) gene segments from circulating influenza viruses to induce protective immune responses, and the six internal gene segments from a cold-adapted Master Donor Virus (MDV). LAIV candidate viruses for the 2012-2013 seasons, A/Victoria/361/2011-CDC-LV1 (LV1) and B/Texas/06/2011-CDC-LV2B (LV2B), were created by classical reassortment of A/Victoria/361/2011 and MDV-A A/Leningrad/134/17/57 (H2N2) or B/Texas/06/2011 and MDV-B B/USSR/60/69. In an attempt to provide better identity and stability testing for quality control of LV1 and LV2B, sensitive real-time RT-PCR assays (rRT-PCR) were developed to detect the presence of undesired gene segments (HA and NA from MDV and the six internal genes from the seasonal influenza viruses). The sensitivity of rRT-PCR assays designed for each gene segment ranged from 0.08 to 0.8EID50 (50% of Egg Infectious Dose) per reaction for the detection of undesired genes in LV1 and from 0.1 to 1EID50 per reaction for the detection of undesired genes in LV2B. No undesired genes were detected either before or after five passages of LV1 or LV2B in eggs. The complete genome sequencing of LV1 and LV2B confirmed the results of rRT-PCR, demonstrating the utility of the new rRT-PCR assays to provide the evidence for the homogeneity of the prepared vaccine candidate. Published by Elsevier B.V.

  19. Tests of the Dynamic Field Theory and the Spatial Precision Hypothesis: Capturing a Qualitative Developmental Transition in Spatial Working Memory

    PubMed Central

    Schutte, Anne R.; Spencer, John P.

    2009-01-01

    This study tested a dynamic field theory (DFT) of spatial working memory and an associated spatial precision hypothesis (SPH). Between three and six years of age there is a qualitative shift in how children use reference axes to remember locations: 3-year-olds’ spatial recall responses are biased toward reference axes after short memory delays, whereas 6-year-olds’ responses are biased away from reference axes. According to the DFT and the SPH, quantitative improvements over development in the precision of excitatory and inhibitory working memory processes lead to this qualitative shift. Simulations of the DFT in Experiment 1 predict that improvements in precision should cause the spatial range of targets attracted toward a reference axis to narrow gradually over development with repulsion emerging and gradually increasing until responses to most targets show biases away from the axis. Results from Experiment 2 with 3- to 5-year-olds support these predictions. Simulations of the DFT in Experiment 3 quantitatively fit the empirical results and offer insights into the neural processes underlying this developmental change. PMID:19968430

  20. General practitioners’ perceptions of introducing near-patient testing for common infections into routine primary care: A qualitative study

    PubMed Central

    Butler, Christopher C.; Simpson, Sharon; Wood, Fiona

    2008-01-01

    Objective Near-patient tests are promoted for guiding management of common infections in primary care with a view to enhancing the effectiveness of prescribing decisions and containing antimicrobial resistance. Changes in clinical practice should be based on appraisals of the factors that might influence change, viewed from the perspective of those expected to implement the change. We therefore explored the views of general practitioners concerning the possible introduction of near-patient tests for managing common infections. Design Qualitative semi-structured interview study. Interviews were recorded and analysed using thematic content analysis. Setting General practices in south-east Wales, UK. Subjects A total of 26 general practitioners (GPs) from high fluroquinolone antibiotics prescribing practices and 14 GPs from practices that prescribed fluroquinolones close to the south-east Wales mean. Results There was strong enthusiasm for a hypothetical near-patient, finger-prick blood tests that could distinguish viral from bacterial infections. Many GPs emphasized that such tests would be valuable in “selling” decisions not to prescribe antibiotics to patients. Concerns included limited additional useful information to guide prescribing above clinical diagnosis alone, that patients might deteriorate even if the tests correctly identified a viral aetiology, and that GPs would need to be convinced by research evidence supporting uptake. Several indicated that tests would be useful only for a limited number of patients and they were concerned by time pressures, apparatus maintenance and quality control, cost, and possible objections from patients, especially children. Conclusions Despite GP enthusiasm for the concept of a rapid test to distinguish viral from bacterial infection, strategies to promote uptake would be enhanced if concerns were addressed regarding the importance and feasibility of such tests in daily practice. PMID:18297558

  1. Stem loop-mediated isothermal amplification test: comparative analysis with classical LAMP and PCR in detection of Entamoeba histolytica in Kenya.

    PubMed

    Mwendwa, Fridah; Mbae, Cecilia K; Kinyua, Johnson; Mulinge, Erastus; Mburugu, Gitonga Nkanata; Njiru, Zablon K

    2017-03-31

    Entamoeba histolytica, the causative agent for amoebiasis is a considerable burden to population in the developing countries where it accounts for over 50 million infections. The tools for detection of amoebiasis are inadequate and diagnosis relies on microscopy which means a significant percent of cases remain undiagnosed. Moreover, tests formats that can be rapidly applied in rural endemic areas are not available. In this study, a loop-mediated isothermal test (LAMP) based on 18S small subunit ribosomal RNA gene was designed with extra reaction accelerating primers (stem primers) and compared with the published LAMP and PCR tests in detection of E. histolytica DNA in clinical samples. The stem LAMP test indicated shorter time to results by an average 11 min and analytical sensitivity of 10(-7) (~30 pg/ml) compared to the standard LAMP and PCR which showed sensitivities levels of 10(-5) (~3 ng/ml) and 10(-4) (~30 ng/ml) respectively using tenfold serial dilution of DNA. In the analysis of clinical specimens positive for Entamoeba spp. trophozoites and cysts using microscopy, the stem LAMP test detected E. histolytica DNA in 36/126, standard LAMP test 20/126 and PCR 17/126 cases respectively. There was 100% agreement in detection of the stem LAMP test product using fluorescence of SYTO-9 dye in real time machine, through addition of 1/10 dilution of SYBR(®) Green I and electrophoresis in 2% agarose gel stained with ethidium bromide. The stem LAMP test developed in this study indicates potential towards detection of E. histolytica.

  2. Collaborative trial for the validation of event-specific PCR detection methods of genetically modified papaya Huanong No.1.

    PubMed

    Wei, Jiaojun; Le, Huangying; Pan, Aihu; Xu, Junfeng; Li, Feiwu; Li, Xiang; Quan, Sheng; Guo, Jinchao; Yang, Litao

    2016-03-01

    For transferring the event-specific PCR methods of genetically modified papaya Huanong No.1 to other laboratories, we validated the previous developed PCR assays of Huanong No.1 according to the international standard organization (ISO) guidelines. A total of 11 laboratories participated and returned their test results in this trial. In qualitative PCR assay, the high specificity and limit of detection as low as 0.1% was confirmed. For the quantitative PCR assay, the limit of quantification was as low as 25 copies. The quantitative biases among ten blind samples were within the range between 0.21% and 10.04%. Furthermore, the measurement uncertainty of the quantitative PCR results was calculated within the range between 0.28% and 2.92% for these ten samples. All results demonstrated that the Huanong No.1 qualitative and quantitative PCR assays were creditable and applicable for identification and quantification of GM papaya Huanong No.1 in further routine lab analysis.

  3. Pre-Clinical Testing of Real-Time PCR Assays for Diarrheal Disease Agents of Genera Escherichia and Shigella

    DTIC Science & Technology

    2014-05-16

    Campylobacter jejuni Negative Negative Negative AF-SAL0085 Salmonella gr.E4 Negative...cross-reactivity Pathogen No. of sample TaqMan PCR Interpretation Acinetobacter Calcoaceticus 1 Negative Arcobacter butzleri 1 Negative Campylobacter ...Negative AF-ETEC966 ETEC-LT Negative AF-ETEC083 ETEC-LT Negative ATCC25922 Escherichia coli Negative ATCC70819 Campylobacter jejuni Negative AF-SAL0085 Salmonella gr. E4 Negative AF-SAL445 Salmonella paratyphi A Negative

  4. Development and Field Testing of a Real-Time PCR Assay for Caprine Arthritis-Encephalitis-Virus (CAEV)

    PubMed Central

    Brajon, Giovanni; Mandas, Daniela; Liciardi, Manuele; Taccori, Flavia; Meloni, Mauro; Corrias, Franco; Montaldo, Caterina; Coghe, Ferdinando; Casciari, Cristina; Giammarioli, Monica; Orrù, Germano

    2011-01-01

    Caprine arthritis/encephalitis (CAE) of goats and occasionally sheep are persistent virus infections caused by a lentivirus (CAEV). This viral infection results in arthritis in adult animals and encephalitis in kids. Prognosis for the encephalitic form is normally poor, with substantial economic loss for the farm. In this context an early/fast laboratory diagnosis for CAEV infection could be useful for effective prophylactic action. In this work we performed a quantitative real time PCR designed on the CAEV env gene to detect/quantify in goat/sheep samples, viral RNA or proviral DNA forms of CAEV. This procedure was validated in 15 sheep, experimentally infected with CAEV or with a highly correlated lentivirus (visna maedi, MVV); in addition, a total of 37 clinical goat specimens recruited in CAEV positive herds were analyzed and compared using serological analysis (Elisa and AGID). All samples infected with MVV resulted negative. In sheep experimentally infected with CAEV, proviral DNA was detectable 15 days post infection, whereas the serological methods revealed an indicative positivity after 40-60 days.This method showed a sensitivity of 102 env fragments/PCR) with a linear dynamic range of quantitation from 103 to 107 env fragments/PCR; the R2 correlation coefficient was 0.98. All subjects with a clinical diagnosis for Caprine Arthritis-Encephalitis (CAE) resulted CAEV DNA positive. PMID:22888382

  5. Impact of Routine Real-Time PCR Testing of Imported Malaria over 4 Years of Implementation in a Clinical Laboratory

    PubMed Central

    Shokoples, Sandra; Mukhi, Shamir N.; Scott, Allison N.

    2013-01-01

    In clinical laboratories, diagnosis of imported malaria is commonly performed by microscopy. However, the volume of specimens is generally low and maintaining proficiency in reading blood smears, particularly at the species level, is challenging in this setting. To address this problem, the Provincial Laboratory for Public Health (ProvLab) in Alberta, Canada, implemented real-time PCR for routine confirmation of all smear-positive samples in the province. Here we report our experience over a 4-year period (2008 to 2012) with this new diagnostic algorithm. While detection of Plasmodium falciparum by microscopy alone was accurate, real-time PCR served as an important adjunct to microscopy for the identification of non-falciparum species. In 18% of cases, the result was reported as non-falciparum or the species could not be identified by microscopy alone, and in all cases, the species was resolved by real-time PCR. In another 4% of cases, the species was misidentified by microscopy. To enhance surveillance for malaria, we integrated our demographic, clinical, and laboratory data into a new system developed by the Canadian Network for Public Health Intelligence, called the Malaria System for Online Surveillance (SOS). Using this application, we characterized our patient populations and travel history to identify risk factors associated with malaria infection abroad. PMID:23554200

  6. A qualitative analysis of inaccurate responses on the Hooper Visual Organization Test.

    PubMed

    Lopez, Michael N; Lazar, Michael D; Imperio, Sherwin M

    2005-06-01

    The present study investigated the types of inaccurate responses, i.e., Don't Know, Semantic, Visual (nonlinguistic), Phonological, Circumlocutory, and Perseverative, made on the Hooper Visual Organization Test by a heterogeneous sample of 68 brain-damaged and 63 substance abuse patients. The mean age of the brain-damaged and substance abuse groups were 46.0 (SD=13.5) and 43.7 (SD=12.9) yr., respectively. Analysis showed that the brain-damaged group made significantly more visual and perseverative responses than the substance abuse group. There was significantly more variance in the Visual responses than the Semantic responses for the brain-damaged group. The authors conclude that visuospatial ability is the primary factor for successful performance on this test.

  7. Detection of malaria infection in blood transfusion: a comparative study among real-time PCR, rapid diagnostic test and microscopy: sensitivity of Malaria detection methods in blood transfusion.

    PubMed

    Hassanpour, Gholamreza; Mohebali, Mehdi; Raeisi, Ahmad; Abolghasemi, Hassan; Zeraati, Hojjat; Alipour, Mohsen; Azizi, Ebrahim; Keshavarz, Hossein

    2011-06-01

    The transmission of malaria by blood transfusion was one of the first transfusion-transmitted infections recorded in the world. Transfusion-transmitted malaria may lead to serious problems because infection with Plasmodium falciparum may cause rapidly fatal death. This study aimed to compare real-time polymerase chain reaction (real-time PCR) with rapid diagnostic test (RDT) and light microscopy for the detection of Plasmodium spp. in blood transfusion, both in endemic and non-endemic areas of malaria disease in Iran. Two sets of 50 blood samples were randomly collected. One set was taken from blood samples donated in blood bank of Bandar Abbas, a city located in a malarious-endemic area, and the other set from Tehran, a non-endemic one. Light microscopic examination on both thin and thick smears, RDTs, and real-time PCR were performed on the blood samples and the results were compared. Thin and thick light microscopic examinations of all samples as well as RDT results were negative for Plasmodium spp. Two blood samples from endemic area were positive only with real-time PCR. It seems that real-time PCR as a highly sensitive method can be helpful for the confirmation of malaria infection in different units of blood transfusion organization especially in malaria-endemic areas where the majority of donors may be potentially infected with malaria parasites.

  8. Detection of Mixed-Species Infections of Plasmodium falciparum and Plasmodium vivax by Nested PCR and Rapid Diagnostic Tests in Southeastern Iran

    PubMed Central

    Ehtesham, Reyhaneh; Fazaeli, Asghar; Raeisi, Ahmad; Keshavarz, Hossein; Heidari, Aliehsan

    2015-01-01

    Coexistence of two species of Plasmodium in a single host has disrupted the diagnosis and treatment of malaria. This study was designed to evaluate the ability of rapid diagnostic test (RDT) kits for the diagnosis of mixed-species malaria infections in southeastern Iran. A total of 100 malaria patients were included in the study out of 164 randomly suspected symptomatic malaria patients from May to November 2012. Nested polymerase chain reaction (PCR) was also used to judge the ability of microscopy versus RDT kits for detecting mixed species. The sensitivity of light microscopy for the detection of mixed-species malaria infections was 16.6% (95% confidence interval [CI] = 3–49.1). Nested PCR revealed 12 patients with mixed-species infection. The CareStart Pv/Pf Combo kit detected 58% of the mixed-species infections, which were determined by nested PCR (sensitivity = 58.3%; 95% CI = 28.5–83.5). For identifying P. falciparum, P. vivax, and mixed-species infections, the concordance rates (kappa statistics) of microscopy and CareStart Pv/Pf Combo kit with nested PCR were 0.76 and 0.79, respectively (P = 0.001). This study underlines the effectiveness of RDT kits to improve the differentiation of mixed-species malaria infections in endemic areas where the prevalence of chloroquine resistance is high. PMID:25962771

  9. Creating more effective health plan quality reports for consumers: lessons from a synthesis of qualitative testing.

    PubMed Central

    Harris-Kojetin, L D; McCormack, L A; Jaël, E F; Sangl, J A; Garfinkel, S A

    2001-01-01

    OBJECTIVE: Social marketing techniques such as consumer testing have only recently been applied to develop effective consumer health insurance information. This article discusses lessons learned from consumer testing to create consumer plan choice materials. DATA SOURCES/STUDY SETTING: Data were collected from 268 publicly and privately insured consumers in three studies between 1994 and 1999. STUDY DESIGN: Iterative testing and revisions were conducted to design seven booklets to help Medicaid, Medicare, and employed consumers choose a health plan. DATA COLLECTION METHODS: Standardized protocols were used in 11 focus groups and 182 interviews to examine the content, comprehension, navigation, and utility of the booklets. PRINCIPAL FINDINGS: A method is suggested to help consumers narrow their plan choices by breaking down the process into smaller decisions using a set of guided worksheets. CONCLUSION: Implementing these lessons is challenging and not often done well. This article gives examples of evidence-based approaches to address cognitive barriers that designers of consumer health insurance information can adapt to their needs. Images Figure. 3 PMID:11482584

  10. Qualitative tests of remote eyetracker recovery and performance during head rotation.

    PubMed

    Hessels, Roy S; Cornelissen, Tim H W; Kemner, Chantal; Hooge, Ignace T C

    2015-09-01

    What are the decision criteria for choosing an eyetracker? Often the choice is based on specifications by the manufacturer of the validity (accuracy) and reliability (precision) of measurements that can be achieved using a particular eyetracker. These specifications are mostly achieved under optimal conditions-for example, by using an artificial eye or trained participants fixed in a chinrest. Research, however, does not always take place in optimal conditions: For instance, when investigating eye movements in infants, school children, and patient groups with disorders such as attention-deficit hyperactivity disorder, it is practically impossible to restrict movement. We modeled movements often seen in infant research in two behaviors: (1) looking away from and back to the screen, to investigate eyetracker recovery, and (2) head orientations, to investigate eyetracker performance with nonoptimal orientations of the eyes. We investigated how eight eyetracking setups by three manufacturers (SMI, Tobii, and LC Technologies) coped with these modeled behaviors in adults. We report that the tested SMI eyetrackers dropped in sampling frequency when the eyes were not visible to the eyetracker, whereas the other systems did not, and discuss the potential consequences thereof. Furthermore, we report that the tested eyetrackers varied in their rates of data loss and systematic offsets during shifted head orientations. We conclude that (prospective) eye-movement researchers who cannot restrict movement or nonoptimal head orientations in their participants might benefit from testing their eyetracker in nonoptimal conditions. Additionally, researchers should be aware of the data loss and inaccuracies that might result from nonoptimal head orientations.

