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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. PMID:23971826

  2. Improved detection of Tritrichomonas foetus in bovine diagnostic specimens using a novel probe-based real time PCR assay.

    PubMed

    McMillen, Lyle; Lew, Ala E

    2006-11-01

    A Tritrichomonas foetus-specific 5' Taq nuclease assay using a 3' minor groove binder-DNA probe (TaqMan MGB) targeting conserved regions of the internal transcribed spacer-1 (ITS-1) was developed and compared to established diagnostic procedures. Specificity of the assay was evaluated using bovine venereal microflora and a range of related trichomonad species. Assay sensitivity was evaluated with log(10) dilutions of known numbers of cells, and compared to that for microscopy following culture (InPouch TF test kit) and the conventional TFR3-TFR4 PCR assay. The 5' Taq nuclease assay detected a single cell per assay from smegma or mucus which was 2500-fold or 250-fold more sensitive than microscopy following selective culture from smegma or mucus respectively, and 500-fold more sensitive than culture followed by conventional PCR assay. The sensitivity of the conventional PCR assay was comparable to the 5' Taq nuclease assay when testing purified DNA extracted from clinical specimens, whereas the 5' Taq nuclease assay sensitivity improved using crude cell lysates, which were not suitable as template for the conventional PCR assay. Urine was evaluated as a diagnostic specimen providing improved and equivalent levels of T. foetus detection in spiked urine by both microscopy following culture and direct 5' Taq nuclease detection, respectively, compared with smegma and mucus, however inconclusive results were obtained with urine samples from the field study. Diagnostic specimens (n=159) were collected from herds with culture positive animals and of the 14 animals positive by 5' Taq nuclease assay, 3 were confirmed by selective culture/microscopy detection (Fisher's exact test P<0.001). The 5' Taq nuclease assay described here demonstrated superior sensitivity to traditional culture/microscopy and offers advantages over the application of conventional PCR for the detection of T. foetus in clinical samples. PMID:16860481

  3. PCR AS A DIAGNOSTIC TOOL FOR BRUCELLOSIS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Numerous PCR-based assays have been developed for the identification of Brucella to improve diagnostic capabilities. Collectively, the repertoire of assays addresses several aspects of the diagnostic process. For some purposes, the simple identification of Brucella is adequate (e.g. diagnosis of ...

  4. Diagnostic PCR of dermatophytes--an overview.

    PubMed

    Gräser, Yvonne; Czaika, Viktor; Ohst, Torsten

    2012-10-01

    The prevalence of onychomycosis is increasing steadily, sevenfold alone in the US within the last twenty years. An important aspect in this development is the demographic development of the human population of the industrial countries like Germany. A fast and accurate laboratory diagnosis is essential for successful treatment because 50% of the cases are misdiagnosed when relying on the clinical appearance only. The current diagnosis of dermatophytosis, based on direct microscopy and culture of the clinical specimen, is problematic given the lacking specificity of the former and the length of time needed for the latter. Molecular techniques can help to solve these problems. In recent years, a number of in-house PCR assays have been developed to identify dermatophytes directly from clinical specimens. Based on the "Mikrobiologisch-infektiologischen Qualitätsstandards (MIQ) für Nukleinsäure-Amplifikationstechniken" and the MIQE guideline (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) 11 studies are reviewed which were published between 2007 and 2010. The present article evaluates the quality of the PCR assays regarding false positive and false negative results due to contamination, PCR format, statistical analysis, and diagnostic performance of the studies. It shows that we are only at the beginning of providing high quality PCR diagnosis of dermatophytes. PMID:23013298

  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. PMID:23513039

  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

    2016-01-01

    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

  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. Viral diagnostics in the era of digital PCR

    PubMed Central

    Sedlak, Ruth Hall; Jerome, Keith R.

    2012-01-01

    Unlike quantitative PCR (qPCR), digital PCR (dPCR) achieves sensitive and accurate absolute quantitation of a DNA sample without the need for a standard curve. A single PCR reaction is divided into many separate reactions that each have a positive or negative signal. By applying Poisson statistics, the number of DNA molecules in the original sample is directly calculated from the number of positive and negative reactions. The recent availability of multiple commercial dPCR platforms has led to increased interest in clinical diagnostic applications, such as low viral load detection and low abundance mutant detection, where dPCR could be superior to traditional qPCR.Here we review current literature that demonstrates dPCR’s potential utility in viral diagnostics, particularly through absolute quantification of target DNA sequences and rare mutant allele detection. PMID:23182074

  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. Rapid Leptospira identification by direct sequencing of the diagnostic PCR products in New Caledonia

    PubMed Central

    2010-01-01

    Background Most of the current knowledge of leptospirosis epidemiology originates from serological results obtained with the reference Microscopic Agglutination Test (MAT). However, inconsistencies and weaknesses of this diagnostic technique are evident. A growing use of PCR has improved the early diagnosis of leptospirosis but a drawback is that it cannot provide information on the infecting Leptospira strain which provides important epidemiologic data. Our work is aimed at evaluating if the sequence polymorphism of diagnostic PCR products could be used to identify the infecting Leptospira strains in the New Caledonian environment. Results Both the lfb1 and secY diagnostic PCR products displayed a sequence polymorphism that could prove useful in presumptively identifying the infecting leptospire. Using both this polymorphism and MLST results with New Caledonian isolates and clinical samples, we confirmed the epidemiological relevance of the sequence-based identification of Leptospira strains. Additionally, we identified one cluster of L. interrogans that contained no reference strain and one cluster of L. borgpetersenii found only in the introduced Rusa deer Cervus timorensis russa that is its probable reservoir. Conclusions The sequence polymorphism of diagnostic PCR products proved useful in presumptively identifying the infecting Leptospira strains. This could contribute to a better understanding of leptospirosis epidemiology by providing epidemiological information that cannot be directly attained from the use of PCR as an early diagnostic test for leptospirosis. PMID:21176235

  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. New PCR diagnostic systems for the detection and quantification of porcine cytomegalovirus (PCMV).

    PubMed

    Morozov, Vladimir A; Morozov, Alexey V; Denner, Joachim

    2016-05-01

    Pigs are frequently infected with porcine cytomegalovirus (PCMV). Infected adult animals may not present with symptoms of disease, and the virus remains latent. However, the virus may be transmitted to human recipients receiving pig transplants. Recently, it was shown that pig-to-non-human-primate xenotransplantations showed 2 to 3 times lower transplant survival when the donor pig was infected with PCMV. Therefore, highly sensitive methods are required to select virus-free pigs and to examine xenotransplants. Seven previously established PCR detection systems targeting the DNA polymerase gene of PCMV were examined by comparison of thermodynamic parameters of oligonucleotides, and new diagnostic nested PCR and real-time PCR systems with improved parameters and high sensitivity were established. The detection limit of conventional PCR was estimated to be 15 copies, and that of the nested PCR was 5 copies. The sensitivity of the real-time PCR with a TaqMan probe was two copies. An equal efficiency of the newly established detection systems was shown by parallel testing of DNA from sera and blood of six pigs, identifying the same animals as PCMV infected. These new diagnostic PCR systems will improve the detection of PCMV and therefore increase the safety of porcine xenotransplants. PMID:26839086

  14. Chemically modified primers for improved multiplex PCR

    PubMed Central

    Shum, Jonathan; Paul, Natasha

    2009-01-01

    Multiplexed PCR, the amplification of multiple targets in a single reaction, presents a new set of challenges that further complicate more traditional PCR set-ups. These complications include a greater probability for non-specific amplicon formation and for imbalanced amplification of different targets, each of which can compromise quantification and detection of multiple targets. Despite these difficulties, multiplex PCR is frequently used in such applications as pathogen detection, RNA quantification, mutation analysis and now, next generation DNA sequencing. Herein, we investigate the utility of primers with one or two thermolabile 4-oxo-1-pentyl phosphotriester modifications in improving multiplex PCR performance. Initial endpoint and real-time analyses reveal a decrease in off-target amplification and subsequent increase in amplicon yield. Furthermore, the use of modified primers in multiplex set-ups revealed a greater limit of detection and more uniform amplification of each target as compared to unmodified primers. Overall, the thermolabile modified primers present a novel and exciting avenue in improving multiplex PCR performance. PMID:19258004

  15. 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.

  16. 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

  17. Rapid detection of Serpulina hyodysenteriae in diagnostic specimens by PCR.

    PubMed Central

    Elder, R O; Duhamel, G E; Schafer, R W; Mathiesen, M R; Ramanathan, M

    1994-01-01

    A PCR assay for the detection of Serpulina hyodysenteriae in diagnostic specimens was developed on the basis of sequence analysis of a recombinant clone designated pRED3C6. Clone pRED3C6, which contained a 2.3-kb DNA fragment unique to S. hyodysenteriae, was identified by screening a plasmid library of S. hyodysenteriae isolate B204 genomic DNA in Escherichia coli by colony immunoblot with the mouse monoclonal antibody 10G6/G10, which was produced against cell-free supernatant antigens from the same isolate. Southern blot analysis of HindIII-digested genomic DNA of S. hyodysenteriae serotypes 1 through 7 and of four weakly beta-hemolytic intestinal spirochetes, including Serpulina innocens, with the 2.3-kb DNA fragment of pRED3C6 indicated that the cloned sequence was present exclusively in the seven serotypes of S. hyodysenteriae. An oligonucleotide primer pair for PCR amplification of a 1.55-kb fragment and an internal oligonucleotide probe were designed and synthesized on the basis of sequence analysis of the 2.3-kb DNA fragment of pRED3C6. Purified genomic DNAs from reference isolates of S. hyodysenteriae serotypes 1 through 9, S. innocens, weakly beta-hemolytic intestinal spirochetes belonging to genotypic groups distinct from those of reference Serpulina spp., other cultivable reference isolates of the order Spirochaetales, and enteric bacteria including Escherichia coli, Salmonella spp., Campylobacter spp., and Bacteroides vulgatus were amplified with the oligonucleotide primer pair in a hot-start PCR. The 1.55-kb products were obtained only in the presence of genomic DNA from each of the nine serotypes of S. hyodysenteriae. The specificity of the 1.55-kb products for S. hyodysenteriae was confirmed on the basis of production of a restriction endonuclease pattern of the PCR products identical to the predicted restriction map analysis of pRED3C6 and positive hybridization signal with the S. hyodysenteriae-specific internal oligonucleotide probe. By using

  18. 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

  19. PCR diagnostic system in the treatment of prosthetic joint infections.

    PubMed

    Jahoda, D; Landor, I; Benedík, J; Pokorný, D; Judl, T; Barták, V; Jahodová, I; Fulín, P; Síbek, M

    2015-09-01

    In our prospective study, we examined whether a multiplex PCR diagnostic method is suitable for the primary detection of pathogens. We also examined the possibility and sensitivity of detecting genes responsible for biofilm production and methicillin resistance. From 2007 to 2009, 94 patients were included in the study. A UNB (universal detection of 16S ribosomal bacterial DNA) and UNF (universal detection of pathogenic fungi) were used in the primary detection. A multiplex assay for biofilm production, methicillin resistance allowed us to distinguish between Gram positivity and negativity and to detect Staphylococci. From all the samples, the culture was positive in 53.2 % of cases, and by using the UNB method, we detected bacteria in 79.8 % of cases-the UNF detection of fungi was positive in 10.6 % of cases. In 75 % of positive findings, we detected a Gram-negative bacterium in 65.3 % of cases. In 47.2 % of Staphylococci detected, the ability to produce biofilm was confirmed. 61.1 % of the Staphylococci exhibited a methicillin resistance. Our multiplex scheme cannot yet fully replace microbial cultivation but can be a rational guide when choosing an appropriate antibiotic therapy in cases where the microbial culture is negative. PMID:25523034

  20. DNA Barcode-Based PCR-RFLP and Diagnostic PCR for Authentication of Jinqian Baihua She (Bungarus Parvus)

    PubMed Central

    Li, Xiaolei; Zeng, Weiping; Liao, Jing; Liang, Zhenbiao; Huang, Shuhua

    2015-01-01

    We established polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and diagnostic PCR based on cytochrome C oxidase subunit I (COI) barcodes of Bungarus multicinctus, genuine Jinqian Baihua She (JBS), and adulterant snake species. The PCR-RFLP system utilizes the specific restriction sites of SpeI and BstEII in the COI sequence of B. multicinctus to allow its cleavage into 3 fragments (120 bp, 230 bp, and 340 bp); the COI sequences of the adulterants do not contain these restriction sites and therefore remained intact after digestion with SpeI and BstEII (except for that of Zaocys dhumnades, which could be cleaved into a 120 bp and a 570 bp fragment). For diagnostic PCR, a pair of species-specific primers (COI37 and COI337) was designed to amplify a specific 300 bp amplicon from the genomic DNA of B. multicinctus; no such amplicons were found in other allied species. We tested the two methods using 11 commercial JBS samples, and the results demonstrated that barcode-based PCR-RFLP and diagnostic PCR both allowed effective and accurate authentication of JBS. PMID:26078770

  1. DNA Barcode-Based PCR-RFLP and Diagnostic PCR for Authentication of Jinqian Baihua She (Bungarus Parvus).

    PubMed

    Li, Xiaolei; Zeng, Weiping; Liao, Jing; Liang, Zhenbiao; Huang, Shuhua; Chao, Zhi

    2015-01-01

    We established polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and diagnostic PCR based on cytochrome C oxidase subunit I (COI) barcodes of Bungarus multicinctus, genuine Jinqian Baihua She (JBS), and adulterant snake species. The PCR-RFLP system utilizes the specific restriction sites of SpeI and BstEII in the COI sequence of B. multicinctus to allow its cleavage into 3 fragments (120 bp, 230 bp, and 340 bp); the COI sequences of the adulterants do not contain these restriction sites and therefore remained intact after digestion with SpeI and BstEII (except for that of Zaocys dhumnades, which could be cleaved into a 120 bp and a 570 bp fragment). For diagnostic PCR, a pair of species-specific primers (COI37 and COI337) was designed to amplify a specific 300 bp amplicon from the genomic DNA of B. multicinctus; no such amplicons were found in other allied species. We tested the two methods using 11 commercial JBS samples, and the results demonstrated that barcode-based PCR-RFLP and diagnostic PCR both allowed effective and accurate authentication of JBS. PMID:26078770

  2. 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

  3. 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

  4. 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.

  5. Quantitative nucleic acid amplification by digital PCR for clinical viral diagnostics.

    PubMed

    Zhang, Kuo; Lin, Guigao; Li, Jinming

    2016-09-01

    In the past few years, interest in the development of digital PCR (dPCR) as a direct nucleic acid amplification technique for clinical viral diagnostics has grown. The main advantages of dPCR over qPCR include: quantification of nucleic acid concentrations without a calibration curve, comparable sensitivity, superior quantitative precision, greater resistance to perturbations by inhibitors, and increased robustness to the variability of the target sequence. In this review, we address the application of dPCR to viral nucleic acid quantification in clinical applications and for nucleic acid quantification standardization. Further development is required to overcome the current limitations of dPCR in order to realize its widespread use for viral load measurements in clinical diagnostic applications. PMID:26845722

  6. 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 ...

  7. 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

  8. Enhanced Diagnostic Yields of Bacteremia and Candidemia in Blood Specimens by PCR-Electrospray Ionization Mass Spectrometry

    PubMed Central

    Laffler, Thomas G.; Cummins, Lendell L.; McClain, Colt M.; Quinn, Criziel D.; Toro, Michelle A.; Carolan, Heather E.; Toleno, Donna M.; Rounds, Megan A.; Eshoo, Mark W.; Stratton, Charles W.; Sampath, Rangarajan; Blyn, Lawrence B.; Ecker, David J.

    2013-01-01

    A prospective study was performed to determine the value of direct molecular testing of whole blood for detecting the presence of culturable and unculturable bacteria and yeasts in patients with suspected bloodstream infections. A total of 464 adult and pediatric patients with positive blood cultures matched with 442 patients with negative blood cultures collected during the same period were recruited during a 10-month study. PCR amplification coupled with electrospray ionization mass spectrometry (PCR-ESI-MS) plus blood culture reached an overall agreement of 78.6% in the detection and species-level identification of bacterial and candidal pathogens. Of 33 culture-negative/PCR-ESI-MS-positive specimens, 31 (93.9%) were judged to be truly bacteremic and/or candidemic based on a medical chart review and analytical metrics. Among the 15 culture-positive specimens in which PCR-ESI-MS detected additional bacterial or yeast species, 66.7% and 20.0% of the additional positive specimens by PCR-ESI-MS were judged to be truly or possibly bacteremic and/or candidemic, respectively. Direct analysis of blood samples by PCR-ESI-MS rapidly detects bacterial and yeast pathogens in patients with bloodstream infections. When used in conjunction with blood culture, PCR-ESI-MS enhances the diagnostics of septicemia by shortening test turnaround time and improving yields. PMID:23966503

  9. 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

  10. Use of PCR in resolving diagnostic difficulties potentially caused by genetic variation of hepatitis B virus.

    PubMed Central

    van Deursen, F J; Hino, K; Wyatt, D; Molyneaux, P; Yates, P; Wallace, L A; Dow, B C; Carman, W F

    1998-01-01

    AIMS: To assess the relevance of genetic variants of hepatitis B virus (HBV) and to demonstrate the usefulness of the polymerase chain reaction (PCR) in cases of HBV diagnostic difficulty. METHODS: Five serum samples from patients that presented diagnostic difficulty in routine laboratories were sent to a research laboratory for PCR, and if appropriate, S gene sequencing, in vitro expression, and antigenic analysis. RESULTS: The demonstration of HBV in serum by PCR allowed a definitive diagnosis of current infection. One serum sample with poor reactivity in a diagnostic assay had a minor hepatitis B surface antigen (HBsAg) variant and another with very poor reactivity had multiple variants of HBsAg. Transient HBsAg reactivity was observed in a recently vaccinated patient. A hepatitis Be antigen (HBeAg) false positive reaction was noted in a patient from a well defined risk group for HBV. One patient who was strongly HBsAg/HBeAg positive, but anti-hepatitis B core antibody negative, was viraemic. CONCLUSIONS: PCR may become the gold standard for the diagnosis of current HBV infection. HBV variants are responsible for a proportion of diagnostically difficult cases. Modification of commercial assays is necessary to increase the sensitivity of detection of such variants. PMID:9602690

  11. Malaria surveillance in the Democratic Republic of the Congo: comparison of microscopy, PCR, and rapid diagnostic test.

    PubMed

    Doctor, Stephanie M; Liu, Yunhao; Whitesell, Amy; Thwai, Kyaw L; Taylor, Steve M; Janko, Mark; Emch, Michael; Kashamuka, Melchior; Muwonga, Jérémie; Tshefu, Antoinette; Meshnick, Steven R

    2016-05-01

    Malaria surveillance is critical for control efforts, but diagnostic methods frequently disagree. Here, we compare microscopy, PCR, and a rapid diagnostic test in 7137 samples from children in the Democratic Republic of the Congo using latent class analysis. PCR had the highest sensitivity (94.6%) and microscopy had the lowest (76.7%). PMID:26915637

  12. 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. PMID:26303619

  13. Biomarker-based diagnostic work-up of invasive pulmonary aspergillosis in immunocompromised paediatric patients--is Aspergillus PCR appropriate?

    PubMed

    Buchheidt, Dieter; Reinwald, Mark; Spiess, Birgit; Boch, Tobias; Hofmann, Wolf-Karsten; Groll, Andreas H; Lehrnbecher, Thomas

    2016-02-01

    Invasive aspergillosis (IA) is an important cause of morbidity and mortality in children and adults with haematologic malignancies or undergoing allogeneic haematopoietic stem cell transplantation, and early diagnosis and adequate antifungal treatment improve outcome. However, important differences exist between children and adults regarding epidemiology, underlying disease, and comorbidities, and the value of diagnostic tools to detect IA may also differ between these patient populations. Imaging studies are important to detect IA early, but typical findings of IA in chest computed tomography of adults are not detected in the majority of children. Whereas the value of the serum marker galactomannan seems to be comparable in children and adults, data on the performance of beta-d-glucan in children are too limited for firm conclusions. PCR-based assays are a promising diagnostic approach to rapidly and reliably detect and identify Aspergillus species in various clinical samples. However, as the majority of data on PCR-based approaches has been obtained in adult patients, the value of this method in paediatric patients has not been defined to date. The present review focuses on studies of PCR-based methods to diagnose IA in immunocompromised paediatric patients. PMID:26756571

  14. Improved purification and PCR amplification of DNA from environmental samples.

    PubMed

    Arbeli, Ziv; Fuentes, Cilia L

    2007-07-01

    Purification and PCR amplification procedures for DNA extracted from environmental samples (soil, compost, and river sediment) were improved by introducing three modifications: precipitation of DNA with 5% polyethylene glycol 8000 (PEG) and 0.6 M NaCl; filtration with a Sepharose 4B-polyvinylpolypyrrolidone (PVPP) spin column; and addition of skim milk (0.3% w/v) to the PCR reaction solution. Humic substances' concentration after precipitation with 5% PEG was 2.57-, 5.3-, and 78.9-fold lower than precipitation with 7.5% PEG, 10% PEG, and isopropanol, respectively. After PEG precipitation, Sepharose, PVPP and the combined (Sepharose-PVPP) column removed 92.3%, 89.5%, and 98%, respectively, of the remaining humic materials. Each of the above-mentioned modifications improved PCR amplification of the 16S rRNA gene. DNA extracted by the proposed protocol is cleaner than DNA extracted by a commercial kit. Nevertheless, the improvement of DNA purification did not improve the detection limit of atrazine degradation gene atzA. PMID:17521406

  15. 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

  16. 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...

  17. RT-PCR is a more accurate diagnostic tool for detection of BCR-ABL rearrangement

    SciTech Connect

    Zehnbauer, B.A.; Allen, A.P.; McGrath, S.D.

    1994-09-01

    Detection of the Philadelphia chromosome (Ph1) or genomic Southern hybridization for clonal gene rearrangement (GSH-R) has provided very specific identification of BCR-ABL gene rearrangement. Reverse transcriptase-polymerase chain reaction (RT-PCR) is diagnostic for patterns of BCR-ABL expression which are undetected by GSH-R and/or Ph1 and provides increased sensitivity both at diagnosis and in detection of minimal residual leukemia. Fifty-three specimens (of 150 tested from 119 consecutive leukemia patients) were RT-PCR positive for BCR-ABL gene expression confirmed by hybridization of PCR products with b{sub 3}a{sub 2}, b{sub 2}a{sub 2}, or e{sub 1}a{sub 2} junction-specific oligonucleotides. In 6 cases of CML with GSH-R{sup {minus}}at diagnosis, RT-PCR provided specific BCR-ABL identification. Deletion of BCR regions, low mitotic index, or e{sub 1}a{sub 2} expression caused failure to detect GSH-R or Ph1 translocation.

  18. 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.

  19. 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.

  20. 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

  1. Performance of a real-time PCR assay in routine bovine mastitis diagnostics compared with in-depth conventional culture.

