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Sample records for improved diagnostic pcr

  1. Pre-PCR processing in bioterrorism preparedness: improved diagnostic capabilities for laboratory response networks.

    PubMed

    Hedman, Johannes; Knutsson, Rickard; Ansell, Ricky; Rådström, Peter; Rasmusson, Birgitta

    2013-09-01

    Diagnostic DNA analysis using polymerase chain reaction (PCR) has become a valuable tool for rapid detection of biothreat agents. However, analysis is often challenging because of the limited size, quality, and purity of the biological target. Pre-PCR processing is an integrated concept in which the issues of analytical limit of detection and simplicity for automation are addressed in all steps leading up to PCR amplification--that is, sampling, sample treatment, and the chemical composition of PCR. The sampling method should maximize target uptake and minimize uptake of extraneous substances that could impair the analysis--so-called PCR inhibitors. In sample treatment, there is a trade-off between yield and purity, as extensive purification leads to DNA loss. A cornerstone of pre-PCR processing is to apply DNA polymerase-buffer systems that are tolerant to specific sample impurities, thereby lowering the need for expensive purification steps and maximizing DNA recovery. Improved awareness among Laboratory Response Networks (LRNs) regarding pre-PCR processing is important, as ineffective sample processing leads to increased cost and possibly false-negative or ambiguous results, hindering the decision-making process in a bioterrorism crisis. This article covers the nature and mechanisms of PCR-inhibitory substances relevant for agroterrorism and bioterrorism preparedness, methods for quality control of PCR reactions, and applications of pre-PCR processing to optimize and simplify the analysis of various biothreat agents. Knowledge about pre-PCR processing will improve diagnostic capabilities of LRNs involved in the response to bioterrorism incidents.

  2. Overcoming inhibition in real-time diagnostic PCR.

    PubMed

    Hedman, Johannes; Rådström, Peter

    2013-01-01

    PCR is an important and powerful tool in several fields, including clinical diagnostics, food analysis, and forensic analysis. In theory, PCR enables the detection of one single cell or DNA molecule. However, the presence of PCR inhibitors in the sample affects the amplification efficiency of PCR, thus lowering the detection limit, as well as the precision of sequence-specific nucleic acid quantification in real-time PCR. In order to overcome the problems caused by PCR inhibitors, all the steps leading up to DNA amplification must be optimized for the sample type in question. Sampling and sample treatment are key steps, but most of the methods currently in use were developed for conventional diagnostic methods and not for PCR. Therefore, there is a need for fast, simple, and robust sample preparation methods that take advantage of the accuracy of PCR. In addition, the thermostable DNA polymerases and buffer systems used in PCR are affected differently by inhibitors. During recent years, real-time PCR has developed considerably and is now widely used as a diagnostic tool. This technique has greatly improved the degree of automation and reduced the analysis time, but has also introduced a new set of PCR inhibitors, namely those affecting the fluorescence signal. The purpose of this chapter is to view the complexity of PCR inhibition from different angles, presenting both molecular explanations and practical ways of dealing with the problem. Although diagnostic PCR brings together scientists from different diagnostic fields, end-users have not fully exploited the potential of learning from each other. Here, we have collected knowledge from archeological analysis, clinical diagnostics, environmental analysis, food analysis, and forensic analysis. The concept of integrating sampling, sample treatment, and the chemistry of PCR, i.e., pre-PCR processing, will be addressed as a general approach to overcoming real-time PCR inhibition and producing samples optimal for PCR

  3. PCR-based diagnostics for anaerobic infections.

    PubMed

    Song, Yuli

    2005-01-01

    Conventional methods to identify anaerobic bacteria have often relied on unique clinical findings, isolation of organisms, and laboratory identification by morphology and biochemical tests (phenotypic tests). Although these methods are still fundamental, there is an increasing move toward molecular diagnostics of anaerobes. In this review, some of the molecular approaches to anaerobic diagnostics based on the polymerase chain reaction (PCR) are discussed. This includes several technological advances in PCR-based methods for the detection, identification, and quantitation of anaerobes including real-time PCR which has been successfully used to provide rapid, quantitative data on anaerobic species on clinical samples. Since its introduction in the mid-1980s, PCR has provided many molecular diagnostic tools, some of which are discussed within this review. With the advances in micro-array technology and real-time PCR methods, the future is bright for the development of accurate, quantitative diagnostic tools that can provide information not only on individual anaerobic species but also on whole communities.

  4. Real-time PCR in Food Science: PCR Diagnostics.

    PubMed

    Rodriguez-Lazaro, David; Cook, Nigel; Hernandez, Marta

    2013-01-01

    A principal consumer demand is a guarantee of the safety and quality of food. The presence of foodborne pathogens and their potential hazard, the use of genetically modified organisms (GMOs) in food production, and the correct labelling in foods suitable for vegetarians are among the subjects where society demands total transparency. The application of controls within the quality assessment programmes of the food industry is a way to satisfy these demands, and is necessary to ensure efficient analytical methodologies are possessed and correctly applied by the Food Sector. The use of real-time PCR has become a promising alternative approach in food diagnostics. It possesses a number of advantages over conventional culturing approaches, including rapidity, excellent analytical sensitivity and selectivity, and potential for quantification. However, the use of expensive equipment and reagents, the need for qualified personnel, and the lack of standardized protocols are impairing its practical implementation for food monitoring and control. PMID:23513039

  5. Real-time PCR in Food Science: PCR Diagnostics.

    PubMed

    Rodriguez-Lazaro, David; Cook, Nigel; Hernandez, Marta

    2013-01-01

    A principal consumer demand is a guarantee of the safety and quality of food. The presence of foodborne pathogens and their potential hazard, the use of genetically modified organisms (GMOs) in food production, and the correct labelling in foods suitable for vegetarians are among the subjects where society demands total transparency. The application of controls within the quality assessment programmes of the food industry is a way to satisfy these demands, and is necessary to ensure efficient analytical methodologies are possessed and correctly applied by the Food Sector. The use of real-time PCR has become a promising alternative approach in food diagnostics. It possesses a number of advantages over conventional culturing approaches, including rapidity, excellent analytical sensitivity and selectivity, and potential for quantification. However, the use of expensive equipment and reagents, the need for qualified personnel, and the lack of standardized protocols are impairing its practical implementation for food monitoring and control.

  6. Multiplex PCR: Optimization and Application in Diagnostic Virology

    PubMed Central

    Elnifro, Elfath M.; Ashshi, Ahmed M.; Cooper, Robert J.; Klapper, Paul E.

    2000-01-01

    PCR has revolutionized the field of infectious disease diagnosis. To overcome the inherent disadvantage of cost and to improve the diagnostic capacity of the test, multiplex PCR, a variant of the test in which more than one target sequence is amplified using more than one pair of primers, has been developed. Multiplex PCRs to detect viral, bacterial, and/or other infectious agents in one reaction tube have been described. Early studies highlighted the obstacles that can jeopardize the production of sensitive and specific multiplex assays, but more recent studies have provided systematic protocols and technical improvements for simple test design. The most useful of these are the empirical choice of oligonucleotide primers and the use of hot start-based PCR methodology. These advances along with others to enhance sensitivity and specificity and to facilitate automation have resulted in the appearance of numerous publications regarding the application of multiplex PCR in the diagnosis of infectious agents, especially those which target viral nucleic acids. This article reviews the principles, optimization, and application of multiplex PCR for the detection of viruses of clinical and epidemiological importance. PMID:11023957

  7. The Use of NS1 Rapid Diagnostic Test and qRT-PCR to Complement IgM ELISA for Improved Dengue Diagnosis from Single Specimen.

    PubMed

    Teoh, Boon-Teong; Sam, Sing-Sin; Tan, Kim-Kee; Johari, Jefree; Abd-Jamil, Juraina; Hooi, Poh-Sim; AbuBakar, Sazaly

    2016-06-09

    Timely and accurate dengue diagnosis is important for differential diagnosis and immediate implementation of appropriate disease control measures. In this study, we compared the usefulness and applicability of NS1 RDT (NS1 Ag Strip) and qRT-PCR tests in complementing the IgM ELISA for dengue diagnosis on single serum specimen (n = 375). The NS1 Ag Strip and qRT-PCR showed a fair concordance (κ = 0.207, p = 0.001). While the NS1 Ag Strip showed higher positivity than qRT-PCR for acute (97.8% vs. 84.8%) and post-acute samples (94.8% vs. 71.8%) of primary infection, qRT-PCR showed higher positivity for acute (58.1% vs. 48.4%) and post-acute (50.0% vs.41.4%) samples in secondary infection. IgM ELISA showed higher positivity in samples from secondary dengue (74.2-94.8%) than in those from primary dengue (21.7-64.1%). More primary dengue samples showed positive with combined NS1 Ag Strip/IgM ELISA (99.0% vs. 92.8%) whereas more secondary samples showed positive with combined qRT-PCR/IgM ELISA (99.4% vs. 96.2%). Combined NS1 Ag Strip/IgM ELISA is a suitable combination tests for timely and accurate dengue diagnosis on single serum specimen. If complemented with qRT-PCR, combined NS1 Ag Strip/IgM ELISA would improve detection of secondary dengue samples.

  8. The Use of NS1 Rapid Diagnostic Test and qRT-PCR to Complement IgM ELISA for Improved Dengue Diagnosis from Single Specimen

    PubMed Central

    Teoh, Boon-Teong; Sam, Sing-Sin; Tan, Kim-Kee; Johari, Jefree; Abd-Jamil, Juraina; Hooi, Poh-Sim; AbuBakar, Sazaly

    2016-01-01

    Timely and accurate dengue diagnosis is important for differential diagnosis and immediate implementation of appropriate disease control measures. In this study, we compared the usefulness and applicability of NS1 RDT (NS1 Ag Strip) and qRT-PCR tests in complementing the IgM ELISA for dengue diagnosis on single serum specimen (n = 375). The NS1 Ag Strip and qRT-PCR showed a fair concordance (κ = 0.207, p = 0.001). While the NS1 Ag Strip showed higher positivity than qRT-PCR for acute (97.8% vs. 84.8%) and post-acute samples (94.8% vs. 71.8%) of primary infection, qRT-PCR showed higher positivity for acute (58.1% vs. 48.4%) and post-acute (50.0% vs.41.4%) samples in secondary infection. IgM ELISA showed higher positivity in samples from secondary dengue (74.2–94.8%) than in those from primary dengue (21.7–64.1%). More primary dengue samples showed positive with combined NS1 Ag Strip/IgM ELISA (99.0% vs. 92.8%) whereas more secondary samples showed positive with combined qRT-PCR/IgM ELISA (99.4% vs. 96.2%). Combined NS1 Ag Strip/IgM ELISA is a suitable combination tests for timely and accurate dengue diagnosis on single serum specimen. If complemented with qRT-PCR, combined NS1 Ag Strip/IgM ELISA would improve detection of secondary dengue samples. PMID:27278716

  9. Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates.

    PubMed

    Baums, Christoph G; Schotte, Ulrich; Amtsberg, Gunter; Goethe, Ralph

    2004-05-20

    In this study we provide a protocol for genotyping Clostridium perfringens with a new multiplex PCR. This PCR enables reliable and specific detection of the toxin genes cpa, cpb, etx, iap, cpe and cpb2 from heat lysed bacterial suspensions. The efficiency of the protocol was demonstrated by typing C. perfringens reference strains and isolates from veterinary bacteriological routine diagnostic specimens.

  10. Diagnostic utility of droplet digital PCR for HIV reservoir quantification.

    PubMed

    Trypsteen, Wim; Kiselinova, Maja; Vandekerckhove, Linos; De Spiegelaere, Ward

    2016-01-01

    Quantitative real-time PCR (qPCR) is implemented in many molecular laboratories worldwide for the quantification of viral nucleic acids. However, over the last two decades, there has been renewed interest in the concept of digital PCR (dPCR) as this platform offers direct quantification without the need for standard curves, a simplified workflow and the possibility to extend the current detection limit. These benefits are of great interest in terms of the quantification of low viral levels in HIV reservoir research because changes in the dynamics of residual HIV reservoirs will be important to monitor HIV cure efforts. Here, we have implemented a systematic literature screening and text mining approach to map the use of droplet dPCR (ddPCR) in the context of HIV quantification. In addition, several technical aspects of ddPCR were compared with qPCR: accuracy, sensitivity, precision and reproducibility, to determine its diagnostic utility. We have observed that ddPCR was used in different body compartments in multiple HIV-1 and HIV-2 assays, with the majority of reported assays focusing on HIV-1 DNA-based applications (i.e. total HIV DNA). Furthermore, ddPCR showed a higher accuracy, precision and reproducibility, but similar sensitivity when compared to qPCR due to reported false positive droplets in the negative template controls with a need for standardised data analysis (i.e. threshold determination). In the context of a low level of detection and HIV reservoir diagnostics, ddPCR can offer a valid alternative to qPCR-based assays but before this platform can be clinically accredited, some remaining issues need to be resolved. PMID:27482456

  11. Diagnostic approaches for oculoglandular tularemia: advantages of PCR

    PubMed Central

    Kantardjiev, Todor; Padeshki, Plamen; Ivanov, Ivan N

    2007-01-01

    Aim The authors describe a diagnostic approach that proved to be particularly valuable in rare cases of ocular tularemia registered during the tularemia outbreak in 1997–2005 in Bulgaria. The authors describe the laboratory findings and diagnosis of four cases with an oculoglandular form of infection. Methods Several different specimens from each patient were analysed. Oculoglandular tularemia was diagnosed in four patients either by culture, immunofluorescent antibody analysis (IFA), serology or by a polymerase chain reaction (PCR) assay. Results and Discussion Three F tularensis strains were isolated and characterised. One of these was isolated from a conjuctival swab specimen obtained from a seronegative patient. The authors report for the first time a successful application of diagnostic PCR performed directly on conjuctival swab specimen. From all analysed specimens IFA was diagnostically effective only in the case of lymph node aspirates and was not sensitive enough for conjuctival swabs or blood samples. The authors also describe the histological picture of a conjunctival granuloma in course of infection. All patients were successfully treated with ciprofloxacin. Conclusions Some of the proposed laboratory diagnostic strategies (swab PCR) are not invasive and could represent a new approach for resolving rare and hard‐to‐diagnose cases of oculoglandular tularemia. PMID:17475710

  12. A Ribeiroia spp. (Class: Trematoda) - Specific PCR-based diagnostic

    USGS Publications Warehouse

    Reinitz, D.M.; Yoshino, T.P.; Cole, R.A.

    2007-01-01

    Increased reporting of amphibian malformations in North America has been noted with concern in light of reports that amphibian numbers and species are declining worldwide. Ribeiroia ondatrae has been shown to cause a variety of types of malformations in amphibians. However, little is known about the prevalence of R. ondatrae in North America. To aid in conducting field studies of Ribeiroia spp., we have developed a polymerase chain reaction (PCR)-based diagnostic. Herein, we describe the development of an accurate, rapid, simple, and cost-effective diagnostic for detection of Ribeiroia spp. infection in snails (Planorbella trivolvis). Candidate oligonucleotide primers for PCR were designed via DNA sequence analyses of multiple ribosomal internal transcribed spacer-2 regions from Ribeiroia spp. and Echinostoma spp. Comparison of consensus sequences determined from both genera identified areas of sequence potentially unique to Ribeiroia spp. The PCR reliably produced a diagnostic 290-base pair (bp) product in the presence of a wide concentration range of snail or frog DNA. Sensitivity was examined with DNA extracted from single R. ondatrae cercaria. The single-tube PCR could routinely detect less than 1 cercariae equivalent, because DNA isolated from a single cercaria could be diluted at least 1:50 and still yield a positive result via gel electrophoresis. An even more sensitive nested PCR also was developed that routinely detected 100 fg of the 290-bp fragment. The assay did not detect furcocercous cercariae of certain Schistosomatidae, Echinostoma sp., or Sphaeridiotrema globulus nor adults of Clinostomum sp. or Cyathocotyle bushiensis. Field testing of 137 P. trivolvis identified 3 positives with no overt environmental cross-reactivity, and results concurred with microscopic examinations in all cases. ?? American Society of Parasitologists 2007.

  13. Lab-on-a-chip PCR: real time PCR in miniaturized format for HLA diagnostics

    NASA Astrophysics Data System (ADS)

    Gaertner, Claudia; Becker, Holger; Hlawatsch, Nadine; Klemm, Richard; Moche, Christian; Sewart, René; Frank, Rainer; Willems, Andreas

    2014-05-01

    In case of transplantation or the identification of special metabolic diseases like coeliac disease, HLA typing has to be done fast and reliably with easy-to-handle devices by using limited amount of sample. Against this background a lab-on-a-chip device was realized enabling a fast HLA typing via miniaturized Real-time PCR. Hereby, two main process steps were combined, namely the extraction of DNA from whole blood and the amplification of the target DNA by Real-time PCR giving rise-to a semi-quantitative analysis. For the implementation of both processes on chip, a sample preparation and a real-time module were used. Sample preparation was carried out by using magnetic beads that were stored directly on chip as dry powder, together with all lysis reagents. After purification of the DNA by applying a special buffer regime, the sample DNA was transferred into the PCR module for amplification and detection. Coping with a massively increased surface-to-volume ratio, which results in a higher amount of unspecific binding on the chip surface, special additives needed to be integrated to compensate for this effect. Finally the overall procedure showed a sensitivity comparable to standard Real-time PCR but reduced the duration of analysis to significantly less than one hour. The presented work demonstrates that the combination of lab-on-a-chip PCR with direct optical read-out in a real-time fashion is an extremely promising tool for molecular diagnostics.

  14. Advances in Microfluidic PCR for Point-of-Care Infectious Disease Diagnostics

    PubMed Central

    Park, Seungkyung; Zhang, Yi; Lin, Shin; Wang, Tza-Huei; Yang, Samuel

    2011-01-01

    Global burdens from existing or emerging infectious diseases emphasize the need for point-of-care (POC) diagnostics to enhance timely recognition and intervention. Molecular approaches based on PCR methods have made significant inroads by improving detection time and accuracy but are still largely hampered by resource-intensive processing in centralized laboratories, thereby precluding their routine bedside- or field-use. Microfluidic technologies have enabled miniaturization of PCR processes onto a chip device with potential benefits including speed, cost, portability, throughput, and automation. In this review, we provide an overview of recent advances in microfluidic PCR technologies and discuss practical issues and perspectives related to implementing them into infectious disease diagnostics. PMID:21741465

  15. PCR diagnostic methods for Ascosphaera infections in bees.

    PubMed

    James, R R; Skinner, J S

    2005-10-01

    Fungi in the genus Ascosphaera are the causative agents of chalkbrood, a major disease affecting bee larval viability. Identification of individual Ascosphaera species based on morphological features has been difficult due to a lack of distinguishing characteristics. Most identifications are based on the size and shape of the ascomata, spore balls and conidia. Unfortunately, much overlap occurs in the size of these structures, and some Ascosphaera species will not produce sexual structures in vitro. We report a quick and reliable diagnostic method for identifying Ascosphaera infections in Megachile bees (leafcutting bees) using PCR markers that employ genus-specific primers for Ascosphaera, and species-specific primers for species known to be associated with Megachile spp. Using these methods, species identifications can be performed directly on bees, including asymptomatic individuals. Furthermore, the PCR markers can detect co-infections of multiple Ascosphaera species in a single host. We also identified a marker for Ascosphaera apis, the predominant cause of chalkbrood in Apis mellifera, the honey bee. Our diagnostic methods eliminate the need for culturing samples, and could be used to process a large number of field collected bee larvae. PMID:16214164

  16. TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR

    PubMed Central

    Zhang, Qian; Wang, Jing; Deng, Fang; Yan, Zhengjian; Xia, Yinglin; Wang, Zhongliang; Ye, Jixing; Deng, Youlin; Zhang, Zhonglin; Qiao, Min; Li, Ruifang; Denduluri, Sahitya K.; Wei, Qiang; Zhao, Lianggong; Lu, Shun; Wang, Xin; Tang, Shengli; Liu, Hao; Luu, Hue H.; Haydon, Rex C.; He, Tong-Chuan; Jiang, Li

    2015-01-01

    The advent of fluorescence-based quantitative real-time PCR (qPCR) has revolutionized the quantification of gene expression analysis in many fields, including life sciences, agriculture, forensic science, molecular diagnostics, and medicine. While SYBR Green-based qPCR is the most commonly-used platform due to its inexpensive nature and robust chemistry, quantifying the expression of genes with low abundance or RNA samples extracted from highly restricted or limited sources can be challenging because the detection sensitivity of SYBR Green-based qPCR is limited. Here, we develop a novel and effective touchdown qPCR (TqPCR) protocol by incorporating a 4-cycle touchdown stage prior to the quantification amplification stage. Using the same cDNA templates, we find that TqPCR can reduce the average Cq values for Gapdh, Rps13, and Hprt1 reference genes by 4.45, 5.47, and 4.94 cycles, respectively, when compared with conventional qPCR; the overall average Cq value reduction for the three reference genes together is 4.95. We further find that TqPCR can improve PCR amplification efficiency and thus increase detection sensitivity. When the quantification of Wnt3A-induced target gene expression in mesenchymal stem cells is analyzed, we find that, while both conventional qPCR and TqPCR can detect the up-regulation of the relatively abundant target Axin2, only TqPCR can detect the up-regulation of the lowly-expressed targets Oct4 and Gbx2. Finally, we demonstrate that the MRQ2 and MRQ3 primer pairs derived from mouse reference gene Tbp can be used to validate the RNA/cDNA integrity of qPCR samples. Taken together, our results strongly suggest that TqPCR may increase detection sensitivity and PCR amplification efficiency. Overall, TqPCR should be advantageous over conventional qPCR in expression quantification, especially when the transcripts of interest are lowly expressed, and/or the availability of total RNA is highly restricted or limited. PMID:26172450

  17. Large scale multiplex PCR improves pathogen detection by DNA microarrays

    PubMed Central

    2009-01-01

    Background Medium density DNA microchips that carry a collection of probes for a broad spectrum of pathogens, have the potential to be powerful tools for simultaneous species identification, detection of virulence factors and antimicrobial resistance determinants. However, their widespread use in microbiological diagnostics is limited by the problem of low pathogen numbers in clinical specimens revealing relatively low amounts of pathogen DNA. Results To increase the detection power of a fluorescence-based prototype-microarray designed to identify pathogenic microorganisms involved in sepsis, we propose a large scale multiplex PCR (LSplex PCR) for amplification of several dozens of gene-segments of 9 pathogenic species. This protocol employs a large set of primer pairs, potentially able to amplify 800 different gene segments that correspond to the capture probes spotted on the microarray. The LSplex protocol is shown to selectively amplify only the gene segments corresponding to the specific pathogen present in the analyte. Application of LSplex increases the microarray detection of target templates by a factor of 100 to 1000. Conclusion Our data provide a proof of principle for the improvement of detection of pathogen DNA by microarray hybridization by using LSplex PCR. PMID:19121223

  18. Improvement in early diagnosis of Japanese spotted fever by using a novel Rick PCR system.

    PubMed

    Kondo, Makoto; Akachi, Shigehiro; Kawano, Mitsuo; Yamanaka, Keiichi; Yamagiwa, Akisa; Gabazza, Esteban C; Ando, Katsuhiko; Mizutani, Hitoshi

    2015-11-01

    Rickettsia diseases, including Japanese spotted fever (JSF), are serious infections. Delayed diagnosis occasionally results in life-threatening liver disorders and disseminated intravascular coagulation (DIC). Because of the shortness of the latent period, serological diagnosis is not preferable for early diagnosis of JSF. Until now, a polymerase chain reaction (PCR)-based diagnosis method has been used for early diagnosis, and the sensitivity reaches as high as 90% using skin biopsy samples as we previously reported. On the other hand, the sensitivity of the same PCR method using blood samples is limited at less than 50%. In the present study, using peripheral blood samples, we developed a novel diagnostic method for JSF using a Rick PCR system with original PCR primers, showing improved sensitivity compared with the conventional nested PCR. It may constitute a preferable diagnostic tool for early and sensitive diagnosis of Rickettsia infection.

  19. A Rapid and Low-Cost PCR Thermal Cycler for Infectious Disease Diagnostics

    PubMed Central

    Chan, Kamfai; Wong, Pui-Yan; Yu, Peter; Hardick, Justin; Wong, Kah-Yat; Wilson, Scott A.; Wu, Tiffany; Hui, Zoe; Gaydos, Charlotte; Wong, Season S.

    2016-01-01

    The ability to make rapid diagnosis of infectious diseases broadly available in a portable, low-cost format would mark a great step forward in global health. Many molecular diagnostic assays are developed based on using thermal cyclers to carry out polymerase chain reaction (PCR) and reverse-transcription PCR for DNA and RNA amplification and detection, respectively. Unfortunately, most commercial thermal cyclers are expensive and need continuous electrical power supply, so they are not suitable for uses in low-resource settings. We have previously reported a low-cost and simple approach to amplify DNA using vacuum insulated stainless steel thermoses food cans, which we have named it thermos thermal cycler or TTC. Here, we describe the use of an improved set up to enable the detection of viral RNA targets by reverse-transcription PCR (RT-PCR), thus expanding the TTC’s ability to identify highly infectious, RNA virus-based diseases in low resource settings. The TTC was successful in demonstrating high-speed and sensitive detection of DNA or RNA targets of sexually transmitted diseases, HIV/AIDS, Ebola hemorrhagic fever, and dengue fever. Our innovative TTC costs less than $200 to build and has a capacity of at least eight tubes. In terms of speed, the TTC’s performance exceeded that of commercial thermal cyclers tested. When coupled with low-cost endpoint detection technologies such as nucleic acid lateral-flow assay or a cell-phone-based fluorescence detector, the TTC will increase the availability of on-site molecular diagnostics in low-resource settings. PMID:26872358

  20. A Rapid and Low-Cost PCR Thermal Cycler for Infectious Disease Diagnostics.

    PubMed

    Chan, Kamfai; Wong, Pui-Yan; Yu, Peter; Hardick, Justin; Wong, Kah-Yat; Wilson, Scott A; Wu, Tiffany; Hui, Zoe; Gaydos, Charlotte; Wong, Season S

    2016-01-01

    The ability to make rapid diagnosis of infectious diseases broadly available in a portable, low-cost format would mark a great step forward in global health. Many molecular diagnostic assays are developed based on using thermal cyclers to carry out polymerase chain reaction (PCR) and reverse-transcription PCR for DNA and RNA amplification and detection, respectively. Unfortunately, most commercial thermal cyclers are expensive and need continuous electrical power supply, so they are not suitable for uses in low-resource settings. We have previously reported a low-cost and simple approach to amplify DNA using vacuum insulated stainless steel thermoses food cans, which we have named it thermos thermal cycler or TTC. Here, we describe the use of an improved set up to enable the detection of viral RNA targets by reverse-transcription PCR (RT-PCR), thus expanding the TTC's ability to identify highly infectious, RNA virus-based diseases in low resource settings. The TTC was successful in demonstrating high-speed and sensitive detection of DNA or RNA targets of sexually transmitted diseases, HIV/AIDS, Ebola hemorrhagic fever, and dengue fever. Our innovative TTC costs less than $200 to build and has a capacity of at least eight tubes. In terms of speed, the TTC's performance exceeded that of commercial thermal cyclers tested. When coupled with low-cost endpoint detection technologies such as nucleic acid lateral-flow assay or a cell-phone-based fluorescence detector, the TTC will increase the availability of on-site molecular diagnostics in low-resource settings. PMID:26872358

  1. Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic laboratory setting

    USGS Publications Warehouse

    Purcell, Maureen K.; Getchell, Rodman G.; McClure, Carol A.; Weber, S.E.; Garver, Kyle A.

    2011-01-01

    Real-time, or quantitative, polymerase chain reaction (qPCR) is quickly supplanting other molecular methods for detecting the nucleic acids of human and other animal pathogens owing to the speed and robustness of the technology. As the aquatic animal health community moves toward implementing national diagnostic testing schemes, it will need to evaluate how qPCR technology should be employed. This review outlines the basic principles of qPCR technology, considerations for assay development, standards and controls, assay performance, diagnostic validation, implementation in the diagnostic laboratory, and quality assurance and control measures. These factors are fundamental for ensuring the validity of qPCR assay results obtained in the diagnostic laboratory setting.

  2. Assessing the Validity of Diagnostic Quantitative PCR Assays for Phakopsora pachyrhizi and P. meibomiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There are 123 confirmed species in the genus Phakopsora worldwide, with 19 species reported in the continental United States. In 2002, a quantitative PCR (qPCR) diagnostic assay was developed by Frederick et al. that has been used for detecting Phakopsora pachyrhizi in spore trapping studies. Based ...

  3. Engineered DNA polymerase improves PCR results for plastid DNA1

    PubMed Central

    Schori, Melanie; Appel, Maryke; Kitko, AlexaRae; Showalter, Allan M.

    2013-01-01

    • Premise of the study: Secondary metabolites often inhibit PCR and sequencing reactions in extractions from plant material, especially from silica-dried and herbarium material. A DNA polymerase that is tolerant to inhibitors improves PCR results. • Methods and Results: A novel DNA amplification system, including a DNA polymerase engineered via directed evolution for improved tolerance to common plant-derived PCR inhibitors, was evaluated and PCR parameters optimized for three species. An additional 31 species were then tested with the engineered enzyme and optimized protocol, as well as with regular Taq polymerase. • Conclusions: PCR products and high-quality sequence data were obtained for 96% of samples for rbcL and 79% for matK, compared to 29% and 21% with regular Taq polymerase. PMID:25202519

  4. Internal Amplification Control for a Cryptosporidium Diagnostic PCR: Construction and Clinical Evaluation.

    PubMed

    Hawash, Yousry; Ghonaim, M M; Al-Hazmi, Ayman S

    2015-04-01

    Various constituents in clinical specimens, particularly feces, can inhibit the PCR assay and lead to false-negative results. To ensure that negative results of a diagnostic PCR assay are true, it should be properly monitored by an inhibition control. In this study, a cloning vector harboring a modified target DNA sequence (≈375 bp) was constructed to be used as a competitive internal amplification control (IAC) for a conventional PCR assay that detects ≈550 bp of the Cryptosporidium oocyst wall protein (COWP) gene sequence in human feces. Modification of the native PCR target was carried out using a new approach comprising inverse PCR and restriction digestion techniques. IAC was included in the assay, with the estimated optimum concentration of 1 fg per reaction, as duplex PCR. When applied on fecal samples spiked with variable oocysts counts, ≈2 oocysts were theoretically enough for detection. When applied on 25 Cryptosporidium-positive fecal samples of various infection intensities, both targets were clearly detected with minimal competition noticed in 2-3 samples. Importantly, both the analytical and the diagnostic sensitivities of the PCR assay were not altered with integration of IAC into the reactions. When tried on 180 randomly collected fecal samples, 159 were Cryptosporidium-negatives. Although the native target DNA was absent, the IAC amplicon was obviously detected on gel of all the Cryptosporidium-negative samples. These results imply that running of the diagnostic PCR, inspired with the previously developed DNA extraction protocol and the constructed IAC, represents a useful tool for Cryptosporidium detection in human feces.

  5. Diagnostic Real-Time PCR for Detection of Salmonella in Food

    PubMed Central

    Malorny, Burkhard; Paccassoni, Elisa; Fach, Patrick; Bunge, Cornelia; Martin, Annett; Helmuth, Reiner

    2004-01-01

    A robust 5′ nuclease (TaqMan) real-time PCR was developed and validated in-house for the specific detection of Salmonella in food. The assay used specifically designed primers and a probe target within the ttrRSBCA locus, which is located near the Salmonella pathogenicity island 2 at centisome 30.5. It is required for tetrathionate respiration in Salmonella. The assay correctly identified all 110 Salmonella strains and 87 non-Salmonella strains tested. An internal amplification control, which is coamplified with the same primers as the Salmonella DNA, was also included in the assay. The detection probabilities were 70% when a Salmonella cell suspension containing 103 CFU/ml was used as a template in the PCR (5 CFU per reaction) and 100% when a suspension of 104 CFU/ml was used. A pre-PCR sample preparation protocol including a preenrichment step in buffered peptone water followed by DNA extraction-purification was applied when 110 various food samples (chicken rinses, minced meat, fish, and raw milk) were investigated for Salmonella. The diagnostic accuracy was shown to be 100% compared to the traditional culture method. The overall analysis time of the PCR method was approximately 24 h, in contrast to 4 to 5 days of analysis time for the traditional culture method. This methodology can contribute to meeting the increasing demand of quality assurance laboratories for standard diagnostic methods. Studies are planned to assess the interlaboratory performance of this diagnostic PCR method. PMID:15574899

  6. A real-time PCR diagnostic method for detection of Naegleria fowleri.

    PubMed

    Madarová, Lucia; Trnková, Katarína; Feiková, Sona; Klement, Cyril; Obernauerová, Margita

    2010-09-01

    Naegleria fowleri is a free-living amoeba that can cause primary amoebic meningoencephalitis (PAM). While, traditional methods for diagnosing PAM still rely on culture, more current laboratory diagnoses exist based on conventional PCR methods; however, only a few real-time PCR processes have been described as yet. Here, we describe a real-time PCR-based diagnostic method using hybridization fluorescent labelled probes, with a LightCycler instrument and accompanying software (Roche), targeting the Naegleria fowleriMp2Cl5 gene sequence. Using this method, no cross reactivity with other tested epidemiologically relevant prokaryotic and eukaryotic organisms was found. The reaction detection limit was 1 copy of the Mp2Cl5 DNA sequence. This assay could become useful in the rapid laboratory diagnostic assessment of the presence or absence of Naegleria fowleri.

  7. Improved PCR Amplification of Broad Spectrum GC DNA Templates

    PubMed Central

    Guido, Nicholas; Starostina, Elena; Leake, Devin; Saaem, Ishtiaq

    2016-01-01

    Many applications in molecular biology can benefit from improved PCR amplification of DNA segments containing a wide range of GC content. Conventional PCR amplification of DNA sequences with regions of GC less than 30%, or higher than 70%, is complex due to secondary structures that block the DNA polymerase as well as mispriming and mis-annealing of the DNA. This complexity will often generate incomplete or nonspecific products that hamper downstream applications. In this study, we address multiplexed PCR amplification of DNA segments containing a wide range of GC content. In order to mitigate amplification complications due to high or low GC regions, we tested a combination of different PCR cycling conditions and chemical additives. To assess the fate of specific oligonucleotide (oligo) species with varying GC content in a multiplexed PCR, we developed a novel method of sequence analysis. Here we show that subcycling during the amplification process significantly improved amplification of short template pools (~200 bp), particularly when the template contained a low percent of GC. Furthermore, the combination of subcycling and 7-deaza-dGTP achieved efficient amplification of short templates ranging from 10–90% GC composition. Moreover, we found that 7-deaza-dGTP improved the amplification of longer products (~1000 bp). These methods provide an updated approach for PCR amplification of DNA segments containing a broad range of GC content. PMID:27271574

  8. Improved PCR Amplification of Broad Spectrum GC DNA Templates.

    PubMed

    Guido, Nicholas; Starostina, Elena; Leake, Devin; Saaem, Ishtiaq

    2016-01-01

    Many applications in molecular biology can benefit from improved PCR amplification of DNA segments containing a wide range of GC content. Conventional PCR amplification of DNA sequences with regions of GC less than 30%, or higher than 70%, is complex due to secondary structures that block the DNA polymerase as well as mispriming and mis-annealing of the DNA. This complexity will often generate incomplete or nonspecific products that hamper downstream applications. In this study, we address multiplexed PCR amplification of DNA segments containing a wide range of GC content. In order to mitigate amplification complications due to high or low GC regions, we tested a combination of different PCR cycling conditions and chemical additives. To assess the fate of specific oligonucleotide (oligo) species with varying GC content in a multiplexed PCR, we developed a novel method of sequence analysis. Here we show that subcycling during the amplification process significantly improved amplification of short template pools (~200 bp), particularly when the template contained a low percent of GC. Furthermore, the combination of subcycling and 7-deaza-dGTP achieved efficient amplification of short templates ranging from 10-90% GC composition. Moreover, we found that 7-deaza-dGTP improved the amplification of longer products (~1000 bp). These methods provide an updated approach for PCR amplification of DNA segments containing a broad range of GC content.

  9. Suitability of stx-PCR directly from fecal samples in clinical diagnostics of STEC.

    PubMed

    Tunsjø, Hege S; Kvissel, Anne K; Follin-Arbelet, Benoit; Brotnov, Beth-Marie; Ranheim, Trond E; Leegaard, Truls M

    2015-10-01

    PCR-based testing for Shiga toxin producing Escherichia coli (STEC) directly from fecal samples is increasingly being implemented in routine diagnostic laboratories. These methods aim to detect clinically relevant amounts of microbes and not stx-carrying phages or low backgrounds of STEC. We present a diagnostic procedure and results from 1 year of stx-targeted real-time PCR of fecal samples from patients with gastrointestinal symptoms in Norway. A rapid stx2 subtyping strategy is described, which aims to quickly reveal the virulence potential of the microbe. stx was detected in 22 of 3320 samples, corresponding to a PCR positive rate of 0.66%. STEC were cultured from 72% of the PCR positive samples. Four stx1 isolates, eight stx2 isolates, and four isolates with both stx1 and stx2 were identified. With the method presented, stx-carrying phages are not commonly detected. Our results support the use of molecular testing combined with classical culture techniques for routine diagnostic purposes.

  10. Digital Mammography: Improvements in Breast Cancer Diagnostic

    NASA Astrophysics Data System (ADS)

    Montaño Zetina, Luis Manuel

    2006-01-01

    X-ray mammography is the most sensitive imaging technique for early detection of breast cancer (diagnostics). It is performed by a radiological system equipped with a rotating molybdenum (Mo) anode tube with an additional Mo filter. In the production of X-ray, bremsstrahlung photons produce an intense diffuse radiation, affecting the contrast between normal and cancerous tissue. So it is known that a good mammographic imaging can help to detect cancer in the first stages avoiding surgery, amputation or even death. In the last years there has been some developments in new imaging techniques to improve the contrast spatial resolution between different tissues: digital imaging, or the so call digital mammography. Digital mammographic imaging is considered an improvement in the prevention of breast cancer due to the advantages it offers.

  11. Digital Mammography: Improvements in Breast Cancer Diagnostic

    SciTech Connect

    Montano Zetina, Luis Manuel

    2006-01-06

    X-ray mammography is the most sensitive imaging technique for early detection of breast cancer (diagnostics). It is performed by a radiological system equipped with a rotating molybdenum (Mo) anode tube with an additional Mo filter. In the production of X-ray, bremsstrahlung photons produce an intense diffuse radiation, affecting the contrast between normal and cancerous tissue. So it is known that a good mammographic imaging can help to detect cancer in the first stages avoiding surgery, amputation or even death. In the last years there has been some developments in new imaging techniques to improve the contrast spatial resolution between different tissues: digital imaging, or the so call digital mammography. Digital mammographic imaging is considered an improvement in the prevention of breast cancer due to the advantages it offers.

  12. Harmonization of Bordetella pertussis real-time PCR diagnostics in the United States in 2012.

    PubMed

    Williams, Margaret M; Taylor, Thomas H; Warshauer, David M; Martin, Monte D; Valley, Ann M; Tondella, M Lucia

    2015-01-01

    Real-time PCR (rt-PCR) is an important diagnostic tool for the identification of Bordetella pertussis, Bordetella holmesii, and Bordetella parapertussis. Most U.S. public health laboratories (USPHLs) target IS481, present in 218 to 238 copies in the B. pertussis genome and 32 to 65 copies in B. holmesii. The CDC developed a multitarget PCR assay to differentiate B. pertussis, B. holmesii, and B. parapertussis and provided protocols and training to 19 USPHLs. The 2012 performance exercise (PE) assessed the capability of USPHLs to detect these three Bordetella species in clinical samples. Laboratories were recruited by the Wisconsin State Proficiency Testing program through the Association of Public Health Laboratories, in partnership with the CDC. Spring and fall PE panels contained 12 samples each of viable Bordetella and non-Bordetella species in saline. Fifty and 53 USPHLs participated in the spring and fall PEs, respectively, using a variety of nucleic acid extraction methods, PCR platforms, and assays. Ninety-six percent and 94% of laboratories targeted IS481 in spring and fall, respectively, in either singleplex or multiplex assays. In spring and fall, respectively, 72% and 79% of USPHLs differentiated B. pertussis and B. holmesii and 68% and 72% identified B. parapertussis. IS481 cycle threshold (CT) values for B. pertussis samples had coefficients of variation (CV) ranging from 10% to 28%. Of the USPHLs that differentiated B. pertussis and B. holmesii, sensitivity was 96% and specificity was 95% for the combined panels. The 2012 PE demonstrated increased harmonization of rt-PCR Bordetella diagnostic protocols in USPHLs compared to that of the previous survey.

  13. An improved PCR method for gender identification of eagles.

    PubMed

    Chang, Hsueh-Wei; Chou, Ta-Ching; Gu, De-Leung; Cheng, Chun-An; Chang, Chia-Che; Yao, Cheng-Te; Chuang, Li-Yeh; Wen, Cheng-Hao; Chou, Yii-Cheng; Tan, Kock-Yee; Cheng, Chien-Chung

    2008-06-01

    Eagles are sexually monomorphic and therefore it is difficult to determine their gender, which is a crucial need for management purposes. In this study, we have developed an improved gender identification method by exploiting length differences between the Chromo-Helicase-DNA binding protein (CHD)-Z and CHD-W genes of Spilornis cheela hoya. By comparing DNA sequences for CHD-W and CHD-Z from 10 species of Falconiformes eagles we designed universal gender identification PCR primers that exploit differences in product size. Standard agarose gels were shown to easily distinguish between the 148-bp CHD-ZW and the 258-bp CHD-W PCR products. When used with 28 samples of S. cheela hoya, our improved universal primers provided a fast and precise gender identification assay. PMID:18385011

  14. Molecular diagnostics via mass spectrometry of PCR-amplified DNA products

    SciTech Connect

    Buchanan, M.; Doktycz, M.; Hurst, G.

    1995-12-31

    Identifying the presence of a specific DNA fragment is becoming increasingly critical to many applications in medical, clinical, forensic and other research laboratories. At present, regions of interest in DNA are amplified using the Polymerase Chain Reaction (PCR) or other reactions to produce fragments containing a specific number of nucleotide units that are diagnostic for the targeted genetic disease, person, or pathogen. These fragments are then typically analyzed by slab gel electrophoresis. Mass spectrometry has the potential of characterizing the DNA fragments faster and more confidently than chromatography-based methods. The authors have evaluated matrix assisted laser desorption (MALDI) time-of-flight (TOF) and electrospray (ES) quadrupole ion trap (QIT) mass spectrometry for the rapid analysis of PCR fragments.

  15. DEVELOPMENT OF AN IMPROVED PCR-BASED TECHNIQUE FOR DETECTION OF PHYTOPHTHORA CACTORUM IN STRAWBERRY PLANTS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Specific and rapid plant pathogen detection methods can aid in strawberry disease management decisions. PCR-based diagnostics for Phytophthora cactorum and other strawberry pathogens are hindered by PCR inhibitors and lack of species-specific PCR primers. We developed a DNA extraction and purificati...

  16. PCR for Detection of Herpes Simplex Virus in Cerebrospinal Fluid: Alternative Acceptance Criteria for Diagnostic Workup

    PubMed Central

    Alonso, Roberto; de Egea, Viviana; Usubillaga, Rafael; Muñoz, Patricia; Bouza, Emilio

    2013-01-01

    The determination of herpes simplex virus (HSV) infection using a PCR assay is one of the most commonly requested tests for analysis of cerebrospinal fluid (CSF), although only a very low proportion of results are positive. A previously reported study showed that selecting only those CSF samples with >5 leukocytes/mm3 or a protein level of >50 mg/dl was adequate for the diagnostic workup. The aim of the present study was to assess the reliability of alternative acceptance criteria based on elevated CSF white blood cell counts (>10 cells/mm3). We analyzed all requests for HSV PCR received between January 2008 and December 2011. CSF samples were accepted for analysis if they had >10 cells/mm3 or if the sample was from an immunocompromised patient or a child aged <2 years. In order to evaluate our selection criteria, we identified those CSF samples with a leukocyte count of 5 to 10 cells/mm3 or protein levels of >50 mg/dl in order to test them for HSV type 1 and 2 (HSV-1 and HSV-2) DNA. During the study period, 466 CSF samples were submitted to the microbiology laboratory for HSV PCR. Of these, 268 (57.5%) were rejected, and 198 (42.5%) were tested according to our routine criteria. Of the tested samples, 11 (5.5%) were positive for HSV DNA (7 for HSV-1 and 4 for HSV-2). Of the 268 rejected specimens, 74 met the criteria of >5 cells/mm3 and/or protein levels of >50 mg/dl. Of these, 70 (94.6%) were available for analysis. None of the samples yielded a positive HSV PCR result. Acceptance criteria based on CSF leukocyte counts, host immune status, and age can help to streamline the application of HSV PCR without reducing sensitivity. PMID:23804382

  17. The Diagnostic Utility of Bact/ALERT and Nested PCR in the Diagnosis of Tuberculous Meningitis

    PubMed Central

    Sastry, Apurba Sankar; Bhat K, Sandhya; Kumudavathi

    2013-01-01

    Objective: The early laboratory diagnosis of Tuberculous Meningitis (TBM) is crucial, to start the antitubercular chemotherapy and to prevent its complications. However, the conventional methods are either less sensitive or time consuming. Hence, the diagnostic potentials of BacT/ALERT and Polymerase Chain Reaction (PCR) was evaluated in this study. Material and Method: The study group comprised of 62 cases and 33 controls. The cases were divided according to Ahuja’s criteria into the confirmed (two cases), highly probable (19 cases), probable (26 cases) and the possible (15 cases) subgroups. Ziehl Neelsen’s (ZN) and Auramine Phenol (AP) staining, Lowenstein Jensen (LJ) medium culture, BacT/ALERT and nested Polymerase Chain Reaction (PCR) which targeted IS6110 were carried out on all the patients. Observation and Results: The sensitivity of the LJ culture was 3.22%. BacT/ALERT showed a sensitivity and a specificity of 25.80% and 100% and those of nested PCR were found to be 40.32% and 96.97% respectively. The mean detection time of growth of the LJ culture was 31.28 days, whereas that of BacT/ALERT was 20.68 days. The contamination rate in the LJ culture and BacT/ALERT were 7.2% and 5.8% respectively. Conclusion: Nested PCR was found to be more sensitive, followed by BacT/ALERT as compared to the LJ culture and smear microscopy. As both false negative and false positive results have been reported for nested PCR, so it should not be used alone as a criterion for initiating or terminating the therapy, but it should be supported by clinical, radiological, cytological and other microbiological findings. PMID:23450650

  18. Improved sensitivity of PCR for Chlamydophila using pmp genes.

    PubMed

    Laroucau, K; Souriau, A; Rodolakis, A

    2001-09-20

    Primers targeting the conserved pmp gene family of Chlamydophila abortus were evaluated for their ability to improve the polymerase chain reaction (PCR) sensitivity. In purified DNA, specific pmp primers (named CpsiA and CpsiB) allowed at least a 10-fold increase of the PCR sensitivity compared to the specific ompA primers for C. abortus, but also for C. psittaci and C. caviae strains. No amplification was observed on C. felis, C. pecorum, C. pneumoniae and Chlamydia trachomatis strains. Tested on contaminated specimens such as genital swabs, the PCR sensitivity observed with CpsiA/CpsiB was also better than with the ompA primers. This study demonstrated that these specific pmp primers could serve as valuable, sensitive and common tools for a specific Chlamydophila diagnosis in ruminant, avian and human diseases. Digestion by AluI of the CpsiA/CpsiB fragments allowed a specific discrimination of the strains in function of their hosts and/or their serotypes.

  19. Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design

    PubMed Central

    Pilotte, Nils; Papaiakovou, Marina; Grant, Jessica R.; Bierwert, Lou Ann; Llewellyn, Stacey; McCarthy, James S.; Williams, Steven A.

    2016-01-01

    Background The soil transmitted helminths are a group of parasitic worms responsible for extensive morbidity in many of the world’s most economically depressed locations. With growing emphasis on disease mapping and eradication, the availability of accurate and cost-effective diagnostic measures is of paramount importance to global control and elimination efforts. While real-time PCR-based molecular detection assays have shown great promise, to date, these assays have utilized sub-optimal targets. By performing next-generation sequencing-based repeat analyses, we have identified high copy-number, non-coding DNA sequences from a series of soil transmitted pathogens. We have used these repetitive DNA elements as targets in the development of novel, multi-parallel, PCR-based diagnostic assays. Methodology/Principal Findings Utilizing next-generation sequencing and the Galaxy-based RepeatExplorer web server, we performed repeat DNA analysis on five species of soil transmitted helminths (Necator americanus, Ancylostoma duodenale, Trichuris trichiura, Ascaris lumbricoides, and Strongyloides stercoralis). Employing high copy-number, non-coding repeat DNA sequences as targets, novel real-time PCR assays were designed, and assays were tested against established molecular detection methods. Each assay provided consistent detection of genomic DNA at quantities of 2 fg or less, demonstrated species-specificity, and showed an improved limit of detection over the existing, proven PCR-based assay. Conclusions/Significance The utilization of next-generation sequencing-based repeat DNA analysis methodologies for the identification of molecular diagnostic targets has the ability to improve assay species-specificity and limits of detection. By exploiting such high copy-number repeat sequences, the assays described here will facilitate soil transmitted helminth diagnostic efforts. We recommend similar analyses when designing PCR-based diagnostic tests for the detection of other

  20. Rational primer design greatly improves differential display-PCR (DD-PCR).

    PubMed Central

    Graf, D; Fisher, A G; Merkenschlager, M

    1997-01-01

    Since its conception in 1992, differential display PCR (DD-PCR) has attracted widespread interest. Theoretically an attractive cloning approach, it combines the comparative analysis of several samples with the sensitivity of PCR. Although a large number of studies embracing this technology have been initiated, few novel genes of interest have been identified, suggesting that the method has not realised its potential. The present report shows that by modifying primer design, sampling of differentially expressed genes can be greatly enhanced and relevant genes can be isolated. Using our modified conditions DD-PCR efficiently screens a wide range of gene expression levels, in which differences are represented on a linear scale. PMID:9153330

  1. Diagnostic PCR can be used to illuminate meiofaunal diets and trophic relationships

    PubMed Central

    Maghsoud, Hanna; Weiss, Austin; Smith, Julian P.S.; Litvaitis, Marian K.; Fegley, Stephen R.

    2014-01-01

    Analysis of the meiofaunal food web is hampered because few prey have features that persist long enough in a predator’s digestive tract to allow identification to species. Hence, at least for platyhelminth predators, direct observations of prey preference are almost nonexistent, and where they occur, prey identification is often limited to phylum. Studies using an in vitro approach are rare because they are extremely time-consuming and are subject to the criticism that predators removed from their natural environment may exhibit altered behaviors. Although PCR-based approaches have achieved wide application in food-web analysis, their application to meiofaunal flatworms suffers from a number of limitations. Most importantly, the microscopic size of both the predator and prey does not allow for removal of prey material from the digestive tract of the predator, and thus the challenge is to amplify prey sequences in the presence of large quantities of predator sequence. Here, we report on the successful use of prey-taxon-specific primers in diagnostic PCR to identify, to species level, specific prey items of 13 species of meiofaunal flatworms. Extension of this method will allow, for the first time, the development of a species-level understanding of trophic interactions among the meiofauna. PMID:25071364

  2. Performance of a new gelled nested PCR test for the diagnosis of imported malaria: comparison with microscopy, rapid diagnostic test, and real-time PCR.

    PubMed

    Iglesias, Nuria; Subirats, Mercedes; Trevisi, Patricia; Ramírez-Olivencia, Germán; Castán, Pablo; Puente, Sabino; Toro, Carlos

    2014-07-01

    Microscopy and rapid diagnostic tests (RDTs) are the techniques commonly used for malaria diagnosis but they are usually insensitive at very low levels of parasitemia. Nested PCR is commonly used as a reference technique in the diagnosis of malaria due to its high sensitivity and specificity. However, it is a cumbersome assay only available in reference centers. We evaluated a new nested PCR-based assay, BIOMALAR kit (Biotools B&M Labs, Madrid, Spain) which employs ready-to-use gelled reagents and allows the identification of the main four species of Plasmodium. Blood samples were obtained from patients with clinical suspicion of malaria. A total of 94 subjects were studied. Fifty-two (55.3%) of them were malaria-infected subjects corresponding to 48 cases of Plasmodium falciparum, 1 Plasmodium malariae, 2 Plasmodium vivax, and 1 Plasmodium ovale. The performance of the BIOMALAR test was compared with microscopy, rapid diagnostic test (RDT) (BinaxNOW® Malaria) and real-time quantitative PCR (qPCR). The BIOMALAR test showed a sensitivity of 98.1% (95% confidence interval [CI], 89.7-100), superior to microscopy (82.7% [95% CI, 69.7-91.8]) and RDT (94.2% [95% CI, 84.1-98.8]) and similar to qPCR (100% [95% CI, 93.2-100]). In terms of specificity, the BIOMALAR assay showed the same value as microscopy and qPCR (100% [95% CI, 93.2-100]). Nine subjects were submicroscopic carriers of malaria. The BIOMALAR test identified almost all of them (8/9) in comparison with RDT (6/9) and microscopy (0/9). In conclusion, the BIOMALAR is a PCR-based assay easy to use with an excellent performance and especially useful for diagnosis submicroscopic malaria. PMID:24770719

  3. Better Tests, Better Care: Improved Diagnostics for Infectious Diseases

    PubMed Central

    Caliendo, Angela M.; Gilbert, David N.; Ginocchio, Christine C.; Hanson, Kimberly E.; May, Larissa; Quinn, Thomas C.; Tenover, Fred C.; Alland, David; Blaschke, Anne J.; Bonomo, Robert A.; Carroll, Karen C.; Ferraro, Mary Jane; Hirschhorn, Lisa R.; Joseph, W. Patrick; Karchmer, Tobi; MacIntyre, Ann T.; Reller, L. Barth; Jackson, Audrey F.

    2013-01-01

    In this IDSA policy paper, we review the current diagnostic landscape, including unmet needs and emerging technologies, and assess the challenges to the development and clinical integration of improved tests. To fulfill the promise of emerging diagnostics, IDSA presents recommendations that address a host of identified barriers. Achieving these goals will require the engagement and coordination of a number of stakeholders, including Congress, funding and regulatory bodies, public health agencies, the diagnostics industry, healthcare systems, professional societies, and individual clinicians. PMID:24200831

  4. Aspergillus PCR-Based Investigation of Fresh Tissue and Effusion Samples in Patients with Suspected Invasive Aspergillosis Enhances Diagnostic Capabilities

    PubMed Central

    Reinwald, M.; Spiess, B.; Heinz, W. J.; Heussel, C. P.; Bertz, H.; Cornely, O. A.; Hahn, J.; Lehrnbecher, T.; Kiehl, M.; Laws, H. J.; Wolf, H. H.; Schwerdtfeger, R.; Schultheis, B.; Burchardt, A.; Klein, M.; Dürken, M.; Claus, B.; Schlegel, F.; Hummel, M.; Hofmann, W.-K.

    2013-01-01

    Although it is a severe complication in immunocompromised patients, diagnosing invasive fungal disease (IFD), especially invasive aspergillosis (IA), remains difficult. In certain clinical scenarios, examining tissue samples for identification of the infectious organism becomes important. As culture-based methods rarely yield results, the performance of an Aspergillus-specific nested PCR in fresh tissue or pleural effusion samples was evaluated. Fresh tissue (n = 59) and effusion (n = 47) specimens from 79 immunocompromised patients were subjected to an Aspergillus-specific PCR assay. Twenty-six patients had proven (n = 20) or probable (n = 6) IFD, according to the 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) criteria, while the remaining patients were classified as having either possible IFD (n = 30) or no IFD (n = 23). IA was identified as the underlying IFD in 21/26 proven/probable cases. PCR positivity was observed for 18/21 proven/probable and 6 possible IA cases; cases classified as no IA did not show positive signals. Patients with proven IFD (n = 5) with cultures positive for non-Aspergillus molds also had negative Aspergillus PCR results. Aspergillus PCR performance analysis yielded sensitivity and specificity values of 86% (95% confidence interval [CI], 65% to 95%) and 100% (95% CI, 86% to 100%), respectively, thus leading to a diagnostic odds ratio of >200. In this analysis, good diagnostic performance of the PCR assay for detection of IA was observed for tissue samples, while effusion samples showed lower sensitivity rates. PCR testing represents a complementary tool; a positive PCR result strengthens the likelihood of IA, whereas IA seems unlikely in cases with negative results but findings could indicate non-Aspergillus IFD. Thus, PCR testing of these specimens enhances the diagnostic capabilities. PMID

  5. An improved molecular diagnostic assay for canine and feline dermatophytosis.

    PubMed

    Cafarchia, Claudia; Gasser, Robin B; Figueredo, Luciana A; Weigl, Stefania; Danesi, Patrizia; Capelli, Gioia; Otranto, Domenico

    2013-02-01

    The few studies attempting to specifically characterize dermatophytes from hair samples of dogs and cats using PCR-based methodology relied on sequence-based analysis of selected genetic markers. The aim of the present investigation was to establish and evaluate a PCR-based approach employing genetic markers of nuclear DNA for the specific detection of dermatophytes on such specimens. Using 183 hair samples, we directly compared the test results of our one-step and nested-PCR assays with those based on conventional microscopy and in vitro culture techniques (using the latter as the reference method). The one step-PCR was highly accurate (AUC > 90) for the testing of samples from dogs, but only moderately accurate (AUC = 78.6) for cats. A nested-PCR was accurate (AUC = 93.6) for samples from cats, and achieved higher specificity (94.1 and 94.4%) and sensitivity (100 and 94.9%) for samples from dogs and cats, respectively. In addition, the nested-PCR allowed the differentiation of Microsporum canis from Trichophyton interdigitale (zoophilic) and geophilic dermatophytes (i.e., Microsporum gypseum or Trichophyton terrestre), which was not possible using the one step-assay. The PCRs evaluated here provide practical tools for diagnostic applications to support clinicians in initiating prompt and targeted chemotherapy of dermatophytoses. PMID:22686247

  6. Evaluation of real-time PCR for Strongyloides stercoralis and hookworm as diagnostic tool in asymptomatic schoolchildren in Cambodia.

    PubMed

    Schär, Fabian; Odermatt, Peter; Khieu, Virak; Panning, Marcus; Duong, Socheat; Muth, Sinuon; Marti, Hanspeter; Kramme, Stefanie

    2013-05-01

    Diagnosis of soil-transmitted helminths such as Strongyloides stercoralis and hookworms (Ancylostoma duodenale and Necator americanus) is challenging due to irregular larval and egg output in infected individuals and insensitive conventional diagnostic procedures. Sensitive novel real-time PCR assays have been developed. Our study aimed to evaluate the real-time PCR assays as a diagnostic tool for detection of Strongyloides spp. and hookworms in a random stool sample of 218 asymptomatic schoolchildren in Cambodia. Overall prevalence of 17.4% (38/218) and 34.9% (76/218) were determined by real-time PCR for S. stercoralis and hookworms, respectively. Sensitivity and specificity of S. stercoralis specific real-time PCR as compared to the combination of Baermann/Koga Agar as gold standard were 88.9% and 92.7%, respectively. For hookworm specific real-time PCR a sensitivity of 78.9% and specificity of 78.9% were calculated. Co-infections were detectable by PCR in 12.8% (28/218) of individuals. S. stercoralis real-time PCR applied in asymptomatic cases showed a lower sensitivity compared to studies undertaken with symptomatic patients with the same molecular tool, yet it proved to be a valid supplement in the diagnosis of STH infection in Cambodia.

  7. Direct PCR Improves the Recovery of DNA from Various Substrates.

    PubMed

    Templeton, Jennifer E L; Taylor, Duncan; Handt, Oliva; Skuza, Pawel; Linacre, Adrian

    2015-11-01

    This study reports on the comparison of a standard extraction process with the direct PCR approach of processing low-level DNA swabs typical in forensic investigations. Varying concentrations of control DNA were deposited onto three commonly encountered substrates, brass, plastic, and glass, left to dry, and swabbed using premoistened DNA-free nylon FLOQswabs(™) . Swabs (n = 90) were either processed using the DNA IQ(™) kit or, for direct PCR, swab fibers (~2 mm(2) ) were added directly to the PCR with no prior extraction. A significant increase in the height of the alleles (p < 0.005) was observed when using the direct PCR approach over the extraction methodology when controlling for surface type and mass of DNA deposited. The findings indicate the potential use of direct PCR for increasing the PCR product obtained from low-template DNA samples in addition to minimizing contamination and saving resources.

  8. Rapid and Sensitive Detection of Yersinia pestis Using Amplification of Plague Diagnostic Bacteriophages Monitored by Real-Time PCR

    PubMed Central

    Sergueev, Kirill V.; He, Yunxiu; Borschel, Richard H.; Nikolich, Mikeljon P.; Filippov, Andrey A.

    2010-01-01

    Background Yersinia pestis, the agent of plague, has caused many millions of human deaths and still poses a serious threat to global public health. Timely and reliable detection of such a dangerous pathogen is of critical importance. Lysis by specific bacteriophages remains an essential method of Y. pestis detection and plague diagnostics. Methodology/Principal Findings The objective of this work was to develop an alternative to conventional phage lysis tests – a rapid and highly sensitive method of indirect detection of live Y. pestis cells based on quantitative real-time PCR (qPCR) monitoring of amplification of reporter Y. pestis-specific bacteriophages. Plague diagnostic phages ϕA1122 and L-413C were shown to be highly effective diagnostic tools for the detection and identification of Y. pestis by using qPCR with primers specific for phage DNA. The template DNA extraction step that usually precedes qPCR was omitted. ϕA1122-specific qPCR enabled the detection of an initial bacterial concentration of 103 CFU/ml (equivalent to as few as one Y. pestis cell per 1-µl sample) in four hours. L-413C-mediated detection of Y. pestis was less sensitive (up to 100 bacteria per sample) but more specific, and thus we propose parallel qPCR for the two phages as a rapid and reliable method of Y. pestis identification. Importantly, ϕA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR. Conclusions/Significance Thus, we developed a novel assay for detection and identification of Y. pestis using amplification of specific phages monitored by qPCR. The method is simple, rapid, highly sensitive, and specific and allows the detection of only live bacteria. PMID:20596528

  9. An improved, PCR-based strategy for the detection of Trypanosoma cruzi in human blood samples.

    PubMed

    Ribeiro-dos-Santos, G; Nishiya, A S; Sabino, E C; Chamone, D F; Saez-Alquézar, A

    1999-10-01

    Attempts were made to improve the PCR-based detection of Trypanosoma cruzi in blood samples, primarily for screening blood donors. Samples were obtained from candidate donors who were reactive in one or two of three serological tests for Chagas disease (and therefore considered 'indeterminate') or in all three tests (3+). Each sample was then examined using three different, PCR-based techniques: 'PCR-I' (in which the target DNA is a nuclear repetitive sequence); 'PCR-II' [amplifying a conserved region of the T. cruzi kinetoplast DNA (kDNA)]; and 'PCR-III' (a new strategy in which the target kDNA is amplified by 'nested' PCR). Among the samples from 3+ individuals, PCR-I, PCR-II and PCR-III amplified two (3.8%) out of 52, four (4.5%) out of 88, and 27 (25.7%) out of 105 samples tested, respectively. Seven, 69 and 70 samples from 'indeterminate' subjects were tested by PCR-I, PCR-II and PCR-III, respectively; there was not a single positive result by PCR-I or PCR-II, but three (4.3%) of the samples tested by PCR-III were positive. In a reconstruction experiment, in conditions in which PCR-I and PCR-II could not detect 10,000 parasites/ml, PCR-III was able to detect one parasite/ml. Although all three PCR-based strategies examined had rather poor sensitivities, PCR-III was far more sensitive than PCR-I or PCR-II. PMID:10715696

  10. 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

  11. 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

  12. Rapid detection and high occurrence of porcine rotavirus A, B, and C by RT-qPCR in diagnostic samples.

    PubMed

    Marthaler, Douglas; Homwong, Nitipong; Rossow, Kurt; Culhane, Marie; Goyal, Sagar; Collins, James; Matthijnssens, Jelle; Ciarlet, Max

    2014-12-01

    Rotaviruses are important cause of diarrhea in animals, including humans. Currently, rotavirus species A, B, C, E, and H (RVA-RVC, RVE, and RVH) have been identified in pigs. Traditionally, RVA has been considered the primary cause of diarrhea in pigs, and RVB and RVC had been described sporadically in pigs until recently. Qualitative porcine RVA, RVB, and RVC RT-PCR (RT-qPCR) assays were designed and 7508 porcine diarrheic samples, submitted to University of Minnesota, were tested to estimate the percentage of RVA, RVB, and RVC over a period of approximately 2 years (from 2009 to 2011). The individual RVA and RVC RT-qPCR assays were multiplex into a single RT-qPCR while the RVB RT-qPCR assay remained as an individual RT-qPCR. In total, 83% of the samples were positive for RVA, RVB, or RVC. As expected, RVA was detected at the highest overall percentage (62%). However, 33% and 53% of the samples were positive for RVB and RVC, respectively, indicating that both RVB and RVC are also epidemiologically important in the swine population. RVC was most predominant in young pigs (1-20 days of age), while RVA and RVB were most predominant in ≥21 day old pigs. As diagnostic tools, the developed RT-qPCR assays could successfully discriminate among infecting RV species, which could lead to better surveillance and epidemiological studies for ultimately better prevention and control strategies.

  13. Rapid detection and high occurrence of porcine rotavirus A, B, and C by RT-qPCR in diagnostic samples.

    PubMed

    Marthaler, Douglas; Homwong, Nitipong; Rossow, Kurt; Culhane, Marie; Goyal, Sagar; Collins, James; Matthijnssens, Jelle; Ciarlet, Max

    2014-12-01

    Rotaviruses are important cause of diarrhea in animals, including humans. Currently, rotavirus species A, B, C, E, and H (RVA-RVC, RVE, and RVH) have been identified in pigs. Traditionally, RVA has been considered the primary cause of diarrhea in pigs, and RVB and RVC had been described sporadically in pigs until recently. Qualitative porcine RVA, RVB, and RVC RT-PCR (RT-qPCR) assays were designed and 7508 porcine diarrheic samples, submitted to University of Minnesota, were tested to estimate the percentage of RVA, RVB, and RVC over a period of approximately 2 years (from 2009 to 2011). The individual RVA and RVC RT-qPCR assays were multiplex into a single RT-qPCR while the RVB RT-qPCR assay remained as an individual RT-qPCR. In total, 83% of the samples were positive for RVA, RVB, or RVC. As expected, RVA was detected at the highest overall percentage (62%). However, 33% and 53% of the samples were positive for RVB and RVC, respectively, indicating that both RVB and RVC are also epidemiologically important in the swine population. RVC was most predominant in young pigs (1-20 days of age), while RVA and RVB were most predominant in ≥21 day old pigs. As diagnostic tools, the developed RT-qPCR assays could successfully discriminate among infecting RV species, which could lead to better surveillance and epidemiological studies for ultimately better prevention and control strategies. PMID:25194889

  14. Improving early cycle economic evaluation of diagnostic technologies.

    PubMed

    Steuten, Lotte M G; Ramsey, Scott D

    2014-08-01

    The rapidly increasing range and expense of new diagnostics, compels consideration of a different, more proactive approach to health economic evaluation of diagnostic technologies. Early cycle economic evaluation is a decision analytic approach to evaluate technologies in development so as to increase the return on investment as well as patient and societal impact. This paper describes examples of 'early cycle economic evaluations' as applied to diagnostic technologies and highlights challenges in its real-time application. It shows that especially in the field of diagnostics, with rapid technological developments and a changing regulatory climate, early cycle economic evaluation can have a guiding role to improve the efficiency of the diagnostics innovation process. In the next five years the attention will move beyond the methodological and analytic challenges of early cycle economic evaluation towards the challenge of effectively applying it to improve diagnostic research and development and patient value. Future work in this area should therefore be 'strong on principles and soft on metrics', that is, the metrics that resonate most clearly with the various decision makers in this field. PMID:24766321

  15. Use of Universal 16S rRNA Gene PCR as a Diagnostic Tool for Venous Access Port-Related Bloodstream Infections

    PubMed Central

    Marín, M.; Martín-Rabadán, P.; Echenagusia, A.; Camúñez, F.; Rodríguez-Rosales, G.; Simó, G.; Echenagusia, M.; Bouza, E.

    2013-01-01

    Amplification of the universal 16S rRNA gene using PCR has improved the diagnostic yield of microbiological samples. However, no data have been reported on the reliability of this technique with venous access ports (VAPs). We assessed the utility of 16S rRNA PCR for the prediction of VAP-related bloodstream infection (VAP-RBSI). During a 2-year period, we prospectively received all VAPs removed by interventional radiologists. PCR and conventional cultures were performed using samples from the different VAP sites. We compared the results of PCR with those of conventional culture for patients with confirmed VAP-RBSI. We collected 219 VAPs from 219 patients. Conventional VAP culture revealed 15 episodes of VAP-RBSI. PCR revealed a further 4 episodes in patients undergoing antibiotic therapy which would have gone undetected using conventional culture. Moreover, it had a negative predictive value of 97.8% for the prediction of VAP-RBSI when it was performed using biofilm from the internal surface of the port. In conclusion, universal 16S rRNA PCR performed with samples from the inside of VAPs proved to be a useful tool for the diagnosis of VAP-RBSI. It increased detection of VAP-RBSI episodes by 21.1% in patients undergoing antibiotic therapy whose episodes would have gone undetected using conventional culture. Therefore, we propose a new application of 16S rRNA PCR as a useful tool for the diagnosis of VAP-RBSI in patients receiving antibiotic therapy. PMID:23254136

  16. 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.

  17. 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

  18. Improved diagnostic testing and malaria treatment practices in Zambia

    PubMed Central

    Hamer, Davidson H; Ndhlovu, Micky; Zurovac, Dejan; Fox, Matthew; Yeboah-Antwi, Kojo; Chanda, Pascalina; Sipilinyambe, Naawa; Simon, Jonathon L; Snow, Robert W

    2009-01-01

    Context Improving the accuracy of malaria diagnosis using rapid diagnostic tests (RDT) has been proposed as an approach for reducing over-treatment of malaria in the current era of widespread implementation of artemisinin-based combination therapy in sub-Saharan Africa. Objective To assess the impact of microscopy and RDT use on prescription of antimalarials. Design, Setting, and Participants Cross-sectional, cluster sample survey of all sick outpatients seen at a health facility during one working day that included all public and mission health facilities in four sentinel districts in Zambia. Main Outcome Measures Proportions of patients undergoing malaria diagnostic procedures and receiving anti-malarial treatment. Results 17% of the 104 health facilities surveyed had functional microscopy, 63% had RDTs available, and 73% had at least one type of malaria diagnostics. 27.8% of subjects with fever (suspected malaria) seen in health facilities with malaria diagnostics were tested and 44.6% were positive. 58.4% of patients with negative blood smears were prescribed an antimalarial as were 35.5% of those with a negative RDT result. 65.9% of the subjects with fever who did not have diagnostic tests done were also prescribed antimalarials. In facilities with artemether-lumefantrine in stock, this antimalarial was prescribed to a larger proportion of febrile patients with a positive diagnostic test (blood smear 75.0%; RDT 70.4%) than those with a negative diagnostic test (blood smear 30.4%; RDT 26.7%). Conclusion Despite efforts to scale up the provision of malaria diagnostics in Zambia they continue to be under-utilized and patients with negative test results frequently receive antimalarials. The provision of new tools to reduce the inappropriate use of new expensive antimalarial treatments must be accompanied by a paradigm shift in clinical management of patients without evidence of malaria infection. PMID:17519412

  19. Molecular diagnostics of the honey bee parasites Lotmaria passim and Crithidia spp. (Trypanosomatidae) using multiplex PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lotmaria passim Schwarz is a recently described trypanosome parasite of honey bees in continental United States, Europe, and Japan. We developed a multiplex PCR technique using a PCR primer specific for L. passim to distinguish this species from C. mellificae. We report the presence of L. passim in ...

  20. Evaluation of Diagnostic Value and Epidemiological Implications of PCR for Pneumocystis carinii in Different Immunosuppressed and Immunocompetent Patient Groups

    PubMed Central

    Sing, Andreas; Trebesius, Karlheinz; Roggenkamp, Andreas; Rüssmann, Holger; Tybus, Karin; Pfaff, Friederike; Bogner, Johannes R.; Emminger, Christoph; Heesemann, Jürgen

    2000-01-01

    To evaluate the value of single and nested PCRs for diagnosis of Pneumocystis carinii pneumonia (PCP) in a variety of respiratorily distressed patient groups, 574 respiratory samples from 334 patients (89 human immunodeficiency virus [HIV]-positive patients, 61 transplant recipients, 66 malignancy patients, 34 otherwise immunosuppressed patients, and 84 immunocompetent patients) were prospectively examined by microscopy and single and nested PCRs. The resulting data were correlated with clinical evidence of PCP. Microscopy and single PCR of bronchoalveolar lavage (BAL) specimens from HIV patients were 100% sensitive and specific in detecting PCP, whereas nested PCR, although being 100% sensitive, reached a specificity of only 97.5%. In the three non-HIV immunosuppressed patient groups, both single and nested PCR invariably produced lower positive predictive values than microscopy. Among immunocompetent patients, the positive predictive values of both PCRs were 0%. Therefore, the diagnostic values of the PCR methods tested do not seem to offer any additional advantage compared to that of conventional microscopy for these patient groups. However, nested PCR identified a significant percentage of clinically silent P. carinii colonizations in about 17 to 20% of immunocompetent and immunosuppressed non-HIV patients. PMID:10747126

  1. The diagnostic utility of combination of HMGA2 and IMP3 qRT-PCR testing in thyroid neoplasms.

    PubMed

    Jin, Long; Lloyd, Ricardo V; Henry, Michael R; Erickson, Lori A; Sebo, Thomas J; Rumilla, Kandelaria M; Zhang, Jun

    2015-01-01

    The diagnosis of malignant thyroid tumors in some cytologic and histologic specimens remains challenging. High-mobility group A2 (HMGA2) expression and insulin-like growth factor II mRNA-binding protein-3 (IMP3) expression were evaluated by relative quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The aim of this study was to evaluate whether the combination of HMGA2 and IMP3 qRT-PCR was diagnostically useful in differentiating benign from malignant thyroid neoplasms. Fine-needle aspiration (FNA) specimens from 120 patients including 56 benign lesions and 64 carcinomas were used. The available 80 corresponding formalin-fixed paraffin-embedded (FFPE) thyroid tissues from 66 patients were also included in this study. HMGA2 and IMP3 expression levels were detected by qRT-PCR and reported as relative fold change after normalizing with a calibrator. The diagnostic utilities of HMGA2 and IMP3 qRT-PCR tests were evaluated individually and in combination. In FNA specimens, HMGA2 and IMP3 expression was consistently higher in thyroid malignancies compared with benign lesions in all subgroups except in Hürthle cell tumors. After exclusion of Hürthle cell tumors, the sensitivity was 90.2% for HMGA2, 88.2% for IMP3, and 98% for HMGA2+IMP3; the specificity was 97.1% for HMGA2, 79.4% for IMP3, and 79.4% for HMGA+IMP3. qRT-PCR data showed similar results in FFPE tissues: the sensitivity was 84.2% for HMGA2, 85.7% for IMP3, and 94.7% for HMGA2+IMP3; the specificity was 96.9% for HMGA2, 91.2% for IMP3, and 90.6% for HMGA2+IMP3. qRT-PCR data were concordant between FNA and FFPE samples for HMGA2 (97.4%) and IMP3 (96.9%). The results indicate that HMGA2 qRT-PCR with high specificity may be a useful ancillary technique to assist in the classification of difficult thyroid specimens, excluding Hürthle cell tumors. The HMGA2 and IMP3 qRT-PCR combination model with increased sensitivity and negative predictive value (96.4%) may be useful in screening thyroid

  2. Diagnostic real-time RT-PCR for the simultaneous detection of Citrus exocortis viroid and Hop stunt viroid.

    PubMed

    Papayiannis, Lambros C

    2014-02-01

    Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) are two important viroids known to infect several plant species worldwide. In this study, a real-time reverse transcription (RT) TaqMan polymerase chain reaction (PCR) assay was developed and optimized for the simultaneous detection of CEVd and HSVd. The assay's analytical and diagnostic sensitivity and specificity were evaluated using reference isolates. Two different RNA extraction methods and one rapid crude template preparation procedure were compared in terms of extraction purity and efficiency for PCR applications. Extraction method Q included a commercially available kit, whereas method C was a modified chloroform-phase extraction in house protocol. Procedure S involved blotting the sap extract on a positively charged nylon membrane and elution. The multiplex RT-TaqMan PCR assay successfully discriminated the two viroid species from all reference samples and its recorded diagnostic sensitivity (Dse) and specificity (Dsp) was 100%. On the contrary, in conventional RT-PCR tests, the overall Dse and Dsp were lower and estimated at 94 and 95% for CEVd, and 97 and 98% for HSVd, respectively. In a direct comparison, the developed assay presented 1000-fold more analytical sensitivity. Spectrophotometric results showed that RNA extraction methods Q and C, yielded the purest RNA, and gave the lowest mean Ct values. Alternative template preparation method S resulted in Ct values statistically similar to those obtained with methods Q to C when tested by RT-TaqMan PCR. The developed assay, using crude template preparation S, allows the simple, accurate and cost-effective testing of a large number of plant samples, and can be applied in surveys and certification schemes.