  11. Combined Use of PCR-Based TCRG and TCRB Clonality Tests on Paraffin-Embedded Skin Tissue in the Differential Diagnosis of Mycosis Fungoides and Inflammatory Dermatoses

    PubMed Central

    Zhang, Bing; Beck, Andrew H.; Taube, Janis M.; Kohler, Sabine; Seo, Katie; Zwerner, Jeffrey; Viakhereva, Natalie; Sundram, Uma; Kim, Youn H.; Schrijver, Iris; Arber, Daniel A.; Zehnder, James L.

    2010-01-01

    The distinction between mycosis fungoides (MF) and inflammatory dermatoses (ID) by clinicopathologic criteria can be challenging. There is limited information regarding the performance characteristics and utility of TCRG and TCRB clonality assays in diagnosis of MF and ID from paraffin-embedded tissue sections. In this study, PCR tests were performed with both TCRG and TCRB BIOMED-2 clonality methods followed by capillary electrophoresis and Genescan analysis using DNA samples from 35 MF and 96 ID patients with 69 and 133 paraffin-embedded specimens, respectively. Performance characteristics were determined for each test individually and in combination. TCRG and TCRB tests demonstrated identical sensitivity (64%) and specificity (84%) when analyzed as individual assays. The positive predictive value, negative predictive value, and change of posttest MF probability over a range of MF pretest probabilities were obtained. These data were used to construct an algorithm for sequential use of TCRG and TCRB. As single tests, commercially available BIOMED-2 PCR-based TCRG and TCRB clonality tests on paraffin-embedded tissue have no significant difference in terms of sensitivity and specificity. Combined use of the two tests in patients with intermediate pretest probabilities as proposed in the algorithm could improve test utility. PMID:20203005

  12. A Qualitative Study of Healthcare Providers’ Perspectives on the Implications of Genome-Wide Testing in Pediatric Clinical Practice

    PubMed Central

    Reiff, Marian; Mueller, Rebecca; Mulchandani, Surabhi; Spinner, Nancy B.; Pyeritz, Reed E.; Bernhardt, Barbara A.

    2013-01-01

    The utilization of genome-wide chromosomal microarray analysis (CMA) in pediatric clinical practice provides an opportunity to consider how genetic diagnostics is evolving, and to prepare for the clinical integration of genome-wide sequencing technologies. We conducted semi-structured interviews with 15 healthcare providers (7 genetic counselors, 4 medical geneticists, and 4 non-genetics providers) to investigate the impact of CMA on clinical practice, and implications for providers, patients and families. Interviews were analyzed qualitatively using content analysis. Most providers reported that genomic testing enhanced their professional experience and was beneficial to patients, primarily due to the improved diagnostic rate compared with earlier chromosomal studies. Other effects on practice included moving towards genotype-first diagnosis and broadening indications for chromosomal testing. Opinions varied concerning informed consent and disclosure of results. The duty to disclose incidental findings (IFs) was noted; however concerns were raised about potential psychosocial harms of disclosing pre-symptomatic findings. Tensions were revealed between the need for comprehensive informed consent for all families and the challenges of communicating time-consuming and potentially anxiety-provoking information regarding uncertain and incidental findings that may be relevant only in rare cases. Genetic counselors can play an important role in liaising with families, health professionals and testing laboratories, providing education and guidance to non-genetics providers, and enabling families to receive adequate pre- and post-test information and follow-up care. PMID:24037030

  13. A Qualitative Exploration of Sexual Risk and HIV Testing Behaviors among Men Who Have Sex with Men in Beirut, Lebanon

    PubMed Central

    Wagner, Glenn J.; Aunon, Frances M.; Kaplan, Rachel L.; Rana, Yashodhara; Khouri, Danielle; Tohme, Johnny; Mokhbat, Jacques

    2012-01-01

    Men who have sex with men (MSM) may account for most new HIV infections in Lebanon, yet little is known about the factors that influence sexual risk behavior and HIV testing in this population. Qualitative interviews were conducted with 31 MSM living in Beirut, and content analysis was used to identify emergent themes. Mean age of the participants was 28.4 years, and all identified as either gay (77%) or bisexual (23%). Half reported not using condoms consistently and one quarter had not been HIV-tested. Many described not using condoms with a regular partner in the context of a meaningful relationship, mutual HIV testing, and a desire to not use condoms, suggesting that trust, commitment and intimacy play a role in condom use decisions. Condoms were more likely to be used with casual partners, partners believed to be HIV-positive, and with partners met online where men found it easier to candidly discuss HIV risk. Fear of infection motivated many to get HIV tested and use condoms, but such affect also led some to avoid HIV testing in fear of disease and social stigma if found to be infected. Respondents who were very comfortable with their sexual orientation and who had disclosed their sexuality to family and parents tended to be more likely to use condoms consistently and be tested for HIV. These findings indicate that similar factors influence the condom use and HIV testing of MSM in Beirut as those observed in studies elsewhere of MSM; hence, prevention efforts in Lebanon can likely benefit from lessons learned and interventions developed in other regions, particularly for younger, gay-identified men. Further research is needed to determine how prevention efforts may need to be tailored to address the needs of men who are less integrated into or do not identify with the gay community. PMID:23029103

  14. A qualitative exploration of sexual risk and HIV testing behaviors among men who have sex with men in Beirut, Lebanon.

    PubMed

    Wagner, Glenn J; Aunon, Frances M; Kaplan, Rachel L; Rana, Yashodhara; Khouri, Danielle; Tohme, Johnny; Mokhbat, Jacques

    2012-01-01

    Men who have sex with men (MSM) may account for most new HIV infections in Lebanon, yet little is known about the factors that influence sexual risk behavior and HIV testing in this population. Qualitative interviews were conducted with 31 MSM living in Beirut, and content analysis was used to identify emergent themes. Mean age of the participants was 28.4 years, and all identified as either gay (77%) or bisexual (23%). Half reported not using condoms consistently and one quarter had not been HIV-tested. Many described not using condoms with a regular partner in the context of a meaningful relationship, mutual HIV testing, and a desire to not use condoms, suggesting that trust, commitment and intimacy play a role in condom use decisions. Condoms were more likely to be used with casual partners, partners believed to be HIV-positive, and with partners met online where men found it easier to candidly discuss HIV risk. Fear of infection motivated many to get HIV tested and use condoms, but such affect also led some to avoid HIV testing in fear of disease and social stigma if found to be infected. Respondents who were very comfortable with their sexual orientation and who had disclosed their sexuality to family and parents tended to be more likely to use condoms consistently and be tested for HIV. These findings indicate that similar factors influence the condom use and HIV testing of MSM in Beirut as those observed in studies elsewhere of MSM; hence, prevention efforts in Lebanon can likely benefit from lessons learned and interventions developed in other regions, particularly for younger, gay-identified men. Further research is needed to determine how prevention efforts may need to be tailored to address the needs of men who are less integrated into or do not identify with the gay community.

  15. Pregnant women’s experiences of routine counselling and testing for HIV in Eastern Uganda: a qualitative study

    PubMed Central

    2013-01-01

    Background Routine HIV counselling and testing as part of antenatal care has been institutionalized in Uganda as an entry point for pregnant women into the prevention of mother-to-child transmission of HIV (PMTCT) programme. Understanding how women experience this mode of HIV testing is important to generate ideas on how to strengthen the PMTCT programme. We explored pregnant HIV positive and negative women’s experiences of routine counselling and testing in Mbale District, Eastern Uganda and formulated suggestions for improving service delivery. Methods This was a qualitative study conducted at Mbale Regional Referral Hospital in Eastern Uganda between January and May 2010. Data were collected using in-depth interviews with 30 pregnant women (15 HIV positive and 15 HIV negative) attending an antenatal clinic, six key informant interviews with health workers providing antenatal care and observations. Data were analyzed using a content thematic approach. Results Prior to attending their current ANC visit, most women knew that the hospital provided HIV counselling and testing services as part of antenatal care (ANC). HIV testing was perceived as compulsory for all women attending ANC at the hospital but beneficial, for mothers, especially those who test HIV positive and their unborn babies. Most HIV positive women were satisfied with the immediate counselling they received from health workers, but identified the need to provide follow up counselling and support after the test, as areas for improvement. However, most HIV negative women mentioned that they were given inadequate attention during post-test counselling. This left them with unanswered questions and, for some, doubts about the negative test results. Conclusions In this setting, routine HIV counselling and testing services are known and acceptable to mothers. There is need to strengthen post-test and follow up counselling for both HIV positive and negative women in order to maximize opportunities for

  16. Genotoxicity testing: moving beyond qualitative "screen and bin" approach towards characterization of dose-response and thresholds.

    PubMed

    Pottenger, Lynn H; Gollapudi, B Bhaskar

    2010-01-01

    For more than 40+ years, genotoxicity data have been interpreted in a qualitative, binary mode; a chemical is considered either positive or negative for a response in the test system. Although dose-response information is sometimes used in this decision, it is not routine to obtain the amount of information needed to inform risk assessment, for example to determine no-observed-genotoxic-effect-levels, primarily due to the historical view of genotoxic responses as "linear, no-threshold." Only recently have researchers begun to address this issue through robust experimental designs and application of statistical models. A growing body-of-evidence supports the existence of response thresholds for a number of mutagenic agents, in vitro and in vivo. Clearly, simple observation of a "hockey-stick" dose-response curve is not sufficient to establish a threshold. Collection of robust empirical data must be supported with an analysis of biological plausibility for the observed threshold. In this context, a chemical-specific mode-of-action (MOA) approach, which identifies key events responsible for the observed mutagenic effect, is extremely valuable. Biomarkers of key events, providing qualitative and quantitative information, can be integrated in a weight-of-evidence-based assessment of genotoxicity data from multiple test systems and used to identify data gaps to resolve/reduce uncertainties during the risk assessment process. To this end, specific recommendations on study design and data analysis are proposed. As the Environmental Mutagen Society celebrates its 40th anniversary, the field of genetic toxicology is marking a milestone on the path to a new paradigm, using a MOA, data-driven approach to answer questions about thresholds for genotoxic agents.

  17. A qualitative study into the difficulties experienced by healthcare decision makers when reading a Cochrane diagnostic test accuracy review.

    PubMed

    Zhelev, Zhivko; Garside, Ruth; Hyde, Christopher

    2013-05-16

    Cochrane reviews are one of the best known and most trusted sources of evidence-based information in health care. While steps have been taken to make Cochrane intervention reviews accessible to a diverse readership, little is known about the accessibility of the newcomer to the Cochrane library: diagnostic test accuracy reviews (DTARs). The current qualitative study explored how healthcare decision makers, who varied in their knowledge and experience with test accuracy research and systematic reviews, read and made sense of DTARs. A purposive sample of clinicians, researchers and policy makers (n = 21) took part in a series of think-aloud interviews, using as interview material the first three DTARs published in the Cochrane library. Thematic qualitative analysis of the transcripts was carried out to identify patterns in participants' 'reading' and interpretation of the reviews and the difficulties they encountered. Participants unfamiliar with the design and methodology of DTARs found the reviews largely inaccessible and experienced a range of difficulties stemming mainly from the mismatch between background knowledge and level of explanation provided in the text. Experience with systematic reviews of interventions did not guarantee better understanding and, in some cases, led to confusion and misinterpretation. These difficulties were further exacerbated by poor layout and presentation, which affected even those with relatively good knowledge of DTARs and had a negative impact not only on their understanding of the reviews but also on their motivation to engage with the text. Comparison between the readings of the three reviews showed that more accessible presentation, such as presenting the results as natural frequencies, significantly increased participants' understanding. The study demonstrates that authors and editors should pay more attention to the presentation as well as the content of Cochrane DTARs, especially if the reports are aimed at readers with

  18. Training Vegetable Parenting Practices Through a Mobile Game: Iterative Qualitative Alpha Test

    PubMed Central

    Beltran, Alicia; Buday, Richard; Hughes, Sheryl; O'Connor, Teresia; Baranowski, Janice; Dadabhoy, Hafza R; Diep, Cassandra S; Baranowski, Tom

    2015-01-01

    Background Vegetable consumption protects against chronic diseases, but many young children do not eat vegetables. One quest within the mobile application Mommio was developed to train mothers of preschoolers in effective vegetable parenting practices, or ways to approach getting their child to eat and enjoy vegetables. A much earlier version of the game, then called Kiddio, was alpha tested previously, but the game has since evolved in key ways. Objective The purpose of this research was to alpha test the first quest, substantiate earlier findings and obtain feedback on new game features to develop an effective, compelling parenting game. Methods Mothers of preschool children (n=20) played a single quest of Mommio 2 to 4 times, immediately after which a semi-structured interview about their experience was completed. Interviews were transcribed and double coded using thematic analysis methods. Results Mothers generally liked the game, finding it realistic and engaging. Some participants had difficulties with mechanics for moving around the 3-D environment. Tips and hints were well received, and further expansion and customization were desired. Conclusions Earlier findings were supported, though Mommio players reported more enjoyment than Kiddio players. Continued development will include more user-friendly mechanics, customization, opportunities for environment interaction, and food parenting scenarios. PMID:26208899

  19. Empirical testing of 16S rRNA gene PCR primer pairs reveals variance in target specificity and efficacy not suggested by in silico analysis.

    PubMed

    Morales, Sergio E; Holben, William E

    2009-05-01

    Phylogenetic and "fingerprinting" analyses of the 16S rRNA genes of prokaryotes have been a mainstay of microbial ecology during the last two decades. However, many methods and results from studies that rely on the 16S rRNA gene for detection and quantification of specific microbial taxa have seemingly received only cursory or even no validation. To directly examine the efficacy and specificity of 16S rRNA gene-based primers for phylum-, class-, and operational taxonomic unit-specific target amplification in quantitative PCR, we created a collection of primers based solely on an extensive soil bacterial 16S rRNA gene clone library containing approximately 5,000 sequences from a single soil sample (i.e., a closed site-specific library was used to create PCR primers for use at this site). These primers were initially tested in silico prior to empirical testing by PCR amplification of known target sequences and of controls based on disparate phylogenetic groups. Although all primers were highly specific according to the in silico analysis, the empirical analyses clearly exhibited a high degree of nonspecificity for many of the phyla or classes, while other primers proved to be highly specific. These findings suggest that significant care must be taken when interpreting studies whose results were obtained with target specific primers that were not adequately validated, especially where population densities or dynamics have been inferred from the data. Further, we suggest that the reliability of quantification of specific target abundance using 16S rRNA-based quantitative PCR is case specific and must be determined through rigorous empirical testing rather than solely in silico.

  20. Fully automated sample preparation microsystem for genetic testing of hereditary hearing loss using two-color multiplex allele-specific PCR.

    PubMed

    Zhuang, Bin; Gan, Wupeng; Wang, Shuaiqin; Han, Junping; Xiang, Guangxin; Li, Cai-Xia; Sun, Jing; Liu, Peng

    2015-01-20

    A fully automated microsystem consisting of a disposable DNA extraction and PCR microchip, as well as a compact control instrument, has been successfully developed for genetic testing of hereditary hearing loss from human whole blood. DNA extraction and PCR were integrated into a single 15-μL reaction chamber, where a piece of filter paper was embedded for capturing genomic DNA, followed by in-situ PCR amplification without elution. Diaphragm microvalves actuated by external solenoids together with a "one-way" fluidic control strategy operated by a modular valve positioner and a syringe pump were employed to control the fluids and to seal the chamber during thermal cycling. Fully automated DNA extractions from as low as 0.3-μL human whole blood followed by amplifications of 59-bp β-actin fragments can be completed on the microsystem in about 100 min. Negative control tests that were performed between blood sample analyses proved the successful elimination of any contamination or carryover in the system. To more critically test the microsystem, a two-color multiplex allele-specific PCR (ASPCR) assay for detecting c.176_191del16, c.235delC, and c.299_300delAT mutations in GJB2 gene that accounts for hereditary hearing loss was constructed. Two allele-specific primers, one labeled with TAMRA for wild type and the other with FAM for mutation, were designed for each locus. DNA extraction from blood and ASPCR were performed on the microsystem, followed by an electrophoretic analysis on a portable microchip capillary electrophoresis system. Blood samples from a healthy donor and five persons with genetic mutations were all accurately analyzed with only two steps in less than 2 h.