    PubMed

    Hiitiö, Heidi; Riva, Rauna; Autio, Tiina; Pohjanvirta, Tarja; Holopainen, Jani; Pyörälä, Satu; Pelkonen, Sinikka

    2015-05-01

    Reliable identification of the aetiological agent is crucial in mastitis diagnostics. Real-time PCR is a fast, automated tool for detecting the most common udder pathogens directly from milk. In this study aseptically taken quarter milk samples were analysed with a real-time PCR assay (Thermo Scientific PathoProof Mastitis Complete-12 Kit, Thermo Fisher Scientific Ltd.) and by semi-quantitative, in-depth bacteriological culture (BC). The aim of the study was to evaluate the diagnostic performance of the real-time PCR assay in routine use. A total of 294 quarter milk samples from routine mastitis cases were cultured in the national reference laboratory of Finland and examined with real-time PCR. With BC, 251 out of 294 (85.7%) of the milk samples had at least one colony on the plate and 38 samples were considered contaminated. In the PCR mastitis assay, DNA of target species was amplified in 244 samples out of 294 (83.0%). The most common bacterial species detected in the samples, irrespective of the diagnostic method, was the coagulase negative staphylococci (CNS) group (later referred as Staphylococcus spp.) followed by Staphylococcus aureus. Sensitivity (Se) and specificity (Sp) for the PCR assay to provide a positive Staph. aureus result was 97.0 and 95.8% compared with BC. For Staphylococcus spp., the corresponding figures were 86.7 and 75.4%. Our results imply that PCR performed well as a diagnostic tool to detect Staph. aureus but may be too nonspecific for Staphylococcus spp. in routine use with the current cut-off Ct value (37.0). Using PCR as the only microbiological method for mastitis diagnostics, clinical relevance of the results should be carefully considered before further decisions, for instance antimicrobial treatment, especially when minor pathogens with low amount of DNA have been detected. Introducing the concept of contaminated samples should also be considered. PMID:25704849

  2. 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

  3. 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

  4. Design and evaluation of a unique RT-qPCR assay for diagnostic quality control assessment that is applicable to pathogen detection in three species of salmonid fish

    PubMed Central

    2013-01-01

    Background The detection of pathogens at early stages of infection is a key point for disease control in aquaculture. Therefore, accurate diagnostic procedures are a must. Real-time PCR has been a mainstay in diagnostics over the years due to its speed, specificity, sensitivity, reproducibility and throughput; as such, real-time PCR is a target for improvement. Nevertheless, to validate a novel diagnostic tool, correct setup of the assay, including proper endogenous controls to evaluate the quantity and quality of the samples and to detect possible sample degradation, is compulsory. This work aims to design a unique RT-qPCR assay for pathogen detection in the three salmonid species reared in Chile. The assay uses elongation factor 1 alpha as the single endogenous control, thus avoiding the need for multiple endogenous controls, as well as multiple validations and non-comparable quality control parameters. Results The in vivo and in vitro analyses of samples from Salmo salar, Oncorhynchus mykiss and Oncorhynchus kisutch showed that when primers were accurately selected to target conserved regions of the elongation factor 1 alpha (ELF1α) gene, a single novel RT-qPCR assay yielding similar and reproducible Ct values between the three species could be designed. The opposite occurred when an assay originally designed for Salmo salar was tested in samples from the two species of the genus Oncorhynchus. Conclusions Here, we report the design and evaluation of an accurate trans-species RT-qPCR assay that uses the elongation factor 1 alpha (ELF1α) gene as an endogenous control and is applicable for diagnostic purposes in samples obtained from the three salmonid species reared in Chile. PMID:24040749

  5. Improved Specificity for Detection of Mycobacterium bovis in Fresh Tissues Using IS6110 Real-time PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Culture of M. bovis from diagnostic specimens is the gold standard for bovine tuberculosis diagnostics in the US. Detection of M. bovis by PCR in tissue homogenates may provide a simple, rapid method to complement diagnostic culture. A significant impediment to PCR based assays on tissue...

  6. Optimized diagnostic model combination for improving diagnostic accuracy

    NASA Astrophysics Data System (ADS)

    Kunche, S.; Chen, C.; Pecht, M. G.

    Identifying the most suitable classifier for diagnostics is a challenging task. In addition to using domain expertise, a trial and error method has been widely used to identify the most suitable classifier. Classifier fusion can be used to overcome this challenge and it has been widely known to perform better than single classifier. Classifier fusion helps in overcoming the error due to inductive bias of various classifiers. The combination rule also plays a vital role in classifier fusion, and it has not been well studied which combination rules provide the best performance during classifier fusion. Good combination rules will achieve good generalizability while taking advantage of the diversity of the classifiers. In this work, we develop an approach for ensemble learning consisting of an optimized combination rule. The generalizability has been acknowledged to be a challenge for training a diverse set of classifiers, but it can be achieved by an optimal balance between bias and variance errors using the combination rule in this paper. Generalizability implies the ability of a classifier to learn the underlying model from the training data and to predict the unseen observations. In this paper, cross validation has been employed during performance evaluation of each classifier to get an unbiased performance estimate. An objective function is constructed and optimized based on the performance evaluation to achieve the optimal bias-variance balance. This function can be solved as a constrained nonlinear optimization problem. Sequential Quadratic Programming based optimization with better convergence property has been employed for the optimization. We have demonstrated the applicability of the algorithm by using support vector machine and neural networks as classifiers, but the methodology can be broadly applicable for combining other classifier algorithms as well. The method has been applied to the fault diagnosis of analog circuits. The performance of the proposed

  7. PCR and in vitro cultivation for detection of Leishmania spp. in diagnostic samples from humans and dogs.

    PubMed Central

    Mathis, A; Deplazes, P

    1995-01-01

    A PCR assay for the diagnosis of leishmaniosis was developed by using primers that were selected from the sequence of the small-subunit rRNA gene. The assay was optimized for routine diagnostic use. Processing of the clinical samples is rapid and simple (lysis of erythrocytes in Tris-EDTA buffer, digestion with proteinase K directly in PCR buffer, and no further purification steps). Furthermore, an internal control is included in every specimen in order to detect the presence of PCR inhibitors. The PCR was compared with diagnostic in vitro cultivation of promastigote stages for the detection of Leishmania spp. in clinical specimens from humans and dogs with a tentative diagnosis of leishmaniosis. PCR and cultivation gave identical results with all but 1 of the 95 specimens from humans. The PCR result in this case was false negative, possibly because of unequal apportionment of this sample. With 10 skin biopsies from six patients with cutaneous leishmaniosis, the sensitivity was 60%. For six human immunodeficiency virus-positive patients with visceral leishmaniosis, all bone marrow biopsies and 7 of 11 whole blood samples (after isolation of leukocytes by Ficoll-Paque) were positive in both tests. PCR detected one more case with the use of 500 microliters of whole blood with direct lysis of the erythrocytes in Tris-EDTA buffer. With dog lymph node aspirates, the sensitivity was 100% (16 of 16 samples) for both methods; furthermore, PCR was positive for 5 of 13 whole blood samples from dogs with leishmaniosis. The specificity of the PCR was 100% (70 specimens from patients without leishmaniosis).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7615719

  8. Development and Evaluation of a Next-Generation Digital PCR Diagnostic Assay for Ocular Chlamydia trachomatis Infections

    PubMed Central

    Last, Anna; Molina-Gonzalez, Sandra; Cassama, Eunice; Butcher, Robert; Nabicassa, Meno; McCarthy, Elizabeth; Burr, Sarah E.; Mabey, David C.; Bailey, Robin L.; Holland, Martin J.

    2013-01-01

    Droplet digital PCR (ddPCR) is an emulsion PCR process that performs absolute quantitation of nucleic acids. We developed a ddPCR assay for Chlamydia trachomatis infections and found it to be accurate and precise. Using PCR mixtures containing plasmids engineered to include the PCR target sequences, we were able to quantify with a dynamic range between 0.07 and 3,160 targets/μl (r2 = 0.9927) with >95% confidence. Using 1,509 clinical conjunctival swab samples from a population in which trachoma is endemic in Guinea Bissau, we evaluated the specificity and sensitivity of the quantitative ddPCR assay in diagnosing ocular C. trachomatis infections by comparing the performances of ddPCR and the Roche Amplicor CT/NG test. We defined ddPCR tests as positive when we had ≥95% confidence in a nonzero estimate of target load. The sensitivity of ddPCR against Amplicor was 73.3% (95% confidence interval [CI], 67.9 to 78.7%), and specificity was 99.1% (95% CI, 98.6 to 99.6%). Negative and positive predictive values were 94.6% (95% CI, 93.4 to 95.8%) and 94.5% (95% CI, 91.3 to 97.7%), respectively. Based on Amplicor CT/NG testing, the estimated population prevalence of C. trachomatis ocular infection was ∼17.5%. Receiver-operator curve analysis was used to select critical cutoff values for use in clinical settings in which a balance between higher sensitivity and specificity is required. We concluded that ddPCR is an effective diagnostic technology suitable for both research and clinical use in diagnosing ocular C. trachomatis infections. PMID:23637300

  9. 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

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

    PubMed

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

    2016-02-01

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

  11. Does Diagnostic Math Testing Improve Student Learning?

    ERIC Educational Resources Information Center

    Betts, Julian R.; Hahn, Youjin; Zau, Andrew C.

    2011-01-01

    The Mathematics Diagnostic Testing Project (MDTP) offers course-specific assessments that provide teachers with timely feedback on their students' strengths and weaknesses in mathematics, often returning feedback to teachers on individual students and the entire class within a week of testing. In this way, teachers can quickly act on what they…

  12. 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

  13. Extensive multiplex PCR diagnostics reveal new insights into the epidemiology of viral respiratory infections.

    PubMed

    Nickbakhsh, S; Thorburn, F; VON Wissmann, B; McMENAMIN, J; Gunson, R N; Murcia, P R

    2016-07-01

    Viral respiratory infections continue to pose a major global healthcare burden. At the community level, the co-circulation of respiratory viruses is common and yet studies generally focus on single aetiologies. We conducted the first comprehensive epidemiological analysis to encompass all major respiratory viruses in a single population. Using extensive multiplex PCR diagnostic data generated by the largest NHS board in Scotland, we analysed 44230 patient episodes of respiratory illness that were simultaneously tested for 11 virus groups between 2005 and 2013, spanning the 2009 influenza A pandemic. We measured viral infection prevalence, described co-infections, and identified factors independently associated with viral infection using multivariable logistic regression. Our study provides baseline measures and reveals new insights that will direct future research into the epidemiological consequences of virus co-circulation. In particular, our study shows that (i) human coronavirus infections are more common during influenza seasons and in co-infections than previously recognized, (ii) factors associated with co-infection differ from those associated with viral infection overall, (iii) virus prevalence has increased over time especially in infants aged <1 year, and (iv) viral infection risk is greater in the post-2009 pandemic era, likely reflecting a widespread change in the viral population that warrants further investigation. PMID:26931455

  14. 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

  15. 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

  16. 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

  17. Modifications of Commercial Toxigenic Clostridium difficile PCR Resulting in Improved Economy and Workflow Efficiency▿

    PubMed Central

    Munson, Erik; Bilbo, Dorothy; Paul, Mary; Napierala, Maureen; Hryciuk, Jeanne E.

    2011-01-01

    Expense inherent to molecular diagnostics may prevent laboratories from utilizing real-time PCR for Clostridium difficile infection. Frozen master mix and overnight aliquot modifications of the BD GeneOhm Cdiff assay failed to impact performance indices compared to the package insert protocol (P ≥ 0.31), provided accurate results, and decreased reagent expenditure. PMID:21450967

  18. Rapid diagnostic tests as a source of DNA for Plasmodium species-specific real-time PCR

    PubMed Central

    2011-01-01

    Background This study describes the use of malaria rapid diagnostic tests (RDTs) as a source of DNA for Plasmodium species-specific real-time PCR. Methods First, the best method to recover DNA from RDTs was investigated and then the applicability of this DNA extraction method was assessed on 12 different RDT brands. Finally, two RDT brands (OptiMAL Rapid Malaria Test and SDFK60 malaria Ag Plasmodium falciparum/Pan test) were comprehensively evaluated on a panel of clinical samples submitted for routine malaria diagnosis at ITM. DNA amplification was done with the 18S rRNA real-time PCR targeting the four Plasmodium species. Results of PCR on RDT were compared to those obtained by PCR on whole blood samples. Results Best results were obtained by isolating DNA from the proximal part of the nitrocellulose component of the RDT strip with a simple DNA elution method. The PCR on RDT showed a detection limit of 0.02 asexual parasites/μl, which was identical to the same PCR on whole blood. For all 12 RDT brands tested, DNA was detected except for one brand when a low parasite density sample was applied. In RDTs with a plastic seal covering the nitrocellulose strip, DNA extraction was hampered. PCR analysis on clinical RDT samples demonstrated correct identification for single species infections for all RDT samples with asexual parasites of P. falciparum (n = 60), Plasmodium vivax (n = 10), Plasmodium ovale (n = 10) and Plasmodium malariae (n = 10). Samples with only gametocytes were detected in all OptiMAL and in 10 of the 11 SDFK60 tests. None of the negative samples (n = 20) gave a signal by PCR on RDT. With PCR on RDT, higher Ct-values were observed than with PCR on whole blood, with a mean difference of 2.68 for OptiMAL and 3.53 for SDFK60. Mixed infections were correctly identified with PCR on RDT in 4/5 OptiMAL tests and 2/5 SDFK60 tests. Conclusions RDTs are a reliable source of DNA for Plasmodium real-time PCR. This study demonstrates the best method of RDT

  19. 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

  20. DEVELOPMENT OF MULTIPLEX REAL-TIME RT-PCR AS A DIAGNOSTIC TOOL FOR AVIAN INFLUENZA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A multiplex real-time RT-PCR (RRT-PCR) assay for the simultaneous detection of the H5 and H7 hemagglutinin (HA) subtypes was developed with hydrolysis type probes labeled with the FAM (H5 probe) and ROX (H7 probe) dyes. The sensitivity of the H5-H7 subtyping assay was determined, using in vitro tran...

  1. Development a diagnostic pan-dermatophyte TaqMan probe real-time PCR assay based on beta tubulin gene.

    PubMed

    Mirhendi, Hossein; Motamedi, Marjan; Makimura, Koichi; Satoh, Kazuo

    2016-08-01

    Early differentiation of dermatophytosis from other cutaneous mycoses is essential to avoid inaccurate therapy. DNA-based techniques including real-time PCR have increasingly been considered for detection of fungal elements in clinical specimens. In this study, after partial sequence analysis of beta tubulin (BT2) gene in 13 common and rare pathogenic dermatophyte species, a pan-dermatophyte primer and probe set was designed in a TaqMan probe-based PCR format. The sensitivity and specificity of the system was tested with 22 reference strains of dermatophytes, 234 positive clinical specimens, 32 DNA samples extracted from normal nails, several fungi other than dermatophytes and human DNAs. Analytical detection limit of the designed PCR on serially diluted DNAs of prepared recombinant plasmid indicated that only five molecules per sample are the minimum number for reliable detection by the assay. A total of 226 out of 234 (96.5%) DNAs extracted from clinical samples, but none of the 32 nail samples, from healthy volunteers were positive in PCR. The real-time PCR targeted beta tubulin gene established in this study could be a sensitive diagnostic tool which is significantly faster than the conventional culture method and should be useful in the clinical settings, in large-scale epidemiological studies and in clinical trials of antifungal therapy. PMID:27071371

  2. Diagnostic PCR tests for Microsporum audouinii, M. canis and Trichophyton infections.

    PubMed

    Brillowska-Dabrowska, Anna; Swierkowska, Aleksandra; Lindhardt Saunte, Ditte Marie; Arendrup, Maiken Cavling

    2010-05-01

    Since traditional diagnosis of dermatophyte infections is slow, we present a rapid new PCR test for detection of Trichophyton spp., Microsporum canis and M. audouinii infections. The performance of the test was evaluated with: 58 dermatophyte isolates; 10 yeast, mould and human DNA control samples; 25 routine specimens from patients suspected of having dermatophytosis; 10 hair specimens from guinea pigs experimentally infected with M. canis; and two samples from un-infected control animals. DNA was prepared by a 10-min procedure from pure cultures as previously described. The 302 bp PCR product was obtained for 35/35 Trichophyton isolates (10 species included) and the 279 bp for 3/3 M. canis and 4/4 M. audouinii samples. None of the 2 E. floccosum, 11 M. gypseum, 3 M M. persicolor or 12 control samples (yeast, mould, human DNA) were positive with either of the two PCR tests. Among the patient specimens, seven were T. rubrum positive, two for T. mentagrophytes, one was positive for T. tonsurans and 15 were dermatophyte negative by routine investigation (culture and/or pan-dermatophyte + T. rubrum multiplex PCR). The PCR results with our procedures were in 100% agreement with these results. Finally, the Microsporum PCR was positive for 10/10 guinea pig specimens from infected animals but for 0/2 of the control animal samples. The evaluation of the two PCR tests indicated excellent sensitivity and specificity. PMID:19886764

  3. 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. PMID:24252553

  4. Quantitative real-time PCR and phase specific serology are mutually supportive in Q fever diagnostics in goats.

    PubMed

    Sting, Reinhard; Molz, Kerstin; Philipp, Werner; Bothe, Friederike; Runge, Martin; Ganter, Martin

    2013-12-27

    This study presents results of quantitative pathogen detection by real-time PCR (qPCR) and phase-specific serology for complete Q fever diagnostics. For this, samples of 42 goats in total were taken during a Q fever outbreak. In the early phase of the Q-fever infection, 10(4)-10(8)Coxiella (C.) burnetii pathogens per vaginal swab and 10(2)-10(6)C. burnetii per ml milk were detected using quantitative real-time PCR (qPCR). Pathogen excretion decreased continuously within two months to less than 10(4) (vaginal swab) and 10(2) (milk) C. burnetii. At the end of the study there was a shift toward a 10 fold higher excretion of the pathogen via the genital tract and milk. At the start of the study, serological tests showed a dominance of the phase-2 antibody in 76% (22/29) of the goats in the MONA- (Multiple of Normal Activity) ELISA and 79% (23/29) in the IDEXX-ELISA, which was replaced by a phase-1 dominance in 85% (29/34) and 62% (21/34), of the animals respectively at the end of the study. Serum samples from 13 goats before lambing that excreted C. burnetii after lambing showed antibodies against phase 2 of 100% using MONA-ELISA and 77% in the IDEXX-ELISA. The most important diagnostic instrument for Q-fever infection in goats following birth is testing of vaginal swabs using qPCR. Phase-specific serology allows an estimation of possible pathogen excretion even before birth, as well as achieving valuable results for determination of the infection phase. PMID:24095624

  5. 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

  6. 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 ...

  7. 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.

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

    PubMed

    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. PMID:23924819

  9. Real-Time PCR Improves Helicobacter pylori Detection in Patients with Peptic Ulcer Bleeding

    PubMed Central

    Casalots, Alex; Sanfeliu, Esther; Boix, Loreto; García-Iglesias, Pilar; Sánchez-Delgado, Jordi; Montserrat, Antònia; Bella-Cueto, Maria Rosa; Gallach, Marta; Sanfeliu, Isabel; Segura, Ferran; Calvet, Xavier

    2011-01-01

    Background and Aims Histological and rapid urease tests to detect H. pylori in biopsy specimens obtained during peptic ulcer bleeding episodes (PUB) often produce false-negative results. We aimed to examine whether immunohistochemistry and real-time PCR can improve the sensitivity of these biopsies. Patients and Methods We selected 52 histology-negative formalin-fixed paraffin-embedded biopsy specimens obtained during PUB episodes. Additional tests showed 10 were true negatives and 42 were false negatives. We also selected 17 histology-positive biopsy specimens obtained during PUB to use as controls. We performed immunohistochemistry staining and real-time PCR for 16S rRNA, ureA, and 23S rRNA for H. pylori genes on all specimens. Results All controls were positive for H. pylori on all PCR assays and immunohistochemical staining. Regarding the 52 initially negative biopsies, all PCR tests were significantly more sensitive than immunohistochemical staining (p<0.01). Sensitivity and specificity were 55% and 80% for 16S rRNA PCR, 43% and 90% for ureA PCR, 41% and 80% for 23S rRNA PCR, and 7% and 100% for immunohistochemical staining, respectively. Combined analysis of PCR assays for two genes were significantly more sensitive than ureA or 23S rRNA PCR tests alone (p<0.05) and marginally better than 16S rRNA PCR alone. The best combination was 16S rRNA+ureA, with a sensitivity of 64% and a specificity of 80%. Conclusions Real-time PCR improves the detection of H. pylori infection in histology-negative formalin-fixed paraffin-embedded biopsy samples obtained during PUB episodes. The low reported prevalence of H. pylori in PUB may be due to the failure of conventional tests to detect infection. PMID:21625499

  10. 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. PMID:16857272

  11. Diagnostic accuracy of Kato-Katz, FLOTAC, Baermann, and PCR methods for the detection of light-intensity hookworm and Strongyloides stercoralis infections in Tanzania.

    PubMed

    Knopp, Stefanie; Salim, Nahya; Schindler, Tobias; Karagiannis Voules, Dimitrios A; Rothen, Julian; Lweno, Omar; Mohammed, Alisa S; Singo, Raymond; Benninghoff, Myrna; Nsojo, Anthony A; Genton, Blaise; Daubenberger, Claudia

    2014-03-01

    Sensitive diagnostic tools are crucial for an accurate assessment of helminth infections in low-endemicity areas. We examined stool samples from Tanzanian individuals and compared the diagnostic accuracy of a real-time polymerase chain reaction (PCR) with the FLOTAC technique and the Kato-Katz method for hookworm and the Baermann method for Strongyloides stercoralis detection. Only FLOTAC had a higher sensitivity than the Kato-Katz method for hookworm diagnosis; the sensitivities of PCR and the Kato-Katz method were equal. PCR had a very low sensitivity for S. stercoralis detection. The cycle threshold values of the PCR were negatively correlated with the logarithm of hookworm egg and S. stercoralis larvae counts. The median larvae count was significantly lower in PCR false negatives than true positives. All methods failed to detect very low-intensity infections. New diagnostic approaches are needed for monitoring of progressing helminth control programs, confirmation of elimination, or surveillance of disease recrudescence. PMID:24445211

  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. Additional diagnostic yield of adding serology to PCR in diagnosing viral acute respiratory infections in Kenyan patients 5 years of age and older.