  3. 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

    measurement of human leukocyte's telomere length in comparison to qPCR. In conclusion, flow-FISH appears to be a more appropriate method for diagnostic purposes. PMID:25409313

  4. An improved colony PCR procedure for genetic screening of Chlorella and related microalgae.

    PubMed

    Wan, Minxi; Rosenberg, Julian N; Faruq, Junaid; Betenbaugh, Michael J; Xia, Jinlan

    2011-08-01

    A colony PCR technique was applied for both genomic and chloroplast DNA in the green microalgae Chlorella. Of five different lysis buffers, Chelex-100 was superior for DNA extraction, PCR and DNA storage. It also was insensitive to variations in cell density. The conditions established for an improved PCR formulation are applicable for screening of genetically-engineered transformants as well as bioprospecting of natural microalgal isolates. Besides multiple Chlorella species, we also demonstrate the efficacy of Chelex-100 for colony PCR with a number of other microalgal strains, including Chlamydomonas reinhardtii, Dunaliella salina, Nannochloropsis sp., Coccomyxa sp., and Thalassiosira pseudonana.

  5. Improving diagnostic criteria for Propionibacterium acnes osteomyelitis: a retrospective analysis.

    PubMed

    Asseray, Nathalie; Papin, Christophe; Touchais, Sophie; Bemer, Pascale; Lambert, Chantal; Boutoille, David; Tequi, Brigitte; Gouin, François; Raffi, François; Passuti, Norbert; Potel, Gilles

    2010-07-01

    The identification of Propionibacterium acnes in cultures of bone and joint samples is always difficult to interpret because of the ubiquity of this microorganism. The aim of this study was to propose a diagnostic strategy to distinguish infections from contaminations. This was a retrospective analysis of all patient charts of those patients with >or=1 deep samples culture-positive for P. acnes. Every criterion was tested for sensitivity, specificity, and positive likelihood ratio, and then the diagnostic probability of combinations of criteria was calculated. Among 65 patients, 52 (80%) were considered truly infected with P. acnes, a diagnosis based on a multidisciplinary process. The most valuable diagnostic criteria were: >or=2 positive deep samples, peri-operative findings (necrosis, hardware loosening, etc.), and >or=2 surgical procedures. However, no single criterion was sufficient to ascertain the diagnosis. The following combinations of criteria had a diagnostic probability of >90%: >or=2 positive cultures + 1 criterion among: peri-operative findings, local signs of infection, >or=2 previous operations, orthopaedic devices; 1 positive culture + 3 criteria among: peri-operative findings, local signs of infection, >or=2 previous surgical operations, orthopaedic devices, inflammatory syndrome. The diagnosis of P. acnes osteomyelitis was greatly improved by combining different criteria, allowing differentiation between infection and contamination.

  6. Improving diagnostic criteria for Propionibacterium acnes osteomyelitis: a retrospective analysis.

    PubMed

    Asseray, Nathalie; Papin, Christophe; Touchais, Sophie; Bemer, Pascale; Lambert, Chantal; Boutoille, David; Tequi, Brigitte; Gouin, François; Raffi, François; Passuti, Norbert; Potel, Gilles

    2010-07-01

    The identification of Propionibacterium acnes in cultures of bone and joint samples is always difficult to interpret because of the ubiquity of this microorganism. The aim of this study was to propose a diagnostic strategy to distinguish infections from contaminations. This was a retrospective analysis of all patient charts of those patients with >or=1 deep samples culture-positive for P. acnes. Every criterion was tested for sensitivity, specificity, and positive likelihood ratio, and then the diagnostic probability of combinations of criteria was calculated. Among 65 patients, 52 (80%) were considered truly infected with P. acnes, a diagnosis based on a multidisciplinary process. The most valuable diagnostic criteria were: >or=2 positive deep samples, peri-operative findings (necrosis, hardware loosening, etc.), and >or=2 surgical procedures. However, no single criterion was sufficient to ascertain the diagnosis. The following combinations of criteria had a diagnostic probability of >90%: >or=2 positive cultures + 1 criterion among: peri-operative findings, local signs of infection, >or=2 previous operations, orthopaedic devices; 1 positive culture + 3 criteria among: peri-operative findings, local signs of infection, >or=2 previous surgical operations, orthopaedic devices, inflammatory syndrome. The diagnosis of P. acnes osteomyelitis was greatly improved by combining different criteria, allowing differentiation between infection and contamination. PMID:20141491

  7. Further improvement and validation of MagMAX-96 AI/ND viral RNA isolation for efficient removal of RT-PCR inhibitors from cloacal swabs and tissues for rapid diagnosis of avian influenza virus by RT reverse transcription PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real time RT-PCR (RRT-PCR) is a high throughput molecular diagnostic test used for rapid detection of avian influenza virus (AIV) in clinical samples. However the performance of RRT-PCR can be adversely affected by RT-PCR inhibitors present in the sample. The tested commercial RNA extraction kits ...

  8. Improved thermal cycling durability and PCR compatibility of polymer coated quantum dot

    NASA Astrophysics Data System (ADS)

    Xun, Zhe; Zhao, Xiaoyun; Guan, Yifu

    2013-09-01

    Quantum dots have experienced rapid development in imaging, labeling and sensing in medicine and life science. To be suitable for polymerase chain reaction (PCR) assay, we have tested QD thermal cycling durability and compatibility, which have not been addressed in previous reports. In this study, we synthesized CdSe/ZnS QDs with a surface modification with high-MW amphiphilic copolymers and observed that Mg2+ ions in the PCR reaction could induce the QDs to precipitate and reduce their fluorescence signal significantly after thermal cycling. To overcome this problem, we used mPEG2000 to conjugate the QD surface for further protection, and found that this modification enables QDs to endure 40 thermal cycles in the presence of other components essential for PCR reactions. We have also identified that QDs have different effects on rTaq and Ex Taq polymerization systems. A high QD concentration could apparently reduce the PCR efficiency, but this inhibition was relieved significantly in the Ex PCR system as the concentration of Ex Taq polymerase was increased. Real-time PCR amplification results showed that QDs could provide a sufficiently measurable fluorescence signal without excessively inhibiting the DNA amplification. Based on this improved thermal cycling durability and compatibility with the PCR system, QDs have the potential to be developed as stable fluorescent sensors in PCR and real-time PCR amplification.

  9. Improved detection of episomal Banana streak viruses by multiplex immunocapture PCR.

    PubMed

    Le Provost, Grégoire; Iskra-Caruana, Marie-Line; Acina, Isabelle; Teycheney, Pierre-Yves

    2006-10-01

    Banana streak viruses (BSV) are currently the main viral constraint to Musa germplasm movement, genetic improvement and mass propagation. Therefore, it is necessary to develop and implement BSV detection strategies that are both reliable and sensitive, such as PCR-based techniques. Unfortunately, BSV endogenous pararetrovirus sequences (BSV EPRVs) are present in the genome of Musa balbisiana. They interfere with PCR-based detection of episomal BSV in infected banana and plantain, such as immunocapture PCR. Therefore, a multiplex, immunocapture PCR (M-IC-PCR) was developed for the detection of BSV. Musa sequence tagged microsatellite site (STMS) primers were selected and used in combination with BSV species-specific primers in order to monitor possible contamination by Musa genomic DNA, using multiplex PCR. Furthermore, immunocapture conditions were optimized in order to prevent Musa DNA from interfering with episomal BSV DNA during the PCR step. This improved detection method successfully allowed the accurate, specific and sensitive detection of episomal DNA only from distinct BSV species. Its implementation should benefit PCR-based detection of viruses for which homologous sequences are present in the genome of their hosts, including transgenic plants expressing viral sequences.

  10. Cost-effectiveness of diagnostic strategies using quantitative real-time PCR and bacterial culture to identify contagious mastitis cases in large dairy herds.

    PubMed

    Murai, Kiyokazu; Lehenbauer, Terry W; Champagne, John D; Glenn, Kathy; Aly, Sharif S

    2014-03-01

    Diagnostic strategies to detect contagious mastitis caused by Mycoplasma bovis, Staphylococcus aureus, and Streptococcus agalactiae in dairy herds during an outbreak have been minimally studied with regard to cost and diagnostic sensitivity. The objective of this cross-sectional study was to compare the cost-effectiveness of diagnostic strategies for identification of infected cows in two California dairy herds during contagious mastitis outbreaks. M. bovis was investigated in a subset of a herd (n=1210 cows) with an estimated prevalence of 2.8% (95% CI=1.9, 3.7), whereas Staph. aureus and Strep. agalactiae were studied in a second herd (n=351 cows) with an estimated prevalence of 3.4% (95% CI=1.5, 5.3) and 16.8% (95% CI=12.9, 20.7), respectively. Diagnostic strategies involved a combination of testing stages that utilized bacterial culture, quantitative real-time PCR (qPCR), or both. Strategies were applied to individual or pooled samples of 5, 10, 50 or 100 samples. Culture was considered the gold standard for sensitivity estimation of each strategy. The reference strategy was the strategy with the lowest cost per culture-positive cow which for both M. bovis and Strep. agalactiae consisted of 2 stages, culture of samples in pools of 5 followed by culture of individual samples in positive pools with a sensitivity of 73.5% (95% CI: 55.6, 87.1) and 96.6% (95% CI: 27.7, 84.8), respectively. The reference strategy for Staph. aureus consisted of 3 stages, culture of individual samples in pools of 100 (stage 1), culture constituents of those positive from stage 1 in pools of 5 (stage 2), culture constituents of those positive from stage 2 individually (stage 3) which resulted in a sensitivity of 58.3% (95% CI: 88.3, 99.6). The most cost-effective alternative to the reference strategy was whole herd milk culture for all 3 pathogens. QPCR testing was a component of the second most cost-effective alternative for M. bovis and the third most cost-effective alternatives for

  11. Development of a PCR Diagnostic System for Iris yellow spot tospovirus in Quarantine.

    PubMed

    Shin, Yong-Gil; Rho, Jae-Young

    2014-12-01

    Iris yellow spot virus (IYSV) is a plant pathogenic virus which has been reported to continuously occur in onion bulbs, allium field crops, seed crops, lisianthus, and irises. In South Korea, IYSV is a "controlled" virus that has not been reported, and inspection is performed when crops of the genus Iris are imported into South Korea. In this study, reverse-transcription polymerase chain reaction (RT-PCR) and nested PCR inspection methods, which can detect IYSV, from imported crops of the genus Iris at quarantine sites, were developed. In addition, a modified positive plasmid, which can be used as a positive control during inspection, was developed. This modified plasmid can facilitate a more accurate inspection by enabling the examination of a laboratory contamination in an inspection system. The inspection methods that were developed in this study are expected to contribute, through the prompt and accurate inspection of IYSV at quarantine sites to the plant quarantine in South Korea. PMID:25506310

  12. Dielectrophoresis chips improve PCR detection of the food-spoiling yeast Zygosaccharomyces rouxii in apple juice.

    PubMed

    del Carmen Jaramillo, Maria; Huttener, Mario; Alvarez, Juan Manuel; Homs-Corbera, Antoni; Samitier, Josep; Torrents, Eduard; Juárez, Antonio

    2015-07-01

    Dielectrophoretic (DEP) manipulation of cells present in real samples is challenging. We show in this work that an interdigitated DEP chip can be used to trap and wash a population of the food-spoiling yeast Zygosaccharomyces rouxii that contaminates a sample of apple juice. By previously calibrating the chip, the yeast population loaded is efficiently trapped, washed, and recovered in a small-volume fraction that, in turn, can be used for efficient PCR detection of this yeast. DEP washing of yeast cells gets rid of PCR inhibitors present in apple juice and facilitates PCR analysis. This and previous works on the use of DEP chips to improve PCR analysis show that a potential use of DEP is to be used as a treatment of real samples prior to PCR. PMID:25808673

  13. Dielectrophoresis chips improve PCR detection of the food-spoiling yeast Zygosaccharomyces rouxii in apple juice.

    PubMed

    del Carmen Jaramillo, Maria; Huttener, Mario; Alvarez, Juan Manuel; Homs-Corbera, Antoni; Samitier, Josep; Torrents, Eduard; Juárez, Antonio

    2015-07-01

    Dielectrophoretic (DEP) manipulation of cells present in real samples is challenging. We show in this work that an interdigitated DEP chip can be used to trap and wash a population of the food-spoiling yeast Zygosaccharomyces rouxii that contaminates a sample of apple juice. By previously calibrating the chip, the yeast population loaded is efficiently trapped, washed, and recovered in a small-volume fraction that, in turn, can be used for efficient PCR detection of this yeast. DEP washing of yeast cells gets rid of PCR inhibitors present in apple juice and facilitates PCR analysis. This and previous works on the use of DEP chips to improve PCR analysis show that a potential use of DEP is to be used as a treatment of real samples prior to PCR.

  14. Visual aids improve diagnostic inferences and metacognitive judgment calibration

    PubMed Central

    Garcia-Retamero, Rocio; Cokely, Edward T.; Hoffrage, Ulrich

    2015-01-01

    Visual aids can improve comprehension of risks associated with medical treatments, screenings, and lifestyles. Do visual aids also help decision makers accurately assess their risk comprehension? That is, do visual aids help them become well calibrated? To address these questions, we investigated the benefits of visual aids displaying numerical information and measured accuracy of self-assessment of diagnostic inferences (i.e., metacognitive judgment calibration) controlling for individual differences in numeracy. Participants included 108 patients who made diagnostic inferences about three medical tests on the basis of information about the sensitivity and false-positive rate of the tests and disease prevalence. Half of the patients received the information in numbers without a visual aid, while the other half received numbers along with a grid representing the numerical information. In the numerical condition, many patients–especially those with low numeracy–misinterpreted the predictive value of the tests and profoundly overestimated the accuracy of their inferences. Metacognitive judgment calibration mediated the relationship between numeracy and accuracy of diagnostic inferences. In contrast, in the visual aid condition, patients at all levels of numeracy showed high-levels of inferential accuracy and metacognitive judgment calibration. Results indicate that accurate metacognitive assessment may explain the beneficial effects of visual aids and numeracy–a result that accords with theory suggesting that metacognition is an essential part of risk literacy. We conclude that well-designed risk communications can inform patients about healthrelevant numerical information while helping them assess the quality of their own risk comprehension. PMID:26236247

  15. Improved Diagnostics Using Polarization Imaging and Artificial Neural Networks

    PubMed Central

    Xuan, Jianhua; Klimach, Uwe; Zhao, Hongzhi; Chen, Qiushui; Zou, Yingyin; Wang, Yue

    2007-01-01

    In recent years, there has been an increasing interest in studying the propagation of polarized light in biological cells and tissues. This paper presents a novel approach to cell or tissue imaging using a full Stokes imaging system with advanced polarization image analysis algorithms for improved diagnostics. The key component of the Stokes imaging system is the electrically tunable retarder, enabling high-speed operation of the system to acquire four intensity images sequentially. From the acquired intensity images, four Stokes vector images can be computed to obtain complete polarization information. Polarization image analysis algorithms are then developed to analyze Stokes polarization images for cell or tissue classification. Specifically, wavelet transforms are first applied to the Stokes components for initial feature analysis and extraction. Artificial neural networks (ANNs) are then used to extract diagnostic features for improved classification and prediction. In this study, phantom experiments have been conducted using a prototyped Stokes polarization imaging device. In particular, several types of phantoms, consisting of polystyrene latex spheres in various diameters, were prepared to simulate different conditions of epidermal layer of skin. The experimental results from phantom studies and a plant cell study show that the classification performance using Stokes images is significantly improved over that using the intensity image only. PMID:18274657

  16. Increasing the Number of Thyroid Lesions Classes in Microarray Analysis Improves the Relevance of Diagnostic Markers

    PubMed Central

    Fontaine, Jean-Fred; Mirebeau-Prunier, Delphine; Raharijaona, Mahatsangy; Franc, Brigitte; Triau, Stephane; Rodien, Patrice; Goëau-Brissonniére, Olivier; Karayan-Tapon, Lucie; Mello, Marielle; Houlgatte, Rémi; Malthiery, Yves; Savagner, Frédérique

    2009-01-01

    Background Genetic markers for thyroid cancers identified by microarray analysis have offered limited predictive accuracy so far because of the few classes of thyroid lesions usually taken into account. To improve diagnostic relevance, we have simultaneously analyzed microarray data from six public datasets covering a total of 347 thyroid tissue samples representing 12 histological classes of follicular lesions and normal thyroid tissue. Our own dataset, containing about half the thyroid tissue samples, included all categories of thyroid lesions. Methodology/Principal Findings Classifier predictions were strongly affected by similarities between classes and by the number of classes in the training sets. In each dataset, sample prediction was improved by separating the samples into three groups according to class similarities. The cross-validation of differential genes revealed four clusters with functional enrichments. The analysis of six of these genes (APOD, APOE, CLGN, CRABP1, SDHA and TIMP1) in 49 new samples showed consistent gene and protein profiles with the class similarities observed. Focusing on four subclasses of follicular tumor, we explored the diagnostic potential of 12 selected markers (CASP10, CDH16, CLGN, CRABP1, HMGB2, ALPL2, ADAMTS2, CABIN1, ALDH1A3, USP13, NR2F2, KRTHB5) by real-time quantitative RT-PCR on 32 other new samples. The gene expression profiles of follicular tumors were examined with reference to the mutational status of the Pax8-PPARγ, TSHR, GNAS and NRAS genes. Conclusion/Significance We show that diagnostic tools defined on the basis of microarray data are more relevant when a large number of samples and tissue classes are used. Taking into account the relationships between the thyroid tumor pathologies, together with the main biological functions and pathways involved, improved the diagnostic accuracy of the samples. Our approach was particularly relevant for the classification of microfollicular adenomas. PMID:19893615

  17. International Consensus Diagnostic Criteria for Autoimmune Pancreatitis and Its Japanese Amendment Have Improved Diagnostic Ability over Existing Criteria

    PubMed Central

    Maruyama, Masahiro; Watanabe, Takayuki; Kanai, Keita; Oguchi, Takaya; Muraki, Takashi; Hamano, Hideaki; Arakura, Norikazu; Kawa, Shigeyuki

    2013-01-01

    Objectives. The recent International Consensus Diagnostic Criteria (ICDC) for autoimmune pancreatitis (AIP) and its Japanese amendment developed by the Japanese Pancreas Society (JPS 2011) may have overcome the drawbacks of earlier criteria and achieved a higher diagnostic ability for AIP. The aim of the present study is to evaluate this possibility and identify the underlying causes of this change. Methods. We compared the diagnostic abilities of the ICDC and JPS 2011 with those of the Japanese diagnostic criteria 2006 (JPS 2006), Korean diagnostic criteria (Korean), Asian diagnostic criteria (Asian), and HISORt diagnostic criteria in 110 patients with AIP and 31 patients with malignant pancreatic cancer. Results. The ICDC achieved the highest diagnostic ability in terms of accuracy (95.0%), followed by JPS 2011 (92.9%), Korean (92.2%), HISORt (88.7%), Asian (87.2%), and JPS 2006 (85.1%). Nearly all criteria systems exhibited a high specificity of 100%, indicating that the enhanced diagnostic ability of the ICDC and JPS 2011 likely stemmed from increased sensitivity brought about by inclusion of diagnostic items requiring no endoscopic retrograde pancreatography. The diagnostic ability of JPS 2011 was nearly equivalent to that of the ICDC. Conclusions. The ICDC and JPS 2011 have improved diagnostic ability as compared with earlier criteria sets because of an increase in sensitivity. PMID:24348535

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

    PubMed

    Hawash, Yousry

    2014-06-01

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

  19. Comparative evaluation and economic assessment of coprological diagnostic methods and PCR for detection of Cryptosporidium spp. in bovines.

    PubMed

    Paul, S; Chandra, D; Tewari, A K; Banerjee, P S; Ray, D D; Boral, R; Rao, J R

    2009-10-14

    The role of Cryptosporidium spp. as a major cause of diarrhoea and gastrointestinal illness of protozoan origin in neonatal calves has been established. Many coprological and serological techniques have been described for detection of the parasites with the limitations of sensitivity and specificity. Polymerase chain reaction (PCR) technique offers a useful alternative to conventional diagnosis of Cryptosporidium spp. in bovines from both clinical and environmental samples. We compared four conventional coprological techniques, viz., direct faecal smear staining (DFSS), normal saline sedimentation staining (NSSS), Sheather's flotation (SF) and Sheather's flotation sedimentation staining (SFSS) with PCR directed against the 18S SSU rRNA gene as standard reference test for the diagnosis of cryptosporidiosis in bovines. Out of 457 faecal samples collected from neonatal bovine calves, specific PCR amplification was achieved in 138 samples, whereas, 65 samples turned positive by DFSS. Normal saline sedimentation staining, SF and SFSS could detect 92, 82 and 109 samples as positive, respectively. Sheather's flotation sedimentation staining was found to be the most sensitive (82.6%) and specific (98.76%) among the coprological techniques. On per sample processing based cost analysis, DFSS was found to be the most economical method (15 cents) followed by NSSS (19.6 cents), SF (23.6 cents) and SFSS (33.9 cents). The time taken for complete processing and diagnosis varied between 70 and 100 min. PCR based diagnosis of a sample took about 7.5-8h for completion and cost of diagnosis was estimated as approximately 7.604 US$ per sample. Among the conventional coprological methods, SFSS provided the required sensitivity and specificity along with nominal cost for diagnosis on per sample basis, and may be considered as a viable diagnostic alternative when PCR is not an option for a particular laboratory setting, especially in developing countries. This is the first comparative

  20. Action Research to Improve the Learning Space for Diagnostic Techniques†

    PubMed Central

    Ariel, Ellen; Owens, Leigh

    2015-01-01

    The module described and evaluated here was created in response to perceived learning difficulties in diagnostic test design and interpretation for students in third-year Clinical Microbiology. Previously, the activities in lectures and laboratory classes in the module fell into the lower cognitive operations of “knowledge” and “understanding.” The new approach was to exchange part of the traditional activities with elements of interactive learning, where students had the opportunity to engage in deep learning using a variety of learning styles. The effectiveness of the new curriculum was assessed by means of on-course student assessment throughout the module, a final exam, an anonymous questionnaire on student evaluation of the different activities and a focus group of volunteers. Although the new curriculum enabled a major part of the student cohort to achieve higher pass grades (p < 0.001), it did not meet the requirements of the weaker students, and the proportion of the students failing the module remained at 34%. The action research applied here provided a number of valuable suggestions from students on how to improve future curricula from their perspective. Most importantly, an interactive online program that facilitated flexibility in the learning space for the different reagents and their interaction in diagnostic tests was proposed. The methods applied to improve and assess a curriculum refresh by involving students as partners in the process, as well as the outcomes, are discussed. Journal of Microbiology & Biology Education PMID:26753024

  1. Improved detection of mycobacterial DNA by PCR in formalin-fixed, paraffin-embedded tissues using thin sections.

    PubMed

    Loeschke, S; Goldmann, T; Vollmer, E

    2005-01-01

    PCR is a unique methodology allowing for the sensitive detection of mycobacterial DNA-sequences in cases in which no fresh material can be obtained for classic analyses. Despite the limitations of this technique, for example the less satisfactory quality of DNA from paraffin-embedded specimens and the high effort necessary to control contamination, PCR still represents a useful additional tool for routine diagnostic examinations of mycobacterial infections. Fragmentation of the DNA extracted from formalin-fixed, paraffin-embedded samples on the one hand and the rigid cell wall of mycobacteria on the other hand are obstacles to detecting the DNA of these microorganisms by PCR. Here, we describe a simple mechanical procedure that allows us to improve the detection of mycobacterial DNA with the use of thin (1 microm) sections instead of thicker sections. This could be explained by a gentle, mechanical opening of the acid fast mycobacterial cell wall. Thus, even the application of heat/cold shock treatments is not necessary. This inexpensive fast procedure can also be used for the detection of other infectious agents. PMID:15807309

  2. Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth disease virus and look-alike disease viruses

    SciTech Connect

    Hindson, B J; Reid, S M; Baker, B R; Ebert, K; Ferris, N P; Bentley Tammero, L F; Lenhoff, R J; Naraghi-Arani, P; Vitalis, E A; Slezak, T R; Hullinger, P J; King, D P

    2007-07-26

    A high-throughput multiplexed assay was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.

  3. Real-Time Reverse Transcription PCR Assay for Detection of Senecavirus A in Swine Vesicular Diagnostic Specimens

    PubMed Central

    Fabian, Andrew W.; Barrette, Roger W.; Sayed, Abu

    2016-01-01

    Senecavirus A (SV-A), formerly, Seneca Valley virus (SVV), has been detected in swine with vesicular lesions and is thought to be associated with swine idiopathic vesicular disease (SIVD), a vesicular disease syndrome that lacks a defined causative agent. The clinical presentation of SIVD resembles that of other more contagious and economically devastating vesicular diseases, such as foot-and-mouth disease (FMD), swine vesicular disease (SVD), and vesicular stomatitis (VS), that typically require immediate rule out diagnostics to lift restrictions on animal quarantine, movement, and trade. This study presents the development of a sensitive, SYBR Green RT-qPCR assay suitable for detection of SV-A in diagnostic swine specimens. After testing 50 pigs with clinical signs consistent with vesicular disease, 44 (88%) were found to be positive for SV-A by RT-qPCR as compared to none from a negative cohort of 35 animals without vesicular disease, indicating that the assay is able to successfully detect the virus in an endemic population. SV-A RNA was also detectable at a low level in sera from a subset of pigs that presented with (18%) or without (6%) vesicular signs. In 2015, there has been an increase in the occurrence of SV-A in the US, and over 200 specimens submitted to our laboratory for vesicular investigation have tested positive for the virus using this method. SV-A RNA was detectable in all common types of vesicular specimens including swabs and tissue from hoof lesions, oral and snout epithelium, oral swabs, scabs, and internal organ tissues such as liver and lymph node. Genome sequencing analysis from recent virus isolates was performed to confirm target amplicon specificity and was aligned to previous isolates. PMID:26757142

  4. Real-Time Reverse Transcription PCR Assay for Detection of Senecavirus A in Swine Vesicular Diagnostic Specimens.

    PubMed

    Bracht, Alexa J; O'Hearn, Emily S; Fabian, Andrew W; Barrette, Roger W; Sayed, Abu

    2016-01-01

    Senecavirus A (SV-A), formerly, Seneca Valley virus (SVV), has been detected in swine with vesicular lesions and is thought to be associated with swine idiopathic vesicular disease (SIVD), a vesicular disease syndrome that lacks a defined causative agent. The clinical presentation of SIVD resembles that of other more contagious and economically devastating vesicular diseases, such as foot-and-mouth disease (FMD), swine vesicular disease (SVD), and vesicular stomatitis (VS), that typically require immediate rule out diagnostics to lift restrictions on animal quarantine, movement, and trade. This study presents the development of a sensitive, SYBR Green RT-qPCR assay suitable for detection of SV-A in diagnostic swine specimens. After testing 50 pigs with clinical signs consistent with vesicular disease, 44 (88%) were found to be positive for SV-A by RT-qPCR as compared to none from a negative cohort of 35 animals without vesicular disease, indicating that the assay is able to successfully detect the virus in an endemic population. SV-A RNA was also detectable at a low level in sera from a subset of pigs that presented with (18%) or without (6%) vesicular signs. In 2015, there has been an increase in the occurrence of SV-A in the US, and over 200 specimens submitted to our laboratory for vesicular investigation have tested positive for the virus using this method. SV-A RNA was detectable in all common types of vesicular specimens including swabs and tissue from hoof lesions, oral and snout epithelium, oral swabs, scabs, and internal organ tissues such as liver and lymph node. Genome sequencing analysis from recent virus isolates was performed to confirm target amplicon specificity and was aligned to previous isolates. PMID:26757142

  5. Improved Diagnosis of Periprosthetic Joint Infection by Multiplex PCR of Sonication Fluid from Removed Implants▿

    PubMed Central

    Achermann, Yvonne; Vogt, Markus; Leunig, Michael; Wüst, Jürg; Trampuz, Andrej

    2010-01-01

    The microbiological diagnosis of periprosthetic joint infection (PJI) is crucial for successful antimicrobial treatment. Cultures have limited sensitivity, especially in patients receiving antibiotics. We evaluated the value of multiplex PCR for detection of microbial DNA in sonication fluid from removed orthopedic prostheses. Cases of PJI in which the prosthesis (or part of it) was removed were prospectively included. The removed implant was sonicated, and the resulting sonication fluid was cultured and subjected to multiplex PCR. Of 37 PJI cases (17 hip prostheses, 14 knee prostheses, 4 shoulder prostheses, 1 elbow prosthesis, and 1 ankle prosthesis), pathogens were identified in periprosthetic tissue in 24 (65%) cases, in sonication fluid in 23 (62%) cases, and by multiplex PCR in 29 (78%) cases. The pathogen was detected in 5 cases in sonication fluid only (Propionibacterium acnes in all cases; none of these patients had previously received antibiotics) and in 11 cases by multiplex PCR only (all of these patients had previously received antibiotics). After exclusion of 8 cases caused by P. acnes or Corynebacterium species, which cannot be detected due to the absence of specific primers in the PCR kit, sonication cultures were positive in 17 cases and multiplex PCR sonication cultures were positive in 29 cases (59% versus 100%, respectively; P < 0.01). Among 19 cases (51%) receiving antibiotics, multiplex PCR was positive in all 19 (100%), whereas sonication cultures grew the organism in 8 (42%) (P < 0.01). Multiplex PCR of sonication fluid is a promising test for diagnosis of PJI, particularly in patients who previously received antibiotics. With modified primer sets, multiplex PCR has the potential for further improvement of the diagnosis of PJI. PMID:20164283

  6. Comparison of Diagnostic Accuracy of PCR Targeting the 47-Kilodalton Protein Membrane Gene of Treponema pallidum and PCR Targeting the DNA Polymerase I Gene: Systematic Review and Meta-analysis.

    PubMed

    Gayet-Ageron, Angèle; Combescure, Christophe; Lautenschlager, Stephan; Ninet, Béatrice; Perneger, Thomas V

    2015-11-01

    Treponema pallidum PCR (Tp-PCR) testing now is recommended as a valid tool for the diagnosis of primary or secondary syphilis. The objectives were to systematically review and determine the optimal specific target gene to be used for Tp-PCR. Comparisons of the performance of the two main targets are tpp47 and polA genes were done using meta-analysis. Three electronic bibliographic databases, representing abstract books from five conferences specialized in infectious diseases from January 1990 to March 2015, were searched. Search keywords included ("syphilis" OR "Treponema pallidum" OR "neurosyphilis") AND ("PCR" OR "PCR" OR "molecular amplification"). We included diagnostic studies assessing the performance of Tp-PCR targeting tpp47 (tpp47-Tp-PCR) or the polA gene (polA-Tp-PCR) in ulcers from early syphilis. All studies were assessed against quality criteria using the QUADAS-2 tool. Of 37 studies identified, 62.2% were judged at low risk of bias or applicability. Most used the U.S. Centers for Disease Control and Prevention (CDC) case definitions for primary or secondary (early) syphilis (89.2%; n = 33); 15 (40.5%) used darkfield microscopy (DFM). We did not find differences in sensitivity and specificity between the two Tp-PCR methods in the subgroup of studies using adequate reference tests. Among studies using DFM as the reference test, sensitivities were 79.8% (95% confidence intervals [CI], 72.7 to 85.4%) and 71.4% (46.0 to 88.0%) for tpp47-Tp-PCR and polA-Tp-PCR (P = 0.217), respectively; respective specificities were 95.3% (93.5 to 96.6%) and 93.7% (91.8 to 95.2%) (P = 0.304). Our findings suggest that the two Tp-PCR methods have similar accuracy and could be used interchangeably. PMID:26311859

  7. Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth and look-alike disease viruses

    SciTech Connect

    Hindson, B J; Baker, B R; Bentley Tammero, L F; Lenhoff, R J; Naraghi-Arani, P; Vitalis, E A; Slezak, T R; Hullinger, P J; Reid, S M; Ebert, K; Ferris, N P; King, D P

    2007-09-18

    A high-throughput multiplexed assay (Multiplex Version 1.0) was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRTPCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.

  8. Connectivity of diagnostic technologies: improving surveillance and accelerating tuberculosis elimination.

    PubMed

    Andre, E; Isaacs, C; Affolabi, D; Alagna, R; Brockmann, D; de Jong, B C; Cambau, E; Churchyard, G; Cohen, T; Delmee, M; Delvenne, J-C; Farhat, M; Habib, A; Holme, P; Keshavjee, S; Khan, A; Lightfoot, P; Moore, D; Moreno, Y; Mundade, Y; Pai, M; Patel, S; Nyaruhirira, A U; Rocha, L E C; Takle, J; Trébucq, A; Creswell, J; Boehme, C

    2016-08-01

    In regard to tuberculosis (TB) and other major global epidemics, the use of new diagnostic tests is increasing dramatically, including in resource-limited countries. Although there has never been as much digital information generated, this data source has not been exploited to its full potential. In this opinion paper, we discuss lessons learned from the global scale-up of these laboratory devices and the pathway to tapping the potential of laboratory-generated information in the field of TB by using connectivity. Responding to the demand for connectivity, innovative third-party players have proposed solutions that have been widely adopted by field users of the Xpert(®) MTB/RIF assay. The experience associated with the utilisation of these systems, which facilitate the monitoring of wide laboratory networks, stressed the need for a more global and comprehensive approach to diagnostic connectivity. In addition to facilitating the reporting of test results, the mobility of digital information allows the sharing of information generated in programme settings. When they become easily accessible, these data can be used to improve patient care, disease surveillance and drug discovery. They should therefore be considered as a public health good. We list several examples of concrete initiatives that should allow data sources to be combined to improve the understanding of the epidemic, support the operational response and, finally, accelerate TB elimination. With the many opportunities that the pooling of data associated with the TB epidemic can provide, pooling of this information at an international level has become an absolute priority. PMID:27393530

  9. Connectivity of diagnostic technologies: improving surveillance and accelerating tuberculosis elimination.

    PubMed

    Andre, E; Isaacs, C; Affolabi, D; Alagna, R; Brockmann, D; de Jong, B C; Cambau, E; Churchyard, G; Cohen, T; Delmee, M; Delvenne, J-C; Farhat, M; Habib, A; Holme, P; Keshavjee, S; Khan, A; Lightfoot, P; Moore, D; Moreno, Y; Mundade, Y; Pai, M; Patel, S; Nyaruhirira, A U; Rocha, L E C; Takle, J; Trébucq, A; Creswell, J; Boehme, C

    2016-08-01

    In regard to tuberculosis (TB) and other major global epidemics, the use of new diagnostic tests is increasing dramatically, including in resource-limited countries. Although there has never been as much digital information generated, this data source has not been exploited to its full potential. In this opinion paper, we discuss lessons learned from the global scale-up of these laboratory devices and the pathway to tapping the potential of laboratory-generated information in the field of TB by using connectivity. Responding to the demand for connectivity, innovative third-party players have proposed solutions that have been widely adopted by field users of the Xpert(®) MTB/RIF assay. The experience associated with the utilisation of these systems, which facilitate the monitoring of wide laboratory networks, stressed the need for a more global and comprehensive approach to diagnostic connectivity. In addition to facilitating the reporting of test results, the mobility of digital information allows the sharing of information generated in programme settings. When they become easily accessible, these data can be used to improve patient care, disease surveillance and drug discovery. They should therefore be considered as a public health good. We list several examples of concrete initiatives that should allow data sources to be combined to improve the understanding of the epidemic, support the operational response and, finally, accelerate TB elimination. With the many opportunities that the pooling of data associated with the TB epidemic can provide, pooling of this information at an international level has become an absolute priority.

  10. Connectivity of diagnostic technologies: improving surveillance and accelerating tuberculosis elimination

    PubMed Central

    Isaacs, C.; Affolabi, D.; Alagna, R.; Brockmann, D.; de Jong, B. C.; Cambau, E.; Churchyard, G.; Cohen, T.; Delmee, M.; Delvenne, J-C.; Farhat, M.; Habib, A.; Holme, P.; Keshavjee, S.; Khan, A.; Lightfoot, P.; Moore, D.; Moreno, Y.; Mundade, Y.; Pai, M.; Patel, S.; Nyaruhirira, A. U.; Rocha, L. E. C.; Takle, J.; Trébucq, A.; Creswell, J.; Boehme, C.

    2016-01-01

    SUMMARY In regard to tuberculosis (TB) and other major global epidemics, the use of new diagnostic tests is increasing dramatically, including in resource-limited countries. Although there has never been as much digital information generated, this data source has not been exploited to its full potential. In this opinion paper, we discuss lessons learned from the global scale-up of these laboratory devices and the pathway to tapping the potential of laboratory-generated information in the field of TB by using connectivity. Responding to the demand for connectivity, innovative third-party players have proposed solutions that have been widely adopted by field users of the Xpert® MTB/RIF assay. The experience associated with the utilisation of these systems, which facilitate the monitoring of wide laboratory networks, stressed the need for a more global and comprehensive approach to diagnostic connectivity. In addition to facilitating the reporting of test results, the mobility of digital information allows the sharing of information generated in programme settings. When they become easily accessible, these data can be used to improve patient care, disease surveillance and drug discovery. They should therefore be considered as a public health good. We list several examples of concrete initiatives that should allow data sources to be combined to improve the understanding of the epidemic, support the operational response and, finally, accelerate TB elimination. With the many opportunities that the pooling of data associated with the TB epidemic can provide, pooling of this information at an international level has become an absolute priority. PMID:27393530

  11. Real-Time PCR for Diagnosing Helicobacter pylori Infection in Patients with Upper Gastrointestinal Bleeding: Comparison with Other Classical Diagnostic Methods

    PubMed Central

    Saez, Jesús; Belda, Sofía; Santibáñez, Miguel; Sola-Vera, Javier; Galiana, Antonio; Ruiz-García, Montserrat; Brotons, Alicia; López-Girona, Elena; Girona, Eva; Sillero, Carlos; Royo, Gloria

    2012-01-01

    The aim of this study was to determine the diagnostic usefulness of quantification of the H. pylori genome in detection of infection in patients with upper gastrointestinal bleeding (UGB). A total of 158 consecutive patients with digestive disorders, 80 of whom had clinical presentation of UGB, were studied. The number of microorganisms was quantified using a real-time PCR system which amplifies the urease gene with an internal control for eliminating the false negatives. A biopsy sample from the antrum and corpus of each patient was processed. The rapid urease test, culture, histological study, stool antigen test, and breath test were done. The gold standard was a positive culture or positive results in at least two of the other techniques. When a positive result was defined as any number of microorganisms/human cell, the sensitivity of real-time PCR was greater in bleeding patients, especially in the gastric corpus: 68.4% (95% confidence interval [CI], 52.3 to 84.5%) in non-UGB patients versus 91.5% (95% CI, 79.6 to 97.6%) in UGB patients. When a positive result was defined as a number of microorganisms/human cell above the optimal value that maximizes the Youden index (>3.56 microorganisms/human cell in the antrum and >2.69 in the corpus), the sensitivity and specificity in UGB patients were over 80% in both antrum and corpus. Our findings suggest that some bleeding patients with infection caused by H. pylori may not be correctly diagnosed by classical methods, and such patients could benefit from the improved diagnosis provided by real-time PCR. However, the clinical significance of a small number of microorganisms in patients with negative results in classical tests should be evaluated. PMID:22837325

  12. Stratified computed tomography findings improve diagnostic accuracy for appendicitis

    PubMed Central

    Park, Geon; Lee, Sang Chul; Choi, Byung-Jo; Kim, Say-June

    2014-01-01

    AIM: To improve the diagnostic accuracy in patients with symptoms and signs of appendicitis, but without confirmative computed tomography (CT) findings. METHODS: We retrospectively reviewed the database of 224 patients who had been operated on for the suspicion of appendicitis, but whose CT findings were negative or equivocal for appendicitis. The patient population was divided into two groups: a pathologically proven appendicitis group (n = 177) and a non-appendicitis group (n = 47). The CT images of these patients were re-evaluated according to the characteristic CT features as described in the literature. The re-evaluations and baseline characteristics of the two groups were compared. RESULTS: The two groups showed significant differences with respect to appendiceal diameter, and the presence of periappendiceal fat stranding and intraluminal air in the appendix. A larger proportion of patients in the appendicitis group showed distended appendices larger than 6.0 mm (66.3% vs 37.0%; P < 0.001), periappendiceal fat stranding (34.1% vs 8.9%; P = 0.001), and the absence of intraluminal air (67.6% vs 48.9%; P = 0.024) compared to the non-appendicitis group. Furthermore, the presence of two or more of these factors increased the odds ratio to 6.8 times higher than baseline (95%CI: 3.013-15.454; P < 0.001). CONCLUSION: Appendiceal diameter and wall thickening, fat stranding, and absence of intraluminal air can be used to increased diagnostic accuracy for appendicitis with equivocal CT findings. PMID:25320531

  13. Emulating a crowded intracellular environment in vitro dramatically improves RT-PCR performance

    SciTech Connect

    Lareu, Ricky R.; Harve, Karthik S.; Raghunath, Michael

    2007-11-09

    The polymerase chain reaction's (PCR) phenomenal success in advancing fields as diverse as Medicine, Agriculture, Conservation, or Paleontology is based on the ability of using isolated prokaryotic thermostable DNA polymerases in vitro to copy DNA irrespective of origin. This process occurs intracellularly and has evolved to function efficiently under crowded conditions, namely in an environment packed with macromolecules. However, current in vitro practice ignores this important biophysical parameter of life. In order to more closely emulate conditions of intracellular biochemistry in vitro we added inert macromolecules into reverse transcription (RT) and PCR. We show dramatic improvements in all parameters of RT-PCR including 8- to 10-fold greater sensitivity, enhanced polymerase processivity, higher specific amplicon yield, greater primer annealing and specificity, and enhanced DNA polymerase thermal stability. The faster and more efficient reaction kinetics was a consequence of the cumulative molecular and thermodynamic effects of the excluded volume effect created by macromolecular crowding.

  14. Improved Genotyping Vaccine and Wild-Type Poliovirus Strains by Restriction Fragment Length Polymorphism Analysis: Clinical Diagnostic Implications

    PubMed Central

    Georgopoulou, Amalia; Markoulatos, Panayotis; Spyrou, Niki; Vamvakopoulos, Nicholas C.

    2000-01-01

    The combination of preventive vaccination and diagnostic typing of viral isolates from patients with clinical poliomyelitis constitutes our main protective shield against polioviruses. The restriction fragment length polymorphism (RFLP) adaptation of the reverse transcriptase (RT)-PCR methodology has advanced diagnostic genotyping of polioviruses, although further improvements are definitely needed. We report here on an improved RFLP procedure for the genotyping of polioviruses. A highly conserved segment within the 5′ noncoding region of polioviruses was selected for RT-PCR amplification by the UC53-UG52 primer pair with the hope that it would be most resistant to the inescapable genetic alteration-drift experienced by the other segments of the viral genome. Complete inter- and intratypic genotyping of polioviruses by the present RFLP method was accomplished with a minimum set of four restriction endonucleases (HaeIII, DdeI, NcoI, and AvaI). To compensate for potential genetic drift within the recognition sites of HaeIII, DdeI, or NcoI in atypical clinical samples, the RFLP patterns generated with HpaII and StyI as replacements were analyzed. The specificity of the method was also successfully assessed by RFLP analysis of 55 reference nonpoliovirus enterovirus controls. The concerted implementation of these conditional protocols for diagnostic inter- and intratypic genotyping of polioviruses was evaluated with 21 clinical samples with absolute success. PMID:11101561

  15. Diagnostic Performance of a Multiple Real-Time PCR Assay in Patients with Suspected Sepsis Hospitalized in an Internal Medicine Ward

    PubMed Central

    Pasqualini, Leonella; Leli, Christian; Montagna, Paolo; Cardaccia, Angela; Cenci, Elio; Montecarlo, Ines; Pirro, Matteo; di Filippo, Francesco; Cistaro, Emma; Schillaci, Giuseppe; Bistoni, Francesco; Mannarino, Elmo

    2012-01-01

    Early identification of causative pathogen in sepsis patients is pivotal to improve clinical outcome. SeptiFast (SF), a commercially available system for molecular diagnosis of sepsis based on PCR, has been mostly used in patients hospitalized in hematology and intensive care units. We evaluated the diagnostic accuracy and clinical usefulness of SF, compared to blood culture (BC), in 391 patients with suspected sepsis, hospitalized in a department of internal medicine. A causative pathogen was identified in 85 patients (22%). Sixty pathogens were detected by SF and 57 by BC. No significant differences were found between the two methods in the rates of pathogen detection (P = 0.74), even after excluding 9 pathogens which were isolated by BC and were not included in the SF master list (P = 0.096). The combination of SF and BC significantly improved the diagnostic yield in comparison to BC alone (P < 0.001). Compared to BC, SF showed a significantly lower contamination rate (0 versus 19 cases; P < 0.001) with a higher specificity for pathogen identification (1.00, 95% confidence interval [CI] of 0.99 to 1.00, versus 0.94, 95% CI of 0.90 to 0.96; P = 0.005) and a higher positive predictive value (1.00, 95% CI of 1.00 to 0.92%, versus 0.75, 95% CI of 0.63 to 0.83; P = 0.005). In the subgroup of patients (n = 191) who had been receiving antibiotic treatment for ≥24 h, SF identified more pathogens (16 versus 6; P = 0.049) compared to BC. These results suggest that, in patients with suspected sepsis, hospitalized in an internal medicine ward, SF could be a highly valuable adjunct to conventional BC, particularly in patients under antibiotic treatment. PMID:22322348

  16. Systematic use of universal 16S rRNA gene polymerase chain reaction (PCR) and sequencing for processing pleural effusions improves conventional culture techniques.

    PubMed

    Insa, Rosario; Marín, Mercedes; Martín, Adoración; Martín-Rabadán, Pablo; Alcalá, Luís; Cercenado, Emilia; Calatayud, Laura; Liñares, Josefina; Bouza, Emilio

    2012-03-01

    Conventional culture of pleural fluid samples frequently provides false-negative results. Universal polymerase chain reaction (PCR) of the 16S ribosomal ribonucleic acid (rRNA) gene (16S PCR) has proven useful in the diagnosis of various bacterial infections. We conducted a prospective study to assess the value of 16S PCR in the etiologic diagnosis of pleural effusion. All pleural fluid samples received for culture were also studied using 16S PCR. Positive samples were sequenced for identification. Clinical records and conventional culture results were analyzed to classify pleural fluid samples as infected or not infected. We studied 723 samples. We excluded 188 samples because they were obtained from a long-term chest tube, there was a diagnosis of mycobacterial infection, or there were insufficient data to classify the episode. Finally, 535 pleural fluid samples were analyzed. According to our criteria, 82 (15.3%) were infected and 453 (84.7%) were not infected. In the infected samples, 16S PCR was positive in 67 samples (81.7%) while conventional culture was positive in 45 (54.9%). There were 4 false positives with 16S PCR (0.9%) and 12 with culture (2.6%). The values for the etiologic diagnosis of bacterial pleural effusion of conventional culture compared with 16S PCR were as follows: sensitivity, 54.9%/81.7%; specificity, 97.4%/99.1%; positive predictive value, 76.3%/94.4%; negative predictive value, 92.6%/96.8%; and accuracy, 90.8%/96.5%.When compared with conventional culture, 16S PCR plus sequencing substantially improves the etiologic diagnosis of infectious pleural effusion. In our opinion, this technique should be added to the routine diagnostic armamentarium of clinical microbiology laboratories.

  17. Designing multiplex PCR system of Campylobacter jejuni for efficient typing by improving monoplex PCR binary typing method.

    PubMed

    Yamada, Kazuhiro; Ibata, Ami; Suzuki, Masahiro; Matsumoto, Masakado; Yamashita, Teruo; Minagawa, Hiroko; Kurane, Ryuichiro

    2015-01-01

    Campylobacter jejuni is responsible for the majority of Campylobacter infections. As the molecular epidemiological study of outbreaks, pulsed-field gel electrophoresis (PFGE) is performed in general. But PFGE has several problems. PCR binary typing (P-BIT) method is a typing method for Campylobacter spp. that was recently developed, and was reported to have a similar discriminatory power and stability to those of PFGE. We modified the P-BIT method from 18 monoplex PCRs to two multiplex PCR systems (mP-BIT). The same results were obtained from monoplex PCRs using original primers and multiplex PCR in the representative isolates. The mP-BIT can analyze 48 strains at a time by using 96-well PCR systems and can identify C. jejuni because mP-BIT includes C. jejuni marker. The typing of the isolates by the mP-BIT and PFGE demonstrated generally concordant results and the mP-BIT method (D = 0.980) has a similar discriminatory power to that of PFGE with SmaI digest (D = 0.975) or KpnI digest (D = 0.987) as with original article. The mP-BIT method is quick, simple and easy, and comes to be able to perform it at low cost by having become a multiplex PCR system. Therefore, the mP-BIT method with two multiplex PCR systems has high potential for a rapid first-line surveillance typing assay of C. jejuni and can be used for routine surveillance and outbreak investigations of C. jejuni in the future.

  18. Designing multiplex PCR system of Campylobacter jejuni for efficient typing by improving monoplex PCR binary typing method.

    PubMed

    Yamada, Kazuhiro; Ibata, Ami; Suzuki, Masahiro; Matsumoto, Masakado; Yamashita, Teruo; Minagawa, Hiroko; Kurane, Ryuichiro

    2015-01-01

    Campylobacter jejuni is responsible for the majority of Campylobacter infections. As the molecular epidemiological study of outbreaks, pulsed-field gel electrophoresis (PFGE) is performed in general. But PFGE has several problems. PCR binary typing (P-BIT) method is a typing method for Campylobacter spp. that was recently developed, and was reported to have a similar discriminatory power and stability to those of PFGE. We modified the P-BIT method from 18 monoplex PCRs to two multiplex PCR systems (mP-BIT). The same results were obtained from monoplex PCRs using original primers and multiplex PCR in the representative isolates. The mP-BIT can analyze 48 strains at a time by using 96-well PCR systems and can identify C. jejuni because mP-BIT includes C. jejuni marker. The typing of the isolates by the mP-BIT and PFGE demonstrated generally concordant results and the mP-BIT method (D = 0.980) has a similar discriminatory power to that of PFGE with SmaI digest (D = 0.975) or KpnI digest (D = 0.987) as with original article. The mP-BIT method is quick, simple and easy, and comes to be able to perform it at low cost by having become a multiplex PCR system. Therefore, the mP-BIT method with two multiplex PCR systems has high potential for a rapid first-line surveillance typing assay of C. jejuni and can be used for routine surveillance and outbreak investigations of C. jejuni in the future. PMID:25455748

  19. An improved method of gene synthesis based on DNA works software and overlap extension PCR.

    PubMed

    Dong, Bingxue; Mao, Runqian; Li, Baojian; Liu, Qiuyun; Xu, Peilin; Li, Gang

    2007-11-01

    A bottleneck in recent gene synthesis technologies is the high cost of oligonucleotide synthesis and post-synthesis sequencing. In this article, a simple and rapid method for low-cost gene synthesis technology was developed based on DNAWorks program and an improved single-step overlap extension PCR (OE-PCR). This method enables any DNA sequence to be synthesized with few errors, then any mutated sites could be corrected by site-specific mutagenesis technology or PCR amplification-assembly method, which can amplify different DNA fragments of target gene followed by assembly into an entire gene through their overlapped region. Eventually, full-length DNA sequence without error was obtained via this novel method. Our method is simple, rapid and low-cost, and also easily amenable to automation based on a DNAWorks design program and defined set of OE-PCR reaction conditions suitable for different genes. Using this method, several genes including Manganese peroxidase gene (Mnp) of Phanerochaete chrysosporium (P. chrysosporium), Laccase gene (Lac) of Trametes versicolor (T. versicolor) and Cip1 peroxidase gene (cip 1) of Coprinus cinereus (C. cinereus) with sizes ranging from 1.0 kb to 1.5 kb have been synthesized successfully.

  20. Recent improvements of the JET lithium beam diagnostic

    SciTech Connect

    Brix, M.; Morgan, P.; Stamp, M.; Zastrow, K.-D.; Dunai, D.; Meszaros, B.; Petravich, G.; Refy, D. I.; Szabolics, T.; Zoletnik, S.; Lupelli, I.; Marsen, S.; Melson, T. F.; Silva, C. [EURATOM Collaboration: JET-EFDA Contributors

    2012-10-15

    A 60 kV neutral lithium diagnostic beam probes the edge plasma of JET for the measurement of electron density profiles. This paper describes recent enhancements of the diagnostic setup, new procedures for calibration and protection measures for the lithium ion gun during massive gas puffs for disruption mitigation. New light splitting optics allow in parallel beam emission measurements with a new double entrance slit CCD spectrometer (spectrally resolved) and a new interference filter avalanche photodiode camera (fast density and fluctuation studies).

  1. Drugs and diagnostic innovations to improve global health.

    PubMed

    Peeling, Rosanna W; Nwaka, Solomon

    2011-09-01

    Infectious diseases remain the major cause of morbidity and mortality in the developing world. Affordable effective drugs and diagnostics are critical for patient management and disease control but the development of new drugs and diagnostics is too slow to keep up with the emergence and spread of infectious diseases around the world. Innovative collaborative research and development involving disease endemic countries and developed countries are urgently needed to accelerate progress along the path from discovery to product adoption. These emerging approaches and the need for increased investment in human and financial resources to support them are discussed.

  2. The Autism Diagnostic Observation Schedule: Revised Algorithms for Improved Diagnostic Validity

    ERIC Educational Resources Information Center

    Gotham, Katherine; Risi, Susan; Pickles, Andrew; Lord, Catherine

    2007-01-01

    Autism Diagnostic Observation Schedule (ADOS) Modules 1-3 item and domain total distributions were reviewed for 1,630 assessments of children aged 14 months to 16 years with an autism spectrum disorder (ASD) or with heterogeneous non-spectrum disorders. Children were divided by language level and age to yield more homogeneous cells. Items were…

  3. Use of Quantitative Fluorescent Polymerase Chain Reaction (QF PCR) in Prenatal Diagnostic of Fetal Aneuploidies in a 17 Month Period in Parallel with Karyotyping

    PubMed Central

    Konjhodzic, Rijad; Dervovic, Edina; Kurtovic-Basic, Ilvana; Stomornjak-Vukadin, Meliha; Muhic, Adis; Baljevic, Sumeja; Pirnat-Gegic, Aida; Basic, Ejub; Bilalovic, Nurija

    2014-01-01

    Introduction: QF PCR has recently entered diagnostic practice as a possible way to bypass culturing of the fetal cells, as well as to provide a rapid response following amniocentesis. Material and methods: The effective value of the QF PCR remains a much debated issue, positions ranging from that it makes classic kayotyping obsolete except in special occasions, to that it is no more than a guideline for a mandatory karyotype. Current practices of the gynecology specialists generates samples in such fashion that kariotyping of samples quickly falls behind to the point of obsoleteness, because, by the time a karyotype has been finished, a window of opportunity for termination of pregnancy has closed. Results: QF PCR provides a rapid response alternative, but it is necessary to establish its reproducibility, as well as an algorithm of its use along classic kariotyping. This study contains samples processed in a period from August 1, 2012 to December 31 2013 in both QF PCR and classic karyotype. Object of this study was compare results obtained by two methods, and establish confidence interval of the QF PCR testing. Overall, 661 amniotic fluid samples were processed and typed with QF PCR, out of which 221 were done in parallel with karyiotyping, as an confirmation of results. PMID:24825930

  4. Controlled Trial Using Computerized Feedback to Improve Physicians' Diagnostic Judgments.

    ERIC Educational Resources Information Center

    Poses, Roy M.; And Others

    1992-01-01

    A study involving 14 experienced physicians investigated the effectiveness of a computer program (providing statistical feedback to teach a clinical diagnostic rule that predicts the probability of streptococcal pharyngitis), in conjunction with traditional lecture and periodic disease-prevalence reports. Results suggest the integrated method is a…

  5. Diagnostic Bias and Conduct Disorder: Improving Culturally Sensitive Diagnosis

    ERIC Educational Resources Information Center

    Mizock, Lauren; Harkins, Debra

    2011-01-01

    Disproportionately high rates of Conduct Disorder are diagnosed in African American and Latino youth of color. Diagnostic bias contributes to overdiagnosis of Conduct Disorder in these adolescents of color. Following a diagnosis of Conduct Disorder, adolescents of color face poorer outcomes than their White counterparts. These negative outcomes…

  6. High diagnostic yield by CSF-PCR for entero- and herpes simplex viruses and TBEV serology in adults with acute aseptic meningitis in Stockholm.

    PubMed

    Franzen-Rohl, Elisabeth; Larsson, Kenny; Skoog, Eva; Tiveljung-Lindell, Annika; Grillner, Lena; Aurelius, Elisabeth; Glimåker, Martin

    2008-01-01

    Acute aseptic meningitis (AAM) affects 10-20/100,000 inhabitants per years in Sweden. Up to the beginning of the 1980s the diagnoses were made by virus isolation and/or determination of viral antibodies in serum. The development of PCR for detection of viruses in CSF samples has increased the sensitivity and diagnostic efficiency considerably. We investigated the aetiology of AAM and the diagnostic efficiency in an adult population in Stockholm, using a limited first-line combination of microbiological assays. CSF and serum samples, consecutively collected in 419 patients with clinical symptoms of AAM in northern Stockholm during 1999-2004, were included. PCR assays for herpes simplex virus (HSV) DNA and enterovirus (EV) RNA in the CSF as well as ELISA for IgM in serum to tick-borne encephalitis virus (TBEV) were performed routinely. A viral diagnosis was obtained in 255 of the 419 cases (62%) with these routinely performed assays. Clinical findings in combination with additional diagnostic tests resulted in an overall aetiological yield of 72%. EV was the major causative agent (27%) followed by TBEV (21%) and HSV-2 (19%). We conclude that consistent use of CSF-PCR for EV and HSV and TBEV serology established a diagnosis in the majority of AAM patients.

  7. Comparison of Diagnostic Accuracy of PCR Targeting the 47-Kilodalton Protein Membrane Gene of Treponema pallidum and PCR Targeting the DNA Polymerase I Gene: Systematic Review and Meta-analysis

    PubMed Central

    Combescure, Christophe; Lautenschlager, Stephan; Ninet, Béatrice; Perneger, Thomas V.

    2015-01-01

    Treponema pallidum PCR (Tp-PCR) testing now is recommended as a valid tool for the diagnosis of primary or secondary syphilis. The objectives were to systematically review and determine the optimal specific target gene to be used for Tp-PCR. Comparisons of the performance of the two main targets are tpp47 and polA genes were done using meta-analysis. Three electronic bibliographic databases, representing abstract books from five conferences specialized in infectious diseases from January 1990 to March 2015, were searched. Search keywords included (“syphilis” OR “Treponema pallidum” OR “neurosyphilis”) AND (“PCR” OR “PCR” OR “molecular amplification”). We included diagnostic studies assessing the performance of Tp-PCR targeting tpp47 (tpp47-Tp-PCR) or the polA gene (polA-Tp-PCR) in ulcers from early syphilis. All studies were assessed against quality criteria using the QUADAS-2 tool. Of 37 studies identified, 62.2% were judged at low risk of bias or applicability. Most used the U.S. Centers for Disease Control and Prevention (CDC) case definitions for primary or secondary (early) syphilis (89.2%; n = 33); 15 (40.5%) used darkfield microscopy (DFM). We did not find differences in sensitivity and specificity between the two Tp-PCR methods in the subgroup of studies using adequate reference tests. Among studies using DFM as the reference test, sensitivities were 79.8% (95% confidence intervals [CI], 72.7 to 85.4%) and 71.4% (46.0 to 88.0%) for tpp47-Tp-PCR and polA-Tp-PCR (P = 0.217), respectively; respective specificities were 95.3% (93.5 to 96.6%) and 93.7% (91.8 to 95.2%) (P = 0.304). Our findings suggest that the two Tp-PCR methods have similar accuracy and could be used interchangeably. PMID:26311859

  8. Risk of Misdiagnosis Due to Allele Dropout and False-Positive PCR Artifacts in Molecular Diagnostics: Analysis of 30,769 Genotypes.

    PubMed

    Blais, Jonatan; Lavoie, Sébastien B; Giroux, Sylvie; Bussières, Johanne; Lindsay, Carmen; Dionne, Jacqueline; Laroche, Mélissa; Giguère, Yves; Rousseau, François

    2015-09-01

    Quality control is a complex issue for clinical molecular diagnostic applications. In the case of genotyping assays, artifacts such as allele dropout represent a risk of misdiagnosis for amplification-based methods. However, its frequency of occurrence in PCR-based diagnostic assays remains unknown. To maximize the likelihood of detecting allele dropout, our clinical genotyping PCR-based assays are designed with two independent assays for each allele (nonoverlapping primers on each DNA strand). To estimate the incidence of allelic dropout, we took advantage of the capacity of our clinical assays to detect such events. We retrospectively studied their occurrence in the initial PCR assay for 30,769 patient reports for mutations involved in four diseases produced over 8 years. Ninety-three allele dropout events were detected and all were solved before reporting. In addition, 42 cases of artifacts caused by amplification of an allele ultimately confirmed to not be part of the genotype (drop-in events) were detected and solved. These artifacts affected 1:227 genotypes, 94% of which were due to nonreproducible PCR failures rather than sequence variants interfering with the assay, suggesting that careful primer design cannot prevent most of these errors. This provides a quantitative estimate for clinical laboratories to take this phenomenon into account in quality management and to favor assay designs that can detect (and minimize) occurrence of these artifacts in routine clinical use.

  9. Risk of Misdiagnosis Due to Allele Dropout and False-Positive PCR Artifacts in Molecular Diagnostics: Analysis of 30,769 Genotypes.

    PubMed

    Blais, Jonatan; Lavoie, Sébastien B; Giroux, Sylvie; Bussières, Johanne; Lindsay, Carmen; Dionne, Jacqueline; Laroche, Mélissa; Giguère, Yves; Rousseau, François

    2015-09-01

    Quality control is a complex issue for clinical molecular diagnostic applications. In the case of genotyping assays, artifacts such as allele dropout represent a risk of misdiagnosis for amplification-based methods. However, its frequency of occurrence in PCR-based diagnostic assays remains unknown. To maximize the likelihood of detecting allele dropout, our clinical genotyping PCR-based assays are designed with two independent assays for each allele (nonoverlapping primers on each DNA strand). To estimate the incidence of allelic dropout, we took advantage of the capacity of our clinical assays to detect such events. We retrospectively studied their occurrence in the initial PCR assay for 30,769 patient reports for mutations involved in four diseases produced over 8 years. Ninety-three allele dropout events were detected and all were solved before reporting. In addition, 42 cases of artifacts caused by amplification of an allele ultimately confirmed to not be part of the genotype (drop-in events) were detected and solved. These artifacts affected 1:227 genotypes, 94% of which were due to nonreproducible PCR failures rather than sequence variants interfering with the assay, suggesting that careful primer design cannot prevent most of these errors. This provides a quantitative estimate for clinical laboratories to take this phenomenon into account in quality management and to favor assay designs that can detect (and minimize) occurrence of these artifacts in routine clinical use. PMID:26146130

  10. 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

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

    PubMed

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

    2013-01-01

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

  12. Can we improve the diagnostic yield of small bowel video-capsule endoscopy?

    PubMed

    Triantafyllou, Konstantinos

    2010-05-16

    Video-capsule endoscopy has revolutionized the examination of small bowel mucosa. However, this modality is relatively young and its diagnostic yield is low. Herein, we discuss different approaches to improve examination's diagnostic yield. There are strong data supporting some of them while there is speculation about the rest. As capsule endoscopy continues to evolve there is also a strong belief that technology will overcome at least some of the obstacles that hamper capsule endoscopy's diagnostic yield sometime in the near future.

  13. Comparison of Established Diagnostic Methodologies and a Novel Bacterial smpB Real-Time PCR Assay for Specific Detection of Haemophilus influenzae Isolates Associated with Respiratory Tract Infections

    PubMed Central

    Reddington, Kate; Schwenk, Stefan; Tuite, Nina; Platt, Gareth; Davar, Danesh; Coughlan, Helena; Personne, Yoann; Gant, Vanya; Enne, Virve I.; Zumla, Alimuddin

    2015-01-01

    Haemophilus influenzae is a significant causative agent of respiratory tract infections (RTI) worldwide. The development of a rapid H. influenzae diagnostic assay that would allow for the implementation of infection control measures and also improve antimicrobial stewardship for patients is required. A number of nucleic acid diagnostics approaches that detect H. influenzae in RTIs have been described in the literature; however, there are reported specificity and sensitivity limitations for these assays. In this study, a novel real-time PCR diagnostic assay targeting the smpB gene was designed to detect all serogroups of H. influenzae. The assay was validated using a panel of well-characterized Haemophilus spp. Subsequently, 44 Haemophilus clinical isolates were collected, and 36 isolates were identified as H. influenzae using a gold standard methodology that combined the results of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and a fucK diagnostic assay. Using the novel smpB diagnostic assay, 100% concordance was observed with the gold standard, demonstrating a sensitivity of 100% (95% confidence interval [CI], 90.26% to 100.00%) and a specificity of 100% (95% CI, 63.06% to 100.00%) when used on clinical isolates. To demonstrate the clinical utility of the diagnostic assay presented, a panel of lower RTI samples (n = 98) were blindly tested with the gold standard and smpB diagnostic assays. The results generated were concordant for 94/98 samples tested, demonstrating a sensitivity of 90.91% (95% CI, 78.33% to 97.47%) and a specificity of 100% (95% CI, 93.40% to 100.00%) for the novel smpB assay when used directly on respiratory specimens. PMID:26109443

  14. Comparison of Established Diagnostic Methodologies and a Novel Bacterial smpB Real-Time PCR Assay for Specific Detection of Haemophilus influenzae Isolates Associated with Respiratory Tract Infections.

    PubMed

    Reddington, Kate; Schwenk, Stefan; Tuite, Nina; Platt, Gareth; Davar, Danesh; Coughlan, Helena; Personne, Yoann; Gant, Vanya; Enne, Virve I; Zumla, Alimuddin; Barry, Thomas

    2015-09-01

    Haemophilus influenzae is a significant causative agent of respiratory tract infections (RTI) worldwide. The development of a rapid H. influenzae diagnostic assay that would allow for the implementation of infection control measures and also improve antimicrobial stewardship for patients is required. A number of nucleic acid diagnostics approaches that detect H. influenzae in RTIs have been described in the literature; however, there are reported specificity and sensitivity limitations for these assays. In this study, a novel real-time PCR diagnostic assay targeting the smpB gene was designed to detect all serogroups of H. influenzae. The assay was validated using a panel of well-characterized Haemophilus spp. Subsequently, 44 Haemophilus clinical isolates were collected, and 36 isolates were identified as H. influenzae using a gold standard methodology that combined the results of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and a fucK diagnostic assay. Using the novel smpB diagnostic assay, 100% concordance was observed with the gold standard, demonstrating a sensitivity of 100% (95% confidence interval [CI], 90.26% to 100.00%) and a specificity of 100% (95% CI, 63.06% to 100.00%) when used on clinical isolates. To demonstrate the clinical utility of the diagnostic assay presented, a panel of lower RTI samples (n = 98) were blindly tested with the gold standard and smpB diagnostic assays. The results generated were concordant for 94/98 samples tested, demonstrating a sensitivity of 90.91% (95% CI, 78.33% to 97.47%) and a specificity of 100% (95% CI, 93.40% to 100.00%) for the novel smpB assay when used directly on respiratory specimens. PMID:26109443

  15. Improving the diagnostic stage of the suspected colorectal cancer pathway: A quality improvement project.

    PubMed

    Haddow, James B; Walshe, Maria; Aggarwal, Dinesh; Thapar, Ankur; Hardman, John; Wilson, Jonathan; Oshowo, Ayo; Bhan, Chetan; Mukhtar, Hasan

    2016-09-01

    We aimed to improve the lead-time and the patient experience of the diagnostic stage of the suspected colorectal cancer pathway. This project worked within the constraints of limited resources and an austere environment. The core team included a project manager trained in quality improvement methodologies. Senior and Fleming's planned change model was used as the overall framework. Baseline data supported the case for change and highlighted targets for improvement. A stakeholder workshop employed social movement theory, lean thinking, experience-based design and patient stories to engage influential leaders and secure support and commitment. Solutions that arose from the workshop were then researched. A "Genchi Genbutsu" ethos took the team to Northumbria to learn about another unit's pathway innovations. Subsequently, our new pathway employed solutions aimed at increasing the proportion of patients who went straight-to-test. Consensus on the design was achieved using Schein's process consultation theory. Implementation of the new pathway resulted in a significant reduction in the median time from referral to endoscopy from 26 days to 14 days (P<0.001), and a significant increase in the proportion going straight-to-test from 6% to 43%. Changes to improve patient experience were also implemented, however data to evidence this has not yet been collected. Going forward, further standardisation is required and issues around sustainability need to be tackled. This project exemplified, amongst others, the value of working from data from the beginning and a comprehensive early stakeholder engagement. PMID:27637830

  16. Improving the diagnostic stage of the suspected colorectal cancer pathway: A quality improvement project.

    PubMed

    Haddow, James B; Walshe, Maria; Aggarwal, Dinesh; Thapar, Ankur; Hardman, John; Wilson, Jonathan; Oshowo, Ayo; Bhan, Chetan; Mukhtar, Hasan

    2016-09-01

    We aimed to improve the lead-time and the patient experience of the diagnostic stage of the suspected colorectal cancer pathway. This project worked within the constraints of limited resources and an austere environment. The core team included a project manager trained in quality improvement methodologies. Senior and Fleming's planned change model was used as the overall framework. Baseline data supported the case for change and highlighted targets for improvement. A stakeholder workshop employed social movement theory, lean thinking, experience-based design and patient stories to engage influential leaders and secure support and commitment. Solutions that arose from the workshop were then researched. A "Genchi Genbutsu" ethos took the team to Northumbria to learn about another unit's pathway innovations. Subsequently, our new pathway employed solutions aimed at increasing the proportion of patients who went straight-to-test. Consensus on the design was achieved using Schein's process consultation theory. Implementation of the new pathway resulted in a significant reduction in the median time from referral to endoscopy from 26 days to 14 days (P<0.001), and a significant increase in the proportion going straight-to-test from 6% to 43%. Changes to improve patient experience were also implemented, however data to evidence this has not yet been collected. Going forward, further standardisation is required and issues around sustainability need to be tackled. This project exemplified, amongst others, the value of working from data from the beginning and a comprehensive early stakeholder engagement.

  17. Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions

    PubMed Central

    Belmonte, Frances R.; Martin, James L.; Frescura, Kristin; Damas, Joana; Pereira, Filipe; Tarnopolsky, Mark A.; Kaufman, Brett A.

    2016-01-01

    Mitochondrial DNA (mtDNA) mutations are a common cause of primary mitochondrial disorders, and have also been implicated in a broad collection of conditions, including aging, neurodegeneration, and cancer. Prevalent among these pathogenic variants are mtDNA deletions, which show a strong bias for the loss of sequence in the major arc between, but not including, the heavy and light strand origins of replication. Because individual mtDNA deletions can accumulate focally, occur with multiple mixed breakpoints, and in the presence of normal mtDNA sequences, methods that detect broad-spectrum mutations with enhanced sensitivity and limited costs have both research and clinical applications. In this study, we evaluated semi-quantitative and digital PCR-based methods of mtDNA deletion detection using double-stranded reference templates or biological samples. Our aim was to describe key experimental assay parameters that will enable the analysis of low levels or small differences in mtDNA deletion load during disease progression, with limited false-positive detection. We determined that the digital PCR method significantly improved mtDNA deletion detection sensitivity through absolute quantitation, improved precision and reduced assay standard error. PMID:27122135

  18. EGFR Mutation Analysis of Circulating Tumor DNA Using an Improved PNA-LNA PCR Clamp Method

    PubMed Central

    Watanabe, Kana; Fukuhara, Tatsuro; Tsukita, Yoko; Morita, Mami; Suzuki, Aya; Tanaka, Nobuyuki; Terasaki, Hiroshi; Nukiwa, Toshihiro

    2016-01-01

    Introduction. Rebiopsies have become more crucial in non-small cell lung cancer (NSCLC). Instead of invasive biopsies, development of collecting biological data of the tumor from blood samples is expected. We conducted a prospective study to assess the feasibility of detection of epidermal growth factor receptor (EGFR) mutation in plasma samples. Method. NSCLC patients harboring EGFR activating mutations, who were going to receive EGFR-tyrosine kinase inhibitors (TKIs) as first-line treatment, were enrolled in this study. Plasma EGFR activating mutations and the T790M resistance mutation were analyzed by an improved PNA-LNA PCR clamp method, characterized by a 10-fold or more sensitivity compared with the original methods. Result. Six patients with wild-type EGFR and 24 patients with EGFR mutations were enrolled in this study. Pretreatment plasma samples achieved sensitivity of 79%. The 6 patients with wild-type EGFR were all negative for plasma EGFR mutations. At the time of disease progression, plasma T790M mutation was detected in 8 of 16 cases. Absence of T790M before and during TKI treatment and disappearance of activating mutations during TKI treatment were considered as predictors of EGFR-TKIs efficacy. Conclusion. We were able to detect EGFR mutations in plasma samples by using an improved PNA-LNA PCR clamp method. PMID:27478396

  19. Detection of Echinococcus multilocularis by MC-PCR: evaluation of diagnostic sensitivity and specificity without gold standard

    PubMed Central

    Wahlström, Helene; Comin, Arianna; Isaksson, Mats; Deplazes, Peter

    2016-01-01

    Introduction A semi-automated magnetic capture probe-based DNA extraction and real-time PCR method (MC-PCR), allowing for a more efficient large-scale surveillance of Echinococcus multilocularis occurrence, has been developed. The test sensitivity has previously been evaluated using the sedimentation and counting technique (SCT) as a gold standard. However, as the sensitivity of the SCT is not 1, test characteristics of the MC-PCR was also evaluated using latent class analysis, a methodology not requiring a gold standard. Materials and methods Test results, MC-PCR and SCT, from a previous evaluation of the MC-PCR using 177 foxes shot in the spring (n=108) and autumn 2012 (n=69) in high prevalence areas in Switzerland were used. Latent class analysis was used to estimate the test characteristics of the MC-PCR. Although it is not the primary aim of this study, estimates of the test characteristics of the SCT were also obtained. Results and discussion This study showed that the sensitivity of the MC-PCR was 0.88 [95% posterior credible interval (PCI) 0.80–0.93], which was not significantly different than the SCT, 0.83 (95% PCI 0.76–0.88), which is currently considered as the gold standard. The specificity of both tests was high, 0.98 (95% PCI 0.94–0.99) for the MC-PCR and 0.99 (95% PCI 0.99–1) for the SCT. In a previous study, using fox scats from a low prevalence area, the specificity of the MC-PCR was higher, 0.999% (95% PCI 0.997–1). One reason for the lower estimate of the specificity in this study could be that the MC-PCR detects DNA from infected but non-infectious rodents eaten by foxes. When using MC-PCR in low prevalence areas or areas free from the parasite, a positive result in the MC-PCR should be regarded as a true positive. Conclusion The sensitivity of the MC-PCR (0.88) was comparable to the sensitivity of SCT (0.83). PMID:26968153

  20. Improved Diagnostic Validity of the ADOS Revised Algorithms: A Replication Study in an Independent Sample

    ERIC Educational Resources Information Center

    Oosterling, Iris; Roos, Sascha; de Bildt, Annelies; Rommelse, Nanda; de Jonge, Maretha; Visser, Janne; Lappenschaar, Martijn; Swinkels, Sophie; van der Gaag, Rutger Jan; Buitelaar, Jan

    2010-01-01

    Recently, Gotham et al. ("2007") proposed revised algorithms for the Autism Diagnostic Observation Schedule (ADOS) with improved diagnostic validity. The aim of the current study was to replicate predictive validity, factor structure, and correlations with age and verbal and nonverbal IQ of the ADOS revised algorithms for Modules 1 and 2 in a…

  1. Improvement of the edge rotation diagnostic spectrum analysis via simulation

    SciTech Connect

    Luo, J.; Zhuang, G. Cheng, Z. F.; Zhang, X. L.; Hou, S. Y.; Cheng, C.