  1. Field evaluation of Abbott Real Time HIV-1 Qualitative test for early infant diagnosis using dried blood spots samples in comparison to Roche COBAS Ampliprep/COBAS TaqMan HIV-1 Qual test in Kenya.

    PubMed

    Chang, Joy; Omuomo, Kenneth; Anyango, Emily; Kingwara, Leonard; Basiye, Frank; Morwabe, Alex; Shanmugam, Vedapuri; Nguyen, Shon; Sabatier, Jennifer; Zeh, Clement; Ellenberger, Dennis

    2014-08-01

    Timely diagnosis and treatment of infants infected with HIV are critical for reducing infant mortality. High-throughput automated diagnostic tests like Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Qual Test (Roche CAPCTM Qual) and the Abbott Real Time HIV-1 Qualitative (Abbott Qualitative) can be used to rapidly expand early infant diagnosis testing services. In this study, the performance characteristics of the Abbott Qualitative were evaluated using two hundred dried blood spots (DBS) samples (100 HIV-1 positive and 100 HIV-1 negative) collected from infants attending the antenatal facilities in Kisumu, Kenya. The Abbott Qualitative results were compared to the diagnostic testing completed using the Roche CAPCTM Qual in Kenya. The sensitivity and specificity of the Abbott Qualitative were 99.0% (95% CI: 95.0-100.0) and 100.0% (95% CI: 96.0-100.0), respectively, and the overall reproducibility was 98.0% (95% CI: 86.0-100.0). The limits of detection for the Abbott Qualitative and Roche CAPCTM Qual were 56.5 and 6.9copies/mL at 95% CIs (p=0.005), respectively. The study findings demonstrate that the Abbott Qualitative test is a practical option for timely diagnosis of HIV in infants.

  2. Field evaluation of Abbott Real Time HIV-1 Qualitative test for early infant diagnosis using dried blood spots samples in comparison to Roche COBAS Ampliprep/COBAS TaqMan HIV-1 Qual Test in Kenya

    PubMed Central

    Chang, Joy; Omuomo, Kenneth; Anyango, Emily; Kingwara, Leonard; Basiye, Frank; Morwabe, Alex; Shanmugam, Vedapuri; Nguyen, Shon; Sabatier, Jennifer; Zeh, Clement; Ellenberger, Dennis

    2016-01-01

    Timely diagnosis and treatment of infants infected with HIV are critical for reducing infant mortality. High-throughput automated diagnostic tests like Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Qual Test (Roche CAPCTM Qual) and the Abbott Real Time HIV-1 Qualitative (Abbott Qualitative) can be used to rapidly expand early infant diagnosis testing services. In this study, the performance characteristics of the Abbott Qualitative were evaluated using two hundred dried blood spots (DBS) samples (100 HIV-1 positive and 100 HIV-1 negative) collected from infants attending the antenatal facilities in Kisumu, Kenya. The Abbott Qualitative results were compared to the diagnostic testing completed using the Roche CAPCTM Qual in Kenya. The sensitivity and specificity of the Abbott Qualitative were 99.0% (95% CI: 95.0–100.0) and 100.0% (95% CI: 96.0–100.0), respectively, and the overall reproducibility was 98.0% (95% CI: 86.0–100.0). The limits of detection for the Abbott Qualitative and Roche CAPCTM Qual were 56.5 and 6.9 copies/mL at 95% CIs (p = 0.005), respectively. The study findings demonstrate that the Abbott Qualitative test is a practical option for timely diagnosis of HIV in infants. PMID:24726703

  3. RNA and DNA bacteriophages as molecular diagnosis controls in clinical virology: a comprehensive study of more than 45,000 routine PCR tests.

    PubMed

    Ninove, Laetitia; Nougairede, Antoine; Gazin, Celine; Thirion, Laurence; Delogu, Ilenia; Zandotti, Christine; Charrel, Remi N; De Lamballerie, Xavier

    2011-02-09

    Real-time PCR techniques are now commonly used for the detection of viral genomes in various human specimens and require for validation both external and internal controls (ECs and ICs). In particular, ICs added to clinical samples are necessary to monitor the extraction, reverse transcription, and amplification steps in order to detect false-negative results resulting from PCR-inhibition or errors in the technical procedure. Here, we performed a large scale evaluation of the use of bacteriophages as ICs in routine molecular diagnosis. This allowed to propose simple standardized procedures (i) to design specific ECs for both DNA and RNA viruses and (ii) to use T4 (DNA) or MS2 (RNA) phages as ICs in routine diagnosis. Various technical formats for using phages as ICs were optimised and validated. Subsequently, T4 and MS2 ICs were evaluated in routine real-time PCR or RT-PCR virological diagnostic tests, using a series of 8,950 clinical samples (representing 36 distinct specimen types) sent to our laboratory for the detection of a variety of DNA and RNA viruses. The frequency of inefficient detection of ICs was analyzed according to the nature of the sample. Inhibitors of enzymatic reactions were detected at high frequency in specific sample types such as heparinized blood and bone marrow (>70%), broncho-alveolar liquid (41%) and stools (36%). The use of T4 and MS2 phages as ICs proved to be cost-effective, flexible and adaptable to various technical procedures of real-time PCR detection in virology. It represents a valuable strategy for enhancing the quality of routine molecular diagnosis in laboratories that use in-house designed diagnostic systems, which can conveniently be associated to the use of specific synthetic ECs. The high rate of inhibitors observed in a variety of specimen types should stimulate the elaboration of improved technical protocols for the extraction and amplification of nucleic acids.

  4. Decision-making about healthcare related tests and diagnostic strategies: a qualitative study with experts suggests that test accuracy data alone is rarely sufficient for decision-making.

    PubMed

    Mustafa, Reem A; Wiercioch, Wojtek; Ventresca, Matthew; Brozek, Jan; Schünemann, Holger J

    2017-09-13

    To identify the critical factors that determine recommendations and other decisions about health care related tests and diagnostic strategies (HCTDS). We used a qualitative descriptive approach and conducted semi-structured in-depth interviews with 24 international experts (informants) in evidence and decisions about HCTDS. Although test accuracy (TA) was the factor most commonly considered by organizations when developing recommendations about HCTDS, informants agreed that TA is necessary but rarely, if ever, sufficient and may be misleading when solely considered. The informants identified factors that are important for developing recommendations about HCTDS. Informants largely agreed that laying out the potential care pathways based on the test result is an essential early step but is rarely done in developing recommendations about HCTDS. Most informants also agreed that decision analysis could be useful for organizing the clinical, cost, and preference data relevant to the use of tests in the absence of direct evidence. However, they noted that utilizing models is limited by the lack of resources and expertise required. Developing guidelines about HCTDS requires consideration of factors beyond TA, but implementing this may be challenging. Further development and testing of "frameworks" that can guide this process is a priority for decision makers. Copyright © 2017. Published by Elsevier Inc.

  5. [Application of real-time PCR analysis of the SMN1gene in the carrier testing of spinal muscular atrophy].

    PubMed

    Lu, Li-Ping; Ma, Hong-Wei; Jiang, Jun; Wang, Tao; Hu, Bin

    2007-10-01

    Spinal muscular atrophy (SMA) is one of common autosomal recessive diseases and is characterized by degeneration of the anterior horn cells of the spinal cord. The reported prevalence is 1/10,000 live births with a carrier rate of one in 50. It is important in genetic counseling to identify the carriers with one copy deletion for the survival motor neuron (SMN(1)) gene. However, the duplication of the SMA locus makes the detection of SMA carriers difficult. This study aimed to determine the potential of the quantitative PCR analysis in the identification of SMA carriers. The SMN(1) gene copy number was detected by realdouble ended arrowtime PCR with TaqMan technology in 109 SMA parents of affected children and 40 normal controls. The average copy numbers of SMN(1) in the individuals with known one copy of the SMN(1) gene and with the two copies were 0.777 +/-0.035 (CV=4.5%) and 2.064 +/-0.120 (CV= 5.8%) respectively. The average copy number of SMN(1) in all of the parents with affected individuals was 0.798 +/-0.108 (CV=13.5%), and that of normal controls was 2.106 +/-0.18 (CV=8.5%). About 98% of SMA patients' parents carried 1 copy SMN(1), and 95% of normal controls carried 2 copies. The gene copy numbers for SMN(1) were one and two for SMA carriers and non-carriers, respectively. Our results suggested that the quantitative PCR analysis can distinguish the SMN(1) deletion carriers from non-carriers.

  6. How well does physician selection of microbiologic tests identify Clostridium difficile and other pathogens in paediatric diarrhoea? Insights using multiplex PCR-based detection.

    PubMed

    Stockmann, C; Rogatcheva, M; Harrel, B; Vaughn, M; Crisp, R; Poritz, M; Thatcher, S; Korgenski, E K; Barney, T; Daly, J; Pavia, A T

    2015-02-01

    The objective of this study was to compare the aetiologic yield of standard-of-care microbiologic testing ordered by physicians with that of a multiplex PCR platform. Stool specimens obtained from children and young adults with gastrointestinal illness were evaluated by standard laboratory methods and a developmental version of the FilmArray Gastrointestinal (GI) Diagnostic System (FilmArray GI Panel), a rapid multiplex PCR platform that detects 23 bacterial, viral and protozoal agents. Results were classified according to the microbiologic tests requested by the treating physician. A median of three (range 1-10) microbiologic tests were performed by the clinical laboratory during 378 unique diarrhoeal episodes. A potential aetiologic agent was identified in 46% of stool specimens by standard laboratory methods and in 65% of specimens tested using the FilmArray GI Panel (p < 0.001). For those patients who only had Clostridium difficile testing requested, an alternative pathogen was identified in 29% of cases with the FilmArray GI Panel. Notably, 11 (12%) cases of norovirus were identified among children who only had testing for Clostridium difficile ordered. Among those who had C. difficile testing ordered in combination with other tests, an additional pathogen was identified in 57% of stool specimens with the FilmArray GI Panel. For patients who had no C. difficile testing performed, the FilmArray GI Panel identified a pathogen in 63% of cases, including C. difficile in 8%. Physician-specified laboratory testing may miss important diarrhoeal pathogens. Additionally, standard laboratory testing is likely to underestimate co-infections with multiple infectious diarrhoeagenic agents.

  7. Test me and treat me”—attitudes to vitamin D deficiency and supplementation: a qualitative study

    PubMed Central

    Kotta, Siddharth; Gadhvi, Dev; Jakeways, Niki; Saeed, Maryum; Sohanpal, Ratna; Hull, Sally; Famakin, Olufunke; Martineau, Adrian; Griffiths, Chris

    2015-01-01

    Objective Lay interest in vitamin D and the potential benefits of supplementation is considerable, but little information exists concerning lay knowledge, beliefs and attitudes towards vitamin D to inform public health initiatives and professional guidance. Design Qualitative focus group study. Participants 58 adults capturing diversity in disease status, gender, age and ethnicity. Setting A large general practice in east London. Results Many respondents lacked knowledge about vitamin D, including dietary sources and government recommendations. Most were positive about sun exposure, but confused by ambiguous health messages about risks and benefits of sunshine. Medicalised views of vitamin D were prominent, notably from those in favour of supplementation, who talked of “doses”, “side effects” and “regular testing.” Fortification of food with vitamin D was controversial, with opposing utilitarian (better overall for the majority) and libertarian (freedom to choose) views. Conclusions Knowledge about vitamin D was limited. Clearer messages are needed about risks and benefits of sun exposure. Testing and supplementation by health professionals, while potentially useful in some high-risk groups, have contributed to a medicalised view of vitamin D. Health policy should address the public's need for clear information on sources and effects of vitamin D, including risks and benefits of sun exposure, and take account of divergent views on fortification. Professional guidance is needed on testing and supplementation to counter inappropriate medicalisation. PMID:26173717

  8. Impact of Birth HIV-PCR Testing on the Uptake at Follow-Up Early Infant Diagnosis Services in Cape Town, South Africa.

    PubMed

    Dunning, Lorna; Kroon, Max; Fourie, Lezanne; Ciaranello, Andrea; Myer, Landon

    2017-07-27

    PCR testing at birth ('birth-testing') is suggested by new World Health Organization guidelines for rapid diagnosis of infants infected with HIV in utero. However, there are few data on the implementation of this approach in sub-Saharan Africa and whether birth-testing affects uptake of subsequent routine early infant diagnosis (EID) testing at 6-10 weeks of age is unknown. We reviewed 575 consecutive infants undergoing targeted high-risk birth-testing in Cape Town, South Africa, and matched those testing HIV-negative at birth (n=551) to HIV-exposed infants who did not receive birth-testing (n=551). Maternal and infant clinical and demographic data, including EID testing uptake, were abstracted from routine records. Overall 3.8% of all birth-tests conducted were positive, while later EID testing positivity rates were 0.5% for those infants testing HIV-negative at birth and 0.4% for those without birth-testing. Infants who underwent birth-testing were less likely to present for later EID compared with those without a birth-test (73% vs 85%; p<0.001). This difference persisted after adjusting for maternal and infant characteristics (adjusted odds ratio,0.60 95% confidence interval, 0.41-0.86) and across demographic and clinical subgroups. Infants undergoing birth-testing also presented for later EID at a significantly older age (mean age 60 vs 50 days, p<0.001). While the yield of targeted high-risk birth testing in this setting appears high, neonates testing HIV-negative at birth may be less likely to present for subsequent EID testing. For birth-testing implementation to contribute to overall EID program goals, structured interventions are required to support follow-up EID services after negative birth-test results.

  9. Crafting Appealing Text Messages to Encourage Colorectal Cancer Screening Test Completion: A Qualitative Study

    PubMed Central

    Ellis, Shellie D; Denizard-Thompson, Nancy; Kronner, Donna; Miller, David P

    2015-01-01

    texting shorthand phrases and complicated replies); they did not want messages that contain bad news or test results. They wanted the ability to choose alternative options such as email or phone calls. Conclusions Older adults are receptive to receiving cancer screening text messages from health care providers. Sharing sample messages with patients may increase acceptance of this tool in the clinic setting. Supportive tailored text messaging reminders could enhance uptake of colorectal cancer screening by enhancing patient self-efficacy and providing cues to action to complete colonoscopy or fecal occult blood testing. PMID:26537553

  10. Evaluation of applied biosystems MicroSeq real-time PCR system for detection of Listeria monocytogenes in food. Performance Tested Method 011002.

    PubMed

    Tebbs, Robert S; Balachandran, Priya; Wong, Lily Y; Zoder, Patrick; Furtado, Manohar R; Petrauskene, Olga V; Cao, Yanxiang

    2011-01-01

    Increasingly, more food companies are relying on molecular methods, such as PCR, for pathogen detection due to their improved simplicity, sensitivity, and rapid time to results. This report describes the validation of a new Real-Time PCR method to detect Listeria monocytogenes in nine different food matrixes. The complete system consists of the MicroSEQ L. monocytogenes Detection Kit, sample preparation, the Applied Biosystems 7500 Fast Real-Time PCR instrument, and RapidFinder Express software. Two sample preparation methods were validated: the PrepSEQ Nucleic Acid extraction kit and the PrepSEQ Rapid Spin sample preparation kit. The test method was compared to the ISO 11290-1 reference method using an unpaired-study design to detect L. monocytogenes in roast beef, cured bacon, lox (smoked salmon), lettuce, whole cow's milk, dry infant formula, ice cream, salad dressing, and mayonnaise. The MicroSEQ L. monocytogenes Detection Kit and the ISO 11290-1 reference method showed equivalent detection based on Chi-square analysis for all food matrixes when the samples were prepared using either of the two sample preparation methods. An independent validation confirmed these findings on smoked salmon and whole cow's milk. The MicroSEQ kit detected all 50 L. monocytogenes strains tested, and none of the 30 nontargeted bacteria strains.

  11. easyPAC: A Tool for Fast Prediction, Testing and Reference Mapping of Degenerate PCR Primers from Alignments or Consensus Sequences

    PubMed Central

    Rosenkranz, David

    2012-01-01

    The PCR-amplification of unknown homologous or paralogous genes generally relies on PCR primers predicted from multi sequence alignments. But increasing sequence divergence can induce the need to use degenerate primers which entails the problem of testing the characteristics, unwanted interactions and potential mispriming of degenerate primers. Here I introduce easyPAC, a new software for the prediction of degenerate primers from multi sequence alignments or single consensus sequences. As a major innovation, easyPAC allows to apply all customary primer test procedures to degenerate primer sequences including fast mapping to reference files. Thus, easyPAC simplifies and expedites the designing of specific degenerate primers enormously. Degenerate primers suggested by easyPAC were used in PCR amplification with subsequent de novo sequencing of TDRD1 exon 11 homologs from several representatives of the haplorrhine primate phylogeny. The results demonstrate the efficient performance of the suggested primers and therefore show that easyPAC can advance upcoming comparative genetic studies.

  12. Sequence-modified primers for the differential RT-PCR detection of Andean potato latent and Andean potato mild mosaic viruses in quarantine tests.