    PubMed

    Feikin, Daniel R; Njenga, M Kariuki; Bigogo, Godfrey; Aura, Barrack; Gikunju, Stella; Balish, Amanda; Katz, Mark A; Erdman, Dean; Breiman, Robert F

    2013-01-01

    The role of serology in the setting of PCR-based diagnosis of acute respiratory infections (ARIs) is unclear. We found that acute- and convalescent-phase paired-sample serologic testing increased the diagnostic yield of naso/oropharyngeal swabs for influenza virus, respiratory syncytial virus (RSV), human metapneumovirus, adenovirus, and parainfluenza viruses beyond PCR by 0.4% to 10.7%. Although still limited for clinical use, serology, along with PCR, can maximize etiologic diagnosis in epidemiologic studies. PMID:23114699

  14. Low-stringency PCR with diagnostically useful primers for identification of Leptospira serovars.

    PubMed Central

    de Caballero, O L; Dias Neto, E; Koury, M C; Romanha, A J; Simpson, A J

    1994-01-01

    Primers proposed for the diagnosis of the pathogenic spirochete Leptospira spp. (C. Gravekamp, H. V. D. Kemp, M. Franzen, D. Carrington, G.J. Schoone, G.J.J.M. Van Eys, C. O. R. Everard, R.A. Hartskeel, and W.J. Terpstra, J. Gen. Microbiol. 139:1691-1700, 1993) have been found to produce complex serovar-specific patterns under low-stringency PCR conditions. Such patterns obtained by low-stringency PCR, which maintain the specific band as an internal control, offer, an approach to the standardized identification of Leptospira serovars in clinical laboratories. Images PMID:8051272

  15. 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

  16. 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

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

    PubMed Central

    Shin, Yong-Gil; Rho, Jae-Young

    2014-01-01

    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

  18. Single-Cell Quantitative PCR: Advances and Potential in Cancer Diagnostics.

    PubMed

    Ok, Chi Young; Singh, Rajesh R; Salim, Alaa A

    2016-01-01

    Tissues are heterogeneous in their components. If cells of interest are a minor population of collected tissue, it would be difficult to obtain genetic or genomic information of the interested cell population with conventional genomic DNA extraction from the collected tissue. Single-cell DNA analysis is important in the analysis of genetics of cell clonality, genetic anticipation, and single-cell DNA polymorphisms. Single-cell PCR using Single Cell Ampligrid/GeXP platform is described in this chapter. PMID:26843054

  19. Multiplex Real-Time PCR Diagnostic of Relapsing Fevers in Africa

    PubMed Central

    Elbir, Haitham; Henry, Mireille; Diatta, Georges; Mediannikov, Oleg; Sokhna, Cheikh; Tall, Adama; Socolovschi, Cristina; Cutler, Sally J.; Bilcha, Kassahum D.; Ali, Jemal; Campelo, Dayana; Barker, Steven C.; Raoult, Didier; Drancourt, Michel

    2013-01-01

    Background In Africa, relapsing fever borreliae are neglected arthropod-borne pathogens causing mild to deadly septicemia and miscarriage. The closely related Borrelia crocidurae, Borrelia duttonii, Borrelia recurrentis and Borrelia hispanica are rarely diagnosed at the species level, hampering refined epidemiological and clinical knowledge of the relapsing fevers. It would be hugely beneficial to have simultaneous detection and identification of Borrelia to species level directly from clinical samples. Methodology/Principal Findings We designed a multiplex real-time PCR protocol targeting the 16S rRNA gene detecting all four Borrelia, the glpQ gene specifically detecting B. crocidurae, the recN gene specifically detecting B. duttonii/B. recurrentis and the recC gene specifically detecting B. hispanica. Compared to combined 16S rRNA gene and flaB gene sequencing as the gold standard, multiplex real-time PCR analyses of 171 Borrelia-positive and 101 Borrelia-negative control blood specimens yielded 100% sensitivity and specificity for B. duttonii/B. recurrentis and B. hispanica and 99% sensitivity and specificity for B. crocidurae. Conclusions/Significance The multiplex real-time PCR developed in this study is a rapid technique for both molecular detection and speciation of relapsing fever borreliae from blood in Africa. It could be incorporated in point-of-care laboratory to confirm diagnosis and provide evidence of the burden of infection attributed to different species of known or potentially novel relapsing fever borreliae. PMID:23390560

  20. Diagnostic usefulness of PCR profiling of the differentially expressed marker genes in thyroid papillary carcinomas.

    PubMed

    Hamada, Aiko; Mankovskaya, Svetlana; Saenko, Vladimir; Rogounovitch, Tatiana; Mine, Mariko; Namba, Hiroyuki; Nakashima, Masahiro; Demidchik, Yuri; Demidchik, Eugeny; Yamashita, Shunichi

    2005-06-28

    The study was set out to determine whether characteristic changes in the gene expression profile in papillary thyroid carcinoma (PTC) discovered by microarray assays can be used for conventional molecular diagnosis. Expression levels of five reported to be overexpressed and three underexpressed genes were examined in PTC and normal human tissues by real-time PCR and semi-quantitative duplex PCR. Stepwise logistic regression analysis, duplex PCR data evaluation with recursive partition machine algorithm and hierarchical cluster analysis identified SFTPB (upregulated) and TFF3 (downregulated) gene combination as most favorable for differential molecular diagnosis of PTC. Sensitivity, specificity and accuracy obtained in a series of histologically characterized thyroid tumor and normal tissue samples were 88.9, 96.7 and 94.9%, respectively. Applicability of the method to fine needle aspiration biopsy (FNAB) samples was demonstrated using a collection of needle washouts. In spite individual thyroid tumor and normal tissues as well as FNAB samples displayed a substantial degree of variability in the expression levels of analyzed genes, simultaneous molecular analysis of a panel of optimal markers allows making a high probability predictive estimate and may be considered as an informative method of preoperative PTC diagnosis. PMID:15914279

  1. PCR diagnostics and monitoring of adenoviral infections in hematopoietic stem cell transplantation recipients.

    PubMed

    Bil-Lula, Iwona; Ussowicz, Marek; Rybka, Blanka; Wendycz-Domalewska, Danuta; Ryczan, Renata; Gorczyńska, Ewa; Kałwak, Krzysztof; Woźniak, Mieczysław

    2010-12-01

    After stem cell transplantation, human patients are prone to life-threatening opportunistic infections with a plethora of microorganisms. We report a retrospective study on 116 patients (98 children, 18 adults) who were transplanted in a pediatric bone marrow transplantation unit. Blood, urine and stool samples were collected and monitored for adenovirus (AdV) DNA using polymerase chain reaction (PCR) and real-time PCR (RT-PCR) on a regular basis. AdV DNA was detected in 52 (44.8%) patients, with mortality reaching 19% in this subgroup. Variables associated with adenovirus infection were transplantations from matched unrelated donors and older age of the recipient. An increased seasonal occurrence of adenoviral infections was observed in autumn and winter. Analysis of immune reconstitution showed a higher incidence of AdV infections during periods of low T-lymphocyte count. This study also showed a strong interaction between co-infections of AdV and BK polyomavirus in patients undergoing hematopoietic stem cell transplantations. PMID:20848295

  2. PCR diagnostics and monitoring of adenoviral infections in hematopoietic stem cell transplantation recipients

    PubMed Central

    Ussowicz, Marek; Rybka, Blanka; Wendycz-Domalewska, Danuta; Ryczan, Renata; Gorczyńska, Ewa; Kałwak, Krzysztof; Woźniak, Mieczysław

    2010-01-01

    After stem cell transplantation, human patients are prone to life-threatening opportunistic infections with a plethora of microorganisms. We report a retrospective study on 116 patients (98 children, 18 adults) who were transplanted in a pediatric bone marrow transplantation unit. Blood, urine and stool samples were collected and monitored for adenovirus (AdV) DNA using polymerase chain reaction (PCR) and real-time PCR (RT-PCR) on a regular basis. AdV DNA was detected in 52 (44.8%) patients, with mortality reaching 19% in this subgroup. Variables associated with adenovirus infection were transplantations from matched unrelated donors and older age of the recipient. An increased seasonal occurrence of adenoviral infections was observed in autumn and winter. Analysis of immune reconstitution showed a higher incidence of AdV infections during periods of low T-lymphocyte count. This study also showed a strong interaction between co-infections of AdV and BK polyomavirus in patients undergoing hematopoietic stem cell transplantations. PMID:20848295

  3. PCR diagnostics of Mycobacterium tuberculosis in historic human long bone remains from 18th century burials in Kaiserebersdorf, Austria

    PubMed Central

    Bachmann, Lutz; Däubl, Barbara; Lindqvist, Charlotte; Kruckenhauser, Luise; Teschler-Nicola, Maria; Haring, Elisabeth

    2008-01-01

    Background In the present pilot study we applied recently published protocols for detecting Mycobacterium tuberculosis in human remains. We screened long bones from an 18th century cemetery and skulls from the anatomical "Weisbach collection" (19th century). In addition, besides the study of abundance of tuberculosis in inmates of the poorhouse itself, we were interested to test whether in this particular instance tuberculosis can be identified from cortical bones, which are rarely affected by tuberculosis, but mostly better preserved than the vertebral bodies or epiphyses. Method The DNA extractions from the bone samples were obtained following established ancient DNA protocols. Subsequently extracts were subjected to a series of PCR amplifications using primer pairs published previously [1,2]. PCR products of the expected size were subsequently sequenced. Results Only primers targeting the repetitive IS6110 insertion sequence yielded PCR products of appropriate size. In one sample only (skull sample WB354 of the "Weisbach collection") sequence analysis revealed an authentic M. tuberculosis sequence that matched to a reference sequence from GenBank. Conclusion With a variety of established PCR approaches we failed to detect M. tuberculosis DNA in historic human femurs from an 18th century cemetery relating to a poor house in Kaiserebersdorf, Austria. Our data may indicate that in this particular case, thoracic or lumbar vertebrae, i.e. bones that are severely affected by the disease, would be more suitable for molecular diagnostics than long bones. However, the unpredictable state of DNA preservation in bones from museum collections does not allow any general recommendation of any type of bone. PMID:18799009

  4. Discovery of estrogen-responsive genes using an improved method which combines subtractive hybridization and PCR.

    PubMed Central

    Liu, W; Su, W; Roberts, T M

    1998-01-01

    Here we describe a reliable method for isolating genes that are differentially expressed in two cell populations. The method is a combination of subtractive hybridization and PCR. Among many improvements to previously described methods is the incorporation of a new technology into the procedure which sterilizes(inactivates) PCR amplicons, and thereby overcomes the limitation of similar procedures. To test this improved method, we conducted a search for estrogen-responsive genes. Estrogen-regulated genes dominated the subtracted libraries after four rounds of subtractive hybridizations. Four estrogen-regulated genes were identified from the initial screening. PMID:9671829

  5. Improved specificity for detection of Mycobacterium bovis in fresh tissues using IS6110 real-time PCR

    PubMed Central

    2011-01-01

    Background Culture of M. bovis from diagnostic specimens is the gold standard for bovine tuberculosis diagnostics in the USA. Detection of M. bovis by PCR in tissue homogenates may provide a simple rapid method to complement bacterial culture. A significant impediment to PCR based assays on tissue homogenates is specificity since mycobacteria other than M. bovis may be associated with the tissues. Results Previously published IS6110 based PCR diagnostic assays, along with one developed in house, were tested against environmental mycobacteria commonly isolated from diagnostic tissues submitted to the National Veterinary Services Laboratory. A real-time PCR assay was developed (IS6110_T) that had increased specificity over other IS6110 based assays. Of the 13 non-tuberculous mycobacteria tested with IS6110_T only M. wolinskyi was positive. Thirty M. bovis infected tissue homogenates and 18 control tissues were used to evaluate the potential for the assay as a diagnostic test. In this small sample, IS6110_T detected 20/30 samples from M. bovis infected animals and 0/18 control tissues. Conclusions The IS6110_T assay provides a PCR based assay system that is compatible with current diagnostic protocols for the detection of M. bovis in the USA and compliments current testing strategies. PMID:21867516

  6. 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. PMID:25031466

  7. Improving qPCR telomere length assays: Controlling for well position effects increases statistical power

    PubMed Central

    Eisenberg, Dan T.A.; Kuzawa, Christopher W.; Hayes, M. Geoffrey

    2015-01-01

    Objectives Telomere length (TL) is commonly measured using quantitative PCR (qPCR). Although easier than the southern blot of terminal restriction fragments (TRF) TL measurement method, one drawback of qPCR is that it introduces greater measurement error and thus reduces the statistical power of analyses. To address a potential source of measurement error, we consider the effect of well position on qPCR TL measurements. Methods qPCR TL data from 3,638 people run on a Bio-Rad iCycler iQ are reanalyzed here. To evaluate measurement validity, correspondence with TRF, age and between mother and offspring are examined. Results First, we present evidence for systematic variation in qPCR TL measurements in relation to thermocycler well position. Controlling for these well-position effects consistently improves measurement validity and yields estimated improvements in statistical power equivalent to increasing sample sizes by 16%. We additionally evaluated the linearity of the relationships between telomere and single copy gene control amplicons and between qPCR and TRF measures. We find that, unlike some previous reports, our data exhibit linear relationships. We introduce the standard error in percent, a superior method for quantifying measurement error compared to the commonly used coefficient of variation. Using this measure, we find that excluding samples with high measurement error does not improve measurement validity. Conclusions Future studies using block-based thermocyclers should consider well position effects. Since additional information can be gleaned from well position corrections, re-running analyses of previous results with well position correction could serve as an independent test of the validity of these results. PMID:25757675

  8. High-throughput Method of One-Step DNA Isolation for PCR Diagnostics of Mycobacterium tuberculosis.

    PubMed

    Kapustin, D V; Prostyakova, A I; Alexeev, Ya I; Varlamov, D A; Zubov, V P; Zavriev, S K

    2014-04-01

    The efficiency of one-step and multi-step protocols of DNA isolation from lysed sputum samples containing the Mycobacterium tuberculosis complex has been compared. DNA was isolated using spin-cartridges containing a special silica-based sorbent modified with fluoroplast and polyaniline, or using an automated isolation system. One-step isolation using the obtained sorbent has been shown to ensure a significantly lower DNA loss and higher sensitivity in the PCR detection of Mycobacterium tuberculosis as compared to a system based on sorption and desorption of nucleic acids during the isolation. PMID:25093111

  9. Improvement and automation of a real-time PCR assay for vaginal fluids.

    PubMed

    De Vittori, E; Giampaoli, S; Barni, F; Baldi, M; Berti, A; Ripani, L; Romano Spica, V

    2016-05-01

    The identification of vaginal fluids is crucial in forensic science. Several molecular protocols based on PCR amplification of mfDNA (microflora DNA) specific for vaginal bacteria are now available. Unfortunately mfDNA extraction and PCR reactions require manual optimization of several steps. The aim of present study was the verification of a partial automatization of vaginal fluids identification through two instruments widely diffused in forensic laboratories: EZ1 Advanced robot and Rotor Gene Q 5Plex HRM. Moreover, taking advantage of 5-plex thermocycler technology, the ForFluid kit performances were improved by expanding the mfDNA characterization panel with a new bacterial target for vaginal fluids and with an internal positive control (IPC) to monitor PCR inhibition. Results underlined the feasibility of a semi-automated extraction of mfDNA using a BioRobot and demonstrated the analytical improvements of the kit. PMID:27022861

  10. 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

  11. Improved PCR primers to amplify 16S rRNA genes from NC10 bacteria.

    PubMed

    He, Zhanfei; Wang, Jiaqi; Hu, Jiajie; Zhang, Hao; Cai, Chaoyang; Shen, Jiaxian; Xu, Xinhua; Zheng, Ping; Hu, Baolan

    2016-06-01

    Anaerobic oxidation of methane (AOM) coupled to nitrite reduction (AOM-NIR) is ecologically significant for mitigating the methane-induced greenhouse effect. The microbes responsible for this reaction, NC10 bacteria, have been widely detected in diverse ecosystems. However, some defects were discovered in the commonly used NC10-specific primers, 202F and qP1F. In the present work, the primers were redesigned and improved to overcome the defects found in the previous primers. A new nested PCR method was developed using the improved primers to amplify 16S ribosomal RNA (rRNA) genes from NC10 bacteria. In the new nested PCR method, the qP1mF/1492R and 1051F/qP2R primer sets were used in the first and second rounds, respectively. The PCR products were sequenced, and more operational taxonomic units (OTUs) of the NC10 phylum were obtained using the new primers compared to the previous primers. The sensitivity of the new nested PCR was tested by the serial dilution method, and the limit of detection was approximately 10(3) copies g(-1) dry sed. for the environmental samples compared to approximately 10(5) copies g(-1) dry sed. by the previous method. Finally, the improved primer, qP1mF, was used in quantitative PCR (qPCR) to determine the abundance of NC10 bacteria, and the results agreed well with the activity of AOM-NIR measured by isotope tracer experiments. The improved primers are able to amplify NC10 16S rRNA genes more efficiently than the previous primers and useful to explore the microbial community of the NC10 phylum in different systems. PMID:27020287

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. Bayesian reclassification statistics for assessing improvements in diagnostic accuracy.

    PubMed

    Huang, Zhipeng; Li, Jialiang; Cheng, Ching-Yu; Cheung, Carol; Wong, Tien-Yin

    2016-07-10

    We propose a Bayesian approach to the estimation of the net reclassification improvement (NRI) and three versions of the integrated discrimination improvement (IDI) under the logistic regression model. Both NRI and IDI were proposed as numerical characterizations of accuracy improvement for diagnostic tests and were shown to retain certain practical advantage over analysis based on ROC curves and offer complementary information to the changes in area under the curve. Our development is a new contribution towards Bayesian solution for the estimation of NRI and IDI, which eases computational burden and increases flexibility. Our simulation results indicate that Bayesian estimation enjoys satisfactory performance comparable with frequentist estimation and achieves point estimation and credible interval construction simultaneously. We adopt the methodology to analyze a real data from the Singapore Malay Eye Study. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26875442

  19. 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

  20. Visual aids improve diagnostic inferences and metacognitive judgment calibration.

    PubMed

    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

  1. Quantitative PCR for Plasma Epstein-Barr Virus Loads in Cancer Diagnostics.

    PubMed

    Loghavi, Sanam

    2016-01-01

    Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and is associated with posttransplant lymphoproliferative disease (PTLD), Hodgkin's lymphoma, Burkitt's lymphoma, nasopharyngeal carcinoma, and HIV-related lymphomas. It infects nearly all humans and then persists for the life of the host in a small proportion of benign B lymphocytes. EBV reactivation, usually in the setting of immunosuppression, is the main risk factor for development of EBV-associated malignancies. EBV reactivation can be detected in tissue specimens using EBV-encoded RNA (EBER) in situ hybridization (ISH), which is routinely used for diagnosis of PTLD and nasopharyngeal carcinoma. However, EBER ISH cannot be routinely used for screening asymptomatic or monitoring posttreatment outcome due to difficulty in obtaining tissue specimens for testing and the nonquantitative nature of the assay. Recent studies have shown that EBV genomic DNA can be detected in blood of patients with EBV-associated diseases, and that monitoring of EBV viral load in blood could be an effective method of distinguishing disease-associated EBV reactivation from incidental EBV present in benign B lymphocytes, and could be used for diagnostic screening and monitoring of EBV-associated diseases. In this chapter we discuss a protocol for quantitative plasma EBV DNA analysis. PMID:26843046

  2. Improved Amplification of Microbial DNA from Blood Cultures by Removal of the PCR Inhibitor Sodium Polyanetholesulfonate

    PubMed Central

    Fredricks, David N.; Relman, David A.

    1998-01-01

    Molecular methods are increasingly used to identify microbes in clinical samples. A common technical problem with PCR is failed amplification due to the presence of PCR inhibitors. Initial attempts at amplification of the bacterial 16S rRNA gene from inoculated blood culture media failed for this reason. The inhibitor persisted, despite numerous attempts to purify the DNA, and was identified as sodium polyanetholesulfonate (SPS), a common additive to blood culture media. Like DNA, SPS is a high-molecular-weight polyanion that is soluble in water but insoluble in alcohol. Accordingly, SPS tends to copurify with DNA. An extraction method was designed for purification of DNA from blood culture media and removal of SPS. Blood culture media containing human blood and spiked with Escherichia coli was subjected to an organic extraction procedure with benzyl alcohol, and removal of SPS was documented spectrophotometrically. Successful amplification of the extracted E. coli 16S rRNA gene was achieved by adding 5 μl of undiluted processed sample DNA to a 50-μl PCR mixture. When using other purification methods, the inhibitory effect of SPS could be overcome only by dilution of these samples. By our extraction technique, even uninoculated blood culture media were found to contain bacterial DNA when they were subjected to broad-range 16S rRNA gene consensus PCR. We conclude that the blood culture additive SPS is a potent inhibitor of PCR, is resistant to removal by traditional DNA purification methods, but can be removed by a benzyl alcohol extraction protocol that results in improved PCR performance. PMID:9738025

  3. Action Research to Improve the Learning Space for Diagnostic Techniques.

    PubMed

    Ariel, Ellen; Owens, Leigh

    2015-12-01

    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

  4. 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

  5. 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. PMID:17952664

  6. Diagnostic Accuracy and Applicability of a PCR System for the Detection of Schistosoma mansoni DNA in Human Urine Samples from an Endemic Area

    PubMed Central

    Enk, Martin Johannes; Oliveira e Silva, Guilherme; Rodrigues, Nilton Barnabé

    2012-01-01

    Schistosomiasis caused by Schistosoma mansoni, one of the most neglected human parasitoses in Latin America and Africa, is routinely confirmed by microscopic visualization of eggs in stool. The main limitation of this diagnostic approach is its lack of sensitivity in detecting individual low worm burdens and consequently data on infection rates in low transmission settings are little reliable. According to the scientific literature, PCR assays are characterized by high sensitivity and specificity in detecting parasite DNA in biological samples. A simple and cost effective extraction method for DNA of Schistosoma mansoni from urine samples in combination with a conventional PCR assay was developed and applied in an endemic area. This urine based PCR system was tested for diagnostic accuracy among a population of a small village in an endemic area, comparing it to a reference test composed of three different parasitological techniques. The diagnostic parameters revealed a sensitivity of 100%, a specificity of 91.20%, positive and negative predictive values of 86.25% and 100%, respectively, and a test accuracy of 94.33%. Further statistical analysis showed a k index of 0.8806, indicating an excellent agreement between the reference test and the PCR system. Data obtained from the mouse model indicate the infection can be detected one week after cercariae penetration, opening a new perspective for early detection and patient management during this stage of the disease. The data indicate that this innovative PCR system provides a simple to handle and robust diagnostic tool for the detection of S. mansoni DNA from urine samples and a promising approach to overcome the diagnostic obstacles in low transmission settings. Furthermore the principals of this molecular technique, based on the examination of human urine samples may be useful for the diagnosis of other neglected tropical diseases that can be detected by trans-renal DNA. PMID:22701733

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

    PubMed

    Enk, Martin Johannes; Oliveira e Silva, Guilherme; Rodrigues, Nilton Barnabé

    2012-01-01

    Schistosomiasis caused by Schistosoma mansoni, one of the most neglected human parasitoses in Latin America and Africa, is routinely confirmed by microscopic visualization of eggs in stool. The main limitation of this diagnostic approach is its lack of sensitivity in detecting individual low worm burdens and consequently data on infection rates in low transmission settings are little reliable. According to the scientific literature, PCR assays are characterized by high sensitivity and specificity in detecting parasite DNA in biological samples. A simple and cost effective extraction method for DNA of Schistosoma mansoni from urine samples in combination with a conventional PCR assay was developed and applied in an endemic area. This urine based PCR system was tested for diagnostic accuracy among a population of a small village in an endemic area, comparing it to a reference test composed of three different parasitological techniques. The diagnostic parameters revealed a sensitivity of 100%, a specificity of 91.20%, positive and negative predictive values of 86.25% and 100%, respectively, and a test accuracy of 94.33%. Further statistical analysis showed a k index of 0.8806, indicating an excellent agreement between the reference test and the PCR system. Data obtained from the mouse model indicate the infection can be detected one week after cercariae penetration, opening a new perspective for early detection and patient management during this stage of the disease. The data indicate that this innovative PCR system provides a simple to handle and robust diagnostic tool for the detection of S. mansoni DNA from urine samples and a promising approach to overcome the diagnostic obstacles in low transmission settings. Furthermore the principals of this molecular technique, based on the examination of human urine samples may be useful for the diagnosis of other neglected tropical diseases that can be detected by trans-renal DNA. PMID:22701733

  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 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

  10. Improved determination of plasmid copy number using quantitative real-time PCR for monitoring fermentation processes

    PubMed Central

    Škulj, Mihaela; Okršlar, Veronika; Jalen, Špela; Jevševar, Simona; Slanc, Petra; Štrukelj, Borut; Menart, Viktor

    2008-01-01

    Background Recombinant protein production in Escherichia coli cells is a complex process, where among other parameters, plasmid copy number, structural and segregational stability of plasmid have an important impact on the success of productivity. It was recognised that a method for accurate and rapid quantification of plasmid copy number is necessary for optimization and better understanding of this process. Lately, qPCR is becoming the method of choice for this purpose. In the presented work, an improved qPCR method adopted for PCN determination in various fermentation processes was developed. Results To avoid experimental errors arising from irreproducible DNA isolation, whole cells, treated by heating at 95°C for 10 minutes prior to storage at -20°C, were used as a template source. Relative quantification, taking into account different amplification efficiencies of amplicons for chromosome and plasmid, was used in the PCN calculation. The best reproducibility was achieved when the efficiency estimated for specific amplicon, obtained within one run, was averaged. It was demonstrated that the quantification range of 2 log units (100 to 10000 bacteria per well) enable quantification in each time point during fermentation. The method was applied to study PCN variation in fermentation at 25°C and the correlation between PCN and protein accumulation was established. Conclusion Using whole cells as a template source and relative quantification considering different PCR amplification efficiencies are significant improvements of the qPCR method for PCN determination. Due to the approaches used, the method is suitable for PCN determination in fermentation processes using various media and conditions. PMID:18328094

  11. Removal of real-time reverse transcription polymerase chain reaction (RT-PCR) inhibitors associated with cloacal swab samples and tissues for improved diagnosis of avian influenza virus by RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real time reverse transcriptase polymerase chain reaction (RRT-PCR) is routinely used for the rapid detection of Avian Influenza virus (AIV) in clinical samples. The usefulness of diagnostic RRT-PCR can be limited, in part, by the inhibitory substances present in some clinical specimens, which can ...