    2014-11-15

    The edge rotation diagnostic (ERD) system has been developed on the Joint Texas Experimental Tokamak to measure the edge toroidal rotation velocity by observing the shifted wavelength of carbon V (C V 227.09 nm). Since the measured spectrum is an integrated result along the viewing line from the plasma core to the edge, a method via simulation has been developed to analyze the ERD spectrum. With the necessary parameters such as C V radiation profile and the ion temperature profile, a local rotation profile at the normalized minor radius of 0.5-1 is obtained.

  2. Adaptive optics for improved retinal surgery and diagnostics

    SciTech Connect

    Humayun, M S; Sadda, S R; Thompson, C A; Olivier, S S; Kartz, M W

    2000-08-21

    It is now possible to field a compact adaptive optics (AO) system on a surgical microscope for use in retinal diagnostics and surgery. Recent developments in integrated circuit technology and optical photonics have led to the capability of building an AO system that is compact and significantly less expensive than traditional AO systems. It is foreseen that such an AO system can be integrated into a surgical microscope while maintaining a package size of a lunchbox. A prototype device can be developed in a manner that lends itself well to large-scale manufacturing.

  3. Improvement of the edge rotation diagnostic spectrum analysis via simulationa)

    NASA Astrophysics Data System (ADS)

    Luo, J.; Zhuang, G.; Cheng, Z. F.; Zhang, X. L.; Hou, S. Y.; Cheng, C.

    2014-11-01

    The edge rotation diagnostic (ERD) system has been developed on the Joint Texas Experimental Tokamak to measure the edge toroidal rotation velocity by observing the shifted wavelength of carbon V (C V 227.09 nm). Since the measured spectrum is an integrated result along the viewing line from the plasma core to the edge, a method via simulation has been developed to analyze the ERD spectrum. With the necessary parameters such as C V radiation profile and the ion temperature profile, a local rotation profile at the normalized minor radius of 0.5-1 is obtained.

  4. Evaluation of Three Diagnostic Methods, Including Real-Time PCR, for Detection of Dientamoeba fragilis in Stool Specimens

    PubMed Central

    Stark, D.; Beebe, N.; Marriott, D.; Ellis, J.; Harkness, J.

    2006-01-01

    Dientamoeba fragilis is a protozoan parasite of humans that infects the mucosa of the large intestine and is associated with gastrointestinal disease. We developed a 5′ nuclease (TaqMan)-based real-time PCR assay, targeting the small subunit rRNA gene, for the detection of D. fragilis in human stool specimens and compared its sensitivity and specificity to conventional PCR and microscopic examination by a traditional modified iron-hematoxylin staining procedure. Real-time PCR exhibited 100% sensitivity and specificity. PMID:16390978

  5. Polyethersulfone improves isothermal nucleic acid amplification compared to current paper-based diagnostics

    PubMed Central

    Linnes, J. C.; Rodriguez, N. M.; Liu, L.

    2016-01-01

    Devices based on rapid, paper-based, isothermal nucleic acid amplification techniques have recently emerged with the potential to fill a growing need for highly sensitive point-of-care diagnostics throughout the world. As this field develops, such devices will require optimized materials that promote amplification and sample preparation. Herein, we systematically investigated isothermal nucleic acid amplification in materials currently used in rapid diagnostics (cellulose paper, glass fiber, and nitrocellulose) and two additional porous membranes with upstream sample preparation capabilities (polyethersulfone and polycarbonate). We compared amplification efficiency from four separate DNA and RNA targets (Bordetella pertussis, Chlamydia trachomatis, Neisseria gonorrhoeae, and Influenza A H1N1) within these materials using two different isothermal amplification schemes, helicase dependent amplification (tHDA) and loop-mediated isothermal amplification (LAMP), and traditional PCR. We found that the current paper-based diagnostic membranes inhibited nucleic acid amplification when compared to membrane-free controls; however, polyethersulfone allowed for efficient amplification in both LAMP and tHDA reactions. Further, observing the performance of traditional PCR amplification within these membranes was not predicative of their effects on in situ LAMP and tHDA. Polyethersulfone is a new material for paper-based nucleic acid amplification, yet provides an optimal support for rapid molecular diagnostics for point-of-care applications. PMID:26906904

  6. Polyethersulfone improves isothermal nucleic acid amplification compared to current paper-based diagnostics.

    PubMed

    Linnes, J C; Rodriguez, N M; Liu, L; Klapperich, C M

    2016-04-01

    Devices based on rapid, paper-based, isothermal nucleic acid amplification techniques have recently emerged with the potential to fill a growing need for highly sensitive point-of-care diagnostics throughout the world. As this field develops, such devices will require optimized materials that promote amplification and sample preparation. Herein, we systematically investigated isothermal nucleic acid amplification in materials currently used in rapid diagnostics (cellulose paper, glass fiber, and nitrocellulose) and two additional porous membranes with upstream sample preparation capabilities (polyethersulfone and polycarbonate). We compared amplification efficiency from four separate DNA and RNA targets (Bordetella pertussis, Chlamydia trachomatis, Neisseria gonorrhoeae, and Influenza A H1N1) within these materials using two different isothermal amplification schemes, helicase dependent amplification (tHDA) and loop-mediated isothermal amplification (LAMP), and traditional PCR. We found that the current paper-based diagnostic membranes inhibited nucleic acid amplification when compared to membrane-free controls; however, polyethersulfone allowed for efficient amplification in both LAMP and tHDA reactions. Further, observing the performance of traditional PCR amplification within these membranes was not predicative of their effects on in situ LAMP and tHDA. Polyethersulfone is a new material for paper-based nucleic acid amplification, yet provides an optimal support for rapid molecular diagnostics for point-of-care applications. PMID:26906904

  7. Ages of celiac disease: From changing environment to improved diagnostics

    PubMed Central

    Tommasini, Alberto; Not, Tarcisio; Ventura, Alessandro

    2011-01-01

    From the time of Gee’s landmark writings, the recent history of celiac disease (CD) can be divided into many ages, each driven by a diagnostic advance and a deeper knowledge of disease pathogenesis. At the same time, these advances were paralleled by the identification of new clinical patterns associated with CD and by a continuous redefinition of the prevalence of the disease in population. In the beginning, CD was considered a chronic indigestion, even if the causative food was not known; later, the disease was proven to depend on an intolerance to wheat gliadin, leading to typical mucosal changes in the gut and to a malabsorption syndrome. This knowledge led to curing the disease with a gluten-free diet. After the identification of antibodies to gluten (AGA) in the serum of patients and the identification of gluten-specific lymphocytes in the mucosa, CD was described as an immune disorder, resembling a chronic “gluten infection”. The use of serological testing for AGA allowed identification of the higher prevalence of this disorder, revealing atypical patterns of presentation. More recently, the characterization of autoantibodies to endomysium and to transglutaminase shifted the attention to a complex autoimmune pathogenesis and to the increased risk of developing autoimmune disorders in untreated CD. New diagnostic assays, based on molecular technologies, will introduce new changes, with the promise of better defining the spectrum of gluten reactivity and the real burden of gluten related-disorders in the population. Herein, we describe the different periods of CD experience, and further developments for the next celiac age will be proposed. PMID:21990947

  8. 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.

  9. Improved HF183 Quantitative Real-Time PCR Assay for Characterization of Human Fecal Pollution in Ambient Surface Water Samples

    PubMed Central

    Green, Hyatt C.; Haugland, Richard A.; Varma, Manju; Millen, Hana T.; Borchardt, Mark A.; Field, Katharine G.; Walters, William A.; Knight, R.; Sivaganesan, Mano; Kelty, Catherine A.

    2014-01-01

    Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus Bacteroides are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters. PMID:24610857

  10. Improved HF183 quantitative real-time PCR assay for characterization of human fecal pollution in ambient surface water samples.

    PubMed

    Green, Hyatt C; Haugland, Richard A; Varma, Manju; Millen, Hana T; Borchardt, Mark A; Field, Katharine G; Walters, William A; Knight, R; Sivaganesan, Mano; Kelty, Catherine A; Shanks, Orin C

    2014-05-01

    Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus Bacteroides are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters.

  11. Bovine mastitis: the diagnostic properties of a PCR-based assay to monitor the Staphylococcus aureus genotype B status of a herd, using bulk tank milk.

    PubMed

    Syring, C; Boss, R; Reist, M; Bodmer, M; Hummerjohann, J; Gehrig, P; Graber, H U

    2012-07-01

    Staphylococcus aureus genotype B (GTB) is a contagious mastitis pathogen in cattle, occurring in up to 87% of individuals. Because treatment is generally insufficient, culling is often required, leading to large economic loss in the Swiss dairy industry. As the detection of this pathogen in bulk tank milk (BTM) would greatly facilitate its control, a novel real-time quantitative PCR-based assay for BTM has previously been developed and is now being evaluated for its diagnostic properties at the herd level. Herds were initially classified as to their Staph. aureus GTB status by a reference method. Using BTM and herd pools of single-quarter and 4-quarter milk, the herds were then grouped by the novel assay, and the resulting classifications were compared. A total of 54 dairy herds were evaluated. Using the reference method, 21 herds were found to be GTB positive, whereas 33 were found to be negative. Considering the novel assay using both herd pools, all herds were grouped correctly, resulting in maximal diagnostic sensitivities (100%) and specificities (100%). For BTM samples, diagnostic sensitivities and specificities were 90 and 100%, respectively. Two herds were false negative in BTM, because cows with clinical signs of mastitis were not milked into the tank. Besides its excellent diagnostic properties, the assay is characterized by its low detection level, high efficiency, and its suitability for automation. Using the novel knowledge and assay, eradication of Staph. aureus GTB from a dairy herd may be considered as a realistic goal.

  12. Application of modern diagnostic methods to environmental improvement. Annual progress report, October 1994--September 1995

    SciTech Connect

    Shepard, W.S.

    1995-12-01

    The Diagnostic Instrumentation and Analysis Laboratory (DIAL), an interdisciplinary research department in the College of Engineering at Mississippi State University (MSU), is under contract with the US Department of Energy (DOE) to develop and apply advanced diagnostic instrumentation and analysis techniques to aid in solving DOE`s nuclear waste problem. The program is a comprehensive effort which includes five focus areas: advanced diagnostic systems; development/application; torch operation and test facilities; process development; on-site field measurement and analysis; technology transfer/commercialization. As part of this program, diagnostic methods will be developed and evaluated for characterization, monitoring and process control. Also, the measured parameters, will be employed to improve, optimize and control the operation of the plasma torch and the overall plasma treatment process. Moreover, on-site field measurements at various DOE facilities are carried out to aid in the rapid demonstration and implementation of modern fieldable diagnostic methods. Such efforts also provide a basis for technology transfer.

  13. Lyophilization to improve the sensitivity of qPCR for bacterial DNA detection in serum: the Q fever paradigm.

    PubMed

    Edouard, Sophie; Raoult, Didier

    2016-06-01

    Quantitative real-time PCR (qPCR) on serum provides significant added value to the diagnosis of Q fever, mainly at the acute stage of the disease in seronegative patients and in patients with endocarditis. We evaluated the benefits of Coxiella burnetii DNA concentration in serum by lyophilization to improve qPCR sensitivity. The detection limit of qPCR was determined by comparing six 10-fold dilutions of serum (calibrated with 104 bacteria ml-1) with and without lyophilization. We also tested, after lyophilization, 73 sera from patients with acute Q fever and 10 sera from patients with endocarditis for which specific qPCR for C. burnetii performed under our usual conditions remained negative. Lyophilization of DNA was found to improve sensitivity of the qPCR; the limit of detection of C. burnetii DNA by qPCR was 100-fold lower in lyophilized sera (1 bacterium ml-1) than in non-lyophilized sera (102 bacteria ml-1). Among the 73 sera from patients with acute Q fever, 26 (36 %) were positive after lyophilization, demonstrating a sensitivity gain of 44 % for early negative sera and 30 % for positive sera compared to our usual qPCR conditions. Sensitivity was also higher in sera from patients with endocarditis for which 8/10 (80 %) were positive after lyophilization. Our results serve as a proof of concept that lyophilization increases the sensitivity of qPCR in serum by concentrating bacterial DNA. This technique may be applied for the earlier diagnosis of other fastidious bacteria or viruses and extended to other clinical samples. PMID:27008653

  14. Development of a novel PCR-RFLP assay for improved detection and typing of bovine papillomaviruses.

    PubMed

    Kawauchi, Kyoko; Takahashi, Chiaki; Ishihara, Ryoko; Hatama, Shinichi

    2015-06-15

    A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed to detect and type bovine papillomaviruses (BPVs) from tumors in cattle. Two degenerate primer sets targeting the BPV L1 gene, subAup/subAdw and subBup/subBdw, and one restriction enzyme RsaI were used in this assay. In silico analyses of the restriction enzyme sites in the PCR fragments of 13 BPV sequences (BPV-1 to -13) revealed that all known BPVs are differentiated by the PCR-RFLP assay. Analyses of 63 previously typed clinical samples, that included teat papillomas and both esophageal and urinary bladder cancer biopsies, show that the assay clearly differentiates between eight clinically important BPV types (BPV-1 to -6, -9, -10), and discriminates between single and multiple infections. To further assess the reliability of the PCR-RFLP method amplified fragments were sequenced. A high correlation (95%) was observed when the results of the PCR-RFLP method were compared with PCR-sequencing. Differences in typing occurred for 3 of 63 specimens; PCR-RFLP identified additional BPV types in these specimens, while the PCR-sequencing identified only one. These results indicate that the PCR-RFLP method reported here is simpler and more reliable in the detection and typing of BPVs from bovine tumor samples than PCR-sequencing.

  15. Online Monitoring to Enable Improved Diagnostics, Prognostics and Maintenance

    SciTech Connect

    Bond, Leonard J.

    2011-02-01

    For both existing and new plant designs there are increasing opportunities and needs for the application of advanced online surveillance, diagnostic and prognostic techniques. These methods can continuously monitor and assess the health of nuclear power plant systems and components. The added effectiveness of such programs has the potential to enable holistic plant management, and minimize exposure to future and unknown risks. The 'NDE & On-line Monitoring' activities within the Advanced Instrumentation, Information and Control Systems (II&CS) Pathway are developing R&D to establish advanced condition monitoring and prognostics technologies to understand and predict future phenomena, derived from plant aging in systems, structures, and components (SSC). This research includes utilization of the enhanced functionality and system condition awareness that becomes available through the application of digital technologies at existing nuclear power plants for online monitoring and prognostics. The current state-of-the-art for on-line monitoring applied to active components (eg pumps, valves, motors) and passive structure (eg core internals, primary piping, pressure vessel, concrete, cables, buried pipes) is being reviewed. This includes looking at the current deployment of systems that monitor reactor noise, acoustic signals and vibration in various forms, leak monitoring, and now increasingly condition-based maintenance (CBM) for active components. The NDE and on-line monitoring projects are designed to look beyond locally monitored CBM. Current trends include centralized plant monitoring of SSC, potential fleet-based CBM and technology that will enable operation and maintenance to be performed with limited on-site staff. Attention is also moving to systems that use online monitoring to permit longer term operation (LTO), including a prognostic or predictive element that estimates a remaining useful life (RUL). Many, if not all, active components (pumps, valves, motors

  16. Requisite analytic and diagnostic performance characteristics for the clinical detection of BRAF V600E in hairy cell leukemia: a comparison of 2 allele-specific PCR assays.

    PubMed

    Brown, Noah A; Weigelin, Helmut C; Bailey, Nathanael; Laliberte, Julie; Elenitoba-Johnson, Kojo S J; Lim, Megan S; Betz, Bryan L

    2015-09-01

    Detection of high-frequency BRAF V600E mutations in hairy cell leukemia (HCL) has important diagnostic utility. However, the requisite analytic performance for a clinical assay to routinely detect BRAF V600E mutations in HCL has not been clearly defined. In this study, we sought to determine the level of analytic sensitivity needed for formalin-fixed, paraffin-embedded (FFPE) and frozen samples and to compare the performance of 2 allele-specific polymerase chain reaction (PCR) assays. Twenty-nine cases of classic HCL, including 22 FFPE bone marrow aspirates and 7 frozen specimens from blood or bone marrow were evaluated using a laboratory-developed allele-specific PCR assay and a commercially available allele-specific quantitative PCR assay-myT BRAF Ultra. Also included were 6 HCL variant and 40 non-HCL B-cell lymphomas. Two cases of classic HCL, 1 showing CD5 expression, were truly BRAF V600E-negative based on negative results by PCR and sequencing despite high-level leukemic involvement. Among the remaining 27 specimens, V600E mutations were detected in 88.9% (17/20 FFPE; 7/7 frozen) and 81.5% (15/20 FFPE; 7/7 frozen), for the laboratory-developed and commercial assays, respectively. No mutations were detected among the 46 non-HCL lymphomas. Both assays showed an analytic sensitivity of 0.3% involvement in frozen specimens and 5% in FFPE tissue. On the basis of these results, an assay with high analytic sensitivity is required for the clinical detection of V600E mutations in HCL specimens. Two allele-specific PCR assays performed well in both frozen and FFPE bone marrow aspirates, although detection in FFPE tissue required 5% or more involvement.

  17. Novel multiplex real-time PCR diagnostic assay for identification and differentiation of Mycobacterium tuberculosis, Mycobacterium canettii, and Mycobacterium tuberculosis complex strains.

    PubMed

    Reddington, Kate; O'Grady, Justin; Dorai-Raj, Siobhan; Maher, Majella; van Soolingen, Dick; Barry, Thomas

    2011-02-01

    Tuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosis complex (MTC). Rapid detection of the MTC is necessary for the timely initiation of antibiotic treatment, while differentiation between members of the complex may be important to guide the appropriate antibiotic treatment and provide epidemiological information. In this study, a multiplex real-time PCR diagnostics assay using novel molecular targets was designed to identify the MTC while simultaneously differentiating between M. tuberculosis and M. canettii. The lepA gene was targeted for the detection of members of the MTC, the wbbl1 gene was used for the differentiation of M. tuberculosis and M. canettii from the remainder of the complex, and a unique region of the M. canettii genome, a possible novel region of difference (RD), was targeted for the specific identification of M. canettii. The multiplex real-time PCR assay was tested using 125 bacterial strains (64 MTC isolates, 44 nontuberculosis mycobacteria [NTM], and 17 other bacteria). The assay was determined to be 100% specific for the mycobacteria tested. Limits of detection of 2.2, 2.17, and 0.73 cell equivalents were determined for M. tuberculosis/M. canettii, the MTC, and M. canettii, respectively, using probit regression analysis. Further validation of this diagnostics assay, using clinical samples, should demonstrate its potential for the rapid, accurate, and sensitive diagnosis of TB caused by M. tuberculosis, M. canettii, and the other members of the MTC.

  18. Formal Art Observation Training Improves Medical Students’ Visual Diagnostic Skills

    PubMed Central

    Naghshineh, Sheila; Hafler, Janet P.; Miller, Alexa R.; Blanco, Maria A.; Lipsitz, Stuart R.; Dubroff, Rachel P.; Khoshbin, Shahram

    2008-01-01

    Background Despite evidence of inadequate physical examination skills among medical students, teaching these skills has declined. One method of enhancing inspection skills is teaching “visual literacy,” the ability to reason physiology and pathophysiology from careful and unbiased observation. Objective To improve students’ visual acumen through structured observation of artworks, understanding of fine arts concepts and applying these skills to patient care. Design Prospective, partially randomized pre- vs. post-course evaluation using mixed-methods data analysis. Participants Twenty-four pre-clinical student participants were compared to 34 classmates at a similar stage of training. Intervention Training the Eye: Improving the Art of Physical Diagnosis consists of eight paired sessions of art observation exercises with didactics that integrate fine arts concepts with physical diagnosis topics and an elective life drawing session. Measurements The frequency of accurate observations on a 1-h visual skills examination was used to evaluate pre- vs. post-course descriptions of patient photographs and art imagery. Content analysis was used to identify thematic categories. All assessments were blinded to study group and pre- vs. post-course evaluation. Results Following the course, class participants increased their total mean number of observations compared to controls (5.41 ± 0.63 vs. 0.36 ± 0.53, p < 0.0001) and had increased sophistication in their descriptions of artistic and clinical imagery. A ‘dose-response’ was found for those who attended eight or more sessions, compared to participants who attended seven or fewer sessions (6.31 + 0.81 and 2.76 + 1.2, respectively, p = 0.03). Conclusions This interdisciplinary course improved participants’ capacity to make accurate observations of art and physical findings. Electronic supplementary material The online version of this article (doi:10.1007/s11606-008-0667-0) contains

  19. Optimization of diagnostic RT-PCR protocols and sampling procedures for the reliable and cost-effective detection of Cassava brown streak virus.

    PubMed

    Abarshi, M M; Mohammed, I U; Wasswa, P; Hillocks, R J; Holt, J; Legg, J P; Seal, S E; Maruthi, M N

    2010-02-01

    Sampling procedures and diagnostic protocols were optimized for accurate diagnosis of Cassava brown streak virus (CBSV) (genus Ipomovirus, family Potyviridae). A cetyl trimethyl ammonium bromide (CTAB) method was optimized for sample preparation from infected cassava plants and compared with the RNeasy plant mini kit (Qiagen) for sensitivity, reproducibility and costs. CBSV was detectable readily in total RNAs extracted using either method. The major difference between the two methods was in the cost of consumables, with the CTAB 10x cheaper (0.53 pounds sterling=US$0.80 per sample) than the RNeasy method (5.91 pounds sterling=US$8.86 per sample). A two-step RT-PCR (1.34 pounds sterling=US$2.01 per sample), although less sensitive, was at least 3-times cheaper than a one-step RT-PCR (4.48 pounds sterling=US$6.72). The two RT-PCR tests revealed consistently the presence of CBSV both in symptomatic and asymptomatic leaves and indicated that asymptomatic leaves can be used reliably for virus diagnosis. Depending on the accuracy required, sampling 100-400 plants per field is an appropriate recommendation for CBSD diagnosis, giving a 99.9% probability of detecting a disease incidence of 6.7-1.7%, respectively. CBSV was detected at 10(-4)-fold dilutions in composite sampling, indicating that the most efficient way to index many samples for CBSV will be to screen pooled samples. The diagnostic protocols described below are reliable and the most cost-effective methods available currently for detecting CBSV.

  20. A PCR-Based Diagnostic System for Differentiating Two Weevil Species (Coleoptera: Curculionidae) of Economic Importance to the Chilean Citrus Industry.

    PubMed

    Aguirre, C; Olivares, N; Luppichini, P; Hinrichsen, P

    2015-02-01

    A PCR-based method was developed to identify Naupactus cervinus (Boheman) and Naupactus xanthographus (Germar), two curculionids affecting the citrus industry in Chile. The quarantine status of these two species depends on the country to which fruits are exported. This identification method was developed because it is not possible to discriminate between these two species at the egg stage. The method is based on the species-specific amplification of sequences of internal transcribed spacers, for which we cloned and sequenced these genome fragments from each species. We designed an identification system based on two duplex-PCR reactions. Each one contains the species-specific primer set and a second generic primer set that amplify a short 18S region common to coleopterans, to avoid false negatives. The marker system is able to differentiate each Naupactus species at any life stage, and with a diagnostic sensitivity to 0.045 ng of genomic DNA. This PCR kit was validated by samples collected from different citrus production areas throughout Chile and showed 100% accuracy in differentiating the two Naupactus species. PMID:26470110

  1. Molecular Detection and Typing of Dengue Viruses from Archived Tissues of Fatal Cases by RT-PCR and Sequencing: Diagnostic and Epidemiologic Implications

    PubMed Central

    Bhatnagar, Julu; Blau, Dianna M.; Shieh, Wun-Ju; Paddock, Christopher D.; Drew, Clifton; Liu, Lindy; Jones, Tara; Patel, Mitesh; Zaki, Sherif R.

    2012-01-01

    Diagnosis of dengue virus (DENV) infection in fatal cases is challenging because of the frequent unavailability of blood or fresh tissues. For formalin-fixed, paraffin-embedded (FFPE) tissues immunohistochemistry (IHC) can be used; however, it may not be as sensitive and serotyping is not possible. The application of reverse transcription-polymerase chain reaction (RT-PCR) for the detection of DENV in FFPE tissues has been very limited. We evaluated FFPE autopsy tissues of 122 patients with suspected DENV infection by flavivirus and DENV RT-PCR, sequencing, and DENV IHC. The DENV was detected in 61 (50%) cases by RT-PCR or IHC. The RT-PCR and sequencing detected DENV in 60 (49%) cases (DENV-1 in 16, DENV-2 in 27, DENV-3 in 8, and DENV-4 in 6 cases). No serotype could be identified in three cases. The IHC detected DENV antigens in 50 (40%) cases. The RT-PCR using FFPE tissue improves detection of DENV in fatal cases and provides sequence information useful for typing and epidemiologic studies. PMID:22302871

  2. Improving Building Energy Simulation Programs Through Diagnostic Testing (Fact Sheet)

    SciTech Connect

    Not Available

    2012-02-01

    New test procedure evaluates quality and accuracy of energy analysis tools for the residential building retrofit market. Reducing the energy use of existing homes in the United States offers significant energy-saving opportunities, which can be identified through building simulation software tools that calculate optimal packages of efficiency measures. To improve the accuracy of energy analysis for residential buildings, the National Renewable Energy Laboratory's (NREL) Buildings Research team developed the Building Energy Simulation Test for Existing Homes (BESTEST-EX), a method for diagnosing and correcting errors in building energy audit software and calibration procedures. BESTEST-EX consists of building physics and utility bill calibration test cases, which software developers can use to compare their tools simulation findings to reference results generated with state-of-the-art simulation tools. Overall, the BESTEST-EX methodology: (1) Tests software predictions of retrofit energy savings in existing homes; (2) Ensures building physics calculations and utility bill calibration procedures perform to a minimum standard; and (3) Quantifies impacts of uncertainties in input audit data and occupant behavior. BESTEST-EX is helping software developers identify and correct bugs in their software, as well as develop and test utility bill calibration procedures.

  3. Improving Communication of Diagnostic Radiology Findings through Structured Reporting

    PubMed Central

    Panicek, David M.; Berk, Alexandra R.; Li, Yuelin; Hricak, Hedvig

    2011-01-01

    Purpose: To compare the content, clarity, and clinical usefulness of conventional (ie, free-form) and structured radiology reports of body computed tomographic (CT) scans, as evaluated by referring physicians, attending radiologists, and radiology fellows at a tertiary care cancer center. Materials and Methods: The institutional review board approved the study as a quality improvement initiative; no written consent was required. Three radiologists, three radiology fellows, three surgeons, and two medical oncologists evaluated 330 randomly selected conventional and structured radiology reports of body CT scans. For nonradiologists, reports were randomly selected from patients with diagnoses relevant to the physician’s area of specialization. Each physician read 15 reports in each format and rated both the content and clarity of each report from 1 (very dissatisfied or very confusing) to 10 (very satisfied or very clear). By using a previously published radiology report grading scale, physicians graded each report’s effectiveness in advancing the patient’s position on the clinical spectrum. Mixed-effects models were used to test differences between report types. Results: Mean content satisfaction ratings were 7.61 (95% confidence interval [CI]: 7.12, 8.16) for conventional reports and 8.33 (95% CI: 7.82, 8.86) for structured reports, and the difference was significant (P < .0001). Mean clarity satisfaction ratings were 7.45 (95% CI: 6.89, 8.02) for conventional reports and 8.25 (95% CI: 7.68, 8.82) for structured reports, and the difference was significant (P < .0001). Grade ratings did not differ significantly between conventional and structured reports. Conclusion: Referring clinicians and radiologists found that structured reports had better content and greater clarity than conventional reports. © RSNA, 2011 Supplemental material: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.11101913/-/DC1 PMID:21518775

  4. Modified DOP-PCR for improved STR typing of degraded DNA from human skeletal remains and bloodstains.

    PubMed

    Ambers, Angie; Turnbough, Meredith; Benjamin, Robert; Gill-King, Harrell; King, Jonathan; Sajantila, Antti; Budowle, Bruce

    2016-01-01

    Forensic and ancient DNA samples often are damaged and in limited quantity as a result of exposure to harsh environments and the passage of time. Several strategies have been proposed to address the challenges posed by degraded and low copy templates, including a PCR based whole genome amplification method called degenerate oligonucleotide-primed PCR (DOP-PCR). This study assessed the efficacy of four modified versions of the original DOP-PCR primer that retain at least a portion of the 5' defined sequence and alter the number of bases on the 3' end. The use of each of the four modified primers resulted in improved STR profiles from environmentally-damaged bloodstains, contemporary human skeletal remains, American Civil War era bone samples, and skeletal remains of WWII soldiers over those obtained by previously described DOP-PCR methods and routine STR typing. Additionally, the modified DOP-PCR procedure allows for a larger volume of DNA extract to be used, reducing the need to concentrate the sample and thus mitigating the effects of concurrent concentration of inhibitors. PMID:26832369

  5. Improved Detection of Lassa Virus by Reverse Transcription-PCR Targeting the 5′ Region of S RNA▿

    PubMed Central

    Ölschläger, Stephan; Lelke, Michaela; Emmerich, Petra; Panning, Marcus; Drosten, Christian; Hass, Meike; Asogun, Danny; Ehichioya, Deborah; Omilabu, Sunday; Günther, Stephan

    2010-01-01

    The method of choice for the detection of Lassa virus is reverse transcription (RT)-PCR. However, the high degree of genetic variability of the virus poses a problem with the design of RT-PCR assays that will reliably detect all strains. Recently, we encountered difficulties in detecting some strains from Liberia and Nigeria in a commonly used glycoprotein precursor (GPC) gene-specific RT-PCR assay (A. H. Demby, J. Chamberlain, D. W. Brown, and C. S. Clegg, J. Clin. Microbiol. 32:2898-2903, 1994), which prompted us to revise the protocol. The design of the new assay, the GPC RT-PCR/2007 assay, took into account 62 S RNA sequences from all countries where Lassa fever is endemic, including 40 sequences generated from the strains in our collection. The analytical sensitivity of the new assay was determined with 11 strains from Sierra Leone, Liberia, Ivory Coast, and Nigeria by probit analysis; the viral loads detectable with a probability of 95% ranged from 342 to 2,560 S RNA copies/ml serum, which corresponds to 4 to 30 S RNA copies/assay. The GPC RT-PCR/2007 assay was validated with 77 serum samples and 1 cerebrospinal fluid sample from patients with laboratory-confirmed Lassa fever. The samples mainly originated from Liberia and Nigeria and included strains difficult to detect in the assay of 1994. The GPC RT-PCR/2007 assay detected virus in all clinical specimens (100% sensitivity). In conclusion, a new RT-PCR assay, based in part on the protocol developed by Demby et al. in 1994, for the detection of Lassa virus is described. Compared to the assay developed in 1994, the GPC RT-PCR/2007 assay offers improved sensitivity for the detection of Liberian and Nigerian Lassa virus strains. PMID:20351210

  6. Point-of-Care Diagnostics for Improving Maternal Health in South Africa

    PubMed Central

    Mashamba-Thompson, Tivani P.; Sartorius, Benn; Drain, Paul K.

    2016-01-01

    Improving maternal health is a global priority, particularly in high HIV-endemic, resource-limited settings. Failure to use health care facilities due to poor access is one of the main causes of maternal deaths in South Africa. “Point-of-care” (POC) diagnostics are an innovative healthcare approach to improve healthcare access and health outcomes in remote and resource-limited settings. In this review, POC testing is defined as a diagnostic test that is carried out near patients and leads to rapid clinical decisions. We review the current and emerging POC diagnostics for maternal health, with a specific focus on the World Health Organization (WHO) quality-ASSURED (Affordability, Sensitivity, Specificity, User friendly, Rapid and robust, Equipment free and Delivered) criteria for an ideal point-of-care test in resource-limited settings. The performance of POC diagnostics, barriers and challenges related to implementing POC diagnostics for maternal health in rural and resource-limited settings are reviewed. Innovative strategies for overcoming these barriers are recommended to achieve substantial progress on improving maternal health outcomes in these settings. PMID:27589808

  7. Point-of-Care Diagnostics for Improving Maternal Health in South Africa.

    PubMed

    Mashamba-Thompson, Tivani P; Sartorius, Benn; Drain, Paul K

    2016-01-01

    Improving maternal health is a global priority, particularly in high HIV-endemic, resource-limited settings. Failure to use health care facilities due to poor access is one of the main causes of maternal deaths in South Africa. "Point-of-care" (POC) diagnostics are an innovative healthcare approach to improve healthcare access and health outcomes in remote and resource-limited settings. In this review, POC testing is defined as a diagnostic test that is carried out near patients and leads to rapid clinical decisions. We review the current and emerging POC diagnostics for maternal health, with a specific focus on the World Health Organization (WHO) quality-ASSURED (Affordability, Sensitivity, Specificity, User friendly, Rapid and robust, Equipment free and Delivered) criteria for an ideal point-of-care test in resource-limited settings. The performance of POC diagnostics, barriers and challenges related to implementing POC diagnostics for maternal health in rural and resource-limited settings are reviewed. Innovative strategies for overcoming these barriers are recommended to achieve substantial progress on improving maternal health outcomes in these settings. PMID:27589808

  8. The clinical diagnostic accuracy of rapid detection of healthcare-associated bloodstream infection in intensive care using multipathogen real-time PCR technology

    PubMed Central

    Dunn, Graham; Chadwick, Paul; Young, Duncan; Bentley, Andrew; Carlson, Gordon; Warhurst, Geoffrey

    2011-01-01

    Background There is growing interest in the potential utility of real-time PCR in diagnosing bloodstream infection by detecting pathogen DNA in blood samples within a few hours. SeptiFast is a multipathogen probe-based real-time PCR system targeting ribosomal DNA sequences of bacteria and fungi. It detects and identifies the commonest pathogens causing bloodstream infection and has European regulatory approval. The SeptiFast pathogen panel is suited to identifying healthcare-associated bloodstream infection acquired during complex healthcare, and the authors report here the protocol for the first detailed health-technology assessment of multiplex real-time PCR in this setting. Methods/design A Phase III multicentre double-blinded diagnostic study will determine the clinical validity of SeptiFast for the rapid detection of healthcare-associated bloodstream infection, against the current service standard of microbiological culture, in an adequately sized population of critically ill adult patients. Results from SeptiFast and standard microbiological culture procedures in each patient will be compared at study conclusion and the metrics of clinical diagnostic accuracy of SeptiFast determined in this population setting. In addition, this study aims to assess further the preliminary evidence that the detection of pathogen DNA in the bloodstream using SeptiFast may have value in identifying the presence of infection elsewhere in the body. Furthermore, differences in circulating immune-inflammatory markers in patient groups differentiated by the presence/absence of culturable pathogens and pathogen DNA will help elucidate further the patho-physiology of infection developing in the critically ill. Ethics and dissemination Ethical approval has been granted by the North West 6 Research Ethics Committee (09/H1003/109). Based on the results of this first non-commercial study, independent recommendations will be made to The Department of Health (open-access health technology

  9. Rapid concentration of bacteria using submicron magnetic anion exchangers for improving PCR-based multiplex pathogen detection.

    PubMed

    Yang, Kun; Jenkins, Daniel M; Su, Wei Wen

    2011-07-01

    Rapid concentration of bacterial targets from dilute solutions to improve subsequent PCR detection is investigated in this study. Submicron (average size 500nm) superparamagnetic anion-exchangers (SiMAG-DEAE) were used successfully to concentrate target bacteria from very dilute solutions. A mass-balance model predicted that for Escherichia coli, the extent of cell concentrating increases almost linearly with increasing sample/SiMAG volume ratio up to about 2000, accompanied by only a slight decrease in the capture efficiency (<10%). Our experimental data generally support this analysis in that the SiMAG beads concentrated bacterial targets by two to three orders of magnitude using a sample/bead volume ratio of about 1000, and lowered the PCR detection limit to a level of 10(2)CFU/mL, from 10(4) to 10(5)CFU/mL without concentrating. Several target bacteria can be concentrated concurrently and detected via multiplex PCR, as illustrated using E. coli and Agrobacterium tumefaciens as model bacteria. Finally, concentration and detection of bacteria in fresh produce samples were demonstrated. The integration of submicron magnetic ion exchangers and PCR detection provides an appealing alternative to immunomagnetic separation/PCR in improving pathogen detection.

  10. Improved Strategies and Optimization of Calibration Models for Real-time PCR Absolute Quantification

    EPA Science Inventory

    Real-time PCR absolute quantification applications rely on the use of standard curves to make estimates of DNA target concentrations in unknown samples. Traditional absolute quantification approaches dictate that a standard curve must accompany each experimental run. However, t...

  11. [Diagnostics of phytopathogen fungi Septoria tritici and Stagonospora nodorum by fluorescent amplification-based specific hybridization (FLASH) PCR].

    PubMed

    Abramova, S L; Riazantsev, D Iu; Voinova, T M; Zavriev, S K

    2008-01-01

    A PCR system in the fluorescent amplification-based specific hybridization (FLASH) format was developed for the detection and identification of two important wheat pathogenic fungi Septoria tritici (teleomorph of Mycosphaerella graminicola and Stagonospora nodorum (teleomorph of Phaeosphaeria nodorum), which cause spots on leaves and glumes, respectively. The pathogen detection system is based on the amplification of a genome fragment in the internal transcribed spacer 1 (ITS 1) region and a site encoding the 5.8S ribosomal RNA. The forward primers to ITS1 and a universal reverse primer and a Beacon type probe to the 5.8S ribosomal RNA region were chosen to provide the detection of the products in the FLASH format. This system was tested on different isolates of the pathogens, and on infected soil, leaf, and seed samples.

  12. Improved repetitive-element PCR fingerprinting of Salmonella enterica with the use of extremely elevated annealing temperatures.

    PubMed

    Johnson, J R; Clabots, C

    2000-03-01

    Modified thermal cycling conditions were explored in an effort to improve the reproducibility and resolving power of repetitive-element PCR (rep-PCR) fingerprinting. Assay performance was rigorously evaluated under standard and modified cycling conditions, using as a test set 12 strains putatively representing 12 serovars of Salmonella enterica. For all three fingerprint types (ERIC2, BOXA1R, and composite fingerprints), the use of extremely elevated annealing temperatures plus an initial "touchdown" cycling routine yielded significant improvements in day-to-day reproducibility and discriminating power despite the somewhat sparser appearance of the fingerprints. Modified cycling conditions markedly reduced the variability of fingerprints between cyclers, allowing fingerprints from different cyclers to be analyzed together without the degradation of assay performance that occurred with between-cycler analyses under standard cycling conditions. With modified cycling, composite fingerprints exhibited the lowest reproducibility but the highest net discriminating power of the three fingerprint types. rep-PCR fingerprints led to the discovery of a serotyping error involving one of the 12 test strains. These data demonstrate that modified cycling regimens that incorporate elevated annealing temperatures (with or without an initial touchdown routine) may markedly improve the performance of rep-PCR fingerprinting as a bacterial typing tool.

  13. A PCR-based diagnostic assay for detecting DNA of the olive fruit fly, Bactrocera oleae, in the gut of soil-living arthropods.

    PubMed

    Rejili, M; Fernandes, T; Dinis, A M; Pereira, J A; Baptista, P; Santos, S A P; Lino-Neto, T

    2016-10-01

    Bactrocera oleae (Rossi) (Diptera: Tephritidae) is considered the most devastating pest of the olive tree worldwide. In an effort to develop management and biological control strategies against this pest, new molecular tools are urgently needed. In this study, we present the design of B. oleae-specific primers based on mitochondrial DNA sequences of cytochrome oxidase subunit I (COI) gene. Two pairs of B. oleae-specific primers were successfully designed and named as SBo1-F/SBo1-R and SBo2-F/SBo1-R, being able to amplify 108 and 214 bp COI fragments, respectively. The specificity of designed primers was tested by amplifying DNA from phylogenetically related (i.e. Diptera order) and other non-pest insects living in olive groves from the Mediterranean region. When using these primers on a PCR-based diagnostic assay, B. oleae DNA was detected in the gut content of a soil-living insect, Pterostichus globosus (Fabricius) (Coleoptera: Carabidae). The detection of B. oleae DNA in the guts of arthropods was further optimized by adding bovine serum albumin enhancer to the PCR reaction, in order to get a fast, reproducible and sensitive tool for detecting B. oleae remains in the guts of soil-living arthropods. This molecular tool could be useful for understanding pest-predator relationships and establishing future biological control strategies for this pest. PMID:27296773

  14. A PCR-based diagnostic assay for detecting DNA of the olive fruit fly, Bactrocera oleae, in the gut of soil-living arthropods.

    PubMed

    Rejili, M; Fernandes, T; Dinis, A M; Pereira, J A; Baptista, P; Santos, S A P; Lino-Neto, T

    2016-10-01

    Bactrocera oleae (Rossi) (Diptera: Tephritidae) is considered the most devastating pest of the olive tree worldwide. In an effort to develop management and biological control strategies against this pest, new molecular tools are urgently needed. In this study, we present the design of B. oleae-specific primers based on mitochondrial DNA sequences of cytochrome oxidase subunit I (COI) gene. Two pairs of B. oleae-specific primers were successfully designed and named as SBo1-F/SBo1-R and SBo2-F/SBo1-R, being able to amplify 108 and 214 bp COI fragments, respectively. The specificity of designed primers was tested by amplifying DNA from phylogenetically related (i.e. Diptera order) and other non-pest insects living in olive groves from the Mediterranean region. When using these primers on a PCR-based diagnostic assay, B. oleae DNA was detected in the gut content of a soil-living insect, Pterostichus globosus (Fabricius) (Coleoptera: Carabidae). The detection of B. oleae DNA in the guts of arthropods was further optimized by adding bovine serum albumin enhancer to the PCR reaction, in order to get a fast, reproducible and sensitive tool for detecting B. oleae remains in the guts of soil-living arthropods. This molecular tool could be useful for understanding pest-predator relationships and establishing future biological control strategies for this pest.

  15. A vibroacoustic diagnostic system as an element improving road transport safety.

    PubMed

    Komorska, Iwona

    2013-01-01

    Mechanical defects of a vehicle driving system can be dangerous on the road. Diagnostic systems, which monitor operations of electric and electronic elements and devices of vehicles, are continuously developed and improved, while defects of mechanical systems are still not managed properly. This article proposes supplementing existing on-board diagnostics with a system of diagnosing selected defects to minimize their impact. It presents a method of diagnosing mechanical defects of the engine, gearbox and other elements of the driving system on the basis of a model of the vibration signal obtained adaptively. This method is suitable for engine valves, engine head gasket, main gearbox, joints, etc.

  16. Diagnostic performance of a multiplex PCR assay for meningitis in an HIV-infected population in Uganda.

    PubMed

    Rhein, Joshua; Bahr, Nathan C; Hemmert, Andrew C; Cloud, Joann L; Bellamkonda, Satya; Oswald, Cody; Lo, Eric; Nabeta, Henry; Kiggundu, Reuben; Akampurira, Andrew; Musubire, Abdu; Williams, Darlisha A; Meya, David B; Boulware, David R

    2016-03-01

    Meningitis remains a worldwide problem, and rapid diagnosis is essential to optimize survival. We evaluated the utility of a multiplex PCR test in differentiating possible etiologies of meningitis. Cerebrospinal fluid (CSF) from 69 HIV-infected Ugandan adults with meningitis was collected at diagnosis (n=51) and among persons with cryptococcal meningitis during therapeutic lumbar punctures (n=68). Cryopreserved CSF specimens were analyzed with BioFire FilmArray® Meningitis/Encephalitis panel, which targets 17 pathogens. The panel detected Cryptococcus in the CSF of patients diagnosed with a first episode of cryptococcal meningitis by fungal culture with 100% sensitivity and specificity and differentiated between fungal relapse and paradoxical immune reconstitution inflammatory syndrome in recurrent episodes. A negative FilmArray result was predictive of CSF sterility on follow-up lumbar punctures for cryptococcal meningitis. EBV was frequently detected in this immunosuppressed population (n=45). Other pathogens detected included: cytomegalovirus (n=2), varicella zoster virus (n=2), human herpes virus 6 (n=1), and Streptococcus pneumoniae (n=1). The FilmArray Meningitis/Encephalitis panel offers a promising platform for rapid meningitis diagnosis. PMID:26711635

  17. Advances in multiplex PCR: balancing primer efficiencies and improving detection success

    PubMed Central

    Sint, Daniela; Raso, Lorna; Traugott, Michael

    2012-01-01

    1. Multiplex PCR is a valuable tool in many biological studies but it is a multifaceted procedure that has to be planned and optimised thoroughly to achieve robust and meaningful results. In particular, primer concentrations have to be adjusted to assure an even amplification of all targeted DNA fragments. Until now, total DNA extracts were used for balancing primer efficiencies; however, the applicability for comparisons between taxa or different multiple-copy genes was limited owing to the unknown number of template molecules present per total DNA. 2. Based on a multiplex system developed to track trophic interactions in high Alpine arthropods, we demonstrate a fast and easy way of generating standardised DNA templates. These were then used to balance the amplification success for the different targets and to subsequently determine the sensitivity of each primer pair in the multiplex PCR. 3. In the current multiplex assay, this approach led to an even amplification success for all seven targeted DNA fragments. Using this balanced multiplex PCR, methodological bias owing to variation in primer efficiency will be avoided when analysing field-derived samples. 4. The approach outlined here allows comparing multiplex PCR sensitivity, independent of the investigated species, genome size or the targeted genes. The application of standardised DNA templates not only makes it possible to optimise primer efficiency within a given multiplex PCR, but it also offers to adjust and/or to compare the sensitivity between different assays. Along with other factors that influence the success of multiplex reactions, and which we discuss here in relation to the presented detection system, the adoption of this approach will allow for direct comparison of multiplex PCR data between systems and studies, enhancing the utility of this assay type. PMID:23549328

  18. Improved diagnosis of central nervous system tuberculosis by MPB64-Target PCR

    PubMed Central

    Dil-Afroze; Mir, Abdul Waheed; Kirmani1, Altaf; Shakeel-ul-Rehman; Eachkoti, Rafiqa; Siddiqi, Mushtaq A.

    2008-01-01

    Central nervous system (CNS) tuberculosis is a serious clinical problem, the treatment of which is sometimes hampered by delayed diagnosis. Clearly, prompt laboratory diagnosis is of vital importance as the spectrum of disease is wide and abnormalities of the cerebrospinal fluid (CSF) are incredibly variable. Since delayed hypersensitivity is the underlying immune response, bacterial load is very low. The conventional bacteriological methods rarely detect Mycobacterium tuberculosis in CSF and are of limited use in diagnosis of tuberculous meningitis (TBM). This double blind study was, therefore, directed to the molecular analysis of CNS tuberculosis by an in-house-developed PCR targeted for amplification of a 240bp nucleotide sequence coding for MPB64 protein specific for Mycobacterium tuberculosis. Based on the clinical criteria, 47 patients with CNS tuberculosis and a control group of 10 patients having non-tubercular lesions of the CNS were included in the study. Analyses were done in three groups; one group consisting of 27 patients of TBM, a second group of 20 patients with intracranial tuberculomas and a third group of 10 patients having nontubercular lesions of the CNS acted as control. There were no false positive results by PCR and the specificity worked out to be 100%. In the three study groups, routine CSF analysis (cells and chemistry), CSF for AFB smear and culture were negative in all cases. PCR was positive for 21/27 patients (77.7% sensitivity) of the first group of TBM patients, 6/20 patients (30% sensitivity) of the second group with intracranial tuberculomas were positive by PCR and none was PCR-positive (100% specificity) in the third group. Thus, PCR was found to be more sensitive than any other conventional method in the diagnosis of clinically suspected tubercular meningitis. PMID:24031203

  19. Service program diagnostics and decision support technology for improving health technology service efficiency and productivity.

    PubMed

    Blumberg, D F

    1998-01-01

    Our in-depth survey of both the state of the art and the results of application, implementation, and rollout of problem resolution diagnostics in control help desks and field service clearly shows the potential of online remote diagnostics technology (Figure 9) to improve service force productivity and efficiency and to make more effective use of service resources (people and parts). Our survey of the current and planned future expenditures for diagnostics used in field service clearly indicates that the overall service diagnostics market is currently sizable, in the range of $2 billion as of 1997, and is projected to grow to approximately $2.6 billion by 2000. This current expenditure and growth will occur primarily in the electronics arena, but there are still substantial development and application investments occurring in both electromechanical and mechanical areas. We believe that the pace of investment will fall off as the service industry shifts from development and experimental research to the application and rollout of standard off-the-shelf technology. Thus, the overall pattern of the diagnostics market indicates a continuing increase in expenditures by the field service industry for electronics-oriented service diagnostics and some increase with respect to use of this technology in electromechanical and mechanical applications. The application and use of diagnostics technology will also be affected by the ability of the field service management community to recognize the need to search for an optimum as opposed to a feasible solution to dispatch, assignment, call screening, call avoidance, and logistics deployment. The further rollout of affordable wireless communications technology such as Ardis and Ram mobile and the new cellular-based CPDP technology, coupled with the increased availability of inexpensive wireless-based laptops, portables, and CD-ROM-based problem resolution and diagnostics technology clearly shows the value of this technology in

  20. Improvements to a PCR-based serogrouping scheme for Salmonella enterica from dairy farm samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The PCR method described by Herrera-León, et al. (Research in Microbiology 158:122-127, 2007) has proved to be a simple and useful technique for characterizing isolates of Salmonella enterica enterica belonging to serogroups B, C1, C2, D1, and E1, groups which encompass a majority of the isolates fr...

  1. Application of COLD-PCR for improved detection of NF2 mosaic mutations.

    PubMed

    Paganini, Irene; Mancini, Irene; Baroncelli, Marta; Arena, Guido; Gensini, Francesca; Papi, Laura; Sestini, Roberta

    2014-07-01

    Somatic mosaicism represents the coexistence of two or more cell populations with different genotypes in one person, and it is involved in >30 monogenic disorders. Somatic mosaicism characterizes approximately 25% to 33% of patients with de novo neurofibromatosis type 2 (NF2). The identification of mosaicism is crucial to patients and their families because the clinical course of the disease and its transmission risk is influenced by the degree and distribution of mutated cells. Moreover, in NF2, the capability of discriminating patients with mosaicism is especially important to make differential diagnosis with schwannomatosis. However, the identification of mosaic variants is considerably difficult, and the development of specific molecular techniques to detect low levels of unknown molecular alterations is required. Co-amplification at lower denaturation temperature (COLD)-PCR has been described as a powerful method to selectively amplify minority alleles from mixtures of wild-type and mutation-containing sequences. Here, we applied COLD-PCR to molecular analysis of patients with NF2 mosaicism. With the use of COLD-PCR, followed by direct sequencing, we were able to detect NF2 mutations in blood DNA of three patients with NF2 mosaicism. Our study has shown the capability of COLD-PCR in enriching low-represented mutated allele in blood DNA sample, making it usable for molecular diagnosis of patients with mosaicism.

  2. Microfluidic based multiplex qRT-PCR identifies diagnostic and prognostic microRNA signatures in sera of prostate cancer patients

    PubMed Central

    Moltzahn, Felix; Olshen, Adam B.; Baehner, Lauren; Peek, Andrew; Fong, Lawrence; Stöppler, Hubert; Simko, Jeffry; Hilton, Joan F.; Carroll, Peter; Blelloch, Robert

    2010-01-01

    Recent prostate specific antigen (PSA) based screening trials indicate an urgent need for novel and non-invasive biomarker identification strategies to improve the prediction of prostate cancer behavior. Non-coding microRNAs (miRNAs) in the serum and plasma have been shown to have potential as non-invasive markers for physiological and pathological conditions. To identify serum miRNAs that diagnose and correlate with prognosis of prostate cancer, we developed a multiplex quantitative reverse transcription PCR (qRT-PCR) method involving purification of multiplex PCR products followed by uniplex analysis on a microfluidics chip to evaluate 384 human miRNAs. Using Dgcr8 and Dicer knockout (small RNA - deficient) mouse ES cells (mESC) as the benchmark, we confirmed the validity of our technique, while uncovering a significant lack of accuracy in previously published methods. Profiling 48 sera from healthy men and untreated prostate cancer patients with differing CAPRA (Cancer of the Prostate Risk Assessment) scores, we identified miRNA signatures that allow to diagnose cancer patients and correlate with prognosis. These serum signatures include oncogenic and tumor suppressive miRNAs suggesting functional roles in prostate cancer progression. PMID:21098088

  3. Design of molecular beacons: 3' couple quenchers improve fluorogenic properties of a probe in real-time PCR assay.

    PubMed

    Ryazantsev, Dmitry Y; Kvach, Maksim V; Tsybulsky, Dmitry A; Prokhorenko, Igor A; Stepanova, Irina A; Martynenko, Yury V; Gontarev, Sergey V; Shmanai, Vadim V; Zavriev, Sergey K; Korshun, Vladimir A

    2014-06-01

    Convenient preparation of fluorogenic hairpin DNA probes (molecular beacons) carrying a pair of FAM fluorophores (located close to 5'-terminus of the probe) or a pair of BHQ1 quenchers on 3'-terminus (with (BHQ1)2 or BHQ1-BHQ1 composition) is reported. These probes were used for the first time in a real-time PCR assay and showed considerable improvements in fluorogenic properties (the total fluorescence increase or signal-to-background ratio) in assay conditions vs. conventional one-FAM-one-BHQ1 molecular beacon probes as well as vs. hydrolyzable one-FAM-one-BHQ1 TaqMan probes. At the same time, such multiple modifications of the probe do not influence its Cq (a fractional PCR cycle used for quantification). The probe MB14 containing a BHQ1-BHQ1 pair showed a PCR fluorescence/background value of 9.6 which is more than two times higher than that of a regular probe MB2 (4.6). This study demonstrates prospects for the design of highly fluorogenic molecular beacon probes suitable for quantitative real-time PCR and for other potential applications (e.g. intracellular RNA detection and SNP/mutation analysis).

  4. Detection of allergen walnut component in food by an improved real-time PCR method.

    PubMed

    Wang, Haiyan; Yuan, Fei; Wu, Yajun; Yang, Hairong; Xu, Baoliang; Liu, Zhongxue; Chen, Ying

    2009-11-01

    A real-time PCR method aimed at the gene sequence of the walnut vicilin-like seed storage protein was established for the detection of the allergen walnut in food. The primers and probe were designed based on published methods. The method provided positive results for walnut and negative results for other tested agricultural plant materials including pecan. The intrinsic detection limit of the method was 0.00125 ng of walnut DNA, and the practical detection limit was 0.001% (wt/wt) walnut content in wheat; both of these values are lower than that of previously published methods. Therefore, this real-time PCR method is sufficiently specific and sensitive for the detection of walnut component in food.

  5. [Epidemics of schistosomiasis in military staff assigned to endemic areas: standard diagnostic techniques and the development of real-time PCR techniques].

    PubMed

    Biance-Valero, E; De Laval, F; Delerue, M; Savini, H; Cheinin, S; Leroy, P; Soullié, B

    2013-05-01

    The authors report the results of molecular biology techniques for the early diagnosis of cases (invasion phase) of schistosomiasis during two epidemics occurring during French military projects in the Central African Republic and Madagascar. The use of these techniques in real time for subjects not residing in the endemic area significantly improves the sensitivity of screening. The attack rates of these episodes, according to a case definition that took positive specific PCR results into account, were 59% and 26%. These results are a concrete illustration of the proverb that "yaws begin where the trail stops".

  6. Can rapid integrated polymerase chain reaction-based diagnostics for gastrointestinal pathogens improve routine hospital infection control practice? A diagnostic study.

    PubMed Central

    Pankhurst, Louise; Macfarlane-Smith, Louissa; Buchanan, James; Anson, Luke; Davies, Kerrie; O'Connor, Lily; Ashwin, Helen; Pike, Graham; Dingle, Kate E; Peto, Timothy Ea; Wordsworth, Sarah; Walker, A Sarah; Wilcox, Mark H; Crook, Derrick W

    2014-01-01

    BACKGROUND Every year approximately 5000-9000 patients are admitted to a hospital with diarrhoea, which in up to 90% of cases has a non-infectious cause. As a result, single rooms are 'blocked' by patients with non-infectious diarrhoea, while patients with infectious diarrhoea are still in open bays because of a lack of free side rooms. A rapid test for differentiating infectious from non-infectious diarrhoea could be very beneficial for patients. OBJECTIVE To evaluate MassCode multiplex polymerase chain reaction (PCR) for the simultaneous diagnosis of multiple enteropathogens directly from stool, in terms of sensitivity/specificity to detect four common important enteropathogens: Clostridium difficile, Campylobacter spp., Salmonella spp. and norovirus. DESIGN A retrospective study of fixed numbers of samples positive for C. difficile (n = 200), Campylobacter spp. (n = 200), Salmonella spp. (n = 100) and norovirus (n = 200) plus samples negative for all these pathogens (n = 300). Samples were sourced from NHS microbiology laboratories in Oxford and Leeds where initial diagnostic testing was performed according to Public Health England methodology. Researchers carrying out MassCode assays were blind to this information. A questionnaire survey, examining current practice for infection control teams and microbiology laboratories managing infectious diarrhoea, was also carried out. SETTING MassCode assays were carried out at Oxford University Hospitals NHS Trust. Further multiplex assays, carried out using Luminex, were run on the same set of samples at Leeds Teaching Hospitals NHS Trust. The questionnaire was completed by various NHS trusts. MAIN OUTCOME MEASURES Sensitivity and specificity to detect C. difficile, Campylobacter spp., Salmonella spp., and norovirus. RESULTS Nucleic acids were extracted from 948 clinical samples using an optimised protocol (200 Campylobacter spp., 199 C. difficile, 60 S. enterica, 199 norovirus and 295 negative

  7. Microfluidic purification and concentration of malignant pleural effusions for improved molecular and cytomorphological diagnostics.

    PubMed

    Che, James; Mach, Albert J; Go, Derek E; Talati, Ish; Ying, Yong; Rao, Jianyu; Kulkarni, Rajan P; Di Carlo, Dino

    2013-01-01

    Evaluation of pleural fluids for metastatic cells is a key component of diagnostic cytopathology. However, a large background of smaller leukocytes and/or erythrocytes can make accurate diagnosis difficult and reduce specificity in identification of mutations of interest for targeted anti-cancer therapies. Here, we describe an automated microfluidic system (Centrifuge Chip) which employs microscale vortices for the size-based isolation and concentration of cancer cells and mesothelial cells from a background of blood cells. We are able to process non-diluted pleural fluids at 6 mL/min and enrich target cells significantly over the background; we achieved improved purity in all patient samples analyzed. The resulting isolated and viable cells are readily available for immunostaining, cytological analysis, and detection of gene mutations. To demonstrate the utility towards aiding companion diagnostics, we also show improved detection accuracy of KRAS gene mutations in lung cancer cells processed using the Centrifuge Chip, leading to an increase in the area under the curve (AUC) of the receiver operating characteristic from 0.90 to 0.99. The Centrifuge Chip allows for rapid concentration and processing of large volumes of bodily fluid samples for improved cytological diagnosis and purification of cells of interest for genetic testing, which will be helpful for enhancing diagnostic accuracy. PMID:24205153

  8. Application of modern diagnostic methods to environmental improvement. Annual progress report, January--October 1994

    SciTech Connect

    Shepard, W.S.

    1994-12-01

    The Diagnostic Instrumentation and Analysis Laboratory (DIAL), a research department in the College of Engineering at Mississippi State University (MSU), is under contract with the US Department of Energy (DOE) to develop and apply advanced diagnostic instrumentation and analysis techniques to real world processes; measurements are made in hot, highly corrosive atmospheres in which conventional measurement devices are ineffective. Task 1 of this agreement is concerned with the development and application of various diagnostic methods to characterize the plasma properties, the melt properties and the downstream emissions from a plasma torch facility designed to vitrify mixed waste. Correlation of the measured properties with the operating parameters of the torch will be sought to improve, optimize and control the overall operation of the plasma treatment process. As part of this program, diagnostic methods will be developed and evaluated for characterization, monitoring and control purposes of treatment processes in general. Task 2 of this agreement is concerned with the development of a system to monitor and control the combustion stoichiometry in real time in order to minimize environmental impact and maximize process efficiency. Staged fuel injection is also being studied to minimize NO{sub x} formation.

  9. Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates.

    PubMed

    Sanchis, Joaquin; Fernández, Layla; Carballeira, J Daniel; Drone, Jullien; Gumulya, Yosephine; Höbenreich, Horst; Kahakeaw, Daniel; Kille, Sabrina; Lohmer, Renate; Peyralans, Jérôme J-P; Podtetenieff, John; Prasad, Shreenath; Soni, Pankaj; Taglieber, Andreas; Wu, Sheng; Zilly, Felipe E; Reetz, Manfred T

    2008-11-01

    Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene's QuikChange sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.

  10. Improved diagnostic differentiation of renal cystic lesions with phase-contrast computed tomography (PCCT)

    NASA Astrophysics Data System (ADS)

    Noel, Peter B.; Willner, Marian; Fingerle, Alexander; Herzen, Julia; Münzel, Daniela; Hahn, Dieter; Rummeny, Ernst J.; Pfeiffer, Franz

    2012-03-01

    The diagnostic quality of phase-contrast computed tomography (PCCT) is one the unexplored areas in medical imaging; at the same time, it seems to offer the opportunity as a fast and highly sensitive diagnostic tool. Conventional computed tomography (CT) has had an enormous impact on medicine, while it is limited in soft-tissue contrast. One example that portrays this challenge is the differentiation between benign and malignant renal cysts. In this work we report on a feasibility study to determine the usefulness of PCCT in differentiation of renal cysts. A renal phantom was imaged with a grating-based PCCT system consisting of a standard rotating anode x-ray tube (40 kV, 70 mA) and a Pilatus II photoncounting detector (pixel size: 172 μm). The phantom is composed of a renal equivalent soft-tissue and cystic lesions grouped in non-enhancing cyst and hemorrhage series and an iodine enhancing series. The acquired projection images (absorption and phase-contrast) are reconstructed with a standard filtered backprojection algorithm. For evaluation both reconstructions are compared in respect to contrast-to-noise ratio (CNR), signal-to-noise ratio (SNR), and subjective image quality. We found that with PCCT a significantly improved differentiation between hemorrhage renal cysts from contrast enhancing malignant cysts is possible. If comparing PCCT and CT with respect to CNR and SNR, PCCT shows significant improvements. In conclusion, PCCT has the potential to improve the diagnostics and characterization of renal cysts without using any contrast agents. These results in combination with a non-synchrotron setup indicate a future paradigm shift in diagnostic computed tomography.

  11. Modification of an HLA-B PCR-SSOP typing system leading to improved allele determination.

    PubMed

    Middleton, D; Williams, F; Cullen, C; Mallon, E

    1995-04-01

    Modifications have been introduced to a previously reported HLA-B PCR-SSOP typing system. This has enabled further definition of alleles, determination of the probe pattern of some alleles not previously examined and identification of patterns of possible new alleles. However there are still some alleles that cannot be differentiated and there are several alleles which when present as a homozygote have the same pattern as in combination with another allele. When the method was applied to the typing of 66 consecutive cadaveric donors there were three donors whose type differed from the serological type.

  12. Real-time magnetic resonance imaging guidance improves the diagnostic yield of endomyocardial biopsy

    PubMed Central

    Rogers, Toby; Ratnayaka, Kanishka; Karmarkar, Parag; Campbell-Washburn, Adrienne E.; Schenke, William H.; Mazal, Jonathan R.; Kocaturk, Ozgur; Faranesh, Anthony Z.; Lederman, Robert J.

    2016-01-01

    Background Diagnostic yield of endomyocardial biopsy is low, particularly in disease that affects the myocardium in a non-uniform distribution. We hypothesized that real-time MRI guidance could improve the yield through targeted biopsy of focal myocardial pathology. Methods An animal model of focal myocardial pathology was created by infusing 3mL of fluorescent microspheres (NuFlow Hydrocoat, 15μm diameter, 5 million spheres/mL) followed by 2mL of 100% ethanol to a branch coronary artery. Animals were survived for minimum 14days, before undergoing MRI guided endomyocardial biopsy using a custom 6.5Fr active visualization MRI-conditional bioptome and X-ray guided biopsy using a commercial bioptome. Specimens were analyzed using a dissecting microscope under ultraviolet light to determine the proportion of ‘on-target’ specimens containing fluorescent microspheres. Results A total of 77 specimens were obtained using real-time MRI guidance and 87 using X-ray guidance, in five animals. Specimens obtained with the MRI-conditional bioptome were smaller compared with the commercial X-ray bioptome. Real-time MRI guidance significantly increased the diagnostic yield of endomyocardial biopsy (82% vs. 56% on-target biopsy specimens with real-time MRI vs. X-ray guidance, p<0.01). Conclusions Endomyocardial biopsy performed using real-time MRI guidance is feasible and significantly improves the diagnostic yield compared with X-ray fluoroscopy guidance. PMID:27631028

  13. Real-time magnetic resonance imaging guidance improves the diagnostic yield of endomyocardial biopsy

    PubMed Central

    Rogers, Toby; Ratnayaka, Kanishka; Karmarkar, Parag; Campbell-Washburn, Adrienne E.; Schenke, William H.; Mazal, Jonathan R.; Kocaturk, Ozgur; Faranesh, Anthony Z.; Lederman, Robert J.

    2016-01-01

    Background Diagnostic yield of endomyocardial biopsy is low, particularly in disease that affects the myocardium in a non-uniform distribution. We hypothesized that real-time MRI guidance could improve the yield through targeted biopsy of focal myocardial pathology. Methods An animal model of focal myocardial pathology was created by infusing 3mL of fluorescent microspheres (NuFlow Hydrocoat, 15μm diameter, 5 million spheres/mL) followed by 2mL of 100% ethanol to a branch coronary artery. Animals were survived for minimum 14days, before undergoing MRI guided endomyocardial biopsy using a custom 6.5Fr active visualization MRI-conditional bioptome and X-ray guided biopsy using a commercial bioptome. Specimens were analyzed using a dissecting microscope under ultraviolet light to determine the proportion of ‘on-target’ specimens containing fluorescent microspheres. Results A total of 77 specimens were obtained using real-time MRI guidance and 87 using X-ray guidance, in five animals. Specimens obtained with the MRI-conditional bioptome were smaller compared with the commercial X-ray bioptome. Real-time MRI guidance significantly increased the diagnostic yield of endomyocardial biopsy (82% vs. 56% on-target biopsy specimens with real-time MRI vs. X-ray guidance, p<0.01). Conclusions Endomyocardial biopsy performed using real-time MRI guidance is feasible and significantly improves the diagnostic yield compared with X-ray fluoroscopy guidance.

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

    PubMed

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

    2015-10-01

    Human infections with the helminth species Strongyloides stercoralis encompass a wide clinical spectrum, ranging from asymptomatic carriage to life-threatening disease. The diagnosis of S. stercoralis is cumbersome and the sensitivity of conventional stool microscopy is low. New molecular tools have been developed to increase sensitivity. We compared the diagnostic accuracy of real-time PCR with microscopy for the detection of S. stercoralis and hookworm in human stool samples, and investigated the inter-laboratory agreement of S. stercoralis-specific real-time PCR in two European laboratories. Stool specimens from 256 randomly selected individuals in rural Côte d'Ivoire were examined using three microscopic techniques (i.e. Kato-Katz, Koga agar plate (KAP) and Baermann (BM)). Additionally, ethanol-fixed stool aliquots were subjected to molecular diagnosis. The prevalence of S. stercoralis and hookworm infection was 21.9% and 52.0%, respectively, whilst co-infections were detected in 35 (13.7%) participants. The diagnostic agreement between real-time PCR and microscopy was excellent when both KAP and BM tested positive for S. stercoralis, but was considerably lower when only one microscopic technique was positive. The sensitivity of KAP, BM and real-time PCR for detection of S. stercoralis as compared to a combination of all diagnostic techniques was 21.4%, 37.5% and 76.8%, respectively. The inter-laboratory agreement of S. stercoralis-specific PCR was substantial (κ=0.63, p<0.001). We conclude that a combination of real-time PCR and stool microscopy shows high accuracy for S. stercoralis diagnosis. Besides high sensitivity, PCR may also enhance specificity by reducing microscopic misdiagnosis of morphologically similar helminth larvae (i.e. hookworm and S. stercoralis) in settings where both helminth species co-exist.

  15. Improved Signal Processing Technique Leads to More Robust Self Diagnostic Accelerometer System

    NASA Technical Reports Server (NTRS)

    Tokars, Roger; Lekki, John; Jaros, Dave; Riggs, Terrence; Evans, Kenneth P.

    2010-01-01

    The self diagnostic accelerometer (SDA) is a sensor system designed to actively monitor the health of an accelerometer. In this case an accelerometer is considered healthy if it can be determined that it is operating correctly and its measurements may be relied upon. The SDA system accomplishes this by actively monitoring the accelerometer for a variety of failure conditions including accelerometer structural damage, an electrical open circuit, and most importantly accelerometer detachment. In recent testing of the SDA system in emulated engine operating conditions it has been found that a more robust signal processing technique was necessary. An improved accelerometer diagnostic technique and test results of the SDA system utilizing this technique are presented here. Furthermore, the real time, autonomous capability of the SDA system to concurrently compensate for effects from real operating conditions such as temperature changes and mechanical noise, while monitoring the condition of the accelerometer health and attachment, will be demonstrated.

  16. Urine proteomics for discovery of improved diagnostic markers of Kawasaki disease

    PubMed Central

    Kentsis, Alex; Shulman, Andrew; Ahmed, Saima; Brennan, Eileen; Monuteaux, Michael C; Lee, Young-Ho; Lipsett, Susan; Paulo, Joao A; Dedeoglu, Fatma; Fuhlbrigge, Robert; Bachur, Richard; Bradwin, Gary; Arditi, Moshe; Sundel, Robert P; Newburger, Jane W; Steen, Hanno; Kim, Susan

    2013-01-01

    Kawasaki disease (KD) is a systemic vasculitis of unknown etiology. Absence of definitive diagnostic markers limits the accuracy of clinical evaluations of suspected KD with significant increases in morbidity. In turn, incomplete understanding of its molecular pathogenesis hinders the identification of rational targets needed to improve therapy. We used high-accuracy mass spectrometry proteomics to analyse over 2000 unique proteins in clinical urine specimens of patients with KD. We discovered that urine proteomes of patients with KD, but not those with mimicking conditions, were enriched for markers of cellular injury such as filamin and talin, immune regulators such as complement regulator CSMD3, immune pattern recognition receptor muclin, and immune cytokine protease meprin A. Significant elevations of filamin C and meprin A were detected in both the serum and urine in two independent cohorts of patients with KD, comprised of a total of 236 patients. Meprin A and filamin C exhibited superior diagnostic performance as compared to currently used markers of disease in a blinded case-control study of 107 patients with suspected KD, with receiver operating characteristic areas under the curve of 0.98 (95% confidence intervals [CI] of 0.97–1 and 0.95–1, respectively). Notably, meprin A was enriched in the coronary artery lesions of a mouse model of KD. In all, urine proteome profiles revealed novel candidate molecular markers of KD, including filamin C and meprin A that exhibit excellent diagnostic performance. These disease markers may improve the diagnostic accuracy of clinical evaluations of children with suspected KD, lead to the identification of novel therapeutic targets, and allow the development of a biological classification of Kawasaki disease. PMID:23281308

  17. Selection of more appropriate PCR primer pairs for improved efficiency in detecting Norwalk-like virus (NLV) RNA.

    PubMed

    Naitou, Hirotaka; Morita, Tamotsu

    2001-10-01

    A pair of primers, NV35 and NV36, and another pair of primers, NV81 and NV82/SM82, are commonly used for polymerase chain reaction (PCR) detection of Norwalk-like virus (NLV) genome RNA sequences in authorized test laboratories in Japan. However, the efficiency of NLV genome RNA detection with these primer pairs has been less than satisfactory. In the present study, we attempted to establish more appropriately matched primer pairs for improved detection of NLV genome RNA sequences using a combination of primers including NV35, NV36, NV81, NV82/SM82, SR33, and SRs (a mixture of 4 primers SR46, SR48, SR50, and SR52). We also evaluated appropriate primers for improved reverse transcription of NLV genome RNA. Stool samples used for detection of NLV included 18 samples collected from NLV-infected patients who ingested oysters (group 1) and 13 samples collected from those who did not ingest oysters (group 2). Reverse transcription of RNA genome with primer NV35 was less efficient compared with that with primer SR33 or NV81. When PCR products obtained with NV35 and NV36 as a pair of primers were subjected to gel electrophoresis, a strong extra band was detected compared with those obtained with other primer pairs. Since this extra band may represent heterodimeric or homodimeric hybrids, or intramolecular hybrids derived from these primers, this template-independent hybridization could lower the efficiency of primer-dependent polymerase reaction. Of 18 primer pairs, a pair of NV81 and SRs provided the best detection of PCR products following reverse transcription of NLV RNA with SR33 or NV81. The detection rate was 61% for both reverse transcription with SR33 and that with NV81. After reverse transcription using SR33 as a primer, nested PCR using a pair of NV81 and SRs following primary PCR using a pair of NV81 and NV82/SM82 increased the detection rate to 89% in group 1 and 100% in group 2.

  18. An improved PCR-restriction fragment length polymorphism (RFLP) method for the identification of cry1-type genes.

    PubMed

    Shu, Changlong; Liu, Dongming; Zhou, Zishan; Cai, Jilin; Peng, Qi; Gao, Jiguo; Song, Fuping; Zhang, Jie

    2013-11-01

    The cry1-type genes of Bacillus thuringiensis represent the largest cry gene family, which contains 50 distinct holotypes. It is becoming more and more difficult to identify cry1-type genes using current methods because of the increasing number of cry1-type genes. In the present study, an improved PCR-restriction fragment length polymorphism (PCR-RFLP) method which can distinguish 41 holotypes of cry1-type genes was developed. This improved method was used to identify cry1-type genes in 20 B. thuringiensis strains that are toxic to lepidoptera. The results showed that the improved method can efficiently identify single and clustered cry1-type genes and can be used to evaluate cry1-type genes in novel strain collections of B. thuringiensis. Among the detected cry1-type genes, we identified four novel genes, cry1Ai, cry1Bb, cry1Ja, and cry1La. The bioassay results from the expressed products of the four novel cry genes showed that Cry1Ai2, Cry1Bb2, and Cry1Ja2 were highly toxic against Plutella xylostella, whereas Cry1La2 exhibited no activity. Moreover, Cry1Ai2 had good lethal activity against Ostrinia furnacalis, Hyphantria cunea, Chilo suppressalis, and Bombyx mori larvae and considerable weight loss activity against Helicoverpa armigera.

  19. Realization of process improvement at a diagnostic radiology department with aid of simulation modeling.

    PubMed

    Oh, Hong-Choon; Toh, Hong-Guan; Giap Cheong, Eddy Seng

    2011-11-01

    Using the classical process improvement framework of Plan-Do-Study-Act (PDSA), the diagnostic radiology department of a tertiary hospital identified several patient cycle time reduction strategies. Experimentation of these strategies (which included procurement of new machines, hiring of new staff, redesign of queue system, etc.) through pilot scale implementation was impractical because it might incur substantial expenditure or be operationally disruptive. With this in mind, simulation modeling was used to test these strategies via performance of "what if" analyses. Using the output generated by the simulation model, the team was able to identify a cost-free cycle time reduction strategy, which subsequently led to a reduction of patient cycle time and achievement of a management-defined performance target. As healthcare professionals work continually to improve healthcare operational efficiency in response to rising healthcare costs and patient expectation, simulation modeling offers an effective scientific framework that can complement established process improvement framework like PDSA to realize healthcare process enhancement.