    PubMed

    Koenig, Renate; Ziebell, Heiko

    2014-05-01

    To enable the differential PCR detection of Andean potato latent virus (APLV) and Andean potato mild mosaic virus (APMMV) strains, sense primers were designed that correspond to regions directly upstream of the coat protein genes. Their differentiating power was increased by A->C or T->C replacements in their 3'-terminal parts. Together with the broad-specificity antisense primer EM3, primer AL-a-mod3C detected all APLV strains tested, but none of the APMMV strains. Primer AM-a-mod4C yielded PCR products with all APMMV preparations, but also with some APLV preparations. Sequence analysis revealed that this was not due to a lack of primer specificity, but to the sensitive detection of contaminating APMMV in some of our APLV preparations.

  13. The identification of point mutations in Duchenne muscular dystrophy patients by using reverse-transcription PCR and the protein truncation test

    SciTech Connect

    Gardner, R.J.; Bobrow, M.; Roberts, R.G.

    1995-08-01

    The protein truncation test (PTT) is a mutation-detection method that monitors the integrity of the open reading frame (ORF). More than 60% of cases of Duchenne muscular dystrophy (DMD) result from gross frameshifting deletions in the dystrophin gene that are detectable by multiplex PCR system. It has become apparent that virtually all of the remaining DMD mutations also disrupt the translational reading frame, making the PTT a logical next step toward a comprehensive strategy for the identification of all DMD mutations. We report here a pilot study involving 22 patients and describe the mutations characterized. These constitute 12 point mutations or small insertions/deletions and 4 gross rearrangements. We also have a remaining five patients in whom there does not appear to be mutation in the ORF. We believe that reverse-transcription-PCR/PTT is an efficient method by which to screen for small mutations in DMD patients with no deletion. 29 refs., 2 figs., 3 tabs.

  14. Evaluation of PCR testing of ethanol-fixed nasal swab specimens as an augmented surveillance strategy for influenza virus and adenovirus identification.

    PubMed

    Krafft, A E; Russell, K L; Hawksworth, A W; McCall, S; Irvine, M; Daum, L T; Connoly, J L; Reid, A H; Gaydos, J C; Taubenberger, J K

    2005-04-01

    Viral culture isolation has been widely accepted as the "gold standard" for laboratory confirmation of viral infection; however, it requires ultralow temperature specimen storage. Storage of specimens in ethanol at room temperature could expand our ability to conduct active surveillance and retrospective screenings of viruses with rapid and inexpensive real-time PCR tests, including isolates from remote regions where freezing specimens for culture is not feasible. Molecular methods allow for rapid identification of viral pathogens without the need to maintain viability. We hypothesized that ethanol, while inactivating viruses, can preserve DNA and RNA for PCR-based methods. To evaluate the use of ethanol-stored specimens for augmenting surveillance for detection of influenza viruses A and B and adenoviruses (AdV), paired nasal swab specimens were collected from 384 recruits with febrile respiratory illness at Fort Jackson, S.C., in a 2-year study. One swab was stored at ambient temperature in 100% ethanol for up to 6 months, and the other swab was stored at -70 degrees C in viral medium. For viral detection, frozen specimens were cultured for a variety of respiratory viruses, and ethanol-fixed specimens were tested with TaqMan (TM) probe and LightCycler SYBR green (SG) melting curve assays with at least two different PCR targets for each virus. The sensitivities of the TM and SG assays on specimens stored in ethanol for 1 month were 75% and 58% for influenza A, 89% and 67% for influenza B, and 93 to 98% and 57% for AdV, respectively. Lower specificities of the real-time assays corresponded to the increased detection of PCR-positive but culture-negative specimens. Influenza virus RNA was detected as well or better after 6 months of storage in ethanol.

  15. First one-step nucleic acid amplification testing in papillary thyroid cancer lymph nodes - a comparison with histopathology and real-time PCR.

    PubMed

    Kaczka, Krzysztof; Fendler, Wojciech; Borowiec, Maciej; Młynarski, Wojciech; Pomorski, Lech

    2014-01-01

    The significance of lymph node metastases and the optimal extent of lymphadenectomy remain matters of controversy in papillary thyroid cancer. This study was designed to assess the feasibility and reliability of OSNA and real-time PCR for CK19 and TG mRNA in papillary thyroid cancer lymph nodes evaluation compared to standard histopathology. Each of 92 randomised lymph nodes from 32 papillary thyroid cancer patients were divided into representative parts and assessed using the three studied methods. Eighteen (19.6%) lymph nodes from ten (31.3%) patients were positive according to histopathology. When the cut-off value distinguishing metastatic from non-metastatic lymph nodes in the OSNA assay was set at 250 copies per microlitre, the results were positive in 16 (17.4%) lymph nodes from 11 (34.4%) patients. Twenty three (25%) lymph nodes were tested positive in real-time PCR for TG mRNA. Real-time PCR for CK19 mRNA was positive in 18 (19.6%) lymph nodes from 13 (40.6%) patients. No statistically significant differences were noted between the diagnostic accuracy of either molecular method compared to the histopathological examination (p = 0.81). Overall, 20 positive molecular biology results were noted in patients with negative histopathology results. Conversely, in 18 lymph nodes, despite a metastasis finding in histopathology, at least one molecular test yielded a negative result. It was revealed that OSNA is a reliable technique for the evaluation of lymph node metastases in papillary thyroid cancer. This method was shown to have equivalent accuracy to histopathology and real-time PCR.

  16. HIV testing experiences and their implications for patient engagement with HIV care and treatment on the eve of 'test and treat': findings from a multicountry qualitative study.

    PubMed

    Wringe, Alison; Moshabela, Mosa; Nyamukapa, Constance; Bukenya, Dominic; Ondenge, Ken; Ddaaki, William; Wamoyi, Joyce; Seeley, Janet; Church, Kathryn; Zaba, Basia; Hosegood, Victoria; Bonnington, Oliver; Skovdal, Morten; Renju, Jenny

    2017-07-01

    In view of expanding 'test and treat' initiatives, we sought to elicit how the experience of HIV testing influenced subsequent engagement in HIV care among people diagnosed with HIV. As part of a multisite qualitative study, we conducted in-depth interviews in Uganda, South Africa, Tanzania, Kenya, Malawi and Zimbabwe with 5-10 health workers and 28-59 people living with HIV, per country. Topic guides covered patient and provider experiences of HIV testing and treatment services. Themes were derived through deductive and inductive coding. Various practices and techniques were employed by health workers to increase HIV testing uptake in line with national policies, some of which affected patients' subsequent engagement with HIV services. Provider-initiated testing was generally appreciated, but rarely considered voluntary, with instances of coercion and testing without consent, which could lead to disengagement from care.Conflicting rationalities for HIV testing between health workers and their clients caused tensions that undermined engagement in HIV care among people living with HIV. Although many health workers helped clients to accept their diagnosis and engage in care, some delivered static, morally charged messages regarding sexual behaviours and expectations of clinic use which discouraged future care seeking. Repeat testing was commonly reported, reflecting patients' doubts over the accuracy of prior results and beliefs that antiretroviral therapy may cure HIV. Repeat testing provided an opportunity to develop familiarity with clinical procedures, address concerns about HIV services and build trust with health workers. The principles of consent and confidentiality that should underlie HIV testing and counselling practices may be modified or omitted by health workers to achieve perceived public health benefits and policy expectations. While such actions can increase HIV testing rates, they may also jeopardise efforts to connect people diagnosed with HIV to

  17. Why do marital partners of people living with HIV not test for HIV? A qualitative study in Lusaka, Zambia.

    PubMed

    Musheke, Maurice; Merten, Sonja; Bond, Virginia

    2016-08-25

    Knowledge of HIV status is crucial for HIV prevention and management in marital relationships. Yet some marital partners of people living with HIV decline HIV testing despite knowing the HIV-positive status of their partners. To date, little research has explored the reasons for this. An exploratory qualitative study was undertaken in Lusaka, Zambia, between March 2010 and September 2011, nested within a larger ethnographic study. In-depth interviews were held with individuals who knew the HIV-positive status of their marital partners but never sought HIV testing (n = 30) and HIV service providers of a public sector clinic (n = 10). A focus group discussion was also conducted with eight (8) lay HIV counsellors. Data was transcribed, coded and managed using ATLAS.ti and analysed using latent content analysis. The overarching barrier to uptake of HIV testing was study participants' perception of their physical health, reinforced by uptake of herbal remedies and conventional non-HIV medication to mitigate perceived HIV-related symptoms. They indicated willingness to test for HIV if they noticed a decline in physical health and other alternative forms of care became ineffective. Also, some study participants viewed themselves as already infected with HIV on account of the HIV-positive status of their marital partners, with some opting for faith healing to get 'cured'. Other barriers were the perceived psychological burden of living with HIV, modulated by lay belief that knowledge of HIV-positive status led to rapid physical deterioration of health. Perceived inability to sustain uptake of life-long treatment - influenced by a negative attitude towards treatment - further undermined uptake of HIV testing. Self-stigma, which manifested itself through fear of blame and a need to maintain moral credibility in marital relationships, also undermined uptake of HIV testing. Improving uptake of HIV testing requires a multi-pronged approach that addresses self-stigma, lay risk

  18. The education and practice program for medical students with quantitative and qualitative fit test for respiratory protective equipment

    PubMed Central

    MYONG, Jun-Pyo; BYUN, JunSu; CHO, YounMo; SEO, Hye-Kyung; BAEK, Jung-Eun; KOO, Jung-Wan; KIM, Hyunwook

    2015-01-01

    Tuberculosis infection is prevalent in Korea and health care workers are vulnerable to tuberculosis infection in the hospital. The aims of this study were to develop and validate an education program that teaches senior medical students how to wear and choose the proper size and type of respiratory protective equipment (RPE), which may help reduce the risk of contracting Mycobacterium tuberculosis (MTB) from patients. Overall, 50 senior medical students participated in this education program. Methods of choosing the proper type of RPE, performing a fit check of the RPE, and choosing a suitable mask size were taught by certified instructors using the real-time quantitative fit test (QNFT). The validity of education program was evaluated with qualitative fit test (QLFT) before and after the education as pass or fail. The education program was effective, as shown by the significantly pass rate (increased 30 to 74%) in the QLFT after the education program (p<0.05). Among study participants, changing mask size from medium to small significantly increased the pass rate (p<0.001). Incorporation of this program into the medical school curriculum may help reduce risk of MTB infection in medical students working in the hospital. PMID:26538001

  19. Qualitative and Quantitative Detection of Botulinum Neurotoxins from Complex Matrices: Results of the First International Proficiency Test.

    PubMed

    Worbs, Sylvia; Fiebig, Uwe; Zeleny, Reinhard; Schimmel, Heinz; Rummel, Andreas; Luginbühl, Werner; Dorner, Brigitte G

    2015-11-26

    In the framework of the EU project EQuATox, a first international proficiency test (PT) on the detection and quantification of botulinum neurotoxins (BoNT) was conducted. Sample materials included BoNT serotypes A, B and E spiked into buffer, milk, meat extract and serum. Different methods were applied by the participants combining different principles of detection, identification and quantification. Based on qualitative assays, 95% of all results reported were correct. Successful strategies for BoNT detection were based on a combination of complementary immunological, MS-based and functional methods or on suitable functional in vivo/in vitro approaches (mouse bioassay, hemidiaphragm assay and Endopep-MS assay). Quantification of BoNT/A, BoNT/B and BoNT/E was performed by 48% of participating laboratories. It turned out that precise quantification of BoNT was difficult, resulting in a substantial scatter of quantitative data. This was especially true for results obtained by the mouse bioassay which is currently considered as "gold standard" for BoNT detection. The results clearly demonstrate the urgent need for certified BoNT reference materials and the development of methods replacing animal testing. In this context, the BoNT PT provided the valuable information that both the Endopep-MS assay and the hemidiaphragm assay delivered quantitative results superior to the mouse bioassay.

  20. Culture qualitatively but not quantitatively influences performance in the Boston naming test in a chinese-speaking population.

    PubMed

    Chen, Ting-Bin; Lin, Chi-Ying; Lin, Ker-Neng; Yeh, Yen-Chi; Chen, Wei-Ta; Wang, Kuo-Shu; Wang, Pei-Ning

    2014-01-01

    The Boston Naming Test (BNT) is the most frequently administered confrontational naming test, but the cultural background of the patients may influence their performance in the BNT. The aim of this study was to identify differences in performance in the BNT between a Chinese population in Taiwan, Chinese populations in other areas and a Caucasian population. A total of 264 native, Chinese-speaking, cognitively normal elders aged >60 years were enrolled in our study and conducted the 30-item Chinese version of the BNT. Another 10 BNT studies were categorized, analyzed and compared with the present study. Higher education was associated with higher scores, whereas age and gender had no effect on performance in the BNT. The score of the Chinese-speaking population was equivalent to the English-speaking population. A disparity in difficulties with items was not only apparent between the Taiwanese and Caucasian populations, but also between the Chinese-speaking populations in the different geographic areas. For the most part, the impact of culture on performance in the BNT may not be quantitative but qualitative. Attention should be paid to a potential effect of culture on difficulties with items when administering the BNT to non-English-speaking populations. Understanding differences in performance in the BNT in distinct cultural settings improves the clinical application of the BNT.

  1. Culture Qualitatively but Not Quantitatively Influences Performance in the Boston Naming Test in a Chinese-Speaking Population

    PubMed Central

    Chen, Ting-Bin; Lin, Chi-Ying; Lin, Ker-Neng; Yeh, Yen-Chi; Chen, Wei-Ta; Wang, Kuo-Shu; Wang, Pei-Ning

    2014-01-01

    Background/Aims The Boston Naming Test (BNT) is the most frequently administered confrontational naming test, but the cultural background of the patients may influence their performance in the BNT. The aim of this study was to identify differences in performance in the BNT between a Chinese population in Taiwan, Chinese populations in other areas and a Caucasian population. Methods A total of 264 native, Chinese-speaking, cognitively normal elders aged >60 years were enrolled in our study and conducted the 30-item Chinese version of the BNT. Another 10 BNT studies were categorized, analyzed and compared with the present study. Results Higher education was associated with higher scores, whereas age and gender had no effect on performance in the BNT. The score of the Chinese-speaking population was equivalent to the English-speaking population. A disparity in difficulties with items was not only apparent between the Taiwanese and Caucasian populations, but also between the Chinese-speaking populations in the different geographic areas. Conclusion For the most part, the impact of culture on performance in the BNT may not be quantitative but qualitative. Attention should be paid to a potential effect of culture on difficulties with items when administering the BNT to non-English-speaking populations. Understanding differences in performance in the BNT in distinct cultural settings improves the clinical application of the BNT. PMID:24847347

  2. How to Make Feedback More Effective? Qualitative Findings from Pilot Testing of an Audit and Feedback Report for Endoscopists

    PubMed Central

    Webster, Fiona; Patel, Jigisha; Rice, Kathleen; Baxter, Nancy; Paszat, Lawrence; Rabeneck, Linda

    2016-01-01

    Background. Audit and feedback (A/F) reports are one of the few knowledge translation activities that can effect change in physician behavior. In this study, we pilot-tested an endoscopist A/F report to elicit opinions about the proposed report's usability, acceptability and usefulness, and implications for knowledge translation. Methods. Semi-structured qualitative interviews were conducted with eleven endoscopists in Ontario, Canada. We tested an A/F report template comprising 9 validated, accepted colonoscopy quality indicators populated with simulated data. Interview transcripts were coded using techniques such as constant comparison and themes were identified inductively over several team meetings. Results. Four interrelated themes were identified: (1) overall perceptions of the A/F report; (2) accountability and consequences for poor performance; (3) motivation to change/improve skills; and (4) training for performance enhancement and available resources. The A/F report was well received; however, participants cited some possible threats to the report's effectiveness including the perceived threat of loss of privileges or licensing and the potential for the data to be dismissed. Conclusions. Participants agreed that A/F has the potential to improve colonoscopy performance. However, in order to be effective in changing physician behavior, A/F must be thoughtfully implemented with attention to the potential concerns of its recipients. PMID:27722149

  3. Attracting female sex workers to HIV testing and counselling in Ethiopia: a qualitative study with sex workers in Addis Ababa.

    PubMed

    Ameyan, Wole; Jeffery, Caroline; Negash, Kassahun; Biruk, Etsegenet; Taegtmeyer, Miriam

    2015-01-01

    Despite growing efforts to increase HIV testing and counselling (HTC) services for most at risk populations in Ethiopia, the use of these services by female sex workers (FSWs) remains low. With rising numbers of FSWs in Addis Ketema and concerns about their high risk behaviours, exploring and addressing the barriers to uptake is crucial. This qualitative study explores the barriers to utilising HTC facilities and identifies the motives and motivations of FSWs who seek HTC through in-depth and semi-structured interviews with female sex workers, healthcare workers and key informants. Results indicate that FSWs face numerous barriers including inability to seek treatment if found to be positive due to the requirement of an identity (ID) card many do not own. Many FSWs reported discriminatory behaviour from healthcare workers and a lack of dedicated services. What is clear from the findings is that distinct strategies, which differ from those of the broader population, are required to attract FSWs--strategies which take into account the barriers and maximise the reported motives and motivations for testing.