  12. 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

  13. Improved genotyping vaccine and wild-type poliovirus strains by restriction fragment length polymorphism analysis: clinical diagnostic implications.

    PubMed

    Georgopoulou, A; Markoulatos, P; Spyrou, N; Vamvakopoulos, N C

    2000-12-01

    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 UC(53)-UG(52) 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

  14. Diagnostic Value of PCR for Detection of Borrelia burgdorferi in Skin Biopsy and Urine Samples from Patients with Skin Borreliosis

    PubMed Central

    Brettschneider, S.; Bruckbauer, H.; Klugbauer, N.; Hofmann, H.

    1998-01-01

    Skin biopsies of 36 patients with erythema migrans and acrodermatitis chronica atrophicans (ACA) before therapy and those of 8 patients after therapy were examined for Borrelia burgdorferi DNA by PCR. Skin biopsies of 27 patients with dermatological diseases other than Lyme borreliosis and those of 10 healthy persons were examined as controls. Two different primer sets targeting 23S rRNA (PCR I) and 66-kDa protein (PCR II) genes were used. PCR was performed with freshly frozen tissue (FFT) and paraffin-embedded tissue (PET). For FFT specimens of erythema migrans, 73% were positive by PCR I, 79% were positive by PCR II, and 88% were positive by combining PCR I and II. For PET specimens, PCR was less sensitive (PCR I, 44%; PCR II, 52%). For FFT specimens of ACA, PCR I was positive for two of five patients and PCR II was positive for four of five patients. B. burgdorferi was cultured from 79% of the erythema migrans specimens but not from any of the ACA lesions. Elevated B. burgdorferi antibodies were detected in sera of 74% of erythema migrans patients and 100% of ACA patients. All urine samples were negative by PCR II, whereas PCR I was positive for 27%. However, hybridization of these amplicons was negative. Sequencing of three amplicons identified nonborrelial DNA. In conclusion, urine PCR is not suitable for the diagnosis of skin borreliosis. A combination of two different primer sets achieves high sensitivity with skin biopsies. In early erythema migrans infection, culture and PCR are more sensitive than serology. PMID:9705410

  15. Evaluation of a novel PCR-based diagnostic assay for detection of Mycobacterium tuberculosis in sputum samples.

    PubMed Central

    Maher, M; Glennon, M; Martinazzo, G; Turchetti, E; Marcolini, S; Smith, T; Dawson, M T

    1996-01-01

    We report on a PCR-based assay we have developed for the detection of Mycobacterium tuberculosis in sputum samples. One hundred sputum specimens, which included 34 culture-positive and 66 culture-negative specimens, were evaluated with this system. Of the 34 culture-positive specimens, 31 were PCR positive, and 60 of the culture-negative specimens were PCR negative. An internal standard has been included in the assay system to monitor PCR inhibition and to confirm the reliability of the PCR assay. PMID:8862607

  16. Measurement of Phospholipids May Improve Diagnostic Accuracy in Ovarian Cancer

    PubMed Central

    Davis, Lorelei; Han, Gang; Zhu, Weiwei; Molina, Ashley D.; Arango, Hector; LaPolla, James P.; Hoffman, Mitchell S.; Sellers, Thomas; Kirby, Tyler; Nicosia, Santo V.; Sutphen, Rebecca

    2012-01-01

    Background More than two-thirds of women who undergo surgery for suspected ovarian neoplasm do not have cancer. Our previous results suggest phospholipids as potential biomarkers of ovarian cancer. In this study, we measured the serum levels of multiple phospholipids among women undergoing surgery for suspected ovarian cancer to identify biomarkers that better predict whether an ovarian mass is malignant. Methodology/Principal Findings We obtained serum samples preoperatively from women with suspected ovarian cancer enrolled through a prospective, population-based rapid ascertainment system. Samples were analyzed from all women in whom a diagnosis of epithelial ovarian cancer (EOC) was confirmed and from benign disease cases randomly selected from the remaining (non-EOC) samples. We measured biologically relevant phospholipids using liquid chromatography/electrospray ionization mass spectrometry. We applied a powerful statistical and machine learning approach, Hybrid huberized support vector machine (HH-SVM) to prioritize phospholipids to enter the biomarker models, and used cross-validation to obtain conservative estimates of classification error rates. Results The HH-SVM model using the measurements of specific combinations of phospholipids supplements clinical CA125 measurement and improves diagnostic accuracy. Specifically, the measurement of phospholipids improved sensitivity (identification of cases with preoperative CA125 levels below 35) among two types of cases in which CA125 performance is historically poor - early stage cases and those of mucinous histology. Measurement of phospholipids improved the identification of early stage cases from 65% (based on CA125) to 82%, and mucinous cases from 44% to 88%. Conclusions/Significance Levels of specific serum phospholipids differ between women with ovarian cancer and those with benign conditions. If validated by independent studies in the future, these biomarkers may serve as an adjunct at the time of clinical

  17. 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

  18. 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

  19. Brief Report: Excluding the ADI-R Behavioral Domain Improves Diagnostic Agreement in Toddlers

    ERIC Educational Resources Information Center

    Wiggins, Lisa D.; Robins, Diana L.

    2008-01-01

    Past research shows poor agreement between the Autism Diagnostic Interview-Revised (ADI-R) and other diagnostic measures in toddlers. Our goal was to examine whether exclusion of the ADI-R behavioral domain results in improved diagnostic agreement. Toddlers aged 16-37 months (M = 26 months) received an evaluation because they failed the Modified…

  20. An improved real-time PCR assay for the detection of Old World screwworm flies.

    PubMed

    Morgan, Jess A T; Urech, Rudolf

    2014-10-01

    The Old World screwworm (OWS) fly, Chrysomya bezziana, is a serious pest of livestock, wildlife and humans in tropical Africa, parts of the Middle East, the Indian subcontinent, south-east Asia and Papua New Guinea. Although to date Australia remains free of OWS flies, an incursion would have serious economic and animal welfare implications. For these reasons Australia has an OWS fly preparedness plan including OWS fly surveillance with fly traps. The recent development of an improved OWS fly trap and synthetic attractant and a specific and sensitive real-time PCR molecular assay for the detection of OWS flies in trap catches has improved Australia's OWS fly surveillance capabilities. Because all Australian trap samples gave negative results in the PCR assay, it was deemed necessary to include a positive control mechanism to ensure that fly DNA was being successfully extracted and amplified and to guard against false negative results. A new non-competitive internal amplification control (IAC) has been developed that can be used in conjunction with the OWS fly PCR assay in a multiplexed single-tube reaction. The multiplexed assay provides an indicator of the performance of DNA extraction and amplification without greatly increasing labour or reagent costs. The fly IAC targets a region of the ribosomal 16S mitochondrial DNA which is conserved across at least six genera of commonly trapped flies. Compared to the OWS fly assay alone, the multiplexed OWS fly and fly IAC assay displayed no loss in sensitivity or specificity for OWS fly detection. The multiplexed OWS fly and fly IAC assay provides greater confidence for trap catch samples returning negative OWS fly results. PMID:24613153

  1. 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

  2. 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

  3. Optimising the diagnostic strategy for onychomycosis from sample collection to FUNGAL identification evaluation of a diagnostic kit for real-time PCR.

    PubMed

    Petinataud, Dimitri; Berger, Sibel; Ferdynus, Cyril; Debourgogne, Anne; Contet-Audonneau, Nelly; Machouart, Marie

    2016-05-01

    Onychomycosis is a common nail disorder mainly due to dermatophytes for which the conventional diagnosis requires direct microscopic observation and culture of a biological sample. Nevertheless, antifungal treatments are commonly prescribed without a mycological examination having been performed, partly because of the slow growth of dermatophytes. Therefore, molecular biology has been applied to this pathology, to support a quick and accurate distinction between onychomycosis and other nail damage. Commercial kits are now available from several companies for improving traditional microbiological diagnosis. In this paper, we present the first evaluation of the real-time PCR kit marketed by Bio Evolution for the diagnosis of dermatophytosis. Secondly, we compare the efficacy of the kit on optimal and non-optimal samples. This study was conducted on 180 nails samples, processed by conventional methods and retrospectively analysed using this kit. According to our results, this molecular kit has shown high specificity and sensitivity in detecting dermatophytes, regardless of sample quality. On the other hand, and as expected, optimal samples allowed the identification of a higher number of dermatophytes by conventional mycological diagnosis, compared to non-optimal samples. Finally, we have suggested several strategies for the practical use of such a kit in a medical laboratory for quick pathogen detection. PMID:26806228

  4. 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

  5. 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

  6. 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

  7. 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).

  8. Comparison and evaluation of Renibacterium salmoninarum quantitative PCR diagnostic assays using field samples of Chinook and coho salmon.

    PubMed

    Sandell, Todd A; Jacobson, Kym C

    2011-01-21

    Renibacterium salmoninarum is a Gram-positive bacterium causing bacterial kidney disease (BKD) in susceptible salmonid fishes. Several quantitative PCR (qPCR) assays to measure R. salmoninarum infection intensity have been reported, but comparison and evaluation of these assays has been limited. Here, we compared 3 qPCR primer/probe sets for detection of R. salmoninarum in field samples of naturally exposed Chinook and coho salmon first identified as positive by nested PCR (nPCR). Additional samples from a hatchery population of Chinook salmon with BKD were included to serve as strong positive controls. The 3 qPCR assays targeted either the multiple copy major soluble antigen (msa) genes or the single copy abc gene. The msa/non-fluorescent quencher (NFQ) assay amplified R. salmoninarum DNA in 53.2% of the nPCR positive samples, whereas the abc/NFQ assay amplified 21.8% of the samples and the abc/TAMRA assay 18.2%. The enzyme-linked immunosorbent assay (ELISA) successfully quantified only 16.4% of the nPCR positive samples. Although the msa/NFQ assay amplified a greater proportion of nPCR positive samples, the abc/NFQ assay better amplified those samples with medium and high ELISA values. A comparison of the geometric mean quantity ratios highlighted limitations of the assays, and the abc/NFQ assay strongly amplified some samples that were negative in other tests, in contrast to its performance among the sample group as a whole. These data demonstrate that both the msa/NFQ and abc/NFQ qPCR assays are specific and effective at higher infection levels and outperform the ELISA. However, most pathogen studies will continue to require multiple assays to both detect and quantify R. salmoninarum infection. PMID:21381519

  9. Seronegative invasive gastro-intestinal cytomegalovirus disease in renal allograft recipients a diagnostic dilemma! - Tissue PCR the saviour?

    PubMed

    Kaul, A; Bhadauria, D; Agarwal, V; Ruhela, V; Kumar, A; Mohendra, S; Barai, S; Prasad, N; Gupta, A; Sharma, R K

    2015-01-01

    Seronegative Invasive Gastro-intestinal cytomegalovirus disease in renal allograft recipients Background -CMV as oppurtunistic infection affecting the gastrointerstinal tract is the most common cause for tissue invasive CMV disease occuring in 10-30% of organ transplant recepients. Gastrointerstinal CMV disease can be diagnosed in presence of clinical suspecion along with histopathological findings (CMV inclusions) and presence of mucosal lesion(s) on endoscopic examination with collaborative evidences via molecular technique. Aims-Few cases of CMV infection affecting the gastrointerstinal tract show no evidences of dissemintion despite use of highly sensitive molecular techniques. We encountered 6 cases where in despite strong clinical suspecion of Gastrointerstinal CMV disease there were seronegative and endoscopic negative evidences for CMV, blind tissue biopsy yeilded positive results for CMV disease with excellent improvement with antiviral therapy. Conclusions-Blind biopsy specimen for tissue PCR could serve as saviour in an immunocompromised individiual who has a strong clinical symptomatology for GI-CMV disease in absence of viremia, normal endoscopy and histopathology, so that the early therapeutic interventions could help in excellent patient and graft survival. PMID:26068358

  10. Diagnostic Accuracy of In-House PCR for Pulmonary Tuberculosis in Smear-Positive Patients: Meta-Analysis and Metaregression▿ †

    PubMed Central

    Greco, S.; Rulli, M.; Girardi, E.; Piersimoni, C.; Saltini, C.

    2009-01-01

    In-house PCR (hPCR) could speed differential diagnosis between tuberculosis (TB) and nontuberculous mycobacterial disease in patients with positive smears and pulmonary infiltrates, but its reported accuracy fluctuates across studies. We conducted a systematic review and meta-analysis of hPCR sensitivity and specificity for smear-positive TB diagnosis, using culture as the reference standard. After searching English language studies in MEDLINE and EMBASE, we estimated cumulative accuracy by means of summary receiver operating characteristic analysis. The possible influence of hPCR procedures and study methodological features on accuracy was explored by univariate metaregression, followed by multivariate adjustment of items selected as significant. Thirty-five articles (1991 to 2006) met the inclusion criteria. The pooled estimates of the diagnostic odds ratio, sensitivity, and specificity (random-effect model) were, respectively, 60 (confidence interval [CI], 29 to 123), 0.96 (CI, 0.95 to 0.97), and 0.81 (CI, 0.78 to 0.84), but significant variations (mainly in specificity) limit their clinical applicability. The quality of the reference test, the detection method, and real-time PCR use explained some of the observed heterogeneity. Probably due to the limited study power of our meta-analysis and to the wide differences in both laboratory techniques and methodological quality, only real-time PCR also displayed a positive impact on accuracy in the multivariate model. Currently, hPCR can be confidently used to exclude TB in smear-positive patients, but its low specificity could lead to erroneous initiation of therapy, isolation, and contact investigation. As the inclusion of samples from treated patients could have artificially reduced specificity, future studies should report mycobacterial-culture results for each TB and non-TB sample analyzed. PMID:19144797

  11. 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

  12. 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

  13. 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

  14. 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. PMID:24610857

  15. 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

  16. Recent improvements of the JET lithium beam diagnostic.

    PubMed

    Brix, M; Dodt, D; Dunai, D; Lupelli, I; Marsen, S; Melson, T F; Meszaros, B; Morgan, P; Petravich, G; Refy, D I; Silva, C; Stamp, M; Szabolics, T; Zastrow, K-D; Zoletnik, S

    2012-10-01

    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). PMID:23130794

  17. 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. PMID:21896368

  18. 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

  19. 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…

  20. 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…

  1. 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…

  2. PcrV antibody-antibiotic combination improves survival in Pseudomonas aeruginosa-infected mice.

    PubMed

    Song, Y; Baer, M; Srinivasan, R; Lima, J; Yarranton, G; Bebbington, C; Lynch, S V

    2012-08-01

    The type III secretion system (TTSS) of Pseudomonas aeruginosa, associated with acute infection, facilitates the direct injection of cytotoxins into the host cell cytoplasm. Mab166, a murine monoclonal antibody against PcrV, a protein located at the tip of the injectisome, has demonstrated efficacy against P. aeruginosa infection, resulting in reduced lung injury and increased survival in murine models of infection. We hypothesised that the administration of Mab166 in combination with an antibiotic would further improve the survival of P. aeruginosa-infected mice. A murine model of P. aeruginosa acute infection, three clinically relevant antibiotics (ciprofloxacin, tobramycin and ceftazidime) and the Mab166 antibody were used for this study. Consistently, compared to other treatment groups (antibiotic or antibody administered in isolation), the combination of Mab166 and antibiotic significantly improved the survival of mice infected with three times the lethal dose (LD(90)) of the highly cytotoxic ExoU-secreting strain, PA103. This synergistic effect was primarily due to enhanced bactericidal effect and protection against lung injury, which prevented bacterial dissemination to other organs. Hence, the combination of Mab166 with antibiotic administration provides a new, more effective strategy against P. aeruginosa airway infection, especially when large numbers of highly virulent strains are present. PMID:22187351

  3. Linear combinations of biomarkers to improve diagnostic accuracy with three ordinal diagnostic categories

    PubMed Central

    Kang, Le; Xiong, Chengjie; Crane, Paul; Tian, Lili

    2015-01-01

    Many researchers have addressed the problem of finding the optimal linear combination of biomarkers to maximize the area under receiver operating characteristic (ROC) curves for scenarios with binary disease status. In practice, many disease processes such as Alzheimer can be naturally classified into three diagnostic categories such as normal, mild cognitive impairment and Alzheimer’s disease (AD), and for such diseases the volume under the ROC surface (VUS) is the most commonly used index of diagnostic accuracy. In this article, we propose a few parametric and nonparametric approaches to address the problem of finding the optimal linear combination to maximize the VUS. We carried out simulation studies to investigate the performance of the proposed methods. We apply all of the investigated approaches to a real data set from a cohort study in early stage AD. PMID:22865796

  4. Development of a diagnostic one-tube RT-PCR for the detection of Rift Valley fever virus.

    PubMed

    Espach, A; Romito, M; Nel, L H; Viljoen, G J

    2002-09-01

    Diagnosis of Rift Valley fever (RVF) is based on serology and virus isolation. The disadvantages of the former include poor sensitivity, high cost, risks associated with using infectious virus as antigen, the lengthy duration of ELISA as well as cross-reactivity with other Phleboviruses. We developed, optimised and evaluated a one-tube reverse-transcription-polymerase chain reaction (RT-PCR) for the detection of Rift Valley fever virus (RVFV) in ruminants. The PCR primers for this assay were designed to anneal to a region within the M segment of the virus genome, encoding glycoproteins G1 and G2. A PCR amplicon of 363 bp was obtained. The sensitivity of the assay was determined to be 0.25 TCID50. This test should allow for the early and rapid detection of RVFV in both serum and whole blood. In addition, it could facilitate the quantification of antigen for the manufacture of current vaccines. PMID:12356173

  5. Molecular characterization with RAPD-PCR: Application of genetic diagnostics to biological control of the sweetpotato whitefly

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The application of genetic diagnostics under the umbrella of classical taxonomy was imperative for successful development and delivery of the biological control program against the sweet potato whitefly, Bemisia tabaci Gennadius biotype B (= silverleaf whitefly, B. argentifolii Bellows and Perring)....

  6. An improved multiplex IC-RT-PCR assay distinguishes nine strains of Potato virus Y

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A multiplex RT-PCR assay was previously developed to identify a group of PVY isolates with unusual recombinant structures, e.g. PVYNTN-NW and SYR-III, and to differentiate them from other PVY strains. In the present study, the efficiency of this multiplex RT-PCR assay was validated and extended cons...

  7. 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

  8. 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

  9. Nested PCR-SSCP assay for the detection of p53 mutations in paraffin wax embedded bone tumours: improvement of sensitivity and fidelity.

    PubMed

    Wang, L T; Smith, A; Iacopetta, B; Wood, D J; Papadimitriou, J M; Zheng, M H

    1996-06-01

    DNA extraction and PCR amplification from paraffin wax embedded bone tumour specimens present several difficulties, firstly, because of the abundant matrix they contain and, secondly, because decalcification often causes degradation of DNA. In this report, comparative studies were carried out to determine the most efficient method for DNA extraction and PCR amplification from such specimens. The results indicated that nested PCR produced appropriate strong reaction products with minimal background contamination. A method for DNA extraction from paraffin wax embedded bone tissue and a nested PCR-SSCP technique have been developed for use in such diagnostic specimens. PMID:16696068

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

    PubMed

    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. PMID:25430334

  11. 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.

  12. 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.

  13. 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.

  14. 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

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

    SciTech Connect

    Bond, Leonard J.

    2011-08-31

    Only time will tell what the implications of the Fukushima incident will be. Discussions are on-going with regard to continued operation and life extension of the existing fleet, new build, and the wider policy issues including technologies needed to address spent fuel storage and ensure energy security, and the related desires to provide sustainable energy systems while at the same time limiting greenhouse gas emissions. The science base for advanced diagnostics and prognostics to support its use in nuclear power plants (NPPs) for active components (pumps, valves etc) has been demonstrated. A challenge is enabling adaption of these technologies for NPP deployment and the validation of the data from these technologies. Advanced diagnostics, monitoring and prognostics applied to passive structures, which in the USA context of longer term operation is up to 80 years, are being researched. Early laboratory work is demonstrating the potential for these methods, although technical challenges remain. It can be expected that there will be an increased need for and use of on-line monitoring for a wide range of both active and passive systems in all types of nuclear power plants.

  16. Novel Multiplex Real-Time PCR Diagnostic Assay for Identification and Differentiation of Mycobacterium tuberculosis, Mycobacterium canettii, and Mycobacterium tuberculosis Complex Strains▿†

    PubMed Central

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

    2011-01-01

    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. PMID:21123525

  17. Immunohistochemistry and real-time PCR as diagnostic tools for detection of Borrelia burgdorferi sensu lato in ticks collected from humans.