  20. COMPREHENSIVE DIAGNOSTIC AND IMPROVEMENT TOOLS FOR HVAC-SYSTEM INSTALLATIONS IN LIGHT COMMERCIAL BUILDINGS

    SciTech Connect

    Abram Conant; Mark Modera; Joe Pira; John Proctor; Mike Gebbie

    2004-10-31

    Proctor Engineering Group, Ltd. (PEG) and Carrier-Aeroseal LLP performed an investigation of opportunities for improving air conditioning and heating system performance in existing light commercial buildings. Comprehensive diagnostic and improvement tools were created to address equipment performance parameters (including airflow, refrigerant charge, and economizer operation), duct-system performance (including duct leakage, zonal flows and thermal-energy delivery), and combustion appliance safety within these buildings. This investigation, sponsored by the National Energy Technology Laboratory, a division of the U.S. Department of Energy, involved collaboration between PEG and Aeroseal in order to refine three technologies previously developed for the residential market: (1) an aerosol-based duct sealing technology that allows the ducts to be sealed remotely (i.e., without removing the ceiling tiles), (2) a computer-driven diagnostic and improvement-tracking tool for residential duct installations, and (3) an integrated diagnosis verification and customer satisfaction system utilizing a combined computer/human expert system for HVAC performance. Prior to this work the aerosol-sealing technology was virtually untested in the light commercial sector--mostly because the savings potential and practicality of this or any other type of duct sealing had not been documented. Based upon the field experiences of PEG and Aeroseal, the overall product was tailored to suit the skill sets of typical HVAC-contractor personnel.

  1. Upgrades to improve the usability, reliability, and spectral range of the MST Thomson scattering diagnostic

    NASA Astrophysics Data System (ADS)

    Kubala, S. Z.; Borchardt, M. T.; Den Hartog, D. J.; Holly, D. J.; Jacobson, C. M.; Morton, L. A.; Young, W. C.

    2016-11-01

    The Thomson scattering diagnostic on MST records both equilibrium and fluctuating electron temperature with a range capability of 10 eV-5 keV. Standard operation with two modified commercial Nd:YAG lasers allows measurements at rates of 1 kHz-25 kHz. Several subsystems of the diagnostic are being improved. The power supplies for the avalanche photodiode detectors (APDs) that record the scattered light are being replaced to improve usability, reliability, and maintainability. Each of the 144 APDs will have an individual rack mounted switching supply, with bias voltage adjustable to match the APD. Long-wavelength filters (1140 nm center, 80 nm bandwidth) have been added to the polychromators to improve capability to resolve non-Maxwellian distributions and to enable directed electron flow measurements. A supercontinuum (SC) pulsed white light source has replaced the tungsten halogen lamp previously used for spectral calibration of the polychromators. The SC source combines substantial brightness produced in nanosecond pulses with a spectrum that covers the entire range of the polychromators.

  2. An improved validated SYBR green-based real-time quantitative PCR assay for the detection of the Penaeus stylirostris densovirus in penaeid shrimp.

    PubMed

    Encinas-García, Trinidad; Mendoza-Cano, Fernando; Enríquez-Espinoza, Tania; Luken-Vega, Leonardo; Vichido-Chávez, Rodrigo; Sánchez-Paz, Arturo

    2015-02-01

    The Penaeus stylirostris densovirus (PstDV) (also known as infectious hypodermal and hematopoietic necrosis virus, IHHNV), one of the major shrimp pathogens, has a worldwide distribution in farmed and wild shrimp populations. Outbreaks of IHHNV have been associated with substantial economic losses which are accompanied by a negative social impact. Current diagnostic PCR tests may result in false-positive results as several parts of PstDV genome may be endogenized in the nuclear genome of the shrimp P. stylirostris. A one-step qPCR SYBR-Green based quantitative real-time polymerase chain reaction (qPCR) assay to detect different isolates of the IHHNV in shrimp samples was developed. The detection limit of the assay was 81 viral copies of targeted DNA per reaction. The specificity of the assay was evaluated by melting curve analysis, which showed that the IHHNV product generated a single melt peak at 81.4±0.044°C. The assay was more sensitive than conventional PCR. The standardized PCR was shown to be highly sensible, specific, robust, and reproducible, which makes it an economical and powerful tool for both diagnostic applications and general research of IHHNV.

  3. 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.

  4. 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.

  5. Clinical applications of autoimmunity to citrullinated proteins in rheumatoid arthritis, from improving diagnostics to future therapies.

    PubMed

    Kinloch, Andrew J; Ng, Karen; Wright, Graham P

    2011-05-01

    Rheumatoid arthritis (RA), although widely considered to be the most commonly occurring autoimmune disease, has only truly been substantiated as a distinct autoimmune disease very recently. The lack of understanding of the specific autoimmune system/s at work in rheumatoid patients resulted in an absence of robust diagnostic tools and had meant that the rational choice for use and design of therapy was based on broad-spectrum immunosuppression. The revelation that the autoimmune response specific for patients with RA is to particular protein antigens bearing the post-translational modification 'citrulline' has therefore revolutionized diagnostics and has helped explain why patients carrying particular MHC alleles are predisposed to the disease. The last two decades have seen the characterization of citrullinated antigens targeted by both antibodies and T cells in rheumatoid patients. In more recent years, we have also witnessed the success of biological therapies in the treatment of RA that specifically target T cells and B cells. Ongoing mapping of antibody targets is increasing the percentage of patients who can be definitively diagnosed with, and prognosed to develop, RA. These advances have led to a great number of patents for citrullinated peptides that have been and may be, in the coming years, used in diagnostic test kits. More recently, characterization of T cell targets (citrullinated peptides) has resulted in the patenting of peptides that could be used in antigen specific therapy. This review focuses on the characterization of the autoimmune response to citrullinated protein targets in RA and how the community is translating this knowledge to improve diagnostics, prognostics and therapy. PMID:21453269

  6. Early-Onset Neonatal Sepsis: Still Room for Improvement in Procalcitonin Diagnostic Accuracy Studies.

    PubMed

    Chiesa, Claudio; Pacifico, Lucia; Osborn, John F; Bonci, Enea; Hofer, Nora; Resch, Bernhard

    2015-07-01

    To perform a systematic review assessing accuracy and completeness of diagnostic studies of procalcitonin (PCT) for early-onset neonatal sepsis (EONS) using the Standards for Reporting of Diagnostic Accuracy (STARD) initiative.EONS, diagnosed during the first 3 days of life, remains a common and serious problem. Increased PCT is a potentially useful diagnostic marker of EONS, but reports in the literature are contradictory. There are several possible explanations for the divergent results including the quality of studies reporting the clinical usefulness of PCT in ruling in or ruling out EONS.We systematically reviewed PubMed, Scopus, and the Cochrane Library databases up to October 1, 2014. Studies were eligible for inclusion in our review if they provided measures of PCT accuracy for diagnosing EONS. A data extraction form based on the STARD checklist and adapted for neonates with EONS was used to appraise the quality of the reporting of included studies.We found 18 articles (1998-2014) fulfilling our eligibility criteria which were included in the final analysis. Overall, the results of our analysis showed that the quality of studies reporting diagnostic accuracy of PCT for EONS was suboptimal leaving ample room for improvement. Information on key elements of design, analysis, and interpretation of test accuracy were frequently missing.Authors should be aware of the STARD criteria before starting a study in this field. We welcome stricter adherence to this guideline. Well-reported studies with appropriate designs will provide more reliable information to guide decisions on the use and interpretations of PCT test results in the management of neonates with EONS.

  7. Improved PCR assay for the species-specific identification and quantitation of Legionella pneumophila in water.

    PubMed

    Cho, Min Seok; Ahn, Tae-Young; Joh, Kiseong; Lee, Eui Seok; Park, Dong Suk

    2015-11-01

    Legionellosis outbreak is a major global health care problem. However, current Legionella risk assessments may be compromised by uncertainties in Legionella detection methods, infectious dose, and strain infectivity. These limitations may place public health at significant risk, leading to significant monetary losses in health care. However, there are still unmet needs for its rapid identification and monitoring of legionellae in water systems. Therefore, in the present study, a primer set was designed based on a LysR-type transcriptional regulator (LTTR) family protein gene of Legionella pneumophila subsp. pneumophila str. Philadelphia 1 because it was found that this gene is structurally diverse among species through BLAST searches. The specificity of the primer set was evaluated using genomic DNA from 6 strains of L. pneumophila, 5 type strains of other related Legionella species, and other 29 reference pathogenic bacteria. The primer set used in the PCR assay amplified a 264-bp product for only targeted six strains of L. pneumophila. The assay was also able to detect at least 1.39 × 10(3) copies/μl of cloned amplified target DNA using purified DNA or 7.4 × 10(0) colony-forming unit per reaction when using calibrated cell suspension. In addition, the sensitivity and specificity of this assay were confirmed by successful detection of Legionella pneumophila in environmental water samples. PMID:26142386

  8. Improved PCR assay for the species-specific identification and quantitation of Legionella pneumophila in water.

    PubMed

    Cho, Min Seok; Ahn, Tae-Young; Joh, Kiseong; Lee, Eui Seok; Park, Dong Suk

    2015-11-01

    Legionellosis outbreak is a major global health care problem. However, current Legionella risk assessments may be compromised by uncertainties in Legionella detection methods, infectious dose, and strain infectivity. These limitations may place public health at significant risk, leading to significant monetary losses in health care. However, there are still unmet needs for its rapid identification and monitoring of legionellae in water systems. Therefore, in the present study, a primer set was designed based on a LysR-type transcriptional regulator (LTTR) family protein gene of Legionella pneumophila subsp. pneumophila str. Philadelphia 1 because it was found that this gene is structurally diverse among species through BLAST searches. The specificity of the primer set was evaluated using genomic DNA from 6 strains of L. pneumophila, 5 type strains of other related Legionella species, and other 29 reference pathogenic bacteria. The primer set used in the PCR assay amplified a 264-bp product for only targeted six strains of L. pneumophila. The assay was also able to detect at least 1.39 × 10(3) copies/μl of cloned amplified target DNA using purified DNA or 7.4 × 10(0) colony-forming unit per reaction when using calibrated cell suspension. In addition, the sensitivity and specificity of this assay were confirmed by successful detection of Legionella pneumophila in environmental water samples.

  9. Improved PCR primers for the detection and identification of arbuscular mycorrhizal fungi.

    PubMed

    Lee, Jaikoo; Lee, Sangsun; Young, J Peter W

    2008-08-01

    A set of PCR primers that should amplify all subgroups of arbuscular mycorrhizal fungi (AMF, Glomeromycota), but exclude sequences from other organisms, was designed to facilitate rapid detection and identification directly from field-grown plant roots. The small subunit rRNA gene was targeted for the new primers (AML1 and AML2) because phylogenetic relationships among the Glomeromycota are well understood for this gene. Sequence comparisons indicate that the new primers should amplify all published AMF sequences except those from Archaeospora trappei. The specificity of the new primers was tested using 23 different AMF spore morphotypes from trap cultures and Miscanthus sinensis, Glycine max and Panax ginseng roots sampled from the field. Non-AMF DNA of 14 plants, 14 Basidiomycota and 18 Ascomycota was also tested as negative controls. Sequences amplified from roots using the new primers were compared with those obtained using the established NS31 and AM1 primer combination. The new primers have much better specificity and coverage of all known AMF groups. PMID:18631176

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

    PubMed

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

    2014-03-01

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

  11. Low cost HIV-1 quantitative RT-PCR assay in resource-limited settings: improvement and implementation.

    PubMed

    Fibriani, Azzania; Farah, Nadya; Kusumadewi, Inri; Pas, Suzan D; van Crevel, Reinout; van der Ven, Andre; Boucher, Charles A B; Schutten, Martin

    2012-10-01

    Monitoring of HIV viral load in low and middle income settings is limited by high cost of the commercial assays. Therefore, we developed a novel RT-PCR quantitative assay was developed. This assay targets the HIV-1 pol integrase gene (INT). Subsequently, the performance of the INT assay, described previously as a Long Terminal Repeat (LTR) assay and a combined INT/LTR dual target RT-PCR assay was compared. The LTR-assay was found to be sensitive and cost-effective (50-70% cheaper than commercial assays) with the lowest coefficient of variation (%CV). Introduction of an internal standard further improved assay reliability. Therefore, this LTR assay was implemented in West Java, Indonesia. Linearity and precision of the LTR assay were good: %CV ranged from 1.0% to 10.4%. The limit of quantitation was 616 copies/ml. Performance was comparable with the commercial assay (Abbott assay) (r(2)=0.01), although on average the viral loads were 0.39 log(10)copies/ml lower. In clinical practice, it had excellent capability for monitoring treatment failure, the positive predictive value was 99% and the negative predictive value was 93%. In conclusion, the implementation of the improved HIV-1 viral load LTR-assay for routine diagnosis in resource poor settings can be a good alternative when commercial assays are unaffordable.

  12. Diagnostics in a digital age: an opportunity to strengthen health systems and improve health outcomes.

    PubMed

    Peeling, Rosanna W

    2015-11-01

    Diagnostics play a critical role in clinical decision making, and in disease control and prevention. Rapid point-of-care (POC) tests for infectious diseases can improve access to diagnosis and patient management, but the quality of these tests vary, quality of testing is often not assured and there are few mechanisms to capture test results for surveillance when the testing is so decentralised. A new generation of POC molecular tests that are highly sensitive and specific, robust and easy to use are now available for deployment in low resource settings. Decentralisation of testing outside of the laboratory can put tremendous stress on the healthcare system and presents challenges for training and quality assurance. A feature of many of these POC molecular devices is that they are equipped with data transmission capacities. In a digital age, it is possible to link data from diagnostic laboratories and POC test readers and devices to provide data on testing coverage, disease trends and timely information for early warning of infectious disease outbreaks to inform design or optimisation of disease control and elimination programmes. Data connectivity also allows control programmes to monitor the quality of tests and testing, and optimise supply chain management; thus, increasing the efficiency of healthcare systems and improving patient outcomes. PMID:26553825

  13. Diagnostics in a digital age: an opportunity to strengthen health systems and improve health outcomes.

    PubMed

    Peeling, Rosanna W

    2015-11-01

    Diagnostics play a critical role in clinical decision making, and in disease control and prevention. Rapid point-of-care (POC) tests for infectious diseases can improve access to diagnosis and patient management, but the quality of these tests vary, quality of testing is often not assured and there are few mechanisms to capture test results for surveillance when the testing is so decentralised. A new generation of POC molecular tests that are highly sensitive and specific, robust and easy to use are now available for deployment in low resource settings. Decentralisation of testing outside of the laboratory can put tremendous stress on the healthcare system and presents challenges for training and quality assurance. A feature of many of these POC molecular devices is that they are equipped with data transmission capacities. In a digital age, it is possible to link data from diagnostic laboratories and POC test readers and devices to provide data on testing coverage, disease trends and timely information for early warning of infectious disease outbreaks to inform design or optimisation of disease control and elimination programmes. Data connectivity also allows control programmes to monitor the quality of tests and testing, and optimise supply chain management; thus, increasing the efficiency of healthcare systems and improving patient outcomes.

  14. 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

  15. Developmental validation of the AmpFℓSTR® Identifiler® Plus PCR Amplification Kit: an established multiplex assay with improved performance.

    PubMed

    Wang, Dennis Y; Chang, Chien-Wei; Lagacé, Robert E; Calandro, Lisa M; Hennessy, Lori K

    2012-03-01

    Analysis of length polymorphism at short tandem repeat (STR) loci utilizing multiplex polymerase chain reaction (PCR) remains the primary method for genotyping forensic samples. The AmpFℓSTR(®) Identifiler(®) Plus PCR Amplification Kit is an improved version of the AmpFℓSTR(®) Identifiler(®) PCR Amplification Kit and amplifies the core CODIS loci: D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, TH01, TPOX, and vWA. Additional loci amplified in the multiplex reaction are the sex-determinant, amelogenin, and two internationally accepted loci, D2S1338 and D19S433. While the primer sequences and dye configurations were unchanged, the AmpFℓSTR(®) Identifiler(®) Plus PCR Amplification Kit features an enhanced buffer formulation and an optimized PCR cycling protocol that increases sensitivity, provides better tolerance to PCR inhibitors, and improves performance on mixture samples. The AmpFℓSTR(®) Identifiler(®) Plus PCR Amplification Kit has been validated according to the FBI/National Standards and Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. The validation results support the use of the AmpFℓSTR(®) Identifiler(®) Plus PCR Amplification Kit for human identity and parentage testing.

  16. A fast-and-robust profiler for improving polymerase chain reaction diagnostics.

    PubMed

    Besseris, George J

    2014-01-01

    Polymerase chain reaction (PCR) is an in vitro technology in molecular genetics that progressively amplifies minimal copies of short DNA sequences in a fast and inexpensive manner. However, PCR performance is sensitive to suboptimal processing conditions. Compromised PCR conditions lead to artifacts and bias that downgrade the discriminatory power and reproducibility of the results. Promising attempts to resolve the PCR performance optimization issue have been guided by quality improvement tactics adopted in the past for industrial trials. Thus, orthogonal arrays (OAs) have been employed to program quick-and-easy structured experiments. Profiling of influences facilitates the quantification of effects that may counteract the detectability of amplified DNA fragments. Nevertheless, the attractive feature of reducing greatly the amount of work and expenditures by planning trials with saturated-unreplicated OA schemes is known to be relinquished in the subsequent analysis phase. This is because of an inherent incompatibility of ordinary multi-factorial comparison techniques to convert small yet dense datasets. Treating unreplicated-saturated data with either the analysis of variance (ANOVA) or regression models destroys the information extraction process. Both of those mentioned approaches are rendered blind to error since the examined effects absorb all available degrees of freedom. Therefore, in lack of approximating an experimental uncertainty, any outcome interpretation is rendered subjective. We propose a profiling method that permits the non-linear maximization of amplicon resolution by eliminating the necessity for direct error estimation. Our approach is distribution-free, calibration-free, simulation-free and sparsity-free with well-known power properties. It is also user-friendly by promoting rudimentary analytics. Testing our method on published amplicon count data, we found that the preponderant effect is the concentration of MgCl2 (p<0.05) followed by the

  17. Estimation of the diagnostic accuracy of the invA-gene-based PCR technique and a bacteriological culture for the detection of Salmonella spp. in caecal content from slaughtered pigs using Bayesian analysis.

    PubMed

    Mainar-Jaime, R C; Atashparvar, N; Chirino-Trejo, M

    2008-01-01

    The goal of this study was to estimate the accuracy of the invA-gene-based polymerase chain reaction (PCR) and a culture technique based on pre-enrichment with buffered peptone water, three selective enrichment media (selenite, tetrathionate and Rappaport-Vassiliadis broths) and four selective, solid media (Xylose-Lysine-Tergitol-4, Salmonella/Shigella, Hekton-Enteric and MacConkey), for the detection of Salmonella organisms from caecal samples from slaughter pigs. For this purpose a latent-class (Bayesian) approach was used. Two hundred and three slaughtered pigs were used after grouping them into two groups of 96 and 107 animals. Sensitivity (Se) was estimated to be 56% (95% probability interval 40, 76) for culture and 91% (81, 97) for PCR. The specificity (Sp) of the PCR was 88% (80, 95) while the Sp of the culture had been considered 100% in the statistical analysis as all culture-positive samples were confirmed by serotyping. PCR Se was not affected by the Salmonella serotypes present in the samples analysed. Accordingly, a minimum of 25.5% of the pigs was estimated to harbour Salmonella organisms in their faeces. It was concluded that bacteriology on caecal samples alone was a poor diagnostic method, and that the PCR method could be considered a cost-effective alternative to culture in Salmonella monitoring programmes. However, given the moderate Sp of this molecular technique, PCR-positive samples should be further confirmed through bacteriology.

  18. IMPROVED DETECTION OF HUMAN ENTERIC VIRUSES IN FOODS BY RT-PCR. (R826139)

    EPA Science Inventory

    Human enteric viruses (including hepatitis A virus (HAV) and Norwalk-like viruses (NLVs)) are now recognized as common causes of foodborne disease. While methods to detect these agents in clinical specimens have improved significantly over the last 10 years, applications to fo...

  19. Optimized PCR Conditions and Increased shRNA Fold Representation Improve Reproducibility of Pooled shRNA Screens

    PubMed Central

    Strezoska, Žaklina; Licon, Abel; Haimes, Josh; Spayd, Katie Jansen; Patel, Kruti M.; Sullivan, Kevin; Jastrzebski, Katarzyna; Simpson, Kaylene J.; Leake, Devin; van Brabant Smith, Anja; Vermeulen, Annaleen

    2012-01-01

    RNAi screening using pooled shRNA libraries is a valuable tool for identifying genetic regulators of biological processes. However, for a successful pooled shRNA screen, it is imperative to thoroughly optimize experimental conditions to obtain reproducible data. Here we performed viability screens with a library of ∼10 000 shRNAs at two different fold representations (100- and 500-fold at transduction) and report the reproducibility of shRNA abundance changes between screening replicates determined by microarray and next generation sequencing analyses. We show that the technical reproducibility between PCR replicates from a pooled screen can be drastically improved by ensuring that PCR amplification steps are kept within the exponential phase and by using an amount of genomic DNA input in the reaction that maintains the average template copies per shRNA used during library transduction. Using these optimized PCR conditions, we then show that higher reproducibility of biological replicates is obtained by both microarray and next generation sequencing when screening with higher average shRNA fold representation. shRNAs that change abundance reproducibly in biological replicates (primary hits) are identified from screens performed with both 100- and 500-fold shRNA representation, however a higher percentage of primary hit overlap between screening replicates is obtained from 500-fold shRNA representation screens. While strong hits with larger changes in relative abundance were generally identified in both screens, hits with smaller changes were identified only in the screens performed with the higher shRNA fold representation at transduction. PMID:22870320

  20. Clinical Evaluation of Rapid Diagnostic Test Kit for Scrub Typhus with Improved Performance

    PubMed Central

    2016-01-01

    Diagnosis of scrub typhus is challenging due to its more than twenty serotypes and the similar clinical symptoms with other acute febrile illnesses including leptospirosis, murine typhus and hemorrhagic fever with renal syndrome. Accuracy and rapidity of a diagnostic test to Orientia tsutsugamushi is an important step to diagnose this disease. To discriminate scrub typhus from other diseases, the improved ImmuneMed Scrub Typhus Rapid Diagnostic Test (RDT) was evaluated in Korea and Sri Lanka. The sensitivity at the base of each IgM and IgG indirect immunofluorescent assay (IFA) in Korean patients was 98.6% and 97.1%, and the specificity was 98.2% and 97.7% respectively. The sensitivity and specificity for retrospective diagnosis at the base of IFA in Sri Lanka was 92.1% and 96.1%. ImmuneMed RDT was not reactive to any serum from seventeen diseases including hemorrhagic fever with renal syndrome (n = 48), leptospirosis (n = 23), and murine typhus (n = 48). ImmuneMed RDT shows superior sensitivity (98.6% and 97.1%) compared with SD Bioline RDT (84.4% at IgM and 83.3% at IgG) in Korea. The retrospective diagnosis of ImmuneMed RDT exhibits 94.0% identity with enzyme-linked Immunosorbent assay (ELISA) using South India patient serum samples. These results suggest that this RDT can replace other diagnostic tests and is applicable for global diagnosis of scrub typhus. This rapid and accurate diagnosis will be beneficial for diagnosing and managing scrub typhus. PMID:27478327

  1. Clinical Evaluation of Rapid Diagnostic Test Kit for Scrub Typhus with Improved Performance.

    PubMed

    Kim, Young-Jin; Park, Sungman; Premaratna, Ranjan; Selvaraj, Stephen; Park, Sang-Jin; Kim, Sora; Kim, Donghwan; Kim, Min Soo; Shin, Dong Hoon; Choi, Kyung-Chan; Kwon, Soon-Hwan; Seo, Wonjun; Lee, Nam Taek; Kim, Seung-Han; Kang, Heui Keun; Kim, Yoon-Won

    2016-08-01

    Diagnosis of scrub typhus is challenging due to its more than twenty serotypes and the similar clinical symptoms with other acute febrile illnesses including leptospirosis, murine typhus and hemorrhagic fever with renal syndrome. Accuracy and rapidity of a diagnostic test to Orientia tsutsugamushi is an important step to diagnose this disease. To discriminate scrub typhus from other diseases, the improved ImmuneMed Scrub Typhus Rapid Diagnostic Test (RDT) was evaluated in Korea and Sri Lanka. The sensitivity at the base of each IgM and IgG indirect immunofluorescent assay (IFA) in Korean patients was 98.6% and 97.1%, and the specificity was 98.2% and 97.7% respectively. The sensitivity and specificity for retrospective diagnosis at the base of IFA in Sri Lanka was 92.1% and 96.1%. ImmuneMed RDT was not reactive to any serum from seventeen diseases including hemorrhagic fever with renal syndrome (n = 48), leptospirosis (n = 23), and murine typhus (n = 48). ImmuneMed RDT shows superior sensitivity (98.6% and 97.1%) compared with SD Bioline RDT (84.4% at IgM and 83.3% at IgG) in Korea. The retrospective diagnosis of ImmuneMed RDT exhibits 94.0% identity with enzyme-linked Immunosorbent assay (ELISA) using South India patient serum samples. These results suggest that this RDT can replace other diagnostic tests and is applicable for global diagnosis of scrub typhus. This rapid and accurate diagnosis will be beneficial for diagnosing and managing scrub typhus. PMID:27478327

  2. Diagnostic Molecular Mycobacteriology in Regions With Low Tuberculosis Endemicity: Combining Real-time PCR Assays for Detection of Multiple Mycobacterial Pathogens With Line Probe Assays for Identification of Resistance Mutations.

    PubMed

    Deggim-Messmer, Vanessa; Bloemberg, Guido V; Ritter, Claudia; Voit, Antje; Hömke, Rico; Keller, Peter M; Böttger, Erik C

    2016-07-01

    Molecular assays have not yet been able to replace time-consuming culture-based methods in clinical mycobacteriology. Using 6875 clinical samples and a study period of 35months we evaluated the use of PCR-based assays to establish a diagnostic workflow with a fast time-to-result of 1-2days, for 1. detection of Mycobacterium tuberculosis complex (MTB), 2. detection and identification of nontuberculous mycobacteria (NTM), and 3. identification of drug susceptible MTB. MTB molecular-based detection and culture gave concordant results for 97.7% of the specimens. NTM PCR-based detection and culture gave concordant results for 97.0% of the specimens. Defining specimens on the basis of combined laboratory data as true positives or negatives with discrepant results resolved by clinical chart reviews, we calculated sensitivity, specificity, PPV and NPV for PCR-based MTB detection as 84.7%, 100%, 100%, and 98.7%; the corresponding values for culture-based MTB detection were 86.3%, 100%, 100%, and 98.8%. PCR-based detection of NTM had a sensitivity of 84.7% compared to 78.0% of that of culture-based NTM detection. Molecular drug susceptibility testing (DST) by line-probe assay was found to predict phenotypic DST results in MTB with excellent accuracy. Our findings suggest a diagnostic algorithm to largely replace lengthy culture-based techniques by rapid molecular-based methods.

  3. Testing for cattle allergy: modified diagnostic cutoff levels improve sensitivity in symptomatic claw trimmers

    PubMed Central

    Dik, Natalja; Hallier, Ernst; Zuberbier, Torsten; Bergmann, Karl-Christian

    2010-01-01

    Background The diagnosis of cattle-related sensitization is complicated by the variability and complexity of cattle allergen extracts. Objective To evaluate a modified diagnostic procedure leading to more accurate results especially in the early phase of sensitization. Methods We tested 27 claw trimmers with and 65 without cattle-related symptoms using two commercially available cattle allergen extracts. We also used a self-prepared cattle allergen mix designed to represent the full spectrum of cattle allergens from a typical agricultural workplace. Results More than 50% of symptomatic claw trimmers showed negative test results with commercial extracts and a sensitization cutoff point of 0.35 kU/l. In contrast, with the self-prepared cattle allergen mix, positive results were observed for almost all of them. Evaluating the results of the commercial test kits at different cutoff levels, we found an ideal cutoff point to improve the sensitivity at 0.2 kU/l. Conclusion Additional tests with self-made cattle hair extracts can help to bridge the diagnostic gap seen in patients showing cattle-related symptoms, but negative results in commercially available tests. For early-stage sensitization screening, we propose to lower the cutoff level indicating sensitization to 0.2 kU/l. PMID:20658147

  4. Improving the reliability of diagnostic tests in population-based agreement studies

    PubMed Central

    Nelson, Kerrie P.; Edwards, Don

    2016-01-01

    Many large-scale studies have recently been carried out to assess the reliability of diagnostic procedures, such as mammography for the detection of breast cancer. The large numbers of raters and subjects involved raise new challenges in how to measure agreement in these types of studies. An important motivator of these studies is the identification of factors that contribute to the often wide discrepancies observed between raters’ classifications, such as a rater’s experience, in order to improve the reliability of the diagnostic process of interest. Incorporating covariate information into the agreement model is a key component in addressing these questions. Few agreement models are currently available that jointly model larger numbers of raters and subjects and incorporate covariate information. In this paper, we extend a recently developed population-based model and measure of agreement for binary ratings to incorporate covariate information using the class of generalized linear mixed models with a probit link function. Important information on factors related to the subjects and raters can be included as fixed and/or random effects in the model. We demonstrate how agreement can be assessed between subgroups of the raters and/or subjects, for example, comparing agreement between experienced and less experienced raters. Simulation studies are carried out to test the performance of the proposed models and measures of agreement. Application to a large-scale breast cancer study is presented. PMID:20128018

  5. Network Analysis of Force Concept Inventory Responses to Improve Diagnostic Utility

    NASA Astrophysics Data System (ADS)

    Brewe, Eric; Bruun, Jesper

    2015-04-01

    The Force Concept Inventory (FCI) is a diagnostic instrument designed to investigate students' understanding of Newtonian Mechanics and is widely used in Physics Education Research. One of the strengths of the FCI is that the distractors are drawn from student conceptions based in their experiences. The distractors chosen are often more informative about student's understanding as they identify the particular nature of students' alternative conceptions. We propose a network based analysis of the FCI which will enhance the utility of the FCI as a diagnostic tool for identifying student conceptions. In this approach, student responses are treated as a bipartite network which is then projected into two networks - students and responses. The response network includes all responses that are shared among students. We use the LANS backbone extraction algorithm to identify patterns in student responses. We use community detection algorithms on the backbone networks to identify clusters of common responses which map to models held by students, for example, ``force is needed for movement'' and ``the active agent uses the most force.'' This method has utility across a variety of instruments and could be used to improve instruction by providing in-depth knowledge of student conceptions. Supported in part by NSF Grant #PHY 134424.

  6. “A New Tool Improves Diagnostic Test Performance for Transmission EM Evaluation of Axonemal Dynein Arms”

    PubMed Central

    Funkhouser, W. Keith; Niethammer, Marc; Carson, Johnny L.; Burns, Kimberlie A.; Knowles, Michael R.; Leigh, Margaret W.; Zariwala, Maimoona A.; Funkhouser, William K.

    2014-01-01

    Diagnosis of primary ciliary dyskinesia (PCD) by identification of dynein arm loss in transmission electron microscopy (TEM) images can be confounded by high background noise due to random electron-dense material within the ciliary matrix, leading to diagnostic uncertainty even for experienced morphologists. We developed a novel image analysis tool to average the axonemal peripheral microtubular doublets, thereby increasing microtubular signal and reducing random background noise. In a randomized, double-blinded study that compared two experienced morphologists and three different diagnostic approaches, we found that use of this tool led to improvement in diagnostic TEM test performance. PMID:23957500

  7. Efforts to improve the diagnostic accuracy of endoscopic ultrasound-guided fine-needle aspiration for pancreatic tumors

    PubMed Central

    Yamabe, Akane; Irisawa, Atsushi; Bhutani, Manoop S.; Shibukawa, Goro; Fujisawa, Mariko; Sato, Ai; Yoshida, Yoshitsugu; Arakawa, Noriyuki; Ikeda, Tsunehiko; Igarashi, Ryo; Maki, Takumi; Yamamoto, Shogo

    2016-01-01

    Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) is widely used to obtain a definitive diagnosis of pancreatic tumors. Good results have been reported for its diagnostic accuracy, with high sensitivity and specificity of around 90%; however, technological developments and adaptations to improve it still further are currently underway. The endosonographic technique can be improved when several tips and tricks useful to overcome challenges of EUS-FNA are known. This review provides various techniques and equipment for improvement in the diagnostic accuracy in EUS-FNA. PMID:27503153

  8. Bayesian estimation of the diagnostic accuracy of a multiplex real-time PCR assay and bacteriological culture for 4 common bovine intramammary pathogens.

    PubMed

    Paradis, M-È; Haine, D; Gillespie, B; Oliver, S P; Messier, S; Comeau, J; Scholl, D T

    2012-11-01

    Bacteriological culture (BC) is the traditional method for intramammary infection diagnosis but lacks sensitivity and is time consuming. Multiplex real-time PCR (mr-PCR) enables testing the presence of several bacteria and reduces diagnosis time. Our objective was to estimate bacterial species-specific sensitivity (Se) and specificity of both BC and mr-PCR tests for detecting bacteria in milk samples from clinical mastitis cases and from apparently normal quarters, using a Bayesian latent class model. Milk samples from 1,014 clinical mastitis cases and 1,495 samples from apparently normal quarters were analyzed by BC and mr-PCR. Two positive culture definitions were used: ≥1 cfu/0.01 mL and ≥10 cfu/0.01 mL of the specified bacteria. The mr-PCR was designed to simultaneously detect Staphylococcus aureus, Streptococcus uberis, Escherichia coli, and Streptococcus agalactiae. The priors used in our Bayesian model were weakly informative, with BC priors using the best available error data. Results were compared with those obtained using uniform priors for mr-PCR to test robustness. Weak and uniform priors gave about the same posterior distributions except for Strep. uberis from normal quarters and Strep. agalactiae. Multiplex real-time PCR Se on milk from clinical mastitis were lower than mr-PCR Se on milk from normal quarters. Multiplex real-time PCR Se was higher than BC on milk from normal quarters. Multiplex real-time PCR Se was generally lower than BC on milk from clinical mastitis and it varied by clinical severity. The estimate specificities of detection for all pathogens were ≥99%, regardless of sample type. The effect of milk sample preservation before testing was evaluated and may have been a factor that affected our observed results. A significant association was observed between sample age and mr-PCR results leading to reduced detection of E. coli and Strep. agalactiae in nonclinical samples. Differences in sample age between conduct of BC and of mr-PCR

  9. NREL Develops Diagnostic Test Cases to Improve Building Energy Simulation Programs (Fact Sheet)

    SciTech Connect

    Not Available

    2011-12-01

    This technical highlight describes NREL research to develop a set of diagnostic test cases for building energy simulations in order to achieve more accurate energy use and savings predictions. The National Renewable Energy Laboratory (NREL) Residential and Commercial Buildings research groups developed a set of diagnostic test cases for building energy simulations. Eight test cases were developed to test surface conduction heat transfer algorithms of building envelopes in building energy simulation programs. These algorithms are used to predict energy flow through external opaque surfaces such as walls, ceilings, and floors. The test cases consist of analytical and vetted numerical heat transfer solutions that have been available for decades, which increases confidence in test results. NREL researchers adapted these solutions for comparisons with building energy simulation results. Testing the new cases with EnergyPlus identified issues with the conduction finite difference (CondFD) heat transfer algorithm in versions 5 and 6. NREL researchers resolved these issues for EnergyPlus version 7. The new test cases will help users and developers of EnergyPlus and other building energy tools to identify and fix problems associated with solid conduction heat transfer algorithms of building envelopes and their boundary conditions. In the long term, improvements to software algorithms will result in more accurate energy use and savings predictions. NREL researchers plan to document the set of test cases and make them available for future consideration by validation standards such as ASHRAE Standard 140: Standard Method of Test for the Evaluation of Building Energy Analysis Computer Programs. EnergyPlus users will also have access to the improved CondFD model in version 7 after its next scheduled release.

  10. The possibilities of improvement in the sensitivity of cancer fluorescence diagnostics by computer image processing

    NASA Astrophysics Data System (ADS)

    Ledwon, Aleksandra; Bieda, Robert; Kawczyk-Krupka, Aleksandra; Polanski, Andrzej; Wojciechowski, Konrad; Latos, Wojciech; Sieron-Stoltny, Karolina; Sieron, Aleksander

    2008-02-01

    Background: Fluorescence diagnostics uses the ability of tissues to fluoresce after exposition to a specific wavelength of light. The change in fluorescence between normal and progression to cancer allows to see early cancer and precancerous lesions often missed by white light. Aim: To improve by computer image processing the sensitivity of fluorescence images obtained during examination of skin, oral cavity, vulva and cervix lesions, during endoscopy, cystoscopy and bronchoscopy using Xillix ONCOLIFE. Methods: Function of image f(x,y):R2 --> R 3 was transformed from original color space RGB to space in which vector of 46 values refers to every point labeled by defined xy-coordinates- f(x,y):R2 --> R 46. By means of Fisher discriminator vector of attributes of concrete point analalyzed in the image was reduced according to two defined classes defined as pathologic areas (foreground) and healthy areas (background). As a result the highest four fisher's coefficients allowing the greatest separation between points of pathologic (foreground) and healthy (background) areas were chosen. In this way new function f(x,y):R2 --> R 4 was created in which point x,y corresponds with vector Y, H, a*, c II. In the second step using Gaussian Mixtures and Expectation-Maximisation appropriate classificator was constructed. This classificator enables determination of probability that the selected pixel of analyzed image is a pathologically changed point (foreground) or healthy one (background). Obtained map of probability distribution was presented by means of pseudocolors. Results: Image processing techniques improve the sensitivity, quality and sharpness of original fluorescence images. Conclusion: Computer image processing enables better visualization of suspected areas examined by means of fluorescence diagnostics.

  11. Polymeric LabChip Real-Time PCR as a Point-of-Care-Potential Diagnostic Tool for Rapid Detection of Influenza A/H1N1 Virus in Human Clinical Specimens

    PubMed Central

    Song, Hyun-Ok; Kim, Je-Hyoung; Ryu, Ho-Sun; Lee, Dong-Hoon; Kim, Sun-Jin; Kim, Deog-Joong; Suh, In Bum; Choi, Du Young; In, Kwang-Ho; Kim, Sung-Woo; Park, Hyun

    2012-01-01

    It is clinically important to be able to detect influenza A/H1N1 virus using a fast, portable, and accurate system that has high specificity and sensitivity. To achieve this goal, it is necessary to develop a highly specific primer set that recognizes only influenza A viral genes and a rapid real-time PCR system that can detect even a single copy of the viral gene. In this study, we developed and validated a novel fluidic chip-type real-time PCR (LabChip real-time PCR) system that is sensitive and specific for the detection of influenza A/H1N1, including the pandemic influenza strain A/H1N1 of 2009. This LabChip real-time PCR system has several remarkable features: (1) It allows rapid quantitative analysis, requiring only 15 min to perform 30 cycles of real-time PCR. (2) It is portable, with a weight of only 5.5 kg. (3) The reaction cost is low, since it uses disposable plastic chips. (4) Its high efficiency is equivalent to that of commercially available tube-type real-time PCR systems. The developed disposable LabChip is an economic, heat-transferable, light-transparent, and easy-to-fabricate polymeric chip compared to conventional silicon- or glass-based labchip. In addition, our LabChip has large surface-to-volume ratios in micro channels that are required for overcoming time consumed for temperature control during real-time PCR. The efficiency of the LabChip real-time PCR system was confirmed using novel primer sets specifically targeted to the hemagglutinin (HA) gene of influenza A/H1N1 and clinical specimens. Eighty-five human clinical swab samples were tested using the LabChip real-time PCR. The results demonstrated 100% sensitivity and specificity, showing 72 positive and 13 negative cases. These results were identical to those from a tube-type real-time PCR system. This indicates that the novel LabChip real-time PCR may be an ultra-fast, quantitative, point-of-care-potential diagnostic tool for influenza A/H1N1 with a high sensitivity and specificity

  12. Identification of CD70 as a diagnostic biomarker for clear cell renal cell carcinoma by gene expression profiling, real-time RT-PCR and immunohistochemistry.

    PubMed

    Diegmann, Julia; Junker, Kerstin; Gerstmayer, Bernhard; Bosio, Andreas; Hindermann, Winfried; Rosenhahn, Julia; von Eggeling, Ferdinand

    2005-08-01

    The underlying molecular mechanisms of renal cell carcinoma (RCC) are poorly understood and more reliable markers for early diagnosis are needed. Hence, alternative strategies for biomarker discovery with appropriate validation technologies have to be performed. To elucidate genesis and progression of RCC we used high parallel chip based gene expression profiling comparing normal and tumour tissues. We compared corresponding control and tumour tissue samples from 10 patients with clear cell RCC. We isolated RNA from histologically well characterised tissue sections and performed reverse transcription, labelling and linear RNA amplification. Samples were hybridised on microarrays containing 642 human cDNAs. Of the 352 differentially expressed genes found, CD70 and FRA2 were selected for further evaluation by real-time RT-PCR. The analysis all showed a high potential to discriminate between normal and tumour tissue. Moreover, increased CD70 mRNA expression in tumour cells could be correlated to its expression at the protein level. Immunohistochemistry (IHC) showed very strong expression of CD70 in all tumour samples but no expression in adjacent normal kidney tissue. With our combined approach we were able to identify CD70 as a new marker for RCC, which may be useful in the future for improved immunohistochemical diagnosis. PMID:16043348

  13. Diagnostic performance and application of a real-time PCR assay for the detection of Salmonella in fecal samples collected from hospitalized horses with or without signs of gastrointestinal tract disease.

    PubMed

    Ekiri, A B; Long, M T; Hernandez, J A

    2016-02-01

    The main objective of this study was to assess the diagnostic performance of a real-time polymerase chain reaction (PCR) assay for the detection of Salmonella in fecal samples collected from hospitalized horses with or without signs of gastrointestinal (GI) tract disease. The PCR assay used primers and a probe that targeted the invA gene of Salmonella. Assuming a sensitivity of 100% and a specificity of 96.6%, and a disease prevalence of 2%, 5%, and 10-15% in study horses, the PCR assay had a high (100%) negative predictive value, and a positive predictive value that ranged from 37% in horses without signs of GI disease that tested Salmonella culture-negative, to 60% in horses with signs of GI disease that tested Salmonella culture-negative, to 76-83% in horses with signs of GI disease that tested Salmonella culture-positive. This study provides evidence that the real-time PCR that targets the Salmonella invA gene can be used as a screening test for the detection of Salmonella in feces of hospitalized horses with signs of GI disease. Horses that test PCR-positive can be tested in series using bacteriologic culture to reduce false positive results or to provide additional data (e.g., antibiogram and serotyping data) that can be used to identify potential nosocomial Salmonella infections.

  14. PCR amplification of a multi-copy mitochondrial gene (cox3) improves detection of Cytauxzoon felis infection as compared to a ribosomal gene (18S).

    PubMed

    Schreeg, Megan E; Marr, Henry S; Griffith, Emily H; Tarigo, Jaime L; Bird, David M; Reichard, Mason V; Cohn, Leah A; Levy, Michael G; Birkenheuer, Adam J

    2016-07-30

    Cytauxzoon felis is a tick-transmitted protozoan parasite that infects felids. Clinical disease caused by acute C. felis infection rapidly progresses in domestic cats, leading to high morbidity and mortality. Accurately diagnosing cytauxzoonosis as soon as possible during acute infection would allow for earlier initiation of antiprotozoal therapy which could lead to higher survival rates. Molecular detection of parasite rRNA genes (18S) by PCR has previously been shown to be a sensitive method of diagnosing C. felis infections. Based on evidence from related apicomplexan species, we hypothesized that C. felis mitochondrial genes would exist at higher copy numbers than 18S and would be a more sensitive diagnostic target. In this study we have designed a PCR assay targeting the C. felis mitochondrial gene cytochrome c oxidase subunit III (cox3). Herein we demonstrate that (1) the cox3 PCR can detect as low as 1 copy of DNA target and can detect C. felis in samples with known mitochondrial sequence heterogeneity, (2) cox3 copy number is increased relative to 18S in blood and tissue samples from acutely infected cats, and (3) the cox3 PCR is more sensitive than 18S PCR for detection of C. felis during early infections.

  15. Evaluation of tcdB real-time PCR in a three-step diagnostic algorithm for detection of toxigenic Clostridium difficile.

    PubMed

    Larson, Ann M; Fung, Angela M; Fang, Ferric C

    2010-01-01

    Clostridium difficile is the most common infectious cause of diarrhea in hospitalized patients. The optimal approach for the detection of toxigenic C. difficile remains controversial because no single test is sensitive, specific, and affordable. We have developed a real-time PCR method (direct stool PCR [DPCR]) to detect the tcdB gene encoding toxin B directly from stool specimens and have combined it with enzyme immunoassays (EIAs) in a three-step protocol. DPCR was performed on 699 specimens that were positive for C. difficile glutamate dehydrogenase (GDH) by Wampole C Diff Quik Chek EIA (GDH-Q) and negative for toxins A and B by Wampole Tox A/B Quik Chek EIA (AB-Q), performed sequentially. The performance of this three-step algorithm was compared with a modified "gold standard" that combined tissue culture cytotoxicity (CYT) and DPCR. A separate investigation was performed to evaluate the sensitivity of the GDH-Q as a screening test, and toxigenic C. difficile was found in 1.9% of 211 GDH-Q-negative specimens. The overall sensitivity, specificity, and positive and negative predictive values, respectively, were as follows for an algorithm combining GDH-Q, AB-Q, and DPCR: 83.8%, 99.7%, 97.1%, and 97.9%. Those for CYT alone were 58.8%, 100%, 100%, and 94.9%, respectively. In comparison, the sensitivity and specificity of DPCR were estimated to be 97.5% and 99.7%, respectively, using the same modified gold standard. Neither CYT nor toxin EIA was sufficiently sensitive to exclude toxigenic C. difficile, and combining EIAs with CYT in a three-step algorithm failed to substantially improve sensitivity. DPCR is a sensitive and specific method for the detection of toxigenic C. difficile that can provide same-day results at a cost-per-positive test comparable to those of other methods. A three-step algorithm in which DPCR is used to analyze GDH EIA-positive, toxin EIA-negative specimens provides a convenient and specific alternative with rapid results for 87.7% of

  16. Evaluation of tcdB Real-Time PCR in a Three-Step Diagnostic Algorithm for Detection of Toxigenic Clostridium difficile▿

    PubMed Central

    Larson, Ann M.; Fung, Angela M.; Fang, Ferric C.

    2010-01-01

    Clostridium difficile is the most common infectious cause of diarrhea in hospitalized patients. The optimal approach for the detection of toxigenic C. difficile remains controversial because no single test is sensitive, specific, and affordable. We have developed a real-time PCR method (direct stool PCR [DPCR]) to detect the tcdB gene encoding toxin B directly from stool specimens and have combined it with enzyme immunoassays (EIAs) in a three-step protocol. DPCR was performed on 699 specimens that were positive for C. difficile glutamate dehydrogenase (GDH) by Wampole C Diff Quik Chek EIA (GDH-Q) and negative for toxins A and B by Wampole Tox A/B Quik Chek EIA (AB-Q), performed sequentially. The performance of this three-step algorithm was compared with a modified “gold standard” that combined tissue culture cytotoxicity (CYT) and DPCR. A separate investigation was performed to evaluate the sensitivity of the GDH-Q as a screening test, and toxigenic C. difficile was found in 1.9% of 211 GDH-Q-negative specimens. The overall sensitivity, specificity, and positive and negative predictive values, respectively, were as follows for an algorithm combining GDH-Q, AB-Q, and DPCR: 83.8%, 99.7%, 97.1%, and 97.9%. Those for CYT alone were 58.8%, 100%, 100%, and 94.9%, respectively. In comparison, the sensitivity and specificity of DPCR were estimated to be 97.5% and 99.7%, respectively, using the same modified gold standard. Neither CYT nor toxin EIA was sufficiently sensitive to exclude toxigenic C. difficile, and combining EIAs with CYT in a three-step algorithm failed to substantially improve sensitivity. DPCR is a sensitive and specific method for the detection of toxigenic C. difficile that can provide same-day results at a cost-per-positive test comparable to those of other methods. A three-step algorithm in which DPCR is used to analyze GDH EIA-positive, toxin EIA-negative specimens provides a convenient and specific alternative with rapid results for 87.7% of

  17. Evaluation of PCR Based Assays for the Improvement of Proportion Estimation of Bacterial and Viral Pathogens in Diarrheal Surveillance.

    PubMed

    Guan, Hongxia; Zhang, Jingyun; Xiao, Yong; Sha, Dan; Ling, Xia; Kan, Biao

    2016-01-01

    Diarrhea can be caused by a variety of bacterial, viral and parasitic organisms. Laboratory diagnosis is essential in the pathogen-specific burden assessment. In the pathogen spectrum monitoring in the diarrheal surveillance, culture methods are commonly used for the bacterial pathogens' detection whereas nucleic acid based amplification, the non-cultural methods are used for the viral pathogens. Different methodology may cause the inaccurate pathogen spectrum for the bacterial pathogens because of their different culture abilities with the different media, and for the comparison of bacterial vs. viral pathogens. The application of nucleic acid-based methods in the detection of viral and bacterial pathogens will likely increase the number of confirmed positive diagnoses, and will be comparable since all pathogens will be detected based on the same nucleic acid extracts from the same sample. In this study, bacterial pathogens, including diarrheagenic Escherichia coli (DEC), Salmonella spp., Shigella spp., Vibrio parahaemolyticus and V. cholerae, were detected in 334 diarrheal samples by PCR-based methods using nucleic acid extracted from stool samples and associated enrichment cultures. A protocol was established to facilitate the consistent identification of bacterial pathogens in diarrheal patients. Five common enteric viruses were also detected by RT-PCR, including rotavirus, sapovirus, norovirus (I and II), human astrovirus, and enteric adenovirus. Higher positive rates were found for the bacterial pathogens, showing the lower proportion estimation if only using culture methods. This application will improve the quality of bacterial diarrheagenic pathogen survey, providing more accurate information pertaining to the pathogen spectrum associated with finding of food safety problems and disease burden evaluation. PMID:27065958

  18. Evaluation of PCR Based Assays for the Improvement of Proportion Estimation of Bacterial and Viral Pathogens in Diarrheal Surveillance

    PubMed Central

    Guan, Hongxia; Zhang, Jingyun; Xiao, Yong; Sha, Dan; Ling, Xia; Kan, Biao

    2016-01-01

    Diarrhea can be caused by a variety of bacterial, viral and parasitic organisms. Laboratory diagnosis is essential in the pathogen-specific burden assessment. In the pathogen spectrum monitoring in the diarrheal surveillance, culture methods are commonly used for the bacterial pathogens' detection whereas nucleic acid based amplification, the non-cultural methods are used for the viral pathogens. Different methodology may cause the inaccurate pathogen spectrum for the bacterial pathogens because of their different culture abilities with the different media, and for the comparison of bacterial vs. viral pathogens. The application of nucleic acid-based methods in the detection of viral and bacterial pathogens will likely increase the number of confirmed positive diagnoses, and will be comparable since all pathogens will be detected based on the same nucleic acid extracts from the same sample. In this study, bacterial pathogens, including diarrheagenic Escherichia coli (DEC), Salmonella spp., Shigella spp., Vibrio parahaemolyticus and V. cholerae, were detected in 334 diarrheal samples by PCR-based methods using nucleic acid extracted from stool samples and associated enrichment cultures. A protocol was established to facilitate the consistent identification of bacterial pathogens in diarrheal patients. Five common enteric viruses were also detected by RT-PCR, including rotavirus, sapovirus, norovirus (I and II), human astrovirus, and enteric adenovirus. Higher positive rates were found for the bacterial pathogens, showing the lower proportion estimation if only using culture methods. This application will improve the quality of bacterial diarrheagenic pathogen survey, providing more accurate information pertaining to the pathogen spectrum associated with finding of food safety problems and disease burden evaluation. PMID:27065958

  19. Evaluation of PCR Based Assays for the Improvement of Proportion Estimation of Bacterial and Viral Pathogens in Diarrheal Surveillance.

    PubMed

    Guan, Hongxia; Zhang, Jingyun; Xiao, Yong; Sha, Dan; Ling, Xia; Kan, Biao

    2016-01-01

    Diarrhea can be caused by a variety of bacterial, viral and parasitic organisms. Laboratory diagnosis is essential in the pathogen-specific burden assessment. In the pathogen spectrum monitoring in the diarrheal surveillance, culture methods are commonly used for the bacterial pathogens' detection whereas nucleic acid based amplification, the non-cultural methods are used for the viral pathogens. Different methodology may cause the inaccurate pathogen spectrum for the bacterial pathogens because of their different culture abilities with the different media, and for the comparison of bacterial vs. viral pathogens. The application of nucleic acid-based methods in the detection of viral and bacterial pathogens will likely increase the number of confirmed positive diagnoses, and will be comparable since all pathogens will be detected based on the same nucleic acid extracts from the same sample. In this study, bacterial pathogens, including diarrheagenic Escherichia coli (DEC), Salmonella spp., Shigella spp., Vibrio parahaemolyticus and V. cholerae, were detected in 334 diarrheal samples by PCR-based methods using nucleic acid extracted from stool samples and associated enrichment cultures. A protocol was established to facilitate the consistent identification of bacterial pathogens in diarrheal patients. Five common enteric viruses were also detected by RT-PCR, including rotavirus, sapovirus, norovirus (I and II), human astrovirus, and enteric adenovirus. Higher positive rates were found for the bacterial pathogens, showing the lower proportion estimation if only using culture methods. This application will improve the quality of bacterial diarrheagenic pathogen survey, providing more accurate information pertaining to the pathogen spectrum associated with finding of food safety problems and disease burden evaluation.

  20. Burn injury diagnostic imaging device's accuracy improved by outlier detection and removal

    NASA Astrophysics Data System (ADS)

    Li, Weizhi; Mo, Weirong; Zhang, Xu; Lu, Yang; Squiers, John J.; Sellke, Eric W.; Fan, Wensheng; DiMaio, J. Michael; Thatcher, Jeffery E.

    2015-05-01

    Multispectral imaging (MSI) was implemented to develop a burn diagnostic device that will assist burn surgeons in planning and performing burn debridement surgery by classifying burn tissue. In order to build a burn classification model, training data that accurately represents the burn tissue is needed. Acquiring accurate training data is difficult, in part because the labeling of raw MSI data to the appropriate tissue classes is prone to errors. We hypothesized that these difficulties could be surmounted by removing outliers from the training dataset, leading to an improvement in the classification accuracy. A swine burn model was developed to build an initial MSI training database and study an algorithm's ability to classify clinically important tissues present in a burn injury. Once the ground-truth database was generated from the swine images, we then developed a multi-stage method based on Z-test and univariate analysis to detect and remove outliers from the training dataset. Using 10-fold cross validation, we compared the algorithm's accuracy when trained with and without the presence of outliers. The outlier detection and removal method reduced the variance of the training data from wavelength space, and test accuracy was improved from 63% to 76%. Establishing this simple method of conditioning for the training data improved the accuracy of the algorithm to match the current standard of care in burn injury assessment. Given that there are few burn surgeons and burn care facilities in the United States, this technology is expected to improve the standard of burn care for burn patients with less access to specialized facilities.

  1. [Improved quality of coronary diagnostics and interventions by virtual reality simulation].

    PubMed

    Voelker, W; Maier, S; Lengenfelder, B; Schöbel, W; Petersen, J; Bonz, A; Ertl, G

    2011-08-01

    Currently, more than 800,000 diagnostic procedures and 300,000 percutaneous coronary interventions are performed annually in 556 catheter laboratories in Germany. These numbers document the importance of training programs in interventional cardiology. However, this need is in sharp contrast to the time constraints for continuing medical education in Germany due to personnel and financial restrictions. A possible solution for this dilemma could be new training programs which partially supplement conventional clinical training by simulation-based medical education. Currently five virtual reality simulators for diagnostic procedures and percutaneous coronary interventions are available. These simulators provide a realistic hands-on training comparable to flight simulation in aviation.The simulator of choice for a defined training program depending on the underlying learning objectives could either be a simple mechanical model (for puncture training) or even a combination of virtual reality simulator and a full-scale mannequin (for team training and crisis resource management). For the selection of the adequate training program the basic skills of the trainee, the learning objectives and the underlying curriculum have to be taken into account. Absolutely mandatory for the success of simulation-based training is a dedicated teacher providing feedback and guidance. This teacher should be an experienced interventional cardiologist who knows both the simulator and the selected training cases which serve as a vehicle for transferring knowledge and skills.In this paper the potential of virtual reality simulation in cardiology will be discussed and the conditions which must be fulfilled to achieve quality improvement by simulation-based training will be defined. PMID:21748387

  2. Improving the molecular diagnosis of Chlamydia psittaci and Chlamydia abortus infection with a species-specific duplex real-time PCR.

    PubMed

    Opota, Onya; Jaton, Katia; Branley, James; Vanrompay, Daisy; Erard, Veronique; Borel, Nicole; Longbottom, David; Greub, Gilbert

    2015-10-01

    Chlamydia psittaci and Chlamydia abortus are closely related intracellular bacteria exhibiting different tissue tropism that may cause severe but distinct infection in humans. C. psittaci causes psittacosis, a respiratory zoonotic infection transmitted by birds. C. abortus is an abortigenic agent in small ruminants, which can also colonize the human placenta and lead to foetal death and miscarriage. Infections caused by C. psittaci and C. abortus are underestimated mainly due to diagnosis difficulties resulting from their strict intracellular growth. We developed a duplex real-time PCR to detect and distinguish these two bacteria in clinical samples. The first PCR (PCR1) targeted a sequence of the 16S-23S rRNA operon allowing the detection of both C. psittaci and C. abortus. The second PCR (PCR2) targeted the coding DNA sequence CPSIT_0607 unique to C. psittaci. The two PCRs showed 100 % detection for ≥ 10 DNA copies per reaction (1000 copies ml(- 1)). Using a set of 120 samples, including bacterial reference strains, clinical specimens and infected cell culture material, we monitored 100 % sensitivity and 100 % specificity for the detection of C. psittaci and C. abortus for PCR1. When PCR1 was positive, PCR2 could discriminate C. psittaci from C. abortus with a positive predictive value of 100 % and a negative predictive value of 88 %. In conclusion, this new duplex PCR represents a low-cost and time-saving method with high-throughput potential, expected to improve the routine diagnosis of psittacosis and pregnancy complication in large-scale screening programs and also during outbreaks.

  3. Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial

    PubMed Central

    Kapp, Joshua R; Diss, Tim; Spicer, James; Gandy, Michael; Schrijver, Iris; Jennings, Lawrence J; Li, Marilyn M; Tsongalis, Gregory J; de Castro, David Gonzalez; Bridge, Julia A; Wallace, Andrew; Deignan, Joshua L; Hing, Sandra; Butler, Rachel; Verghese, Eldo; Latham, Gary J; Hamoudi, Rifat A

    2015-01-01

    Aims Mutation detection accuracy has been described extensively; however, it is surprising that pre-PCR processing of formalin-fixed paraffin-embedded (FFPE) samples has not been systematically assessed in clinical context. We designed a RING trial to (i) investigate pre-PCR variability, (ii) correlate pre-PCR variation with EGFR/BRAF mutation testing accuracy and (iii) investigate causes for observed variation. Methods 13 molecular pathology laboratories were recruited. 104 blinded FFPE curls including engineered FFPE curls, cell-negative FFPE curls and control FFPE tissue samples were distributed to participants for pre-PCR processing and mutation detection. Follow-up analysis was performed to assess sample purity, DNA integrity and DNA quantitation. Results Rate of mutation detection failure was 11.9%. Of these failures, 80% were attributed to pre-PCR error. Significant differences in DNA yields across all samples were seen using analysis of variance (p<0.0001), and yield variation from engineered samples was not significant (p=0.3782). Two laboratories failed DNA extraction from samples that may be attributed to operator error. DNA extraction protocols themselves were not found to contribute significant variation. 10/13 labs reported yields averaging 235.8 ng (95% CI 90.7 to 380.9) from cell-negative samples, which was attributed to issues with spectrophotometry. DNA measurements using Qubit Fluorometry demonstrated a median fivefold overestimation of DNA quantity by Nanodrop Spectrophotometry. DNA integrity and PCR inhibition were factors not found to contribute significant variation. Conclusions In this study, we provide evidence demonstrating that variation in pre-PCR steps is prevalent and may detrimentally affect the patient's ability to receive critical therapy. We provide recommendations for preanalytical workflow optimisation that may reduce errors in down-stream sequencing and for next-generation sequencing library generation. PMID:25430497

  4. Primers with 5' flaps improve the efficiency and sensitivity of multiplex PCR assays for the detection of Salmonella and Escherichia coli O157:H7.

    PubMed

    Timmons, Chris; Dobhal, Shefali; Fletcher, Jacqueline; Ma, Li Maria

    2013-04-01

    Foodborne illnesses caused by Salmonella enterica and Escherichia coli O157:H7 are worldwide health concerns. Rapid, sensitive, and robust detection of these pathogens in foods and in clinical and environmental samples is essential for routine food quality testing, effective surveillance, and outbreak investigations. The aim of this study was to evaluate the effect on PCR sensitivity of adding a short, AT-rich overhanging nucleotide sequence (flap) to the 5' end of PCR primers specific for the detection of Salmonella and E. coli O157:H7. Primers targeting the invA gene of Salmonella and the rfbE gene of E. coli O157:H7 were synthesized with or without a 12-bp, AT-rich 5' flap (5'-AATAAATCATAA-3'). Singleplex PCR, multiplex PCR, and real-time PCR sensitivity assays were conducted using purified bacterial genomic DNA and crude cell lysates of bacterial cells. The effect of background flora on detection was evaluated by spiking tomato and jalapeno pepper surface washes with E. coli O157:H7 and Salmonella Saintpaul. When targeting individual pathogens, end-point PCR assays using flap-amended primers were more efficient than nonamended primers, with 20.4 and 23.5% increases in amplicon yield for Salmonella and E. coli O157:H7, respectively. In multiplex PCR assays, a 10- to 100-fold increase in detection sensitivity was observed when the primer flap sequence was incorporated. This improvement in both singleplex and multiplex PCR efficiency and sensitivity can lead to improved Salmonella and E. coli O157:H7 detection.

  5. Real-time PCR improves detection of Trichomonas vaginalis infection compared with culture using self-collected vaginal swabs.

    PubMed Central

    Caliendo, A M; Jordan, J A; Green, A M; Ingersoll, J; Diclemente, R J; Wingood, G M

    2005-01-01

    OBJECTIVE: To compare a real-time polymerase chain reaction (PCR) assay with broth culture for the detection of Trichomonas vaginalis using self-collected vaginal swabs. METHODS: Self-collected vaginal swabs were obtained from adolescent and young adult African-American women participating in HIV-1 prevention programs. T. vaginalis culture was performed using the InPouch TV System. Samples for the real-time PCR assay were collected using the BDProbeTec ET Culturette Direct Dry Swab system and tested in a laboratory-developed assay which targeted a repeated sequence of the genome. Discrepant samples that were culture negative and positive in the real-time PCR assay were tested in a confirmatory PCR which targeted a different region of the T. vaginalis genome, the18S ribosomal DNA gene. RESULTS: Of the 524 specimens tested by both culture and real-time PCR, 36 were culture positive and 54 were positive in the real-time PCR assay; 16 of the 18 discrepant specimens were also positive in the confirmatory PCR assay. Using a modified gold standard of positive by culture or positive in both PCR assays, the sensitivity of the real-time PCR assay was 100% and the specificity was 99.6%, whereas culture had a sensitivity of 69.2% and a specificity of 100%. CONCLUSIONS: The real-time PCR assay was sensitive and specific for the detection of T. vaginalis DNA from self-collected vaginal swab specimens. The ability to use the BDProbeTec dry swab system for the real-time PCR testing allowed for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and T. vaginalis from a single specimen. PMID:16126499

  6. Facing the problem of "false positives": re-assessment and improvement of a multiplex RT-PCR procedure for the diagnosis of A. flavus mycotoxin producers.

    PubMed

    Degola, F; Berni, E; Spotti, E; Ferrero, I; Restivo, F M

    2009-02-28

    The aim of our research project was to consolidate a multiplex RT-PCR protocol to detect aflatoxigenic strains of Aspergillus flavus. Several independent A. flavus strains were isolated from corn and flour samples from the North of Italy and from three European countries. Aflatoxin producing/not producing phenotype was assessed by qualitative and quantitative assays at day five of growth in aflatoxin inducing conditions. Expression of 16 genes belonging to the aflatoxin cluster was assayed by multiplex or monomeric RT-PCR. There is a good correlation between gene expression and aflatoxin production. Strains that apparently transcribed all the relevant genes but did not release aflatoxin in the medium ("false positives") were re-assessed for mycotoxin production after extended growth in inducing condition. All the "false positive" strains in actual fact were positive when aflatoxin determination was performed after 10 days of growth. These strains should then be re-classified as "slow aflatoxin accumulators". To optimise the diagnostic procedure, a quintuplex RT-PCR procedure was designed consisting of a primer set directed against four informative aflatoxin cluster genes and the beta-tubulin gene as an internal amplification control. In conclusion we have provided evidence for the robustness and reliability of our RT-PCR protocol in discriminating mycotoxin producer from non-producer strains of A. flavus. and the molecular procedure we devised is a promising tool with which to screen and control the endemic population of A. flavus colonising different areas of the World.

  7. Improvements for comparative analysis of changes in diversity of microbial communities using internal standards in PCR-DGGE.

    PubMed

    Petersen, Dorthe Groth; Dahllöf, Ingela

    2005-08-01

    The use of internal standards both during DNA extraction and PCR-DGGE procedure gives the opportunity to analyse the relative abundance of individual species back to the original sample, thereby facilitating relative comparative analysis of diversity. Internal standards were used throughout the DNA extraction and PCR-DGGE to compensate for experimental variability. Such variability causes decreased reproducibility among replicate samples as well as compromise comparisons between samples, since experimental errors cannot be differentiated from actual changes in the community abundance and structure. The use of internal standards during DNA extraction and PCR-DGGE is suitable for ecological and ecotoxicological experiments with microbial communities, where relative changes in the community abundance and structure are studied. We have developed a protocol Internal Standards in Molecular Analysis of Diversity (ISMAD) that is simple to use, inexpensive, rapid to perform and it does not require additional samples to be processed. The internal standard for DNA extraction (ExtrIS) is a fluorescent 510-basepair PCR product which is added to the samples prior to DNA extraction, recovered together with the extracted DNA from the samples and analysed with fluorescence spectrophotometry. The use of ExtrIS during isolation of sample DNA significantly reduced variation among replicate samples. The PCR internal standard (PCR(IS)) originates from the Drosophila melanogaster genome and is a 140-basepair long PCR product, which is amplified by non-competitive primers in the same PCR reaction tubes as the target DNA and analysed together with the target PCR product on the same DGGE gel. The use of PCR(IS) during PCR significantly reduced variation among replicate samples both when assessing total PCR product and when comparing bands representing species on a DGGE gel. The entire ISMAD protocol was shown to accurately describe changes in relative abundance in an environmental sample

  8. Improved Diagnostic Multimodal Biomarkers for Alzheimer's Disease and Mild Cognitive Impairment.

    PubMed

    Martínez-Torteya, Antonio; Treviño, Víctor; Tamez-Peña, José G

    2015-01-01

    The early diagnosis of Alzheimer's disease (AD) and mild cognitive impairment (MCI) is very important for treatment research and patient care purposes. Few biomarkers are currently considered in clinical settings, and their use is still optional. The objective of this work was to determine whether multimodal and nonpreviously AD associated features could improve the classification accuracy between AD, MCI, and healthy controls, which may impact future AD biomarkers. For this, Alzheimer's Disease Neuroimaging Initiative database was mined for case-control candidates. At least 652 baseline features extracted from MRI and PET analyses, biological samples, and clinical data up to February 2014 were used. A feature selection methodology that includes a genetic algorithm search coupled to a logistic regression classifier and forward and backward selection strategies was used to explore combinations of features. This generated diagnostic models with sizes ranging from 3 to 8, including well documented AD biomarkers, as well as unexplored image, biochemical, and clinical features. Accuracies of 0.85, 0.79, and 0.80 were achieved for HC-AD, HC-MCI, and MCI-AD classifications, respectively, when evaluated using a blind test set. In conclusion, a set of features provided additional and independent information to well-established AD biomarkers, aiding in the classification of MCI and AD.

  9. Luminex xMAP combined with Western blot improves HIV diagnostic sensitivity.

    PubMed

    Kong, Weiwei; Li, Yan; Cheng, Shaohui; Yan, Chen; An, Shiping; Dong, Zheng; Yan, Lina; Yuan, Yuhua

    2016-01-01

    Currently, Western blot is used to confirm the initial serodiagnosis of HIV infection by antibody detection. However, a major deficiency of the Western blot relates to a lack of sufficient sensitivity in detecting HIV antibodies. This report describes a simple, sensitive and inexpensive bead-based assay for detection of early HIV infection. A panel of 138 positive specimens including 105 blood donors and 33 MSM with known Western blot results were evaluated using Luminex xMAP at Tianjin Centers for Disease Control and Prevention (CDC). We demonstrate a superior sensitivity of Luminex xMAP compared with Western blot. Of the 87 confirmed HIV positive blood donors, Western blot only confirmed 65 cases with 74.7% (65/87) sensitivity while Luminex xMAP identified 72 cases with 82.8% (72/87) sensitivity (p<0.05). Western blot and Luminex xMAP verified 13 and 19 of 33 MSM specimens, respectively. The sensitivity was 39.4% (13/33) for Western blot and 57.6% (19/33) for Luminex xMAP (p<0.1). Luminex xMAP combined with Western blot improves the diagnostic sensitivity of HIV infection at an early stage, and reduces the chances of missed diagnosis.

  10. Using Targeted Active-Learning Exercises and Diagnostic Question Clusters to Improve Students' Understanding of Carbon Cycling in Ecosystems

    ERIC Educational Resources Information Center

    Maskiewicz, April Cordero; Griscom, Heather Peckham; Welch, Nicole Turrill

    2012-01-01

    In this study, we used targeted active-learning activities to help students improve their ways of reasoning about carbon flow in ecosystems. The results of a validated ecology conceptual inventory (diagnostic question clusters [DQCs]) provided us with information about students' understanding of and reasoning about transformation of inorganic and…

  11. Can rapid integrated polymerase chain reaction-based diagnostics for gastrointestinal pathogens improve routine hospital infection control practice? A diagnostic study.

    PubMed Central

    Pankhurst, Louise; Macfarlane-Smith, Louissa; Buchanan, James; Anson, Luke; Davies, Kerrie; O'Connor, Lily; Ashwin, Helen; Pike, Graham; Dingle, Kate E; Peto, Timothy Ea; Wordsworth, Sarah; Walker, A Sarah; Wilcox, Mark H; Crook, Derrick W

    2014-01-01

    BACKGROUND Every year approximately 5000-9000 patients are admitted to a hospital with diarrhoea, which in up to 90% of cases has a non-infectious cause. As a result, single rooms are 'blocked' by patients with non-infectious diarrhoea, while patients with infectious diarrhoea are still in open bays because of a lack of free side rooms. A rapid test for differentiating infectious from non-infectious diarrhoea could be very beneficial for patients. OBJECTIVE To evaluate MassCode multiplex polymerase chain reaction (PCR) for the simultaneous diagnosis of multiple enteropathogens directly from stool, in terms of sensitivity/specificity to detect four common important enteropathogens: Clostridium difficile, Campylobacter spp., Salmonella spp. and norovirus. DESIGN A retrospective study of fixed numbers of samples positive for C. difficile (n = 200), Campylobacter spp. (n = 200), Salmonella spp. (n = 100) and norovirus (n = 200) plus samples negative for all these pathogens (n = 300). Samples were sourced from NHS microbiology laboratories in Oxford and Leeds where initial diagnostic testing was performed according to Public Health England methodology. Researchers carrying out MassCode assays were blind to this information. A questionnaire survey, examining current practice for infection control teams and microbiology laboratories managing infectious diarrhoea, was also carried out. SETTING MassCode assays were carried out at Oxford University Hospitals NHS Trust. Further multiplex assays, carried out using Luminex, were run on the same set of samples at Leeds Teaching Hospitals NHS Trust. The questionnaire was completed by various NHS trusts. MAIN OUTCOME MEASURES Sensitivity and specificity to detect C. difficile, Campylobacter spp., Salmonella spp., and norovirus. RESULTS Nucleic acids were extracted from 948 clinical samples using an optimised protocol (200 Campylobacter spp., 199 C. difficile, 60 S. enterica, 199 norovirus and 295 negative

  12. Establishment of an improved high-efficiency thermal asymmetric interlaced PCR for identification of genomic integration sites mediated by phiC31 integrase.

    PubMed

    Zhou, Zaiwei; Ma, Haiyan; Qu, Lijuan; Xie, Fei; Ma, Qingwen; Ren, Zhaorui

    2012-03-01

    Streptomyces phage phiC31 integrase is widely used to mediate the integration of exogenous genes into host genomes for gene therapy and genomic modification, as it autonomously performs efficient, unidirectional, site-specific integration into pseudo attP sites of the host genome. Although pseudo attP sites are rarely found within exons, it is necessary to map their precise locations to avoid the risk of insertion mutagenesis. High-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR) is a technique that has been developed to recover genomic sequences that flank insertion tags. We have found, however, that this technique is poorly efficient, as it amplifies many non-specific targets and frequently does not generate sufficient product for downstream analysis. Therefore, we have modified the hiTAIL-PCR procedure and re-designed the random primers. As a result, both the amount and specificity of the reaction product were enhanced for each integration site. Restriction analysis of known sequences within the integrated vector, which co-amplified with the flanking genomic sequences, validated 90% of these bands for sequencing. In contrast, only 30% of the bands produced by previous hiTAIL-PCR could be validated. Compared with the original hiTAIL-PCR, our improved hiTAIL-PCR procedure identified phiC31 integration sites more accurately and efficiently.

  13. Nanofluidic Digital PCR and Extended Genotyping of RAS and BRAF for Improved Selection of Metastatic Colorectal Cancer Patients for Anti-EGFR Therapies.

    PubMed

    Azuara, Daniel; Santos, Cristina; Lopez-Doriga, Adriana; Grasselli, Julieta; Nadal, Marga; Sanjuan, Xavier; Marin, Fátima; Vidal, Joana; Montal, Robert; Moreno, Victor; Bellosillo, Beatriz; Argiles, Guillem; Elez, Elena; Dienstmann, Rodrigo; Montagut, Clara; Tabernero, Josep; Capellá, Gabriel; Salazar, Ramon

    2016-05-01

    The clinical significance of low-frequent RAS pathway-mutated alleles and the optimal sensitivity cutoff value in the prediction of response to anti-EGFR therapy in metastatic colorectal cancer (mCRC) patients remains controversial. We aimed to evaluate the added value of genotyping an extended RAS panel using a robust nanofluidic digital PCR (dPCR) approach. A panel of 34 hotspots, including RAS (KRAS and NRAS exons 2/3/4) and BRAF (V600E), was analyzed in tumor FFPE samples from 102 mCRC patients treated with anti-EGFR therapy. dPCR was compared with conventional quantitative PCR (qPCR). Response rates, progression-free survival (PFS), and overall survival (OS) were correlated to the mutational status and the mutated allele fraction. Tumor response evaluations were not available in 9 patients and were excluded for response rate analysis. Twenty-two percent of patients were positive for one mutation with qPCR (mutated alleles ranged from 2.1% to 66.6%). Analysis by dPCR increased the number of positive patients to 47%. Mutated alleles for patients only detected by dPCR ranged from 0.04% to 10.8%. An inverse correlation between the fraction of mutated alleles and radiologic response was observed. ROC analysis showed that a fraction of 1% or higher of any mutated alleles offered the best predictive value for all combinations of RAS and BRAF analysis. In addition, this threshold also optimized prediction both PFS and OS. We conclude that mutation testing using an extended gene panel, including RAS and BRAF with a threshold of 1% improved prediction of response to anti-EGFR therapy. Mol Cancer Ther; 15(5); 1106-12. ©2016 AACR.