  4. Qualitative and Quantitative Detection of Botulinum Neurotoxins from Complex Matrices: Results of the First International Proficiency Test

    PubMed Central

    Worbs, Sylvia; Fiebig, Uwe; Zeleny, Reinhard; Schimmel, Heinz; Rummel, Andreas; Luginbühl, Werner; Dorner, Brigitte G.

    2015-01-01

    In the framework of the EU project EQuATox, a first international proficiency test (PT) on the detection and quantification of botulinum neurotoxins (BoNT) was conducted. Sample materials included BoNT serotypes A, B and E spiked into buffer, milk, meat extract and serum. Different methods were applied by the participants combining different principles of detection, identification and quantification. Based on qualitative assays, 95% of all results reported were correct. Successful strategies for BoNT detection were based on a combination of complementary immunological, MS-based and functional methods or on suitable functional in vivo/in vitro approaches (mouse bioassay, hemidiaphragm assay and Endopep-MS assay). Quantification of BoNT/A, BoNT/B and BoNT/E was performed by 48% of participating laboratories. It turned out that precise quantification of BoNT was difficult, resulting in a substantial scatter of quantitative data. This was especially true for results obtained by the mouse bioassay which is currently considered as “gold standard” for BoNT detection. The results clearly demonstrate the urgent need for certified BoNT reference materials and the development of methods replacing animal testing. In this context, the BoNT PT provided the valuable information that both the Endopep-MS assay and the hemidiaphragm assay delivered quantitative results superior to the mouse bioassay. PMID:26703724

  5. Salmonella contamination in pigs at slaughter and on the farm: a field study using an antibody ELISA test and a PCR technique.

    PubMed

    Nowak, Bernhard; von Müffling, Theda; Chaunchom, Sujate; Hartung, Jörg

    2007-04-20

    An antibody ELISA test and a PCR method for identifying the risk of Salmonella contamination were compared in a field study on the same lots of animals in a slaughterhouse. The results were compared to investigations carried out on two farms with different prevalences of Salmonella antibody-positive animals. Salmonella antibody ELISA testing was carried out on all 383 meat juice samples derived from the diaphragm pillar muscle of each pig. Salmonella DNA analysis was performed by PCR technique on small intestine samples with lymph nodes from all 383 pigs, and on tonsils from the last 129 pigs. The 383 animals tested came from 32 different pig farms. Furthermore, the herd antibody blood serum status against Salmonella spp. of weaners was determined on two selected pig fattening farms, one with low and one with high seroprevalence in meat juice. A total of 7.0% (ELISA cut-off OD% > or =40) of the slaughtered pigs from 6 of 32 fattening farms were seropositive. Salmonella DNA was found in 16.4% of the jejunum/lymph nodes (383 animals) and in 15.5% of the tonsils (129 animals). Salmonella DNA was found in the jejunum/lymph nodes of 41% of the seropositive pigs. However, serotitres were also positive in only 17.5% of all pigs positive in the jejunum DNA test. Two farms were selected for further investigation: farm 13 (F13), with a high prevalence of seropositive pigs, 29.0%, Category II; and F11, with 9.4%, Category I. However, categorization according to the blood serum tests of the fattening pigs after on-farm testing was very different: F13 had 5% positive animals (Category I); and F11, 23.3% (Category II). The study led to the following results and recommendations: First, ELISA tests are useful for the detection of farms that are regularly contaminated with Salmonella, but such tests cannot give information on the infectious status of a single animal (or a group) at the point of slaughter. Second, it is crucial that management measures are taken to prevent the

  6. Modification of the BAX System PCR assay for detecting Salmonella in beef, produce, and soy protein isolate. Performance Tested Method 100201.

    PubMed

    Peng, Linda X; Wallace, Morgan; Andaloro, Bridget; Fallon, Dawn; Fleck, Lois; Delduco, Dan; Tice, George

    2011-01-01

    The BAX System PCR assay for Salmonella detection in foods was previously validated as AOAC Research Institute (RI) Performance Tested Method (PTM) 100201. New studies were conducted on beef and produce using the same media and protocol currently approved for the BAX System PCR assay for E. coli O157:H7 multiplex (MP). Additionally, soy protein isolate was tested for matrix extension using the U.S. Food and Drug Administration-Bacteriological Analytical Manual (FDA-BAM) enrichment protocols. The studies compared the BAX System method to the U.S. Department of Agriculture culture method for detecting Salmonella in beef and the FDA-BAM culture method for detecting Salmonella in produce and soy protein isolate. Method comparison studies on low-level inoculates showed that the BAX System assay for Salmonella performed as well as or better than the reference method for detecting Salmonella in beef and produce in 8-24 h enrichment when the BAX System E. coli O157:H7 MP media was used, and soy protein isolate in 20 h enrichment with lactose broth followed by 3 h regrowth in brain heart infusion broth. An inclusivity panel of 104 Salmonella strains with diverse serotypes was tested by the BAX System using the proprietary BAX System media and returned all positive results. Ruggedness factors involved in the enrichment phase were also evaluated by testing outside the specified parameters, and none of the factors examined affected the performance of the assay.

  7. Variation in MRSA identification results from different generations of Xpert MRSA real-time PCR testing kits from nasal swabs.

    PubMed

    Rabaan, Ali A; Bazzi, Ali M

    2017-02-06

    GeneXpert MRSA kits (Cepheid) are based on a multiplex, real-time PCR method for methicillin-resistant Staphylococcus aureus (MRSA) detection, with primers to detect each SCCmec type and the chromosomal orfX-SCCmec junction. Modifications in recent kit versions were proposed to help overcome false-positive issues in earlier kit versions. The main objective of this study was to determine whether use of any version of the GeneXpert MRSA multiplex, real-time PCR kits yielded higher than expected MRSA+ results. We also estimated the level of MRSA in our healthcare facility as a proportion of total S. aureus between 2010 and 2015. We examined results from five generations of the kits used between 2008 and 2015. Results were from nasal swab samples from 16,431 patients in the Johns Hopkins Aramco Healthcare facility in Saudi Arabia. The percentage of isolates scored as MRSA+ for the original Xpert MRSA kit was 18.57%, compared to 6.93±1.12% (mean±SD) for the other four kits. The Xpert MRSA-SA Nasal kit yielded 6.48% Invalid results, compared to 0.73±0.28% for the other four kits. The succeeding Xpert MRSA-SA Nasal G3 and Xpert MRSA-SA Nasal Complete G3 kits yielded Invalid results rates of 0.29% and 1.04% respectively. Levels of MRSA-positive isolates as a percentage of total S. aureus-containing samples ranged between 19.81% and 26.74%. In conclusion, the original Xpert MRSA kit yielded higher than expected rates of MRSA+. Issues with over-estimation of MRSA+ and/or numerous Invalid results have been overcome in the most recent modified kits.

  8. Development and evaluation of a test for tuberculosis in live European badgers (Meles meles) based on measurement of gamma interferon mRNA by real-time PCR.

    PubMed

    Sawyer, J; Mealing, D; Dalley, D; Davé, D; Lesellier, S; Palmer, S; Bowen-Davies, J; Crawshaw, T R; Chambers, M A

    2007-08-01

    A real-time PCR assay for the measurement of gamma interferon (IFN-gamma) mRNA in European badger (Meles meles) blood cultures was developed. The levels of IFN-gamma mRNA in blood cultures stimulated with either bovine or avian tuberculin or specific mycobacterial antigens were compared with those in a nonstimulated control blood culture as the basis for determining the tuberculosis (TB) status of live badgers. The assay was validated by testing 247 animals for which there were matching data from postmortem examination and culture of tissues. Relative changes in the levels of IFN-gamma mRNA in response to bovine tuberculin and specific antigens were found to be greater among badgers with tissues positive for TB on culture. The test was at its most accurate (87% of test results were correct) by using blood cultures containing bovine tuberculin as the antigen and when the response to avian tuberculin was taken into account by subtracting the avian tuberculin response from the bovine tuberculin response. At a specificity of 90.7%, the test was 70.6% sensitive. At the same specificity, the current serological enzyme-linked immunosorbent assay for TB in badgers was only 53% sensitive. This work demonstrates that measurement of IFN-gamma mRNA by real-time PCR is a valid method for the detection of TB in live badgers and may provide an alternative to the current serological methods of diagnosis, the Brock test. The testing procedure can be completed within 5 h of receipt of the blood culture samples. In addition, the use of a molecular biology-based test offers the potential to fully automate the testing procedure through the use of robotics.

  9. Development of a Multiplex PCR Test with Automated Genotyping Targeting E7 for Detection of Six High-Risk Human Papillomaviruses.

    PubMed

    Paes, Eliana Ferreira; de Assis, Angela Maria; Teixeira, Cirbia S Campos; Aoki, Francisco Hideo; Teixeira, Julio Cesar

    2015-01-01

    Cervical cancer is caused by high-risk human papillomaviruses (HPV) and viral detection tests aid in the diagnosis of precursor lesions. In the present study, a molecular test for detection of high-risk HPV DNA, called E7-HPV, was standardized and assessed in samples from women with pre-cancerous lesions. The development of the E7-HPV test for detection and genotyping of six high-risk HPV (types 16, 18, 31, 33, 45 and 52), consisted of evaluating primer quality and adjusting the multiplex PCR conditions. Primer design was based on the E7 region of each HPV, and the fluorochrome 6-FAM was added to PCR primers. Viral detection was performed by capillary electrophoresis in automated sequencer in samples obtained from 60 women (55 with ASC-H/HSIL cytology) from August to September 2013. A non-inferiority analysis was conducted with the cobas HPV test as a reference and following international guidelines for the development of new tests. The two tests had a high concordance rate in HPV16 detection (kappa=0.972), with only one discordant case (cervical intraepithelial neoplasia grade 3, negative with cobas and positive for HPV16 by E7-HPV) and complete agreement in HPV18 detection. When comparing detection of all high-risk HPV, three cases were positive with cobas but negative with E7-HPV, and another three cases were negative with cobas but positive with E7-HPV (HPV16, 31 and 52). When we evaluate the cases initially suspected by cytology, the two tests had the same sensitivity in detection CIN2 or worse. In conclusion, the E7-HPV test has satisfactory initial results, and its development can be continued.

  10. A qualitative analysis of the barriers and facilitators of HIV counselling and testing perceived by adolescents in South Africa.

    PubMed

    Strauss, Michael; Rhodes, Bruce; George, Gavin

    2015-06-30

    Youth in South Africa have been identified as a high-risk group for contracting HIV. In response, the South African Integrated School Health Policy (ISHP) has been developed with the aim of guiding the provision of comprehensive healthcare services within South African schools. Accordingly, the scale-up of HIV counselling and testing (HCT) in high schools is a priority. This study examines the factors affecting the utilisation of HCT services amongst learners in high schools in the KwaZulu-Natal province of South Africa. Focus group discussions were conducted in 12 rural schools in the Vulindlela sub-district of uMgungundlovu in KwaZulu-Natal. A total of 158 randomly selected learners took part, aged 16 years and older from grades 10, 11 and 12. Qualitative analysis was conducted using the framework approach, providing a systematic structure allowing for a priori and emergent codes, with social cognitive theory as a theoretical framework. The stigma and discrimination attached to testing, along with the inherent fear of a positive result were the biggest barriers to HCT uptake. Fear and the subsequent negative beliefs around HCT were borne out of insufficient knowledge. These fears were exacerbated by the perceived or real attitudes of peers, partners and family towards HIV. The prospect of a positive result and the possible resultant societal backlash hinders high and regular uptake of HCT. Stigma and discrimination remain the foremost barriers to HIV testing despite the presence of localised and convenient testing. Interventions aimed at addressing these challenges could increase the demand for HIV testing amongst adolescents. Increasing education about the importance of HCT and creating awareness about available HCT services will not be enough to increase uptake in schools in South Africa. Efforts to decrease stigma around HIV and HCT by integrating testing into general and sexual reproductive health services offered to youth, and normalising the epidemic within

  11. Rapid visual identification of PCR amplified nucleic acids by centrifugal gel separation: Potential use for molecular point-of-care tests.

    PubMed

    Hwang, Sang-Hyun; Kim, Dong-Eun; Im, Ji-Hyun; Kang, Su-Jin; Lee, Do-Hoon; Son, Sang Jun

    2016-05-15

    Recently, nucleic acid amplification and detection techniques have progressed based on advances in in microfluidics, microelectronics, and optical systems. Nucleic acids amplification based point-of-care test (POCT) in resource-limited settings requires simple visual detection methods. Several biosensing methods including lateral flow immunoassays (LFIA) were previously used to visually detect nucleic acids. However, prolonged assay time, several washing steps, and a need for specific antibodies limited their use. Here we developed a novel, rapid method to visualize amplified nucleic acids with naked eyes in clinical samples. First, we optimized conditions based on separation using very low centrifugal force and a density medium to detect human papillomavirus (HPV)-16 DNA in cervical specimens. After DNA extraction, HPV16 PCR was performed with biotin-labeled forward primer and Cy3-labeled reverse primer. PCR amplicon was mixed with streptavidin-magnetic beads, introduced into the density medium. After two-minute centrifugation, the result was visually identified. This system showed identical results with commercial HPV real-time PCR for 30 clinical samples and could detect up to 10(2)copies/mL of HPV DNA without any optical instruments. This robust and sensitive visual detection system is suitable for non-specialist personnel and point-of-care diagnosis in low-resource settings. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. A qualitative study into the difficulties experienced by healthcare decision makers when reading a Cochrane diagnostic test accuracy review

    PubMed Central

    2013-01-01

    Background Cochrane reviews are one of the best known and most trusted sources of evidence-based information in health care. While steps have been taken to make Cochrane intervention reviews accessible to a diverse readership, little is known about the accessibility of the newcomer to the Cochrane library: diagnostic test accuracy reviews (DTARs). The current qualitative study explored how healthcare decision makers, who varied in their knowledge and experience with test accuracy research and systematic reviews, read and made sense of DTARs. Methods A purposive sample of clinicians, researchers and policy makers (n = 21) took part in a series of think-aloud interviews, using as interview material the first three DTARs published in the Cochrane library. Thematic qualitative analysis of the transcripts was carried out to identify patterns in participants’ ‘reading’ and interpretation of the reviews and the difficulties they encountered. Results Participants unfamiliar with the design and methodology of DTARs found the reviews largely inaccessible and experienced a range of difficulties stemming mainly from the mismatch between background knowledge and level of explanation provided in the text. Experience with systematic reviews of interventions did not guarantee better understanding and, in some cases, led to confusion and misinterpretation. These difficulties were further exacerbated by poor layout and presentation, which affected even those with relatively good knowledge of DTARs and had a negative impact not only on their understanding of the reviews but also on their motivation to engage with the text. Comparison between the readings of the three reviews showed that more accessible presentation, such as presenting the results as natural frequencies, significantly increased participants’ understanding. Conclusions The study demonstrates that authors and editors should pay more attention to the presentation as well as the content of Cochrane DTARs

  13. Parasitological diagnosis combining an internally controlled real-time PCR assay for the detection of four protozoa in stool samples with a testing algorithm for microscopy.

    PubMed

    Bruijnesteijn van Coppenraet, L E S; Wallinga, J A; Ruijs, G J H M; Bruins, M J; Verweij, J J

    2009-09-01

    Molecular detection of gastrointestinal protozoa is more sensitive and more specific than microscopy but, to date, has not routinely replaced time-consuming microscopic analysis. Two internally controlled real-time PCR assays for the combined detection of Entamoeba histolytica, Giardia lamblia, Cryptosporidium spp. and Dientamoeba fragilis in single faecal samples were compared with Triple Faeces Test (TFT) microscopy results from 397 patient samples. Additionally, an algorithm for complete parasitological diagnosis was created. Real-time PCR revealed 152 (38.3%) positive cases, 18 of which were double infections: one (0.3%) sample was positive for E. histolytica, 44 (11.1%) samples were positive for G. lamblia, 122 (30.7%) samples were positive for D. fragilis, and three (0.8%) samples were positive for Cryptosporidium. TFT microscopy yielded 96 (24.2%) positive cases, including five double infections: one sample was positive for E. histolytica/Entamoeba dispar, 29 (7.3%) samples were positive for G. lamblia, 69 (17.4%) samples were positive for D. fragilis, and two (0.5%) samples were positive for Cryptosporidium hominis/Cryptosporidium parvum. Retrospective analysis of the clinical patient information of 2887 TFT sets showed that eosinophilia, elevated IgE levels, adoption and travelling to (sub)tropical areas are predisposing factors for infection with non-protozoal gastrointestinal parasites. The proposed diagnostic algorithm includes application of real-time PCR to all samples, with the addition of microscopy on an unpreserved faecal sample in cases of a predisposing factor, or a repeat request for parasitological examination. Application of real-time PCR improved the diagnostic yield by 18%. A single stool sample is sufficient for complete parasitological diagnosis when an algorithm based on clinical information is applied.