    PubMed

    Briciu, Violeta T; Sebah, Daniela; Coroiu, Georgiana; Lupşe, Mihaela; Cârstina, Dumitru; Ţăţulescu, Doina F; Mihalca, Andrei D; Gherman, Călin M; Leucuţa, Daniel; Meyer, Fabian; Hizo-Teufel, Cecilia; Fingerle, Volker; Huber, Ingrid

    2016-05-01

    The objective of this study was to evaluate different methods used for detection of Borrelia burgdorferi sensu lato (s.l.) in ticks: immunohistochemistry followed by focus floating microscopy (FFM) and real-time polymerase chain reaction (real-time PCR) targeting the ospA and hbb genes. Additionally, an optimized ospA real-time PCR assay was developed with an integrated internal amplification control (IAC) for the detection of inhibition in the PCR assay and was validated as an improved screening tool for B. burgdorferi. One hundred and thirty-six ticks collected from humans in a hospital from Cluj-Napoca, Romania, were investigated regarding genus, stage of development and sex, and then tested by all three assays. A poor quality of agreement was found between FFM and each of the two real-time PCR assays, as assessed by concordance analysis (Cohen's kappa), whereas the agreement between the two real-time PCR assays was moderate. The present study argues for a low sensitivity of FFM and underlines that discordant results of different assays used for detection of B. burgdorferi in ticks are frequent. PMID:26801157

  18. 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

  19. Diagnostic Utility of a Clonality Test for Lymphoproliferative Diseases in Koreans Using the BIOMED-2 PCR Assay

    PubMed Central

    Kim, Young; Choi, Yoo Duk; Choi, Chan

    2013-01-01

    Background A clonality test for immunoglobulin (IG) and T cell receptor (TCR) is a useful adjunctive method for the diagnosis of lymphoproliferative diseases (LPDs). Recently, the BIOMED-2 multiplex polymerase chain reaction (PCR) assay has been established as a standard method for assessing the clonality of LPDs. We tested clonality in LPDs in Koreans using the BIOMED-2 multiplex PCR and compared the results with those obtained in European, Taiwanese, and Thai participants. We also evaluated the usefulness of the test as an ancillary method for diagnosing LPDs. Methods Two hundred and nineteen specimens embedded in paraffin, including 78 B cell lymphomas, 80 T cell lymphomas and 61 cases of reactive lymphadenitis, were used for the clonality test. Results Mature B cell malignancies showed 95.7% clonality for IG, 2.9% co-existing clonality, and 4.3% polyclonality. Mature T cell malignancies exhibited 83.8% clonality for TCR, 8.1% co-existing clonality, and 16.2% polyclonality. Reactive lymphadenitis showed 93.4% polyclonality for IG and TCR. The majority of our results were similar to those obtained in Europeans. However, the clonality for IGK of B cell malignancies and TCRG of T cell malignancies was lower in Koreans than Europeans. Conclusions The BIOMED-2 multiplex PCR assay was a useful adjunctive method for diagnosing LPDs. PMID:24255634

  20. 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

  1. 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...

  2. PCR Analysis of IgH and TCR-γ Gene Rearrangements as a Confirmatory Diagnostic Tool for Lymphoproliferative Disorders.

    PubMed

    Poopak, Behzad; Valeshabad, Ali Kord; Elahi, Fazel; Rezvani, Hamid; Khosravipour, Gelareh; Jahangirpour, Mohammad Ali; Bolouri, Shirin; Golkar, Tolou; Salari, Fatemeh; Shahjahani, Mohammad; Saki, Najmaldin

    2015-03-01

    This study investigates PCR analysis of immunoglobulin heavy chain (IgH) and T cell receptor (TCR) gene rearrangements on paraffin-embedded tissue sections and bone marrow aspirates of patients suspected to have lymphoproliferative disorders but with inconclusive diagnosis in histopathological examination. 130 samples of patients with inconclusive immunohistochemistry results were evaluated for clonal rearrangement of IgH and TCR genes. Based on histopathology examination, the patients were divided into three groups: the first group without any definite diagnosis of lymphoproliferative disorders (60 cases, 46.2 %), the second group suspected to have a lymphoproliferative disorder but in favor of benign disorders (19 cases, 14.6 %) and the third group suspect to lymphoproliferative disorders but relatively in favor of malignant disorders (51 cases, 39.2 %). After DNA extraction and quality control, semi-nested PCR was performed using consensus primers for amplification of TCR-γ and CDR-3 regions of IgH genes. PCR products were analyzed after heteroduplex analysis using polyacrylamide gel electrophoresis, and were subject to silver staining. Totally, in over half of the cases (55.4 %), a monoclonal pattern was found in IgH or TCR-γ genes rearrangements. Monoclonal IgH gene rearrangement was detected in 48.1 % of patients, whereas monoclonal TCR-γ gene rearrangement was found in 33.6 % of them, which was not statistically significant (P = 0.008). Only in 32 patients (24.6 %) were the results of TCR-γ and IgH gene rearrangements consistent with respect to the presence (2.3 %) or absence (22.3 %) of monoclonality. Finally, PCR analysis of TCR-γ and IgH gene rearrangements led to definite diagnosis in 105 patients (80.8 %), and only 25 cases (19.2 %) remained inconclusive. Our results emphasize the usefulness of gene rearrangement study in cases without a definite diagnosis in immunohistochemistry studies. Multiple PCR analysis results when combined

  3. Clinical Utility of Droplet Digital PCR for Human Cytomegalovirus

    PubMed Central

    Sedlak, Ruth Hall; Cook, Linda; Cheng, Anqi; Magaret, Amalia

    2014-01-01

    Human cytomegalovirus (CMV) has historically been the major infectious cause of morbidity and mortality among patients receiving hematopoietic cell or organ transplant. Standard care in a transplant setting involves frequent monitoring of CMV viral load over weeks to months to determine when antiviral treatment may be required. Quantitative PCR (qPCR) is the standard molecular diagnostic method for monitoring. Recently, digital PCR (dPCR) has shown promise in viral diagnostics, although current dPCR systems have lower throughput than qPCR systems. Here, we compare qPCR and droplet digital PCR (ddPCR) for CMV detection in patient plasma samples. Droplet digital PCR exhibits increased precision over qPCR at viral loads of ≥4 log10 with equivalent sensitivity. However, retrospective analysis of longitudinal samples from transplant patients with CMV viral loads near therapeutic thresholds did not provide evidence that the improved precision of ddPCR would be of clinical benefit. Given the throughput advantages of current qPCR systems, a widespread switch to dPCR for CMV monitoring would appear premature. PMID:24871214

  4. Improving qPCR efficiency in environmental samples by selective removal of humic acids with DAX-8.

    PubMed

    Schriewer, A; Wehlmann, A; Wuertz, S

    2011-04-01

    Quantitative PCR is becoming the method of choice for the detection of pathogenic microorganisms and other targets in the environment. A major obstacle when amplifying DNA is the presence of inhibiting substances like humic acids that decrease the efficiency of PCR. We combined the polymeric adsorbent Supelite™ DAX-8 with a large-volume (10 mL) nucleic acid extraction method to decrease the humic acid content prior to qPCR quantification in water samples. The method was tested by spiking with humic acid standards and the bacterial surrogate Acinetobacter baylyi ADP1. Improvements in qPCR detection of ADP1 after application of DAX-8 resin (5 and 10 w/v%) were compared with the effects of added bovine serum albumin (BSA) (50, 100 and 200 ng/μL). Both additions improved detection of ADP1 by counteracting inhibitory effects. There were no changes in mean cycle threshold difference (ΔC(T)) after application of DAX-8 compared to the control despite some loss of DNA, whereas significant increases occurred for BSA, irrespective of BSA concentration applied. The use of DAX-8 leads to an increase in qPCR amplification efficiency in contrast to BSA. The commonly used method to calculate genomic sample concentrations by comparing measured CT values relative to standard curves is only valid if amplification efficiencies of both are sufficiently similar. DAX-8 can provide this efficiency by removing humic acids permanently from nucleic acid extracts and has the potential to significantly increase the reliability of reported non-detects and measured results obtained by qPCR in environmental monitoring. PMID:21256890

  5. 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.

  6. Use of DNA melting simulation software for in silico diagnostic assay design: targeting regions with complex melting curves and confirmation by real-time PCR using intercalating dyes

    PubMed Central

    Rasmussen, John P; Saint, Christopher P; Monis, Paul T

    2007-01-01

    Background DNA melting curve analysis using double-stranded DNA-specific dyes such as SYTO9 produce complex and reproducible melting profiles, resulting in the detection of multiple melting peaks from a single amplicon and allowing the discrimination of different species. We compare the melting curves of several Naegleria and Cryptosporidium amplicons generated in vitro with in silico DNA melting simulations using the programs POLAND and MELTSIM., then test the utility of these programs for assay design using a genetic marker for toxin production in cyanobacteria. Results The SYTO9 melting curve profiles of three species of Naegleria and two species of Cryptosporidium were similar to POLAND and MELTSIM melting simulations, excepting some differences in the relative peak heights and the absolute melting temperatures of these peaks. MELTSIM and POLAND were used to screen sequences from a putative toxin gene in two different species of cyanobacteria and identify regions exhibiting diagnostic melting profiles. For one of these diagnostic regions the POLAND and MELTSIM melting simulations were observed to be different, with POLAND more accurately predicting the melting curve generated in vitro. Upon further investigation of this region with MELTSIM, inconsistencies between the melting simulation for forward and reverse complement sequences were observed. The assay was used to accurately type twenty seven cyanobacterial DNA extracts in vitro. Conclusion Whilst neither POLAND nor MELTSIM simulation programs were capable of exactly predicting DNA dissociation in the presence of an intercalating dye, the programs were successfully used as tools to identify regions where melting curve differences could be exploited for diagnostic melting curve assay design. Refinements in the simulation parameters would be required to account for the effect of the intercalating dye and salt concentrations used in real-time PCR. The agreement between the melting curve simulations for

  7. 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

  8. An improved PCR method for direct identification of Porphyra (Bangiales, Rhodophyta) using conchocelis based on a RUBISCO intergenic spacer

    NASA Astrophysics Data System (ADS)

    Wang, Chao; Dong, Dong; Wang, Guangce; Zhang, Baoyu; Peng, Guang; Xu, Pu; Tang, Xiaorong

    2009-09-01

    An improved method of PCR in which the small segment of conchocelis is amplified directly without DNA extraction was used to amplify a RUBISCO intergenic spacer DNA fragment from nine species of red algal genus Porphyra (Bangiales, Rhodophyta), including Porphyra yezoensis (Jiangsu, China), P. haitanensis (Fujian, China), P. oligospermatangia (Qingdao, China), P. katadai (Qingdao, China), P. tenera (Qingdao, China), P. suborboculata (Fujian, China), P. pseudolinearis (Kogendo, Korea), P. linearis (Devon, England), and P. fallax (Seattle, USA). Standard PCR and the method developed here were both conducted using primers specific for the RUBISCO spacer region, after which the two PCR products were sequenced. The sequencing data of the amplicons obtained using both methods were identical, suggesting that the improved PCR method was functional. These findings indicate that the method developed here may be useful for the rapid identification of species of Porphyra in a germplasm bank. In addition, a phylogenetic tree was constructed using the RUBISCO spacer and partial rbcS sequence, and the results were in concordant with possible alternative phylogenies based on traditional morphological taxonomic characteristics, indicating that the RUBISCO spacer is a useful region for phylogenetic studies.

  9. 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

  10. 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

  11. A new tool improves diagnostic test performance for transmission em evaluation of axonemal dynein arms.

    PubMed

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

    2014-08-01

    Abstract 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. The authors 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, they found that use of this tool led to improvement in diagnostic TEM test performance. PMID:23957500

  12. 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...

  13. 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. PMID:24815379

  14. Towards Q-PCR of pathogenic bacteria with improved electrochemical double-tagged genosensing detection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A very sensitive assay for the rapid detection of pathogenic bacteria based on electrochemical genosensing has been designed. The assay was performed by the PCR specific amplification of the eaeA gene, related with the pathogenic activity of Escherichia coli O157:H7. The efficiency and selectivity o...

  15. 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

  16. Barcoding the kingdom Plantae: new PCR primers for ITS regions of plants with improved universality and specificity.

    PubMed

    Cheng, Tao; Xu, Chao; Lei, Li; Li, Changhao; Zhang, Yu; Zhou, Shiliang

    2016-01-01

    The internal transcribed spacer (ITS) of nuclear ribosomal DNA is one of the most commonly used DNA markers in plant phylogenetic and DNA barcoding analyses, and it has been recommended as a core plant DNA barcode. Despite this popularity, the universality and specificity of PCR primers for the ITS region are not satisfactory, resulting in amplification and sequencing difficulties. By thoroughly surveying and analysing the 18S, 5.8S and 26S sequences of Plantae and Fungi from GenBank, we designed new universal and plant-specific PCR primers for amplifying the whole ITS region and a part of it (ITS1 or ITS2) of plants. In silico analyses of the new and the existing ITS primers based on these highly representative data sets indicated that (i) the newly designed universal primers are suitable for over 95% of plants in most groups; and (ii) the plant-specific primers are suitable for over 85% of plants in most groups without amplification of fungi. A total of 335 samples from 219 angiosperm families, 11 gymnosperm families, 24 fern and lycophyte families, 16 moss families and 17 fungus families were used to test the performances of these primers. In vitro PCR produced similar results to those from the in silico analyses. Our new primer pairs gave PCR improvements up to 30% compared with common-used ones. The new universal ITS primers will find wide application in both plant and fungal biology, and the new plant-specific ITS primers will, by eliminating PCR amplification of nonplant templates, significantly improve the quality of ITS sequence information collections in plant molecular systematics and DNA barcoding. PMID:26084789

  17. 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.

  18. A two-step protocol for isolation of influenza A (H7N7) virions and their RNA for PCR diagnostics based on modified paramagnetic particles.

    PubMed

    Michalek, Petr; Dostalova, Simona; Buchtelova, Hana; Cernei, Natalia; Krejcova, Ludmila; Hynek, David; Milosavljevic, Vedran; Jimenez, Ana Maria Jimenez; Kopel, Pavel; Heger, Zbynek; Adam, Vojtech

    2016-07-01

    Annual epidemics of influenza cause death of hundreds of thousands people and they also have a significant economic impact. Hence, a need for fast and cheap influenza diagnostic method is arising. The conventional methods for an isolation of the viruses are time-consuming and require expensive instrumentation as well as trained personnel. In this study, we modified the surface of nanomaghemite (γ-Fe2 O3 ) paramagnetic core with tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane and the resulting particles were utilized for the isolation of H7N7 influenza virions. Consequently, we designed γ-Fe2 O3 paramagnetic core modified with calcium tripolyphosphate which was employed for the isolation of viral nucleic acid after virion's lysis. Both of these procedures can be performed rapidly in less than 10 min and, in combination with the RT-PCR, the whole influenza detection can be shortened to few hours. Moreover, the whole protocol could be easily automated and/or miniaturized, and thus can serve as a basis for use in a lab-on-a-chip device. We assume that magnetic isolation is an exceptional procedure which can significantly accelerate the diagnostic possibilities of a broad spectrum of diseases. PMID:27130152

  19. 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

  20. A New Real-Time PCR Assay for Improved Detection of the Parasite Babesia microti

    PubMed Central

    Teal, Allen E.; Habura, Andrea; Ennis, Jill; Keithly, Janet S.

    2012-01-01

    Babesiosis is an emerging zoonosis with important public health implications, as the incidence of the disease has risen dramatically over the past decade. Because the current gold standard for detection of Babesia is microscopic examination of blood smears, accurate identification requires trained personnel. Species in the genus cannot be distinguished microscopically, and Babesia can also be confused with the early trophozoite stage (ring forms) of Plasmodium parasites. To allow more accurate diagnosis in a format that is accessible to a wider variety of laboratories, we developed a real-time PCR assay targeting the 18S rRNA gene of Babesia microti, the dominant babesiosis pathogen in the United States. The real-time PCR is performed on DNA extracted from whole-blood specimens and detects Babesia microti with a limit of detection of ∼100 gene copies in 5 μl of blood. The real-time PCR assay was shown to be 100% specific when tested against a panel of 24 organisms consisting of Babesia microti, other Babesia species, Plasmodium species, tick-borne and other pathogenic bacteria, and other blood-borne parasites. The results using clinical specimens show that the assay can detect infections of lower parasitemia than can be detected by microscopic examination. This method is therefore a rapid, sensitive, and accurate method for detection of Babesia microti in patient specimens. PMID:22170915

  1. Improved coverage of fungal diversity in polluted groundwaters by semi-nested PCR.

    PubMed

    Solé, M; Chatzinotas, A; Sridhar, K R; Harms, H; Krauss, G

    2008-11-15

    Traditional methods used for studying communities of aquatic hyphomycetes are based on the detection and identification of their asexual spores under a microscope. These techniques limit detection to aquatic fungi present in sufficient quantity and capable of sporulating under laboratory conditions. Our objective was to develop a molecular approach to detect and monitor all types of fungi (i.e. strictly or facultatively aquatic) in harsh habitats (i.e. groundwater wells and heavily polluted surface water) where fungal biomass may become limited. We developed a semi-nested PCR protocol for fungal 18S ribosomal RNA genes coupled to subsequent analysis of the PCR products by Temperature Gradient Gel Electrophoresis (TGGE) to monitor the fungal community structure in aquatic habitats characterized by a pollution gradient. Our TGGE-protocol was compared with the traditional morphological approach and revealed a higher diversity in groundwaters and in some polluted surface waters. Thus, PCR-TGGE is a promising alternative in particular in habitats with low fungal biomass. The dynamics of fungal biomass and sporulation rates during the first weeks of leaf colonization showed that habitats with adverse ecological conditions allow only reduced fungal growth, which might subsequently impact upper trophic levels and thus interfere with key ecological processes of leaf decomposition. PMID:18715627

  2. Multicenter Evaluation of a Commercial PCR-Enzyme-Linked Immunosorbent Assay Diagnostic Kit (Onychodiag) for Diagnosis of Dermatophytic Onychomycosis▿

    PubMed Central

    Savin, C.; Huck, S.; Rolland, C.; Benderdouche, M.; Faure, O.; Noacco, G.; Menotti, J.; Candolfi, E.; Pelloux, H.; Grillot, R.; Coupe, S.; Derouin, F.

    2007-01-01

    We prospectively evaluated a new PCR-enzyme-linked immunosorbent assay kit (Onychodiag; BioAdvance, France) for the diagnosis of dermatophytic onychomycosis by testing nail samples from 438 patients with suspected onychomycosis and from 108 healthy controls in three independent laboratories. In two laboratories, samples were collected by trained mycologists as close as possible to the lesions (proximal samples). In one laboratory, samples were collected by other physicians. All samples were processed by conventional mycological techniques and by Onychodiag, blindly to the mycological results. An additional distal sample, collected by clipping the nail plate, was obtained from 75 patients and tested with Onychodiag alone. In patients with culture-proven dermatophytic onychomycosis, the sensitivity of Onychodiag was 83.6% (87.9% including the gray zone) and ranged from 75 to 100% according to the laboratory and the sampling conditions. The specificity was 100% when healthy subjects were considered true negative controls. Onychodiag was positive on 68 patient samples that were sterile or yielded nondermatophyte species in culture. Based on the results of Onychodiag for mycologically proven positive samples and true-negative samples, these results were considered true positives, and the poor performance of mycology on these samples was attributed to inconvenient sampling conditions or to contaminants. When tested on distal samples, Onychodiag was positive in 49/53 (92%) cases of proven dermatophytic onychomycosis. Finally, with either proximal or distal samples, Onychodiag provided a diagnosis of dermatophytic onychomycosis within 24 to 48 h after sampling, and its sensitivity was close to that of mycological techniques applied to proximal samples. PMID:17287330

  3. 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

  4. 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

  5. One-Step RT-PCR protocols improve the rate of dengue diagnosis compared to Two-Step RT-PCR approaches.

    PubMed

    De Paula, Sérgio Oliveira; de Melo Lima, Cristiane; Torres, Maria Paula; Pereira, Márcia Rodrigues Garbin; Lopes da Fonseca, Benedito Antônio

    2004-08-01

    Dengue is the most important arboviral disease transmitted to humans. In our laboratory, we have been working on the standardization of the polymerase chain reaction (PCR) diagnosis of this disease. In this work, we compared five commercial kits regularly used on reverse-transcription polymerase chain reaction (RT-PCR) protocols: two Two-Step kits (SuperScript II RT/Super Mix kit and reverse transcription system/Taq DNA polymerase) and three One-Step kits (ready-to-go RT-PCR Beads kit, QIAGEN One-Step RT-PCR kit, and AcessQuick RT-PCR system). Thirty-one serum samples of patients with clinical diagnosis of dengue fever (DF) were analyzed by RT-PCR and serology. RNA extraction was done with the QIAamp Viral RNA kit, and cDNA synthesis and PCR done according to the manufacturer's protocol for the five kits. Out of the 31 serum samples collected from patients suspected of having dengue, 27 were IgM-positive, confirming the dengue diagnosis. Out of those, 24 were positive by the ready-to-go RT-PCR Beads kit, 25 were positive by AcessQuick RT-PCR system and 27 were positive by QIAGEN One-Step RT-PCR kit. On the other hand, only six samples were positive by the SuperScript II RT/Super Mix kits and 10 were positive by reverse transcription system/Taq DNA polymerase kit. The best performance observed with the One-Step kits was confirmed in spiked samples with known quantities of dengue-1 virus since they detected up 1 x 10(2) PFU/ml, while the most sensitive Two-Step kit detected up 1 x 10(4) PFU/ml. These data show that One-Step RT-PCR kits yielded a higher rate of dengue virus detection than the Two-Step kits and correlated well with the serological diagnosis. PMID:15163417

  6. [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". PMID:24001641

  7. Identification of Helicobacter pylori and the cagA genotype in gastric biopsies using highly sensitive real-time PCR as a new diagnostic tool.

    PubMed

    Yamazaki, Shiho; Kato, Shunji; Matsukura, Norio; Ohtani, Masahiro; Ito, Yoshiyuki; Suto, Hiroyuki; Yamazaki, Yukinao; Yamakawa, Akiyo; Tokudome, Shinkan; Higashi, Hideaki; Hatakeyama, Masanori; Azuma, Takeshi

    2005-06-01

    The CagA protein is one of the virulence factors of Helicobacter pylori, and two major subtypes of CagA have been observed, the Western and East Asian type. CagA is injected from the bacteria into gastric epithelial cells, undergoes tyrosine phosphorylation, and binds to Src homology 2 domain-containing protein-tyrosine phosphatase SHP-2. The East Asian type CagA binds to SHP-2 more strongly than the Western type CagA. Here, we tried to distinguish the CagA type by highly sensitive real-time PCR with the objective of establishing a system to detect H. pylori and CagA subtypes from gastric biopsies. We designed primers and probe sets for Western or East Asian-cagA at Western-specific or East Asian-specific sequence regions, respectively, and H. pylori 16S rRNA. We could detect the H. pylori 16S rRNA gene, Western and East Asian-cagA gene from DNA of gastric biopsies. The sensitivity and specificity for H. pylori infection was 100% in this system. In Thai patients, 87.8% (36/41) were cagA-positive; 26.8% (11/41) were Western-cagA positive and 53.7% (22/41) were East Asian-cagA positive, while 7.3% (3/41) reacted with both types of cagA. These results suggest that this real-time PCR system provides a highly sensitive assessment of CagA type as a new diagnostic tool for the pathogenicity of H. pylori infection. PMID:15907447

  8. 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.

  9. 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.