  14. Using the full spectral capacity (six channels) of a real‐time PCR instrument can simplify diagnostic laboratory screening and typing protocols for pandemic H1N1 influenza

    PubMed Central

    Hopkins, Mark J.; Moorcroft, Jay F.; Correia, Jailson B; Hart, Ian J.

    2010-01-01

    Please cite this paper as: Hopkins et al. (2011) Using the full spectral capacity (six channels) of a real‐time PCR instrument can simplify diagnostic laboratory screening and typing protocols for pandemic H1N1 influenza. Influenza and Other Respiratory Viruses 5(2), 110–114. Background  Timely reporting of influenza A virus subtype affects patient management. Real‐time PCR is a rapid and sensitive method routinely used to characterise viral nucleic acid, but the full spectral capability of the instruments is not employed. Objectives  To evaluate a hexaplex real‐time PCR assay (Flu‐6plx assay) capable of detecting influenza A and B, hMPV, respiratory syncytial virus (RSV) and distinguishing 2008 ‘human’ influenza A/H1 from 2009 pandemic A/H1 subtypes. Methods  Respiratory specimens (n = 213) were tested using the Flu‐6plx assay and a further four monoplex PCRs targeting hMPV, RSV, influenza A and B. The FDA‐approved ProFlu ST test was used to validate the Flu‐6plx PCR influenza A/H1 subtyping components. Discrepant 2009 pandemic A/H1 results were further tested using the CDC swine H1 assay. Results  The Flu‐6plx assay had excellent sensitivity identifying 106/106 influenza A RNA–positive samples. The ProFlu ST test was a less sensitive subtyping test, and discrepant analysis could not confirm A/H1 status for four samples resulting in Flu‐6plx PCR specificities of 98% and 95% for human A/H1 and 2009 pandemic A/H1, respectively. Co‐infection affected the sensitivity of the Flu‐6plx PCR hMPV component whereby low‐level hMPV RNA could be masked by much higher concentrations of influenza A virus RNA. Conclusions  The Flu‐6plx assay is a sensitive and specific test for the universal detection of influenza A infection and determination of A/H1 subtype. Concomitant detection of influenza B, hMPV and RSV demonstrates the utility of hexaplex real‐time PCRs in viral diagnostics. PMID:21306574

  15. Improved detection of malaria cases in island settings of Vanuatu and Kenya by PCR that targets the Plasmodium mitochondrial cytochrome c oxidase III (cox3) gene.

    PubMed

    Isozumi, Rie; Fukui, Mayumi; Kaneko, Akira; Chan, Chim W; Kawamoto, Fumihiko; Kimura, Masatsugu

    2015-06-01

    Detection of sub-microscopic parasitemia is crucial for all malaria elimination programs. PCR-based methods have proven to be sensitive, but two rounds of amplification (nested PCR) are often needed to detect the presence of Plasmodium DNA. To simplify the detection process, we designed a nested PCR method whereby only the primary PCR is required for the detection of the four major human Plasmodium species. Primers designed for the detection of the fifth species, Plasmodium knowlesi, were not included in this study due to the absence of appropriate field samples. Compared to the standard 18S rDNA PCR method, our cytochrome c oxidase III (cox3) method detected 10-50% more cases while maintaining high sensitivities (1.00) for all four Plasmodium species in our samples from Vanuatu (n=77) and Kenya (n=76). Improvement in detection efficiency was more substantial for samples with sub-microscopic parasitemia (54%) than those with observable parasitemia (10-16%). Our method will contribute to improved malaria surveillance in low endemicity settings.

  16. Quantitation of rare circulating tumor cells by folate receptor α ligand-targeted PCR in bladder transitional cell carcinoma and its potential diagnostic significance.

    PubMed

    Qi, Fuming; Liu, Yuchen; Zhao, Rongchang; Zou, Xiangjun; Zhang, Lei; Li, Jiaqiang; Wang, Yongqiang; Li, Feiyang; Zou, Xiaowen; Xia, Ye; Wang, Xuliang; Xing, Li; Li, Cailing; Lu, Jingxiao; Tang, Junlong; Zhou, Fangjian; Liu, Chunxiao; Gui, Yaoting; Cai, Zhiming; Sun, Xiaojuan

    2014-07-01

    Numerous attempts for detection of circulating tumor cells (CTC) have been made to develop reliable assays for early diagnosis of cancers. In this study, we validated the application of folate receptor α (FRα) as the tumor marker to detect CTC through tumor-specific ligand PCR (LT-PCR) and assessed its utility for diagnosis of bladder transitional cell carcinoma (TCC). Immunohistochemistry for FRα was performed on ten bladder TCC tissues. Enzyme-linked immunosorbent assay (ELISA) for FRα was performed on both urine and serum specimens from bladder TCC patients (n = 64 and n = 20, respectively) and healthy volunteers (n = 20 and n = 23, respectively). Western blot analysis and qRT-PCR were performed to confirm the expression of FRα in bladder TCC cells. CTC values in 3-mL peripheral blood were measured in 57 bladder TCC patients, 48 healthy volunteers, and 15 subjects with benign urologic pathologies by the folate receptor α ligand-targeted PCR. We found that FRα protein was overexpressed in both bladder TCC cells and tissues. The levels of FRα mRNA were also much higher in bladder cancer cell lines 5637 and SW780 than those of leukocyte. Values of FRα were higher in both serum and urine specimens of bladder TCC patients than those of control. CTC values were also higher in 3-mL peripheral blood of bladder TCC patients than those of control (median 26.5 Cu/3 mL vs 14.0 Cu/3 mL). Area under the receiver operating characteristic (ROC) curve for bladder TCC detection was 0.819, 95 % CI (0.738-0.883). At the cutoff value of 15.43 Cu/3 mL, the sensitivity and the specificity for detecting bladder cancer are 82.14 and 61.9 %, respectively. We concluded that quantitation of CTCs through FRα ligand-PCR could be a promising method for noninvasive diagnosis of bladder TCC.

  17. Improvement of an invA-based PCR for the specific detection of Salmonella typhimurium in organs of pigs.

    PubMed

    Scholz, H C; Arnold, T; Marg, H; Rösler, U; Hensel, A

    2001-01-01

    The aim of this study was to investigate the suitability of the invA-based polymerase chain reaction (PCR) assay for the specific detection of Salmonella in organs of experimentally infected pigs and to compare these results to classical bacterial culture. While the PCR conditions specified in the "Deutsche Industrie Norm", DIN 10135 (section 35 LMBG, 1999), cutle based on the publication of Rahn et al. 1992, revealed various unspecific amplification products, modifications of the PCR conditions allowed the specific amplification of the invA fragment from inner organs. The modified PCR assay correlates exactly with cultivation results (as required by DIN Norm 6579) and enables the detection of Salmonella within 48 hours with equal sensitivity compared to routine cultivation.

  18. Improvement in the Diagnostic Evaluation of a Positive Fecal Occult Blood Test in an Integrated Health Care Organization

    PubMed Central

    Miglioretti, Diana L.; Rutter, Carolyn M.; Bradford, Susan Carol; Zauber, Ann G.; Kessler, Larry G.; Feuer, Eric J.; Grossman, David C.

    2014-01-01

    Background Screening for fecal occult blood can be effective in reducing colorectal cancer mortality only if positive tests are appropriately followed up with complete diagnostic evaluation (i.e., colonoscopy or flexible sigmoidoscopy with double contrast barium enema) and treatment. Objectives To examine whether rates of complete diagnostic evaluation following a positive fecal occult blood test (FOBT) have improved over time after the implementation of tracking systems and physician guidelines within a large integrated health care organization. Research Design From 1993 to 2005, 8513 positive FOBTs were identified on 8291 enrollees aged 50–79 of a large health care system. Automated records were used to identify repeat FOBTs, colonoscopy, flexible sigmoidoscopy, and double-contrast barium enema within one year after the positive FOBT. National rates of complete diagnostic evaluation were estimated from the 2005 National Health Interview Survey. Results In this integrated health care organization, the percentage of positive FOBTs followed by complete diagnostic evaluation within one year increased from 57%–64% in 1993–1996 to 82%–86% from 2000–2005. Use of repeat FOBT following a positive FOBT decreased from 28–31% in 1993–1996 to 6–11% in 2000–2005. Based on the National Health Interview Survey, only 52% of positive FOBTs from 2000–2005 were followed by complete diagnostic evaluation nationally. Conclusions Adherence to recommendations for complete diagnostic evaluation following a positive FOBT has greatly improved over time in an integrated group medical practice. Through the use of tracking systems and screening guidelines, it may be possible to reach levels of follow-up that are comparable to those observed in randomized trials. PMID:18725839

  19. Diagnostic moléculaire d'helicobacter pylori par PCR chez les patients en consultation gastroentérologique au Centre Médical Saint Camille de Ouagadougou

    PubMed Central

    Werme, Karidia; Bisseye, Cyrille; Ouedraogo, Issiaka; Yonli, Albert Théophane; Ouermi, Djénèba; Djigma, Florencia; Moret, Rémy; Gnoula, Charlemagne; Nikiema, Jean-Baptiste; Simpore, Jacques

    2015-01-01

    Introduction L'infection par Helicobacter pylori constitue un problème de santé publique notamment dans les pays en développement. Elle entraine une gastrite pouvant évoluer vers des formes sévères d'ulcération et de transformation maligne. La présenté étude avait pour objectif de diagnostiquer H. pylori par des techniques sérologique et moléculaire au Burkina Faso. Méthodes L’étude prospective a été conduite de mars à juin 2012 sur 70 patients venus en consultation dans le service de gastroentérologie au Centre Médical Saint Camille. Le diagnostic de H. pylori a été réalisé par le test ELISA Immunocomb (ORGENICS Ltd, Yavne, Israël) et la PCR sur des biopsies gastriques prélevées sur les patients. Résultats Les pathologies gastroduodénales étaient plus fréquentes chez les patients de plus de 45 ans. Les prévalences de H. pylori étaient respectivement de 88,57% et de 91,43% par sérologie Immunocomb et par PCR. La différence entre les deux techniques n’était pas significative (P = 0,573). La performance de la PCR a été comparée à celle de la technique Immunocomb. Les résultats montrent une sensibilité et une spécificité de 92,2% et 50,0% pour la technique Immunocomb. Conclusion Le diagnostic de H. pylori par PCR est plus spécifique que le test sérologique Immunocomb et devrait être introduit dans le diagnostic de routine de cette bactérie pathogène au Burkina Faso. PMID:26327960

  20. Examining heterogeneity in the diagnostic accuracy of culture and PCR for Salmonella spp. in swine: a systematic review/meta-regression approach.

    PubMed

    Wilkins, W; Rajić, A; Parker, S; Waddell, L; Sanchez, J; Sargeant, J; Waldner, C

    2010-11-01

    The accuracy of bacterial culture and PCR for Salmonella in swine was examined through systematic review of existing primary research in this field. A replicable search was conducted in 10 electronic databases. All steps of the review were conducted by two reviewers: to identify relevant publications, to assess their methodological soundness and reporting, and to extract raw data or reported test accuracy estimates. Meta-analyses and meta-regression were performed: to evaluate pooled estimates of test sensitivity (Se) and specificity (Sp), to identify variables explaining the variation in reported test estimates, and to evaluate the association between these variables and reported test Se and Sp. Twenty-nine studies were included in the review. Unique test evaluations reported in these 29 studies were categorized according to the type of test comparison: culture versus culture (n = 134 test evaluations) and PCR versus culture (n = 21). We identified significant heterogeneity among evaluations for each test category. For culture, more heterogeneity was caused by differences in individual test protocols (52%) than overall differences between studies (16%). Enrichment temperature, study population, agar and enrichment type were significantly associated with variation in culture Se. Furthermore, interaction between enrichment temperature and enrichment type was detected. For PCR, most of the heterogeneity was caused by overall differences between studies (65-70%); sample type and study size were associated with variation in reported PCR Se and Sp. The overall methodological soundness and/or reporting of primary studies included in this review were poor, with variable use of reference standards, and consistent lack of the use or reporting of blinding, randomization and subject (sample) selection criteria. Consequently, the food safety and veterinary public health research community should formally consider ways for standardizing the conduct and reporting of this type of

  1. Development of a rapid diagnostic method for identification of Staphylococcus aureus and antimicrobial resistance in positive blood culture bottles using a PCR-DNA-chromatography method.

    PubMed

    Ohshiro, Takeya; Miyagi, Chihiro; Tamaki, Yoshikazu; Mizuno, Takuya; Ezaki, Takayuki

    2016-06-01

    Blood culturing and the rapid reporting of results are essential for infectious disease clinics to obtain bacterial information that can affect patient prognosis. When gram-positive coccoid cells are observed in blood culture bottles, it is important to determine whether the strain is Staphylococcus aureus and whether the strain has resistance genes, such as mecA and blaZ, for proper antibiotic selection. Previous work led to the development of a PCR method that is useful for rapid identification of bacterial species and antimicrobial susceptibility. However, that method has not yet been adopted in community hospitals due to the high cost and methodological complexity. We report here the development of a quick PCR and DNA-chromatography test, based on single-tag hybridization chromatography, that permits detection of S. aureus and the mecA and blaZ genes; results can be obtained within 1 h for positive blood culture bottles. We evaluated this method using 42 clinical isolates. Detection of S. aureus and the resistance genes by the PCR-DNA-chromatography method was compared with that obtained via the conventional identification method and actual antimicrobial susceptibility testing. Our method had a sensitivity of 97.0% and a specificity of 100% for the identification of the bacterial species. For the detection of the mecA gene of S. aureus, the sensitivity was 100% and the specificity was 95.2%. For the detection of the blaZ gene of S. aureus, the sensitivity was 100% and the specificity was 88.9%. The speed and simplicity of this PCR-DNA-chromatography method suggest that our method will facilitate rapid diagnoses. PMID:27056092

  2. Treatment of PCR products with exonuclease I and heat-labile alkaline phosphatase improves the visibility of combined bisulfite restriction analysis

    SciTech Connect

    Watanabe, Kousuke; Emoto, Noriko; Sunohara, Mitsuhiro; Kawakami, Masanori; Kage, Hidenori; Nagase, Takahide; Ohishi, Nobuya; Takai, Daiya

    2010-08-27

    Research highlights: {yields} Incubating PCR products at a high temperature causes smears in gel electrophoresis. {yields} Smears interfere with the interpretation of methylation analysis using COBRA. {yields} Treatment with exonuclease I and heat-labile alkaline phosphatase eliminates smears. {yields} The elimination of smears improves the visibility of COBRA. -- Abstract: DNA methylation plays a vital role in the regulation of gene expression. Abnormal promoter hypermethylation is an important mechanism of inactivating tumor suppressor genes in human cancers. Combined bisulfite restriction analysis (COBRA) is a widely used method for identifying the DNA methylation of specific CpG sites. Here, we report that exonuclease I and heat-labile alkaline phosphatase can be used for PCR purification for COBRA, improving the visibility of gel electrophoresis after restriction digestion. This improvement is observed when restriction digestion is performed at a high temperature, such as 60 {sup o}C or 65 {sup o}C, with BstUI and TaqI, respectively. This simple method can be applied instead of DNA purification using spin columns or phenol/chloroform extraction. It can also be applied to other situations when PCR products are digested by thermophile-derived restriction enzymes, such as PCR restriction fragment length polymorphism (RFLP) analysis.

  3. A PCR-based diagnostic assay for the detection of Roseovarius crassostreae in Crassostrea virginica affected by juvenile oyster disease (JOD)

    USGS Publications Warehouse

    Maloy, A.P.; Barber, B.J.; Boettcher, K.J.

    2005-01-01

    We have developed a PCR-assay for the diagnosis of juvenile oyster disease (JOD) based on the detection of Roseovarius crassostreae directly from affected oysters. Species-specific primers are used to amplify the 16S-23S rDNA internal transcribed spacer (ITS) of R. crassostreae, and confirmation of product identity is accomplished by restriction enzyme analysis. No false positives were obtained with either closely related bacterial species or from other DNAs present in oyster samples. The assay has the potential to detect as few as 10 cells of R. crassostreae per oyster when samples are taken from the inner valve surfaces of the animal. Inclusion of material from soft body surfaces is not necessary, and may reduce sensitivity approximately 10-fold. In a JOD-affected population, a positive PCR result was obtained from all oysters from which these bacteria were subsequently cultured. The assay also detected the presence of R. crassostreae in 2 oysters from which no R. crassostreae isolates were recovered. No R. crassostreae was detected by either PCR or bacteriology in oysters from a population that was not exhibiting JOD-signs. This assay is expected to advance regional disease management efforts and provide valuable insights into the disease process and epizootiology of JOD. ?? Inter-Research 2005.

  4. Audit and improve! Evaluation of a real-time probe-based PCR assay with internal control for the direct detection of Mycobacterium tuberculosis complex.

    PubMed

    Inoue, M; Tang, W Y; Wee, S Y; Barkham, T

    2011-01-01

    We retrospectively audited the performance of the commercial kit in use in our laboratory for the detection of Mycobacterium tuberculosis complex (MTBC) and found the sensitivity to be unacceptably low at 69% (52/75). We developed an in-house end-point polymerase chain reaction (PCR) detecting IS6110, an IS-like element of MTBC, and achieved a sensitivity of 90% (66/73) with the same DNA samples, re-emphasising the poor performance of the commercial kit. In order to avoid specificity issues surrounding gel-based PCR, we developed a probe-based real-time PCR assay with an internal control and achieved a sensitivity of 84%, specificity of 97% and diagnostic odds ratio (DOR) of 207. The evaluation was performed on clinically requested samples, so we expect the performance of the assay in real life to match the data from this evaluation. Centers for Disease Control and Prevention (CDC) guidelines recommending nucleic acid tests for the investigation of possible cases of tuberculosis are expected to promote the use of molecular assays. It is important that clinical laboratories do not assume that assays, in-house or commercial, will perform well or that they will continue to perform well. Audit at regular intervals is necessary to maintain confidence and to demonstrate that the assay works to specification in the real test population.

  5. 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

  6. Accurate Point-of-Care Detection of Ruptured Fetal Membranes: Improved Diagnostic Performance Characteristics with a Monoclonal/Polyclonal Immunoassay

    PubMed Central

    Rogers, Linda C.; Scott, Laurie; Block, Jon E.

    2016-01-01

    OBJECTIVE Accurate and timely diagnosis of rupture of membranes (ROM) is imperative to allow for gestational age-specific interventions. This study compared the diagnostic performance characteristics between two methods used for the detection of ROM as measured in the same patient. METHODS Vaginal secretions were evaluated using the conventional fern test as well as a point-of-care monoclonal/polyclonal immunoassay test (ROM Plus®) in 75 pregnant patients who presented to labor and delivery with complaints of leaking amniotic fluid. Both tests were compared to analytical confirmation of ROM using three external laboratory tests. Diagnostic performance characteristics were calculated including sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy. RESULTS Diagnostic performance characteristics uniformly favored ROM detection using the immunoassay test compared to the fern test: sensitivity (100% vs. 77.8%), specificity (94.8% vs. 79.3%), PPV (75% vs. 36.8%), NPV (100% vs. 95.8%), and accuracy (95.5% vs. 79.1%). CONCLUSIONS The point-of-care immunoassay test provides improved diagnostic accuracy for the detection of ROM compared to fern testing. It has the potential of improving patient management decisions, thereby minimizing serious complications and perinatal morbidity. PMID:27199579

  7. Improvement of diagnostic agreement among pathologists in resolving an "atypical glands suspicious for cancer" diagnosis in prostate biopsies using a novel "Disease-Focused Diagnostic Review" quality improvement process.

    PubMed

    Shah, Rajal B; Leandro, Gioacchino; Romerocaces, Gloria; Bentley, James; Yoon, Jiyoon; Mendrinos, Savvas; Tadros, Yousef; Tian, Wei; Lash, Richard

    2016-10-01

    One of the major goals of an anatomic pathology laboratory quality program is to minimize unwarranted diagnostic variability and equivocal reporting. This study evaluated the utility of Miraca Life Sciences' "Disease-Focused Diagnostic Review" (DFDR) quality program in improving interobserver diagnostic reproducibility associated with classification of "atypical glands suspicious for adenocarcinoma" (ATYP) in prostate biopsies. Seventy-one selected prostate biopsies with a focus of ATYP were reviewed by 8 pathologists. Participants were blinded to the original diagnosis and were first asked to classify the ATYP as benign, atypical, or limited adenocarcinoma. DFDR comprised a "theoretical consensus" (in which pathologists first reached consensus on the morphological features they considered relevant for the diagnosis of limited prostatic adenocarcinoma), a didactic review including relevant literature, and "practical consensus" (pathologists performed joint microscopic sessions, reconciling each other's observations and positions evaluating a separate unique slide set). Participants were finally asked to reclassify the original 71 ATYP cases based on knowledge gleaned from DFDR. Pre- and post-DFDR interobserver reproducibility of overall diagnostic agreement was assessed. Interobserver reproducibility measured by Fleiss κ values of pre- and post-DFDR was 0.36 and 0.59, respectively (P=.006). Post-DFDR, there were significant improvement for "100% concordance" (P=.011) and reduction for "no consensus" (P=.0004) categories. Despite a lower pre-DFDR reproducibility for non-uropathology fellowship-trained (n=3, κ=0.38) versus uropathology fellowship-trained (n=5, κ=0.43) pathologists, both groups achieved similarly high post-DFDR κ levels (κ=0.58 and 0.56, respectively). DFDR represents an effective tool to formally achieve diagnostic consensus and reduce variability associated with critical diagnoses in an anatomic pathology practice.

  8. Microradiography of Microcalcifications in Breast Specimen: A New Histological Correlation Procedure and the Effect of Improved Resolution on Diagnostic Validity

    PubMed Central

    Langen, H.-J.; Koehler, S.; Bielmeier, J.; Jocher, R.; Kranzfelder, D.; Jagusch, N.; Treutlein, G.; Wetzler, Th.; Müller, J.; Ott, G.

    2012-01-01

    Introduction. Does high-resolution visualization of microcalcifications improve diagnostic reliability? Method. X-rays were taken of mamma specimens with microcalcifications in 32 patients (10 malignant; 22 benign) using conventional radiography (12 Lp/mm) and high-resolution radiography (2000 Lp/mm). Histological sections were subsequently prepared and correlated to the microradiographic image and every calcification was assigned an exact malignant or benign histological diagnosis. Five radiologists classified single groups of calcifications in both methods according to the BIRADS classification system. Results. Using microradiography microcalcifications can be shown in high resolution at the cell level including histological correlation. In some cases, the diagnostic validity was improved by the high resolution in microradiography. In other cases, the high resolution resulted in more visible calcifications, thus giving benign calcifications a malignant appearance. In the BIRADS 2 and 3 group, the probability of malignancy was 28.6% in the conventional radiography evaluation and 37.8% in the microradiography evaluation. In the BIRADS 4 and 5 group, the probability of malignancy was 34.2% in the conventional radiography evaluation and 24.4% in the microradiography evaluation. The differences were not significant. Summary. Overall, the improved resolution in microradiography did not show an improvement in diagnostic accuracy compared to conventional radiography. PMID:23097699

  9. Diagnostic accuracy of quantitative PCR (Xpert MTB/RIF) for tuberculous pericarditis compared to adenosine deaminase and unstimulated interferon-γ in a high burden setting: a prospective study

    PubMed Central

    2014-01-01

    Background Tuberculous pericarditis (TBP) is associated with high morbidity and mortality, and is an important treatable cause of heart failure in developing countries. Tuberculous aetiology of pericarditis is difficult to diagnose promptly. The utility of the new quantitative PCR test (Xpert MTB/RIF) for the diagnosis of TBP is unknown. This study sought to evaluate the diagnostic accuracy of the Xpert MTB/RIF test compared to pericardial adenosine deaminase (ADA) and unstimulated interferon-gamma (uIFNγ) in suspected TBP. Methods From October 2009 through September 2012, 151 consecutive patients with suspected TBP were enrolled at a single centre in Cape Town, South Africa. Mycobacterium tuberculosis culture and/or pericardial histology served as the reference standard for definite TBP. Receiver-operating-characteristic curve analysis was used for selection of ADA and uIFNγ cut-points. Results Of the participants, 49% (74/151) were classified as definite TBP, 33% (50/151) as probable TBP and 18% (27/151) as non TBP. A total of 105 (74%) participants were human immunodeficiency virus (HIV) positive. Xpert-MTB/RIF had a sensitivity and specificity (95% confidence interval (CI)) of 63.8% (52.4% to 75.1%) and 100% (85.6% to 100%), respectively. Concentration of pericardial fluid by centrifugation and using standard sample processing did not improve Xpert MTB/RIF accuracy. ADA (≥35 IU/L) and uIFNγ (≥44 pg/ml) both had a sensitivity of 95.7% (88.1% to 98.5%) and a negative likelihood ratio of 0.05 (0.02 to 0.10). However, the specificity and positive likelihood ratio of uIFNγ was higher than ADA (96.3% (81.7% to 99.3%) and 25.8 (3.6 to 183.4) versus 84% (65.4% to 93.6%) and 6.0 (3.7 to 9.8); P = 0.03) at an estimated background prevalence of TB of 30%. The sensitivity and negative predictive value of both uIFNγ and ADA were higher than Xpert-MT/RIF (P < 0.001). Conclusions uIFNγ offers superior accuracy for the diagnosis of microbiologically

  10. Soft x-ray power diagnostic improvements at the Omega Laser Facility

    SciTech Connect

    Sorce, C.; Schein, J.; Weber, F.; Widmann, K.; Campbell, K.; Dewald, E.; Turner, R.; Landen, O.; Jacoby, K.; Torres, P.; Pellinen, D.

    2006-10-15

    Soft x-ray power diagnostics are essential for evaluating high temperature laser plasma experiments. The Dante soft x-ray spectrometer, a core diagnostic for radiation flux and temperature measurements of Hohlraums, installed on the Omega Laser Facility at the Laboratory for Laser Energetics has recently undergone a series of upgrades. Work performed at Brookhaven National Laboratory for the development of the National Ignition Facility (NIF) Dante spectrometer enables the Omega Dante to offer a total of 18 absolutely calibrated channels in the energy range from 50 eV to 20 keV. This feature provides Dante with the capability to measure higher, NIF relevant, radiation temperatures with increased accuracy including a differentiation of higher energy radiation such as the Au M and L bands. Diagnostic monitoring using experimental data from directly driven Au spherical shots is discussed.

  11. Explanation of the mechanism by which CAD assistance improves diagnostic performance when reading CT images

    NASA Astrophysics Data System (ADS)

    Matsumoto, Toru; Wada, Shinichi; Yamamoto, Shinji; Murao, Kohei; Furukawa, Akira; Endo, Masahiro; Matsumoto, Mitsuomi; Sone, Shusuke

    2006-03-01

    The purpose of our research is to make clear the mechanism that a reader (physician or radiological technologist) effectively identify abnormal findings in CT images of lung cancer screening by using with CAD system. A method guessing the 2X2 decision matrix between reader / CAD and reader / reader with CAD was investigated. We suppose the next scene to be it. At first, a reader judges whether abnormal findings per one patient per one CT image are present (1) or absent (0) without CAD results. The second, a reader judges whether abnormal findings are present (1) or absent (0) with CAD results. We expresses the correlation between diagnoses by a reader and CAD system for abnormal cases and for normal cases by following formula using phi correlation coefficient:φ=(cd-ab)/√(a+c)(b+d)(b+c)(a+d). a,b,c,d: 2X2 decision matrix parameters. If TPR1=(a+c)/n, TPR2=(b+c)/n and TPR3=(a+b+c)/n for abnormal cases, TPR3=TPR1+TPR2 - TPR1×TRR2 - φ√TPR1(1-TPR1)TPR2(1-TPR2). Therefore, a=n (TPR3 - TPR1), b=n (TPR3 - TPR2), c=n (TPR1 + TPR2 -TPR3), d=n (1.0 - TPR3). This theory was applied for the experimental data. The 41 students interpreted the same CT images [no training]. A second interpretation was performed after they had been instructed on how to interpret CT images [training], and third was assisted by a virtual CAD [training + CAD]. The mechanism that makes up for a good point of a reader and a CAD with CAD in interpreting CT images was theoretically and experimentally investigated. We concluded that a method guessing the decision matrix (2X2) between a reader and a CAD decided the "presence" or "absence" of abnormal findings explain the improvement mechanism of diagnostic performance with CAD system.

  12. Bartonellae in domestic and stray cats from Israel: comparison of bacterial cultures and high-resolution melt real-time PCR as diagnostic methods.

    PubMed

    Gutiérrez, Ricardo; Morick, Danny; Gross, Ifat; Winkler, Ronen; Abdeen, Ziad; Harrus, Shimon

    2013-12-01

    To determine the occurrence of feline bartonellosis in Israel, blood samples were collected from 179 stray and 155 domestic cats from 18 cities or villages in central and northcentral Israel. Samples were screened for Bartonella infection by culture isolation and molecular detection using high-resolution melt (HRM) real-time PCR assay targeting the 16S-23S rRNA internal transcribed spacer (ITS). All positive samples were confirmed by two additional HRM real-time PCR assays targeting two fragments of the β-subunit of RNA polymerase (rpoB) and the 16S rRNA genes. The prevalence of Bartonella spp. infection in the general tested population was 25.1% (84/334). A higher prevalence was detected in the stray (30.7%; 55/179) than the domestic cats (18.7%; 29/155). Bartonella henselae, Bartonella clarridgeiae, and Bartonella koehlerae were highly prevalent in both cat populations, however their distribution among the two populations varied significantly (p=0.016). B. clarridgeiae and B. koehlerae were found to be more prevalent in stray than domestic cats, whereas B. henselae was evenly distributed. Co-infection with two or more different Bartonella spp. was determined in 2.1% (7) of the cats. The ITS HRM real-time PCR assay used in this study was shown to have a greater screening power than bacterial isolation, detecting 94.0% (79/84) compared to 35.7% (30/84), respectively, of all positive samples. The high prevalence of these zoonotic Bartonella species, coupled with the overpopulation of stray cats, and increased numbers of domestic cats in the major urban centers in Israel represent a significant threat for the public health in this country.

  13. Quantitative PCR for detection of Shigella improves ascertainment of Shigella burden in children with moderate-to-severe diarrhea in low-income countries.

    PubMed

    Lindsay, Brianna; Ochieng, John B; Ikumapayi, Usman N; Toure, Aliou; Ahmed, Dilruba; Li, Shan; Panchalingam, Sandra; Levine, Myron M; Kotloff, Karen; Rasko, David A; Morris, Carolyn R; Juma, Jane; Fields, Barry S; Dione, Michel; Malle, Dramane; Becker, Stephen M; Houpt, Eric R; Nataro, James P; Sommerfelt, Halvor; Pop, Mihai; Oundo, Joe; Antonio, Martin; Hossain, Anowar; Tamboura, Boubou; Stine, O Colin

    2013-06-01

    Estimates of the prevalence of Shigella spp. are limited by the suboptimal sensitivity of current diagnostic and surveillance methods. We used a quantitative PCR (qPCR) assay to detect Shigella in the stool samples of 3,533 children aged <59 months from the Gambia, Mali, Kenya, and Bangladesh, with or without moderate-to-severe diarrhea (MSD). We compared the results from conventional culture to those from qPCR for the Shigella ipaH gene. Using MSD as the reference standard, we determined the optimal cutpoint to be 2.9 × 10(4) ipaH copies per 100 ng of stool DNA for set 1 (n = 877). One hundred fifty-eight (18%) specimens yielded >2.9 × 10(4) ipaH copies. Ninety (10%) specimens were positive by traditional culture for Shigella. Individuals with ≥ 2.9 × 10(4) ipaH copies have 5.6-times-higher odds of having diarrhea than those with <2.9 × 10(4) ipaH copies (95% confidence interval, 3.7 to 8.5; P < 0.0001). Nearly identical results were found using an independent set of samples. qPCR detected 155 additional MSD cases with high copy numbers of ipaH, a 90% increase from the 172 cases detected by culture in both samples. Among a subset (n = 2,874) comprising MSD cases and their age-, gender-, and location-matched controls, the fraction of MSD cases that were attributable to Shigella infection increased from 9.6% (n = 129) for culture to 17.6% (n = 262) for qPCR when employing our cutpoint. We suggest that qPCR with a cutpoint of approximately 1.4 × 10(4) ipaH copies be the new reference standard for the detection and diagnosis of shigellosis in children in low-income countries. The acceptance of this new standard would substantially increase the fraction of MSD cases that are attributable to Shigella. PMID:23536399

  14. Lab-on-a-chip enabled HLA diagnostic: combined sample preparation and real time PCR for HLA-B57 diagnosis

    NASA Astrophysics Data System (ADS)

    Gärtner, Claudia; Becker, Holger; Hlawatsch, Nadine; Klemm, Richard; Moche, Christian; Schattschneider, Sebastian; Frank, Rainer; Willems, Andreas

    2015-05-01

    The diverse human HLA (human leukocyte antigen) system is responsible for antigen presentation and recognition. It is essential for the immune system to maintain a stable defense line, but also is also involved in autoimmunity as well as metabolic disease. HLA-haplotype (HLA-B27), for instance, is associated with inflammatory diseases such as Bechterew's disease. The administration of the HIV drug Abacavir in combination with another HLA-haplotype (HLAB57) is associated with severe hypersensitivity reactions. Accordingly, the HLA status has to be monitored for diagnosis or prior to start of therapy. Along this line, a miniaturized microfluidic platform has been developed allowing performing the complete analytical process from "sample-in" to "answer-out" in a point-of-care environment. The main steps of the analytical cascade inside the integrated system are blood cell lysis and DNA isolation, DNA purification, real-time PCR and quantitative monitoring of the rise of a fluorescent signal appearing during the PCR based sequence amplification. All bio-analytical steps were intended to be performed inside one chip and will be actuated, controlled and monitored by a matching device. This report will show that all required processes are established and tested and all device components work well and interact with the functional modules on the chips in a harmonized fashion.

  15. Astronomy Diagnostic Test Results Reflect Course Goals and Show Room for Improvement

    ERIC Educational Resources Information Center

    LoPresto, Michael C.

    2007-01-01

    The results of administering the Astronomy Diagnostic Test (ADT) to introductory astronomy students at Henry Ford Community College over three years have shown gains comparable with national averages. Results have also accurately corresponded to course goals, showing greater gains in topics covered in more detail, and lower gains in topics covered…

  16. EPSE Project 1: Using Diagnostic Assessment To Improve Science Teaching and Learning.

    ERIC Educational Resources Information Center

    Millar, Robin; Hames, Vicky

    2002-01-01

    Reports on Project 1 from the Evidence-based Practice in Science Education (EPSE) Research Network. In this project, a group of teachers develop banks of diagnostic questions in four science topic areas: electric circuits, force and motion, matter and chemical change, and biochemical life processes. (DDR)

  17. Combining watershed attributes with culture- and PCR-based methods for improved characterization and management of fecal pollution

    EPA Science Inventory

    Culture- and PCR-based methods for characterization of fecal pollution were evaluated in relation to physiographic, biotic, and chemical indicators of stream condition. Stream water samples (n = 235) were collected monthly over a two year period from ten channels draining subwat...

  18. The Use of Collagenase to Improve the Detection of Plant Viruses in Vector Nematodes by RT/PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tomato ringspot virus (ToRSV) Tobacco ringspot virus (TRSV) and Tobacco rattle virus (TRV) are transmitted to healthy plants by viruliferous nematodes in the soil. We developed a method for extraction of genomic viral RNA from virus particles carried within nematodes and a sensitive nested RT/PCR ...

  19. Improved HF183 quantitative real-time PCR assay for characterization of human fecal pollution in ambient surface water samples

    EPA Science Inventory

    Real-time quantitative PCR assays that target the human-associated HF183 bacterial cluster are considered to be some of the top performing methods for the characterization of human fecal pollution in ambient surface waters. In response, the United States Environmental Protectio...

  20. Improving molecular detection of fungal DNA in formalin-fixed paraffin-embedded tissues: comparison of five tissue DNA extraction methods using panfungal PCR.

    PubMed

    Muñoz-Cadavid, C; Rudd, S; Zaki, S R; Patel, M; Moser, S A; Brandt, M E; Gómez, B L

    2010-06-01

    DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues is difficult and requires special protocols in order to extract small amounts of DNA suitable for amplification. Most described methods report an amplification success rate between 60 and 80%; therefore, there is a need to improve molecular detection and identification of fungi in FFPE tissue. Eighty-one archived FFPE tissues with a positive Gomori methenamine silver (GMS) stain were evaluated using five different commercial DNA extraction kits with some modifications. Three different panfungal PCR assays were used to detect fungal DNA, and two housekeeping genes were used to assess the presence of amplifiable DNA and to detect PCR inhibitors. The sensitivities of the five extraction protocols were compared, and the quality of DNA detection (calculated for each kit as the number of housekeeping gene PCR-positive samples divided by the total number of samples) was 60 to 91% among the five protocols. The efficiencies of the three different panfungals used (calculated as the number of panfungal-PCR-positive samples divided by the number of housekeeping gene PCR-positive samples) were 58 to 93%. The panfungal PCR using internal transcribed spacer 3 (ITS3) and ITS4 primers yielded a product in most FFPE tissues. Two of the five DNA extraction kits (from TaKaRa and Qiagen) showed similar and promising results. However, one method (TaKaRa) could extract fungal DNA from 69 of the 74 FFPE tissues from which a housekeeping gene could be amplified and was also cost-effective, with a nonlaborious protocol. Factors such as sensitivity, cost, and labor will help guide the selection of the most appropriate method for the needs of each laboratory.

  1. Heating, Ventilating, and Air-Conditioning: Recent Advances in Diagnostics and Controls to Improve Air-Handling System Performance

    NASA Astrophysics Data System (ADS)

    Wray, C. P.; Sherman, M. H.; Walker, I. S.; Dickerhoff, D. J.; Federspiel, C. C.

    2008-09-01

    The performance of air-handling systems in buildings needs to be improved. Many of the deficiencies result from myths and lore and a lack of understanding about the non-linear physical principles embedded in the associated technologies. By incorporating these principles, a few important efforts related to diagnostics and controls have already begun to solve some of the problems. This paper illustrates three novel solutions: one rapidly assesses duct leakage, the second configures ad hoc duct-static-pressure reset strategies, and the third identifies useful intermittent ventilation strategies. By highlighting these efforts, this paper seeks to stimulate new research and technology developments that could further improve air-handling systems.

  2. Comparative in silico analysis of PCR primers suited for diagnostics and cloning of ammonia monooxygenase genes from ammonia-oxidizing bacteria.

    PubMed

    Junier, Pilar; Kim, Ok-Sun; Molina, Verónica; Limburg, Petra; Junier, Thomas; Imhoff, Johannes F; Witzel, Karl-Paul

    2008-04-01

    Over recent years, several PCR primers have been described to amplify genes encoding the structural subunits of ammonia monooxygenase (AMO) from ammonia-oxidizing bacteria (AOB). Most of them target amoA, while amoB and amoC have been neglected so far. This study compared the nucleotide sequence of 33 primers that have been used to amplify different regions of the amoCAB operon with alignments of all available sequences in public databases. The advantages and disadvantages of these primers are discussed based on the original description and the spectrum of matching sequences obtained. Additionally, new primers to amplify the almost complete amoCAB operon of AOB belonging to Betaproteobacteria (betaproteobacterial AOB), a primer pair for DGGE analysis of amoA and specific primers for gammaproteobacterial AOB, are also described. The specificity of these new primers was also evaluated using the databases of the sequences created during this study. PMID:18248438

  3. Cryphonectria naterciae: a new species in the Cryphonectria-Endothia complex and diagnostic molecular markers based on microsatellite-primed PCR.

    PubMed

    Bragança, Helena; Rigling, Daniel; Diogo, Eugénio; Capelo, Jorge; Phillips, Alan; Tenreiro, Rogério

    2011-09-01

    In a recent study intended to assess the distribution of Cryphonectria parasitica in Portugal, 22 morphologically atypical orange isolates were collected in the Midwestern regions. Eleven isolates were recovered from Castanea sativa, in areas severely affected by chestnut blight and eleven isolates from Quercus suber in areas with cork oak decline. These isolates were compared with known C. parasitica and Cryphonectria radicalis isolates using an integrated approach comprising morphological and molecular methods. Morphologically the atypical isolates were more similar to C. radicalis than to C. parasitica. Phylogenetic analyses based on internal transcribed spacer (ITS) and β-tubulin sequence data grouped the isolates in a well-supported clade separate from C. radicalis. Combining morphological, cultural, and molecular data Cryphonectria naterciae is newly described in the Cryphonectria-Endothia complex. Microsatellite-primed PCR fingerprinting with (GACA)(4) primer discriminated between C. naterciae, C. radicalis, and C. parasitica.

  4. Development of an improved PCR-ICT hybrid assay for direct detection of Legionellae and Legionella pneumophila from cooling tower water specimens.

    PubMed

    Horng, Yu-Tze; Soo, Po-Chi; Shen, Bin-Jon; Hung, Yu-Li; Lo, Kai-Yin; Su, Hsun-Pi; Wei, Jun-Rong; Hsieh, Shang-Chen; Hsueh, Po-Ren; Lai, Hsin-Chih

    2006-06-01

    A novelly improved polymerase chian reaction and immunochromatography test (PCR-ICT) hybrid assay comprising traditional multiplex-nested PCR and ICT, (a lateral-flow device) was developed for direct detection of Legionella bacteria from environmental cooling tower samples. The partial 16S rDNA (specific for Legionella spp.) and dnaJ (specific for Legionella pneumophila) genes from Legionella chromosome were first specifically amplified by multiplex-nested PCR, respectively, followed by detection using ICT strip. Reading of results was based on presence or absence of the two test lines on the strips. Presence of test line 1 indicated existence of Legionella spp. specific 16S rDNA and identified Legionella spp. Presence of test line 2 further indicated existence of dnaJ and thus specifically identified L. pneumophila. In contrast, for non-Legionellae bacteria no test line formation was observed. Results of direct detection of Legionella bacteria and L. pneumophila from water tower specimens by this assay showed 100% sensitivity, and 96.6% and 100% specificity, respectively compared with traditional culture, biochemical and serological identification methods. The PCR-ICT hybrid assay does not require sophisticated equipment and was proved to be practically useful in rapid and direct Legionellae detection from environmental water samples. PMID:16713613

  5. Development of the Zebra load region for increased capability plasma diagnostics and improved Leopard laser access

    NASA Astrophysics Data System (ADS)

    Astanovitskiy, Alexey; Presura, R.; Ivanov, V. V.; Haboub, A.; Plachaty, C.; Kindel, J. M.

    2008-11-01

    A new geometry for the load area in the Zebra (1MA pulse generator) is developed. It will form the basis for future experiments requiring Leopard (1057nm, 50TW laser) to Zebra coupling and give extended capability to z-pinch diagnostics. This required the development of a new current return, which allows laser access and installation of the OD 4'' parabolic mirror for the x-ray radiography, isochoric heating and magnetized plasma experiments, and accommodates wire-array z-pinch loads, to which the laser may then be coupled. In addition, this configuration allows diagnostics access close to the plasma, leading to a significant increase of the spatial resolution for imaging of z-pinches, as well as the photon flux in imaging and spectroscopy of laser produced plasmas. These diagnostics will allow coupling of the Leopard beam for x-ray laser probing of the pinch plasma and we will test point-projection x-ray backlighting of the pinch plasma.

  6. A Diagnostic System for Improving Biomass Quality Based on a Sensor Network

    PubMed Central

    Bochtis, Dionysis D.; Sørensen, Claus G.; Green, Ole; Bartzanas, Thomas

    2011-01-01

    Losses during storage of biomass are the main parameter that defines the profitability of using preserved biomass as feed for animal husbandry. In order to minimize storage losses, potential changes in specific physicochemical properties must be identified to subsequently act as indicators of silage decomposition and form the basis for preventive measures. This study presents a framework for a diagnostic system capable of detecting potential changes in specific physicochemical properties, i.e., temperature and the oxygen content, during the biomass storage process. The diagnostic system comprises a monitoring tool based on a wireless sensors network and a prediction tool based on a validated computation fluid dynamics model. It is shown that the system can provide the manager (end-user) with continuously updated information about specific biomass quality parameters. The system encompasses graphical visualization of the information to the end-user as a first step and, as a second step, the system identifies alerts depicting real differences between actual and predicted values of the monitored properties. The perspective is that this diagnostic system will provide managers with a solid basis for necessary preventive measures. PMID:22163886

  7. Expressed Repeat Elements Improve RT-qPCR Normalization across a Wide Range of Zebrafish Gene Expression Studies

    PubMed Central

    Vanhauwaert, Suzanne; Van Peer, Gert; Rihani, Ali; Janssens, Els; Rondou, Pieter; Lefever, Steve; De Paepe, Anne; Coucke, Paul J.; Speleman, Frank; Vandesompele, Jo; Willaert, Andy

    2014-01-01

    The selection and validation of stably expressed reference genes is a critical issue for proper RT-qPCR data normalization. In zebrafish expression studies, many commonly used reference genes are not generally applicable given their variability in expression levels under a variety of experimental conditions. Inappropriate use of these reference genes may lead to false interpretation of expression data and unreliable conclusions. In this study, we evaluated a novel normalization method in zebrafish using expressed repetitive elements (ERE) as reference targets, instead of specific protein coding mRNA targets. We assessed and compared the expression stability of a number of EREs to that of commonly used zebrafish reference genes in a diverse set of experimental conditions including a developmental time series, a set of different organs from adult fish and different treatments of zebrafish embryos including morpholino injections and administration of chemicals. Using geNorm and rank aggregation analysis we demonstrated that EREs have a higher overall expression stability compared to the commonly used reference genes. Moreover, we propose a limited set of ERE reference targets (hatn10, dna15ta1 and loopern4), that show stable expression throughout the wide range of experiments in this study, as strong candidates for inclusion as reference targets for qPCR normalization in future zebrafish expression studies. Our applied strategy to find and evaluate candidate expressed repeat elements for RT-qPCR data normalization has high potential to be used also for other species. PMID:25310091

  8. The use of collagenase to improve the detection of plant viruses in vector nematodes by RT-PCR.

    PubMed

    Martin, Robert R; Pinkerton, Jack N; Kraus, Jennifer

    2009-01-01

    Tomato ringspot virus (ToRSV), Tobacco ringspot virus (TRSV) and Tobacco rattle virus (TRV) are transmitted to healthy plants by viruliferous nematodes in the soil. A method was developed for extraction of genomic viral RNA from virus particles carried within nematodes and a sensitive nested RT-PCR detection assay. The procedure has been adapted to microscale for handling multiple samples. This assay is effective for detection of ToRSV or TRSV in Xiphinema americanum or TRV in Paratrichodorus allius. With this method, viruses can be detected in nematodes fed on infected plants or from field-collected nematodes where the percentage of viruliferous nematodes is unknown. Soil samples from four red raspberry fields infected with ToRSV were collected in 2003 and 2004. Nematodes isolated from these samples were assayed for ToRSV by RT-PCR and compared to cucumber baiting bioassay for virus transmission from the same soil samples. ToRSV was detected in nematodes throughout the season with similar frequencies by the RT-PCR assay and the transmission bioassay.

  9. Improving Patient Care Via Development of a Protein-Based Diagnostic Test for Microbe-Specific Detection of Chronic Rhinosinusitis

    PubMed Central

    Das, Subinoy; Rosas, Lucia E.; Jurcisek, Joseph A.; Novotny, Laura A.; Green, Kari B.; Bakaletz, Lauren O.

    2014-01-01

    Objectives The hypothesis is that signature bacterial proteins can be identified in sinus secretions via high-throughput, proteomic based techniques. Non-typeable Haemophilus influenzae (NTHI) is the most common bacterial pathogen associated with sinusitis and will serve as a proof of principle pathogen for identifying biomarkers. Study Design In vitro and in vivo studies using proteomic based analysis of cultures of NTHI and a novel, experimental chinchilla polymicrobial sinusitis model. Methods Nano-liquid chromatography/tandem mass spectrometry (nano-LC-MS/MS) was performed to annotate the secretome from an NTHI biofilm. A model of NTHI induced sinusitis was developed in a chinchilla and NTHI proteins were detected in chinchilla secretions. A reference standard RT-PCR based assay was adapted to allow for sensitivity and specificity testing of the identified signature biomarkers in human patients. Results Outer membrane proteins P2 (OMP-P2) and P5 (OMP-P5) were identified as promising candidates for detection of NTHI biofilms and positively detected in nasopharyngeal secretions of chinchillas experimentally infected with NTHI. An RT-PCR based test for the presence of NTHI biofilms demonstrated 100% sensitivity and 100% specificity when tested against 8 unique strains commonly found in human bacterial rhinosinusitis. Conclusions Proteomic analysis was successful in identifying signature proteins for possible use as a biomarker for CRS. OMP-P2 and OMP-P5 were validated as promising candidates and were positively detected from nasopharyngeal secretions from chinchillas experimentally infected with NTHI. Collectively, these data support the use of OMP-P2 and OMP-P5 as biomarkers for a human clinical trial to develop a point of care medical diagnostic test to assist in the diagnosis and treatment of CRS. PMID:23904321

  10. Identification of Chabertiidae (Nematoda, Strongylida) by PCR-RFLP based method: a new diagnostic tool for cross transmission investigation between domestic and wild ruminants in France.

    PubMed

    Patrelle, Cécile; Ferté, Hubert; Jouet, Damien

    2014-12-01

    We describe a PCR-RFLP-based method that allows reliable identification of four species of nematode parasites presenting similar infective third-stage larvae (L3) with a flagelliform tail and more than 16 intestinal cells, commonly observed in gastrointestinal tract of ruminants in France. Molecular analysis of the second internal transcribed spacer (ITS2) of ribosomal DNA, considered as a specific marker for Strongylida, revealed four robust monophyletic clades corresponding to species Chabertia ovina, Oesophagostomum sikae, Oesophagostomum radiatum and Oesophagostomum venulosum. One restriction enzyme (DdeI) was used to digest this domain, and we observed four different and clear digestion patterns according to these species (adults or larvae). Hence, this new method is a good tool easy to use for veterinary laboratories to characterize the different species, and allows considering possible cross transmission between domestic and wild ruminants, especially cervids often incriminated as potential reservoir of parasites for cattle. Moreover, thanks to this new tool, necroscopic analyses could be substituted by coprological methods, a non-invasive approach.

  11. Clinical utility of machine-learning approaches in schizophrenia: improving diagnostic confidence for translational neuroimaging.

    PubMed

    Iwabuchi, Sarina J; Liddle, Peter F; Palaniyappan, Lena

    2013-01-01

    Machine-learning approaches are becoming commonplace in the neuroimaging literature as potential diagnostic and prognostic tools for the study of clinical populations. However, very few studies provide clinically informative measures to aid in decision-making and resource allocation. Head-to-head comparison of neuroimaging-based multivariate classifiers is an essential first step to promote translation of these tools to clinical practice. We systematically evaluated the classifier performance using back-to-back structural MRI in two field strengths (3- and 7-T) to discriminate patients with schizophrenia (n = 19) from healthy controls (n = 20). Gray matter (GM) and white matter images were used as inputs into a support vector machine to classify patients and control subjects. Seven Tesla classifiers outperformed the 3-T classifiers with accuracy reaching as high as 77% for the 7-T GM classifier compared to 66.6% for the 3-T GM classifier. Furthermore, diagnostic odds ratio (a measure that is not affected by variations in sample characteristics) and number needed to predict (a measure based on Bayesian certainty of a test result) indicated superior performance of the 7-T classifiers, whereby for each correct diagnosis made, the number of patients that need to be examined using the 7-T GM classifier was one less than the number that need to be examined if a different classifier was used. Using a hypothetical example, we highlight how these findings could have significant implications for clinical decision-making. We encourage the reporting of measures proposed here in future studies utilizing machine-learning approaches. This will not only promote the search for an optimum diagnostic tool but also aid in the translation of neuroimaging to clinical use. PMID:24009589

  12. Clinical Utility of Machine-Learning Approaches in Schizophrenia: Improving Diagnostic Confidence for Translational Neuroimaging

    PubMed Central

    Iwabuchi, Sarina J.; Liddle, Peter F.; Palaniyappan, Lena

    2013-01-01

    Machine-learning approaches are becoming commonplace in the neuroimaging literature as potential diagnostic and prognostic tools for the study of clinical populations. However, very few studies provide clinically informative measures to aid in decision-making and resource allocation. Head-to-head comparison of neuroimaging-based multivariate classifiers is an essential first step to promote translation of these tools to clinical practice. We systematically evaluated the classifier performance using back-to-back structural MRI in two field strengths (3- and 7-T) to discriminate patients with schizophrenia (n = 19) from healthy controls (n = 20). Gray matter (GM) and white matter images were used as inputs into a support vector machine to classify patients and control subjects. Seven Tesla classifiers outperformed the 3-T classifiers with accuracy reaching as high as 77% for the 7-T GM classifier compared to 66.6% for the 3-T GM classifier. Furthermore, diagnostic odds ratio (a measure that is not affected by variations in sample characteristics) and number needed to predict (a measure based on Bayesian certainty of a test result) indicated superior performance of the 7-T classifiers, whereby for each correct diagnosis made, the number of patients that need to be examined using the 7-T GM classifier was one less than the number that need to be examined if a different classifier was used. Using a hypothetical example, we highlight how these findings could have significant implications for clinical decision-making. We encourage the reporting of measures proposed here in future studies utilizing machine-learning approaches. This will not only promote the search for an optimum diagnostic tool but also aid in the translation of neuroimaging to clinical use. PMID:24009589

  13. Clinical utility of machine-learning approaches in schizophrenia: improving diagnostic confidence for translational neuroimaging.

    PubMed

    Iwabuchi, Sarina J; Liddle, Peter F; Palaniyappan, Lena

    2013-01-01

    Machine-learning approaches are becoming commonplace in the neuroimaging literature as potential diagnostic and prognostic tools for the study of clinical populations. However, very few studies provide clinically informative measures to aid in decision-making and resource allocation. Head-to-head comparison of neuroimaging-based multivariate classifiers is an essential first step to promote translation of these tools to clinical practice. We systematically evaluated the classifier performance using back-to-back structural MRI in two field strengths (3- and 7-T) to discriminate patients with schizophrenia (n = 19) from healthy controls (n = 20). Gray matter (GM) and white matter images were used as inputs into a support vector machine to classify patients and control subjects. Seven Tesla classifiers outperformed the 3-T classifiers with accuracy reaching as high as 77% for the 7-T GM classifier compared to 66.6% for the 3-T GM classifier. Furthermore, diagnostic odds ratio (a measure that is not affected by variations in sample characteristics) and number needed to predict (a measure based on Bayesian certainty of a test result) indicated superior performance of the 7-T classifiers, whereby for each correct diagnosis made, the number of patients that need to be examined using the 7-T GM classifier was one less than the number that need to be examined if a different classifier was used. Using a hypothetical example, we highlight how these findings could have significant implications for clinical decision-making. We encourage the reporting of measures proposed here in future studies utilizing machine-learning approaches. This will not only promote the search for an optimum diagnostic tool but also aid in the translation of neuroimaging to clinical use.

  14. Diagnostic Marker for Improving Treatment Outcomes of Hepatitis C | NCI Technology Transfer Center | TTC

    Cancer.gov

    NCI Researchers have discovered Interferon-lambda 4 (IFNL4), a protein found through analysis of genomic data. Preliminary studies indicate that this protein may play a role in the clearance of HCV and may be a new target for diagnosing and treating HCV infection. The National Cancer Institute (NCI) Division of Cancer Epidemiology and Genetics (DCEG) Immunoepidemiology Branch is seeking statements of capability or interest from parties interested in in-licensing or collaborative research to further co-develop a gene-based diagnostic for Hepatitis C virus (HepC, HCV).

  15. Edge Thomson scattering diagnostic on COMPASS tokamak: Installation, calibration, operation, improvements

    SciTech Connect

    Bohm, P. Bilkova, P.; Melich, R.; Sestak, D.; Weinzettl, V.; Stockel, J.; Hron, M.; Panek, R.; Mikulin, O.; Scannell, R.; Naylor, G.; Frassinetti, L.; Fassina, A.; Walsh, M. J.

    2014-11-15

    The core Thomson scattering diagnostic (TS) on the COMPASS tokamak was put in operation and reported earlier. Implementation of edge TS, with spatial resolution along the laser beam up to ∼1/100 of the tokamak minor radius, is presented now. The procedure for spatial calibration and alignment of both core and edge systems is described. Several further upgrades of the TS system, like a triggering unit and piezo motor driven vacuum window shutter, are introduced as well. The edge TS system, together with the core TS, is now in routine operation and provides electron temperature and density profiles.

  16. Edge Thomson scattering diagnostic on COMPASS tokamak: installation, calibration, operation, improvements.

    PubMed

    Bohm, P; Aftanas, M; Bilkova, P; Stefanikova, E; Mikulin, O; Melich, R; Janky, F; Havlicek, J; Sestak, D; Weinzettl, V; Stockel, J; Hron, M; Panek, R; Scannell, R; Frassinetti, L; Fassina, A; Naylor, G; Walsh, M J

    2014-11-01

    The core Thomson scattering diagnostic (TS) on the COMPASS tokamak was put in operation and reported earlier. Implementation of edge TS, with spatial resolution along the laser beam up to ∼1/100 of the tokamak minor radius, is presented now. The procedure for spatial calibration and alignment of both core and edge systems is described. Several further upgrades of the TS system, like a triggering unit and piezo motor driven vacuum window shutter, are introduced as well. The edge TS system, together with the core TS, is now in routine operation and provides electron temperature and density profiles.

  17. Improved Beam Diagnostic Spatial Calibration Using In-Situ Measurements of Beam Emission

    NASA Astrophysics Data System (ADS)

    Chrystal, C.; Burrell, K. H.; Pace, D. C.; Grierson, B. A.; Pablant, N. A.

    2014-10-01

    A new technique has been developed for determining the measurement geometry of the charge exchange recombination spectroscopy diagnostic (CER) on DIII-D. This technique removes uncertainty in the measurement geometry related to the position of the neutral beams when they are injecting power. This has been accomplished by combining standard measurements that use in-vessel calibration targets with spectroscopic measurements of Doppler shifted and Stark split beam emission to fully describe the neutral beam positions and CER views. A least squares fitting routine determines the measurement geometry consistent with all the calibration data. The use of beam emission measurements allows the position of the neutral beams to be determined in-situ by the same views that makeup the CER diagnostic. Results indicate that changes in the measurement geometry are required to create a consistent set of calibration measurements. However, changes in quantities derived from the geometry, e.g. ion temperature gradient and poloidal rotation, are small. Work supported by the US DOE under DE-FG02-07ER54917, DE-FC02-04ER54698, and DE-AC02-09H11466.

  18. BREAST: a novel method to improve the diagnostic efficacy of mammography

    NASA Astrophysics Data System (ADS)

    Brennan, P. C.; Tapia, K.; Ryan, J.; Lee, W.

    2013-03-01

    High quality breast imaging and accurate image assessment are critical to the early diagnoses, treatment and management of women with breast cancer. Breast Screen Reader Assessment Strategy (BREAST) provides a platform, accessible by researchers and clinicians world-wide, which will contain image data bases, algorithms to assess reader performance and on-line systems for image evaluation. The platform will contribute to the diagnostic efficacy of breast imaging in Australia and beyond on two fronts: reducing errors in mammography, and transforming our assessment of novel technologies and techniques. Mammography is the primary diagnostic tool for detecting breast cancer with over 800,000 women X-rayed each year in Australia, however, it fails to detect 30% of breast cancers with a number of missed cancers being visible on the image [1-6]. BREAST will monitor the mistakes, identify reasons for mammographic errors, and facilitate innovative solutions to reduce error rates. The BREAST platform has the potential to enable expert assessment of breast imaging innovations, anywhere in the world where experts or innovations are located. Currently, innovations are often being assessed by limited numbers of individuals who happen to be geographically located close to the innovation, resulting in equivocal studies with low statistical power. BREAST will transform this current paradigm by enabling large numbers of experts to assess any new method or technology using our embedded evaluation methods. We are confident that this world-first system will play an important part in the future efficacy of breast imaging.

  19. An improved method of DNA isolation suitable for PCR-based detection of begomoviruses from jute and other mucilaginous plants.

    PubMed

    Ghosh, Raju; Paul, Sujay; Ghosh, Subrata Kumar; Roy, Anirban

    2009-07-01

    A relatively quick and inexpensive modified cetyl trimethylammonium bromide method for extraction of DNA from leaf materials containing large quantities of mucilage is described. The modification including use of more volume of extraction buffer and dissolving crude nucleic acid pellet in 1 M NaCl, reduced markedly the viscosity of the mucilage and thus in the final purification step yielded a larger quantity of mucilage-free DNA suitable for subsequent PCR-based detection of begomoviruses. The method was standardized with jute samples with yellow mosaic disease and validated with different other mucilaginous-hosts with low titre of begomoviruses. DNA isolated using this method showed consistency in yield and compatibility with PCR for detection of begomoviruses from different mucilaginous plant species. The method was compared for efficacy with other reported methods and it was found to be superior over the existing methods described for isolation of DNA from mucilaginous hosts. Thus the method described could be used on a wider scale for reliable and consistent detection of begomoviruses from mucilaginous hosts for characterization and variability study.

  20. An improved method of DNA isolation suitable for PCR-based detection of begomoviruses from jute and other mucilaginous plants.

    PubMed

    Ghosh, Raju; Paul, Sujay; Ghosh, Subrata Kumar; Roy, Anirban

    2009-07-01

    A relatively quick and inexpensive modified cetyl trimethylammonium bromide method for extraction of DNA from leaf materials containing large quantities of mucilage is described. The modification including use of more volume of extraction buffer and dissolving crude nucleic acid pellet in 1 M NaCl, reduced markedly the viscosity of the mucilage and thus in the final purification step yielded a larger quantity of mucilage-free DNA suitable for subsequent PCR-based detection of begomoviruses. The method was standardized with jute samples with yellow mosaic disease and validated with different other mucilaginous-hosts with low titre of begomoviruses. DNA isolated using this method showed consistency in yield and compatibility with PCR for detection of begomoviruses from different mucilaginous plant species. The method was compared for efficacy with other reported methods and it was found to be superior over the existing methods described for isolation of DNA from mucilaginous hosts. Thus the method described could be used on a wider scale for reliable and consistent detection of begomoviruses from mucilaginous hosts for characterization and variability study. PMID:19442842

  1. Development and improvement of the operating diagnostics systems of NPO CKTI works for turbine of thermal and nuclear power plants

    NASA Astrophysics Data System (ADS)

    Kovalev, I. A.; Rakovskii, V. G.; Isakov, N. Yu.; Sandovskii, A. V.

    2016-03-01

    The work results on the development and improvement of the techniques, algorithms, and software-hardware of continuous operating diagnostics systems of rotating units and parts of turbine equipment state are presented. In particular, to ensure the full remote service of monitored turbine equipment using web technologies, the web version of the software of the automated systems of vibration-based diagnostics (ASVD VIDAS) was developed. The experience in the automated analysis of data obtained by ASVD VIDAS form the basis of the new algorithm of early detection of such dangerous defects as rotor deflection, crack in the rotor, and strong misalignment of supports. The program-technical complex of monitoring and measuring the deflection of medium pressure rotor (PTC) realizing this algorithm will alert the electric power plant staff during a deflection and indicate its value. This will give the opportunity to take timely measures to prevent the further extension of the defect. Repeatedly, recorded cases of full or partial destruction of shrouded shelves of rotor blades of the last stages of low-pressure cylinders of steam turbines defined the need to develop a version of the automated system of blade diagnostics (ASBD SKALA) for shrouded stages. The processing, analysis, presentation, and backup of data characterizing the mechanical state of blade device are carried out with a newly developed controller of the diagnostics system. As a result of the implementation of the works, the diagnosed parameters determining the operation security of rotating elements of equipment was expanded and the new tasks on monitoring the state of units and parts of turbines were solved. All algorithmic solutions and hardware-software implementations mentioned in the article were tested on the test benches and applied at some power plants.

  2. HRP2 and pLDH-Based Rapid Diagnostic Tests, Expert Microscopy, and PCR for Detection of Malaria Infection during Pregnancy and at Delivery in Areas of Varied Transmission: A Prospective Cohort Study in Burkina Faso and Uganda

    PubMed Central

    Cunningham, Jane; Angutoko, Patrick; Ategeka, John; Compaoré, Yves-Daniel; Muehlenbachs, Atis; Somé, Fabrice A.; Ouattara, Aminata; Rouamba, Noél; Ouédraogo, Jean-Bosco; Hopkins, Heidi

    2016-01-01

    Background Intermittent screening and treatment (IST) of malaria during pregnancy has been proposed as an alternative to intermittent preventive treatment in pregnancy (IPTp), where IPTp is failing due to drug resistance. However, the antenatal parasitaemias are frequently very low, and the most appropriate screening test for IST has not been defined. Methodology/Principal Findings We conducted a multi-center prospective study of 990 HIV-uninfected women attending ANC in two different malaria transmission settings at Tororo District Hospital, eastern Uganda and Colsama Health Center in western Burkina Faso. Women were enrolled in the study in the second or third trimester of pregnancy and followed to delivery, generating 2,597 blood samples for analysis. Screening tests included rapid diagnostic tests (RDTs) targeting histidine-rich protein 2 (HRP2) and parasite lactate dehydrogenase (pLDH) and microscopy, compared to nPCR as a reference standard. At enrolment, the proportion of pregnant women who were positive for P. falciparum by HRP2/pan pLDH RDT, Pf pLDH/pan pLDH RDT, microscopy and PCR was 38%, 29%, 36% and 44% in Uganda and 21%, 16%, 15% and 35% in Burkina Faso, respectively. All test positivity rates declined during follow-up. In comparison to PCR, the sensitivity of the HRP2/pan pLDH RDT, Pf pLDH/pan pLDH RDT and microscopy was 75.7%, 60.1% and 69.7% in Uganda, 55.8%, 42.6% and 55.8% in Burkina Faso respectively for all antenatal visits. Specificity was greater than 96% for all three tests. Comparison of accuracy using generalized estimating equation revealed that the HRP2- detecting RDT was the most accurate test in both settings. Conclusions/Significance The study suggests that HRP2-based RDTs are the most appropriate point-of-care test currently available for use during pregnancy especially for symptomatic women, but will still miss some PCR-positive women. The clinical significance of these very low density infections needs to be better defined. PMID

  3. Improved time response for large area microchannel plate photomultiplier tubes in fusion diagnostics

    SciTech Connect

    Milnes, J. S. Conneely, T. M.; Howorth, J.; Horsfield, C. J.

    2014-11-15

    Fusion diagnostics that utilise high speed scintillators often need to capture a large area of light with a high degree of time accuracy. Microchannel plate (MCP) photomultiplier tubes (PMTs) are recognised as the leading device for capturing fast optical signals. However, when manufactured in their traditional proximity focused construction, the time response performance is reduced as the active area increases. This is due to two main factors: the capacitance of a large anode and the difficulty of obtaining small pore MCPs with a large area. Collaboration between Photek and AWE has produced prototype devices that combine the excellent time response of small area MCP-PMTs with a large active area by replacing the traditional proximity-gap front section with an electro-optically focused photocathode to MCP. We present results from both single and double MCP devices with a 40 mm diameter active area and show simulations for the 100 mm device being built this year.

  4. Application of PCR-ELISA in Molecular Diagnosis

    PubMed Central

    Sue, Mei Jean; Yeap, Swee Keong; Omar, Abdul Rahman; Tan, Sheau Wei

    2014-01-01

    Polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA) is an immunodetection method that can quantify PCR product directly after immobilization of biotinylated DNA on a microplate. This method, which detects nucleic acid instead of protein, is a much more sensitive method compared to conventional PCR method, with shorter analytical time and lower detection limit. Its high specificity and sensitivity, together with its semiquantitative ability, give it a huge potential to serve as a powerful detection tool in various industries such as medical, veterinary, and agricultural industries. With the recent advances in PCR-ELISA, it is envisaged that the assay is more widely recognized for its fast and sensitive detection limit which could improve overall diagnostic time and quality. PMID:24971343

  5. The improved PCR of the fstA (ferric siderophore receptor) gene differentiates the fish pathogen Aeromonas salmonicida from other Aeromonas species.

    PubMed

    Beaz-Hidalgo, Roxana; Latif-Eugenín, Fadua; Figueras, María José

    2013-10-25

    The members of the genus Aeromonas are autochthonous of aquatic ecosystems and several species have been associated to septicaemia, ulcerative and haemorrhagic diseases in fish, causing significant mortality in both wild and farmed, freshwater and marine fish species. The species Aeromonas salmonicida is generally recognized as the most important fish pathogen responsible for epidemic outbreaks of furunculosis in salmonids, also being able to produce infections in other cultured fish such as turbot, halibut, sea bream or goldfish. New species, i.e. Aeromonas aquariorum, Aeromonas tecta and Aeromonas piscicola, have recently been discovered and isolated from diseased fish. The species A. piscicola and Aeromonas bestiarum are practically impossible to differentiate phenotypically and genetically (when using the 16S rRNA gene) from each other and from A. salmonicida. In the present study, two previously described PCR protocols, based on the fstA and gyrB genes, for the specific detection of A. salmonicida were re-evaluated with the type strains of all Aeromonas species and with a set of A. piscicola and A. bestiarum strains. Contrary to what had been published previously it was demonstrated that the gyrB-PCR is not specific for A. salmonicida because of cross-reactions with other Aeromonas species. However, in agreement with previous results, A. salmonicida was detected on the basis of the fstA-PCR, for which an improved protocol was proposed. PMID:23890674

  6. Quantitative single-cell ion-channel gene expression profiling through an improved qRT-PCR technique combined with whole cell patch clamp.

    PubMed

    Veys, K; Labro, A J; De Schutter, E; Snyders, D J

    2012-07-30

    Cellular excitability originates from a concerted action of different ion channels. The genomic diversity of ion channels (over 100 different genes) underlies the functional diversity of neurons in the central nervous system (CNS) and even within a specific type of neurons large differences in channel expression have been observed. Patch-clamp is a powerful technique to study the electrophysiology of excitability at the single cell level, allowing exploration of cell-to-cell variability. Only a few attempts have been made to link electrophysiological profiling to mRNA transcript levels and most suffered from experimental noise precluding conclusive quantitative correlations. Here we describe a refinement to the technique that combines patch-clamp analysis with quantitative real-time (qRT) PCR at the single cell level. Hereto the expression of a housekeeping gene was used to normalize for cell-to-cell variability in mRNA isolation and the subsequent processing steps for performing qRT-PCR. However, the mRNA yield from a single cell was insufficient for performing a valid qRT-PCR assay; this was resolved by including a RNA amplification step. The technique was validated on a stable Ltk(-) cell line expressing the Kv2.1 channel and on embryonic dorsal root ganglion (DRG) cells probing for the expression of Kv2.1. Current density and transcript quantity displayed a clear correlation when the qRT-PCR assay was done in twofold and the data normalized to the transcript level of the housekeeping gene GAPD. Without this normalization no significant correlation was obtained. This improved technique should prove very valuable for studying the molecular background of diversity in cellular excitability.

  7. Droplet digital PCR for simultaneous quantification of general and human-associated fecal indicators for water quality assessment.

    PubMed

    Cao, Yiping; Raith, Meredith R; Griffith, John F

    2015-03-01

    Despite wide application to beach water monitoring and microbial source identification, results produced by quantitative PCR (qPCR) methods are subject to bias introduced by reliance on quantitative standards. Digital PCR technology provides direct, standards-free quantification and may potentially alleviate or greatly reduce other qPCR limitations such as difficulty in multiplexing and susceptibility to PCR inhibition. This study examined the efficacy of employing a duplex droplet digital PCR (ddPCR) assay that simultaneously quantifies Enterococcus spp. and the human fecal-associated HF183 marker for water quality assessment. Duplex ddPCR performance was evaluated side-by-side with qPCR and simplex ddPCR using reference material and 131 fecal and water samples. Results for fecal and water samples were highly correlated between ddPCR and simplex qPCR (coefficients > 0.93, p < 0.001). Duplexing Enterococcus and HF183 in qPCR led to competition and resulted in non-detection or underestimation of the target with low concentration relative to the other, while results produced by simplex and duplex ddPCR were consistent and often indistinguishable from one another. ddPCR showed greater tolerance for inhibition, with no discernable effect on quantification at inhibitor concentrations one to two orders of magnitude higher than that tolerated by qPCR. Overall, ddPCR also exhibited improved precision, higher run-to-run repeatability, similar diagnostic sensitivity and specificity on the HF183 marker, but a lower upper limit of quantification than qPCR. Digital PCR has the potential to become a reliable and economical alternative to qPCR for recreational water monitoring and fecal source identification. Findings from this study may also be of interest to other aspects of water research such as detection of pathogens and antibiotic resistance genes.

  8. Improvement in data processing of Thomson scattering diagnostic on HL-2A tokamak

    NASA Astrophysics Data System (ADS)

    Liu, C. H.; Wang, Y. Q.; Feng, Z.; Huang, Y.

    2015-12-01

    There are two types of digitizers to acquire the values of Thomson scattering signals in HL-2A tokamak. One is charge-sensitive analogue-to-digital converters (Q-ADCs) which simply integrates the signal over a gate interval, and the other is transient recorders with 12 bits resolution and 1 GHz sampling rate at each channel. Because the Thomson scattering diagnostic is prone to electrical noisy environment, in which Q-switched Nd:YAG lasers and polychromators are located closely to the HL-2A device, the high speed transient digitizers are found helpful to reduce noise overlapped in Thomson scattering signals. After triggered by the front of TTL pulse generated by laser light, data acquisition is fulfilled from -250 ns to 250 ns, so that the temporal evolution of Thomson scattering signals is obtained. A Gaussian function is utilized to fit the pulse shape of the digitized scattering signal by nonlinear least square methods. By pulse fitting and data processing, the influence of background perturbations is substantially reduced.

  9. Comparison of MRSASelect Agar, CHROMagar Methicillin-Resistant Staphylococcus aureus (MRSA) Medium, and Xpert MRSA PCR for Detection of MRSA in Nares: Diagnostic Accuracy for Surveillance Samples with Various Bacterial Densities ▿

    PubMed Central

    Wolk, D. M.; Marx, J. L.; Dominguez, L.; Driscoll, D.; Schifman, R. B.

    2009-01-01

    Rapid laboratory methods provide optimal support for active surveillance efforts to screen for methicillin-resistant Staphylococcus aureus (MRSA). Most laboratories struggle to determine the optimal use of resources, considering options to balance cost, speed, and diagnostic accuracy. To assess the performance of common methods, the first comparison of MRSASelect agar (MS) and CHROMagar MRSA (CA), with and without broth enrichment followed by a 24-h subculture to MS, was performed. Results were compared to those of the Xpert MRSA assay. For direct culture methods, the agreement between MS and CA was 98.8%. At 18 h, direct MS identified 93% of all positive samples from direct culture and 84% of those identified by the Xpert MRSA. For Trypticase soy broth-enriched MS culture, incubated overnight and then subcultured for an additional 24 h, the agreement with Xpert MRSA was 96%. The agreement between direct MS and Xpert MRSA was 100% when semiquantitative culture revealed a bacterial density of 2+ or greater; however, discrepancies between culture and Xpert MRSA arose for MRSA bacterial densities of 1+ or less, indicating low density as a common cause of false-negative culture results. Since 1+ or less was established as the most common MRSA carrier state, broth enrichment or PCR may be critical for the identification of all MRSA carriers who may be reservoirs for transmission. In this active-surveillance convenience sample, the use of broth enrichment followed by subculture to MS offered a low-cost but sensitive method for MRSA screening, with performance similar to that of Xpert MRSA PCR. PMID:19828738

  10. Evaluation of the Sensitivity of a pLDH-Based and an Aldolase-Based Rapid Diagnostic Test for Diagnosis of Uncomplicated and Severe Malaria Caused by PCR-Confirmed Plasmodium knowlesi, Plasmodium falciparum, and Plasmodium vivax

    PubMed Central

    William, Timothy; Grigg, Matthew J.; Piera, Kim; Yeo, Tsin W.; Anstey, Nicholas M.