  14. Design and Performance Testing of a DNA Extraction Assay for Sensitive and Reliable Quantification of Acetic Acid Bacteria Directly in Red Wine Using Real Time PCR

    PubMed Central

    Longin, Cédric; Guilloux-Benatier, Michèle; Alexandre, Hervé

    2016-01-01

    Although strategies exist to prevent AAB contamination, the increased interest for wines with low sulfite addition leads to greater AAB spoilage. Hence, there is a real need for a rapid, specific, sensitive, and reliable method for detecting these spoilage bacteria. All these requirements are met by real time Polymerase Chain Reaction (or quantitative PCR; qPCR). Here, we compare existing methods of isolating DNA and their adaptation to a red wine matrix. Two different protocols for isolating DNA and three PCR mix compositions were tested to select the best method. The addition of insoluble polyvinylpolypyrrolidone (PVPP) at 1% (v/v) during DNA extraction using a protocol succeeded in eliminating PCR inhibitors from red wine. We developed a bacterial internal control which was efficient in avoiding false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The specificity, linearity, repeatability, and reproducibility of the method were evaluated. A standard curve was established for the enumeration of AAB inoculated into red wines. The limit of quantification in red wine was 3.7 log AAB/mL and about 2.8 log AAB/mL when the volume of the samples was increased from 1 to 10 mL. Thus, the DNA extraction method developed in this paper allows sensitive and reliable AAB quantification without underestimation thanks to the presence of an internal control. Moreover, monitoring of both the AAB population and the amount of acetic acid in ethanol medium and red wine highlighted that a minimum about 6.0 log cells/mL of AAB is needed to significantly increase the production of acetic acid leading to spoilage. PMID:27313572

  15. Design and Performance Testing of a DNA Extraction Assay for Sensitive and Reliable Quantification of Acetic Acid Bacteria Directly in Red Wine Using Real Time PCR.

    PubMed

    Longin, Cédric; Guilloux-Benatier, Michèle; Alexandre, Hervé

    2016-01-01

    Although strategies exist to prevent AAB contamination, the increased interest for wines with low sulfite addition leads to greater AAB spoilage. Hence, there is a real need for a rapid, specific, sensitive, and reliable method for detecting these spoilage bacteria. All these requirements are met by real time Polymerase Chain Reaction (or quantitative PCR; qPCR). Here, we compare existing methods of isolating DNA and their adaptation to a red wine matrix. Two different protocols for isolating DNA and three PCR mix compositions were tested to select the best method. The addition of insoluble polyvinylpolypyrrolidone (PVPP) at 1% (v/v) during DNA extraction using a protocol succeeded in eliminating PCR inhibitors from red wine. We developed a bacterial internal control which was efficient in avoiding false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The specificity, linearity, repeatability, and reproducibility of the method were evaluated. A standard curve was established for the enumeration of AAB inoculated into red wines. The limit of quantification in red wine was 3.7 log AAB/mL and about 2.8 log AAB/mL when the volume of the samples was increased from 1 to 10 mL. Thus, the DNA extraction method developed in this paper allows sensitive and reliable AAB quantification without underestimation thanks to the presence of an internal control. Moreover, monitoring of both the AAB population and the amount of acetic acid in ethanol medium and red wine highlighted that a minimum about 6.0 log cells/mL of AAB is needed to significantly increase the production of acetic acid leading to spoilage.

  16. Places and people: the perceptions of men who have sex with men concerning STI testing: a qualitative study.

    PubMed

    Datta, Jessica; Reid, David; Hughes, Gwenda; Mercer, Catherine H; Wayal, Sonali; Weatherburn, Peter

    2017-08-04

    To explore the experiences and views of men who have sex with men (MSM) on attending clinical sexual health services and their preferences regarding service characteristics in the context of the disproportionate burden of STIs experienced by this group. The wider study aim was to develop a risk assessment tool for use in sexual health clinics. Qualitative study comprising eight focus group discussions with 61 MSM in four English cities. Topics included: experience of attending sexual health services, perceptions of norms of attendance among MSM, knowledge of, and attitudes towards, STIs and views on 'being researched.' Discussions were audio-recorded and transcribed and a thematic data analysis conducted. Attending sexual health services for STI testing was described as embarrassing by some and some clinic procedures were thought to compromise confidentiality. Young men seeking STI testing were particularly sensitive to feelings of awkwardness and self-consciousness. Black and ethnic minority men were concerned about being exposed in their communities. The personal qualities of staff were seen as key features of sexual health services. Participants wanted staff to be friendly, professional, discreet, knowledgeable and non-judgemental. A range of opinion on the type of STI service men preferred was expressed with some favouring generic sexual and reproductive health clinics and others favouring specialist community-based services. There was consensus on the qualities they would like to see in healthcare staff. The knowledge, conduct and demeanour of staff could exacerbate or ameliorate unease associated with attending for STI testing. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  17. Plasma epidermal growth factor receptor mutation testing with a chip-based digital PCR system in patients with advanced non-small cell lung cancer.

    PubMed

    Kasahara, Norimitsu; Kenmotsu, Hirotsugu; Serizawa, Masakuni; Umehara, Rina; Ono, Akira; Hisamatsu, Yasushi; Wakuda, Kazushige; Omori, Shota; Nakashima, Kazuhisa; Taira, Tetsuhiko; Naito, Tateaki; Murakami, Haruyasu; Koh, Yasuhiro; Mori, Keita; Endo, Masahiro; Nakajima, Takashi; Yamada, Masanobu; Kusuhara, Masatoshi; Takahashi, Toshiaki

    2017-04-01

    Epidermal growth factor receptor (EGFR) mutation testing is a companion diagnostic to determine eligibility for treatment with EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small cell lung cancer (NSCLC). Recently, plasma-based EGFR testing by digital polymerase chain reaction (dPCR), which enables accurate quantification of target DNA, has shown promise as a minimally invasive diagnostic. Here, we aimed to evaluate the accuracy of a plasma-based EGFR mutation test developed using chip-based dPCR-based detection of 3 EGFR mutations (exon 19 deletions, L858R in exon 21, and T790M in exon 20). Forty-nine patients with NSCLC harboring EGFR-activating mutations were enrolled, and circulating free DNAs (cfDNAs) were extracted from the plasma of 21 and 28 patients before treatment and after progression following EGFR-TKI treatment, respectively. Using reference genomic DNA containing each mutation, the detection limit of each assay was determined to be 0.1%. The sensitivity and specificity of detecting exon 19 deletions and L858R mutations, calculated by comparing the mutation status in the corresponding tumors, were 70.6% and 93.3%, and 66.7% and 100%, respectively, showing similar results compared with previous studies. T790M was detected in 43% of 28 cfDNAs after progression with EGFR-TKI treatment, but in no cfDNAs before the start of the treatment. This chip-based dPCR assay can facilitate detection of EGFR mutations in cfDNA as a minimally invasive method in clinical settings. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Investigation of metallo-beta-lactamase producing strains of Pseudomonas aeruginosa and Acinetobacter baumannii by E-test, disk synergy and PCR.

    PubMed

    Aktaş, Zerrin; Kayacan, Ciğdem Bal

    2008-01-01

    Carbapenem non-susceptible Pseudomonas aeruginosa and Acinetobacter baumannii strains were tested for the presence of metallo-beta-lactamases (MBLs) by EDTA-synergy screening. Imipenem hydrolysis was investigated by a bioassay and IMP-/VIM-encoding genes by PCR. No bla(IMP/VIM) related genes or imipenemase activity were detected although E-test found all strains as MBL-positive. Disk synergy tests with 0.5 M EDTA determined 63.6-100%, while those with 0.1 M EDTA detected 0-7.7% of isolates as MBL producers. Most strains were susceptible to EDTA. In conclusion, for MBL-screening purposes, EDTA-synergy results change with molarity of EDTA, but even if some false positives are encountered, 0.1 M EDTA seems to be acceptable.

  19. Diagnostic accuracy of standardised qualitative sensory test in the detection of lumbar lateral stenosis involving the L5 nerve root.

    PubMed

    Lin, Jiann-Her; Hsieh, Yi-Chen; Chen, Yi-Chen; Wang, Yun; Chen, Chih-Cheng; Chiang, Yung-Hsiao

    2017-09-06

    Misdiagnosis of symptomatic lumbar lateral stenosis (LS) may result in an unfavourable prognosis after surgical treatment. This study investigated the diagnostic accuracy of a standardised qualitative sensory test (SQST) in the detection of symptomatic LS in patients who had degenerative spinal disorders involving the L5 spinal nerve. We prospectively identified 75 patients, of which 60 met the inclusion criteria. Lateral recess stenosis at the L5 level or foraminal stenosis at the L5/S1 level on MRI was identified and graded by a neurosurgeon blinded to any clinical information. The reference criteria for the diagnosis of symptomatic LS were grade III LS on MRI and relevant clinical symptoms. Cutaneous sensory functions of the L5 dermatome on the symptomatic side were evaluated using the SQST. Each item of the SQST showed a satisfactory performance in the diagnosis of LS (sensitivity = 0.455-0.727, specificity = 0.868-1.0). A stepwise selection model identified low-strength von-Frey, high-strength von-Frey, and vibration as the most accurate predictors of symptomatic LS with an area under the receiver operating characteristic curve of 0.9563 (95% confidence interval = 0.9003-1.0). In combination with MRI, the SQST is a promising diagnostic tool for detecting symptomatic LS involving L5 nerve roots.

  20. Zika Virus Testing Considerations: Lessons Learned from the First 80 Real-Time Reverse Transcription-PCR-Positive Cases Diagnosed in New York State

    PubMed Central

    Sohi, Inderbir S.; Dufort, Elizabeth M.; Dean, Amy B.; White, Jennifer L.; Limberger, Ronald; Sommer, Jamie N.; Ostrowski, Stephanie; Wong, Susan J.; Backenson, P. Bryon; Kuhles, Daniel; Blog, Debra; Taylor, Jill; Hutton, Brad; Zucker, Howard A.

    2016-01-01

    ABSTRACT The performance and interpretation of laboratory tests for Zika virus (ZKV) continue to be evaluated. Serology is cross-reactive, laborious, and frequently difficult to interpret, and serum was initially solely recommended for molecular diagnosis. ZKV testing was initiated in January 2016 in New York State for symptomatic patients, pregnant women, their infants, and patients with Guillain-Barré syndrome who had traveled to areas with ZKV transmission. Subsequently, eligibility was expanded to pregnant women with sexual partners with similar travel histories. Serum and urine collected within 4 weeks of symptom onset or within 6 weeks of travel were tested with real-time reverse transcription-PCR (RT-PCR) assays targeting the ZKV envelope and NS2B genes. In this review of lessons learned from the first 80 positive cases in NYS, ZKV RNA was detected in urine only in 50 patients, in serum only in 19 patients, and in both samples concurrently in 11 patients, with average viral loads in urine a log higher than those in serum. Among 93 positive samples from the 80 patients, 41 were positive on both gene assays, 52 were positive on the envelope only, and none were positive on the NS2B only. Of the 80 infected patients, test results for 74 (93%) would have defined their infection status as not detected or equivocal if the requirement for positive results from two assay targets (two-target-positive requirement) in the initial federal guidance to public health laboratories was enforced, if urine was not tested, or if the extended eligibility time for molecular testing was not implemented. These changes facilitated more extensive molecular diagnosis of ZKV, reducing reliance on time-consuming and potentially inconclusive serology. PMID:27927917

  1. Zika Virus Testing Considerations: Lessons Learned from the First 80 Real-Time Reverse Transcription-PCR-Positive Cases Diagnosed in New York State.

    PubMed

    St George, Kirsten; Sohi, Inderbir S; Dufort, Elizabeth M; Dean, Amy B; White, Jennifer L; Limberger, Ronald; Sommer, Jamie N; Ostrowski, Stephanie; Wong, Susan J; Backenson, P Bryon; Kuhles, Daniel; Blog, Debra; Taylor, Jill; Hutton, Brad; Zucker, Howard A

    2017-02-01

    The performance and interpretation of laboratory tests for Zika virus (ZKV) continue to be evaluated. Serology is cross-reactive, laborious, and frequently difficult to interpret, and serum was initially solely recommended for molecular diagnosis. ZKV testing was initiated in January 2016 in New York State for symptomatic patients, pregnant women, their infants, and patients with Guillain-Barré syndrome who had traveled to areas with ZKV transmission. Subsequently, eligibility was expanded to pregnant women with sexual partners with similar travel histories. Serum and urine collected within 4 weeks of symptom onset or within 6 weeks of travel were tested with real-time reverse transcription-PCR (RT-PCR) assays targeting the ZKV envelope and NS2B genes. In this review of lessons learned from the first 80 positive cases in NYS, ZKV RNA was detected in urine only in 50 patients, in serum only in 19 patients, and in both samples concurrently in 11 patients, with average viral loads in urine a log higher than those in serum. Among 93 positive samples from the 80 patients, 41 were positive on both gene assays, 52 were positive on the envelope only, and none were positive on the NS2B only. Of the 80 infected patients, test results for 74 (93%) would have defined their infection status as not detected or equivocal if the requirement for positive results from two assay targets (two-target-positive requirement) in the initial federal guidance to public health laboratories was enforced, if urine was not tested, or if the extended eligibility time for molecular testing was not implemented. These changes facilitated more extensive molecular diagnosis of ZKV, reducing reliance on time-consuming and potentially inconclusive serology.

  2. Qualitative thallium urinary assays are almost as valuable as quantitative tests: implication for outpatient settings in low and middle income countries.

    PubMed

    Ghaderi, Amir; Banafshe, Hamid Reza; Khodabandehlo, Soroor; Mehrzad, Fateme; Mehrpour, Omid; Afshari, Reza

    2017-04-01

    Thallium (Tl), lead and steroid exposures were reported as a result of street drug consumption such as heroin and cocaine. This study aimed to compare the values of qualitative and quantitative assays for detecting Tl as an adulterant in opioid-like compound drug users. This case-control study was conducted throughout a specified time bracket ranging from May 2015 to November 2015 in Imam Reza Hospital, Mashhad, Iran. In general, urine thallium samples for 100 opioid overdosed subjects with a history of chronic opioid use and 50 non-drug users were studied. Qualitative 24 hours-urinary-thallium-level (QL) and quantitative 24 hours-urinary-thallium-level (QT) were conducted in both groups. Independent-samples t-test and Spearman's Coefficient were applied for analytical purposes. SPSS software 16 was used to conduct statistical analyses with P values less than 0.05 regarded as significant. A total of 150 cases were studied. Raw opium users accounted for 66% of the cases followed by mixed (28%) and heroin users (6%). Mean (SD) QT level for QL was 26.8 (1) μg/L, while it was 2.3 μg/L (0.4 μg/L) for negative QL, which was far below QL positive cases (p=0.002). The qualitative test showed more accuracy at higher quantitative levels. In all cases, qualitative test was fully sensitive (100%), highly specific (89%) with a positive likelihood ratio (PLR) of 9.1 and a negative likelihood ratio (NLR) of 0. These results suggest that qualitative assays could be used with confidence in assessing Tl exposure in drug users. Physicians may easily and confidently use Tl qualitative tests in rehabilitation centers, where toxicology laboratories may not be available.

  3. Optimizing Infant HIV Diagnosis in Resource-Limited Settings: Modeling the Impact of HIV DNA PCR Testing at Birth

    PubMed Central

    Chiu, Alexander; Modi, Surbhi; Rivadeneira, Emilia D.; Koumans, Emilia H.

    2017-01-01

    Background Early antiretroviral therapy (ART) initiation in HIV-infected infants significantly improves survival but is often delayed in resource-limited settings. Adding HIV testing of infants at birth to the current recommendation of testing at age 4–6 weeks may improve testing rates and decrease time to ART initiation. We modeled the benefit of adding HIV testing at birth to the current 6-week testing algorithm. Methods Microsoft Excel was used to create a decision-tree model of the care continuum for the estimated 1,400,000 HIV-infected women and their infants in sub-Saharan Africa in 2012. The model assumed average published rates for facility births (42.9%), prevention of mother-to-child HIV transmission utilization (63%), mother-to-child-transmission rates based on prevention of mother-to-child HIV transmission regimen (5%–40%), return of test results (41%), enrollment in HIV care (52%), and ART initiation (54%). We conducted sensitivity analyses to model the impact of key variables and applied the model to specific country examples. Results Adding HIV testing at birth would increase the number of infants on ART by 204% by age 18 months. The greatest increase is seen in early ART initiations (543% by age 3 months). The increase would lead to a corresponding increase in survival at 12 months of age, with 5108 fewer infant deaths (44,550, versus 49,658). Conclusion Adding HIV testing at birth has the potential to improve the number and timing of ART initiation of HIV-infected infants, leading to a decrease in infant mortality. Using this model, countries should investigate a combination of HIV testing at birth and during the early infant period. PMID:27792684

  4. A Qualitative Comparison of the C-Ring Test and the Jones Test as Standard Practice Test Methods for Studying Stress Corrosion Cracking in Ferritic Steels

    SciTech Connect

    Thomson, Jeffery K; Pawel, Steven J

    2015-01-01

    Creep-strength-enhanced-ferritic (CSEF) steels have been widely implemented as water wall alloy materials in the coal-fired power industry for many years. The stress corrosion cracking (SCC) behavior of this class of materials is currently of significant interest to the industry due to recent failures. To better understand the test methods used to characterize SCC behavior in the laboratory, three representative CSEF alloys (T23, T24, and T92) were subjected to two SCC test protocols: the Jones Test set forth in DIN 50915, and the C-ring SCC test set forth in ASTM G38-01. Samples were tested in either the as-received (normalized + tempered) condition or in the normalized condition (quenched from 1065 C). Samples were exposed to aerated water in one test case and de-aerated water in a second test case for a period of 7 days at 200 C. It was found that for both test protocols, the normalized condition with aerated water led to severe cracking for all three alloys, whereas no evidence of cracking was found for the other conditions.