  10. 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. PMID:24034880

  11. Understanding latent tuberculosis: the key to improved diagnostic and novel treatment strategies

    PubMed Central

    Esmail, Hanif; Barry, Clifton E; Wilkinson, Robert J

    2012-01-01

    Treatment of latent tuberculosis (LTBI) is a vital component of tuberculosis elimination but is not efficiently implemented with available diagnostics and therapeutics. The tuberculin skin test and interferon gamma release assays can inform that infection has occurred but do not prove that it persists. Treatment of LTBI with isoniazid targets actively replicating bacilli but not non-replicating populations, prolonging treatment duration. Developing more predictive diagnostic tests and treatments of shorter duration requires a greater understanding of the biology of latent tuberculosis, from both host and bacillary perspectives. In this article we discuss the basis of current diagnosis and treatment of LTBI and review recent developments in understanding the biology of latency that may enable future improved diagnostic and treatment strategies. PMID:22198298

  12. 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

  13. 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

  14. 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

  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. PMID:22074494

  16. Improved real-time PCR assay for detection and quantification of all 54 known types of human adenoviruses in clinical samples

    PubMed Central

    Bil-Lula, Iwona; De Franceschi, Nicola; Pawlik, Krzysztof; WoŸniak, Mieczysław

    2012-01-01

    Summary Background Detection and quantification of adenoviruses (AdVs) causing life-threatening complications are important abilities in recognition of infection and management of immunocompromised patients. Due to the rapid increase in the number of known AdV types, most commercial tests for detection and identification of AdVs are outdated. Material/Methods We designed an improved, easier and faster real-time quantitative polymerase chain reaction (RQ-PCR) method for detection and quantification of 54 types of human AdVs. A wide validation effort was undertaken to ensure confidence in highly sensitive and specific detection of AdVs in compromised patients. The validation process included evaluation of the method’s suitability and reliability for use in routine diagnostics. Results Due to high sensitivity (9.2×102 copies/ml) and broad dynamic range (7 log) we are able to detect specific viral DNA in large amounts of cell-free body fluids. The new assay is characterized by high precision and low variation within and between individual virus tests (CV=0.036%, CV=1.29%), low bias error (4%) and no cross-reactivity with other pathogens. Conclusions The implementation of this new assay in clinical and laboratory practice provides a rapid, reliable and less laborious method for detection and monitoring of AdV replication in immunocompromised patients. Moreover, it offers the ability to distinguish between active and latent infection and assess treatment efficiency. PMID:22648243

  17. Comparative evaluation of serum, FTA filter-dried blood and oral fluid as sample material for PRRSV diagnostics by RT-qPCR in a small-scale experimental study.

    PubMed

    Steinrigl, Adolf; Revilla-Fernández, Sandra; Wodak, Eveline; Schmoll, Friedrich; Sattler, Tatjana

    2014-01-01

    Recently, research into alternative sample materials, such as oral fluid or filter-dried blood has been intensified, in order to facilitate cost-effective and animal-friendly sampling of individuals or groups of pigs for diagnostic purposes. The objective of this study was to compare the sensitivity of porcine reproductive and respiratory syndrome virus (PRRSV)-RNA detection by reverse transcription quantitative real-time PCR (RT-qPCR) in serum, FTA filter-dried blood and oral fluid sampled from individual pigs. Ten PRRSV negative pigs were injected with an EU-type PRRSV live vaccine. Blood and oral fluid samples were taken from each pig before, and 4, 7, 14 and 21 days after vaccination. All samples were then analyzed by PRRSV RT-qPCR. In serum, eight often pigs tested RT-qPCR positive at different time points post infection. Absolute quantification showed low serum PRRSV-RNA loads in most samples. In comparison to serum, sensitivity of PRRSV-RNA detection was strongly reduced in matched FTA filter-dried blood and in oral fluid from the same pigs. These results indicate that with low PRRSV-RNA loads the diagnostic sensitivity of PRRSV-RNA detection by RT-qPCR achieved with serum is currently unmatched by either FTA filter-dried blood or oral fluid. PMID:24881272

  18. Research to Improve the Efficiency of Double Stereo PCR Microfluidic Chip by Passivating the Inner Surface of Steel Capillary with NOA61.

    PubMed

    Wu, Jian; Guo, Wei; Wang, Chunyan; Yu, Kuanxin; Ma, Ying; Chen, Tao; Li, Yinghui

    2015-06-01

    In this paper, we report the improvement of PCR microfluidic chip efficiency achieved by coating the inner surface of steel capillary microchannel with a 22-µm film of the ultraviolet-solidified NOA61 using a device invented by us. Our results indicate that with this treatment, the roughness of the inside wall of steel capillary was improved from Ra = 0.921 to Ra = 0.254. The contact angle was decreased from about 95° to 56°, and the surface hydrophobicity was also increased. The flow pressure for performing the real-time PCR in the microfluidic chip with modified surface was reduced by twofold (2.11/1) and that resulted in a substantially increased efficiency of PCR. A modification of the microchannel interior surface improved the quality of the on-chip integrated PCR procedure. PMID:25582422

  19. 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

  20. 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

  1. 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...

  2. 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.

  3. Automated Quantification of Neuropad Improves Its Diagnostic Ability in Patients with Diabetic Neuropathy.

    PubMed

    Ponirakis, Georgios; Fadavi, Hassan; Petropoulos, Ioannis N; Azmi, Shazli; Ferdousi, Maryam; Dabbah, Mohammad A; Kheyami, Ahmad; Alam, Uazman; Asghar, Omar; Marshall, Andrew; Tavakoli, Mitra; Al-Ahmar, Ahmed; Javed, Saad; Jeziorska, Maria; Malik, Rayaz A

    2015-01-01

    Neuropad is currently a categorical visual screening test that identifies diabetic patients at risk of foot ulceration. The diagnostic performance of Neuropad was compared between the categorical and continuous (image-analysis (Sudometrics)) outputs to diagnose diabetic peripheral neuropathy (DPN). 110 subjects with type 1 and 2 diabetes underwent assessment with Neuropad, Neuropathy Disability Score (NDS), peroneal motor nerve conduction velocity (PMNCV), sural nerve action potential (SNAP), Deep Breathing-Heart Rate Variability (DB-HRV), intraepidermal nerve fibre density (IENFD), and corneal confocal microscopy (CCM). 46/110 patients had DPN according to the Toronto consensus. The continuous output displayed high sensitivity and specificity for DB-HRV (91%, 83%), CNFD (88%, 78%), and SNAP (88%, 83%), whereas the categorical output showed high sensitivity but low specificity. The optimal cut-off points were 90% for the detection of autonomic dysfunction (DB-HRV) and 80% for small fibre neuropathy (CNFD). The diagnostic efficacy of the continuous Neuropad output for abnormal DB-HRV (AUC: 91%, P = 0.0003) and CNFD (AUC: 82%, P = 0.01) was better than for PMNCV (AUC: 60%). The categorical output showed no significant difference in diagnostic efficacy for these same measures. An image analysis algorithm generating a continuous output (Sudometrics) improved the diagnostic ability of Neuropad, particularly in detecting autonomic and small fibre neuropathy. PMID:26064991

  4. 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.

  5. 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.

  6. 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

  7. 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. PMID:27369587

  8. 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. PMID:27333026

  9. Improving medical diagnostic accuracy of ultrasound Doppler signals by combining neural network models.

    PubMed

    Ubeyli, Elif Derya; Güler, Inan

    2005-07-01

    There are a number of different quantitative models that can be used in a medical diagnostic decision support system including parametric methods (linear discriminant analysis or logistic regression), nonparametric models (k nearest neighbor or kernel density) and several neural network models. The complexity of the diagnostic task is thought to be one of the prime determinants of model selection. Unfortunately, there is no theory available to guide model selection. This paper illustrates the use of combined neural network models to guide model selection for diagnosis of ophthalmic and internal carotid arterial disorders. The ophthalmic and internal carotid arterial Doppler signals were decomposed into time-frequency representations using discrete wavelet transform and statistical features were calculated to depict their distribution. The first-level networks were implemented for the diagnosis of ophthalmic and internal carotid arterial disorders using the statistical features as inputs. To improve diagnostic accuracy, the second-level networks were trained using the outputs of the first-level networks as input data. The combined neural network models achieved accuracy rates which were higher than that of the stand-alone neural network models. PMID:15780863

  10. Three-dimensional textural features of conventional MRI improve diagnostic classification of childhood brain tumours.

    PubMed

    Fetit, Ahmed E; Novak, Jan; Peet, Andrew C; Arvanitits, Theodoros N

    2015-09-01

    The aim of this study was to assess the efficacy of three-dimensional texture analysis (3D TA) of conventional MR images for the classification of childhood brain tumours in a quantitative manner. The dataset comprised pre-contrast T1 - and T2-weighted MRI series obtained from 48 children diagnosed with brain tumours (medulloblastoma, pilocytic astrocytoma and ependymoma). 3D and 2D TA were carried out on the images using first-, second- and higher order statistical methods. Six supervised classification algorithms were trained with the most influential 3D and 2D textural features, and their performances in the classification of tumour types, using the two feature sets, were compared. Model validation was carried out using the leave-one-out cross-validation (LOOCV) approach, as well as stratified 10-fold cross-validation, in order to provide additional reassurance. McNemar's test was used to test the statistical significance of any improvements demonstrated by 3D-trained classifiers. Supervised learning models trained with 3D textural features showed improved classification performances to those trained with conventional 2D features. For instance, a neural network classifier showed 12% improvement in area under the receiver operator characteristics curve (AUC) and 19% in overall classification accuracy. These improvements were statistically significant for four of the tested classifiers, as per McNemar's tests. This study shows that 3D textural features extracted from conventional T1 - and T2-weighted images can improve the diagnostic classification of childhood brain tumours. Long-term benefits of accurate, yet non-invasive, diagnostic aids include a reduction in surgical procedures, improvement in surgical and therapy planning, and support of discussions with patients' families. It remains necessary, however, to extend the analysis to a multicentre cohort in order to assess the scalability of the techniques used. PMID:26256809

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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

  16. Enhanced CT images by the wavelet transform improving diagnostic accuracy of chest nodules.

    PubMed

    Guo, Xiuhua; Liu, Xiangye; Wang, Huan; Liang, Zhigang; Wu, Wei; He, Qian; Li, Kuncheng; Wang, Wei

    2011-02-01

    The objective of this study was to compare the diagnostic accuracy in the interpretation of chest nodules using original CT images versus enhanced CT images based on the wavelet transform. The CT images of 118 patients with cancers and 60 with benign nodules were used in this study. All images were enhanced through an algorithm based on the wavelet transform. Two experienced radiologists interpreted all the images in two reading sessions. The reading sessions were separated by a minimum of 1 month in order to minimize the effect of observer's recall. The Mann-Whitney U nonparametric test was used to analyze the interpretation results between original and enhanced images. The Kruskal-Wallis H nonparametric test of K independent samples was used to investigate the related factors which could affect the diagnostic accuracy of observers. The area under the ROC curves for the original and enhanced images was 0.681 and 0.736, respectively. There is significant difference in diagnosing the malignant nodules between the original and enhanced images (z = 7.122, P < 0.001), whereas there is no significant difference in diagnosing the benign nodules (z = 0.894, P = 0.371). The results showed that there is significant difference between original and enhancement images when the size of nodules was larger than 2 cm (Z = -2.509, P = 0.012, indicating the size of the nodules is a critical evaluating factor of the diagnostic accuracy of observers). This study indicated that the image enhancement based on wavelet transform could improve the diagnostic accuracy of radiologists for the malignant chest nodules. PMID:19937084

  17. 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

  18. 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. PMID:26297212

  19. A Fast-and-Robust Profiler for Improving Polymerase Chain Reaction Diagnostics

    PubMed Central

    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

  20. Detection of Treponema pallidum in the vitreous by PCR

    PubMed Central

    Müller, M; Ewert, I; Hansmann, F; Tiemann, C; Hagedorn, H J; Solbach, W; Roider, J; Nölle, B; Laqua, H; Hoerauf, H

    2007-01-01

    Background Ocular involvement of syphilis still poses a clinical challenge due to the chameleonic behaviour of the disease. As the serodiagnosis has significant limitations, the direct detection of Treponema pallidum (TP) in the vitreous represents a desirable diagnostic tool. Methods Real‐time polymerase chain reaction (PCR) for the detection of TP was applied in diagnostic vitrectomies of two patients with acute chorioretinitis. Qualitative verification of TP by real‐time PCR and melting point analysis according to a modified protocol was ruled out. Patients underwent complete ophthalmological examination with fundus photographs, fluorescein angiography, serological examination, antibiotic treatment and follow‐up. Results In two cases of acute chorioretinitis of unknown origin, real‐time PCR of vitreous specimens of both patients provided evidence of TP and was 100% specific. Initial diagnosis of presumed viral retinitis was ruled out by PCR of vitreous specimen. Patients were treated with systemic antibiotics and showed prompt improvement in visual function and resolution of fundus lesions. Conclusions With real‐time PCR, detection of TP in the vitreous was possible and delivered a sensitive, quick and inexpensive answer to a disease rather difficult to assess. In cases of acute chorioretinitis, the use of PCR‐based assays of vitreous specimens in the diagnostic evaluation of patients is advisable. Although syphilitic chorioretinitis is a rare disease, PCR should include search for TP, as diagnostic dilemmas prolong definitive treatment in a sight‐threatening disease. PMID:17108014

  1. 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. PMID:23575131

  2. Development of a diagnostic PCR assay based on novel DNA sequences for the detection of Mycoplasma suis (Eperythrozoon suis) in porcine blood.

    PubMed

    Hoelzle, Ludwig E; Adelt, Dagmar; Hoelzle, Katharina; Heinritzi, Karl; Wittenbrink, Max M

    2003-05-29

    An efficient method of control of porcine eperythrozoonosis (PE) caused by Mycoplasma suis is eradication of infection by detection and removal of infected carrier animals. At present, only a few tests are available for the diagnosis of these latent M. suis infections in pigs. The objective of this study was to develop a PCR assay based on novel DNA sequences for the identification of M. suis-infected pigs. A 1.8 kb EcoRI DNA fragment of the M. suis genome was isolated from the blood of pigs experimentally infected with M. suis. Specificity of the DNA fragment was confirmed by DNA sequence analysis and PCR using primers directed against sequences contained in the 1.8 kb fragment. PCR products of 782 bp in size were amplified only from M. suis particles prepared from the blood of experimentally infected pigs but not from any controls, comprising blood from gnotobiotic piglets and a panel of bacteria including other porcine mycoplasmas. PCR results were confirmed by dot blot hybridisation. The applicability of the PCR assay to diagnose M. suis infections in pigs was evaluated by investigating blood samples from 10 symptomatic pigs with clinical signs typical of porcine eperythrozoonosis and blood samples from 10 healthy pigs. The M. suis-specific PCR product was amplified from all samples taken at episodes of acute disease as well as from samples taken during the latent stage of infection, thus demonstrating the suitability of the PCR assay for detecting latent infected carrier animals. PMID:12695043

  3. 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

  4. Consensus diagnostic histopathological criteria for acute gastrointestinal graft versus host disease improve interobserver reproducibility.

    PubMed

    Kreft, Andreas; Mottok, Anja; Mesteri, Ildiko; Cardona, Diana M; Janin, Anne; Kühl, Anja A; Andrulis, Mindaugas; Brunner, Andrea; Shulman, Howard M; Negri, Giovanni; Tzankov, Alexandar; Huber, Elisabeth

    2015-09-01

    Graft versus host disease (GvHD) is a clinically important complication after allogeneic hematopoietic stem cell transplantation (HSCT). Its diagnosis relies on clinical and histopathological findings. In order to evaluate and improve inter-institutional diagnostic agreement on histological diagnosis and grading of acute gastrointestinal GvHD, we conducted a round robin test, which included 33 biopsies from 23 patients after HSCT. Five pathologists from different institutions independently evaluated the original sections from the biopsies submitted for diagnosis. Based on their results, consensus qualitative criteria for the assessment of typical histological features of GvHD (e.g., apoptosis, crypt destruction, mucosa denudation) were proposed, including detailed descriptions as well as histological images. In a second round robin test with involvement of the same pathologists, the reproducibility of both diagnosis and grading had improved. Remaining differences were mostly related to differential diagnostic considerations, including viral infection or toxic side effects of medication, which should be resolved by integrating histopathological findings with proper clinical information. PMID:26164839

  5. Improved endoscopic retrograde cholangiopancreatography brush increases diagnostic yield of malignant biliary strictures

    PubMed Central

    Shieh, Frederick K; Luong-Player, Adelina; Khara, Harshit S; Liu, Haiyan; Lin, Fan; Shellenberger, Matthew J; Johal, Amitpal S; Diehl, David L

    2014-01-01

    AIM: To determine if a new brush design could improve the diagnostic yield of biliary stricture brushings. METHODS: Retrospective chart review was performed of all endoscopic retrograde cholangiopancreatography procedures with malignant biliary stricture brushing between January 2008 and October 2012. A standard wire-guided cytology brush was used prior to protocol implementation in July 2011, after which, a new 9 French wire-guided cytology brush (Infinity sampling device, US Endoscopy, Mentor, OH) was used for all cases. All specimens were reviewed by blinded pathologists who determined whether the sample was positive or negative for malignancy. Cellular yield was quantified by describing the number of cell clusters seen. RESULTS: Thirty-two new brush cases were compared to 46 historical controls. Twenty-five of 32 (78%) cases in the new brush group showed abnormal cellular findings consistent with malignancy as compared to 17 of 46 (37%) in the historical control group (P = 0.0003). There was also a significant increase in the average number of cell clusters of all sizes (21.1 vs 9.9 clusters, P = 0.0007) in the new brush group compared to historical controls. CONCLUSION: The use of a new brush design for brush cytology of biliary strictures shows increased diagnostic accuracy, likely due to improved cellular yield, as evidenced by an increase in number of cellular clusters obtained. PMID:25031790

  6. Simulation Training Improves Surgical Proficiency and Safety During Diagnostic Shoulder Arthroscopy Performed by Residents.

    PubMed

    Waterman, Brian R; Martin, Kevin D; Cameron, Kenneth L; Owens, Brett D; Belmont, Philip J

    2016-05-01

    Although virtual reality simulators have established construct validity, no studies have proven transfer of skills from a simulator to improved in vivo surgical skill. The current authors hypothesized that simulation training would improve residents' basic arthroscopic performance and safety. Twenty-two orthopedic surgery trainees were randomized into simulation or standard practice groups. At baseline testing, all of the participants performed simulator-based testing and a supervised, in vivo diagnostic shoulder arthroscopy with video recording. The simulation group subsequently received 1 hour of total instruction during a 3-month period, and the standard practice group received no simulator training. After intervention, both groups were reevaluated with simulator testing and a second recorded diagnostic shoulder arthroscopy. Two blinded, independent experts evaluated arthroscopic performance using the anatomic checklist, Arthroscopic Surgery Skill Evaluation Tool (ASSET) score, and total elapsed time. All outcome measures were compared within and between groups. After intervention, mean time required by the simulation group to complete the simulator task (30.64 seconds) was 8±1.2 seconds faster than the time required by the control group (38.64 seconds; P=.001). Probe distance (51.65 mm) was improved by 41.2±6.08 mm compared with the control (92.83 mm; P=.001). When comparing ASSET safety scores, the simulation group was competent (3.29) and significantly better than the control group (3.00; P=.005) during final arthroscopic testing. This study establishes transfer validity for an arthroscopic shoulder simulator model. Simulator training for residents in training can decrease surgical times, improve basic surgical skills, and confer greater patient safety during shoulder arthroscopy. [Orthopedics. 2016; 39(3):e479-e485.]. PMID:27135460

  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. Diagnostic Application of IS900 PCR Using Blood as a Source Sample for the Detection of Mycobacterium avium Subspecies Paratuberculosis in Early and Subclinical Cases of Caprine Paratuberculosis

    PubMed Central

    Singh, P. K.; Singh, S. V.; Kumar, H.; Sohal, J. S.; Singh, A. V.

    2010-01-01

    Efficacy of IS900 blood PCR was evaluated for the presence of MAP infection. Serum, fecal, and blood samples of kids, young, and adult goats from farm and farmer's herds in Mathura district were also screened by ELISA, microscopy and culture. Of 111 goats (kids: 40, young: 14, adults: 57) screened, 77.5% were positive by blood PCR. Of 76 goats, 90.8% (kids: 87.5% and adults: 94.4%) were positive by PCR. From 21 kids and 14 young goats, 42.8 and 57.1% were positive. gDNA from goats was genotyped as MAP “Indian Bison type”. Of 21 fecal samples of kids examined by microscopy, 66.7% were positive. In ELISA, 9.5 and 57.1% kids were positives as “type I” and “type II” reactors, respectively. Screening 14 young goats by culture of blood clots, 28.6% were positive. Agreement was substantial between PCR and microscopy. It was fair and moderate when PCR and microscopy were compared with type I and type II reactors, respectively. Presence of MAP in non-clinical kids and young goats indicate early or subclinical infection. Blood PCR was rapid, sensitive, and specific assay for detection of MAP in any stage (early, subclinical, and clinical) and age (kids, young, and adult) of goats. PMID:20445791

  9. 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

  10. 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

  11. Tissular and soluble miRNAs for diagnostic and therapy improvement in digestive tract cancers.

    PubMed

    Albulescu, Radu; Neagu, Monica; Albulescu, Lucian; Tanase, Cristiana

    2011-01-01

    Digestive cancers (e.g., gastric, colorectal, pancreatic or hepatocarcinoma) are among the most frequently reported cancers in the world, and are characterized by invasivity, metastatic potential and poor outcomes. This group includes some of the most critical cancers (among them, are those ranked second to forth in cancer-related mortality) and, despite all sustained efforts, they maintain a profile of low survival rates and lack successful therapies. Discovery of biomarkers that improve disease characterization may make optimized or personalized therapy possible. Novel biomarkers are expected to provide, hopefully, less-invasive or noninvasive diagnostic tools that make possible earlier detection of disease. Also, they may provide a more reliable selection instrument in the drug discovery process. miRNAs, short noncoding RNAs, have emerged in the last few years as significant regulators of cellular activities, controlling protein expression at the post-transcriptional level, with a significant implication in pathology in general and, of most relevance, in cancers. Deregulation of miRNA expression levels and some genetic alterations were demonstrated in various cancers, including digestive cancers. Investigations in tissue samples have provided a considerable amount of knowledge, identifying altered expressions of miRNAs associated with tumorigenesis and tumor progression. Overexpression of some tumor-inducing or tumor-promoting miRNAs was demonstrated, as well as the downregulation of tumor-suppressor miRNAs. Both individual miRNAs, as well as sets of multiple miRNAs, were set up as candidate biomarkers for diagnostics or monitoring, offering relevant insights into tumorigenic mechanisms. Circulating miRNAs were demonstrated as valuable instruments in tumor diagnosis and the prognosis of digestive cancers (affecting the esophagus, stomach, intestine, colorectum, liver and pancreas), and are being investigated thoroughly in order to generate and validate less

  12. 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

  13. 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.

  14. 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

  15. 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

  16. Improved diagnostics by automated matching and enhancement in fluorescein angiography of the ocular fundus

    NASA Astrophysics Data System (ADS)

    Noordmans, Herke Jan; van den Biesen, Pieter; de Roode, Rowland; Verdaasdonk, Rudolf

    2008-02-01

    An interactive image matching program has been developed to help ophthalmologists in perceiving subtle differences between sequential images obtained during fluorescein angiography. In a pilot experiment, it appeared that the image matching program could effectively correct camera alignment errors. By offering simple tools like image overlay, blinking and image subtraction, differences between angiograms can be greatly enhanced and interpreted. It appeared that newly formed, leaking blood vessels could be detected at an earlier stage of the disease process using these tools. Treatment can be initiated right away, thereby preventing the patient from having additional visual loss. The matching program seems to improve the quality of fundus diagnostics but needs to be validated in future studies.