    2013-01-01

    Plasmodium knowlesi can cause severe and fatal human malaria in Southeast Asia. Rapid diagnosis of all Plasmodium species is essential for initiation of effective treatment. Rapid diagnostic tests (RDTs) are sensitive for detection of uncomplicated and severe falciparum malaria but have not been systematically evaluated in knowlesi malaria. At a tertiary referral hospital in Sabah, Malaysia, we prospectively evaluated the sensitivity of two combination RDTs for the diagnosis of uncomplicated and severe malaria from all three potentially fatal Plasmodium species, using a pan-Plasmodium lactate dehydrogenase (pLDH)-P. falciparum histidine-rich protein 2 (PfHRP2) RDT (First Response) and a pan-Plasmodium aldolase-PfHRP2 RDT (ParaHIT). Among 293 hospitalized adults with PCR-confirmed Plasmodium monoinfection, the sensitivity of the pLDH component of the pLDH-PfHRP2 RDT was 74% (95/129; 95% confidence interval [CI], 65 to 80%), 91% (110/121; 95% CI, 84 to 95%), and 95% (41/43; 95% CI, 85 to 99%) for PCR-confirmed P. knowlesi, P. falciparum, and P. vivax infections, respectively, and 88% (30/34; 95% CI, 73 to 95%), 90% (38/42; 95% CI, 78 to 96%), and 100% (12/12; 95% CI, 76 to 100%) among patients tested before antimalarial treatment was begun. Sensitivity in severe malaria was 95% (36/38; 95% CI, 83 to 99), 100% (13/13; 95% CI, 77 to 100), and 100% (7/7; 95% CI, 65 to 100%), respectively. The aldolase component of the aldolase-PfHRP2 RDT performed poorly in all Plasmodium species. The pLDH-based RDT was highly sensitive for the diagnosis of severe malaria from all species; however, neither the pLDH- nor aldolase-based RDT demonstrated sufficiently high overall sensitivity for P. knowlesi. More sensitive RDTs are needed in regions of P. knowlesi endemicity. PMID:23345297

  11. Evaluation of the sensitivity of a pLDH-based and an aldolase-based rapid diagnostic test for diagnosis of uncomplicated and severe malaria caused by PCR-confirmed Plasmodium knowlesi, Plasmodium falciparum, and Plasmodium vivax.

    PubMed

    Barber, Bridget E; William, Timothy; Grigg, Matthew J; Piera, Kim; Yeo, Tsin W; Anstey, Nicholas M

    2013-04-01

    Plasmodium knowlesi can cause severe and fatal human malaria in Southeast Asia. Rapid diagnosis of all Plasmodium species is essential for initiation of effective treatment. Rapid diagnostic tests (RDTs) are sensitive for detection of uncomplicated and severe falciparum malaria but have not been systematically evaluated in knowlesi malaria. At a tertiary referral hospital in Sabah, Malaysia, we prospectively evaluated the sensitivity of two combination RDTs for the diagnosis of uncomplicated and severe malaria from all three potentially fatal Plasmodium species, using a pan-Plasmodium lactate dehydrogenase (pLDH)-P. falciparum histidine-rich protein 2 (PfHRP2) RDT (First Response) and a pan-Plasmodium aldolase-PfHRP2 RDT (ParaHIT). Among 293 hospitalized adults with PCR-confirmed Plasmodium monoinfection, the sensitivity of the pLDH component of the pLDH-PfHRP2 RDT was 74% (95/129; 95% confidence interval [CI], 65 to 80%), 91% (110/121; 95% CI, 84 to 95%), and 95% (41/43; 95% CI, 85 to 99%) for PCR-confirmed P. knowlesi, P. falciparum, and P. vivax infections, respectively, and 88% (30/34; 95% CI, 73 to 95%), 90% (38/42; 95% CI, 78 to 96%), and 100% (12/12; 95% CI, 76 to 100%) among patients tested before antimalarial treatment was begun. Sensitivity in severe malaria was 95% (36/38; 95% CI, 83 to 99), 100% (13/13; 95% CI, 77 to 100), and 100% (7/7; 95% CI, 65 to 100%), respectively. The aldolase component of the aldolase-PfHRP2 RDT performed poorly in all Plasmodium species. The pLDH-based RDT was highly sensitive for the diagnosis of severe malaria from all species; however, neither the pLDH- nor aldolase-based RDT demonstrated sufficiently high overall sensitivity for P. knowlesi. More sensitive RDTs are needed in regions of P. knowlesi endemicity.

  12. Alterations of epigenetic signatures in hepatocyte nuclear factor 4α deficient mouse liver determined by improved ChIP-qPCR and (h)MeDIP-qPCR assays.

    PubMed

    Zhang, Qinghao; Lei, Xiaohong; Lu, Hong

    2014-01-01

    Hepatocyte nuclear factor 4α (HNF4α) is a liver-enriched transcription factor essential for liver development and function. In hepatocytes, HNF4α regulates a large number of genes important for nutrient/xenobiotic metabolism and cell differentiation and proliferation. Currently, little is known about the epigenetic mechanism of gene regulation by HNF4α. In this study, the global and specific alterations at the selected gene loci of representative histone modifications and DNA methylations were investigated in Hnf4a-deficient female mouse livers using the improved MeDIP-, hMeDIP- and ChIP-qPCR assay. Hnf4a deficiency significantly increased hepatic total IPed DNA fragments for histone H3 lysine-4 dimethylation (H3K4me2), H3K4me3, H3K9me2, H3K27me3 and H3K4 acetylation, but not for H3K9me3, 5-methylcytosine,or 5-hydroxymethylcytosine. At specific gene loci, the relative enrichments of histone and DNA modifications were changed to different degree in Hnf4a-deficient mouse liver. Among the epigenetic signatures investigated, changes in H3K4me3 correlated the best with mRNA expression. Additionally, Hnf4a-deficient livers had increased mRNA expression of histone H1.2 and H3.3 as well as epigenetic modifiers Dnmt1, Tet3, Setd7, Kmt2c, Ehmt2, and Ezh2. In conclusion, the present study provides convenient improved (h)MeDIP- and ChIP-qPCR assays for epigenetic study. Hnf4a deficiency in young-adult mouse liver markedly alters histone methylation and acetylation, with fewer effects on DNA methylation and 5-hydroxymethylation. The underlying mechanism may be the induction of epigenetic enzymes responsible for the addition/removal of the epigenetic signatures, and/or the loss of HNF4α per se as a key coordinator for epigenetic modifiers.

  13. Automated innovative diagnostic, data management and communication tool, for improving malaria vector control in endemic settings.

    PubMed

    Vontas, John; Mitsakakis, Konstantinos; Zengerle, Roland; Yewhalaw, Delenasaw; Sikaala, Chadwick Haadezu; Etang, Josiane; Fallani, Matteo; Carman, Bill; Müller, Pie; Chouaïbou, Mouhamadou; Coleman, Marlize; Coleman, Michael

    2016-01-01

    Malaria is a life-threatening disease that caused more than 400,000 deaths in sub-Saharan Africa in 2015. Mass prevention of the disease is best achieved by vector control which heavily relies on the use of insecticides. Monitoring mosquito vector populations is an integral component of control programs and a prerequisite for effective interventions. Several individual methods are used for this task; however, there are obstacles to their uptake, as well as challenges in organizing, interpreting and communicating vector population data. The Horizon 2020 project "DMC-MALVEC" consortium will develop a fully integrated and automated multiplex vector-diagnostic platform (LabDisk) for characterizing mosquito populations in terms of species composition, Plasmodium infections and biochemical insecticide resistance markers. The LabDisk will be interfaced with a Disease Data Management System (DDMS), a custom made data management software which will collate and manage data from routine entomological monitoring activities providing information in a timely fashion based on user needs and in a standardized way. The ResistanceSim, a serious game, a modern ICT platform that uses interactive ways of communicating guidelines and exemplifying good practices of optimal use of interventions in the health sector will also be a key element. The use of the tool will teach operational end users the value of quality data (relevant, timely and accurate) to make informed decisions. The integrated system (LabDisk, DDMS & ResistanceSim) will be evaluated in four malaria endemic countries, representative of the vector control challenges in sub-Saharan Africa, (Cameroon, Ivory Coast, Ethiopia and Zambia), highly representative of malaria settings with different levels of endemicity and vector control challenges, to support informed decision-making in vector control and disease management.

  14. Automated innovative diagnostic, data management and communication tool, for improving malaria vector control in endemic settings.

    PubMed

    Vontas, John; Mitsakakis, Konstantinos; Zengerle, Roland; Yewhalaw, Delenasaw; Sikaala, Chadwick Haadezu; Etang, Josiane; Fallani, Matteo; Carman, Bill; Müller, Pie; Chouaïbou, Mouhamadou; Coleman, Marlize; Coleman, Michael

    2016-01-01

    Malaria is a life-threatening disease that caused more than 400,000 deaths in sub-Saharan Africa in 2015. Mass prevention of the disease is best achieved by vector control which heavily relies on the use of insecticides. Monitoring mosquito vector populations is an integral component of control programs and a prerequisite for effective interventions. Several individual methods are used for this task; however, there are obstacles to their uptake, as well as challenges in organizing, interpreting and communicating vector population data. The Horizon 2020 project "DMC-MALVEC" consortium will develop a fully integrated and automated multiplex vector-diagnostic platform (LabDisk) for characterizing mosquito populations in terms of species composition, Plasmodium infections and biochemical insecticide resistance markers. The LabDisk will be interfaced with a Disease Data Management System (DDMS), a custom made data management software which will collate and manage data from routine entomological monitoring activities providing information in a timely fashion based on user needs and in a standardized way. The ResistanceSim, a serious game, a modern ICT platform that uses interactive ways of communicating guidelines and exemplifying good practices of optimal use of interventions in the health sector will also be a key element. The use of the tool will teach operational end users the value of quality data (relevant, timely and accurate) to make informed decisions. The integrated system (LabDisk, DDMS & ResistanceSim) will be evaluated in four malaria endemic countries, representative of the vector control challenges in sub-Saharan Africa, (Cameroon, Ivory Coast, Ethiopia and Zambia), highly representative of malaria settings with different levels of endemicity and vector control challenges, to support informed decision-making in vector control and disease management. PMID:27225553

  15. Improved Detection of Staphylococcus intermedius Group in a Routine Diagnostic Laboratory

    PubMed Central

    Murray, Aimee; Bendall, Richard; Gaze, William; Zhang, Lihong; Vos, Michiel

    2014-01-01

    The Staphylococcus intermedius group (SIG) includes zoonotic pathogens traditionally associated with dog bites. We describe a simple scheme for improved detection of SIG using routine laboratory methods, report its effect on isolation rates, and use sequencing to confirm that, apart from one atypical SIG strain, most isolates are Staphylococcus pseudintermedius. PMID:25502532

  16. Improved detection of Staphylococcus intermedius group in a routine diagnostic laboratory.

    PubMed

    Lee, John; Murray, Aimee; Bendall, Richard; Gaze, William; Zhang, Lihong; Vos, Michiel

    2015-03-01

    The Staphylococcus intermedius group (SIG) includes zoonotic pathogens traditionally associated with dog bites. We describe a simple scheme for improved detection of SIG using routine laboratory methods, report its effect on isolation rates, and use sequencing to confirm that, apart from one atypical SIG strain, most isolates are Staphylococcus pseudintermedius.

  17. Teaching Old Dogs New Tricks: Using Cognitive Feedback to Improve Physicians' Diagnostic Judgments on Simulated Cases.

    ERIC Educational Resources Information Center

    Wigton, Robert S.; And Others

    1990-01-01

    An educational intervention was effective in improving the judgment of experienced student-health physicians (N=11) in predicting positive culture in simulated patients with pharyngitis. The intervention was three parts: an initial one-hour lecture; three sessions with computer-based cognitive feedback; and monthly reports of the percentage of…

  18. Using Targeted Active-Learning Exercises and Diagnostic Question Clusters to Improve Students' Understanding of Carbon Cycling in Ecosystems

    PubMed Central

    Maskiewicz, April Cordero; Griscom, Heather Peckham; Welch, Nicole Turrill

    2012-01-01

    In this study, we used targeted active-learning activities to help students improve their ways of reasoning about carbon flow in ecosystems. The results of a validated ecology conceptual inventory (diagnostic question clusters [DQCs]) provided us with information about students' understanding of and reasoning about transformation of inorganic and organic carbon-containing compounds in biological systems. These results helped us identify specific active-learning exercises that would be responsive to students' existing knowledge. The effects of the active-learning interventions were then examined through analysis of students' pre- and postinstruction responses on the DQCs. The biology and non–biology majors participating in this study attended a range of institutions and the instructors varied in their use of active learning; one lecture-only comparison class was included. Changes in pre- to postinstruction scores on the DQCs showed that an instructor's teaching method had a highly significant effect on student reasoning following course instruction, especially for questions pertaining to cellular-level, carbon-transforming processes. We conclude that using targeted in-class activities had a beneficial effect on student learning regardless of major or class size, and argue that using diagnostic questions to identify effective learning activities is a valuable strategy for promoting learning, as gains from lecture-only classes were minimal. PMID:22383618

  19. Biomarkers and Molecular Analysis to Improve Bloodstream Infection Diagnostics in an Emergency Care Unit

    PubMed Central

    Loonen, Anne J. M.; de Jager, Cornelis P. C.; Tosserams, Janna; Kusters, Ron; Hilbink, Mirrian; Wever, Peter C.; van den Brule, Adriaan J. C.

    2014-01-01

    subsequent pathogen identification using molecular diagnostics. PMID:24475269

  20. Acanthamoeba keratitis: improving the Scottish diagnostic service for the rapid molecular detection of Acanthamoeba species.

    PubMed

    Alexander, Claire Low; Coyne, Michael; Jones, Brian; Anijeet, Deepa

    2015-07-01

    Acanthamoeba species are responsible for causing the potentially sight-threatening condition, Acanthamoeba keratitis, which is commonly associated with contact lens use. In this report, we highlight the challenges faced using conventional laboratory identification methods to identify this often under-reported pathogen, and discuss the reasons for introducing the first national service in Scotland for the rapid and sensitive molecular identification of Acanthamoeba species. By comparing culture and molecular testing data from a total of 63 patients (n = 80 samples) throughout Scotland presenting with ocular eye disease, we describe the improvement in detection rates where an additional four positive cases were identified using a molecular assay versus culture. The testing of a further ten patients by confocal imaging is also presented. This report emphasizes the importance of continuing to improve clinical laboratory services to ensure a prompt, correct diagnosis and better prognosis, in addition to raising awareness of this potentially debilitating opportunistic pathogen.

  1. Outlier detection and removal improves accuracy of machine learning approach to multispectral burn diagnostic imaging

    NASA Astrophysics Data System (ADS)

    Li, Weizhi; Mo, Weirong; Zhang, Xu; Squiers, John J.; Lu, Yang; Sellke, Eric W.; Fan, Wensheng; DiMaio, J. Michael; Thatcher, Jeffrey E.

    2015-12-01

    Multispectral imaging (MSI) was implemented to develop a burn tissue classification device to assist burn surgeons in planning and performing debridement surgery. To build a classification model via machine learning, training data accurately representing the burn tissue was needed, but assigning raw MSI data to appropriate tissue classes is prone to error. We hypothesized that removing outliers from the training dataset would improve classification accuracy. A swine burn model was developed to build an MSI training database and study an algorithm's burn tissue classification abilities. After the ground-truth database was generated, we developed a multistage method based on Z-test and univariate analysis to detect and remove outliers from the training dataset. Using 10-fold cross validation, we compared the algorithm's accuracy when trained with and without the presence of outliers. The outlier detection and removal method reduced the variance of the training data. Test accuracy was improved from 63% to 76%, matching the accuracy of clinical judgment of expert burn surgeons, the current gold standard in burn injury assessment. Given that there are few surgeons and facilities specializing in burn care, this technology may improve the standard of burn care for patients without access to specialized facilities.

  2. Outlier detection and removal improves accuracy of machine learning approach to multispectral burn diagnostic imaging.

    PubMed

    Li, Weizhi; Mo, Weirong; Zhang, Xu; Squiers, John J; Lu, Yang; Sellke, Eric W; Fan, Wensheng; DiMaio, J Michael; Thatcher, Jeffrey E

    2015-12-01

    Multispectral imaging (MSI) was implemented to develop a burn tissue classification device to assist burn surgeons in planning and performing debridement surgery. To build a classification model via machine learning, training data accurately representing the burn tissue was needed, but assigning raw MSI data to appropriate tissue classes is prone to error. We hypothesized that removing outliers from the training dataset would improve classification accuracy. A swine burn model was developed to build an MSI training database and study an algorithm’s burn tissue classification abilities. After the ground-truth database was generated, we developed a multistage method based on Z -test and univariate analysis to detect and remove outliers from the training dataset. Using 10-fold cross validation, we compared the algorithm’s accuracy when trained with and without the presence of outliers. The outlier detection and removal method reduced the variance of the training data. Test accuracy was improved from 63% to 76%, matching the accuracy of clinical judgment of expert burn surgeons, the current gold standard in burn injury assessment. Given that there are few surgeons and facilities specializing in burn care, this technology may improve the standard of burn care for patients without access to specialized facilities.

  3. Pitfalls in PCR troubleshooting: Expect the unexpected?

    PubMed

    Schrick, Livia; Nitsche, Andreas

    2016-01-01

    PCR is a well-understood and established laboratory technique often used in molecular diagnostics. Huge experience has been accumulated over the last years regarding the design of PCR assays and their set-up, including in-depth troubleshooting to obtain the optimal PCR assay for each purpose. Here we report a PCR troubleshooting that came up with a surprising result never observed before. With this report we hope to sensitize the reader to this peculiar problem and to save troubleshooting efforts in similar situations, especially in time-critical and ambitious diagnostic settings.

  4. Pitfalls in PCR troubleshooting: Expect the unexpected?

    PubMed Central

    Schrick, Livia; Nitsche, Andreas

    2015-01-01

    PCR is a well-understood and established laboratory technique often used in molecular diagnostics. Huge experience has been accumulated over the last years regarding the design of PCR assays and their set-up, including in-depth troubleshooting to obtain the optimal PCR assay for each purpose. Here we report a PCR troubleshooting that came up with a surprising result never observed before. With this report we hope to sensitize the reader to this peculiar problem and to save troubleshooting efforts in similar situations, especially in time-critical and ambitious diagnostic settings. PMID:27077041

  5. The Henry Ford Production System: LEAN Process Redesign Improves Service in the Molecular Diagnostic Laboratory

    PubMed Central

    Cankovic, Milena; Varney, Ruan C.; Whiteley, Lisa; Brown, Ron; D'Angelo, Rita; Chitale, Dhananjay; Zarbo, Richard J.

    2009-01-01

    Accurate and timely molecular test results play an important role in patient management; consequently, there is a customer expectation of short testing turnaround times. Baseline data analysis revealed that the greatest challenge to timely result generation occurred in the preanalytic phase of specimen collection and transport. Here, we describe our efforts to improve molecular testing turnaround times by focusing primarily on redesign of preanalytic processes using the principles of LEAN production. Our goal was to complete greater than 90% of the molecular tests in less than 3 days. The project required cooperation from different laboratory disciplines as well as individuals outside of the laboratory. The redesigned processes involved defining and standardizing the protocols and approaching blood and tissue specimens as analytes for molecular testing. The LEAN process resulted in fewer steps, approaching the ideal of a one-piece flow for specimens through collection/retrieval, transport, and different aspects of the testing process. The outcome of introducing the LEAN process has been a 44% reduction in molecular test turnaround time for tissue specimens, from an average of 2.7 to 1.5 days. In addition, extending LEAN work principles to the clinician suppliers has resulted in a markedly increased number of properly collected and shipped blood specimens (from 50 to 87%). These continuous quality improvements were accomplished by empowered workers in a blame-free environment and are now being sustained with minimal management involvement. PMID:19661386

  6. Improved cancer diagnostics by different image processing techniques on OCT images

    NASA Astrophysics Data System (ADS)

    Kanawade, Rajesh; Lengenfelder, Benjamin; Marini Menezes, Tassiana; Hohmann, Martin; Kopfinger, Stefan; Hohmann, Tim; Grabiec, Urszula; Klämpfl, Florian; Gonzales Menezes, Jean; Waldner, Maximilian; Schmidt, Michael

    2015-07-01

    Optical-coherence tomography (OCT) is a promising non-invasive, high-resolution imaging modality which can be used for cancer diagnosis and its therapeutic assessment. However, speckle noise makes detection of cancer boundaries and image segmentation problematic and unreliable. Therefore, to improve the image analysis for a precise cancer border detection, the performance of different image processing algorithms such as mean, median, hybrid median filter and rotational kernel transformation (RKT) for this task is investigated. This is done on OCT images acquired from an ex-vivo human cancerous mucosa and in vitro by using cultivated tumour applied on organotypical hippocampal slice cultures. The preliminary results confirm that the border between the healthy and the cancer lesions can be identified precisely. The obtained results are verified with fluorescence microscopy. This research can improve cancer diagnosis and the detection of borders between healthy and cancerous tissue. Thus, it could also reduce the number of biopsies required during screening endoscopy by providing better guidance to the physician.

  7. Assessment of African Swine Fever Diagnostic Techniques as a Response to the Epidemic Outbreaks in Eastern European Union Countries: How To Improve Surveillance and Control Programs

    PubMed Central

    Nieto, R.; Soler, A.; Pelayo, V.; Fernández-Pinero, J.; Markowska-Daniel, I.; Pridotkas, G.; Nurmoja, I.; Granta, R.; Simón, A.; Pérez, C.; Martín, E.; Fernández-Pacheco, P.; Arias, M.

    2015-01-01

    This study represents a complete comparative analysis of the most widely used African swine fever (ASF) diagnostic techniques in the European Union (EU) using field and experimental samples from animals infected with genotype II ASF virus (ASFV) isolates circulating in Europe. To detect ASFV, three different PCRs were evaluated in parallel using 785 field and experimental samples. The results showed almost perfect agreement between the Universal ProbeLibrary (UPL-PCR) and the real-time (κ = 0.94 [95% confidence interval {CI}, 0.91 to 0.97]) and conventional (κ = 0.88 [95% CI, 0.83 to 0.92]) World Organisation for Animal Health (OIE)-prescribed PCRs. The UPL-PCR had greater diagnostic sensitivity for detecting survivors and allows earlier detection of the disease. Compared to the commercial antigen enzyme-linked immunosorbent assay (ELISA), good-to-moderate agreement (κ = 0.67 [95% CI, 0.58 to 0.76]) was obtained, with a sensitivity of 77.2% in the commercial test. For ASF antibody detection, five serological methods were tested, including three commercial ELISAs, the OIE-ELISA, and the confirmatory immunoperoxidase test (IPT). Greater sensitivity was obtained with the IPT than with the ELISAs, since the IPT was able to detect ASF antibodies at an earlier point in the serological response, when few antibodies are present. The analysis of the exudate tissues from dead wild boars showed that IPT might be a useful serological tool for determining whether or not animals had been exposed to virus infection, regardless of whether antibodies were present. In conclusion, the UPL-PCR in combination with the IPT was the most trustworthy method for detecting ASF during the epidemic outbreaks affecting EU countries in 2014. The use of the most appropriate diagnostic tools is critical when implementing effective control programs. PMID:26041901

  8. Assessment of African Swine Fever Diagnostic Techniques as a Response to the Epidemic Outbreaks in Eastern European Union Countries: How To Improve Surveillance and Control Programs.

    PubMed

    Gallardo, C; Nieto, R; Soler, A; Pelayo, V; Fernández-Pinero, J; Markowska-Daniel, I; Pridotkas, G; Nurmoja, I; Granta, R; Simón, A; Pérez, C; Martín, E; Fernández-Pacheco, P; Arias, M

    2015-08-01

    This study represents a complete comparative analysis of the most widely used African swine fever (ASF) diagnostic techniques in the European Union (EU) using field and experimental samples from animals infected with genotype II ASF virus (ASFV) isolates circulating in Europe. To detect ASFV, three different PCRs were evaluated in parallel using 785 field and experimental samples. The results showed almost perfect agreement between the Universal ProbeLibrary (UPL-PCR) and the real-time (κ = 0.94 [95% confidence interval {CI}, 0.91 to 0.97]) and conventional (κ = 0.88 [95% CI, 0.83 to 0.92]) World Organisation for Animal Health (OIE)-prescribed PCRs. The UPL-PCR had greater diagnostic sensitivity for detecting survivors and allows earlier detection of the disease. Compared to the commercial antigen enzyme-linked immunosorbent assay (ELISA), good-to-moderate agreement (κ = 0.67 [95% CI, 0.58 to 0.76]) was obtained, with a sensitivity of 77.2% in the commercial test. For ASF antibody detection, five serological methods were tested, including three commercial ELISAs, the OIE-ELISA, and the confirmatory immunoperoxidase test (IPT). Greater sensitivity was obtained with the IPT than with the ELISAs, since the IPT was able to detect ASF antibodies at an earlier point in the serological response, when few antibodies are present. The analysis of the exudate tissues from dead wild boars showed that IPT might be a useful serological tool for determining whether or not animals had been exposed to virus infection, regardless of whether antibodies were present. In conclusion, the UPL-PCR in combination with the IPT was the most trustworthy method for detecting ASF during the epidemic outbreaks affecting EU countries in 2014. The use of the most appropriate diagnostic tools is critical when implementing effective control programs.

  9. Improving diagnostic capability for HPV disease internationally within the NIH-NIAID-Division of AIDS Clinical Trial Networks

    PubMed Central

    Godfrey, Catherine C.; Michelow, Pamela M.; Godard, Mandana; Sahasrabuddhe, Vikrant V.; Darden, Janice; Firnhaber, Cynthia S.; Wetherall, Neal T.; Bremer, James; Coombs, Robert W.; Wilkin, Timothy

    2014-01-01

    Objectives To evaluate an external quality assurance (EQA) program for the laboratory diagnosis of human papillomavirus (HPV) disease that was established to improve international research capability within the Division of AIDS at the National Institute of Allergy and Infectious Disease–supported Adult AIDS Clinical Trials Group network. Methods A three-component EQA scheme was devised comprising assessments of diagnostic accuracy of cytotechnologists and pathologists using available EQA packages, review of quality and accuracy of clinical slides from local sites by an outside expert, and HPV DNA detection using the commercially available HPV test kit. Results Seven laboratories and 17 pathologists in Africa, India, and South America participated. EQA scores were suboptimal for standard packages in three of seven laboratories. There was good agreement between the local laboratory and the central reader 70% of the time (90% confidence interval, 42%-98%). Performance on the College of American Pathologists’ HPV DNA testing panel was successful in all laboratories tested. Conclusions The prequalifying EQA round identified correctable issues that will improve the laboratory diagnosis of HPV related cervical disease at the international sites and will provide a mechanism for ongoing education and continuous quality improvement. PMID:24225757

  10. A model for improving student confidence and experience in diagnostic sample collection and interpretation.

    PubMed

    Williams, Laurel E; Nettifee-Osborne, Julie A; Johnson, Jeffrey L

    2006-01-01

    Confidence and proficiency in diagnosing and treating a variety of diseases is of obvious importance to veterinary students. Traditional teaching methods relying on live-animal laboratories or teaching-hospital cases may not provide the breadth and depth of experience necessary to promote optimal development of confidence and skills. These settings also raise concerns about expense, about animal welfare when animals are used in teaching laboratories, and about the stress and potential risks associated with client-owned pets in the teaching hospital. A one-week course implemented in our veterinary curriculum provides the opportunity for students to develop self-assurance and experience in sample collection and interpretation skills in a realistic, clinical-model setting. This course provides students with significantly improved levels of confidence when performing procedures and interpreting results from a variety of procedures and helps prepare them to become clinicians entering the practice of veterinary medicine. PMID:16767653

  11. Improving the diagnostic quality and adequacy of shoulder radiographs in a District General Hospital

    PubMed Central

    Richards, Bethany; Riley, James; Saithna, Adnan

    2016-01-01

    A high rate of suboptimal shoulder radiographs was identified during a service evaluation exercise in our orthopaedic outpatient clinics. Inadequate radiographs require a return to the radiology department for further imaging, a resultant increased workload, delays in the clinic, increased radiation for patients, and inconvenience and decreased patient satisfaction. Furthermore, if a sub-optimal radiograph is accepted there is concern that diagnoses may be missed. The aim of this project was to decrease the rate of suboptimal radiographs by delivering a teaching package directed towards quality improvement. Evaluation criteria were set for standard orthopaedic shoulder radiographs (Anterior-posterior, axillary, and Velpeau views). Baseline data collection was performed over three, two-week periods and included all patients attending the shoulder clinic. The percentage of x-rays which were deemed adequate was only 19.4% for anterior-posterior views and 57.9% for axillary views. A comprehensive educational package was delivered to radiographers. This included a formal PowerPoint based teaching session, hands on training with practice using a skeleton, posters with step-by step instructions on how to obtain an adequate image, and PDF aide memoires suitable for viewing on a smartphone. Two subsequent two-week periods of data collection were performed to evaluate the benefit of this intervention. Delivery of focussed training and provision of easily accessible aide memoires to facilitate improved quality of radiographs resulted in a significant (p<0.05) reduction in the rate of inadequate images. There was also a significant decreases in the rate of return to the radiology department for repeat imaging. PMID:27559473

  12. Improving the diagnostic quality and adequacy of shoulder radiographs in a District General Hospital.

    PubMed

    Richards, Bethany; Riley, James; Saithna, Adnan

    2016-01-01

    A high rate of suboptimal shoulder radiographs was identified during a service evaluation exercise in our orthopaedic outpatient clinics. Inadequate radiographs require a return to the radiology department for further imaging, a resultant increased workload, delays in the clinic, increased radiation for patients, and inconvenience and decreased patient satisfaction. Furthermore, if a sub-optimal radiograph is accepted there is concern that diagnoses may be missed. The aim of this project was to decrease the rate of suboptimal radiographs by delivering a teaching package directed towards quality improvement. Evaluation criteria were set for standard orthopaedic shoulder radiographs (Anterior-posterior, axillary, and Velpeau views). Baseline data collection was performed over three, two-week periods and included all patients attending the shoulder clinic. The percentage of x-rays which were deemed adequate was only 19.4% for anterior-posterior views and 57.9% for axillary views. A comprehensive educational package was delivered to radiographers. This included a formal PowerPoint based teaching session, hands on training with practice using a skeleton, posters with step-by step instructions on how to obtain an adequate image, and PDF aide memoires suitable for viewing on a smartphone. Two subsequent two-week periods of data collection were performed to evaluate the benefit of this intervention. Delivery of focussed training and provision of easily accessible aide memoires to facilitate improved quality of radiographs resulted in a significant (p<0.05) reduction in the rate of inadequate images. There was also a significant decreases in the rate of return to the radiology department for repeat imaging. PMID:27559473

  13. Improved Method for the Detection and Quantification of Naegleria fowleri in Water and Sediment Using Immunomagnetic Separation and Real-Time PCR.

    PubMed

    Mull, Bonnie J; Narayanan, Jothikumar; Hill, Vincent R

    2013-01-01

    Primary amebic meningoencephalitis (PAM) is a rare and typically fatal infection caused by the thermophilic free-living ameba, Naegleria fowleri. In 2010, the first confirmed case of PAM acquired in Minnesota highlighted the need for improved detection and quantification methods in order to study the changing ecology of N. fowleri and to evaluate potential risk factors for increased exposure. An immunomagnetic separation (IMS) procedure and real-time PCR TaqMan assay were developed to recover and quantify N. fowleri in water and sediment samples. When one liter of lake water was seeded with N. fowleri strain CDC:V212, the method had an average recovery of 46% and detection limit of 14 amebas per liter of water. The method was then applied to sediment and water samples with unknown N. fowleri concentrations, resulting in positive direct detections by real-time PCR in 3 out of 16 samples and confirmation of N. fowleri culture in 6 of 16 samples. This study has resulted in a new method for detection and quantification of N. fowleri in water and sediment that should be a useful tool to facilitate studies of the physical, chemical, and biological factors associated with the presence and dynamics of N. fowleri in environmental systems. PMID:24228172

  14. Improved Method for the Detection and Quantification of Naegleria fowleri in Water and Sediment Using Immunomagnetic Separation and Real-Time PCR.

    PubMed

    Mull, Bonnie J; Narayanan, Jothikumar; Hill, Vincent R

    2013-01-01

    Primary amebic meningoencephalitis (PAM) is a rare and typically fatal infection caused by the thermophilic free-living ameba, Naegleria fowleri. In 2010, the first confirmed case of PAM acquired in Minnesota highlighted the need for improved detection and quantification methods in order to study the changing ecology of N. fowleri and to evaluate potential risk factors for increased exposure. An immunomagnetic separation (IMS) procedure and real-time PCR TaqMan assay were developed to recover and quantify N. fowleri in water and sediment samples. When one liter of lake water was seeded with N. fowleri strain CDC:V212, the method had an average recovery of 46% and detection limit of 14 amebas per liter of water. The method was then applied to sediment and water samples with unknown N. fowleri concentrations, resulting in positive direct detections by real-time PCR in 3 out of 16 samples and confirmation of N. fowleri culture in 6 of 16 samples. This study has resulted in a new method for detection and quantification of N. fowleri in water and sediment that should be a useful tool to facilitate studies of the physical, chemical, and biological factors associated with the presence and dynamics of N. fowleri in environmental systems.

  15. The ANA-reflex test as a model for improving clinical appropriateness in autoimmune diagnostics.

    PubMed

    Tonutti, Elio; Bizzaro, Nicola; Morozzi, Gabriella; Radice, Antonella; Cinquanta, Luigi; Villalta, Danilo; Tozzoli, Renato; Tampoia, Marilina; Porcelli, Brunetta; Fabris, Martina; Brusca, Ignazio; Alessio, Maria Grazia; Barberio, Giuseppina; Sorrentino, Maria Concetta; Antico, Antonio; Bassetti, Danila; Fontana, Desré Ethel; Imbastaro, Tiziana; Visentini, Daniela; Pesce, Giampaola; Bagnasco, Marcello

    2016-12-01

    Reflex tests are widely used in clinical laboratories, for example, to diagnose thyroid disorders or in the follow-up of prostate cancer. Reflex tests for antinuclear antibodies (ANA) have recently gained attention as a way to improve appropriateness in the immunological diagnosis of autoimmune rheumatic diseases and avoid waste of resources. However, the ANA-reflex test is not as simple as other consolidated reflex tests (the TSH-reflex tests or the PSA-reflex tests) because of the intrinsic complexity of the ANA test performed by the indirect immunofluorescence method on cellular substrates. The wide heterogeneity of the ANA patterns, which need correct interpretation, and the subsequent choice of the most appropriate confirmatory test (ANA subserology), which depend on the pattern feature and on clinical information, hinder any informatics automation, and require the pathologist's intervention. In this review, the Study Group on Autoimmune Diseases of the Italian Society of Clinical Pathology and Laboratory Medicine provides some indications on the configuration of the ANA-reflex test, using two different approaches depending on whether clinical information is available or not. We further give some suggestions on how to report results of the ANA-reflex test. PMID:27423928

  16. The ANA-reflex test as a model for improving clinical appropriateness in autoimmune diagnostics.

    PubMed

    Tonutti, Elio; Bizzaro, Nicola; Morozzi, Gabriella; Radice, Antonella; Cinquanta, Luigi; Villalta, Danilo; Tozzoli, Renato; Tampoia, Marilina; Porcelli, Brunetta; Fabris, Martina; Brusca, Ignazio; Alessio, Maria Grazia; Barberio, Giuseppina; Sorrentino, Maria Concetta; Antico, Antonio; Bassetti, Danila; Fontana, Desré Ethel; Imbastaro, Tiziana; Visentini, Daniela; Pesce, Giampaola; Bagnasco, Marcello

    2016-12-01

    Reflex tests are widely used in clinical laboratories, for example, to diagnose thyroid disorders or in the follow-up of prostate cancer. Reflex tests for antinuclear antibodies (ANA) have recently gained attention as a way to improve appropriateness in the immunological diagnosis of autoimmune rheumatic diseases and avoid waste of resources. However, the ANA-reflex test is not as simple as other consolidated reflex tests (the TSH-reflex tests or the PSA-reflex tests) because of the intrinsic complexity of the ANA test performed by the indirect immunofluorescence method on cellular substrates. The wide heterogeneity of the ANA patterns, which need correct interpretation, and the subsequent choice of the most appropriate confirmatory test (ANA subserology), which depend on the pattern feature and on clinical information, hinder any informatics automation, and require the pathologist's intervention. In this review, the Study Group on Autoimmune Diseases of the Italian Society of Clinical Pathology and Laboratory Medicine provides some indications on the configuration of the ANA-reflex test, using two different approaches depending on whether clinical information is available or not. We further give some suggestions on how to report results of the ANA-reflex test.

  17. Rapid Multiplex Nested PCR for Detection of Respiratory Viruses▿

    PubMed Central

    Lam, W. Y.; Yeung, Apple C. M.; Tang, Julian W.; Ip, Margaret; Chan, Edward W. C.; Hui, Mamie; Chan, Paul K. S.

    2007-01-01

    Respiratory tract infections can be caused by a heterogeneous group of viruses and bacteria that produce similar clinical presentations. Specific diagnosis therefore relies on laboratory investigation. This study developed and evaluated five groups of multiplex nested PCR assays that could simultaneously detect 21 different respiratory pathogens: influenza A virus (H1N1, H3N2, and H5N1); influenza B virus; parainfluenza virus types 1, 2, 3, 4a, and 4b; respiratory syncytial virus A and B; human rhinoviruses; human enteroviruses; human coronaviruses OC43 and 229E; severe acute respiratory syndrome coronavirus; human metapneumoviruses; Mycoplasma pneumoniae; Chlamydophila pneumoniae; Legionella pneumophila; and adenoviruses (A to F). These multiplex nested PCRs adopted fast PCR technology. The high speed of fast PCR (within 35 min) greatly improved the efficiency of these assays. The results show that these multiplex nested PCR assays are specific and more sensitive (100- to 1,000-fold) than conventional methods. Among the 303 clinical specimens tested, the multiplex nested PCR achieved an overall positive rate of 48.5% (95% confidence interval [CI], 42.9 to 54.1%), which was significantly higher than that of virus isolation (20.1% [95% CI, 15.6 to 24.6%]) and that of direct detection by immunofluorescence assay (13.5% [95% CI, 9.7 to 17.4%]). The improved sensitivity was partly due to the higher sensitivity of multiplex nested PCR than that of conventional methods in detecting cultivatable viruses. Moreover, the ability of the multiplex nested PCR to detect noncultivatable viruses, particularly rhinoviruses, coronavirus OC43, and metapneumoviruses, contributed a major gain (15.6%) in the overall positive rate. In conclusion, rapid multiplex nested PCR assays can improve the diagnostic yield for respiratory infections to allow prompt interventive actions to be taken. PMID:17804659

  18. Transthoracic measurement of left coronary artery flow reserve improves the diagnostic value of routine dipyridamole-atropine stress echocardiogram

    PubMed Central

    Wejner-Mik, Paulina; Nouri, Aria; Szymczyk, Ewa; Krzemińska-Pakuła, Maria; Lipiec, Piotr

    2013-01-01

    Introduction We hypothesized that coronary flow reserve (CFR) in the left anterior descending artery (LAD) can be effectively measured during an accelerated dipyridamole-atropine stress echocardiography (DASE) protocol to improve the diagnostic performance of the test. Material and methods In 64 patients with suspected or known coronary artery disease scheduled for coronary angiography DASE with concomitant CFR measurement in LAD was performed. Results Coronary flow reserve measurement and calculation were feasible in 83% of patients. The positive predictive value of undetectable LAD flow was 81% for severe LAD disease. Measured values of CFR were in the range 1.3–4.1 (mean: 2.2 ±0.7). Significantly lower CFR was found in patients with LAD disease (1.97 ±0.62 vs. 2.55 ±0.57, p = 0.0015). The optimal cutoff for detecting ≥ 50% stenosis was CFR ≤ 2.1 (ROC AUC 0.776), corresponding with 68% sensitivity and 84% specificity. In patients with negative DASE results 67% of patients with LAD disease had abnormal CFR, whereas in patients with a positive DASE result 92% of patients with normal LAD had normal CFR. The DASE diagnostic accuracy for the detection of coronary artery disease (CAD) increased from 75% to 85% when CFR measurement was added to wall motion abnormality (WMA) analysis. No test with both abnormalities was false positive for the detection of coronary disease. Conclusions Incorporation of CFR measurement into WMA-based stress echocardiography is feasible even in an accelerated DASE protocol and can be translated into an approximate gain of 10% in overall test accuracy. PMID:24273560

  19. A novel, multi-parallel, real-time polymerase chain reaction approach for eight gastrointestinal parasites provides improved diagnostic capabilities to resource-limited at-risk populations.

    PubMed

    Mejia, Rojelio; Vicuña, Yosselin; Broncano, Nely; Sandoval, Carlos; Vaca, Maritza; Chico, Martha; Cooper, Philip J; Nutman, Thomas B

    2013-06-01

    Diagnosis of gastrointestinal parasites has traditionally relied on stool microscopy, which has low diagnostic sensitivity and specificity. We have developed a novel, rapid, high-throughput quantitative multi-parallel real-time polymerase chain reaction (qPCR) platform. Species-specific primers/probes were used for eight common gastrointestinal parasite pathogens: Ascaris lumbricoides, Necator americanus, Ancylostoma duodenale, Giardia lamblia, Cryptosporidium spp., Entamoeba histolytica, Trichuris trichiura, and Strongyloides stercoralis. Stool samples from 400 13-month-old children in rural Ecuador were analyzed and the qPCR was compared with a standard direct wet mount slide for stool microscopy, as were 125 8-14-year-old children before and after anthelmintic treatment. The qPCR showed higher detection rates for all parasites compared with direct microscopy, Ascaris (7.0% versus 5.5%) and for Giardia (31.5% versus 5.8%). Using an enhanced DNA extraction method, we were able to detect T. trichiura DNA. These assays will be useful to refine treatment options for affected populations, ultimately leading to better health outcomes. PMID:23509117

  20. Improving Access to Malaria Rapid Diagnostic Test in Niger State, Nigeria: An Assessment of Implementation up to 2013.

    PubMed

    Awoleye, Olatunji Joshua; Thron, Chris

    2016-01-01

    Nigeria's 2009-2013 malaria strategic plan adopted WHO diagnosis and treatment guidelines, which include the use of rapid diagnostic tests (RDTs) prior to prescribing treatment with artemisinin combination therapies (ACTs). The current study explores accessibility barriers to the use of RDTs in Niger State and makes recommendations for improving the uptake of RDTs. The study employs literature review, review of data from the Niger State Health Management Information System for January-October 2013, and application of Peters' conceptual framework for assessing access to health services. Data showed that 27 percent of public health facilities (HFs) implemented RDTs, with the aid of donor funds. In these facilities, 77 percent of fever cases presented during the study period were tested with RDTs; 53 percent of fever cases were confirmed cases of malaria, while 60 percent of fever cases were treated. Stockouts of RDTs were a major constraint, and severe fever tended to trigger presumptive treatment. We conclude that although implementation of RDTs led to a reduction in the use of ACTs at HFs, more substantial reduction could be achieved if the state government directed more resources towards the acquisition of RDTs as well as raising the level of awareness of potential users. PMID:27042376

  1. Improving Access to Malaria Rapid Diagnostic Test in Niger State, Nigeria: An Assessment of Implementation up to 2013

    PubMed Central

    Awoleye, Olatunji Joshua; Thron, Chris

    2016-01-01

    Nigeria's 2009–2013 malaria strategic plan adopted WHO diagnosis and treatment guidelines, which include the use of rapid diagnostic tests (RDTs) prior to prescribing treatment with artemisinin combination therapies (ACTs). The current study explores accessibility barriers to the use of RDTs in Niger State and makes recommendations for improving the uptake of RDTs. The study employs literature review, review of data from the Niger State Health Management Information System for January–October 2013, and application of Peters' conceptual framework for assessing access to health services. Data showed that 27 percent of public health facilities (HFs) implemented RDTs, with the aid of donor funds. In these facilities, 77 percent of fever cases presented during the study period were tested with RDTs; 53 percent of fever cases were confirmed cases of malaria, while 60 percent of fever cases were treated. Stockouts of RDTs were a major constraint, and severe fever tended to trigger presumptive treatment. We conclude that although implementation of RDTs led to a reduction in the use of ACTs at HFs, more substantial reduction could be achieved if the state government directed more resources towards the acquisition of RDTs as well as raising the level of awareness of potential users. PMID:27042376

  2. Improved Optical Diagnostic and Microwave Power Supply, final report. An ARRA Supplement to Instabilities in Nonthermal Atmospheric Pressure Plasma

    SciTech Connect

    Hopwood, Jeffrey

    2011-05-30

    This is the final report for the supplemental program ''Improved Optical Diagnostic and Microwave Power Supply'' which has funded the purchase of laboratory instrumentation to enhance the main DOE project, ''Instabilities in Non-thermal Atmospheric Pressure Plasma.'' The main program’s goals include a scientific study of the plasma physics causing large-area plasmas to become unstable at atmospheric pressure. These fundamental scientific discoveries will then allow for the design of controllable cold plasma sources capable of materials processing, including photovoltaic devices, at one atmosphere. This leads to lower costs of energy production. This final report describes only the completion of the supplement. A high-speed spectroscopic camera capable of diagnosing plasma fluctuations and instabilities on time-scales of 2 ns was specified, purchased, installed and tested at the Tufts University Plasma Laboratory. In addition, a 30 watt microwave power system capable of producing short pulses of power in the 0.8 – 4.2 GHz bands was specified, purchased, installed and tested. Scientific experiments are continuing under the funding of the main grant, but a few preliminary examples of scientific discoveries made using these items are included in this report.

  3. Diagnostic performance of HPV E6/E7, hTERT, and Ki67 mRNA RT-qPCR assays on formalin-fixed paraffin-embedded cervical tissue specimens from women with cervical cancer.

    PubMed

    Wang, Hye-Young; Kim, Geehyuk; Cho, Hyemi; Kim, Sunghyun; Lee, Dongsup; Park, Sunyoung; Park, Kwang Hwa; Lee, Hyeyoung

    2015-06-01

    Human papillomavirus (HPV) is a major cause of cervical cancer, which is the third most common cancer in women. Human telomerase reverse transcriptase (hTERT) and Ki67 are tumor cell markers indicating cancer cell proliferation in cancer patients, and activation of hTERT and Ki67 leads to progressive cervical carcinogenesis. In the present study, we evaluated the CervicGen HPVE6/E7 mRNA RT-qDx assay, which detects 16 HPV high-risk (HR) genotypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 and 69), and the CervicGen hTERT and Ki67 mRNA RT-qDx assay using 117 formalin-fixed paraffin-embedded (FFPE) cervical cancer tissue samples. The diagnostic validity of the CervicGen HPV RT-qDx assay for detecting histologically proven prevalent squamous cell carcinoma (SCC) was 94% sensitivity, 100% specificity, 77.8% positive predictive value (PPV), and 78.9% negative predictive value (NPV). The most common HPV genotypes detected in FFPE cervical cancer tissue samples were HPV 16 (56%) and HPV 18 (10%). The positivity rate of hTERT and Ki67 mRNA expressions in FFPE cervical cancer tissue samples on RT-qPCR was 65% and 93% respectively. Moreover, the positivity rates were 92% for a combination of HPV E6/E7 and hTERT mRNA expressions, 97% for HPV E6/E7 and Ki67 mRNA expressions, and 99% (99/100) for the combination of HPV E6/E7, hTERT, and Ki67 mRNA expressions. These data showed that SSC FFPE cervical cancer tissue samples correlated more strongly with high Ki67 mRNA expressions than with hTERT mRNA expressions. Notably, hTERT and Ki67 mRNA expression level was increased in high-grade cervical lesions, but was very low in normal samples. Our findings suggest that the combination of HPV E6/E7, hTERT, and Ki67 mRNA expression levels could be used in a complementary manner in diagnosing high-grade cervical lesions. Further studies are required to evaluate these assays as a useful predictive tool for screening low-grade cervical lesions.

  4. Evaluation of PCR for cutaneous leishmaniasis diagnosis and species identification using filter paper samples in Panama, Central America.

    PubMed

    Miranda, A; Saldaña, A; González, K; Paz, H; Santamaría, G; Samudio, F; Calzada, J E

    2012-09-01

    Cutaneous leishmaniasis (CL) is a major vectorborne disease in Panama. In this study, the diagnostic performance and usefulness of two DNA extraction procedures from skin scraping samples collected on FTA filter paper for subsequent PCR diagnosis of CL was evaluated. A positive CL laboratory diagnosis was based on a positive parasitological test (Giemsa-stained smears or in vitro culture) and/or positive PCR test performed from skin scrapings collected in TE buffer (PCR-TE). Of 100 patients with skin lesions suggestive of CL, 82 (82%) were confirmed as CL positive. The sensitivity was calculated for each of the PCR approaches from samples collected on filter paper. The highest sensitivity was achieved by PCR-FTA processed by Chelex 100 (PCR-Chelex) (0.94). PCR-FTA extracted using the FTA purification reagent presented a lower sensitivity (0.60). Good concordance between routine PCR-TE and PCR-Chelex was observed (percent agreement=0.88, κ index=0.65). In conclusion, use of FTA filter paper for skin scraping collection combined with PCR is a reliable and convenient method for CL diagnosis in Panama, with comparable performance to the routine PCR method and with improved sensitivity compared with those of conventional parasitological methods.

  5. CSF α-synuclein improves diagnostic and prognostic performance of CSF tau and Aβ in Alzheimer's disease.

    PubMed

    Toledo, Jon B; Korff, Ane; Shaw, Leslie M; Trojanowski, John Q; Zhang, Jing

    2013-11-01

    Alzheimer's disease (AD) and Lewy body diseases (LBD), e.g., Parkinson's disease (PD) dementia and dementia with Lewy bodies (DLB), are common causes of geriatric cognitive impairments. In addition, AD and LBD are often found in the same patients at autopsy; therefore, biomarkers that can detect the presence of both pathologies in living subjects are needed. In this investigation, we report the assessment of α-synuclein (α-syn) in cerebrospinal fluid (CSF) and its association with CSF total tau (t-tau), phosphorylated tau181 (p-tau181), and amyloid beta1-42 (Aβ1-42) in subjects of the Alzheimer's Disease Neuroimaging Initiative (ADNI; n = 389), with longitudinal clinical assessments. A strong correlation was noted between α-syn and t-tau in controls, as well as in patients with AD and mild cognitive impairment (MCI). However, the correlation is not specific to subjects in the ADNI cohort, as it was also seen in PD patients and controls enrolled in the Parkinson's Progression Markers Initiative (PPMI; n = 102). A bimodal distribution of CSF α-syn levels was observed in the ADNI cohort, with high levels of α-syn in the subjects with abnormally increased t-tau values. Although a correlation was also noted between α-syn and p-tau181, there was a mismatch (α-syn-p-tau181-Mis), i.e., higher p-tau181 levels accompanied by lower α-syn levels in a subset of ADNI patients. We hypothesize that this α-syn-p-tau181-Mis is a CSF signature of concomitant LBD pathology in AD patients. Hence, we suggest that inclusion of measures of CSF α-syn and calculation of α-syn-p-tau181-Mis improves the diagnostic sensitivity/specificity of classic CSF AD biomarkers and better predicts longitudinal cognitive changes.

  6. Prosthetic Vascular Graft Infections: Bacterial Cultures from Negative-Pressure-Wound-Therapy Foams Do Not Improve Diagnostics.

    PubMed

    Scherrer, Alexandra U; Bloemberg, Guido; Zbinden, Reinhard; Zinkernagel, Annelies S; Fuchs, Claudio; Frauenfelder, Sandra; Rancic, Zoran; Mayer, Dieter; Hasse, Barbara

    2016-08-01

    We analyzed the diagnostic value of microorganisms cultured from negative-pressure-wound-therapy (NPWT) foam samples compared to that of microorganisms cultured from deep tissue samples from patients with vascular graft infections. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 58%, 86%, 81%, and 66%, respectively. The diagnostic value of microbiological cultures from NPWT foams was poor. PMID:27252462

  7. Short communication: Improved method for centrifugal recovery of bacteria from raw milk applied to sensitive real-time quantitative PCR detection of Salmonella spp.

    PubMed

    Brewster, Jeffrey D; Paul, Moushumi

    2016-05-01

    Centrifugation is widely used to isolate and concentrate bacteria from dairy products before assay. We found that more than 98% of common pathogenic bacteria added to pasteurized, homogenized, or pasteurized homogenized milk were recovered in the pellet after centrifugation, whereas less than 7% were recovered from raw milk. The remaining bacteria partitioned into the cream layer of raw milk within 5 min, and half-saturation of the cream layer required a bacterial load of approximately 5×10(8) cfu/mL. Known treatments (e.g., heat, enzymes or solvents) can disrupt cream layer binding and improve recovery from raw milk, but can also damage bacteria and compromise detection. We developed a simple, rapid agitation treatment that disrupted bacteria binding to the cream layer and provided more than 95% recovery without affecting bacteria viability. Combining this simple agitation treatment with a previously developed real-time quantitative PCR assay allowed the detection of Salmonella spp. in raw milk at 4 cfu/mL within 3 h. To our knowledge, this is the first report of an effective method for achieving high centrifugal recovery of bacteria from raw milk without impairing bacterial viability.

  8. Improving estimates of water resources in a semi-arid region by assimilating GRACE data into the PCR-GLOBWB hydrological model

    NASA Astrophysics Data System (ADS)

    Tangdamrongsub, Natthachet; Steele-Dunne, Susan; Gunter, Brian; Ditmar, Pavel; Sutanudjaja, Edwin; Sun, Yu; Xia, Ting; Wang, Zhongjing

    2016-04-01

    An accurate estimate of water resources is critical for proper management of both agriculture and the local ecology, particularly in semi-arid regions where water is scarce. Imperfections in model physics, uncertainties in model land parameters and meteorological data, and the human impact on land changes often limit the accuracy of hydrological models in estimating water storages. To address this problem, this study investigated the assimilation of Terrestrial Water Storage (TWS) estimates derived from the Gravity Recovery And Climate Experiment (GRACE) data using an Ensemble Kalman Filter (EnKF) approach. The region considered was the Hexi Corridor of Northern China. The hydrological model used for the analysis was PCR-GLOBWB, driven by satellite-based forcing data from April 2002 to December 2010. The performance of the GRACE Data Assimilation (DA) scheme was evaluated in terms of its impact on the TWS as well as on the individual hydrological storage estimates. The capability of GRACE DA to adjust the storage level was apparent not only in the TWS but also in the groundwater component, which had annual amplitude, phase, and long-term trend estimates closer to the GRACE observations. This study also assessed the impact of considering correlated errors in GRACE-based estimates. These were derived based on the error propagation approach using the full error variance-covariance matrices provided as a part of the GRACE data product. The assessment was carried out by comparing the EnKF results after excluding (EnKF 1D) and including (EnKF 3D) error correlations with the in situ groundwater data from 5 well sites, and the in situ streamflow data from two river gauges. Both EnKF 1D and 3D improved groundwater and streamflow estimates compared to the results from the PCR-GLOBWB alone (Ensemble Open Loop, EnOL). Although EnKF 3D was inferior to 1D at some groundwater measurement locations, on average, it showed equal or greater improvement in all metrics. For example

  9. Evaluation and Improvement of Real-Time PCR Assays Targeting lytA, ply, and psaA Genes for Detection of Pneumococcal DNA▿

    PubMed Central

    Carvalho, Maria da Gloria S.; Tondella, Maria Lucia; McCaustland, Karen; Weidlich, Luciana; McGee, Lesley; Mayer, Leonard W.; Steigerwalt, Arnold; Whaley, Melissa; Facklam, Richard R.; Fields, Barry; Carlone, George; Ades, Edwin W.; Dagan, Ron; Sampson, Jacquelyn S.

    2007-01-01

    The accurate diagnosis of pneumococcal disease has frequently been hampered not only by the difficulties in obtaining isolates of the organism from patient specimens but also by the misidentification of pneumococcus-like viridans group streptococci (P-LVS) as Streptococcus pneumoniae. This is especially critical when the specimen comes from the respiratory tract. In this study, three novel real-time PCR assays designed for the detection of specific sequence regions of the lytA, ply, and psaA genes were developed (lytA-CDC, ply-CDC, and psaA, respectively). These assays showed high sensitivity (<10 copies for lytA-CDC and ply-CDC and an approximately twofold less sensitivity for psaA). Two additional real-time PCR assays for lytA and ply described previously for pneumococcal DNA detection were also evaluated. A panel of isolates consisting of 67 S. pneumoniae isolates (44 different serotypes and 3 nonencapsulated S. pneumoniae isolates from conjunctivitis outbreaks) and 104 nonpneumococcal isolates was used. The 67 S. pneumoniae isolates were reactive in all five assays. The new real-time detection assays targeting the lytA and psaA genes were the most specific for the detection of isolates confirmed to be S. pneumoniae, with lytA-CDC showing the greatest specificity. Both ply PCRs were positive for all isolates of S. pseudopneumoniae, along with 13 other isolates of other P-LVS isolates confirmed to be non-S. pneumoniae by DNA-DNA reassociation. Thus, the use of the ply gene for the detection of pneumococci can lead to false-positive reactions in the presence of P-LVS. The five assays were applied to 15 culture-positive cerebrospinal fluid specimens with 100% sensitivity; and serum and ear fluid specimens were also evaluated. Both the lytA-CDC and psaA assays, particularly the lytA-CDC assay, have improved specificities compared with those of currently available assays and should therefore be considered the assays of choice for the detection of pneumococcal DNA

  10. Evaluation and improvement of real-time PCR assays targeting lytA, ply, and psaA genes for detection of pneumococcal DNA.

    PubMed

    Carvalho, Maria da Gloria S; Tondella, Maria Lucia; McCaustland, Karen; Weidlich, Luciana; McGee, Lesley; Mayer, Leonard W; Steigerwalt, Arnold; Whaley, Melissa; Facklam, Richard R; Fields, Barry; Carlone, George; Ades, Edwin W; Dagan, Ron; Sampson, Jacquelyn S

    2007-08-01

    The accurate diagnosis of pneumococcal disease has frequently been hampered not only by the difficulties in obtaining isolates of the organism from patient specimens but also by the misidentification of pneumococcus-like viridans group streptococci (P-LVS) as Streptococcus pneumoniae. This is especially critical when the specimen comes from the respiratory tract. In this study, three novel real-time PCR assays designed for the detection of specific sequence regions of the lytA, ply, and psaA genes were developed (lytA-CDC, ply-CDC, and psaA, respectively). These assays showed high sensitivity (<10 copies for lytA-CDC and ply-CDC and an approximately twofold less sensitivity for psaA). Two additional real-time PCR assays for lytA and ply described previously for pneumococcal DNA detection were also evaluated. A panel of isolates consisting of 67 S. pneumoniae isolates (44 different serotypes and 3 nonencapsulated S. pneumoniae isolates from conjunctivitis outbreaks) and 104 nonpneumococcal isolates was used. The 67 S. pneumoniae isolates were reactive in all five assays. The new real-time detection assays targeting the lytA and psaA genes were the most specific for the detection of isolates confirmed to be S. pneumoniae, with lytA-CDC showing the greatest specificity. Both ply PCRs were positive for all isolates of S. pseudopneumoniae, along with 13 other isolates of other P-LVS isolates confirmed to be non-S. pneumoniae by DNA-DNA reassociation. Thus, the use of the ply gene for the detection of pneumococci can lead to false-positive reactions in the presence of P-LVS. The five assays were applied to 15 culture-positive cerebrospinal fluid specimens with 100% sensitivity; and serum and ear fluid specimens were also evaluated. Both the lytA-CDC and psaA assays, particularly the lytA-CDC assay, have improved specificities compared with those of currently available assays and should therefore be considered the assays of choice for the detection of pneumococcal DNA

  11. Detection of Mycobacterium tuberculosis in extrapulmonary biopsy samples using PCR targeting IS6110, rpoB, and nested-rpoB PCR Cloning

    PubMed Central

    Meghdadi, Hossein; Khosravi, Azar D.; Ghadiri, Ata A.; Sina, Amir H.; Alami, Ameneh

    2015-01-01

    Present study was aimed to examine the diagnostic utility of polymerase chain reaction (PCR) and nested PCR techniques for the detection of Mycobacterium tuberculosis (MTB) DNA in samples from patients with extra pulmonary tuberculosis (EPTB). In total 80 formalin-fixed, paraffin-embedded (FFPE) samples comprising 70 samples with definite diagnosis of EPTB and 10 samples from known non- EPTB on the basis of histopathology examination, were included in the study. PCR amplification targeting IS6110, rpoB gene and nested PCR targeting the rpoB gene were performed on the extracted DNAs from 80 FFPE samples. The strong positive samples were directly sequenced. For negative samples and those with weak band in nested-rpoB PCR, TA cloning was performed by cloning the products into the plasmid vector with subsequent sequencing. The 95% confidence intervals (CI) for the estimates of sensitivity and specificity were calculated for each method. Fourteen (20%), 34 (48.6%), and 60 (85.7%) of the 70 positive samples confirmed by histopathology, were positive by rpoB-PCR, IS6110-PCR, and nested-rpoB PCR, respectively. By performing TA cloning on samples that yielded weak (n = 8) or negative results (n = 10) in the PCR methods, we were able to improve their quality for later sequencing. All samples with weak band and 7 out of 10 negative samples, showed strong positive results after cloning. So nested-rpoB PCR cloning revealed positivity in 67 out of 70 confirmed samples (95.7%). The sensitivity of these combination methods was calculated as 95.7% in comparison with histopathology examination. The CI for sensitivity of the PCR methods were calculated as 11.39–31.27% for rpoB-PCR, 36.44–60.83% for IS6110- PCR, 75.29–92.93% for nested-rpoB PCR, and 87.98–99.11% for nested-rpoB PCR cloning. The 10 true EPTB negative samples by histopathology, were negative by all tested methods including cloning and were used to calculate the specificity of the applied methods. The CI for 100

  12. Improving Diagnostic Accuracy of Dermoscopically Equivocal Pink Cutaneous Lesions with Reflectance Confocal Microscopy in Telemedicine Settings: Double Reader Concordance Evaluation of 316 Cases

    PubMed Central

    Roterman-Konieczna, I.; Bassoli, S.

    2016-01-01

    Background Solitary pink lesions in differential diagnosis with hypopigmented/amelanotic melanoma present a diagnostic challenge in daily practice and are regularly referred for second expert opinion. Reflectance confocal microscopy (RCM) has been shown to improve diagnostic accuracy of dermoscopically equivocal pink lesions. No studies have been performed to evaluate the effect of adding a second expert reader and automatic removal of lesions with discordant management recommendations and its potential effect on diagnostic sensitivity and final management of these lesions in retrospective or telemedicine settings. Objective To improve diagnostic accuracy and reduce potential mismanagement of dermoscopically equivocal pink cutaneous lesions by implementing double reader concordance evaluation of RCM images. Methods 316 dermoscopically equivocal pink lesions with dermoscopy-RCM image sets were evaluated retrospectively. Accuracy of three readers was evaluated by single reader evaluation of dermoscopy only and dermoscopy-RCM image sets and finally by double reader evaluation of dermoscopy-RCM image sets. Lesions with discordant diagnosis between two readers were automatically recommended for excision. Results Dermoscopy only evaluation resulted in an overall sensitivity of 95.9% and specificity of 33.6%, with 1 of 12 amelanotic melanomas mismanaged. Dermoscopy-RCM image set single reader evaluation resulted in an overall sensitivity of 93.9% and overall specificity of 54.2%, with 1 of 12 melanomas mismanaged. Dermoscopy-RCM image set double reader concordance evaluation resulted in an overall sensitivity of 98.3% and specificity of 42.7%, with no amelanotic melanoma mismanagement. Conclusion Evaluation of dermoscopy-RCM image sets of equivocal pink lesions by a single reader in telemedicine settings is limited by the potential for misdiagnosis of dangerous malignant lesions. Double reader concordance evaluation with automatic referral of lesions for removal in the

  13. Improvement of Image Quality and Diagnostic Performance by an Innovative Motion-Correction Algorithm for Prospectively ECG Triggered Coronary CT Angiography

    PubMed Central

    Lu, Bin; Yan, Hong-Bing; Mu, Chao-Wei; Gao, Yang; Hou, Zhi-Hui; Wang, Zhi-Qiang; Liu, Kun; Parinella, Ashley H.; Leipsic, Jonathon A.

    2015-01-01

    Objective To investigate the effect of a novel motion-correction algorithm (Snap-short Freeze, SSF) on image quality and diagnostic accuracy in patients undergoing prospectively ECG-triggered CCTA without administering rate-lowering medications. Materials and Methods Forty-six consecutive patients suspected of CAD prospectively underwent CCTA using prospective ECG-triggering without rate control and invasive coronary angiography (ICA). Image quality, interpretability, and diagnostic performance of SSF were compared with conventional multisegment reconstruction without SSF, using ICA as the reference standard. Results All subjects (35 men, 57.6 ± 8.9 years) successfully underwent ICA and CCTA. Mean heart rate was 68.8±8.4 (range: 50–88 beats/min) beats/min without rate controlling medications during CT scanning. Overall median image quality score (graded 1–4) was significantly increased from 3.0 to 4.0 by the new algorithm in comparison to conventional reconstruction. Overall interpretability was significantly improved, with a significant reduction in the number of non-diagnostic segments (690 of 694, 99.4% vs 659 of 694, 94.9%; P<0.001). However, only the right coronary artery (RCA) showed a statistically significant difference (45 of 46, 97.8% vs 35 of 46, 76.1%; P = 0.004) on a per-vessel basis in this regard. Diagnostic accuracy for detecting ≥50% stenosis was improved using the motion-correction algorithm on per-vessel [96.2% (177/184) vs 87.0% (160/184); P = 0.002] and per-segment [96.1% (667/694) vs 86.6% (601/694); P <0.001] levels, but there was not a statistically significant improvement on a per-patient level [97.8 (45/46) vs 89.1 (41/46); P = 0.203]. By artery analysis, diagnostic accuracy was improved only for the RCA [97.8% (45/46) vs 78.3% (36/46); P = 0.007]. Conclusion The intracycle motion correction algorithm significantly improved image quality and diagnostic interpretability in patients undergoing CCTA with prospective ECG triggering and

  14. Nested-quantitative PCR approach with improved sensitivity for the detection of low titer levels of Candidatus Liberibacter asiaticus in the Asian citrus psyllid, Diaphorina citri Kuwayama.

    PubMed

    Coy, M R; Hoffmann, M; Kingdom Gibbard, H N; Kuhns, E H; Pelz-Stelinski, K S; Stelinski, L L

    2014-07-01

    Candidatus Liberibacter asiaticus (CLas) is a phloem-limited bacterium transmitted by the Asian citrus psyllid, Diaphorina citri, and the presumptive causal agent of citrus greening disease. The current method of detection for CLas within plant and insect samples is by a presence/absence qPCR assay using the CLas 16S rDNA gene target. Although qPCR is highly sensitive, low bacterial titers or suboptimal qPCR conditions can result in false-negatives. Using a nested qPCR assay, we determined the false-negative rate of the 16S presence/absence qPCR assay was greater than 50%. Studies to determine the performance parameters of the qPCR assays for CLas 16S and Wingless (Wg), the D. citri endogenous gene, using plasmid and psyllid DNA, revealed suboptimal and variable performance of the 16S assay in psyllid samples. Average efficiencies and sensitivity limits of the plasmid assays were 99.0% and 2.7 copies of template for Wg, respectively, and 98.5% and 2.2-22.1 copies for 16S, respectively. Variability in efficiency was significantly greater in psyllid samples for both gene targets compared to the corresponding plasmid assays, and efficiencies as low as 76% were obtained for 16S. A secondary structure analysis revealed the formation of two stem-loop structures that block the forward and probe binding sites in the 16S template, which could hinder amplification. In summary, our results suggest that suboptimal qPCR efficiency is not uncommon for the 16S presence/absence qPCR assay, which combined with lowCLas titers in some samples, could contribute significantly to the under-reporting of CLas infection in psyllid and plant samples.

  15. Nested-quantitative PCR approach with improved sensitivity for the detection of low titer levels of Candidatus Liberibacter asiaticus in the Asian citrus psyllid, Diaphorina citri Kuwayama.

    PubMed

    Coy, M R; Hoffmann, M; Kingdom Gibbard, H N; Kuhns, E H; Pelz-Stelinski, K S; Stelinski, L L

    2014-07-01

    Candidatus Liberibacter asiaticus (CLas) is a phloem-limited bacterium transmitted by the Asian citrus psyllid, Diaphorina citri, and the presumptive causal agent of citrus greening disease. The current method of detection for CLas within plant and insect samples is by a presence/absence qPCR assay using the CLas 16S rDNA gene target. Although qPCR is highly sensitive, low bacterial titers or suboptimal qPCR conditions can result in false-negatives. Using a nested qPCR assay, we determined the false-negative rate of the 16S presence/absence qPCR assay was greater than 50%. Studies to determine the performance parameters of the qPCR assays for CLas 16S and Wingless (Wg), the D. citri endogenous gene, using plasmid and psyllid DNA, revealed suboptimal and variable performance of the 16S assay in psyllid samples. Average efficiencies and sensitivity limits of the plasmid assays were 99.0% and 2.7 copies of template for Wg, respectively, and 98.5% and 2.2-22.1 copies for 16S, respectively. Variability in efficiency was significantly greater in psyllid samples for both gene targets compared to the corresponding plasmid assays, and efficiencies as low as 76% were obtained for 16S. A secondary structure analysis revealed the formation of two stem-loop structures that block the forward and probe binding sites in the 16S template, which could hinder amplification. In summary, our results suggest that suboptimal qPCR efficiency is not uncommon for the 16S presence/absence qPCR assay, which combined with lowCLas titers in some samples, could contribute significantly to the under-reporting of CLas infection in psyllid and plant samples. PMID:24769405

  16. Improved PCR amplification for molecular analysis using DNA from long-term preserved formalin-fixed, paraffin-embedded lung cancer tissue specimens.

    PubMed

    Taga, Masataka; Eguchi, Hidetaka; Shinohara, Tomoko; Takahashi, Keiko; Ito, Reiko; Yasui, Wataru; Nakachi, Kei; Kusunoki, Yoichiro; Hamatani, Kiyohiro

    2013-01-01

    Archival tissue specimens are valuable resources of materials for molecular biological analyses in retrospective studies, especially for rare diseases or those associated with exposure to uncommon environmental events. Although successful amplification with PCR is essential for analysis of DNA extracted from archival formalin-fixed, paraffin-embedded (FFPE) tissue specimens, we have often encountered problems with poor PCR amplification of target fragments. To overcome this, we examined whether heat treatment in alkaline solution could efficiently restore the PCR template activity of DNA that had already been extracted from FFPE lung cancer tissue specimens. The effect of the heat treatment was assessed by PCR for the TP53 gene and other lung cancer-related gene loci. The heat treatment of DNA samples in borate buffer resulted in successful PCR amplification of DNA fragments ranging from 91 to 152 bp. This technique for restoration of template activity of DNA for PCR amplification is very simple and economical, and requires no special apparatus, so it may be applicable for molecular analysis of DNA samples from FFPE tissue specimens at various laboratories.

  17. Linear-After-The-Exponential (LATE)-PCR: primer design criteria for high yields of specific single-stranded DNA and improved real-time detection.

    PubMed

    Pierce, Kenneth E; Sanchez, J Aquiles; Rice, John E; Wangh, Lawrence J

    2005-06-14

    Traditional asymmetric PCR uses conventional PCR primers at unequal concentrations to generate single-stranded DNA. This method, however, is difficult to optimize, often inefficient, and tends to promote nonspecific amplification. An alternative approach, Linear-After-The-Exponential (LATE)-PCR, solves these problems by using primer pairs deliberately designed for use at unequal concentrations. The present report systematically examines the primer design parameters that affect the exponential and linear phases of LATE-PCR amplification. In particular, we investigated how altering the concentration-adjusted melting temperature (Tm) of the limiting primer (TmL) relative to that of the excess primer (TmX) affects both amplification efficiency and specificity during the exponential phase of LATE-PCR. The highest reaction efficiency and specificity were observed when TmL - TmX 5 degrees C. We also investigated how altering TmX relative to the higher Tm of the double-stranded amplicon (TmA) affects the rate and extent of linear amplification. Excess primers with TmX closer to TmA yielded higher rates of linear amplification and stronger signals from a hybridization probe. These design criteria maximize the yield of specific single-stranded DNA products and make LATE-PCR more robust and easier to implement. The conclusions were validated by using primer pairs that amplify sequences within the cystic fibrosis transmembrane regulator (CFTR) gene, mutations of which are responsible for cystic fibrosis.

  18. A Global Health Diagnostic for Personalized Medicine in Resource-Constrained World Settings: A Simple PCR-RFLP Method for Genotyping CYP2B6 g.15582C>T and Science and Policy Relevance for Optimal Use of Antiretroviral Drug Efavirenz.

    PubMed

    Evans, Jonathan; Swart, Marelize; Soko, Nyarai; Wonkam, Ambroise; Huzair, Farah; Dandara, Collet

    2015-06-01

    The use of pharmacogenomics (PGx) knowledge in treatment of individual patients is becoming a common phenomenon in the developed world. However, poorly resourced countries have thus far been constrained for three main reasons. First, the cost of whole genome sequencing is still considerably high in comparison to other (non-genomics) diagnostics in the developing world where both science and social dynamics create a dynamic and fragile healthcare ecosystem. Second, studies correlating genomic differences with drug pharmacokinetics and pharmacodynamics have not been consistent, and more importantly, often not indexed to impact on societal end-points, beyond clinical practice. Third, ethics regulatory frames over PGx testing require improvements based on nested accountability systems and in ways that address the user community needs. Thus, CYP2B6 is a crucial enzyme in the metabolism of antiretroviral drugs, efavirenz and nevirapine. More than 40 genetic variants have been reported, but only a few contribute to differences in plasma EFV and NVP concentrations. The most widely reported CYP2B6 variants affecting plasma drug levels include c.516G>T, c.983T>C, and to a lesser extent, g.15582C>T, which should be considered in future PGx tests. While the first two variants are easily characterized, the g.15582C>T detection has been performed primarily by sequencing, which is costly, labor intensive, and requires access to barely available expertise in the developing world. We report here on a simple, practical PCR-RFLP method with vast potentials for use in resource-constrained world regions to detect the g.15582C>T variation among South African and Cameroonian persons. The effects of CYP2B6 g.15582C>T on plasma EFV concentration were further evaluated among HIV/AIDS patients. We report no differences in the frequency of the g.15582T variant between the South African (0.08) and Cameroonian (0.06) groups, which are significantly lower than reported in Asians (0.39) and

  19. Improving Cognitive Diagnostic Computerized Adaptive Testing by Balancing Attribute Coverage: The Modified Maximum Global Discrimination Index Method

    ERIC Educational Resources Information Center

    Cheng, Ying

    2010-01-01

    This article proposes a new item selection method, namely, the modified maximum global discrimination index (MMGDI) method, for cognitive diagnostic computerized adaptive testing (CD-CAT). The new method captures two aspects of the appeal of an item: (a) the amount of contribution it can make toward adequate coverage of every attribute and (b) the…

  20. Development and Evaluation of a Novel Real-Time PCR for Pan-Dermatophyte Detection in Nail Specimens.

    PubMed

    Gong, Jie; Ran, Menglong; Wang, Xiaowen; Wan, Zhe; Li, Ruoyu

    2016-02-01

    An accurate diagnosis of tinea unguium is necessary for the selection of antimycotics and successful treatment. To rapidly and accurately identify the aetiological agents causing tinea unguium, we improved upon the conventional boiling method for DNA extraction and developed a novel real-time PCR detection system that includes two assays. The two assays, based on the amplification of ribosomal internal transcribed spacer regions and 28S rDNA, were designed to detect pan-dermatophyte and Trichophyton rubrum, respectively. The analytical sensitivities of both assays permitted the detection of ten copies of plasmid DNA template. The analytical specificity of the detection system was confirmed using 11 dermatophyte strains and 25 non-dermatophyte strains. In total, 165 nail specimens were examined by microscopy, culture, conventional PCR, and the novel real-time PCR method. Real-time PCR gave positive results in 47.3 % of the specimens (78), a rate exceeding those obtained using microscopy (72, 43.6 %), conventional PCR (69, 41.8 %), and culture (49, 29.7 %). All conventional PCR-positive specimens were detected by real-time PCR, and real-time PCR detected nine specimens that were missed by conventional PCR. The results from latent class analysis, and further calculations, showed that real-time PCR was the most sensitive method, but the diagnostic specificity of the four approaches was equivalent. In particular, molecular approaches may be more effective than microscopy and culture when the clinical symptoms of tinea unguium are not evident.