  5. Performance of the Cepheid CT/NG Xpert Rapid PCR Test for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae.

    PubMed

    Gaydos, Charlotte A; Van Der Pol, Barbara; Jett-Goheen, Mary; Barnes, Mathilda; Quinn, Nicole; Clark, Carey; Daniel, Grace E; Dixon, Paula B; Hook, Edward W

    2013-06-01

    Tests for Chlamydia trachomatis and Neisseria gonorrhoeae, which can provide results rapidly to guide therapeutic decision-making, offer patient care advantages over laboratory-based tests that require several days to provide results. We compared results from the Cepheid GeneXpert CT/NG (Xpert) assay to results from two currently approved nucleic acid amplification assays in 1,722 female and 1,387 male volunteers. Results for chlamydia in females demonstrated sensitivities for endocervical, vaginal, and urine samples of 97.4%, 98.7%, and 97.6%, respectively, and for urine samples from males, a sensitivity of 97.5%, with all specificity estimates being ≥ 99.4%. Results for gonorrhea in females demonstrated sensitivities for endocervical, vaginal, and urine samples of 100.0%, 100.0%, and 95.6%, respectively, and for urine samples from males, a sensitivity of 98.0%, with all estimates of specificity being ≥ 99.8%. These results indicate that this short-turnaround-time test can be used to accurately test patients and to possibly do so at the site of care, thus potentially improving chlamydia and gonorrhea control efforts.

  6. PCR thermocycler

    DOEpatents

    Benett, William J.; Richards, James B.

    2003-01-01

    A sleeve-type silicon polymerase chain reaction (PCR) chamber or thermocycler having improved thermal performance. The silicon sleeve reaction chamber is improved in thermal performance by etched features therein that reduce thermal mass and increase the surface area of the sleeve for cooling. This improved thermal performance of the thermocycler enables an increase in speed and efficiency of the reaction chamber. The improvement is accomplished by providing grooves in the faces of the sleeve and a series of grooves on the interior surfaces that connect with grooves on the faces of the sleeve. The grooves can be anisotropically etched in the silicon sleeve simultaneously with formation of the chamber.

  7. PCR thermocycler

    DOEpatents

    Benett, William J.; Richards, James B.

    2005-05-17

    A sleeve-type silicon polymerase chain reaction (PCR) chamber or thermocycler having improved thermal performance. The silicon sleeve reaction chamber is improved in thermal performance by etched features therein that reduce thermal mass and increase the surface area of the sleeve for cooling. This improved thermal performance of the thermocycler enables an increase in speed and efficiency of the reaction chamber. The improvement is accomplished by providing grooves in the faces of the sleeve and a series of grooves on the interior surfaces that connect with grooves on the faces of the sleeve. The grooves can be anisotropically etched in the silicon sleeve simultaneously with formation of the chamber.

  8. Diagnostic Accuracy of a Qualitative Fecal Immunochemical Test Varies With Location of Neoplasia But Not Number of Specimens.

    PubMed

    Wong, Martin C S; Ching, Jessica Y L; Chan, Victor C W; Lam, Thomas Y T; Shum, Jeffrey P; Luk, Arthur K C; Wong, Sunny S H; Ng, Siew C; Ng, Simon S M; Wu, Justin C Y; Chan, Francis K L; Sung, Joseph J Y

    2015-08-01

    We compared the accuracy of a qualitative fecal immunochemical test (FIT) in identifying patients with proximal vs distal advanced neoplasia and evaluated whether analysis of 2 specimens performed better than analysis of 1 specimen. Distal advanced neoplasia was defined as colorectal cancer (CRC), any colorectal adenoma ≥10 mm in diameter, high-grade dysplasia, or a lesion with villous or tubulovillous histologic characteristics in a location distal to the splenic flexure, including the descending colon, the rectosigmoid, and the rectum. We collected data from 5343 subjects (50-70 years old) who received 2 FITs (Hemosure; cutoff value, 10 μg hemoglobin/g feces) before colonoscopy in an invitational CRC screening program in Hong Kong from 2008 through 2012. We calculated the FIT's sensitivity, specificity, positive predictive value (PPV), and negative predictive value in detecting colorectal neoplasia. Of the participants, 13.6%, 12.2%, and 6.0% had distal, proximal, and synchronous distal or proximal neoplasia, respectively. Advanced neoplasia was detected in 291 subjects (5.4%); 22 (0.4%) had CRC. FIT detected distal advanced adenoma with 39.7% sensitivity (95% confidence interval [CI], 32.0%-48.0%) vs proximal advanced adenoma with 25.0% sensitivity (95% CI, 17.3%-34.6%; P = .014), distal advanced neoplasia with 40.0% sensitivity (95% CI, 32.5%-47.9%) vs proximal advanced neoplasia with 27.9% sensitivity (95% CI, 20.0%-37.4%; P = .039), and any distal adenoma ≥10 mm, irrespective of other lesion characteristics, with 39.5% sensitivity (95% CI, 31.0%-48.7%) vs. proximal adenoma with 25.3% sensitivity (95% CI, 16.5%-36.6%; P = .038). The specificity of FIT in detecting CRC was similar between the proximal and distal colon. FIT detected distal lesions with higher PPV than proximal lesions. One FIT detected advanced neoplasia with 31.8% sensitivity (95% CI, 25.9%-38.4%) and 92.4% specificity (95% CI, 91.6%-93.2%), whereas 2 FITs detected advanced neoplasia with

  9. Equal Performance of Self-Collected and Health Care Worker-Collected Pharyngeal Swabs for Group A Streptococcus Testing by PCR

    PubMed Central

    Murray, Martha A.; Schulz, Lorie A.; Furst, Joseph W.; Homme, Jason H.; Jenkins, Sarah M.; Uhl, James R.; Patel, Robin; Cockerill, Franklin C.; Myers, Jane F.

    2014-01-01

    A process employing patient- or parent-collected pharyngeal swabs for group A Streptococcus (GAS) testing would expedite diagnosis and treatment, reduce patient exposure to the health care setting, and decrease health care costs. Our aim was to determine the concordance between patient- or parent-collected (self-collected) and health care worker (HCW)-collected pharyngeal swabs for detection of GAS by PCR. From 9 October 2012 to 21 March 2013, patients presenting with a sore throat meeting criteria for GAS testing and not meeting criteria for severe disease were offered the opportunity to collect their own pharyngeal swab. The HCW also collected a swab. Paired swabs were tested by GAS real-time PCR, allowing semiquantitative comparisons between positive results. Of the 402 participants, 206 had a swab collected by the patient and 196 a swab collected by the parent. The percent positivity results were 33.3% for HCW-collected swabs and 34.3% for self-collected swabs (P = 0.41). The overall concordance between the two collection strategies was 94.0% (95% confidence interval [CI], 91.3 to 96.0). Twenty-four of the paired swabs had discordant results, with 10 and 14 positives detected only with the HCW- and self-collected swabs, respectively (P = 0.41). The person collecting the swab in the self-collected arm, the order of collection, and prior swab collection training did not influence results. Among the 124 specimens that were positive by both collection methods, the amount of GAS DNA was higher in the self-collected versus the HCW-collected swabs (P = 0.008). Self-collected pharyngeal swabs provide a reliable alternative to HCW collection for detection of GAS and offer a strategy for improved health care delivery. PMID:25502528

  10. Simultaneous species-specific PCR detection and viability testing of poly(vinyl alcohol) cryogel-entrapped Rhodococcus spp. after their exposure to petroleum hydrocarbons.

    PubMed

    Kuyukina, Maria S; Ivshina, Irena B; Serebrennikova, Marina K; Rubtsova, Ekaterina V; Krivoruchko, Anastasiya V

    2013-08-01

    A method of simultaneous species-specific PCR detection and viability testing of poly(vinyl alcohol) cryogel-entrapped Rhodococcus spp. was developed that allowed the estimation of immobilized Rhodococcus opacus and Rhodococcus ruber survival after their exposure to petroleum hydrocarbon mixture. Spectrophotometric INT assay revealed high tolerance of gel-immobilized rhodococci to petroleum hydrocarbons, while among two Rhodococcus strains studied, R. ruber tolerated better to hydrocarbons compared to R. opacus. These findings were confirmed by respirometry results that showed increased respiratory activity of gel-immobilized Rhodococcus strains after 10-day incubation with 3% (v/v) petroleum hydrocarbon mixture. Moreover, jointly incubated rhodococcal strains demonstrated higher oxidative activities toward petroleum hydrocarbons than individual strains. Both Rhodococcus species were recovered successfully in cryogel granules using 16S rDNA-targeted PCR, even though the granules were previously stained with INT and extracted with ethanol. The method developed can be used for rapid detection and monitoring of gel-immobilized bacterial inocula in bioreactors or contaminated soil systems. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Detection of Bordetella pertussis using a PCR test in infants younger than one year old hospitalized with whooping cough in five Peruvian hospitals.

    PubMed

    Castillo, María Esther; Bada, Carlos; Del Aguila, Olguita; Petrozzi-Helasvuo, Verónica; Casabona-Ore, Verónica; Reyes, Isabel; Del Valle-Mendoza, Juana

    2015-12-01

    To report the incidence, epidemiology, and clinical features of Bordetella pertussis in Peruvian infants under 1 year old. A prospective cross-sectional study was conducted in five hospitals in Peru from January 2010 to July 2012. A total of 392 infants under 1 year old were admitted with a clinical diagnosis of whooping cough and tested for B. pertussis by PCR. The pertussis toxin and IS481 genes were detected in 39.54% (155/392) of the cases. Infants aged less than 3 months were the most affected, with a prevalence of 73.55% (114/155). The most common household contact was the mother, identified in 20% (31/155) of cases. Paroxysm of coughing (89.03%, 138/155), cyanosis (68.39%, 106/155), respiratory distress (67.09%, 104/155), and breastfeeding difficulties (39.35%, 61/155) were the most frequent symptoms reported. An increase in pertussis cases has been reported in recent years in Peru, despite national immunization efforts. Surveillance with PCR for B. pertussis is essential, especially in infants less than 1 year old, in whom a higher rate of disease-related complications and higher mortality have been reported. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. The identification of point mutations in Duchenne muscular dystrophy patients by using reverse-transcription PCR and the protein truncation test.

    PubMed Central

    Gardner, R J; Bobrow, M; Roberts, R G

    1995-01-01

    The protein truncation test (PTT) is a mutation-detection method that monitors the integrity of the open reading frame (ORF). More than 60% of cases of Duchenne muscular dystrophy (DMD) result from gross frame-shifting deletions in the dystrophin gene that are detectable by a multiplex PCR system. It has become apparent that virtually all of the remaining DMD mutations also disrupt the translational reading frame, making the PTT a logical next step toward a comprehensive strategy for the identification of all DMD mutations. We report here a pilot study involving 22 patients and describe the mutations characterized. These constitute 12 point mutations or small insertions/deletions and 4 gross rearrangements. We also have a remaining five patients in whom there does not appear to be a mutation in the ORF. We believe that reverse-transcription--PCR/PTT is an efficient method by which to screen for small mutations in DMD patients with no deletion. Images Figure 2 PMID:7668256

  13. How home HIV testing and counselling with follow-up support achieves high testing coverage and linkage to treatment and prevention: a qualitative analysis from Uganda

    PubMed Central

    Ware, Norma C; Wyatt, Monique A; Asiimwe, Stephen; Turyamureeba, Bosco; Tumwesigye, Elioda; van Rooyen, Heidi; Barnabas, Ruanne V; Celum, Connie L

    2016-01-01

    Introduction The successes of HIV treatment scale-up and the availability of new prevention tools have raised hopes that the epidemic can finally be controlled and ended. Reduction in HIV incidence and control of the epidemic requires high testing rates at population levels, followed by linkage to treatment or prevention. As effective linkage strategies are identified, it becomes important to understand how these strategies work. We use qualitative data from The Linkages Study, a recent community intervention trial of community-based testing with linkage interventions in sub-Saharan Africa, to show how lay counsellor home HIV testing and counselling (home HTC) with follow-up support leads to linkage to clinic-based HIV treatment and medical male circumcision services. Methods We conducted 99 semi-structured individual interviews with study participants and three focus groups with 16 lay counsellors in Kabwohe, Sheema District, Uganda. The participant sample included both HIV+ men and women (N=47) and HIV-uncircumcised men (N=52). Interview and focus group audio-recordings were translated and transcribed. Each transcript was summarized. The summaries were analyzed inductively to identify emergent themes. Thematic concepts were grouped to develop general constructs and framing propositional statements. Results Trial participants expressed interest in linking to clinic-based services at testing, but faced obstacles that eroded their initial enthusiasm. Follow-up support by lay counsellors intervened to restore interest and inspire action. Together, home HTC and follow-up support improved morale, created a desire to reciprocate, and provided reassurance that services were trustworthy. In different ways, these functions built links to the health service system. They worked to strengthen individuals’ general sense of capability, while making the idea of accessing services more manageable and familiar, thus reducing linkage barriers. Conclusions Home HTC with follow

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

    PubMed

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

    2016-12-01

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

  15. Health care workers’ perceptions of point-of-care testing in a low-income country—A qualitative study in Southwestern Uganda

    PubMed Central

    Karlström, Jonas; Muchunguzi, Charles; Mwanga-Amumpaire, Juliet; Gantelius, Jesper; Mårtensson, Andreas; Rivas, Lourdes; Galban, Francesc; Reuterswärd, Philippa; Andersson Svahn, Helene; Alvesson, Helle M.; Boum, Yap; Alfvén, Tobias

    2017-01-01

    Background Point-of-care (POC) tests have become increasingly available and more widely used in recent years. They have been of particular importance to low-income settings, enabling them with clinical capacities that had previously been limited. POC testing programs hold a great potential for significant improvement in low-income health systems. However, as most POC tests are developed in high-income countries, disengagement between developers and end-users inhibit their full potential. This study explores perceptions of POC test end-users in a low-income setting, aiming to support the development of novel POC tests for low-income countries. Methods A qualitative study was conducted in Mbarara District, Southwestern Uganda, in October 2014. Fifty health care workers were included in seven focus groups, comprising midwives, laboratory technicians, clinical and medical officers, junior and senior nurses, and medical doctors. Discussions were audio-recorded and transcribed verbatim. Transcripts were coded through a data-driven approach for qualitative content analysis. Results Nineteen different POC tests were identified as currently being in use. While participants displayed being widely accustomed to and appreciative of the use of POC tests, they also assessed the use and characteristics of current tests as imperfect. An ideal POC test was characterized as being adapted to local conditions, thoughtfully implemented in the specific health system, and capable of improving the care of patients. Tests for specific medical conditions were requested. Opinions differed with regard to the ideal distribution of POC tests in the local health system. Conclusion POC tests are commonly used and greatly appreciated in this study setting. However, there are dissatisfactions with current POC tests and their use. To maximize benefit, stakeholders need to include end-user perspectives in the development and implementation of POC tests. Insights from this study will influence our

  16. Distinction between persistent and transient infection in a bovine viral diarrhoea (BVD) control programme: appropriate interpretation of real-time RT-PCR and antigen-ELISA test results.