  17. Improving Student Outcomes with mCLASS: Math, a Technology-Enhanced CBM and Diagnostic Interview Assessment

    ERIC Educational Resources Information Center

    Wang, Ye; Gushta, Matthew

    2013-01-01

    The No Child Left Behind Act resulted in increased school-level implementation of assessment-based school interventions that aim to improve student performance. Diagnostic assessments are included among these interventions, designed to help teachers use evidence about student performance to modify and differentiate instruction and improve student…

  18. High-throughput mutation profiling improves diagnostic stratification of sporadic medullary thyroid carcinomas.

    PubMed

    Simbolo, Michele; Mian, Caterina; Barollo, Susi; Fassan, Matteo; Mafficini, Andrea; Neves, Diogo; Scardoni, Maria; Pennelli, Gianmaria; Rugge, Massimo; Pelizzo, Maria Rosa; Cavedon, Elisabetta; Fugazzola, Laura; Scarpa, Aldo

    2014-07-01

    Sporadic medullary thyroid carcinoma (MTC) harbors RET gene somatic mutations in up to 50 % of cases, and RAS family gene mutations occur in about 10 %. A timely and comprehensive characterization of molecular alterations is needed to improve MTC diagnostic stratification and design-tailored therapeutic approaches. Twenty surgically resected sporadic MTCs, previously analyzed for RET mutations by Sanger sequencing using DNA from formalin-fixed paraffin-embedded samples, were investigated for intragenic mutations in 50 cancer-associated genes applying a multigene Ion AmpliSeq next-generation sequencing (NGS) technology. Thirteen (65 %) MTCs harbored a RET mutation; 10 were detected at both Sanger and NGS sequencing, while 3 undetected by Sanger were revealed by NGS. One of the 13 RET-mutated cases also showed an F354L germline mutation in STK11. Of the seven RET wild-type MTCs, four cases (57.1 %) harbored a RAS mutation: three in HRAS (all Q61R) and one in KRAS (G12R). The three remaining MTCs (15 %) resulted as wild-type for all the 50 cancer-related genes. Follow-up was available in all but one RET-mutated case. At the end of follow-up, 7 of 12 (58 %) RET-mutated patients had relapsed, while the 4 RAS-mutated MTC patients were disease-free. Two of the three patients with MTC wild-type for all 50 genes relapsed during the follow-up period. Detection of mutations by NGS has the potential to improve the diagnostic stratification of sporadic MTC. PMID:24828033

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. A Unique Primer with an Inosine Chain at the 5′-Terminus Improves the Reliability of SNP Analysis Using the PCR-Amplified Product Length Polymorphism Method

    PubMed Central

    Shojo, Hideki; Tanaka, Mayumi; Takahashi, Ryohei; Kakuda, Tsuneo; Adachi, Noboru

    2015-01-01

    Polymerase chain reaction-amplified product length polymorphism (PCR-APLP) is one of the most convenient and reliable methods for single nucleotide polymorphism (SNP) analysis. This method is based on PCR, but uses allele-specific primers containing SNP sites at the 3′-terminus of each primer. To use this method at least two allele-specific primers and one “counter-primer”, which serves as a common forward or reverse primer of the allele-specific primers, are required. The allele-specific primers have SNP sites at the 3′-terminus, and another primer should have a few non-complementary flaps at the 5′-terminus to detect SNPs by determining the difference of amplicon length by PCR and subsequent electrophoresis. A major disadvantage of the addition of a non-complementary flap is the non-specific annealing of the primer with non-complementary flaps. However, a design principle for avoiding this undesired annealing has not been fully established, therefore, it is often difficult to design effective APLP primers. Here, we report allele-specific primers with an inosine chain at the 5′-terminus for PCR-APLP analysis. This unique design improves the competitiveness of allele-specific primers and the reliability of SNP analysis when using the PCR-APLP method. PMID:26381262

  5. Diagnostic value of the strand displacement amplification method compared to those of Roche Amplicor PCR and culture for detecting mycobacteria in sputum samples.

    PubMed Central

    Ichiyama, S; Ito, Y; Sugiura, F; Iinuma, Y; Yamori, S; Shimojima, M; Hasegawa, Y; Shimokata, K; Nakashima, N

    1997-01-01

    We compared the ability of the semiautomated BDProbeTec-SDA system, which uses the strand displacement amplification (SDA) method, with that of the Roche Amplicor-PCR system and the Septi-Chek AFB culture system to directly detect Mycobacterium tuberculosis complex (MTB) and other mycobacteria in sputum samples. A total of 530 sputum samples from 299 patients were examined in this study. Of the 530 samples, 129 were culture positive for acid-fast bacilli with the Septi-Chek AFB system; 95 for MTB, 29 for M. avium-M. intracellulare complex (MAC), and 5 for other mycobacteria. The BDProbeTec-SDA system detected 90 of the 95 samples culture positive for MTB (sensitivity, 94.7%), and the Amplicor-PCR system detected 85 of the 95 samples culture positive for MTB (sensitivity, 89.5%). The specificity of each system, based on the clinical diagnosis, was 99.8% for SDA and 100% for PCR, respectively. Among the 29 samples culture positive for MAC, the BDProbeTec-SDA system detected MAC in 24 samples (sensitivity, 82.8%), whereas the Amplicor-PCR system detected MAC in 23 samples (sensitivity, 79.3%). The specificities of the systems were 98.3 and 100%, respectively. The high degrees of sensitivity and specificity of the BDProbeTec-SDA system suggest that it should be very useful in clinical laboratories for the rapid detection of mycobacteria in sputum samples. PMID:9399498

  6. 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.

  7. [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

  8. Improving diagnostic accuracy using EHR in emergency departments: A simulation-based study.

    PubMed

    Ben-Assuli, Ofir; Sagi, Doron; Leshno, Moshe; Ironi, Avinoah; Ziv, Amitai

    2015-06-01

    It is widely believed that Electronic Health Records (EHR) improve medical decision-making by enabling medical staff to access medical information stored in the system. It remains unclear, however, whether EHR indeed fulfills this claim under the severe time constraints of Emergency Departments (EDs). We assessed whether accessing EHR in an ED actually improves decision-making by clinicians. A simulated ED environment was created at the Israel Center for Medical Simulation (MSR). Four different actors were trained to simulate four specific complaints and behavior and 'consulted' 26 volunteer ED physicians. Each physician treated half of the cases (randomly) with access to EHR, and their medical decisions were compared to those where the physicians had no access to EHR. Comparison of diagnostic accuracy with and without access showed that accessing the EHR led to an increase in the quality of the clinical decisions. Physicians accessing EHR were more highly informed and thus made more accurate decisions. The percentage of correct diagnoses was higher and these physicians were more confident in their diagnoses and made their decisions faster. PMID:25817921

  9. Deformability based sorting of red blood cells improves diagnostic sensitivity for malaria caused by Plasmodium falciparum.

    PubMed

    Guo, Quan; Duffy, Simon P; Matthews, Kerryn; Deng, Xiaoyan; Santoso, Aline T; Islamzada, Emel; Ma, Hongshen

    2016-02-21

    The loss of red blood cell (RBC) deformability is part of the pathology of many diseases. In malaria caused by Plasmodium falciparum infection, metabolism of hemoglobin by the parasite results in progressive reduction in RBC deformability that is directly correlated with the growth and development of the parasite. The ability to sort RBCs based on deformability therefore provides a means to isolate pathological cells and to study biochemical events associated with disease progression. Existing methods have not been able to sort RBCs based on deformability or to effectively enrich for P. falciparum infected RBCs at clinically relevant concentrations. Here, we develop a method to sort RBCs based on deformability and demonstrate the ability to enrich the concentration of ring-stage P. falciparum infected RBCs (Pf-iRBCs) by >100× from clinically relevant parasitemia (<0.01%). Deformability based sorting of RBCs is accomplished using ratchet transport through asymmetrical constrictions using oscillatory flow. This mechanism provides dramatically improved selectivity over previous biophysical methods by preventing the accumulation of cells in the filter microstructure to ensure that consistent filtration forces are applied to each cell. We show that our approach dramatically improves the sensitivity of malaria diagnosis performed using both microscopy and rapid diagnostic test by converting samples with difficult-to-detect parasitemia (<0.01%) into samples with easily detectable parasitemia (>0.1%). PMID:26768227

  10. 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

  11. 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

  12. Use of the RFLP-PCR diagnostic test for characterizing MACE and kdr insecticide resistance in the peach potato aphid Myzus persicae.

    PubMed

    Cassanelli, Stefano; Cerchiari, Barbara; Giannini, Sara; Bizzaro, Davide; Mazzoni, Emanuele; Manicardi, Gian Carlo

    2005-01-01

    The peach-potato aphid Myzus persicae (Sulzer) has developed a number of insecticide resistance mechanisms owing to the high selective pressure produced by world-wide insecticide treatments. Knowledge of the geographical distribution and the temporal evolution of these resistant phenotypes helps to develop suitable pest-management programs. Current understanding of the major mechanisms of resistance at the molecular level makes it possible to diagnose the presence of modified acetylcholinesterase (MACE) or knockdown resistance (kdr). This paper describes a rapid method for the identification of both resistance mechanisms in a single molecular assay by using restriction fragment length polymorphism of PCR products (RFLP-PCR) in individual as well as pooled aphids. PMID:15593078

  13. Rapid, Simultaneous Detection of Clostridium sordellii and Clostridium perfringens in Archived Tissues by a Novel PCR-Based Microsphere Assay: Diagnostic Implications for Pregnancy-Associated Toxic Shock Syndrome Cases

    PubMed Central

    Bhatnagar, Julu; DeLeon-Carnes, Marlene; Kellar, Kathryn L.; Bandyopadhyay, Kakali; Antoniadou, Zoi-Anna; Shieh, Wun-Ju; Paddock, Christopher D.; Zaki, Sherif R.

    2012-01-01

    Clostridium sordellii and Clostridium perfringens are infrequent human pathogens; however, the case-fatality rates for the infections are very high, particularly in obstetric C. sordellii infections (>90%). Deaths from Clostridium sordellii and Clostridium perfringens toxic shock (CTS) are sudden, and diagnosis is often challenging. Formalin-fixed, paraffin-embedded (FFPE) tissues usually are the only specimens available for sudden fatal cases, and immunohistochemistry (IHC) for Clostridia is generally performed but it cannot identify species. A clear need exists for a rapid, species-specific diagnostic assay for FFPE tissues. We developed a duplex PCR-based microsphere assay for simultaneous detection of C. sordellii and C. perfringens and evaluated DNA extracted from 42 Clostridium isolates and FFPE tissues of 28 patients with toxic shock/endometritis (20 CTS, 8 non-CTS, as confirmed by PCR and sequencing). The microsphere assay correctly identified C. sordellii and C. perfringens in all known isolates and in all CTS patients (10 C. sordellii, 8 C. perfringens, 2 both) and showed 100% concordance with PCR and sequencing results. The microsphere assay is a rapid, specific, and cost-effective method for the diagnosis of CTS and offers the advantage of simultaneous testing for C. sordellii and C. perfringens in FFPE tissues using a limited amount of DNA. PMID:22536012

  14. 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

  15. 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.

  16. 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 ...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real-time quantitative PCR assays that target the human-associated HF183 bacterial cluster have been found to be some of the top performing methods for the characterization of human fecal pollution in ambient surface waters. The United States Environmental Protection Agency is planning to conduct a ...

  19. Improved real-time PCR diagnosis of citrus stubborn disease by targeting prophage genes of Spiroplasma citri

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Spiroplasma citri is a phloem-limited bacterium causing citrus stubborn disease (CSD). Isolation and culturing of S. citri is difficult and time consuming. Current detection methods use polymerase chain reaction (PCR) assays with primers developed from sequences of S. citri house-keeping genes. In c...

  20. 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...

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

    PubMed Central

    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. PMID:26106620

  2. Molecular investigations to improve diagnostic accuracy in patients with ARC syndrome.

    PubMed

    Cullinane, Andrew R; Straatman-Iwanowska, Anna; Seo, Jeong K; Ko, Jae S; Song, Kyung S; Gizewska, Maria; Gruszfeld, Dariusz; Gliwicz, Dorota; Tuysuz, Beyhan; Erdemir, Gulin; Sougrat, Rachid; Wakabayashi, Yoshiyuki; Hinds, Rupert; Barnicoat, Angela; Mandel, Hanna; Chitayat, David; Fischler, Björn; Garcia-Cazorla, Angels; Knisely, A S; Kelly, Deirdre A; Maher, Eamonn R; Gissen, Paul

    2009-02-01

    Arthrogryposis, Renal dysfunction and Cholestasis (ARC) syndrome is a multi-system autosomal recessive disorder caused by germline mutations in VPS33B. The detection of germline VPS33B mutations removes the need for diagnostic organ biopsies (these carry a>50% risk of life-threatening haemorrhage due to platelet dysfunction); however, VPS33B mutations are not detectable in approximately 25% of patients. In order further to define the molecular basis of ARC we performed mutation analysis and mRNA and protein studies in patients with a clinical diagnosis of ARC. Here we report novel mutations in VPS33B in patients from Eastern Europe and South East Asia. One of the mutations was present in 7 unrelated Korean patients. Reduced expression of VPS33B and cellular phenotype was detected in fibroblasts from patients clinically diagnosed with ARC with and without known VPS33B mutations. One mutation-negative patient was found to have normal mRNA and protein levels. This patient's clinical condition improved and he is alive at the age of 2.5 years. Thus we show that all patients with a classical clinical course of ARC had decreased expression of VPS33B whereas normal VPS33B expression was associated with good prognosis despite initial diagnosis of ARC. PMID:18853461

  3. Molecular Investigations to Improve Diagnostic Accuracy in Patients With ARC Syndrome

    PubMed Central

    Cullinane, Andrew R.; Straatman-Iwanowska, Anna; Seo, Jeong K.; Ko, Jae S.; Song, Kyung S.; Gizewska, Maria; Gruszfeld, Dariusz; Gliwicz, Dorota; Tuysuz, Beyhan; Erdemir, Gulin; Sougrat, Rachid; Wakabayashi, Yoshiyuki; Hinds, Rupert; Barnicoat, Angela; Mandel, Hanna; Chitayat, David; Fischler, Björn; Garcia-Cazorla, Angels; Knisely, A. S.; Kelly, Deirdre A.; Maher, Eamonn R.; Gissen, Paul

    2008-01-01

    Arthrogryposis, Renal dysfunction and Cholestasis (ARC) syndrome is a multi-system autosomal recessive disorder caused by germline mutations in VPS33B. The detection of germline VPS33B mutations removes the need for diagnostic organ biopsies (these carry a >50% risk of life-threatening haemorrhage due to platelet dysfunction); however, VPS33B mutations are not detectable in ∼25% of patients. In order further to define the molecular basis of ARC we performed mutation analysis and mRNA and protein studies in patients with a clinical diagnosis of ARC. Here we report novel mutations in VPS33B in patients from Eastern Europe and South East Asia. One of the mutations was present in 7 unrelated Korean patients. Reduced expression of VPS33B and cellular phenotype was detected in fibroblasts from patients clinically diagnosed with ARC with and without known VPS33B mutations. One mutation-negative patient was found to have normal mRNA and protein levels. This patient's clinical condition improved and he is alive at the age of 2.5 years. Thus we show that all patients with a classical clinical course of ARC had decreased expression of VPS33B whereas normal VPS33B expression was associated with good prognosis despite initial diagnosis of ARC. PMID:18853461

  4. 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…

  5. Improving lateral-flow immunoassay (LFIA) diagnostics via biomarker enrichment for mHealth.

    PubMed

    Lai, James J; Stayton, Patrick S

    2015-01-01

    Optical detection technologies based on mobile devices can be utilized to enable many mHealth applications, including a reader for lateral-flow immunoassay (LFIA). However, an intrinsic challenge associated with LFIA for clinical diagnostics is the limitation in sensitivity. Therefore, rapid and simple specimen processing strategies can directly enable more sensitive LFIA by purifying and concentrating biomarkers. Here, a binary reagent system is presented for concentrating analytes from a larger volume specimen to improve the malaria LFIA's limit of detection (LOD). The biomarker enrichment process utilizes temperature-responsive gold-streptavidin conjugates, biotinylated antibodies, and temperature-responsive magnetic nanoparticles. The temperature-responsive gold colloids were synthesized by modifying the citrate-stabilized gold colloids with a diblock copolymer, containing a thermally responsive poly(N-isopropylacrylamide) (pNIPAAm) segment and a gold-binding block composed of NIPAAm-co-N,N-dimethylaminoethylacrylamide. The gold-streptavidin conjugates were synthesized by conjugating temperature-responsive gold colloids with streptavidin via covalent linkages using carbodiimide chemistry chemistry. The gold conjugates formed half-sandwiches, gold labeled biomarker, by complexing with biotinylated antibodies that were bound to Plasmodium falciparum histidine-rich protein 2 (PfHRP2), a malaria antigen. When a thermal stimulus was applied in conjunction with a magnetic field, the half-sandwiches and temperature-responsive magnetic nanoparticles that were both decorated with pNIPAAm formed large aggregates that were efficiently magnetically separated from human plasma. The binary reagent system was applied to a large volume (500 μL) specimen for concentrating biomarker 50-fold into a small volume and applied directly to an off-the-shelf malaria LFIA to improve the signal-to-noise ratio. PMID:25626532

  6. Measurement of Glycosylated Alpha-Fetoprotein Improves Diagnostic Power over the Native Form in Hepatocellular Carcinoma

    PubMed Central

    Jin, Jonghwa; Park, Jiyoung; Yu, Su Jong; Yoon, Jung-Hwan; Kim, Youngsoo

    2014-01-01

    Serum alpha-fetoprotein (AFP) has long been used as a diagnostic marker for hepatocellular carcinoma (HCC), albeit controversially. Although it remains widely used in clinics, the value of AFP in HCC diagnosis has recently been challenged due to its significant rates of false positive and false negative findings. To improve the efficacy of AFP as HCC diagnostic marker, we developed a method of measuring total and glycosylated AFP by multiple reaction monitoring (MRM)-MS. In this study, we verified the total amount of AFP (nonglycopeptide levels) and the degree of glycosylated AFP (deglycopeptide levels) in 60 normal (41 men and 19 women; mean age 53 years; range 32–74 years), 35 LC (23 men and 12 women; mean age 56 years; range 43–78 years; HBV-related), and 60 HCC subjects (42 men and 18 women; mean age 58 years; range 38–76 years; HBV-related; 30 stage I, 15 stage II, and 10 stage III). By MRM-MS analysis, the nonglycopeptide had 56.7% sensitivity, 68.3% specificity, and an AUC of 0.687 [cutoff value: ≥0.02 (light/heavy ratio)], comparing the normal and HCC group, whereas the deglycopeptide had 93.3% sensitivity, 68.3% specificity, and an AUC of 0.859 [cutoff value: ≥0.02 (light/heavy ratio)]. In comparing the stage I HCC subgroup with the LC group, the nonglycopeptide had a sensitivity of 66.7%, specificity of 80.0%, and an AUC of 0.712 [cutoff value: ≥0.02 (light/heavy ratio)], whereas the deglycopeptide had a sensitivity of 96.7%, specificity of 80.0%, and an AUC of 0.918 [cutoff value: ≥0.02 (light/heavy ratio)]. These data demonstrate that the discriminatory power of the deglycopeptide is greater than that of the nonglycopeptide. We conclude that deglycopeptide can distinguish cancer status between normal subjects and HCC patients better than nonglycopeptide. PMID:25310463

  7. Expressed repeat elements improve RT-qPCR normalization across a wide range of zebrafish gene expression studies.

    PubMed

    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. 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

  9. 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.

  10. Diagnostic Real-Time PCR Assays for the Detection of Emetic Bacillus cereus Strains in Foods and Recent Food-Borne Outbreaks▿ †

    PubMed Central

    Fricker, Martina; Messelhäußer, Ute; Busch, Ulrich; Scherer, Siegfried; Ehling-Schulz, Monika

    2007-01-01

    Cereulide-producing Bacillus cereus can cause an emetic type of food-borne disease that mimics the symptoms provoked by Staphylococcus aureus. Based on the recently discovered genetic background for cereulide formation, a novel 5′ nuclease (TaqMan) real-time PCR assay was developed to provide a rapid and sensitive method for the specific detection of emetic B. cereus in food. The TaqMan assay includes an internal amplification control and primers and a probe designed to target a highly specific part of the cereulide synthetase genes. Additionally, a specific SYBR green I assay was developed and extended to create a duplex SYBR green I assay for the one-step identification and discrimination of the two emesis-causing food pathogens B. cereus and S. aureus. The inclusivity and exclusivity of the assay were assessed using a panel of 100 strains, including 23 emetic B. cereus and 14 S. aureus strains. Different methods for DNA isolation from artificially contaminated foods were evaluated, and established real-time assays were used to analyze two recent emetic food poisonings in southern Germany. One of the food-borne outbreaks included 17 children visiting a day care center who vomited after consuming a reheated rice dish, collapsed, and were hospitalized; the other case concerned a single food-poisoning incident occurring after consumption of cauliflower. Within 2 h, the etiological agent of these food poisonings was identified as emetic B. cereus by using the real-time PCR assay. PMID:17259359

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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.

  17. Multiplex real-time quantitative PCR, microscopy and rapid diagnostic immuno-chromatographic tests for the detection of Plasmodium spp: performance, limit of detection analysis and quality assurance

    PubMed Central

    2009-01-01

    Background Accurate laboratory diagnosis of malaria species in returning travelers is paramount in the treatment of this potentially fatal infectious disease. Materials and methods A total of 466 blood specimens from returning travelers to Africa, Asia, and South/Central America with suspected malaria infection were collected between 2007 and 2009 at the reference public health laboratory. These specimens were assessed by reference microscopy, multipex real-time quantitative polymerase chain reaction (QPCR), and two rapid diagnostic immuno-chromatographic tests (ICT) in a blinded manner. Key clinical laboratory parameters such as limit of detection (LOD) analysis on clinical specimens by parasite stage, inter-reader variability of ICTs, staffing implications, quality assurance and cost analysis were evaluated. Results QPCR is the most analytically sensitive method (sensitivity 99.41%), followed by CARESTART (sensitivity 88.24%), and BINAXNOW (sensitivity 86.47%) for the diagnosis of malaria in returning travelers when compared to reference microscopy. However, microscopy was unable to specifically identify Plasmodia spp. in 18 out of 170 positive samples by QPCR. Moreover, the 17 samples that were negative by microscopy and positive by QPCR were also positive by ICTs. Quality assurance was achieved for QPCR by exchanging a blinded proficiency panel with another reference laboratory. The Kappa value of inter-reader variability among three readers for BINAXNOW and CARESTART was calculated to be 0.872 and 0.898 respectively. Serial dilution studies demonstrated that the QPCR cycle threshold correlates linearly with parasitemia (R2 = 0.9746) in a clinically relevant dynamic range and retains a LOD of 11 rDNA copies/μl for P. falciparum, which was several log lower than reference microscopy and ICTs. LOD for QPCR is affected not only by parasitemia but the parasite stage distribution of each clinical specimen. QPCR was approximately 6-fold more costly than reference

  18. Ice-COLD-PCR enables rapid amplification and robust enrichment for low-abundance unknown DNA mutations.

    PubMed

    Milbury, Coren A; Li, Jin; Makrigiorgos, G Mike

    2011-01-01

    Identifying low-abundance mutations within wild-type DNA is important in several fields of medicine, including cancer, prenatal diagnosis and infectious diseases. However, utilizing the clinical and diagnostic potential of rare mutations is limited by sensitivity of the molecular techniques employed, especially when the type and position of mutations are unknown. We have developed a novel platform that incorporates a synthetic reference sequence within a polymerase chain reaction (PCR) reaction, designed to enhance amplification of unknown mutant sequences during COLD-PCR (CO-amplification at Lower Denaturation temperature). This new platform enables an Improved and Complete Enrichment (ice-COLD-PCR) for all mutation types and eliminates shortcomings of previous formats of COLD-PCR. We evaluated ice-COLD-PCR enrichment in regions of TP53 in serially diluted mutant and wild-type DNA mixtures. Conventional-PCR, COLD-PCR and ice-COLD-PCR amplicons were run in parallel and sequenced to determine final mutation abundance for a range of mutations representing all possible single base changes. Amplification by ice-COLD-PCR enriched all mutation types and allowed identification of mutation abundances down to 1%, and 0.1% by Sanger sequencing or pyrosequencing, respectively, surpassing the capabilities of other forms of PCR. Ice-COLD-PCR will help elucidate the clinical significance of low-abundance mutations and our understanding of cancer origin, evolution, recurrence-risk and treatment diagnostics. PMID:20937629

  19. 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)

  20. 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

  1. Alterations of Epigenetic Signatures in Hepatocyte Nuclear Factor 4α Deficient Mouse Liver Determined by Improved ChIP-qPCR and (h)MeDIP-qPCR Assays

    PubMed Central

    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. PMID:24427299

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

    SciTech Connect

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

    2008-02-01

    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.