  1. A Review of 20 Years of Research on Overdiagnosis and Underdiagnosis in the Rhode Island Methods to Improve Diagnostic Assessment and Services (MIDAS) Project.

    PubMed

    Zimmerman, Mark

    2016-02-01

    The Rhode Island Methods to Improve Diagnostic Assessment and Services (MIDAS) project represents an integration of research methodology into a community-based outpatient practice affiliated with an academic medical centre. The MIDAS project is the largest clinical epidemiological study using semi-structured interviews to assess a wide range of psychiatric disorders in a general clinical outpatient practice. In an early report from the MIDAS project, we found that across diagnostic categories clinicians using unstandardized, unstructured clinical interviews underrecognized diagnostic comorbidity, compared with the results of semi-structured interviews. Moreover, we found that the patients often wanted treatment for symptoms of disorders that were diagnosed as comorbid, rather than principal, conditions. This highlighted the importance, from the patient's perspective, of conducting thorough diagnostic interviews to diagnose disorders that are not related to the patient's chief complaint because patients often desire treatment for these additional diagnoses. While several of the initial papers from the MIDAS project identified problems with the detection of comorbid disorders in clinical practice, regarding the diagnosis of bipolar disorder we observed the emergence of an opposite phenomenon-clinician overdiagnosis. The results from the MIDAS project, along with other studies of diagnosis in routine clinical practice, have brought to the forefront the problem with diagnosis in routine clinical practice. An important question is what do these findings suggest about the community standard of care in making psychiatric diagnoses, and whether and how the standard of care should be changed? The implications are discussed. PMID:27253697

  2. A Review of 20 Years of Research on Overdiagnosis and Underdiagnosis in the Rhode Island Methods to Improve Diagnostic Assessment and Services (MIDAS) Project.

    PubMed

    Zimmerman, Mark

    2016-02-01

    The Rhode Island Methods to Improve Diagnostic Assessment and Services (MIDAS) project represents an integration of research methodology into a community-based outpatient practice affiliated with an academic medical centre. The MIDAS project is the largest clinical epidemiological study using semi-structured interviews to assess a wide range of psychiatric disorders in a general clinical outpatient practice. In an early report from the MIDAS project, we found that across diagnostic categories clinicians using unstandardized, unstructured clinical interviews underrecognized diagnostic comorbidity, compared with the results of semi-structured interviews. Moreover, we found that the patients often wanted treatment for symptoms of disorders that were diagnosed as comorbid, rather than principal, conditions. This highlighted the importance, from the patient's perspective, of conducting thorough diagnostic interviews to diagnose disorders that are not related to the patient's chief complaint because patients often desire treatment for these additional diagnoses. While several of the initial papers from the MIDAS project identified problems with the detection of comorbid disorders in clinical practice, regarding the diagnosis of bipolar disorder we observed the emergence of an opposite phenomenon-clinician overdiagnosis. The results from the MIDAS project, along with other studies of diagnosis in routine clinical practice, have brought to the forefront the problem with diagnosis in routine clinical practice. An important question is what do these findings suggest about the community standard of care in making psychiatric diagnoses, and whether and how the standard of care should be changed? The implications are discussed.

  3. A macro-ergonomic work system analysis of the diagnostic testing process in an outpatient health care facility for process improvement and patient safety.

    PubMed

    Hallock, M L; Alper, S J; Karsh, B

    The diagnosis of illness is important for quality patient care and patient safety and is greatly aided by diagnostic testing. For diagnostic tests, such as pathology and radiology, to positively impact patient care, the tests must be processed and the physician and patient must be notified of the results in a timely fashion. There are many steps in the diagnostic testing process, from ordering to result dissemination, where the process can break down and therefore delay patient care and reduce patient safety. This study was carried out to examine the diagnostic testing process (i.e. from ordering to result notification) and used a macro-ergonomic work system analysis to uncover system design flaws that contributed to delayed physician and patient notification of results. The study was carried out in a large urban outpatient health-care facility made up of 30 outpatient clinics. Results indicated a number of variances that contributed to delays, the majority of which occurred across the boundaries of different systems and were related to poor or absent feedback structures. Recommendations for improvements are discussed.

  4. An Improved PCR-RFLP Assay for Detection and Genotyping of Asymptomatic Giardia lamblia Infection in a Resource-Poor Setting.

    PubMed

    Hawash, Yoursry; Ghonaim, M M; Al-Shehri, S S

    2016-02-01

    Laboratory workers, in resource-poor countries, still consider PCR detection of Giardia lamblia more costly and more time-consuming than the classical parasitological techniques. Based on 2 published primers, an in-house one-round touchdown PCR-RFLP assay was developed. The assay was validated with an internal amplification control included in reactions. Performance of the assay was assessed with DNA samples of various purities, 91 control fecal samples with various parasite load, and 472 samples of unknown results. Two cysts per reaction were enough for PCR detection by the assay with exhibited specificity (Sp) and sensitivity (Se) of 100% and 93%, respectively. Taking a published small subunit rRNA reference PCR test results (6%; 29/472) as a nominated gold standard, G. lamblia was identified in 5.9% (28/472), 5.2%, (25/472), and 3.6% (17/472) by PCR assay, RIDA(®) Quick Giardia antigen detection test (R-Biopharm, Darmstadt, Germany), and iodine-stained smear microscopy, respectively. The percent agreements (kappa values) of 99.7% (0.745), 98.9% (0.900), and 97.7% (0.981) were exhibited between the assay results and that of the reference PCR, immunoassay, and microscopy, respectively. Restriction digestion of the 28 Giardia-positive samples revealed genotype A pattern in 12 and genotype B profile in 16 samples. The PCR assay with the described format and exhibited performance has a great potential to be adopted in basic clinical laboratories as a detection tool for G. lamblia especially in asymptomatic infections. This potential is increased more in particular situations where identification of the parasite genotype represents a major requirement as in epidemiological studies and infection outbreaks.

  5. Semi-quantitative RT-PCR-based assay, improved by Southern hybridization technique, for polarity-specific influenza virus RNAs in cultured cells.

    PubMed

    Uchide, Noboru; Ohyama, Kunio; Bessho, Toshio; Yamakawa, Toshio

    2002-10-01

    Complementary (c) DNAs against viral (v) RNA of negative polarity and complementary and/or messenger (c/m) RNA of positive polarity for influenza virus hemagglutinin (HA) were synthesized from total cellular RNA extracted from influenza virus- and mock-infected cells using polarity-specific primers, respectively. HA vRNA and c/mRNA were amplified readily by polymerase chain reaction (PCR) from influenza virus-infected cells during a virus productive period; however, non-specific PCR product was prone to amplification from mock-infected cells and cells at once after virus infection. Southern blots of the PCR products were hybridized with biotinylated DNA probe, which enabled the generation of specific signals to HA vRNA and c/mRNA. Mock-infected cells produced no signals. Furthermore, titration analyses revealed linear relationships between amount of target RNAs and generated signals. Accordingly, Southern hybridization made possible the quantitation of specific PCR products for HA vRNA and c/mRNA in cell culture and proved the lack of HA RNAs in mock-infected cells in the absence of virus. The RT-PCR based assay combined with Southern hybridization methodology was useful with respect for investigating the processes of replication and transcription of viral genes in cell culture before and during the virus productive period.

  6. The impact of genetic diversity in protozoa on molecular diagnostics.

    PubMed

    Stensvold, C Rune; Lebbad, Marianne; Verweij, Jaco J

    2011-02-01

    Detection of intestinal parasitic protists, commonly referred to as 'intestinal protozoa,' by PCR is increasingly used not only for identification or confirmation but also as a first-line diagnostic tool. Apart from the ability to sample correctly and extract parasite DNA directly from faeces, primer and probe specificity and sensitivity affect predictive values and hence the utility of diagnostic assays. Molecular characterization of intestinal protists is necessary to design primers and probes because this is the basic material for current and future improved diagnostic PCRs for either detecting all genetic variants or specifically differentiating among such variants. As an example, this paper highlights the existence of interspecific and intraspecific genetic diversity among intestinal, unicellular parasites and its implications for nucleic acid-based diagnostic assays.

  7. Survey of diagnostic and typing capacity for Clostridium difficile infection in Europe, 2011 and 2014.

    PubMed

    van Dorp, Sofie M; Notermans, Daan W; Alblas, Jeroen; Gastmeier, Petra; Mentula, Silja; Nagy, Elisabeth; Spigaglia, Patrizia; Ivanova, Katiusha; Fitzpatrick, Fidelma; Barbut, Frédéric; Morris, Trefor; Wilcox, Mark H; Kinross, Pete; Suetens, Carl; Kuijper, Ed J

    2016-07-21

    Suboptimal laboratory diagnostics for Clostridium difficile infection (CDI) impedes its surveillance and control across Europe. We evaluated changes in local laboratory CDI diagnostics and changes in national diagnostic and typing capacity for CDI during the European C. difficile Infection Surveillance Network (ECDIS-Net) project, through cross-sectional surveys in 33 European countries in 2011 and 2014. In 2011, 126 (61%) of a convenience sample of 206 laboratories in 31 countries completed a survey on local diagnostics. In 2014, 84 (67%) of these 126 laboratories in 26 countries completed a follow-up survey. Among laboratories that participated in both surveys, use of CDI diagnostics deemed 'optimal' or 'acceptable' increased from 19% to 46% and from 10% to 15%, respectively (p  < 0.001). The survey of national capacity was completed by national coordinators of 31 and 32 countries in 2011 and 2014, respectively. Capacity for any C. difficile typing method increased from 22/31 countries in 2011 to 26/32 countries in 2014; for PCR ribotyping from 20/31 countries to 23/32 countries, and specifically for capillary PCR ribotyping from 7/31 countries to 16/32 countries. While our study indicates improved diagnostic capability and national capacity for capillary PCR ribotyping across European laboratories between 2011 and 2014, increased use of 'optimal' diagnostics should be promoted. PMID:27469624

  8. Autism according to diagnostic and statistical manual of mental disorders 5(th) edition: The need for further improvements.

    PubMed

    Posar, Annio; Resca, Federica; Visconti, Paola

    2015-01-01

    The fifth edition of the diagnostic and statistical manual of mental disorders (DSM-5) introduced significant changes in the classification of autism spectrum disorders (ASD), including the abolition of the diagnostic subcategories proposed by DSM-IV-Text Revision. DSM-5 describes three levels of increasing severity of ASD. The authors report two explanatory cases with ASD (verbal boys, aged about 7 and a half years, without intellectual disability). According to DSM-5, both cases fall into the lowest severity level of ASD. However, their neuropsychological and neurobehavioral profile varies significantly. While the first boy showed a prevalent impairment of visuoconstructional and visuoperceptual abilities, the second one presented a predominant involvement of verbal functions, with qualitative impairments in communication. A further step forward in the definition and classification of ASD, taking into account both intensity and quality of symptoms, is recommended in order to formulate a reliable prognosis, plan an individualized treatment and monitor the clinical course over time.

  9. Reverse-transcription PCR (RT-PCR).

    PubMed

    Bachman, Julia

    2013-01-01

    RT-PCR is commonly used to test for genetic diseases and to characterize gene expression in various tissue types, cell types, and over developmental time courses. This serves as a form of expression profiling, but typically as a candidate approach. RT-PCR is also commonly used to clone cDNAs for further use with other molecular biology techniques (e.g., see Oligo(dT)-primed RT-PCR isolation of polyadenylated RNA degradation intermediates and Circularized RT-PCR (cRT-PCR): analysis of RNA 5' ends, 3' ends, and poly(A) tails).

  10. Effectiveness of Practices for Improving the Diagnostic Accuracy of Non-ST Elevation Myocardial Infarction in the Emergency Department: A Laboratory Medicine Best Practices Systematic Review

    PubMed Central

    Layfield, Christopher; Rose, John; Alford, Aaron; Snyder, Susan R.; Apple, Fred S.; Chowdhury, Farah M.; Kontos, Michael C.; Newby, L. Kristin; Storrow, Alan B.; Tanasijevic, Milenko; Leibach, Elizabeth; Liebow, Edward B.; Christenson, Robert H.

    2016-01-01

    Objectives This article presents evidence from a systematic review of the effectiveness of four practices (assay selection, decision point cardiac troponin (cTn) threshold selection, serial testing, and point of care testing) for improving the diagnostic accuracy for Non-ST-Segment Elevation Myocardial Infarction (NSTEMI) in the Emergency Department. Design and Methods The CDC-funded Laboratory Medicine Best Practices (LMBP™) Initiative systematic review A6 Method for Laboratory Best Practices was used. Results The current guidelines (e.g., ACC/AHA) recommend using cardiac troponin assays with a 99th percentile upper reference limit (URL) diagnostic threshold to diagnose NSTEMI. The evidence in this systematic review indicates that contemporary sensitive cTn assays meet the assay profile requirements (sensitivity, specificity, PPV, and NPV) to more accurately diagnose NSTEMI than alternate tests. Additional biomarkers did not increase diagnostic effectiveness of cTn assays. Sensitivity, specificity, and negative predictive value (NPV) were consistently high and low positive predictive value (PPV) improved with serial sampling. Evidence for use of cTn point of care testing (POCT) was insufficient to make recommendations, though some evidence suggests cTn POCT may result in reduction to patient length of stay and costs. Conclusions Two best practice recommendations emerged from the systematic review and meta-analysis of literature conducted using the LMBP™ A6 Method criteria: Testing with cardiac troponin assays, using the 99th percentile URL as the clinical diagnostic threshold for the diagnosis of NSTEMI and without additional biomarkers, is recommended. Also recommended is serial cardiac troponin sampling with one sample at presentation and at least one additional sample taken a minimum of 6 hours later to identify a rise or fall in the troponin level. Testing with high-sensitivity cardiac troponin assays, at presentation and again within 6 hours, is the

  11. An improved, high-throughput method for detection of bluetongue virus RNA in Culicoides midges utilizing infrared-dye-labeled primers for reverse transcriptase PCR.

    PubMed

    Kato, Cecilia Y; Mayer, Richard T

    2007-03-01

    A new rapid (less than 6h from insect-to-results) high-throughput assay that is sensitive and specific for detecting BTV RNA in Culicoides biting midges is reported. Homogenization and extraction of nucleic acids from individual Culicoides specimens were performed in a 96-well plate format using specialized beads in a homogenization buffer compatible with cell culture and RNA extraction. A portion of homogenate (10%) from each specimen was retained for confirmatory infectious virus isolation, while the remaining 90% was used for RNA extraction. The RNA was used in a single step reverse transcriptase PCR (RT-PCR) reaction with infrared (IR)-dye-labeled primers. The RT-PCR products were visualized in agarose gels with an infrared scanner. The adaptation of IR-dye-labeled primers in combination with a one step RT-PCR resulted in a detection limit of 0.5 pfu of purified BTV RNA. All 24 serotypes of BTV prototype strains and none of the 8 serotypes of the closely related epizootic hemorrhagic disease virus (EHDV) prototype strains were detected.

  12. Improved methodology for identification of protists and microalgae from plankton samples preserved in Lugol's iodine solution: combining microscopic analysis with single-cell PCR.

    PubMed

    Auinger, Barbara M; Pfandl, Karin; Boenigk, Jens

    2008-04-01

    Here we introduce a method for quantitative analysis of planktonic protists and microalgae from preserved field samples combining morphological and small-subunit (SSU) rRNA gene sequence analysis. We linked a microscopic screening with PCR of single cells using field samples preserved with Lugol's iodine solution. Cells possessing a rigid cell wall were incubated with Viscozyme and subsequently with proteinase K for cell disruption; this was unnecessary for fragile cells. The addition of sodium thiosulfate to the PCR tube considerably decreased the inhibiting effect of the fixative (iodine) on the PCR and thus allowed for successful single-cell PCR even of long DNA fragments (up to as many as 3,000 base pairs). We further applied the protocol to investigate the dominant SSU rRNA genotypes in distinct flagellate morphospecies originating from different samples. We hypothesized that despite the morphological similarity, protist morphospecies in different habitats or sampled during different seasons are represented by different genotypes. Our results indicate species-specific differences: the two species Ochromonas sp. and Dinobryon divergens were represented by several different genotypes each, and for the latter species, the dominating genotype differed with habitat. In contrast, Dinobryon pediforme, Dinobryon bavaricum, and Synura sphagnicola were exclusively represented by a single genotype each, and the respective genotype was the same in different samples. In summary, our results highlight the significance of molecular variation within protist morphospecies.

  13. An Improved Quantitative Real-Time PCR Assay for the Enumeration of Heterosigma akashiwo (Raphidophyceae) Cysts Using a DNA Debris Removal Method and a Cyst-Based Standard Curve.

    PubMed

    Kim, Joo-Hwan; Kim, Jin Ho; Wang, Pengbin; Park, Bum Soo; Han, Myung-Soo

    2016-01-01

    The identification and quantification of Heterosigma akashiwo cysts in sediments by light microscopy can be difficult due to the small size and morphology of the cysts, which are often indistinguishable from those of other types of algae. Quantitative real-time PCR (qPCR) based assays represent a potentially efficient method for quantifying the abundance of H. akashiwo cysts, although standard curves must be based on cyst DNA rather than on vegetative cell DNA due to differences in gene copy number and DNA extraction yield between these two cell types. Furthermore, qPCR on sediment samples can be complicated by the presence of extracellular DNA debris. To solve these problems, we constructed a cyst-based standard curve and developed a simple method for removing DNA debris from sediment samples. This cyst-based standard curve was compared with a standard curve based on vegetative cells, as vegetative cells may have twice the gene copy number of cysts. To remove DNA debris from the sediment, we developed a simple method involving dilution with distilled water and heating at 75°C. A total of 18 sediment samples were used to evaluate this method. Cyst abundance determined using the qPCR assay without DNA debris removal yielded results up to 51-fold greater than with direct counting. By contrast, a highly significant correlation was observed between cyst abundance determined by direct counting and the qPCR assay in conjunction with DNA debris removal (r2 = 0.72, slope = 1.07, p < 0.001). Therefore, this improved qPCR method should be a powerful tool for the accurate quantification of H. akashiwo cysts in sediment samples.

  14. An Improved Quantitative Real-Time PCR Assay for the Enumeration of Heterosigma akashiwo (Raphidophyceae) Cysts Using a DNA Debris Removal Method and a Cyst-Based Standard Curve

    PubMed Central

    Park, Bum Soo; Han, Myung-Soo

    2016-01-01

    The identification and quantification of Heterosigma akashiwo cysts in sediments by light microscopy can be difficult due to the small size and morphology of the cysts, which are often indistinguishable from those of other types of algae. Quantitative real-time PCR (qPCR) based assays represent a potentially efficient method for quantifying the abundance of H. akashiwo cysts, although standard curves must be based on cyst DNA rather than on vegetative cell DNA due to differences in gene copy number and DNA extraction yield between these two cell types. Furthermore, qPCR on sediment samples can be complicated by the presence of extracellular DNA debris. To solve these problems, we constructed a cyst-based standard curve and developed a simple method for removing DNA debris from sediment samples. This cyst-based standard curve was compared with a standard curve based on vegetative cells, as vegetative cells may have twice the gene copy number of cysts. To remove DNA debris from the sediment, we developed a simple method involving dilution with distilled water and heating at 75°C. A total of 18 sediment samples were used to evaluate this method. Cyst abundance determined using the qPCR assay without DNA debris removal yielded results up to 51-fold greater than with direct counting. By contrast, a highly significant correlation was observed between cyst abundance determined by direct counting and the qPCR assay in conjunction with DNA debris removal (r2 = 0.72, slope = 1.07, p < 0.001). Therefore, this improved qPCR method should be a powerful tool for the accurate quantification of H. akashiwo cysts in sediment samples. PMID:26741648

  15. Phased-array ultrasound technology enhances accuracy of dual frequency ultrasound measurements - towards improved ultrasound bone diagnostics.

    PubMed

    Linder, Hans; Malo, Markus K H; Liukkonen, Jukka; Jurvelin, Jukka S; Töyräs, Juha

    2016-08-01

    Overlying soft tissues attenuate ultrasound backscattered from bone, complicating diagnostics of osteoporosis at the most important fracture sites. Dual-frequency ultrasound technique (DFUS) has been proposed to solve this problem through determination of thickness and composition of overlying soft tissue. This study applies DFUS technique for the first time with a phased-array transducer to investigate if the thickness of two interfering layers (oil and water) can be accurately determined in a variety of configurations. Results indicate that DFUS may be used with phased-array ultrasound systems, making them a suitable combination to consider in future development of clinical in vivo ultrasound methodologies. PMID:27187271

  16. Phased-array ultrasound technology enhances accuracy of dual frequency ultrasound measurements - towards improved ultrasound bone diagnostics.

    PubMed

    Linder, Hans; Malo, Markus K H; Liukkonen, Jukka; Jurvelin, Jukka S; Töyräs, Juha

    2016-08-01

    Overlying soft tissues attenuate ultrasound backscattered from bone, complicating diagnostics of osteoporosis at the most important fracture sites. Dual-frequency ultrasound technique (DFUS) has been proposed to solve this problem through determination of thickness and composition of overlying soft tissue. This study applies DFUS technique for the first time with a phased-array transducer to investigate if the thickness of two interfering layers (oil and water) can be accurately determined in a variety of configurations. Results indicate that DFUS may be used with phased-array ultrasound systems, making them a suitable combination to consider in future development of clinical in vivo ultrasound methodologies.

  17. Development and validation of real-time PCR for the detection of Yersinia ruckeri.

    PubMed

    Keeling, S E; Johnston, C; Wallis, R; Brosnahan, C L; Gudkovs, N; McDonald, W L

    2012-02-01

    Yersiniosis (enteric red mouth disease) is a contagious bacterial disease caused by Yersinia ruckeri, which primarily affects salmonids. A real-time PCR assay using a molecular beacon has been developed and validated to improve the detection of the causative biotypes of Y. ruckeri. The assay, which targets the glnA (glutamine synthetase) gene, proved to have 100% analytical specificity and analytical sensitivities of 5 fg and 3 × 10(3) CFU g(-1) for DNA and seeded kidney tissue, respectively. The assay was highly repeatable with low % CV for intra- and inter-run experiments, and the optimized parameters transferred easily between different real-time PCR platforms. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon (n = 750) from 10 farms during 2007/08. The real-time PCR was run in parallel with the bacterial culture detection method, and all fish tested were found to be negative by both methods for Y. ruckeri, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real-time PCR system is specific, sensitive, reproducible and a rapid method for the detection of Y. ruckeri and has the potential to be used for routine diagnostic testing, health certification and active surveillance programmes.

  18. Molecular diagnostics for fungal plant pathogens.

    PubMed

    McCartney, H Alastair; Foster, Simon J; Fraaije, Bart A; Ward, Elaine

    2003-02-01

    Accurate identification of fungal phytopathogens is essential for virtually all aspects of plant pathology, from fundamental research on the biology of pathogens to the control of the diseases they cause. Although molecular methods, such as polymerase chain reaction (PCR), are routinely used in the diagnosis of human diseases, they are not yet widely used to detect and identify plant pathogens. Here we review some of the diagnostic tools currently used for fungal plant pathogens and describe some novel applications. Technological advances in PCR-based methods, such as real-time PCR, allow fast, accurate detection and quantification of plant pathogens and are now being applied to practical problems. Molecular methods have been used to detect several pathogens simultaneously in wheat, and to study the development of fungicide resistance in wheat pathogens. Information resulting from such work could be used to improve disease control by allowing more rational decisions to be made about the choice and use of fungicides and resistant cultivars. Molecular methods have also been applied to the study of variation in plant pathogen populations, for example detection of different mating types or virulence types. PCR-based methods can provide new tools to monitor the exposure of a crop to pathogen inoculum that are more reliable and faster than conventional methods. This information can be used to improve disease control decision making. The development and application of molecular diagnostic methods in the future is discussed and we expect that new developments will increase the adoption of these new technologies for the diagnosis and study of plant disease.

  19. Tear-off patterning: a simple method for patterning nitrocellulose membranes to improve the performance of point-of-care diagnostic biosensors.

    PubMed

    Song, Mun-Bum; Joung, Hyou-Arm; Oh, Young Kyoung; Jung, Kwonyoung; Ahn, Young Deok; Kim, Min-Gon

    2015-07-21

    This article describes a new method, referred to as "tear-off patterning," for patterning nitrocellulose (NC) membranes in order to fabricate NC-based point-of-care (POC) diagnostic devices. Paper-based microfluidic sensors usually employ hydrophobic barrier coatings such as paraffin wax on either paper or membranes. Herein, complex patterns were fabricated by stamping the target area with dimethyl sulfoxide before tearing off the stamped area. Fluid flow and morphological analyses were performed in order to characterize the patterned membranes. Furthermore, the myoglobin and creatine kinase-MB levels in human serum were measured simultaneously using a dual-fluidic-channel-patterned NC membrane in order to confirm the usefulness of the patterning method for fabricating POC biosensors. The proposed method for patterning NC membranes offers clear advantages, such as the ability to fabricate complex designs and patterns without a hydrophobic barrier after protein immobilization in a laboratory and in a simple, low-cost manner. We believe that this method can be used to develop various POC diagnostic biosensors at the research and development stage and can help improve the performance and features of POC diagnostic devices. PMID:26062104

  20. Point-of-Care Multi-Organ Ultrasound Improves Diagnostic Accuracy in Adults Presenting to the Emergency Department with Acute Dyspnea

    PubMed Central

    Mantuani, Daniel; Frazee, Bradley W.; Fahimi, Jahan; Nagdev, Arun

    2016-01-01

    Introduction Determining the etiology of acute dyspnea in emregency department (ED) patients is often difficult. Point-of-care ultrasound (POCUS) holds promise for improving immediate diagnostic accuracy (after history and physical), thus improving use of focused therapies. We evaluate the impact of a three-part POCUS exam, or “triple scan” (TS) – composed of abbreviated echocardiography, lung ultrasound and inferior vena cava (IVC) collapsibility assessment – on the treating physician’s immediate diagnostic impression. Methods A convenience sample of adults presenting to our urban academic ED with acute dyspnea (Emergency Severity Index 1, 2) were prospectively enrolled when investigator sonographers were available. The method for performing components of the TS has been previously described in detail. Treating physicians rated the most likely diagnosis after history and physical but before other studies (except electrocardiogram) returned. An investigator then performed TS and disclosed the results, after which most likely diagnosis was reassessed. Final diagnosis (criterion standard) was based on medical record review by expert emergency medicine faculty blinded to TS result. We compared accuracy of pre-TS and post-TS impression (primary outcome) with McNemar’s test. Test characteristics for treating physician impression were also calculated by dichotomizing acute decompensated heart failure (ADHF), chronic obstructive pulmonary disease (COPD) and pneumonia as present or absent. Results 57 patients were enrolled with the leading final diagnoses being ADHF (26%), COPD/asthma (30%), and pneumonia (28%). Overall accuracy of the treating physician’s impression increased from 53% before TS to 77% after TS (p=0.003). The post-TS impression was 100% sensitive and 84% specific for ADHF. Conclusion In this small study, POCUS evaluation of the heart, lungs and IVC improved the treating physician’s immediate overall diagnostic accuracy for ADHF, COPD

  1. Sex Determination Using PCR

    ERIC Educational Resources Information Center

    Kima, Peter E.; Rasche, Madeline E.

    2004-01-01

    PCR has revolutionized many aspects of biochemistry and molecular biology research. In the following exercise, students learn PCR by isolating their own DNA, amplifying specific segments of the X and Y chromosomes, and estimating the sizes of the PCR products using agarose gel electrophoresis. Based on the pattern of PCR products, students can…

  2. Improved Efficiency and Reliability of NGS Amplicon Sequencing Data Analysis for Genetic Diagnostic Procedures Using AGSA Software

    PubMed Central

    Poulet, Axel; Privat, Maud; Viala, Sandrine; Decousus, Stephanie; Perin, Axel; Lafarge, Laurence; Ollier, Marie; El Saghir, Nagi S.

    2016-01-01

    Screening for BRCA mutations in women with familial risk of breast or ovarian cancer is an ideal situation for high-throughput sequencing, providing large amounts of low cost data. However, 454, Roche, and Ion Torrent, Thermo Fisher, technologies produce homopolymer-associated indel errors, complicating their use in routine diagnostics. We developed software, named AGSA, which helps to detect false positive mutations in homopolymeric sequences. Seventy-two familial breast cancer cases were analysed in parallel by amplicon 454 pyrosequencing and Sanger dideoxy sequencing for genetic variations of the BRCA genes. All 565 variants detected by dideoxy sequencing were also detected by pyrosequencing. Furthermore, pyrosequencing detected 42 variants that were missed with Sanger technique. Six amplicons contained homopolymer tracts in the coding sequence that were systematically misread by the software supplied by Roche. Read data plotted as histograms by AGSA software aided the analysis considerably and allowed validation of the majority of homopolymers. As an optimisation, additional 250 patients were analysed using microfluidic amplification of regions of interest (Access Array Fluidigm) of the BRCA genes, followed by 454 sequencing and AGSA analysis. AGSA complements a complete line of high-throughput diagnostic sequence analysis, reducing time and costs while increasing reliability, notably for homopolymer tracts. PMID:27656653

  3. Routine clinical mutation profiling using next generation sequencing and a customized gene panel improves diagnostic precision in myeloid neoplasms

    PubMed Central

    Bartels, Stephan; Schipper, Elisa; Hasemeier, Britta; Kreipe, Hans; Lehmann, Ulrich

    2016-01-01

    Microscopic examination of myelodysplastic syndromes (MDS) and myelodysplastic-myeloproliferative neoplasms (MDS/MPN) may be challenging because morphological features can overlap with those of reactive states. Demonstration of clonal hematopoiesis provides a diagnostic clue and has become possible by comprehensive mutation profiling of a number of frequently mutated genes, some of them with large coding regions. To emphasize the potential benefit of NGS in hematopathology we present sequencing results from routinely processed formalin-fixed and paraffin-embedded (FFPE) bone marrow trephines (n = 192). A customized amplicon-based gene panel including 23 genes frequently mutated in myeloid neoplasms was established and implemented. Thereby, 629,691 reads per sample (range 179,847–1,460,412) and a mean coverage of 2,702 (range 707–6,327) could be obtained, which are sufficient for comprehensive mutational profiling. Seven samples failed in sequencing (3.6%). In 185 samples we found in total 269 pathogenic variants (mean 1.4 variants per patient, range 0-5), 125 Patients exhibit at least one pathogenic mutation (67.6%). Variants show allele frequencies ranging from 6.7% up to 95.7%. Most frequently mutated genes were TET2 (28.7%), SRSF2 (19.5%), ASXL1 (8.6%) and U2AF1 (8.1%). The mutation profiling increases the diagnostic precision and adds prognostic information. PMID:27029036

  4. Routine clinical mutation profiling using next generation sequencing and a customized gene panel improves diagnostic precision in myeloid neoplasms.

    PubMed

    Bartels, Stephan; Schipper, Elisa; Hasemeier, Britta; Kreipe, Hans; Lehmann, Ulrich

    2016-05-24

    Microscopic examination of myelodysplastic syndromes (MDS) and myelodysplastic-myeloproliferative neoplasms (MDS/MPN) may be challenging because morphological features can overlap with those of reactive states. Demonstration of clonal hematopoiesis provides a diagnostic clue and has become possible by comprehensive mutation profiling of a number of frequently mutated genes, some of them with large coding regions.To emphasize the potential benefit of NGS in hematopathology we present sequencing results from routinely processed formalin-fixed and paraffin-embedded (FFPE) bone marrow trephines (n = 192). A customized amplicon-based gene panel including 23 genes frequently mutated in myeloid neoplasms was established and implemented. Thereby, 629,691 reads per sample (range 179,847-1,460,412) and a mean coverage of 2,702 (range 707-6,327) could be obtained, which are sufficient for comprehensive mutational profiling. Seven samples failed in sequencing (3.6%). In 185 samples we found in total 269 pathogenic variants (mean 1.4 variants per patient, range 0-5), 125 Patients exhibit at least one pathogenic mutation (67.6%). Variants show allele frequencies ranging from 6.7% up to 95.7%. Most frequently mutated genes were TET2 (28.7%), SRSF2 (19.5%), ASXL1 (8.6%) and U2AF1 (8.1%). The mutation profiling increases the diagnostic precision and adds prognostic information. PMID:27029036

  5. Improved Efficiency and Reliability of NGS Amplicon Sequencing Data Analysis for Genetic Diagnostic Procedures Using AGSA Software

    PubMed Central

    Poulet, Axel; Privat, Maud; Viala, Sandrine; Decousus, Stephanie; Perin, Axel; Lafarge, Laurence; Ollier, Marie; El Saghir, Nagi S.

    2016-01-01

    Screening for BRCA mutations in women with familial risk of breast or ovarian cancer is an ideal situation for high-throughput sequencing, providing large amounts of low cost data. However, 454, Roche, and Ion Torrent, Thermo Fisher, technologies produce homopolymer-associated indel errors, complicating their use in routine diagnostics. We developed software, named AGSA, which helps to detect false positive mutations in homopolymeric sequences. Seventy-two familial breast cancer cases were analysed in parallel by amplicon 454 pyrosequencing and Sanger dideoxy sequencing for genetic variations of the BRCA genes. All 565 variants detected by dideoxy sequencing were also detected by pyrosequencing. Furthermore, pyrosequencing detected 42 variants that were missed with Sanger technique. Six amplicons contained homopolymer tracts in the coding sequence that were systematically misread by the software supplied by Roche. Read data plotted as histograms by AGSA software aided the analysis considerably and allowed validation of the majority of homopolymers. As an optimisation, additional 250 patients were analysed using microfluidic amplification of regions of interest (Access Array Fluidigm) of the BRCA genes, followed by 454 sequencing and AGSA analysis. AGSA complements a complete line of high-throughput diagnostic sequence analysis, reducing time and costs while increasing reliability, notably for homopolymer tracts.

  6. Comparison of advanced optical imaging techniques with current otolaryngology diagnostics for improved middle ear assessment (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Nolan, Ryan M.; Shelton, Ryan L.; Monroy, Guillermo L.; Spillman, Darold R.; Novak, Michael A.; Boppart, Stephen A.

    2016-02-01

    Otolaryngologists utilize a variety of diagnostic techniques to assess middle ear health. Tympanometry, audiometry, and otoacoustic emissions examine the mobility of the tympanic membrane (eardrum) and ossicles using ear canal pressure and auditory tone delivery and detection. Laser Doppler vibrometry provides non-contact vibrational measurement, and acoustic reflectometry is used to assess middle ear effusion using sonar. These technologies and techniques have advanced the field beyond the use of the standard otoscope, a simple tissue magnifier, yet the need for direct visualization of middle ear disease for superior detection, assessment, and management remains. In this study, we evaluated the use of portable optical coherence tomography (OCT) and pneumatic low-coherence interferometry (LCI) systems with handheld probe delivery to standard tympanometry, audiometry, otoacoustic emissions, laser Doppler vibrometry, and acoustic reflectometry. Comparison of these advanced optical imaging techniques and current diagnostics was conducted with a case study subject with a history of unilateral eardrum trauma. OCT and pneumatic LCI provide novel dynamic spatiotemporal structural data of the middle ear, such as the thickness of the eardrum and quantitative detection of underlying disease pathology, which could allow for more accurate diagnosis and more appropriate management than currently possible.

  7. Application of real time PCR for diagnosis of Swine Dysentery.

    PubMed

    Akase, Satoru; Uchitani, Yumi; Sohmura, Yoshiko; Tatsuta, Keikichi; Sadamasu, Kenji; Adachi, Yoshikazu

    2009-03-01

    Evaluation of a genetic diagnostic technique using real time PCR of Swine Dysentery (SD) was performed using nox primers. Culture, ordinary PCR and real time PCR were compared in this experiment. Sixty-seven specimens from pigs with clinical signs of SD brought to a slaughterhouse in Shibaura, Tokyo, were used. B. hyodysenteriae was isolated from 49 of the pigs, was detected by ordinary PCR in 49 of the pigs and was detected by real time PCR in 54 of the pigs. Furthermore, we were able to determine the numbers of B. hyodysenteriae cells in all positive specimens by real time PCR. The rapid diagnostic technique established in this experiment was useful for detection of B. hyodysenteriae because it was more effective than ordinary PCR and culture.

  8. Screening for Cognitive Impairment in Parkinson's Disease: Improving the Diagnostic Utility of the MoCA through Subtest Weighting

    PubMed Central

    Fengler, Sophie; Kessler, Josef; Timmermann, Lars; Zapf, Alexandra; Elben, Saskia; Wojtecki, Lars; Tucha, Oliver; Kalbe, Elke

    2016-01-01

    Background Given the high prevalence of cognitive impairment in Parkinson’s disease (PD), cognitive screening is important in clinical practice. The Montreal Cognitive Assessment (MoCA) is a frequently used screening test in PD to detect mild cognitive impairment (PD-MCI) and Parkinson’s disease dementia (PD-D). However, the proportion in which the subtests are represented in the MoCA total score does not seem reasonable. We present the development and preliminary evaluation of an empirically based alternative scoring system of the MoCA which aims at increasing the overall diagnostic accuracy. Methods In study 1, the MoCA was administered to 40 patients with PD without cognitive impairment (PD-N), PD-MCI, or PD-D, as defined by a comprehensive neuropsychological test battery. The new MoCA scoring algorithm was developed by defining Areas under the Curve (AUC) for MoCA subtests in a Receiver Operating Characteristic (ROC) and by weighting the subtests according to their sensitivities and specificities. In study 2, an independent sample of 24 PD patients (PD-N, PD-MCI, or PD-D) was tested with the MoCA. In both studies, diagnostic accuracy of the original and the new scoring procedure was calculated. Results Diagnostic accuracy increased with the new MoCA scoring algorithm. In study 1, the sensitivity to detect cognitive impairment increased from 62.5% to 92%, while specificity decreased only slightly from 77.7% to 73%; in study 2, sensitivity increased from 68.8% to 81.3%, while specificity stayed stable at 75%. Conclusion This pilot study demonstrates that the sensitivity of the MoCA can be enhanced substantially by an empirically based weighting procedure and that the proposed scoring algorithm may serve the MoCA’s actual purpose as a screening tool in the detection of cognitive dysfunction in PD patients better than the original scoring of the MoCA. Further research with larger sample sizes is necessary to establish efficacy of the alternate scoring system

  9. Scientists use GEANIE to Study Isotopes of Iridium and Europium to Improve Radiochemical Diagnostics in Nuclear Devices

    SciTech Connect

    Becker, J A; Nelson, R

    2002-11-21

    Radiochemical diagnostics play an important role in helping scientists understand the detonation of a nuclear device. Sometimes some elements or isotopes are inserted as radiochemical detectors at various locations in the nuclear device. During the detonation of the device, these detectors are subjected for a short time to the intense flux of neutrons emitted through fission and possibly through fusion of light elements (usually deuterium and tritium). After the detonation, the radiochemical detectors and their long-lived activation products are retrieved from the area where the underground explosion took place. These radiochemical samples are analyzed to extract information about how the device operated. A large amount of such radiochemical data exist from past nuclear-device tests.

  10. Improved nonparametric estimation of the optimal diagnostic cut-off point associated with the Youden index under different sampling schemes.

    PubMed

    Yin, Jingjing; Samawi, Hani; Linder, Daniel

    2016-07-01

    A diagnostic cut-off point of a biomarker measurement is needed for classifying a random subject to be either diseased or healthy. However, the cut-off point is usually unknown and needs to be estimated by some optimization criteria. One important criterion is the Youden index, which has been widely adopted in practice. The Youden index, which is defined as the maximum of (sensitivity + specificity -1), directly measures the largest total diagnostic accuracy a biomarker can achieve. Therefore, it is desirable to estimate the optimal cut-off point associated with the Youden index. Sometimes, taking the actual measurements of a biomarker is very difficult and expensive, while ranking them without the actual measurement can be relatively easy. In such cases, ranked set sampling can give more precise estimation than simple random sampling, as ranked set samples are more likely to span the full range of the population. In this study, kernel density estimation is utilized to numerically solve for an estimate of the optimal cut-off point. The asymptotic distributions of the kernel estimators based on two sampling schemes are derived analytically and we prove that the estimators based on ranked set sampling are relatively more efficient than that of simple random sampling and both estimators are asymptotically unbiased. Furthermore, the asymptotic confidence intervals are derived. Intensive simulations are carried out to compare the proposed method using ranked set sampling with simple random sampling, with the proposed method outperforming simple random sampling in all cases. A real data set is analyzed for illustrating the proposed method.

  11. Multimodal optical biopsy probe to improve the safety and diagnostic yield of brain needle biopsies (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Desroches, Joannie; Pichette, Julien; Goyette, Andréanne; Tremblay, Marie-Andrée.; Jermyn, Michael; Petrecca, Kevin; Leblond, Frédéric

    2016-03-01

    Brain needle biopsy (BNB) is performed to collect tissue when precise neuropathological diagnosis is required to provide information about tumor type, grade, and growth patterns. The principal risks associated with this procedure are intracranial hemorrhage (due to clipping blood vessels during tissue extraction), incorrect tumor typing/grading due to non-representative or non-diagnostic samples (e.g. necrotic tissue), and missing the lesion. We present an innovative device using sub-diffuse optical tomography to detect blood vessels and Raman spectroscopy to detect molecular differences between tissue types, in order to reduce the risks of misdiagnosis, incorrect tumour grading, and non-diagnostic samples. The needle probe integrates optical fibers directly onto the external cannula of a commercial BNB needle, and can perform measurements for both optical techniques through the same fibers. This integrated optical spectroscopy system uses diffuse reflectance signals to perform a 360-degree reconstruction of the tissue adjacent to the biopsy needle, based on the optical contrast associated with hemoglobin light absorption, thereby localizing blood vessels. Raman spectra measurements are also performed interstitially for tissue characterization. A detailed sensitivity of the system is presented to demonstrate that it can detect absorbers with diameters <300 µm located up to ˜2 mm from the biopsy needle core, for bulk optical properties consistent with brain tissue. Results from animal experiments are presented to validate blood vessel detection and Raman spectrum measurement without disruption of the surgical workflow. We also present phantom measurements of Raman spectra with the needle probe and a comparison with a clinically validated Raman spectroscopy probe.

  12. Improved nonparametric estimation of the optimal diagnostic cut-off point associated with the Youden index under different sampling schemes.

    PubMed

    Yin, Jingjing; Samawi, Hani; Linder, Daniel

    2016-07-01

    A diagnostic cut-off point of a biomarker measurement is needed for classifying a random subject to be either diseased or healthy. However, the cut-off point is usually unknown and needs to be estimated by some optimization criteria. One important criterion is the Youden index, which has been widely adopted in practice. The Youden index, which is defined as the maximum of (sensitivity + specificity -1), directly measures the largest total diagnostic accuracy a biomarker can achieve. Therefore, it is desirable to estimate the optimal cut-off point associated with the Youden index. Sometimes, taking the actual measurements of a biomarker is very difficult and expensive, while ranking them without the actual measurement can be relatively easy. In such cases, ranked set sampling can give more precise estimation than simple random sampling, as ranked set samples are more likely to span the full range of the population. In this study, kernel density estimation is utilized to numerically solve for an estimate of the optimal cut-off point. The asymptotic distributions of the kernel estimators based on two sampling schemes are derived analytically and we prove that the estimators based on ranked set sampling are relatively more efficient than that of simple random sampling and both estimators are asymptotically unbiased. Furthermore, the asymptotic confidence intervals are derived. Intensive simulations are carried out to compare the proposed method using ranked set sampling with simple random sampling, with the proposed method outperforming simple random sampling in all cases. A real data set is analyzed for illustrating the proposed method. PMID:26756282

  13. [Diagnostic Errors in Medicine].

    PubMed

    Buser, Claudia; Bankova, Andriyana

    2015-12-01

    The recognition of diagnostic errors in everyday practice can help improve patient safety. The most common diagnostic errors are the cognitive errors, followed by system-related errors and no fault errors. The cognitive errors often result from mental shortcuts, known as heuristics. The rate of cognitive errors can be reduced by a better understanding of heuristics and the use of checklists. The autopsy as a retrospective quality assessment of clinical diagnosis has a crucial role in learning from diagnostic errors. Diagnostic errors occur more often in primary care in comparison to hospital settings. On the other hand, the inpatient errors are more severe than the outpatient errors.

  14. [Diagnostic Errors in Medicine].

    PubMed

    Buser, Claudia; Bankova, Andriyana

    2015-12-01

    The recognition of diagnostic errors in everyday practice can help improve patient safety. The most common diagnostic errors are the cognitive errors, followed by system-related errors and no fault errors. The cognitive errors often result from mental shortcuts, known as heuristics. The rate of cognitive errors can be reduced by a better understanding of heuristics and the use of checklists. The autopsy as a retrospective quality assessment of clinical diagnosis has a crucial role in learning from diagnostic errors. Diagnostic errors occur more often in primary care in comparison to hospital settings. On the other hand, the inpatient errors are more severe than the outpatient errors. PMID:26649954

  15. 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

  16. Use of Treponema pallidum PCR in testing of ulcers for diagnosis of primary syphilis.

    PubMed

    Gayet-Ageron, Angèle; 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

  17. Detection of orthopoxvirus DNA by real-time PCR and identification of variola virus DNA by melting analysis.

    PubMed

    Nitsche, Andreas; Ellerbrok, Heinz; Pauli, Georg

    2004-03-01

    Although variola virus was eradicated by the World Health Organization vaccination program in the 1970s, the diagnosis of smallpox infection has attracted great interest in the context of a possible deliberate release of variola virus in bioterrorist attacks. Obviously, fast and reliable diagnostic tools are required to detect variola virus and to distinguish it from orthopoxviruses that have identical morphological characteristics, including vaccinia virus. The advent of real-time PCR for the clinical diagnosis of viral infections has facilitated the detection of minute amounts of viral nucleic acids in a fast, safe, and precise manner, including the option to quantify and to genotype the target reliably. In this study a complete set of four hybridization probe-based real-time PCR assays for the specific detection of orthopoxvirus DNA is presented. Melting analysis following PCR enables the identification of variola virus by the PCR product's characteristic melting temperature, permitting the discrimination of variola virus from other orthopoxviruses. In addition, an assay for the specific amplification of variola virus DNA is presented. All assays can be performed simultaneously in the same cycler, and results of a PCR run are obtained in less than 1 h. The application of more than one assay for the same organism significantly contributes to the diagnostic reliability, reducing the risk of false-negative results due to unknown sequence variations. In conclusion, the assays presented will improve the speed and reliability of orthopoxvirus diagnostics and variola virus identification.

  18. Advances in paper-based point-of-care diagnostics.

    PubMed

    Hu, Jie; Wang, ShuQi; Wang, Lin; Li, Fei; Pingguan-Murphy, Belinda; Lu, Tian Jian; Xu, Feng

    2014-04-15

    Advanced diagnostic technologies, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), have been widely used in well-equipped laboratories. However, they are not affordable or accessible in resource-limited settings due to the lack of basic infrastructure and/or trained operators. Paper-based diagnostic technologies are affordable, user-friendly, rapid, robust, and scalable for manufacturing, thus holding great potential to deliver point-of-care (POC) diagnostics to resource-limited settings. In this review, we present the working principles and reaction mechanism of paper-based diagnostics, including dipstick assays, lateral flow assays (LFAs), and microfluidic paper-based analytical devices (μPADs), as well as the selection of substrates and fabrication methods. Further, we report the advances in improving detection sensitivity, quantification readout, procedure simplification and multi-functionalization of paper-based diagnostics, and discuss the disadvantages of paper-based diagnostics. We envision that miniaturized and integrated paper-based diagnostic devices with the sample-in-answer-out capability will meet the diverse requirements for diagnosis and treatment monitoring at the POC.

  19. Uganda's New National Laboratory Sample Transport System: A Successful Model for Improving Access to Diagnostic Services for Early Infant HIV Diagnosis and Other Programs

    PubMed Central

    Kiyaga, Charles; Sendagire, Hakim; Joseph, Eleanor; McConnell, Ian; Grosz, Jeff; Narayan, Vijay; Esiru, Godfrey; Elyanu, Peter; Akol, Zainab; Kirungi, Wilford; Musinguzi, Joshua; Opio, Alex

    2013-01-01

    Introduction Uganda scaled-up Early HIV Infant Diagnosis (EID) when simplified methods for testing of infants using dried blood spots (DBS) were adopted in 2006 and sample transport and management was therefore made feasible in rural settings. Before this time only 35% of the facilities that were providing EID services were reached through the national postal courier system, Posta Uganda. The transportation of samples during this scale-up, therefore, quickly became a challenge and varied from facility to facility as different methods were used to transport the samples. This study evaluates a novel specimen transport network system for EID testing. Methods A retrospective study was done in mid-2012 on 19 pilot hubs serving 616 health facilities in Uganda. The effect on sample-result turnaround time (TAT) and the cost of DBS sample transport on 876 sample-results was analyzed. Results The HUB network system provided increased access to EID services ranging from 36% to 51%, drastically reduced transportation costs by 62%, reduced turn-around times by 46.9% and by a further 46.2% through introduction of SMS printers. Conclusions The HUB model provides a functional, reliable and efficient national referral network against which other health system strengthening initiatives can be built to increase access to critical diagnostic and treatment monitoring services, improve the quality of laboratory and diagnostic services, with reduced turn-around times and improved quality of prevention and treatment programs thereby reducing long-term costs. PMID:24236026

  20. Electrothermal modeling of silicon PCR chips

    NASA Astrophysics Data System (ADS)

    Cui, Zheng; Zhao, Zhan; Xia, Shanhong

    2001-04-01

    Polymerase chain reaction (PCR) on a microchip has drawn considerable attention in recent years. Although a microchip can have must fast heating and cooling rate, the delicacy in its structure makes the PCR experiment difficult and cracks often occurs particularly for the thin membrane type of PCR chips. Electrothermal modeling of PCR chips is presented using commercial MEMS software tool IntelliSuiteTM, with the aim of identifying the problems encountered in experiment and finding an optimum chip structure. Heating characteristics of four different heater designs have been compared, so have the PCR chambers with fixed frame and with suspended frame. The thermal stress analysis has shown that the structure and heater design can make significant difference in heating characteristics and in reducing the failure of PCR chips. The computer simulation has confirmed what has been found in experiment the reason of membrane cracks. Improvement in PCR chip design has been proposed.

  1. Development of a 16S-23S rRNA intergenic spacer-based quantitative PCR assay for improved detection and enumeration of Lactococcus garvieae.

    PubMed

    Thanh, Hien Dang; Park, Hee Kuk; Kim, Wonyong; Shin, Hyoung-Shik

    2013-02-01

    Lactococcus garvieae is an important foodborne pathogen causing lactococcosis associated with hemorrhagic septicemia in fish worldwide. A real-time quantitative polymerase chain reaction (qPCR) protocol targeting the 16S-23S rRNA intergenic spacer (ITS) region was developed for the detection and enum-eration of L. garvieae. The specificity was evaluated using genomic DNAs extracted from 66 cocci strains. Fourteen L. garvieae strains tested were positive, whereas 52 other strains including Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. hordniae and Lactococcus lactis ssp. cremoris did not show a specific signal. The minimal limit of detection was 2.63 fg of purified genomic DNA, equivalent to 1 genome of L. garvieae. The optimized protocol was applied for the survey of L. garvieae in naturally contaminated fish samples. Our results suggest that the qPCR protocol using ITS is a sensitive and efficient tool for the rapid detection and enumeration of L. garvieae in fish and fish-containing foods.

  2. A naked-eye colorimetric "PCR developer"

    NASA Astrophysics Data System (ADS)

    Valentini, Paola; Pompa, Pier Paolo

    2016-04-01

    Despite several advances in molecular biology and diagnostics, Polymerase Chain Reaction (PCR) is currently the gold standard for nucleic acids amplification and detection, due to its versatility, low-cost and universality, with estimated <10 billion reactions per year and a worldwide market of several billion dollars/year. Nevertheless, PCR still relies on the laborious, time-consuming, and multi-step gel electrophoresis-based detection, which includes gel casting, electrophoretic run, gel staining, and gel visualization. In this work, we propose a "PCR developer", namely a universal one-step, one-tube method, based on controlled aggregation of gold nanoparticles (AuNPs), to detect PCR products by naked eye in few minutes, with no need for any instrumentation. We demonstrated the specificity and sensitivity of the PCR developer on different model targets, suitable for a qualitative detection in real-world diagnostics (i.e., gene rearrangements, genetically modified organisms, and pathogens). The PCR developer proved to be highly specific and ultra-sensitive, discriminating down to few copies of HIV viral DNA, diluted in an excess of interfering human genomic DNA, which is a clinically relevant viral load. Hence, it could be a valuable tool for both academic research and clinical applications.

  3. Combination of pulse volume recording (PVR) parameters and ankle-brachial index (ABI) improves diagnostic accuracy for peripheral arterial disease compared with ABI alone.

    PubMed

    Hashimoto, Tomoko; Ichihashi, Shigeo; Iwakoshi, Shinichi; Kichikawa, Kimihiko

    2016-06-01

    The ankle-brachial index (ABI) measurement is widely used as a screening tool to detect peripheral arterial disease (PAD). With the advent of the oscillometric ABI device incorporating a system for the measurement of pulse volume recording (PVR), not only ABI but also other parameters, such as the percentage of mean arterial pressure (%MAP) and the upstroke time (UT), can be obtained automatically. The purpose of the present study was to compare the diagnostic accuracy for PAD with ABI alone with that of a combination of ABI, %MAP and UT. This study included 108 consecutive patients on whom 216 limb measurements were performed. The sensitivity, specificity and positive and negative predictive values of ABI, %MAP, UT and their combination were evaluated and compared with CT angiography that was used as a gold standard for the detection of PAD. The diagnostic accuracy as well as the optimal cutoff values of %MAP and UT were evaluated using receiver operating characteristic (ROC) curve analysis. The combination of ABI, %MAP and UT achieved higher sensitivity, negative predictive value and accuracy than ABI alone, particularly for mild stenosis. The areas under the ROC curve for the detection of 50% stenosis with UT and %MAP were 0.798 and 0.916, respectively. The optimal UT and %MAP values to detect ≧50% stenosis artery were 183 ms and 45%, respectively. The combination of ABI, %MAP and UT contributed to the improvement of the diagnostic accuracy for PAD. Consideration of the values of %MAP and UT in addition to ABI may have a significant impact on the detection of early PAD lesions.

  4. Sporadic Creutzfeldt-Jakob disease diagnostic accuracy is improved by a new CSF ELISA 14-3-3γ assay.

    PubMed

    Leitão, M J; Baldeiras, I; Almeida, M R; Ribeiro, M H; Santos, A C; Ribeiro, M; Tomás, J; Rocha, S; Santana, I; Oliveira, C R

    2016-05-13

    Protein 14-3-3 is a reliable marker of rapid neuronal damage, specifically increased in cerebrospinal fluid (CSF) of sporadic Creutzfeldt-Jakob disease (sCJD) patients. Its detection is usually performed by Western Blot (WB), prone to methodological issues. Our aim was to evaluate the diagnostic performance of a recently developed quantitative enzyme-linked immunosorbent (ELISA) assay for 14-3-3γ, in comparison with WB and other neurodegeneration markers. CSF samples from 145 patients with suspicion of prion disease, later classified as definite sCJD (n=72) or Non-prion diseases (Non-CJD; n=73) comprised our population. 14-3-3 protein was determined by WB and ELISA. Total Tau (t-Tau) and phosphorylated Tau (p-Tau) were also evaluated. Apolipoprotein E gene (ApoE) and prionic protein gene (PRNP) genotyping was assessed. ELISA 14-3-3γ levels were significantly increased in sCJD compared to Non-CJD patients (p<0.001), showing very good accuracy (AUC=0.982; sensitivity=97%; specificity=94%), and matching WB results in 81% of all cases. It strongly correlated with t-Tau and p-Tau (p<0.0001), showing slightly higher specificity (14-3-3 WB - 63%; Tau - 90%; p-Tau/t-Tau ratio - 88%). From WB inconclusive results (n=44), ELISA 14-3-3γ correctly classified 41 patients. Additionally, logistic regression analysis selected ELISA 14-3-3γ as the best single predictive marker for sCJD (overall accuracy=93%). ApoE and PRNP genotypes did not influence ELISA 14-3-3γ levels. Despite specificity for 14-3-3γ isoform, ELISA results not only match WB evaluation but also help discrimination of inconclusive results. Our results therefore reinforce this assay as a single screening test, allowing higher sample throughput and unequivocal results. PMID:26940479

  5. Soluble recombinant merozoite surface antigen-142kDa of Plasmodium vivax: An improved diagnostic antigen for vivax malaria.

    PubMed

    Mirahmadi, Hadi; Fallahi, Shirzad; Seyyed Tabaei, Seyyed Javad

    2016-04-01

    Enzyme Linked Immunosorbent Assay (ELISA), as a serological test, can be a beneficial tool for epidemiological studies by screening blood donors and diagnosis of specific antibodies from Plasmodium vivax (P. vivax) infected cases. Since P. vivax cannot easily be acquired in vitro, ELISA assays using total or semi-purified antigens are seldom used. On the basis of this restriction, we examined whether recombinant protein 42 kDa related to C-terminal region of the merozoite surface antigen-1 of P. vivax (MSA-1(42)) could be suitable for serological detection of vivax malaria infection. Purified recombinant protein produced in Escherichia coli (E. coli) (GST-MSA-1(42)) was examined for its ability to bind to IgG antibodies of individuals with patent P. vivax infection. The method was tested with 262 serum samples collected from individuals living in the south and southeastern regions of Iran where malaria is endemic. Samples exposed to Plasmodium falciparum (P. falciparum) infection and patients with other infectious disease (toxoplasmosis, Leishmania infantum infection, echinococcosis and FUO (fever with unknown origin)) except for P. falciparum were residing in non- malaria endemic areas in Iran. Generally, the sensitivity of ELISA evaluated with sera from naturally infected individuals was 86.9%. The specificity value of the ELISA determined with sera from healthy individuals and from individuals with other infectious diseases was 94.05%. The positive predictive value (PPV), negative predictive value (NPV) provided, and the diagnostic efficiency of anti-rPvMSA-1(42) antibody using indirect ELISA were determined 93.58, 87.77 and 91.06% respectively. Our study demonstrated that, because MSA-1(42) kDa contains both the 33 and 19 kDa fragments in its structure, it can serve as the basis for the development of a sensitive serological test which can be used for epidemiological studies, screening blood donors and diagnosis of P. vivax malaria. PMID:26851675

  6. Comparison of PCR, culturing and Pap smear microscopy for accurate diagnosis of genital Actinomyces.

    PubMed

    Kaya, Dilek; Demirezen, Şayeste; Hasçelik, Gülşen; Gülmez Kivanç, Dolunay; Beksaç, Mehmet Sinan

    2013-05-01

    Members of the genus Actinomyces, Gram-positive, non-spore-forming anaerobic bacteria, are normal inhabitants of the mucosal surfaces of the oral, gastrointestinal and genital tracts. Identification of these bacteria using conventional methods is generally difficult because of their complex transport and growth requirements and their fastidious and slow-growing nature. However, in recent years, the advancement of molecular techniques has provided much improved identification and differentiation of closely related Actinomyces species. The aim of the present study was to evaluate the efficacy of the PCR technique in the diagnosis of genital Actinomyces in comparison with culturing and Papanicolaou (Pap) smear microscopy. Multiple sampling was conducted from 200 women using smear microscopy, culturing and PCR. Cyto-brushes were smeared on glass slides and stained using the routine Pap technique. Culturing was performed from a sterile swab, and Actinomyces were determined using the BBL Crystal ANR ID kit. PCR was performed from a second swab, and the Actinomyces type was determined using type-specific primers designed in our laboratory. Only one vaginal fluid sample (0.5%) revealed Actinomyces-like organisms on Pap smear examination. Actinomyces were detected in nine samples (4.5%) using the BBL Crystal ANR ID kit. Using PCR, eight samples (4%) were found positive for Actinomyces. No specimens that gave positive results by Pap smear microscopy and culturing could be confirmed by PCR. Pap smear microscopy and culturing were both found to have zero sensitivity for Actinomyces. PCR appears to be a sensitive and reliable diagnostic method for the detection of Actinomyces, which are difficult to cultivate from genital samples. PCR can be used for diagnostic confirmation in cases diagnosed by conventional methods, to prevent false-positive results.

  7. Does Medical Students' Diagnostic Performance Improve by Observing Examples of Self-Explanation Provided by Peers or Experts?

    ERIC Educational Resources Information Center

    Chamberland, Martine; Mamede, Sílvia; St-Onge, Christina; Setrakian, Jean; Schmidt, Henk G.

    2015-01-01

    Educational strategies that promote the development of clinical reasoning in students remain scarce. Generating self-explanations (SE) engages students in active learning and has shown to be an effective technique to improve clinical reasoning in clerks. Example-based learning has been shown to support the development of accurate knowledge…

  8. The international standard ISO/TS 21872-1 to study the occurence of total and pathogenic Vibrio parahaemolyticus and Vibrio cholerae in seafood: ITS improvement by use of a chromogenic medium and PCR.

    PubMed

    Rosec, Jean-Philippe; Causse, Véronique; Cruz, Barbara; Rauzier, Jean; Carnat, Laurence

    2012-07-01

    During two surveys conducted in 2008 and 2009, the culture method described in the international standard ISO/TS 21872-1 was applied to the detection of Vibrio parahaemolyticus and Vibrio cholerae in 112 living bivalve mollusc samples, with a chromogenic medium used in addition to the TCBS agar, as second selective isolation medium and for enumeration of V. parahaemolyticus and V. cholerae by surface inoculation. A PCR method for detection of these 2 Vibrio species and the hemolysin genes tdh and trh, was applied in parallel. In 2009, the survey was extended to finfish fillets and crustaceans. PCR was also used for species confirmation of characteristic colonies. The identity of the PCR products, specifically targeting V. parahaemolyticus, was checked by sequencing. Occurrence of V. parahaemolyticus and V. cholerae isolates in living bivalve molluscs ranged from 30.4% to 32.6% and from 1.4% to 4.7% respectively. In frozen crustaceans (2009 survey) V. parahaemolyticus and V. cholerae isolates were respectively found in 45% and 10% of the samples. No V. parahaemolyticus or V. cholerae was detected in frozen fish fillets, neither by the ISO method nor by PCR. In 2009, enteropathogenic V. parahaemolyticus (trh+) was isolated from 4 out of 43 oyster samples while the trh gene was present in V. alginolyticus strains and in samples where V. parahaemolyticus was not detected (9 over 112 samples). The ISO method failed to isolate V. parahaemolyticus in 44% to 53% of the living bivalve molluscs where PCR detected the toxR gene specific of V. parahaemolyticus (Vp-toxR). Our results highlighted the need for a revision of the ISO/TS 21872-1 standard, at least, for analysis of living bivalve molluscs, and confirmed the increasing concern of enteropathogenic V. parahaemolyticus in French bivalve molluscs. Enrichment at 41.5°C was questioned and some reliable solutions for the improvement of the ISO/TS 21872-1 method, such as the PCR method for screening of positive samples and

  9. Auto-induction for high yield expression of recombinant novel isoallergen tropomyosin from King prawn (Melicertus latisulcatus) for improved diagnostics and immunotherapeutics.

    PubMed

    Koeberl, Martina; Kamath, Sandip D; Saptarshi, Shruti R; Smout, Michael J; Rolland, Jennifer M; O'Hehir, Robyn E; Lopata, Andreas L

    2014-12-15

    Food allergies are increasing worldwide, demonstrating a considerable public health concern. Shellfish allergy is one of the major food groups causing allergic sensitization among adults and children, affecting up to 2% of the general world population. Tropomyosin (TM) is the major allergen in shellfish and frequently used in the diagnosis of allergic sensitization and the detection of cross-contaminated food. To improve and establish better and more sensitive diagnostics for allergies and immunotherapeutics, large quantities of pure allergens are required. To establish a reproducible method for the generation of pure recombinant tropomyosin we utilized in this study different Escherichia coli strains (NM522, TOP10 and BL21(DE3)RIPL). In addition, isopropyl-β-D-thiogalactoside (IPTG) induction was compared with a novel auto-induction system to allow the generation of larger quantities of recombinant allergen. We demonstrated that the B-strain of E. coli is better for the expression of TM compared to the K-strain. Moreover, a higher yield could be achieved when using the auto-induction system, with up to 62 mg/l. High yield expressed recombinant TM from King prawn (KP) was compared to recombinant TM from Black tiger prawn (Pen m 1). We demonstrated that recombinant TM from KP and known isoallergen Pen m 1 have very similar molecular and immunological characteristics. Overall, we demonstrate that auto-induction can be used to express larger quantities of recombinant allergens for the development of diagnostic, to quantify allergens as well as immunotherapeutics employing isoallergens.

  10. PCR and Blood Culture for Detection of Escherichia coli Bacteremia in Rats

    PubMed Central

    Heininger, Alexandra; Binder, Marlies; Schmidt, Sibylle; Unertl, Klaus; Botzenhart, Konrad; Döring, Gerd

    1999-01-01

    Critically ill patients often develop symptoms of sepsis and therefore require microbiological tests for bacteremia that use conventional blood culture (BC) techniques. However, since these patients frequently receive early empirical antibiotic therapy before diagnostic procedures are completed, examination by BC can return false-negative results. We therefore hypothesized that PCR could improve the rate of detection of microbial pathogens over that of BC. To test this hypothesis, male Wistar rats were challenged intravenously with 106 CFU of Escherichia coli. Blood was then taken at several time points for detection of E. coli by BC and by PCR with E. coli-specific primers derived from the uidA gene, encoding β-glucuronidase. In further experiments, cefotaxime (100 or 50 mg/kg of body weight) was administered intravenously to rats 10 min after E. coli challenge. Without this chemotherapy, the E. coli detection rate decreased at 15 min and at 210 min after challenge from 100% to 62% of the animals with PCR and from 100% to 54% of the animals with BC (P, >0.05). Chemotherapy decreased the E. coli detection rate at 25 min and at 55 min after challenge from 100% to 50% with PCR and from 100% to 0% with BC (P, <0.05). Thus, at clinically relevant serum antibiotic levels, PCR affords a significantly higher detection rate than BC in this rat model. The results suggest that PCR could be a useful adjunct tool supplementing conventional BC techniques in diagnosing bacteremia. PMID:10405388

  11. An appraisal of PCR-based technology in the detection of Mycobacterium tuberculosis.

    PubMed

    Sankar, Sathish; Ramamurthy, Mageshbabu; Nandagopal, Balaji; Sridharan, Gopalan

    2011-02-01

    Tuberculosis is an under-recognized yet catastrophic health problem, particularly in developing countries. The HIV pandemic has served to increase the number of susceptible individuals, and multidrug-resistance and poor socioeconomic conditions also augment the prevalence and the consequences of the disease. To control the disease and its spread, it is vital that tuberculosis diagnostics are accurate and rapid. Whereas microscopy and culture have several limitations (low sensitivity is a problem for the former, while the latter has a delayed turnaround time), PCR-based techniques targeting regions of the Mycobacterium tuberculosis genome such as IS6110 have proved to be useful. The purpose of this review is to assess the use of PCR-RFLP, nested PCR and real-time PCR protocols and the choice of target regions for the detection of M. tuberculosis. Real-time PCR for the detection of M. tuberculosis target genes in clinical specimens has contributed to improving diagnosis and epidemiologic surveillance in the past decade. However, targeting one genome sequence such as IS6110 may not by itself be sufficiently sensitive to reach 100% diagnosis, especially in the case of pulmonary tuberculosis. Additional testing for target genome sequences such as hsp65 seems encouraging. An interesting approach would be a multiplex real-time PCR targeting both IS6110 and hsp65 to achieve comprehensive and specific molecular diagnosis. This technology needs development and adequate field testing before it becomes the acceptable gold standard for diagnosis.

  12. Use of conventional PCR and smear microscopy to diagnose pulmonary tuberculosis in the Amazonian rainforest area.

    PubMed

    Carniel, F; Dalla Costa, E R; Lima-Bello, G; Martins, C; Scherer, L C; Rossetti, M L

    2014-12-01

    The diagnostic usefulness of Ziehl-Neelsen (ZN)-stained sputum smears combined with conventional polymerase chain reaction (ZN/PCR) to amplify IS6110 region DNA extracted from ZN slides was evaluated. The objective was to verify if this association could improve tuberculosis (TB) diagnosis in patients at remote sites. The study was carried out in 89 patients with culture-confirmed pulmonary TB as defined by the Brazilian Manual for TB Treatment. The participants were recruited in a reference unit for TB treatment in Rondônia, a state in the Amazonian area in northern Brazil. ZN, PCR, and culture performed in the sputum samples from these patients were analyzed in different combinations (i.e., ZN plus PCR and ZN plus culture). The prevalence rates of pulmonary TB in these patients were 32.6 and 28.1% considering culture and ZN/PCR, respectively. The sensitivity and specificity of ZN/PCR were 86 and 93%, respectively. ZN/PCR was able to detect more TB cases than ZN alone. This method could offer a new approach for accurate tuberculosis diagnosis, especially in remote regions of the world where culture is not available.

  13. Improving the Sensitivity and Functionality of Mobile Webcam-Based Fluorescence Detectors for Point-of-Care Diagnostics in Global Health

    PubMed Central

    Rasooly, Reuven; Bruck, Hugh Alan; Balsam, Joshua; Prickril, Ben; Ossandon, Miguel; Rasooly, Avraham

    2016-01-01

    Resource-poor countries and regions require effective, low-cost diagnostic devices for accurate identification and diagnosis of health conditions. Optical detection technologies used for many types of biological and clinical analysis can play a significant role in addressing this need, but must be sufficiently affordable and portable for use in global health settings. Most current clinical optical imaging technologies are accurate and sensitive, but also expensive and difficult to adapt for use in these settings. These challenges can be mitigated by taking advantage of affordable consumer electronics mobile devices such as webcams, mobile phones, charge-coupled device (CCD) cameras, lasers, and LEDs. Low-cost, portable multi-wavelength fluorescence plate readers have been developed for many applications including detection of microbial toxins such as C. Botulinum A neurotoxin, Shiga toxin, and S. aureus enterotoxin B (SEB), and flow cytometry has been used to detect very low cell concentrations. However, the relatively low sensitivities of these devices limit their clinical utility. We have developed several approaches to improve their sensitivity presented here for webcam based fluorescence detectors, including (1) image stacking to improve signal-to-noise ratios; (2) lasers to enable fluorescence excitation for flow cytometry; and (3) streak imaging to capture the trajectory of a single cell, enabling imaging sensors with high noise levels to detect rare cell events. These approaches can also help to overcome some of the limitations of other low-cost optical detection technologies such as CCD or phone-based detectors (like high noise levels or low sensitivities), and provide for their use in low-cost medical diagnostics in resource-poor settings. PMID:27196933

  14. Improving the Sensitivity and Functionality of Mobile Webcam-Based Fluorescence Detectors for Point-of-Care Diagnostics in Global Health.

    PubMed

    Rasooly, Reuven; Bruck, Hugh Alan; Balsam, Joshua; Prickril, Ben; Ossandon, Miguel; Rasooly, Avraham

    2016-01-01

    Resource-poor countries and regions require effective, low-cost diagnostic devices for accurate identification and diagnosis of health conditions. Optical detection technologies used for many types of biological and clinical analysis can play a significant role in addressing this need, but must be sufficiently affordable and portable for use in global health settings. Most current clinical optical imaging technologies are accurate and sensitive, but also expensive and difficult to adapt for use in these settings. These challenges can be mitigated by taking advantage of affordable consumer electronics mobile devices such as webcams, mobile phones, charge-coupled device (CCD) cameras, lasers, and LEDs. Low-cost, portable multi-wavelength fluorescence plate readers have been developed for many applications including detection of microbial toxins such as C. Botulinum A neurotoxin, Shiga toxin, and S. aureus enterotoxin B (SEB), and flow cytometry has been used to detect very low cell concentrations. However, the relatively low sensitivities of these devices limit their clinical utility. We have developed several approaches to improve their sensitivity presented here for webcam based fluorescence detectors, including (1) image stacking to improve signal-to-noise ratios; (2) lasers to enable fluorescence excitation for flow cytometry; and (3) streak imaging to capture the trajectory of a single cell, enabling imaging sensors with high noise levels to detect rare cell events. These approaches can also help to overcome some of the limitations of other low-cost optical detection technologies such as CCD or phone-based detectors (like high noise levels or low sensitivities), and provide for their use in low-cost medical diagnostics in resource-poor settings. PMID:27196933

  15. Mathematical modelling of the transmission dynamics of contagious bovine pleuropneumonia reveals minimal target profiles for improved vaccines and diagnostic assays.

    PubMed

    Ssematimba, Amos; Jores, Joerg; Mariner, Jeffrey C

    2015-01-01

    Contagious bovine pleuropneumonia (CBPP) is a cattle disease that has hampered the development of the livestock sector in sub-Saharan Africa. Currently, vaccination with a live vaccine strain is its recommended control measure although unofficial antimicrobial use is widely practiced. Here, modelling techniques are used to assess the potential impact of early elimination of infected cattle via accurate diagnosis on CBPP dynamics. A herd-level stochastic epidemiological model explicitly incorporating test sensitivity and specificity is developed. Interventions by annual vaccination, annual testing and elimination and a combination of both are implemented in a stepwise manner and their effectiveness compared by running 1000 simulations per intervention over ten years. The model predicts that among the simulated interventions, the ones likely to eliminate the disease from an isolated herd all involved annual vaccination of more than 75% of the animals with a vaccine that protects for at least 18 months combined with annual testing (and elimination of positive reactors) of 75% of the animals every six months after vaccination. The highest probability of disease elimination was 97.5% and this could occur within a median of 2.3 years. Generally, our model predicts that regular testing and elimination of positive reactors using improved tests will play a significant role in minimizing CBPP burden especially in the current situation where improved vaccines are yet to be developed.

  16. Determining Fungi rRNA Copy Number by PCR

    EPA Science Inventory

    The goal of this project is to improve the quantification of indoor fungal pollutants via the specific application of quantitative PCR (qPCR). Improvement will be made in the controls used in current qPCR applications. This work focuses on the use of two separate controls within ...

  17. Diagnostic vitrectomy for infectious uveitis

    PubMed Central

    Jeroudi, Abdallah; Yeh, Steven

    2014-01-01

    The identification of an infectious or noninfectious uveitis syndrome is important to determine the range of therapeutic and prognostic implications of that disease entity. Diagnostic dilemmas arise with atypical history, atypical clinical presentations, inconclusive diagnostic workup, and persistent or worsened inflammation despite appropriate immunosuppression. More invasive intraocular testing is indicated in these situations particularly in infectious uveitis where a delay in treatment may result in worsening of the patient’s disease and a poor visual outcome. Laboratory analysis of vitreous fluid via diagnostic pars plana vitrectomy is an important technique in the diagnostic armamentarium, but the most important aspects of sample collection include rapid processing, close coordination with an ophthalmic pathology laboratory, and directed testing on this limited collected sample. Culture and staining has utility in bacterial, fungal, and nocardial infection. Polymerase chain reaction (PCR) analysis has shown promising results for bacterial endophthalmitis and infection with mycobacterium tuberculosis whereas PCR testing for viral retinitides and ocular toxoplasmosis has a more established role. Antibody testing is appropriate for toxoplasmosis and toxocariasis, and may be complementary to PCR for viral retinitis. Masquerade syndromes represent neoplastic conditions that clinically appear as infectious or inflammatory conditions and should be considered as part of the differential diagnosis. Diagnostic vitrectomy and chorioretinal biopsy are thus critical tools for the management of patients in whom an infectious etiology of uveitis is suspected. PMID:24613892

  18. Improving Laboratory Efficiency by Automation of Preanalytic Processing of ThinPrep Specimens for Real-Time PCR High-Risk HPV Testing.