    PubMed

    Hanon, J-B; Van der Stede, Y; Antonissen, A; Mullender, C; Tignon, M; van den Berg, T; Caij, B

    2014-04-01

    Control of bovine viral diarrhoea (BVD) in Belgium is currently implemented on a voluntary basis at herd level and mainly relies on detection and culling of persistently infected (PI) animals. The present field study was conducted during the winter of 2010/2011 to assess the performances of diagnostic assays used in the testing scheme for BVD as proposed by the two Belgian regional laboratories. Individual blood samples were collected from 4972 animals, and individual samples from the same herd were pooled (maximum of 30 individual samples per pool) and screened for the presence of Bovine Viral Diarrhoea Virus (BVDV)-specific RNA using a commercial real-time RT-PCR test (ADIAGENE). Individual samples from positive pools were then tested in parallel with the same RT-PCR test and with an antigen-capture ELISA test (IDEXX) to detect viremic animals. This study demonstrated that individual results differed according to the type of assay used (P < 0.001): 140 animals (2.8%) were positive by RT-PCR and 72 (1.4%) by antigen-ELISA. A second blood sample was taken 40 days later from 74 PCR positive animals to detect persistent viremia: 17 (23%) of these were still PCR positive and considered to be PI and the 57 that no longer tested positive were assumed to be transiently infected (TI) animals. All PI animals were positive also by antigen-ELISA at both time points. Among TI animals, 10 (16%) were positive by antigen-ELISA at the first but none at the second sampling. A highly significant difference in cycle threshold (Ct ) values obtained by RT-PCR was observed between PI and TI animals. ROC analysis was performed to establish thresholds to confirm with high probability that an animal is PI, based on the result of RT-PCR test performed on a single individual blood sample.

  17. Comparison of visual motor development in Hong Kong and the USA assessed on the Qualitative Scoring System for the Modified Bender-Gestalt test.

    PubMed

    Chan, P W

    2001-02-01

    This study compared the visuomotor development of young children in Hong Kong and the USA assessed on the Qualitative Scoring System for the Modified Bender-Gestalt test. 744 children aged 4:6 to 8:5 years from 6 kindergartens and 6 primary schools in Hong Kong were administered the Modified Bender-Gestalt test. The Qualitative Scoring System was used to measure the children's visuomotor development. Their visuomotor scores were then compared with norms for children in the USA. Analysis indicated significant differences across all age groups of 4:6 to 8:5 years in 6-mo. units. Consistent with previous research, children in Hong Kong outperformed their western peers. Percentile scores and T scores for children in Hong Kong in each age group were reported.

  18. Qualitative research.

    PubMed

    Gelling, Leslie

    2015-03-25

    Qualitative research has an important role in helping nurses and other healthcare professionals understand patient experiences of health and illness. Qualitative researchers have a large number of methodological options and therefore should take care in planning and conducting their research. This article offers a brief overview of some of the key issues qualitative researchers should consider.

  19. Applications of Digital PCR for Clinical Microbiology.

    PubMed

    Kuypers, Jane; Jerome, Keith R

    2017-03-15

    Digital PCR (dPCR) is an important new tool for use in the clinical microbiology laboratory. Its advantages over quantitative PCR (qPCR), including absolute quantification without a standard curve, improved precision, improved accuracy in the presence of inhibitors, and more accurate quantitation when amplification efficiency is low, make dPCR the assay of choice for several specimen testing applications. This mini-review will discuss the advantages and disadvantages of dPCR compared to qPCR, its applications in clinical microbiology and the considerations for implementation of the method in a clinical laboratory.

  20. Development and testing of real-time PCR assays for determining fecal loading and source identification (cattle, human, etc.) in surface water and groundwater

    NASA Astrophysics Data System (ADS)

    McKay, L. D.; Layton, A.; Gentry, R.

    2004-12-01

    A multi-disciplinary group of researchers at the University of Tennessee is developing and testing a series of microbial assay methods based on real-time PCR to detect fecal bacterial concentrations and host sources in water samples. Real-time PCR is an enumeration technique based on the unique and conserved nucleic acid sequences present in all organisms. The first research task was development of an assay (AllBac) to detect total amount of Bacteroides, which represents up to 30 percent of fecal mass. Subsequent assays were developed to detect Bacteroides from cattle (BoBac) and humans (HuBac) using 16sRNA genes based on DNA sequences in the national GenBank, as well as sequences from local fecal samples. The assays potentially have significant advantages over conventional bacterial source tracking methods because: 1. unlike traditional enumeration methods, they do not require bacterial cultivation; 2. there are no known non-fecal sources of Bacteroides; 3. the assays are quantitative with results for total concentration and for each species expressed in mg/l; and 4. they show little regional variation within host species, meaning that they do not require development of extensive local gene libraries. The AllBac and BoBac assays have been used in a study of fecal contamination in a small rural watershed (Stock Creek) near Knoxville, TN, and have proven useful in identification of areas where cattle represent a significant fecal input and in development of BMPs. It is expected that these types of assays (and future assays for birds, hogs, etc.) could have broad applications in monitoring fecal impacts from Animal Feeding Operations, as well as from wildlife and human sources.

  1. Newborn blood spot screening test using multiplexed real-time PCR to simultaneously screen for spinal muscular atrophy and severe combined immunodeficiency.

    PubMed

    Taylor, Jennifer L; Lee, Francis K; Yazdanpanah, Golriz Khadem; Staropoli, John F; Liu, Mei; Carulli, John P; Sun, Chao; Dobrowolski, Steven F; Hannon, W Harry; Vogt, Robert F

    2015-02-01

    Spinal muscular atrophy (SMA) is a motor neuron disorder caused by the absence of a functional survival of motor neuron 1, telomeric (SMN1) gene. Type I SMA, a lethal disease of infancy, accounts for the majority of cases. Newborn blood spot screening (NBS) to detect severe combined immunodeficiency (SCID) has been implemented in public health laboratories in the last 5 years. SCID detection is based on real-time PCR assays to measure T-cell receptor excision circles (TREC), a byproduct of T-cell development. We modified a multiplexed real-time PCR TREC assay to simultaneously determine the presence or absence of the SMN1 gene from a dried blood spot (DBS) punch in a single reaction well. An SMN1 assay using a locked nucleic acid probe was initially developed with cell culture and umbilical cord blood (UCB) DNA extracts, and then integrated into the TREC assay. DBS punches were placed in 96-well arrays, washed, and amplified directly using reagents specific for TREC, a reference gene [ribonuclease P/MRP 30kDa subunit (RPP30)], and the SMN1 gene. The assay was tested on DBS made from UCB units and from peripheral blood samples of SMA-affected individuals and their family members. DBS made from SMA-affected individuals showed no SMN1-specific amplification, whereas DBS made from all unaffected carriers and UCB showed SMN1 amplification above a well-defined threshold. TREC and RPP30 content in all DBS were within the age-adjusted expected range. SMA caused by the absence of SMN1 can be detected from the same DBS punch used to screen newborns for SCID. © 2014 American Association for Clinical Chemistry.

  2. Qualitative modeling.

    PubMed

    Forbus, Kenneth D

    2011-07-01

    Qualitative modeling concerns the representations and reasoning that people use to understand continuous aspects of the world. Qualitative models formalize everyday notions of causality and provide accounts of how to ground symbolic, relational representations in perceptual processes. This article surveys the basic ideas of qualitative modeling and their applications from a cognitive science perspective. It describes the basic principles of qualitative modeling, and a variety of qualitative representations that have been developed for quantities and for relationships between them, providing a kind of qualitative mathematics. Three ontological frameworks for organizing modeling knowledge (processes, components, and field) are summarized, along with research on automatically assembling models for particular tasks from such knowledge. Qualitative simulation and how it carves up time into meaningful units is discussed. We discuss several accounts of causal reasoning about dynamical systems, based on different choices of qualitative mathematics and ontology. Qualitative spatial reasoning is explored, both in terms of relational systems and visual reasoning. Applications of qualitative models of particular interest to cognitive scientists are described, including how they have been used to capture the expertise of scientists and engineers and how they have been used in education. Open questions and frontiers are also discussed, focusing on relationships between ideas developed in the qualitative modeling community and other areas of cognitive science. WIREs Cogni Sci 2011 2 374-391 DOI: 10.1002/wcs.115 For further resources related to this article, please visit the WIREs website.

  3. RhC Phenotyping, Adsorption/Elution Test, and SSP-PCR: The Combined Test for D-Elute Phenotype Screening in Thai RhD-Negative Blood Donors.

    PubMed

    Srijinda, Songsak; Suwanasophon, Chamaiporn; Visawapoka, Unchalee; Pongsavee, Malinee

    2012-01-01

    The Rhesus (Rh) blood group is the most polymorphic human blood group and it is clinically significant in transfusion medicine. Especially, D antigen is the most important and highly immunogenic antigen. Due to anti-D, it is the cause of the hemolytic disease of the newborn and transfusion reaction. About 0.1%-0.5% of Asian people are RhD-negative, whereas in the Thai population, the RhD-negative blood type only occurs in 0.3%. Approximately 10%-30% of RhD-negative in Eastern Asian people actually were D-elute (DEL) phenotype, the very weak D antigen that cannot be detected by indirect antiglobulin test (IAT). There are many reports about anti-D immunization in RhD-negative recipients through the transfusion of red blood cells from individuals with DEL phenotype. D-elute phenotype screening in Thai RhD-negative blood donors was studied to distinguish true RhD-negative from DEL phenotype. A total of 254 Thai serologically RhD-negative blood donors were tested for RhCE phenotypes and anti-D adsorption/elution test. In addition, RhC(+) samples were tested for RHD 1227A allele by SSP-PCR technique. The RhD-negative phenotype samples consisted of 131 ccee, 4 ccEe, 1 ccEE, 101 Ccee, 16 CCee, and 1 CcEe. The 42 Ccee and 8 CCee phenotype samples were typed as DEL phenotype and 96% of DEL samples were positive for RHD 1227A allele. The incidence of RhC(+) was 46.4%, and 48 of the 118 RhC(+) samples were positive for both anti-D adsorption/elution test and SSP-PCR technique for RHD 1227A allele. The sensitivity and specificity were 96% and 100%, respectively, for RHD 1227A detection as compared with the adsorption/elution test. In conclusion, RhC(+) phenotype can combine with anti-D adsorption/elution test and RHD 1227A allele SSP-PCR technique for distinguishing true RhD-negative from DEL phenotype.

  4. The characterization of four gene expression analysis in circulating tumor cells made by Multiplex-PCR from the AdnaTest kit on the lab-on-a-chip Agilent DNA 1000 platform.

    PubMed

    Škereňová, Markéta; Mikulová, Veronika; Čapoun, Otakar; Zima, Tomáš

    2016-01-01

    Nowadays, on-a-chip capillary electrophoresis is a routine method for the detection of PCR fragments. The Agilent 2100 Bioanalyzer was one of the first commercial devices in this field. Our project was designed to study the characteristics of Agilent DNA 1000 kit in PCR fragment analysis as a part of circulating tumour cell (CTC) detection technique. Despite the common use of this kit a complex analysis of the results from a long-term project is still missing. A commercially available Agilent DNA 1000 kit was used as a final step in the CTC detection (AdnaTest) for the determination of the presence of PCR fragments generated by Multiplex PCR. Data from 30 prostate cancer patients obtained during two years of research were analyzed to determine the trueness and precision of the PCR fragment size determination. Additional experiments were performed to demonstrate the precision (repeatability, reproducibility) and robustness of PCR fragment concentration determination. The trueness and precision of the size determination was below 3% and 2% respectively. The repeatability of the concentration determination was below 15%. The difference in concentration determination increases when Multiplex-PCR/storage step is added between the two measurements of one sample. The characteristics established in our study are in concordance with the manufacturer's specifications established for a ladder as a sample. However, the concentration determination may vary depending on chip preparation, sample storage and concentration. The 15% variation of concentration determination repeatability was shown to be partly proportional and can be suppressed by proper normalization.

  5. Assessment accommodations on tests of academic achievement for students who are deaf or hard of hearing: a qualitative meta-analysis of the research literature.

    PubMed

    Cawthon, Stephanie; Leppo, Rachel

    2013-01-01

    The authors conducted a qualitative meta-analysis of the research on assessment accommodations for students who are deaf or hard of hearing. There were 16 identified studies that analyzed the impact of factors related to student performance on academic assessments across different educational settings, content areas, and types of assessment accommodations. The meta-analysis found that the results of analyses of group effects of accommodated versus unaccommodated test formats are often not significant, test-level factors exist that can affect how students perceive the assessments, and differences exist in how test items function across different conditions. Student-level factors, including educational context and academic proficiency, influence accommodations' role in assessment processes. The results of this analysis highlight the complexity of and intersections between student-level factors, test-level factors, and larger policy contexts. Findings are discussed within the context of larger changes in academic assessment, including computer-based administration and high-stakes testing.

  6. A qualitative study of the complete set of solutions of the differential equation of motion of a test particle in the equatorial plane of the Kerr gravitational field

    NASA Technical Reports Server (NTRS)

    Montgomery, H. E.; Chan, F. K.

    1973-01-01

    A study is made of the mathematical solution of the differential equation of motion of a test particle in the equatorial plane of the Kerr gravitational field, using S (Schwarzschild-like) coordinates. A qualitative solution of this equation leads to the conclusion that there can only be 25 different types of orbits. For each value of a, the results are presented in a master diagram for which h and e are the parameters. A master diagram divides the h, e parameter space into regions such that at each point within one of these regions the types of admissible orbits are qualitatively the same. A pictorial representation of the physical orbits in the r, phi plane is also given.

  7. Sequence homologies between Mycoplasma and Chlamydia spp. lead to false-positive results in chlamydial cell cultures tested for mycoplasma contamination with a commercial PCR assay.

    PubMed

    Maass, Viola; Kern, Jan Marco; Poeckl, Matthias; Maass, Matthias

    2011-10-01

    Mycoplasma contamination is a frequent problem in chlamydial cell culture. After obtaining contradictory contamination results, we compared three commercial PCR kits for mycoplasma detection. One kit signaled contamination in mycoplasma-free Chlamydia pneumoniae cultures. Sequencing of cloned PCR products revealed primer homology with the chlamydial genome as the basis of this false-positive result.

  8. Laboratory Evaluations of the Enterococcus qPCR Method for Recreational Water Quality Testing: Method Performance and Sources of Uncertainty in Quantitative Measurements

    EPA Science Inventory

    The BEACH Act of 2000 directed the U.S. EPA to establish more expeditious methods for the detection of pathogen indicators in coastal waters, as well as new water quality criteria based on these methods. Progress has been made in developing a quantitative PCR (qPCR) method for en...

  9. Testing of the Bio-Seeq (Smiths Detection Handheld PCR Instrument): Sensitivity, Specificity, and Effect of Interferents on Bacillus Assay Performance

    DTIC Science & Technology

    2004-07-01

    Smiths Detection-Edgewood (SDE), Inc., recently developed a handheld PCR instrument, the Bio -Seeq(Trademark), an updated, redesigned version of the...small, portable PCR instrument previously known as the Handheld Advanced Nucleic Acid Analyzer (HANAA). The SDE developed the Bio -Seeq(Trademark) to

  10. Laboratory Evaluations of the Enterococcus qPCR Method for Recreational Water Quality Testing: Method Performance and Sources of Uncertainty in Quantitative Measurements

    EPA Science Inventory

    The BEACH Act of 2000 directed the U.S. EPA to establish more expeditious methods for the detection of pathogen indicators in coastal waters, as well as new water quality criteria based on these methods. Progress has been made in developing a quantitative PCR (qPCR) method for en...

  11. HIV testing preferences in Tanzania: a qualitative exploration of the importance of confidentiality, accessibility, and quality of service.

    PubMed

    Njau, Bernard; Ostermann, Jan; Brown, Derek; Mühlbacher, Axel; Reddy, Elizabeth; Thielman, Nathan

    2014-08-12

    HIV counseling and testing (HCT), an effective preventive strategy and an entry point for care, remains under-utilized in Tanzania. Limited uptake of HCT, despite the widespread availability of varied testing options, suggests that existing options may not align well with population preferences for testing. Between October and December 2011, we conducted an exploratory study in the Kilimanjaro Region to develop a conceptual framework for understanding which characteristics of HIV testing are associated with preferences for testing. Forty individuals (55% women, 53% never having tested) participated in in-depth interviews and focus groups to identify factors that influence whether and where people test for HIV. A variety of discrete characteristics of testing venues, test providers, and testing procedures (e.g. distance to testing, counselor experience, type of HIV test, and availability of antiretroviral therapy) mapped conceptually to three domains: confidentiality of testing and test results, quality of HCT, and accessibility and availability of ancillary services. We noted heterogeneous preferences and demonstrate that while some test characteristics overlap and reinforce across multiple domains, others demand clients to make trade-offs between domains. Testing decisions appear to be influenced by an array of often inter-linked factors across multiple domains, including quality, confidentiality, and accessibility; perceptions of these factors varied greatly across participants and across available testing options. HCT interventions that jointly target barriers spanning the three domains have the potential to increase uptake of HIV testing and deserve further exploration.

  12. Reasons for compliance or noncompliance with advice to test for hepatitis C via an internet-mediated blood screening service: a qualitative study

    PubMed Central

    2011-01-01

    Background Hepatitis C virus (HCV) is mainly transmitted by exposure to infected blood, and can lead to liver cirrhosis and liver cancer. Since the onset of HCV and the development of liver cirrhosis usually are asymptomatic, many HCV-infected individuals are still undiagnosed. To identify individuals infected with HCV in the general population, a low threshold, internet-mediated blood testing service was set up. We performed a qualitative study exam