    PubMed

    Barbieri, Daniela; Venturoli, Simona; Costa, Silvano; Landini, Maria Paola

    2016-06-01

    Cervical specimens collected in liquid-based cytology (LBC) media are the most common sample type used for high-risk human papillomavirus (HPV) testing. Since preanalytic steps such as vortexing and decapping vials, liquid transfer to a sample input tube with matching unique identifier, and recapping the original vials are required for processing LBC samples prior to running the Abbott RealTime High Risk HPV assay (Abbott, Wiesbaden, Germany), a full manual execution can be complicated, especially in high-throughput diagnostic contexts. Here, a custom-configured worktable setup for the Tecan Freedom EVO (Tecan, Männedorf, Switzerland) designed to automate and control preanalytic steps for ThinPrep (Hologic, Marlborough, MA) samples was used to evaluate the impact of automated versus manual preanalytics. Archival results for manual processing of 226 samples were compared with those obtained with the Tecan protocol, observing a very good overall concordance for final assay interpretation (95.6%). High overall agreement (100%) resulted also from retesting 99 samples by both the preanalytical protocols. High reproducibility was observed analyzing 23 randomly selected samples by automated preprocessing in triplicate. Hence, the new configuration of the Tecan platform translates the manual steps required to process ThinPrep specimens into automated operations, controls sample identification, and allows for saving hands-on time, while maintaining assay reproducibility and ensuring reliability of results, making it suitable for screening settings.

  19. Melioidosis Diagnostic Workshop, 20131

    PubMed Central

    AuCoin, David; Baccam, Prasith; Baggett, Henry C.; Baird, Rob; Bhengsri, Saithip; Blaney, David D.; Brett, Paul J.; Brooks, Timothy J.G.; Brown, Katherine A.; Chantratita, Narisara; Cheng, Allen C.; Dance, David A.B.; Decuypere, Saskia; Defenbaugh, Dawn; Gee, Jay E.; Houghton, Raymond; Jorakate, Possawat; Lertmemongkolchai, Ganjana; Limmathurotsakul, Direk; Merlin, Toby L.; Mukhopadhyay, Chiranjay; Norton, Robert; Peacock, Sharon J.; Rolim, Dionne B.; Simpson, Andrew J.; Steinmetz, Ivo; Stoddard, Robyn A.; Stokes, Martha M.; Sue, David; Tuanyok, Apichai; Whistler, Toni; Wuthiekanun, Vanaporn; Walke, Henry T.

    2015-01-01

    Melioidosis is a severe disease that can be difficult to diagnose because of its diverse clinical manifestations and a lack of adequate diagnostic capabilities for suspected cases. There is broad interest in improving detection and diagnosis of this disease not only in melioidosis-endemic regions but also outside these regions because melioidosis may be underreported and poses a potential bioterrorism challenge for public health authorities. Therefore, a workshop of academic, government, and private sector personnel from around the world was convened to discuss the current state of melioidosis diagnostics, diagnostic needs, and future directions. PMID:25626057

  20. Inclusion of Neuropsychological Scores in Atrophy Models Improves Diagnostic Classification of Alzheimer's Disease and Mild Cognitive Impairment.

    PubMed

    Goryawala, Mohammed; Zhou, Qi; Barker, Warren; Loewenstein, David A; Duara, Ranjan; Adjouadi, Malek

    2015-01-01

    Brain atrophy in mild cognitive impairment (MCI) and Alzheimer's disease (AD) are difficult to demarcate to assess the progression of AD. This study presents a statistical framework on the basis of MRI volumes and neuropsychological scores. A feature selection technique using backward stepwise linear regression together with linear discriminant analysis is designed to classify cognitive normal (CN) subjects, early MCI (EMCI), late MCI (LMCI), and AD subjects in an exhaustive two-group classification process. Results show a dominance of the neuropsychological parameters like MMSE and RAVLT. Cortical volumetric measures of the temporal, parietal, and cingulate regions are found to be significant classification factors. Moreover, an asymmetrical distribution of the volumetric measures across hemispheres is seen for CN versus EMCI and EMCI versus AD, showing dominance of the right hemisphere; whereas CN versus LMCI and EMCI versus LMCI show dominance of the left hemisphere. A 2-fold cross-validation showed an average accuracy of 93.9%, 90.8%, and 94.5%, for the CN versus AD, CN versus LMCI, and EMCI versus AD, respectively. The accuracy for groups that are difficult to differentiate like EMCI versus LMCI was 73.6%. With the inclusion of the neuropsychological scores, a significant improvement (24.59%) was obtained over using MRI measures alone.

  1. Does gadolinium-based contrast material improve diagnostic accuracy of local invasion in rectal cancer MRI? A multireader study.

    PubMed

    Gollub, Marc J; Lakhman, Yulia; McGinty, Katrina; Weiser, Martin R; Sohn, Michael; Zheng, Junting; Shia, Jinru

    2015-02-01

    OBJECTIVE. The purpose of this study was to compare reader accuracy and agreement on rectal MRI with and without gadolinium administration in the detection of T4 rectal cancer. MATERIALS AND METHODS. In this study, two radiologists and one fellow independently interpreted all posttreatment MRI studies for patients with locally advanced or recurrent rectal cancer using unenhanced images alone or combined with contrast-enhanced images, with a minimum interval of 4 weeks. Readers evaluated involvement of surrounding structures on a 5-point scale and were blinded to pathology and disease stage. Sensitivity, specificity, negative predictive value, positive predictive value, and AUC were calculated and kappa statistics were used to describe interreader agreement. RESULTS. Seventy-two patients (38 men and 34 women) with a mean age of 61 years (range, 32-86 years) were evaluated. Fifteen patients had 32 organs invaded. Global AUCs without and with gadolinium administration were 0.79 and 0.77, 0.91 and 0.86, and 0.83 and 0.78 for readers 1, 2, and 3, respectively. AUCs before and after gadolinium administration were similar. Kappa values before and after gadolinium administration for pairs of readers ranged from 0.5 to 0.7. CONCLUSION. On the basis of pathology as a reference standard, the use of gadolinium during rectal MRI did not significantly improve radiologists' agreement or ability to detect T4 disease.

  2. Variation in copy number of the 28S rDNA of Aspergillus fumigatus measured by droplet digital PCR and analog quantitative real-time PCR.

    PubMed

    Alanio, Alexandre; Sturny-Leclère, Aude; Benabou, Marion; Guigue, Nicolas; Bretagne, Stéphane

    2016-08-01

    Droplet digital PCR (ddPCR) after DNA digestion yielded a 28S rDNA copy number of 61 to 86 copies/genome when testing 10 unrelated Aspergillus fumigatus isolates, higher than with quantitative PCR. Unfortunately, ddPCR after DNA digestion did not improve the sensitivity of our PCR assay when testing serum patients with invasive aspergillosis. PMID:27316653

  3. Optimising droplet digital PCR analysis approaches for detection and quantification of bacteria: a case study of fire blight and potato brown rot.

    PubMed

    Dreo, Tanja; Pirc, Manca; Ramšak, Živa; Pavšič, Jernej; Milavec, Mojca; Zel, Jana; Gruden, Kristina

    2014-10-01

    Here we report on the first assessment of droplet digital PCR (ddPCR) for detection and absolute quantification of two quarantine plant pathogenic bacteria that infect many species of the Rosaceae and Solanaceae families: Erwinia amylovora and Ralstonia solanacearum. An open-source R script was written for the ddPCR data analysis. Analysis of a set of samples with known health status aided the assessment and selection of different threshold settings (QuantaSoft analysis, definetherain pipeline and manual threshold), which led to optimal diagnostic specificity. The interpretation of the E. amylovora ddPCR was straightforward, and the analysis approach had little influence on the final results and the concentrations determined. The sensitivity and linear range were similar to those for real-time PCR (qPCR), for the analysis of both bacterial suspensions and plant material, making ddPCR a viable choice when both detection and quantification are desired. With the R. solanacearum ddPCR, the use of a high global threshold was necessary to exclude false-positive reactions that are sometimes observed in healthy plant material. ddPCR significantly improved the analytical sensitivity over that of qPCR, and improved the detection of low concentrations of R. solanacearum in potato tuber samples. Accurate and rapid absolute quantification of both of these bacteria in pure culture was achieved by direct ddPCR. Our data confirm the suitability of these ddPCR assays for routine detection and quantification of plant pathogens and for preparation of defined in-house reference materials with known target concentrations.

  4. Integrating Rapid Diagnostics and Antimicrobial Stewardship in Two Community Hospitals Improved Process Measures and Antibiotic Adjustment Time.

    PubMed

    Lockwood, Ashley M; Perez, Katherine K; Musick, William L; Ikwuagwu, Judy O; Attia, Engie; Fasoranti, Oyejoke O; Cernoch, Patricia L; Olsen, Randall J; Musser, James M

    2016-04-01

    OBJECTIVE To assess the impact of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry for rapid pathogen identification directly from early-positive blood cultures coupled with an antimicrobial stewardship program (ASP) in two community hospitals. Process measures and outcomes prior and after implementation of MALDI-TOF/ASP were evaluated. DESIGN Multicenter retrospective study. SETTING Two community hospitals in a system setting, Houston Methodist (HM) Sugar Land Hospital (235 beds) or HM Willowbrook Hospital (241 beds). PATIENTS Patients ≥ 18 years of age with culture-proven Gram-negative bacteremia. INTERVENTION Blood cultures from both hospitals were sent to and processed at our central microbiology laboratory. Clinical pharmacists at respective hospitals were notified of pathogen ID and susceptibility results. RESULTS We evaluated 572 patients for possible inclusion. After pre-defined exclusion criteria, 151 patients were included in the pre-intervention group and 242 were included in the intervention group. After MALDI-TOF/ASP implementation, the mean identification time after culture positivity was significantly reduced from 32 hours (±16 hours) to 6.5 hours (±5.4 hours) (P<.001); mean time to susceptibility results was significantly reduced from 48 (±22) hours to 23 (±14) hours (P<.001); and time to therapy adjustment was significantly reduced from 75 (±59) hours to 30 (±30) hours (P<.001). Mean hospital costs per patient were $3,411 less in the intervention group compared with the pre-intervention group ($18,645 vs $15,234; P=.04). CONCLUSION This study is the first to analyze the impact of MALDI-TOF coupled with an ASP in a community hospital setting. Time to results significantly differed with the use of MALDI-TOF, and time to appropriate therapy was significantly improved with the addition of ASP. PMID:26738993

  5. A method for amplification of unknown flanking sequences based on touchdown PCR and suppression-PCR.

    PubMed

    Gao, Song; He, Dan; Li, Guangquan; Zhang, Yanhua; Lv, Huiying; Wang, Li

    2016-09-15

    Thermal asymmetric staggered PCR is the most widely used technique to obtain the flanking sequences. However, it has some limitations, including a low rate of positivity, and complex operation. In this study, a improved method of it was made based on suppression-PCR and touchdown PCR. The PCR fragment obtained by the amplification was used directly for sequencing after gel purification. Using this improved method, the positive rate of amplified flanking sequences of the ATMT mutants reached 99%. In addition, the time from DNA extraction to flanking sequence analysis was shortened to 2 days with about 6 dollars each sample. PMID:27393656

  6. A method for amplification of unknown flanking sequences based on touchdown PCR and suppression-PCR.

    PubMed

    Gao, Song; He, Dan; Li, Guangquan; Zhang, Yanhua; Lv, Huiying; Wang, Li

    2016-09-15

    Thermal asymmetric staggered PCR is the most widely used technique to obtain the flanking sequences. However, it has some limitations, including a low rate of positivity, and complex operation. In this study, a improved method of it was made based on suppression-PCR and touchdown PCR. The PCR fragment obtained by the amplification was used directly for sequencing after gel purification. Using this improved method, the positive rate of amplified flanking sequences of the ATMT mutants reached 99%. In addition, the time from DNA extraction to flanking sequence analysis was shortened to 2 days with about 6 dollars each sample.

  7. SWOG S0800 (NCI CDR0000636131): addition of bevacizumab to neoadjuvant nab-paclitaxel with dose-dense doxorubicin and cyclophosphamide improves pathologic complete response (pCR) rates in inflammatory or locally advanced breast cancer.

    PubMed

    Nahleh, Z A; Barlow, W E; Hayes, D F; Schott, A F; Gralow, J R; Sikov, W M; Perez, E A; Chennuru, S; Mirshahidi, H R; Corso, S W; Lew, D L; Pusztai, L; Livingston, R B; Hortobagyi, G N

    2016-08-01

    SWOG S0800, a randomized open-label Phase II clinical trial, compared the combination of weekly nab-paclitaxel and bevacizumab followed by dose-dense doxorubicin and cyclophosphamide (AC) with nab-paclitaxel followed or preceded by AC as neoadjuvant treatment for HER2-negative locally advanced breast cancer (LABC) or inflammatory breast cancer (IBC). Patients were randomly allocated (2:1:1) to three neoadjuvant chemotherapy arms: (1) nab-paclitaxel with concurrent bevacizumab followed by AC; (2) nab-paclitaxel followed by AC; or (3) AC followed by nab-paclitaxel. The primary endpoint was pathologic complete response (pCR) with stratification by disease type (non-IBC LABC vs. IBC) and hormone receptor status (positive vs. negative). Overall survival (OS), event-free survival (EFS), and toxicity were secondary endpoints. Analyses were intent-to-treat comparing bevacizumab to the combined control arms. A total of 215 patients were accrued including 11 % with IBC and 32 % with triple-negative breast cancer (TNBC). The addition of bevacizumab significantly increased the pCR rate overall (36 vs. 21 %; p = 0.019) and in TNBC (59 vs. 29 %; p = 0.014), but not in hormone receptor-positive disease (24 vs. 18 %; p = 0.41). Sequence of administration of nab-paclitaxel and AC did not affect the pCR rate. While no significant differences in OS or EFS were seen, a trend favored the addition of bevacizumab for EFS (p = 0.06) in TNBC. Overall, Grade 3-4 adverse events did not differ substantially by treatment arm. The addition of bevacizumab to nab-paclitaxel prior to dose-dense AC neoadjuvant chemotherapy significantly improved the pCR rate compared to chemotherapy alone in patients with triple-negative LABC/IBC and was accompanied by a trend for improved EFS. This suggests reconsideration of the role of bevacizumab in high-risk triple-negative locally advanced breast cancer.

  8. Analytical Comparison of In Vitro-Spiked Human Serum and Plasma for PCR-Based Detection of Aspergillus fumigatus DNA: a Study by the European Aspergillus PCR Initiative.

    PubMed

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

    2015-09-01

    The use of serum or plasma for Aspergillus PCR testing facilitates automated and standardized technology. Recommendations for serum testing are available, and while serum and plasma are regularly considered interchangeable for use in fungal diagnostics, differences in galactomannan enzyme immunoassay (GM-EIA) performance have been reported and are attributed to clot formation. Therefore, it is important to assess plasma PCR testing to determine if previous recommendations for serum are applicable and also to compare analytical performance with that of serum PCR. Molecular methods testing serum and plasma were compared through multicenter distribution of quality control panels, with additional studies to investigate the effect of clot formation and blood fractionation on DNA availability. Analytical sensitivity and time to positivity (TTP) were compared, and a regression analysis was performed to identify variables that enhanced plasma PCR performance. When testing plasma, sample volume, preextraction-to-postextraction volume ratio, PCR volume, duplicate testing, and the use of an internal control for PCR were positively associated with performance. When whole-blood samples were spiked and then fractionated, the analytical sensitivity and TTP were superior when testing plasma. Centrifugation had no effect on DNA availability, whereas the presence of clot material significantly lowered the concentration (P = 0.028). Technically, there are no major differences in the molecular processing of serum and plasma, but the formation of clot material potentially reduces available DNA in serum. During disease, Aspergillus DNA burdens in blood are often at the limits of PCR performance. Using plasma might improve performance while maintaining the methodological simplicity of serum testing.

  9. Improvement of peanut (Arachis hypogaea L.) transformation efficiency and determination of transgene copy number by relative quantitative real-time PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The biolistic method is reliable for delivering genes of interest into various species. Low transformation efficiency has been a limiting factor for its application. The DNA coating agent protamine was shown to improve transformation efficiency in rice, while a reduction of plasmid DNA in the bomb...

  10. Real-time PCR detection chemistry.

    PubMed

    Navarro, E; Serrano-Heras, G; Castaño, M J; Solera, J

    2015-01-15

    Real-time PCR is the method of choice in many laboratories for diagnostic and food applications. This technology merges the polymerase chain reaction chemistry with the use of fluorescent reporter molecules in order to monitor the production of amplification products during each cycle of the PCR reaction. Thus, the combination of excellent sensitivity and specificity, reproducible data, low contamination risk and reduced hand-on time, which make it a post-PCR analysis unnecessary, has made real-time PCR technology an appealing alternative to conventional PCR. The present paper attempts to provide a rigorous overview of fluorescent-based methods for nucleic acid analysis in real-time PCR described in the literature so far. Herein, different real-time PCR chemistries have been classified into two main groups; the first group comprises double-stranded DNA intercalating molecules, such as SYBR Green I and EvaGreen, whereas the second includes fluorophore-labeled oligonucleotides. The latter, in turn, has been divided into three subgroups according to the type of fluorescent molecules used in the PCR reaction: (i) primer-probes (Scorpions, Amplifluor, LUX, Cyclicons, Angler); (ii) probes; hydrolysis (TaqMan, MGB-TaqMan, Snake assay) and hybridization (Hybprobe or FRET, Molecular Beacons, HyBeacon, MGB-Pleiades, MGB-Eclipse, ResonSense, Yin-Yang or displacing); and (iii) analogues of nucleic acids (PNA, LNA, ZNA, non-natural bases: Plexor primer, Tiny-Molecular Beacon). In addition, structures, mechanisms of action, advantages and applications of such real-time PCR probes and analogues are depicted in this review.

  11. Sex determination using PCR.

    PubMed

    Kima, Peter E; Rasche, Madeline E

    2004-03-01

    PCR has revolutionized many aspects of biochemistry and molecular biology research. In the following exercise, students learn PCR by isolating their own DNA, amplifying specific segments of the X and Y chromosomes, and estimating the sizes of the PCR products using agarose gel electrophoresis. Based on the pattern of PCR products, students can distinguish between male and female samples and determine the gender of an unknown DNA donor. The exercise is presented for upper division undergraduate majors in microbiology, biochemistry, and molecular biology, but can be adapted to different academic levels and disciplines. The use of student samples in the exercise can enhance learning of these techniques by making PCR and agarose gel electrophoresis directly relevant to the students.

  12. In silico PCR analysis.

    PubMed

    Yu, Bing; Zhang, Changbin

    2011-01-01

    In silico PCR analysis is a useful and efficient complementary method to ensure primer specificity for an extensive range of PCR applications from gene discovery, molecular diagnosis, and pathogen detection to forensic DNA typing. In silico PCR, SNPCheck, and Primer-BLAST are commonly used web-based in silico PCR tools. Their applications are discussed here in stepwise detail along with several examples, which aim to make it easier for the intended users to apply the tools. This virtual PCR method can assist in the selection of newly designed primers, identify potential mismatches in the primer binding sites due to known SNPs, and avoid the amplification of unwanted amplicons so that potential problems can be prevented before any "wet bench" experiment.

  13. The potential of molecular diagnostics and serum procalcitonin levels to change the antibiotic management of community-acquired pneumonia.

    PubMed

    Gilbert, David; Gelfer, Gita; Wang, Lian; Myers, Jillian; Bajema, Kristina; Johnston, Michael; Leggett, James

    2016-09-01

    Two diagnostic bundles were compared in 127 evaluable patients admitted with community-acquired pneumonia (CAP). Diagnostic modalities in all patients included cultures of sputum (if obtainable) and blood, urine for detection of the antigens of Streptococcus pneumoniae and Legionella pneumophila, and nasal swabs for PCR probes for S. pneumoniae and Staphylococcus aureus. At least one procalcitonin level was measured in all patients. For virus detection, patients were randomized to either a 5-virus, lab-generated PCR panel or the broader and faster FilmArray PCR panel. Overall, an etiologic diagnosis was established in 71% of the patients. A respiratory virus was detected in 39%. The potential for improved antibiotic stewardship was evident in 25 patients with only detectable respiratory virus and normal levels of PCT. PMID:27377675

  14. Molecular Diagnostics

    PubMed Central

    Choe, Hyonmin; Deirmengian, Carl A.; Hickok, Noreen J.; Morrison, Tiffany N.; Tuan, Rocky S.

    2015-01-01

    Orthopaedic infections are complex conditions that require immediate diagnosis and accurate identification of the causative organisms to facilitate appropriate management. Conventional methodologies for diagnosis of these infections sometimes lack accuracy or sufficient rapidity. Current molecular diagnostics are an emerging area of bench-to-bedside research in orthopaedic infections. Examples of promising molecular diagnostics include measurement of a specific biomarker in the synovial fluid, polymerase chain reaction–based detection of bacterial genes, and metabolomic determination of responses to orthopaedic infection. PMID:25808967

  15. An algorithmic approach using kappa/lambda ratios to improve the diagnostic accuracy of urine protein electrophoresis and to reduce the volume required for immunoelectrophoresis.

    PubMed

    Levinson, S S

    1997-06-27

    The most sensitive routine method for identifying urinary monoclonal immunoglobulin kappa and lambda light chains, called Bence Jones proteins (BJPs), in clinical laboratories is immunofixation electrophoresis (IFE), but this procedure is time-consuming and expensive. As a result, many laboratories screen for paraproteins with urine protein electrophoresis (UPE), which is insensitive when low concentrations of BJP are present and is difficult to interpret with severe proteinuria. The purpose of this study was to determine whether kappa/lambda ratios can be used in conjunction with UPE to improve diagnostic reliability in identifying paraproteins, and decrease the need for IFE on all samples. Urine specimens from 243 patients were examined by UPE and kappa/lambda ratios, and compared with IFE. Due to poor analytical sensitivity, the urinary kappa or lambda concentrations could not be determined in many cases. As a result, many specimens showed kappa/lambda ratios that were indeterminate. Nevertheless, when both urinary kappa and lambda concentrations were undetectable, a BJP could be ruled out. A urinary kappa/lambda ratio between 0.75-3 also ruled out a BJP. The use of kappa/lambda ratios, in conjunction with UPE, resulted in a 52% decrease in the volume of IFE during the course of this study, with 100% sensitivity for detecting BJP.

  16. Development of an improved data analysis approach for combined laser extinction and two-angle elastic light scattering diagnostics of soot aggregates.

    PubMed

    Zhang, Tongfeng; Thomson, Murray J

    2016-02-01

    An improved data analysis approach has been developed for the combined laser extinction and two-angle elastic light scattering diagnostics to relate the various measured optical cross sections to soot aggregate properties. The performance of the proposed approach is assessed using the comprehensive dataset of Santoro ethylene-air co-flow diffusion flame. Compared to previously reported studies, the proposed approach can be applied to a wider range of soot sources by removing the assumption made to the scattering regime or moment ratio of aggregate size distribution. The proposed approach also considers the contribution of scattering to extinction in determining the soot volume fraction, and this contribution is shown to increase as soot aggregate size becomes larger. The sensitivity of the calculation to the assumed parameters of the approach is examined and discussed. The mean radius of gyration of soot aggregates and the ratio of scattering intensities at the two measurement angles are shown to be independent of soot refractive index and are therefore recommended for soot model validation purposes. PMID:26836101

  17. Improved tympanic thermometer based on a fiber optic infrared radiometer and an otoscope and its use as a new diagnostic tool for acute otitis media

    NASA Astrophysics Data System (ADS)

    Fishman, Gadi; DeRowe, Ari; Ophir, Eyal; Scharf, Vered; Shabtai, Abraham; Ophir, Dov; Katzir, Abraham

    1999-06-01

    Clinical diagnosis of acute otitis media (AOM) in children is not easy. It was assumed that there is a difference ΔT between the Tympanic Membrane (TM) temperatures in the two ears in unilateral AOM and that an accurate measurement of ΔT may improve the diagnosis accuracy. An IR transmitting fiber, made of AgClBr, was coupled into a hand held otoscope and was used for the non-contact (radiometric) measurements of TT, the TM temperature. Experiments were carried out, first, on a laboratory model that simulated the human ear, including an artificial tympanic membrane and an artificial ear canal. Measurements carried out using commercially available tympanic thermometers shown that the temperature Tc of the ear canal affected the results. Tc did not affect the fiberoptic radiometer, and this device accurately measured the true temperature, TT of the tympanic membrane. A prospective blinded sampling of the TM temperature was then performed on 48 children with suspected AOM. The mean temperature difference between the ears, for children with unilateral AOM was ΔT = (0.68 +/- 0.27)°C. For children with bilateral AOM it was ΔT = (0.14+/-0.10)°C (p<0.001). It was demonstrated that afor unilateral AOM the difference ΔT was proportional to the systemic temperature. In conclusion, the fiberoptic interferometric measurements of the TM can be a useful non-invasive diagnostic tool for AOM, when combined with other data.

  18. Cable Diagnostic Focused Initiative

    SciTech Connect

    Hartlein, R.A.; Hampton, R.N.

    2010-12-30

    This report summarizes an extensive effort made to understand how to effectively use the various diagnostic technologies to establish the condition of medium voltage underground cable circuits. These circuits make up an extensive portion of the electric delivery infrastructure in the United States. Much of this infrastructure is old and experiencing unacceptable failure rates. By deploying efficient diagnostic testing programs, electric utilities can replace or repair circuits that are about to fail, providing an optimal approach to improving electric system reliability. This is an intrinsically complex topic. Underground cable systems are not homogeneous. Cable circuits often contain multiple branches with different cable designs and a range of insulation materials. In addition, each insulation material ages differently as a function of time, temperature and operating environment. To complicate matters further, there are a wide variety of diagnostic technologies available for assessing the condition of cable circuits with a diversity of claims about the effectiveness of each approach. As a result, the benefits of deploying cable diagnostic testing programs have been difficult to establish, leading many utilities to avoid the their use altogether. This project was designed to help address these issues. The information provided is the result of a collaborative effort between Georgia Tech NEETRAC staff, Georgia Tech academic faculty, electric utility industry participants, as well as cable system diagnostic testing service providers and test equipment providers. Report topics include: •How cable systems age and fail, •The various technologies available for detecting potential failure sites, •The advantages and disadvantages of different diagnostic technologies, •Different approaches for utilities to employ cable system diagnostics. The primary deliverables of this project are this report, a Cable Diagnostic Handbook (a subset of this report) and an online

  19. Real-Time PCR

    NASA Astrophysics Data System (ADS)

    Evrard, A.; Boulle, N.; Lutfalla, G. S.

    Over the past few years there has been a considerable development of DNA amplification by polymerase chain reaction (PCR), and real-time PCR has now superseded conventional PCR techniques in many areas, e.g., the quantification of nucleic acids and genotyping. This new approach is based on the detection and quantification of a fluorescent signal proportional to the amount of amplicons generated by PCR. Real-time detection is achieved by coupling a thermocycler with a fluorimeter. This chapter discusses the general principles of quantitative real-time PCR, the different steps involved in implementing the technique, and some examples of applications in medicine. The polymerase chain reaction (PCR) provides a way of obtaining a large number of copies of a double-stranded DNA fragment of known sequence. This DNA amplification technique, developed in 1985 by K. Mullis (Cetus Corporation), saw a spectacular development over the space of a few years, revolutionising the methods used up to then in molecular biology. Indeed, PCR has many applications, such as the detection of small amounts of DNA, cloning, and quantitative analysis (assaying), each of which will be discussed further below.

  20. Development and Preliminary Evaluation of a New Real-Time RT-PCR Assay For Detection of Peste des petits Ruminants Virus Genome.

    PubMed

    Polci, A; Cosseddu, G M; Ancora, M; Pinoni, C; El Harrak, M; Sebhatu, T T; Ghebremeskel, E; Sghaier, S; Lelli, R; Monaco, F

    2015-06-01

    A duplex real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for a simple and rapid diagnosis of Peste des petits ruminants (PPR). qRT-PCR primers and TaqMan probe were designed on a conserved region of nucleocapsid protein (Np) of PPR virus (PPRV) genome. An in vitro transcript of the target region was constructed and tested to determine analytical sensitivity. Commercial heterologous Armored RNA(®) was used as an internal positive control (IPC) for either RNA isolation or RT-PCR steps. The detection limit of the newly designed duplex real-time RT-PCR (qRT-PCR PPR_Np) was approximately 20 copies/μl with a 95% probability. No amplification signals were recorded when the qRT-PCR PPR_Np was applied to viruses closely related or clinically similar to PPRV- or to PPR-negative blood samples. A preliminary evaluation of the diagnostic performance was carried out by testing a group of 43 clinical specimens collected from distinct geographic areas of Africa and Middle East. qRT-PCR PPR_Np showed higher sensitivity than the conventional gel-based RT-PCR assays, which have been used as reference standards. Internal positive control made it possible to identify the occurrence of 5 false-negative results caused by the amplification failure, thus improving the accuracy of PPRV detection.

  1. Fungal molecular diagnostics: a mini review.

    PubMed

    Atkins, Simon D; Clark, Ian M

    2004-01-01

    Conventional methods to identify fungi have often relied on identification of disease symptoms, isolation and culturing of environmental organisms, and laboratory identification by morphology and biochemical tests. Although these methods are still fundamental there is an increasing move towards molecular diagnostics of fungi in all fields. In this review, some of the molecular approaches to fungal diagnostics based on polymerase chain reaction (PCR) and DNA/RNA probe technology are discussed. This includes several technological advances in PCR-based methods for the detection, identification and quantification of fungi including real-time PCR which has been successfully used to provide rapid, quantitative data on fungal species from environmental samples. PCR and probe based methods have provided new tools for the enumeration of fungal species, but it is still necessary to combine the new technology with more conventional methods to gain a fuller understanding of interactions occurring in the environment. Since its introduction in the mid 1980's PCR has provided many molecular diagnostic tools, some of which are discussed within this review, and with the advances in micro-array technology and real-time PCR methods the future is bright for the development of accurate, quantitative diagnostic tools that can provide information not only on individual fungal species but also on whole communities.

  2. Influence of reagents formulation on real-time PCR parameters.

    PubMed

    Burgos, J S; Ramírez, C; Tenorio, R; Sastre, I; Bullido, M J

    2002-08-01

    Real-time polymerase chain reaction (PCR) techniques are increasingly used to quantify target sequences for diagnostic and research purposes. Due to its 'quantitative' character, it is very important to determine the variability of this technique correlating with several experimental conditions. The objective of this study was to analyse the effect of manufacturing lots of PCR reagents on two main PCR parameters, specificity and sensitivity. For this study, we used four different amplicons, using either mouse genomic DNA or viral DNA. Although a PCR product could be obtained in any of the conditions, we observed that there are relevant variations in sensitivity depending on the reagents formulation. We conclude that different lots of reagents may determine the analytical performance of PCR assays indicating that reagents testing are of special importance when the PCR protocol is used for quantitative purposes.

  3. Sensitive, microliter PCR with consensus degenerate primers for Epstein Barr virus amplification

    PubMed Central

    Oh, Kyudam; Pak, Nikita; Saunders, D. Curtis; Conrardy, Christina; Landers, James P.; Tong, Suxiang; Forest, Craig R.

    2016-01-01

    Sensitive identification of the etiology of viral diseases is key to implementing appropriate prevention and treatment. The gold standard for virus identification is the polymerase chain reaction (PCR), a technique that allows for highly specific and sensitive detection of pathogens by exponentially amplifying a specific region of DNA from as little as a single copy through thermocycling a biochemical cocktail. Today, molecular biology laboratories use commercial instruments that operate in 0.5–2 h/analysis using reaction volumes of 5–50 μL contained within polymer tubes or chambers. Towards reducing this volume and maintaining performance, we present a semi-quantitative, systematic experimental study of how PCR yield is affected by tube/chamber substrate, surface-area-to-volume ratio (SA:V), and passivation methods. We perform PCR experiments using traditional PCR tubes as well as using disposable polymer microchips with 1 μL reaction volumes thermocycled using water baths. We report the first oil encapsulation microfluidic PCR method without fluid flow and its application to the first microfluidic amplification of Epstein Barr virus using consensus degenerate primers, a powerful and broad PCR method to screen for both known and novel members of a viral family. The limit of detection is measured as 140 starting copies of DNA from a starting concentration of 3×105 copies/mL, regarded as an accepted sensitivity threshold for diagnostic purposes, and reaction specificity was improved as compared to conventional methods. Also notable, these experiments were conducted with conventional reagent concentrations, rather than commonly spiked enzyme and/or template mixtures. This experimental study of the effects of substrate, SA:V, and passivation, together with sensitive and specific microfluidic PCR with consensus degenerate primers represent advances towards lower cost and higher throughput pathogen screening. PMID:23080522

  4. Real-time quantitative PCR in the diagnosis of tuberculosis in formalin-fixed paraffin-embedded pleural tissue in patients from a high HIV endemic area.

    PubMed

    Baba, Kamaldeen; Pathak, Sharad; Sviland, Lisbeth; Langeland, Nina; Hoosen, Anwar A; Asjo, Birgitta; Dyrhol-Riise, Anne M; Mustafa, Tehmina

    2008-06-01

    The aim of the study was to improve the diagnosis of pleural tuberculosis (TB) based on formalin-fixed biopsies from patients living in high TB and human immunodeficiency virus (HIV) endemic areas. A real-time polymerase chain reaction (real-time PCR) assay targeting a segment of the gene for mycobacterial 65-kd heat shock protein was developed and evaluated on pleural biopsies from 25 patients clinically diagnosed as having TB, on the basis of the good response to treatment, and from 11 controls. A nested polymerase chain reaction (N-PCR) assay for the repetitive genetic sequence insert IS6110, common to Mycobacterium tuberculosis complex organisms, was performed for comparison. When compared with N-PCR, the real-time PCR assay gave a sensitivity and specificity of 83% and 72%, respectively. When compared with clinical diagnosis, the sensitivity and specificity of real-time PCR (68% and 73%, respectively) was comparable with the sensitivity and specificity of the N-PCR assay (64% and 82%, respectively). There were no major differences in the diagnostic validity for the confirmed TB/HIV coinfected patients compared with the results from the whole TB group. In conclusion, the overall accuracy of the real-time PCR assay was comparable with that of the N-PCR and both were equally useful as diagnostic tools in the setting of a HIV coinfection. The real-time PCR has the additional advantage of a short turn-around time, low risk of sample contamination, and offers the possibility to quantify bacterial load, making it a powerful tool for the rapid diagnosis of TB pleuritis.

  5. Real-time quantitative PCR in the diagnosis of tuberculosis in formalin-fixed paraffin-embedded pleural tissue in patients from a high HIV endemic area.

    PubMed

    Baba, Kamaldeen; Pathak, Sharad; Sviland, Lisbeth; Langeland, Nina; Hoosen, Anwar A; Asjo, Birgitta; Dyrhol-Riise, Anne M; Mustafa, Tehmina

    2008-06-01

    The aim of the study was to improve the diagnosis of pleural tuberculosis (TB) based on formalin-fixed biopsies from patients living in high TB and human immunodeficiency virus (HIV) endemic areas. A real-time polymerase chain reaction (real-time PCR) assay targeting a segment of the gene for mycobacterial 65-kd heat shock protein was developed and evaluated on pleural biopsies from 25 patients clinically diagnosed as having TB, on the basis of the good response to treatment, and from 11 controls. A nested polymerase chain reaction (N-PCR) assay for the repetitive genetic sequence insert IS6110, common to Mycobacterium tuberculosis complex organisms, was performed for comparison. When compared with N-PCR, the real-time PCR assay gave a sensitivity and specificity of 83% and 72%, respectively. When compared with clinical diagnosis, the sensitivity and specificity of real-time PCR (68% and 73%, respectively) was comparable with the sensitivity and specificity of the N-PCR assay (64% and 82%, respectively). There were no major differences in the diagnostic validity for the confirmed TB/HIV coinfected patients compared with the results from the whole TB group. In conclusion, the overall accuracy of the real-time PCR assay was comparable with that of the N-PCR and both were equally useful as diagnostic tools in the setting of a HIV coinfection. The real-time PCR has the additional advantage of a short turn-around time, low risk of sample contamination, and offers the possibility to quantify bacterial load, making it a powerful tool for the rapid diagnosis of TB pleuritis. PMID:18382372

  6. Fungal Diagnostics

    PubMed Central

    Kozel, Thomas R.; Wickes, Brian

    2014-01-01

    Early diagnosis of fungal infection is critical to effective treatment. There are many impediments to diagnosis such as a diminishing number of clinical mycologists, cost, time to result, and requirements for sensitivity and specificity. In addition, fungal diagnostics must meet the contrasting needs presented by the increasing diversity of fungi found in association with the use of immunosuppressive agents in countries with high levels of medical care and the need for diagnostics in resource-limited countries where large numbers of opportunistic infections occur in patients with AIDS. Traditional approaches to diagnosis include direct microscopic examination of clinical samples, histopathology, culture, and serology. Emerging technologies include molecular diagnostics and antigen detection in clinical samples. Innovative new technologies that use molecular and immunoassay platforms have the potential to meet the needs of both resource-rich and resource-limited clinical environments. PMID:24692193

  7. Emerging technologies in extracellular vesicle-based molecular diagnostics.

    PubMed

    Jia, Shidong; Zocco, Davide; Samuels, Michael L; Chou, Michael F; Chammas, Roger; Skog, Johan; Zarovni, Natasa; Momen-Heravi, Fatemeh; Kuo, Winston Patrick

    2014-04-01

    Extracellular vesicles (EVs), including exosomes and microvesicles, have been shown to carry a variety of biomacromolecules including mRNA, microRNA and other non-coding RNAs. Within the past 5 years, EVs have emerged as a promising minimally invasive novel source of material for molecular diagnostics. Although EVs can be easily identified and collected from biological fluids, further research and proper validation is needed in order for them to be useful in the clinical setting. In addition, innovative and more efficient means of nucleic acid profiling are needed to facilitate investigations into the cellular and molecular mechanisms of EV function and to establish their potential as useful clinical biomarkers and therapeutic tools. In this article, we provide an overview of recent technological improvements in both upstream EV isolation and downstream analytical technologies, including digital PCR and next generation sequencing, highlighting future prospects for EV-based molecular diagnostics.

  8. PML diagnostic criteria

    PubMed Central

    Aksamit, Allen J.; Clifford, David B.; Davis, Larry; Koralnik, Igor J.; Sejvar, James J.; Bartt, Russell; Major, Eugene O.; Nath, Avindra

    2013-01-01

    Objective: To establish criteria for the diagnosis of progressive multifocal leukoencephalopathy (PML). Methods: We reviewed available literature to identify various diagnostic criteria employed. Several search strategies employing the terms “progressive multifocal leukoencephalopathy” with or without “JC virus” were performed with PubMed, SCOPUS, and EMBASE search engines. The articles were reviewed by a committee of individuals with expertise in the disorder in order to determine the most useful applicable criteria. Results: A consensus statement was developed employing clinical, imaging, pathologic, and virologic evidence in support of the diagnosis of PML. Two separate pathways, histopathologic and clinical, for PML diagnosis are proposed. Diagnostic classification includes certain, probable, possible, and not PML. Conclusion: Definitive diagnosis of PML requires neuropathologic demonstration of the typical histopathologic triad (demyelination, bizarre astrocytes, and enlarged oligodendroglial nuclei) coupled with the techniques to show the presence of JC virus. The presence of clinical and imaging manifestations consistent with the diagnosis and not better explained by other disorders coupled with the demonstration of JC virus by PCR in CSF is also considered diagnostic. Algorithms for establishing the diagnosis have been recommended. PMID:23568998

  9. Prospective assessment of rapid diagnostic tests for the detection of antibodies to hepatitis C virus, a tool for improving access to care.

    PubMed

    Chevaliez, S; Poiteau, L; Rosa, I; Soulier, A; Roudot-Thoraval, F; Laperche, S; Hézode, C; Pawlotsky, J-M

    2016-05-01

    Large-scale hepatitis C screening is required to prevent further spread of the infection, improve access to care in the context of new hepatitis C virus (HCV) drug regimens without interferon-alpha and subsequently reduce the risk of long-term complications of chronic liver disease. Rapid diagnostic tests (RDTs) represent an attractive alternative to enzyme immunoassay using blood from venepuncture. The aim of the present study was to prospectively assess the clinical performance of CE-marked RDTs detecting anti-HCV antibodies in fingerstick capillary whole blood and/or oral fluid. A total of 513 individuals, including 318 patients with chronic HCV infection, 25 patients with resolved HCV infection and 170 HCV-seronegative individuals, were prospectively enrolled. The specificity of RDTs with fingerstick whole blood varied from 98.8% to 100%. The clinical sensitivity was high for the OraQuick(®) and Toyo(®) tests (99.4% and 95.8%, respectively), but low for the Labmen(®) test (63.1%). The specificity and clinical sensitivity in crevicular fluid were both satisfactory for the OraQuick(®) test (100% and 97.6%, respectively). HCV antibody RDTs were easy and rapid to perform in the context of patient care. They were highly specific. Both the OraQuick(®) and Toyo(®) tests reached the expected level of performance for wide-scale use, with a performance advantage for the OraQuick(®) HCV test. RDTs appear to be a promising new tool for wide-scale screening of HCV infection in high-risk to medium-risk populations. Hence, careful assessment of the performance of HCV RDTs must be recommended before they can be implemented in clinical practice. PMID:26806260

  10. Epidemiology of meningitis with a negative CSF Gram stain: under-utilization of available diagnostic tests.

    PubMed

    Nesher, L; Hadi, C M; Salazar, L; Wootton, S H; Garey, K W; Lasco, T; Luce, A M; Hasbun, R

    2016-01-01

    Meningitis with a negative cerebrospinal fluid Gram stain (CSF-GS) poses a diagnostic challenge as more than 50% of patients remain without an aetiology. The introduction of polymerase chain reaction (PCR) and arboviral serologies have increased diagnostic capabilities, yet large scale epidemiological studies evaluating their use in clinical practice are lacking. We conducted a prospective observational study in New Orleans between November 1999 and September 2008 (early era) when PCR was not widely available, and in Houston between November 2008 and June 2013 (modern era), when PCR was commonly used. Patients presenting with meningitis and negative CSF-GS were followed for 4 weeks. All investigations, PCR used, and results were recorded as they became available. In 323 patients enrolled, PCR provided the highest diagnostic yield (24·2%) but was ordered for 128 (39·6%) patients; followed by serology for arboviruses (15%) that was ordered for 100 (31%) of all patients. The yield of blood cultures was (10·3%) and that of CSF cultures was 4%; the yield for all other tests was <10%. Overall, 65% of the patients remained without a diagnosis at 4 weeks: 72·1% in early era vs. 53·4% (P < 0·01) in modern era; this change was attributed to diagnosing more viral pathogens, 8·3% and 26·3% (P < 0·01), respectively. The introduction of PCR and arboviral serologies has improved the yield of diagnosing patients with meningitis and a negative CSF-GS, but both tests are being under-utilized.

  11. Capture and Direct Amplification of DNA on Chitosan Microparticles in a Single PCR-Optimal Solution.

    PubMed

    Pandit, Kunal R; Nanayakkara, Imaly A; Cao, Weidong; Raghavan, Srinivasa R; White, Ian M

    2015-11-01

    While nucleic acid amplification tests have great potential as tools for rapid diagnostics, complicated sample preparation requirements inhibit their use in near-patient diagnostics and low-resource-setting applications. Recent advancements in nucleic acid purification have leveraged pH-modulated charge switching polymers to reduce the number of steps required for sample preparation. The polycation chitosan (pKa 6.4) has been used to efficiently purify DNA by binding nucleic acids in acidic buffers and then eluting them at a pH higher than 8.0. Though it is an improvement over conventional methods, this multistep procedure has not transformed the application of nucleic acid amplification assays. Here we describe a simpler approach using magnetic chitosan microparticles that interact with DNA in a manner that has not been reported before. The microparticles capture DNA at a pH optimal for PCR (8.5) just as efficiently as at low pH. Importantly, the captured DNA is still accessible by polymerase, enabling direct amplification from the microparticles. We demonstrate quantitative PCR from DNA captured on the microparticles, thus eliminating nearly all of the sample preparation steps. We anticipate that this new streamlined method for preparing DNA for amplification will greatly expand the diagnostic applications of nucleic acid amplification tests.

  12. Diagnostic imaging.

    PubMed

    Morris, Peter; Perkins, Alan

    2012-04-21

    Physical techniques have always had a key role in medicine, and the second half of the 20th century in particular saw a revolution in medical diagnostic techniques with the development of key imaging instruments: x-ray imaging and emission tomography (nuclear imaging and PET), MRI, and ultrasound. These techniques use the full width of the electromagnetic spectrum, from gamma rays to radio waves, and sound. In most cases, the development of a medical imaging device was opportunistic; many scientists in physics laboratories were experimenting with simple x-ray images within the first year of the discovery of such rays, the development of the cyclotron and later nuclear reactors created the opportunity for nuclear medicine, and one of the co-inventors of MRI was initially attempting to develop an alternative to x-ray diffraction for the analysis of crystal structures. What all these techniques have in common is the brilliant insight of a few pioneering physical scientists and engineers who had the tenacity to develop their inventions, followed by a series of technical innovations that enabled the full diagnostic potential of these instruments to be realised. In this report, we focus on the key part played by these scientists and engineers and the new imaging instruments and diagnostic procedures that they developed. By bringing the key developments and applications together we hope to show the true legacy of physics and engineering in diagnostic medicine. PMID:22516558

  13. Diagnostic Imaging

    MedlinePlus

    Diagnostic imaging lets doctors look inside your body for clues about a medical condition. A variety of machines and techniques can create pictures of the structures and activities inside your body. The type of imaging your doctor uses depends on your symptoms and ...

  14. Heat-activatable primers for hot-start PCR and hot-start one-step RT-PCR: endpoint and real-time experiments.

    PubMed

    Ashrafi, Elena Hidalgo; Paul, Natasha

    2009-10-01

    Hot-start PCR is a technique that improves PCR performance by reducing nonspecific amplification during the initial setup stages of the PCR. This unit describes hot-start PCR protocols which utilize primers containing temperature-sensitive modifications. The introduction of 4-oxo-tetradecyl (OXT) phosphotriester groups onto the 3' end of the primer allows for primer-based hot-start PCR that is amenable for use in a number of PCR-based applications. The protocols described in this unit utilize OXT-modified primers in applications such as standard thermal cycling PCR, fast thermal cycling PCR, multiplex PCR, and one-step reverse-transcription PCR. This method is also advantageous for instances where improved PCR specificity is desired and a hot-start polymerase suitable for your application is not available.

  15. Real-time PCR in Food Science: Introduction.

    PubMed

    Rodriguez-Lazaro, David; Hernandez, Marta

    2013-01-01

    Food safety and quality control programmes are increasingly applied throughout the production food chain in order to guarantee added value products as well as to minimize the risk of infection for the consumer. The development of real-time PCR has represented one of the most significant advances in food diagnostics as it provides rapid, reliable and quantitative results. These aspects become increasingly important for the agricultural and food industry. Different strategies for real-time PCR diagnostics have been developed including unspecific detection independent of the target sequence using fluorescent dyes such as SYBR Green, or by sequence-specific fluorescent oligonucleotide probes such as TaqMan probes or molecular beacons.

  16. Overlap extension PCR cloning.

    PubMed

    Bryksin, Anton; Matsumura, Ichiro

    2013-01-01

    Rising demand for recombinant proteins has motivated the development of efficient and reliable cloning methods. Here we show how a beginner can clone virtually any DNA insert into a plasmid of choice without the use of restriction endonucleases or T4 DNA ligase. Chimeric primers encoding plasmid sequence at the 5' ends and insert sequence at the 3' ends are designed and synthesized. Phusion(®) DNA polymerase is utilized to amplify the desired insert by PCR. The double-stranded product is subsequently employed as a pair of mega-primers in a PCR-like reaction with circular plasmids. The original plasmids are then destroyed in restriction digests with Dpn I. The product of the overlap extension PCR is used to transform competent Escherichia coli cells. Phusion(®) DNA polymerase is used for both the amplification and fusion reactions, so both steps can be monitored and optimized in the same way. PMID:23996437

  17. Diagnostics development plan for ZR.

    SciTech Connect

    Hanson, David Lester

    2003-09-01

    The Z Refurbishment (ZR) Project is a program to upgrade the Z machine at SNL with modern durable pulsed power technology, providing additional shot capacity and improved reliability as well as advanced capabilities for both pulsed x-ray production and high pressure generation. The development of enhanced diagnostic capabilities is an essential requirement for ZR to meet critical mission needs. This report presents a comprehensive plan for diagnostic instrument and infrastructure development for the first few years of ZR operation. The focus of the plan is on: (1) developing diagnostic instruments with high spatial and temporal resolution, capable of low noise operation and survival in the severe EMP, bremsstrahlung, and blast environments of ZR; and (2) providing diagnostic infrastructure improvements, including reduced diagnostic trigger signal jitter, more and flexible diagnostic line-of-sight access, and the capability for efficient exchange of diagnostics with other laboratories. This diagnostic plan is the first step in an extended process to provide enhanced diagnostic capabilities for ZR to meet the diverse programmatic needs of a broad range of defense, energy, and general science programs of an international user community into the next decade.

  18. Beamlet laser diagnostics

    SciTech Connect

    Burkhart, S.C.; Behrendt, W.C.; Smith, I.

    1996-06-01

    Beamlet is instrumented extensively to monitor the performance of the overall laser system and many of its subsystems. Beam diagnostics, installed in key locations, are used to fully characterize the beam during its propagation through the multipass cavity and the laser`s output section. This article describes the diagnostics stations located on Beamlet and discusses the design, calibration, and performance of the Beamlet calorimeters. The authors used Nova`s diagnostics packages to develop the Beamlet design to determine beam energy, spatial profile, temporal profile, and other beam parameters. Technologic improvements within the last several years in controls, charge-coupled device (CCD) cameras, and fast oscilloscopes have allowed the authors to obtain more accurate measurements on the Beamlet laser system. They briefly cover some of these techniques, including a description of their LabVIEW based data acquisition system.

  19. Identification of weakly beta-hemolytic porcine spirochetes by biochemical reactions, PCR-based restriction fragment length polymorphism analysis and species-specific PCR.

    PubMed

    Ohya, Tatsuo; Araki, Hiroshi; Sueyoshi, Masuo

    2008-08-01

    We examined the usefulness of PCR-based restriction fragment length polymorphism (PCR-RFLP) and species-specific PCR combined with a newly devised rapid biochemical test using microplates for identifying weakly beta-hemolytic intestinal spirochetes (WBHIS) isolated from pigs. WBHIS strains showing atypical biochemical characteristics were decisively identified at the species level by PCR-RFLP and species-specific PCR. Identification of WBHIS at the species level in routine diagnostic work will certainly contribute to clarifying the pathogenicity of WBHIS.

  20. Explanatory chapter: PCR primer design.

    PubMed

    Álvarez-Fernández, Rubén

    2013-01-01

    This chapter is intended as a guide on polymerase chain reaction (PCR) primer design (for information on PCR, see General PCR and Explanatory Chapter: Troubleshooting PCR). In the next section, general guidelines will be provided, followed by a discussion on primer design for specific applications. A list of recommended software tools is shown at the end.

  1. Real-time PCR with internal amplification control for detecting tuberculosis: method design and validation.

    PubMed

    Flores, E; Rodríguez, J C; Garcia-Pachón, E; Soto, J L; Ruiz, M; Escribano, I; Royo, G

    2009-08-01

    Real-time PCR has been a major development in the diagnosis of tuberculosis. However, most tests do not include an internal amplification control (IAC), which therefore limits it clinical application. In this study a new, easy to perform real-time PCR test with IAC was designed and validated in clinical samples. The primers amplified a 163-bp fragment of IS6110 of Mycobacterium tuberculosis and the IAC was designed with a fragment of a different microorganism (Chlamydia trachomatis). The interassay and intraassay variation of this test were very low (0.45-1.65% and 0.18-1.80%, respectively). The detection accuracy was validated in 50 samples (25 urine, 25 sputum) with different concentrations of M. tuberculosis, 18 clinical isolates of non-tuberculous mycobacteria and 148 samples with clinical suspicion of pulmonary tuberculosis. The specificity was 100%. The detection limit of this PCR test without IAC was approximately 15 bacteria and with IAC approximately 32 bacteria. This real-time PCR with IAC assay can improve the detection of M. tuberculosis and contribute to standardization of this diagnostic technique.

  2. QUALITY ASSURANCE FOR PCR

    EPA Science Inventory

    The U.S. Environmental Protection Agency (EPA) held a workshop in January 2003 on the detection of viruses in water using polymerase chain reaction (PCR)-based methods. Speakers were asked to address a series of specific questions, including whether a single standard method coul...

  3. QUALITY CONTROLS FOR PCR

    EPA Science Inventory

    The purpose of this presentation is to present an overview of the quality control (QC) sections of a draft EPA document entitled, "Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on Environmental Samples." This document has been prepared by th...

  4. Moving past serology: Diagnostic options without serum.

    PubMed

    Reichel, Michael P; Lanyon, Sasha R; Hill, Fraser I

    2016-09-01

    Detecting antibodies formed in serum in response to infection is the traditional function of serology. Diagnostic modalities have included complement fixation tests, agar gel immune-diffusion, radioimmunoassay, ELISA and immunofluorescence. More recent technology now allows for the direct detection of pathogens by PCR. This review details the options for diagnostic testing using specimen types other than serum, identifying the advantages and disadvantages of these options and providing evidence for more widespread use of these techniques and specimen types. PMID:27160006

  5. Single-step PCR in molecular diagnosis of hepatitis C virus infection.

    PubMed

    Farma, E; Boeri, E; Bettini, P; Repetto, C M; McDermott, J; Lillo, F B; Varnier, O E

    1996-12-01

    The diagnostic utility of two PCR systems and three PCR detection methods for hepatitis C virus (HCV) RNA was evaluated in serum samples. A nested PCR was considered the reference assay and was compared with two single-step PCR methods: the first is based on the detection of PCR products by liquid hybridization with a 32P-end-labeled probe, and the second is the Roche Amplicor colorimetric assay using microwell plate hybridization with a specific nucleic acid probe. Using the Pelicheck HCV RNA Eurohep genotype 1 proficiency panel, our laboratory achieved medium-high levels of performance with all three methods. The highest sensitivity was, however, observed with the isotopic single-step PCR (ss-PCR) method. The analytical sensitivity of ss-PCR with isotopic detection and ss-PCR with colorimetric detection was identical to that of nested PCR, with a 100% result concordance. Comparison of ss-PCR with enzyme-linked immunosorbent and RIBA assays in the analysis of clinical samples showed a high concordance. ss-PCR methods appear more suitable for diagnostic application. Nevertheless, HCV RNA PCR cannot be considered a screening assay; it should be requested in the presence of reactive serology or specific clinical symptomatology with altered liver parameters, and it is a potential tool for the follow-up of patients with HCV infection.

  6. Evaluation of Digital PCR for Absolute RNA Quantification

    PubMed Central

    Sanders, Rebecca; Mason, Deborah J.; Foy, Carole A.; Huggett, Jim F.

    2013-01-01

    Gene expression measurements detailing mRNA quantities are widely employed in molecular biology and are increasingly important in diagnostic fields. Reverse transcription (RT), necessary for generating complementary DNA, can be both inefficient and imprecise, but remains a quintessential RNA analysis tool using qPCR. This study developed a Transcriptomic Calibration Material and assessed the RT reaction using digital (d)PCR for RNA measurement. While many studies characterise dPCR capabilities for DNA quantification, less work has been performed investigating similar parameters using RT-dPCR for RNA analysis. RT-dPCR measurement using three, one-step RT-qPCR kits was evaluated using single and multiplex formats when measuring endogenous and synthetic RNAs. The best performing kit was compared to UV quantification and sensitivity and technical reproducibility investigated. Our results demonstrate assay and kit dependent RT-dPCR measurements differed significantly compared to UV quantification. Different values were reported by different kits for each target, despite evaluation of identical samples using the same instrument. RT-dPCR did not display the strong inter-assay agreement previously described when analysing DNA. This study demonstrates that, as with DNA measurement, RT-dPCR is capable of accurate quantification of low copy RNA targets, but the results are both kit and target dependent supporting the need for calibration controls. PMID:24073259

  7. A nanofluidic system for massively parallel PCR

    NASA Astrophysics Data System (ADS)

    Brenan, Colin; Morrison, Tom; Roberts, Douglas; Hurley, James

    2008-02-01

    Massively parallel nanofluidic systems are lab-on-a-chip devices where solution phase biochemical and biological analyses are implemented in high density arrays of nanoliter holes micro-machined in a thin platen. Polymer coatings make the interior surfaces of the holes hydrophilic and the exterior surface of the platen hydrophobic for precise and accurate self-metered loading of liquids into each hole without cross-contamination. We have created a "nanoplate" based on this concept, equivalent in performance to standard microtiter plates, having 3072 thirty-three nanoliter holes in a stainless steel platen the dimensions of a microscope slide. We report on the performance of this device for PCR-based single nucleotide polymorphism (SNP) genotyping or quantitative measurement of gene expression by real-time PCR in applications ranging from plant and animal diagnostics, agricultural genetics and human disease research.

  8. [Indications for PCR in travel medicine].

    PubMed

    Chappuis, F

    2011-05-11

    The use of PCR-based molecular diagnosis in travel medicine remains limited to specific indications such as clinical suspicion of some of the viral hemorrhagic fevers (e.g. Ebola, Marburg), differential diagnosis between Entamoeba histolytica (pathogen) and E. dispar (non pathogen) in the stools, and parasitological diagnosis of cutaneous leishmaniasis. The scope of indications is likely to expand in the coming years with the development of techniques (e.g. multiplex PCR) able to identify several pathogens from a single sample. Simplification and cost-reduction of molecular techniques, which would allow for more equitable access to these diagnostic tools in countries where the targeted diseases are highly prevalent, pose major technological and ethical challenges.

  9. Molecular diagnostics in tuberculosis.

    PubMed

    Cheng, V C C; Yew, W W; Yuen, K Y

    2005-11-01

    Molecular diagnostics in tuberculosis has enabled rapid detection of Mycobacterium tuberculosis complex in clinical specimens, identification of mycobacterial species, detection of drug resistance, and typing for epidemiological investigation. In the laboratory diagnosis of tuberculosis, the nucleic acid amplification (NAA) test is rapid and specific but not as sensitive as culture of mycobacteria. The primary determinant of successful NAA testing for tuberculosis depends on the shedding of mycobacterial DNA in secretions from caseating granulomas and its dissemination into sterile body fluids or tissue biopsies. In multibacillary diseases with a high mycobacterial load, a positive Ziehl-Neelsen smear with a positive NAA test is diagnostic of active tuberculosis, whereas a positive Ziehl-Neelsen smear with a negative NAA test in the absence of inhibitors would indicate nontuberculous mycobacterial disease. The role of the NAA test is more important in paucibacillary diseases with low mycobacterial loads. The presence of polymerase chain reaction (PCR) inhibitors, however, especially in extrapulmonary specimens, may produce false-negative results. Although this problem can be overcome to some extent by extra extraction steps, the additional processing invariably leads to the loss of mycobacterial DNA. To circumvent this problem, a brief culture augmentation step is carried out before the NAA test is performed, which can enhance the mycobacterial load while concomitantly diluting inhibitors, thereby maintaining the sensitivity of the test without excessively increasing turnaround time.

  10. A review of Theileria diagnostics and epidemiology

    PubMed Central

    Mans, Ben J.; Pienaar, Ronel; Latif, Abdalla A.

    2015-01-01

    An extensive range of serological and molecular diagnostic assays exist for most of the economically important Theileira species such as T. annulata, T. equi, T. lestoquardi, T. parva, T. uilenbergi and other more benign species. Diagnostics of Theileria is considered with regard to sensitivity and specificity of current molecular and serological assays and their use in epidemiology. In the case of serological assays, cross-reactivity of genetically closely related species reduces the use of the gold standard indirect fluorescent antibody test (IFAT). Development of antigen-specific assays does not necessarily address this problem, since closely related species will potentially have similar antigens. Even so, serological assays remain an important line of enquiry in epidemiological surveys. Molecular based assays have exploded in the last decade with significant improvements in sensitivity and specificity. In this review, the current interpretation of what constitute a species in Theileria and its impact on accurate molecular diagnostics is considered. Most molecular assays based on conventional or real-time PCR technology have proven to be on standard with regard to analytical sensitivity. However, consideration of the limits of detection in regard to total blood volume of an animal indicates that most assays may only detect >400,000 parasites/L blood. Even so, natural parasitaemia distribution in carrier-state animals seems to be above this limit of detection, suggesting that most molecular assays should be able to detect the majority of infected individuals under endemic conditions. The potential for false-negative results can, however, only be assessed within the biological context of the parasite within its vertebrate host, i.e. parasitaemia range in the carrier-state that will support infection of the vector and subsequent transmission. PMID:25830110

  11. Identification of invasive fungal diseases in immunocompromised patients by combining an Aspergillus specific PCR with a multifungal DNA-microarray from primary clinical samples.

    PubMed

    Boch, T; Reinwald, M; Postina, P; Cornely, O A; Vehreschild, J J; Heußel, C P; Heinz, W J; Hoenigl, M; Eigl, S; Lehrnbecher, T; Hahn, J; Claus, B; Lauten, M; Egerer, G; Müller, M C; Will, S; Merker, N; Hofmann, W-K; Buchheidt, D; Spiess, B

    2015-12-01

    The increasing incidence of invasive fungal diseases (IFD), most of all invasive aspergillosis (IA) in immunocompromised patients emphasises the need to improve the diagnostic tools for detection of fungal pathogens. We investigated the diagnostic performance of a multifungal DNA-microarray detecting 15 different fungi [Aspergillus, Candida, Fusarium, Mucor, Rhizopus, Scedosporium and Trichosporon species (spp.)] in addition to an Aspergillus specific polymerase chain reaction (PCR) assay. Biopsies, bronchoalveolar lavage and peripheral blood samples of 133 immunocompromised patients (pts) were investigated by a multifungal DNA-microarray as well as a nested Aspergillus specific PCR assay. Patients had proven (n = 18), probable (n = 29), possible (n = 48) and no IFD (n = 38) and were mostly under antifungal therapy at the time of sampling. The results were compared to culture, histopathology, imaging and serology, respectively. For the non-Aspergillus IFD the microarray analysis yielded in all samples a sensitivity of 64% and a specificity of 80%. Best results for the detection of all IFD were achieved by combining DNA-microarray and Aspergillus specific PCR in biopsy samples (sensitivity 79%; specificity 71%). The molecular assays in combination identify genomic DNA of fungal pathogens and may improve identification of causative pathogens of IFD and help overcoming the diagnostic uncertainty of culture and/or histopathology findings, even during antifungal therapy.

  12. DETECTION AND IDENTIFICATION OF PATHOGENIC CANDIDA SPECIES IN WATER USING FLOW CYTOMETRY COUPLED WITH TAQMAN PCR

    EPA Science Inventory

    As the incidence of human fungal infection increases, the ability to detect and identify pathogenic fungi in potential environmental reservoirs becomes increasingly important for disease control. PCR based assays are widely used for diagnostic purposes, but may be inadequate for...

  13. Polymerase chain reaction: A molecular diagnostic tool in periodontology.

    PubMed

    Maheaswari, Rajendran; Kshirsagar, Jaishree Tukaram; Lavanya, Nallasivam

    2016-01-01

    This review discusses the principles of polymerase chain reaction (PCR) and its application as a diagnostic tool in periodontology. The relevant MEDLINE and PubMed indexed journals were searched manually and electronically by typing PCR, applications of PCR, PCR in periodontics, polymorphism studies in periodontitis, and molecular techniques in periodontology. The searches were limited to articles in English language and the articles describing PCR process and its relation to periodontology were collected and used to prepare a concise review. PCR has now become a standard diagnostic and research tool in periodontology. Various studies reveal that its sensitivity and specificity allow it as a rapid, efficient method of detecting, identifying, and quantifying organism. Different immune and inflammatory markers can be identified at the mRNA expression level, and also the determination of genetic polymorphisms, thus providing the deeper insight into the mechanisms underlying the periodontal disease.

  14. Polymerase chain reaction: A molecular diagnostic tool in periodontology

    PubMed Central

    Maheaswari, Rajendran; Kshirsagar, Jaishree Tukaram; Lavanya, Nallasivam

    2016-01-01

    This review discusses the principles of polymerase chain reaction (PCR) and its application as a diagnostic tool in periodontology. The relevant MEDLINE and PubMed indexed journals were searched manually and electronically by typing PCR, applications of PCR, PCR in periodontics, polymorphism studies in periodontitis, and molecular techniques in periodontology. The searches were limited to articles in English language and the articles describing PCR process and its relation to periodontology were collected and used to prepare a concise review. PCR has now become a standard diagnostic and research tool in periodontology. Various studies reveal that its sensitivity and specificity allow it as a rapid, efficient method of detecting, identifying, and quantifying organism. Different immune and inflammatory markers can be identified at the mRNA expression level, and also the determination of genetic polymorphisms, thus providing the deeper insight into the mechanisms underlying the periodontal disease. PMID:27143822

  15. New diagnostic tools in schistosomiasis.

    PubMed

    Utzinger, J; Becker, S L; van Lieshout, L; van Dam, G J; Knopp, S

    2015-06-01

    Schistosomiasis is a water-based parasitic disease that affects over 250 million people. Control efforts have long been in vain, which is one reason why schistosomiasis is considered a neglected tropical disease. However, since the new millennium, interventions against schistosomiasis are escalating. The initial impetus stems from a 2001 World Health Assembly resolution, urging member states to scale-up deworming of school-aged children with the anthelminthic drug praziquantel. Because praziquantel is safe, efficacious and inexpensive when delivered through the school platform, diagnosis before drug intervention was deemed unnecessary and not cost-effective. Hence, there was little interest in research and development of novel diagnostic tools. With the recent publication of the World Health Organization (WHO) Roadmap to overcome the impact of neglected tropical diseases in 2020, we have entered a new era. Elimination of schistosomiasis has become the buzzword and this has important ramifications for diagnostic tools. Indeed, measuring progress towards the WHO Roadmap and whether local elimination has been achieved requires highly accurate diagnostic assays. Here, we introduce target product profiles for diagnostic tools that are required for different stages of a schistosomiasis control programme. We provide an update of the latest developments in schistosomiasis diagnosis, including microscopic techniques, rapid diagnostic tests for antigen detection, polymerase chain reaction (PCR) assays and proxy markers for morbidity assessments. Particular emphasis is placed on challenges and solutions for new technologies to enter clinical practice.

  16. Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

    NASA Astrophysics Data System (ADS)

    Huang, Su-Hua; Yang, Tsuey-Ching; Tsai, Ming-Hong; Tsai, I.-Shou; Lu, Huang-Chih; Chuang, Pei-Hsin; Wan, Lei; Lin, Ying-Ju; Lai, Chih-Ho; Lin, Cheng-Wen

    2008-10-01

    Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples.

  17. Pathological findings and diagnostic implications of a rhesus macaque (Macacca mulatta) model of aerosol exposure to Burkholderia mallei (glanders).

    PubMed

    Yingst, Samuel L; Facemire, Paul; Chuvala, Lara; Norwood, David; Wolcott, Mark; Huzella, Louis

    2015-06-01

    Burkholderia mallei is a Gram-negative bacillus that causes a pneumonic disease known as glanders in equids and humans, and a lymphatic infection known as farcy, primarily in equids. With the potential to infect humans by the respiratory route, aerosol exposure can result in severe, occasionally fatal, pneumonia. Today, glanders infections in humans are rare, likely due to less frequent contact with infected equids than in the past. Acutely ill humans often have non-specific clinical signs and in order to diagnose cases, especially in scenarios of multiple cases in an unexpected setting, rapid diagnostics for B. mallei may be critical. The pathogenesis of acute glanders in the rhesus macaque (Macaca mulatta) was studied as an initial effort to improve diagnostic methods. In the study described here, the diagnostic techniques of PCR, culture and histopathology were compared. The results indicated that PCR may provide rapid, non-invasive diagnosis of glanders in some cases. As expected, PCR results were positive in lung tissue in 11/12 acutely infected rhesus macaques, but more importantly in terms of diagnostic algorithm development, PCR results were frequently positive in non-invasive samples such as broncho-alveolar lavage or nasal swabs (7/12) and occasionally in blood (3/12). However, conventional bacterial culture failed to recover bacteria in many of these samples. The study showed that the clinical presentation of aerosol-exposed rhesus macaques is similar to descriptions of human glanders and that PCR has potential for rapid diagnosis of outbreaks, if not individual cases.

  18. Pathological findings and diagnostic implications of a rhesus macaque (Macacca mulatta) model of aerosol exposure to Burkholderia mallei (glanders)

    PubMed Central

    Yingst, Samuel L.; Chuvala, Lara; Norwood, David; Wolcott, Mark; Huzella, Louis

    2015-01-01

    Burkholderia mallei is a Gram-negative bacillus that causes a pneumonic disease known as glanders in equids and humans, and a lymphatic infection known as farcy, primarily in equids. With the potential to infect humans by the respiratory route, aerosol exposure can result in severe, occasionally fatal, pneumonia. Today, glanders infections in humans are rare, likely due to less frequent contact with infected equids than in the past. Acutely ill humans often have non-specific clinical signs and in order to diagnose cases, especially in scenarios of multiple cases in an unexpected setting, rapid diagnostics for B. mallei may be critical. The pathogenesis of acute glanders in the rhesus macaque (Macaca mulatta) was studied as an initial effort to improve diagnostic methods. In the study described here, the diagnostic techniques of PCR, culture and histopathology were compared. The results indicated that PCR may provide rapid, non-invasive diagnosis of glanders in some cases. As expected, PCR results were positive in lung tissue in 11/12 acutely infected rhesus macaques, but more importantly in terms of diagnostic algorithm development, PCR results were frequently positive in non-invasive samples such as broncho-alveolar lavage or nasal swabs (7/12) and occasionally in blood (3/12). However, conventional bacterial culture failed to recover bacteria in many of these samples. The study showed that the clinical presentation of aerosol-exposed rhesus macaques is similar to descriptions of human glanders and that PCR has potential for rapid diagnosis of outbreaks, if not individual cases. PMID:25850696

  19. Rapid micro-PCR system for hepatitis C virus amplification

    NASA Astrophysics Data System (ADS)

    Lin, Yu-Cheng; Huang, Ming-Yuan; Young, Kung-Chia; Chang, Ting-Tsung; Wu, Ching-Yi

    2000-08-01

    A rapid micro-polymerase chain reaction ((mu) -PCR) system was integrated to amplify the complementary DNA (cDNA) molecules of hepatitis C virus (HCV). This system consists of a rapid thermal cycling system and a (mu) PCR chip fabricated by MEMS fabrication techniques. This rapid (mu) PCR system is verified by using serum samples from patients with chronic hepatitis C. The HCV amplicon of the rapid (mu) PCR system was analyzed by slab gel electrophoresis with separation of DNA marker in parallel. The (mu) PCR chip was fabricated on silicon wafer and Pyrex glass using photolithography, wet etching, and anodic bonding methods. Using silicon material to fabricate the raction well improves the temperature uniformity of sample and helps to reach the desired temperature faster. The rapid close loop thermal cycling system comprises power supplies, a thermal generator, a computer control PID controller, and a data acquisition subsystem. The thermoelectric (T.E.) cooler is used to work as the thermal generator and a heat sink by controlling the polarity of supplied power. The (mu) PCR system was verified with traditional PCR equipment by loading the same PCR mixture with HCV cDNA and running the same cycle numbers, then comparing both HCV amplicon slab gel electrophoresis. The HCV amplicon from the (mu) PCR system shows a DNA fragment with an expected size of 145 base pairs. The background is lower with the (mu) PCR system than that with the tradional PCR equipment. Comparing the traditional PCR equipment which spends 5.5 hours for 30 cycles to gain the detectable amount of HCV amplicon in slab gel separation, this (mu) PCR system takes 30 minutes to finish the 30 thermal cycles. This work has demonstrated that this rapid (mu) PCR system can provide rapid heat generation and dissipation, improved temperature uniformity in DNA amplification.

  20. ddpcr: an R package and web application for analysis of droplet digital PCR data

    PubMed Central

    Attali, Dean; Bidshahri, Roza; Haynes, Charles; Bryan, Jennifer

    2016-01-01

    Droplet digital polymerase chain reaction (ddPCR) is a novel platform for exact quantification of DNA which holds great promise in clinical diagnostics. It is increasingly popular due to its digital nature, which provides more accurate quantification and higher sensitivity than traditional real-time PCR. However, clinical adoption has been slowed in part by the lack of software tools available for analyzing ddPCR data. Here, we present ddpcr – a new R package for ddPCR visualization and analysis. In addition, ddpcr includes a web application (powered by the Shiny R package) that allows users to analyze ddPCR data using an interactive graphical interface. PMID:27703666

  1. ddpcr: an R package and web application for analysis of droplet digital PCR data

    PubMed Central

    Attali, Dean; Bidshahri, Roza; Haynes, Charles; Bryan, Jennifer

    2016-01-01

    Droplet digital polymerase chain reaction (ddPCR) is a novel platform for exact quantification of DNA which holds great promise in clinical diagnostics. It is increasingly popular due to its digital nature, which provides more accurate quantification and higher sensitivity than traditional real-time PCR. However, clinical adoption has been slowed in part by the lack of software tools available for analyzing ddPCR data. Here, we present ddpcr – a new R package for ddPCR visualization and analysis. In addition, ddpcr includes a web application (powered by the Shiny R package) that allows users to analyze ddPCR data using an interactive graphical interface.

  2. DNA probes and PCR for diagnosis of parasitic infections.

    PubMed Central

    Weiss, J B

    1995-01-01

    DNA probe and PCR-based assays to identify and detect parasites are technically complex; however, they have high sensitivity, directly detect parasites independent of the immunocompetence or previous clinical history of the patient, and can distinguish between organisms that are morphologically similar. Diagnosis of parasites is often based on direct detection by microscopy, which is insensitive and laborious and can lack specificity. Most PCR-based assays were more sensitive than DNA probe assays. The development of PCR-based diagnostic assays requires multiple steps following the initial selection of oligonucleotide primers and reporter probe. Generally, the ability to detect the DNA of one parasite was attained by PCR; however, advances in the preparation of samples for PCR (extraction of DNA while removing PCR inhibitors) will be required to achieve that sensitivity with human specimens. Preliminary PCR systems have been developed for many different parasites, yet few have been evaluated with a large number of clinical specimens and/or under field conditions. Those evaluations are essential for determination of clinical and field utility and performance and of the most appropriate application of the assay. Several situations in which PCR-based diagnosis will result in epidemiologic, medical, or public health advances have been identified. PMID:7704890

  3. MAMMALIAN DNA IN PCR REAGENTS

    EPA Science Inventory

    Ancient DNA analysis is becoming widespread. These studies use polymerase chain reaction (PCR) to amplify minute quantities of heavily damaged template. Unusual steps are taken to achieve the sensitivity necessary to detect ancient DNA, including high- cycle PCR amplification t...

  4. Rotorcraft Diagnostics

    NASA Technical Reports Server (NTRS)

    Haste, Deepak; Azam, Mohammad; Ghoshal, Sudipto; Monte, James

    2012-01-01

    Health management (HM) in any engineering systems requires adequate understanding about the system s functioning; a sufficient amount of monitored data; the capability to extract, analyze, and collate information; and the capability to combine understanding and information for HM-related estimation and decision-making. Rotorcraft systems are, in general, highly complex. Obtaining adequate understanding about functioning of such systems is quite difficult, because of the proprietary (restricted access) nature of their designs and dynamic models. Development of an EIM (exact inverse map) solution for rotorcraft requires a process that can overcome the abovementioned difficulties and maximally utilize monitored information for HM facilitation via employing advanced analytic techniques. The goal was to develop a versatile HM solution for rotorcraft for facilitation of the Condition Based Maintenance Plus (CBM+) capabilities. The effort was geared towards developing analytic and reasoning techniques, and proving the ability to embed the required capabilities on a rotorcraft platform, paving the way for implementing the solution on an aircraft-level system for consolidation and reporting. The solution for rotorcraft can he used offboard or embe