  3. An improved colorimetric PCR-based method for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in feces.

    PubMed Central

    Britten, D; Wilson, S M; McNerney, R; Moody, A H; Chiodini, P L; Ackers, J P

    1997-01-01

    The epidemiological implications of the recent separation of "Entamoeba histolytica" into two separate species, pathogenic E. histolytica sensu stricto and commensal E. dispar, will not become apparent without methods of distinguishing between them which are applicable to large numbers of specimens. We have modified a PCR-based method to produce such a technique which may be completed in 1 day while still identifying 10(-1) E. histolytica and 1 to 10 E. dispar trophozoites per g of feces when present separately and 10 E. histolytica and 100 E. dispar trophozoites per g in the presence of 10(6) trophozoites per g of the other species. Applied to fecal specimens from 18 patients from which E. histolytica or E. dispar had been grown and identified to the species level by hexokinase isoenzyme analysis, the method in every case yielded the correct result. Positive and negative results are easily distinguished by eye, and we are now applying this technique to a large-scale epidemiological study of amebiasis in the eastern Mediterranean region. PMID:9114390

  4. 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.

  5. 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

  6. Improvements are needed in reporting of accuracy studies for diagnostic tests used for detection of finfish pathogens.

    PubMed

    Gardner, Ian A; Burnley, Timothy; Caraguel, Charles

    2014-12-01

    Indices of test accuracy, such as diagnostic sensitivity and specificity, are important considerations in test selection for a defined purpose (e.g., screening or confirmation) and affect the interpretation of test results. Many biomedical journals recommend that authors clearly and transparently report test accuracy studies following the Standards for Reporting of Diagnostic Accuracy (STARD) guidelines ( www.stard-statement.org ). This allows readers to evaluate overall study validity and assess potential bias in diagnostic sensitivity and specificity estimates. The purpose of the present study was to evaluate the reporting quality of studies evaluating test accuracy for finfish diseases using the 25 items in the STARD checklist. Based on a database search, 11 studies that included estimates of diagnostic accuracy were identified for independent evaluation by three reviewers. For each study, STARD checklist items were scored as "yes," "no," or "not applicable." Only 10 of the 25 items were consistently reported in most (≥80%) papers, and reporting of the other items was highly variable (mostly between 30% and 60%). Three items ("number, training, and expertise of readers and testers"; "time interval between index tests and reference standard"; and "handling of indeterminate results, missing data, and outliers of the index tests") were reported in less than 10% of papers. Two items ("time interval between index tests and reference standard" and "adverse effects from testing") were considered minimally relevant to fish health because test samples usually are collected postmortem. Modification of STARD to fit finfish studies should increase use by authors and thereby improve the overall reporting quality regardless of how the study was designed. Furthermore, the use of STARD may lead to the improved design of future studies. PMID:25252270

  7. 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

  8. Triplex real-time PCR--an improved method to detect a wide spectrum of mitochondrial DNA deletions in single cells.

    PubMed

    Rygiel, Karolina A; Grady, John P; Taylor, Robert W; Tuppen, Helen A L; Turnbull, Doug M

    2015-01-01

    Mitochondrial DNA (mtDNA) mutations are commonly found in the skeletal muscle of patients with mitochondrial disease, inflammatory myopathies and sarcopenia. The majority of these mutations are mtDNA deletions, which accumulate to high levels in individual muscle fibres causing a respiratory defect. Most mtDNA deletions are major arc deletions with breakpoints located between the origin of light strand (OL) and heavy strand (OH) replication within the major arc. However, under certain disease conditions, rarer, minor arc deletions are detected. Currently, there are few techniques which would allow the detection and quantification of both types of mtDNA deletions in single muscle fibres. We have designed a novel triplex real-time PCR assay which simultaneously amplifies the MT-ND4 gene in the major arc, the MT-ND1 gene in the minor arc, and the non-coding D-Loop region. We demonstrate that this assay is a highly sensitive and reliable tool for the detection and quantification of a broad range of major and minor arc mtDNA deletions with the potential to investigate the molecular pathogenesis in both research and diagnostic settings. PMID:25989140

  9. Improved Multiplex PCR Using Conserved and Species-Specific 16S rRNA Gene Primers for Simultaneous Detection of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis

    PubMed Central

    Tran, Simon Dangtuan; Rudney, Joel D.

    1999-01-01

    Among putative periodontal pathogens, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis are most convincingly implicated as etiological agents in periodontitis. Therefore, techniques for detection of those three species would be of value. We previously published a description of a multiplex PCR that detects A. actinomycetemcomitans and P. gingivalis. The present paper presents an improvement on that technique, which now allows more sensitive detection of all three periodontal pathogens. Sensitivity was determined by testing serial dilutions of A. actinomycetemcomitans, B. forsythus, and P. gingivalis cells. Primer specificity was tested against (i) all gene sequences from the GenBank-EMBL database, (ii) six A. actinomycetemcomitans, one B. forsythus, and four P. gingivalis strains, (iii) eight different species of oral bacteria, and (iv) supra- and subgingival plaque samples from 20 healthy subjects and subgingival plaque samples from 10 patients with periodontitis. The multiplex PCR had a detection limit of 10 A. actinomycetemcomitans, 10 P. gingivalis, and 100 B. forsythus cells. Specificity was confirmed by the fact that (i) none of our forward primers were homologous to the 16S rRNA genes of other oral species, (ii) amplicons of predicted size were detected for all A. actinomycetemcomitans, B. forsythus, and P. gingivalis strains tested, and (iii) no amplicons were detected for the eight other bacterial species. A. actinomycetemcomitans, B. forsythus, and P. gingivalis were detected in 6 of 20, 1 of 20, and 11 of 20 of supragingival plaque samples, respectively, and 4 of 20, 7 of 20, and 13 of 20 of subgingival plaque samples, respectively, from periodontally healthy subjects. Among patients with periodontitis, the organisms were detected in 7 of 10, 10 of 10, and 7 of 10 samples, respectively. The simultaneous detection of three periodontal pathogens is an advantage of this technique over conventional PCR assays. PMID

  10. 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

  11. 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

  12. 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).

  13. 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. PMID:25430338

  14. 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.

  15. A predictive nomogram improved diagnostic accuracy and interobserver agreement of perirectal lymph nodes metastases in rectal cancer

    PubMed Central

    Ding, Ying; Tu, Shanshan; Liu, Yi; Qian, Youcun; Xu, Linghui; Tong, Tong; Cai, Sanjun; Peng, Junjie

    2016-01-01

    Objective To develop a predictive nomogram to improve the diagnostic accuracy and interobserver agreement of pre-therapeutic lymph nodes metastases in patients with rectal cancer. Materials and Methods An institutional database of 411 patients with rectal cancer was used to develop a nomogram to predict perirectal lymph nodes metastases. Patients' clinicopathological and MRI-assessed imaging variables were included in the multivariate logistic regression analysis. The model was externally validated and the performance was assessed by area under curve (AUC) of the receiver operator characteristics (ROC) curves. The interobserver agreement was measured between two independent radiologists. Results The diagnostic accuracy of the conventional MRI-assessed cN stage was 68%; 14.2% of the patients were over-staged and 17.8% of the patients were under-staged. A total of 35.1% of the patients had disagreed diagnosis for the cN stage between the two radiologists, with a kappa value of 0.295. A nomogram for predicting pathological lymph nodes metastases was successfully developed, with an AUC of 0.78 on the training data and 0.71 on the validation data. The predictors included in the nomogram were MRI cT stage, CRM involvement, preoperative CEA, tumor grade and lymph node size category. This nomogram yielded improved prediction in cN stage than the conventional MRI-based assessment. Conclusions By incorporating clinicopathological and MRI imaging features, we established a nomogram that improved the diagnostic accuracy and remarkably minimized the interobserver disagreement in predicting lymph nodes metastases in rectal cancers. PMID:26910373

  16. Mismatch extension of DNA polymerases and high-accuracy single nucleotide polymorphism diagnostics by gold nanoparticle-improved isothermal amplification.

    PubMed

    Chen, Feng; Zhao, Yue; Fan, Chunhai; Zhao, Yongxi

    2015-09-01

    Sequence mismatches may induce nonspecific extension reaction, causing false results for SNP diagnostics. Herein, we systematically investigated the impact of various 3'-terminal mismatches on isothermal amplification catalyzed by representative DNA polymerases. Despite their diverse efficiencies depending on types of mismatch and kinds of DNA polymerase, all 12 kinds of single 3'-terminal mismatches induced the extension reaction. Generally, only several mismatches (primer-template, C-C, G-A, A-G, and A-A) present an observable inhibitory effect on the amplification reaction, whereas other mismatches trigger amplified signals as high as those of Watson-Crick pairs. The related mechanism was deeply discussed, and a primer-design guideline for specific SNP analysis was summarized. Furthermore, we found that the addition of appropriate gold nanoparticles (AuNPs) can significantly inhibit mismatch extension and enhance the amplification specificity. Also the high-accuracy SNP analysis of human blood genomic DNA has been demonstrated by AuNPs-improved isothermal amplification, the result of which was verified by sequencing (the gold standard method for SNP assay). Collectively, this work provides mechanistic insight into mismatch behavior and achieves accurate SNP diagnostics, holding great potential for the application in molecular diagnostics and personalized medicine. PMID:26249366

  17. 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

  18. Improved Method for the Detection and Quantification of Naegleria fowleri in Water and Sediment Using Immunomagnetic Separation and Real-Time PCR

    PubMed Central

    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

  19. 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.

  20. 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.

  1. An improved method for estimating the entrance exposure in diagnostic radiographic examinations.

    PubMed

    Zamenhof, R G; Shahabi, S; Morgan, H T

    1987-09-01

    There is currently a widespread consensus on the importance of monitoring patient radiation exposures during radiographic examinations. Diagnostic facilities under federal jurisdiction already legislate maximum patient exposure limits for various diagnostic radiologic examinations, while an increasing number of state legislatures have instituted such regulations. Compliance requires that institutions be capable of assessing each patient's entrance exposures. A method is proposed that would facilitate the acquisition of such patient exposure information in a relatively straightforward and accurate manner, requiring a minimum number of measurements and access to a suitable programmable calculator. A standardized set of exposure measurements obtained on an accurately calibrated three-phase radiographic unit has been fitted by an analytic function. The average accuracy of the fit between the limits of 40-140 kVp and 2.5- to 6.0-mm aluminum filtration was 0.3%. The concept of linear scaling was employed to allow the analytic function to accurately reproduce the exposure outputs of different radiographic units. Validation experiments on patients indicated that an overall accuracy of 10% can be expected when using well-calibrated radiographic equipment. The method described permits institutions to verify their compliance with federal and/or state regulations and to confirm that their radiation exposures are consistent with national averages. PMID:3497556

  2. 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.

  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. Improvements of data quality of the LHD Thomson scattering diagnostics in high-temperature plasma experiments

    SciTech Connect

    Yamada, I.; Narihara, K.; Funaba, H.; Hayashi, H.; Kohmoto, T.; Takahashi, H.; Shimozuma, T.; Kubo, S.; Yoshimura, Y.; Igami, H.; Tamura, N.

    2010-10-15

    In Large Helical Device (LHD) experiments, an electron temperature (T{sub e}) more than 15 keV has been observed by the yttrium-aluminum-garnet (YAG) laser Thomson scattering diagnostic. Since the LHD Thomson scattering system has been optimized for the temperature region, 50 eV{<=}T{sub e}{<=}10 keV, the data quality becomes worse in the higher T{sub e} region exceeding 10 keV. In order to accurately determine T{sub e} in the LHD high-T{sub e} experiments, we tried to increase the laser pulse energy by simultaneously firing three lasers. The technique enables us to decrease the uncertainties in the measured T{sub e}. Another signal accumulation method was also tested. In addition, we estimated the influence of high-energy electrons on T{sub e} obtained by the LHD Thomson scattering system.

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

    NASA Astrophysics Data System (ADS)

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

    2014-11-01

    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.

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

    PubMed

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

    2014-11-01

    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. PMID:25430347

  7. The well-being 5: development and validation of a diagnostic instrument to improve population well-being.

    PubMed

    Sears, Lindsay E; Agrawal, Sangeeta; Sidney, James A; Castle, Patricia H; Rula, Elizabeth Y; Coberley, Carter R; Witters, Dan; Pope, James E; Harter, James K

    2014-12-01

    Building upon extensive research from 2 validated well-being instruments, the objective of this research was to develop and validate a comprehensive and actionable well-being instrument that informs and facilitates improvement of well-being for individuals, communities, and nations. The goals of the measure were comprehensiveness, validity and reliability, significant relationships with health and performance outcomes, and diagnostic capability for intervention. For measure development and validation, questions from the Well-being Assessment and Wellbeing Finder were simultaneously administered as a test item pool to over 13,000 individuals across 3 independent samples. Exploratory factor analysis was conducted on a random selection from the first sample and confirmed in the other samples. Further evidence of validity was established through correlations to the established well-being scores from the Well-Being Assessment and Wellbeing Finder, and individual outcomes capturing health care utilization and productivity. Results showed the Well-Being 5 score comprehensively captures the known constructs within well-being, demonstrates good reliability and validity, significantly relates to health and performance outcomes, is diagnostic and informative for intervention, and can track and compare well-being over time and across groups. With this tool, well-being deficiencies within a population can be effectively identified, prioritized, and addressed, yielding the potential for substantial improvements to the health status, performance, and quality of life for individuals and cost savings for stakeholders. PMID:24892873

  8. The Well-Being 5: Development and Validation of a Diagnostic Instrument to Improve Population Well-being

    PubMed Central

    Sears, Lindsay E.; Agrawal, Sangeeta; Sidney, James A.; Castle, Patricia H.; Coberley, Carter R.; Witters, Dan; Pope, James E.; Harter, James K.

    2014-01-01

    Abstract Building upon extensive research from 2 validated well-being instruments, the objective of this research was to develop and validate a comprehensive and actionable well-being instrument that informs and facilitates improvement of well-being for individuals, communities, and nations. The goals of the measure were comprehensiveness, validity and reliability, significant relationships with health and performance outcomes, and diagnostic capability for intervention. For measure development and validation, questions from the Well-being Assessment and Wellbeing Finder were simultaneously administered as a test item pool to over 13,000 individuals across 3 independent samples. Exploratory factor analysis was conducted on a random selection from the first sample and confirmed in the other samples. Further evidence of validity was established through correlations to the established well-being scores from the Well-Being Assessment and Wellbeing Finder, and individual outcomes capturing health care utilization and productivity. Results showed the Well-Being 5 score comprehensively captures the known constructs within well-being, demonstrates good reliability and validity, significantly relates to health and performance outcomes, is diagnostic and informative for intervention, and can track and compare well-being over time and across groups. With this tool, well-being deficiencies within a population can be effectively identified, prioritized, and addressed, yielding the potential for substantial improvements to the health status, performance, and quality of life for individuals and cost savings for stakeholders. (Population Health Management 2014;17:357–365) PMID:24892873

  9. [Serological diagnosis of congenital infections and algorithms to improve diagnostic efficacy].

    PubMed

    García-Bermejo, Isabel; de Ory-Manchón, Fernando

    2015-07-01

    Congenital infection is those transmitted by the mother to the fetus before delivery. It can occur transplacentally or by direct contact with the pathogen during birth or in the immediate postnatal period. Congenital infection can be due to viruses (rubella, cytomegalovirus, herpes simplex, varicella-zoster, hepatitis B and C virus, human inunodeficiencia, erythrovirus B19) as bacteria (Treponema pallidum) and parasites (Toxoplasma gondii and Trypanosoma cruzi). Serological diagnosis of congenital infection is based on both the knowledge of infectious serology in the mother, including the systematic serological screening and diagnostic aspects of the determination of IgM and confirmatory methods, IgG avidity tests, establishment of antibody profiles, and in the diagnosis the neonate. Serological diagnosis of congenital infection in the newborn is mainly based on the detection of specific IgM usually by immunoenzymatic assays or immunochemiluminescence techniques. In some instances it is important to perform the serological follow up of the newborn to confirm the congenital infection. PMID:26320992

  10. 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.

  11. Use of Individual-level Covariates to Improve Latent Class Analysis of Trypanosoma Cruzi Diagnostic Tests

    PubMed Central

    Tustin, Aaron W.; Small, Dylan S.; Delgado, Stephen; Neyra, Ricardo Castillo; Verastegui, Manuela R.; Ancca Juárez, Jenny M.; Quispe Machaca, Víctor R.; Gilman, Robert H.; Bern, Caryn; Levy, Michael Z.

    2013-01-01

    Statistical methods such as latent class analysis can estimate the sensitivity and specificity of diagnostic tests when no perfect reference test exists. Traditional latent class methods assume a constant disease prevalence in one or more tested populations. When the risk of disease varies in a known way, these models fail to take advantage of additional information that can be obtained by measuring risk factors at the level of the individual. We show that by incorporating complex field-based epidemiologic data, in which the disease prevalence varies as a continuous function of individual-level covariates, our model produces more accurate sensitivity and specificity estimates than previous methods. We apply this technique to a simulated population and to actual Chagas disease test data from a community near Arequipa, Peru. Results from our model estimate that the first-line enzyme-linked immunosorbent assay has a sensitivity of 78% (95% CI: 62–100%) and a specificity of 100% (95% CI: 99–100%). The confirmatory immunofluorescence assay is estimated to be 73% sensitive (95% CI: 65–81%) and 99% specific (95% CI: 96–100%). PMID:24083130

  12. Improved allele-specific PCR assays for detection of clarithromycin and fluoroquinolone resistant of Helicobacter pylori in gastric biopsies: identification of N87I mutation in GyrA.

    PubMed

    Trespalacios, Alba A; Rimbara, Emiko; Otero, William; Reddy, Rita; Graham, David Y

    2015-04-01

    Molecular testing can rapidly detect Helicobacter pylori susceptibility using gastric biopsies. Allele-specific polymerase chain reaction (ASP-PCR) was used to identify H. pylori 23S rRNA and gyrA mutation using gastric biopsies from Colombian patients and confirmed by PCR and sequencing of the 23S rRNA and gyrA genes. The sensitivity and specificity of ASP-PCR were compared with susceptibilities measured by agar dilution. Samples included gastric biopsies from 107 biopsies with H. pylori infections and 20 H. pylori negative. The sensitivity and specificity of ASP-PCR for the 23S rRNA gene were both 100%. The sensitivity and specificity of ASP-PCR for the gyrA gene, published in 2007 by Nishizawa et al., were 52% and 92.7%, respectively; the lower sensitivity was due to the presence of mutation N87I in our samples, which were not detected by the test. In this study, we designed new primers to detect the mutation N87I in GyrA. The ASP-PCR was performed with the original primers plus the new primers. The molecular test with the new primers improved the sensitivity to 100%. In conclusion, ASP-PCR provides a specific and rapid means of predicting resistance to clarithromycin and levofloxacin in gastric biopsies. PMID:25600075

  13. A microwave holography methodology for diagnostics and performance improvement for large reflector antennas

    NASA Technical Reports Server (NTRS)

    Rochblatt, D. J.

    1992-01-01

    This microwave holography technique utilizes the Fourier transform relation between the complex far field radiation pattern of an antenna and the complex aperture field distribution. Resulting aperture phase and amplitude distribution data can be used to precisely characterize various crucial performance parameters, including panel alignment, panel shaping, subreflector position, antenna aperture illumination, directivity at various frequencies, and gravity deformation effects. The methodology of data processing presented here was successfully applied to the Deep Space Network (DSN) 34-m beam waveguide antennas. The antenna performance was improved at all operating frequencies by reducing the main reflector mechanical surface rms error to 0.43 mm. At Ka-band (32 GHz), the estimated improvement is 4.1 dB, resulting in an aperture efficiency of 52 percent. The performance improvement was verified by efficiency measurements and additional holographic measurements.

  14. Ultrahigh field MRI in clinical neuroimmunology: a potential contribution to improved diagnostics and personalised disease management.

    PubMed

    Sinnecker, Tim; Kuchling, Joseph; Dusek, Petr; Dörr, Jan; Niendorf, Thoralf; Paul, Friedemann; Wuerfel, Jens

    2015-01-01

    Conventional magnetic resonance imaging (MRI) at 1.5 Tesla (T) is limited by modest spatial resolution and signal-to-noise ratio (SNR), impeding the identification and classification of inflammatory central nervous system changes in current clinical practice. Gaining from enhanced susceptibility effects and improved SNR, ultrahigh field MRI at 7 T depicts inflammatory brain lesions in great detail. This review summarises recent reports on 7 T MRI in neuroinflammatory diseases and addresses the question as to whether ultrahigh field MRI may eventually improve clinical decision-making and personalised disease management. PMID:26312125

  15. 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

  16. Modeling Formamide Denaturation of Probe-Target Hybrids for Improved Microarray Probe Design in Microbial Diagnostics

    PubMed Central

    Yilmaz, L. Safak; Loy, Alexander; Wright, Erik S.; Wagner, Michael; Noguera, Daniel R.

    2012-01-01

    Application of high-density microarrays to the diagnostic analysis of microbial communities is challenged by the optimization of oligonucleotide probe sensitivity and specificity, as it is generally unfeasible to experimentally test thousands of probes. This study investigated the adjustment of hybridization stringency using formamide with the idea that sensitivity and specificity can be optimized during probe design if the hybridization efficiency of oligonucleotides with target and non-target molecules can be predicted as a function of formamide concentration. Sigmoidal denaturation profiles were obtained using fluorescently labeled and fragmented 16S rRNA gene amplicon of Escherichia coli as the target with increasing concentrations of formamide in the hybridization buffer. A linear free energy model (LFEM) was developed and microarray-specific nearest neighbor rules were derived. The model simulated formamide melting with a denaturant m-value that increased hybridization free energy (ΔG°) by 0.173 kcal/mol per percent of formamide added (v/v). Using the LFEM and specific probe sets, free energy rules were systematically established to predict the stability of single and double mismatches, including bulged and tandem mismatches. The absolute error in predicting the position of experimental denaturation profiles was less than 5% formamide for more than 90 percent of probes, enabling a practical level of accuracy in probe design. The potential of the modeling approach for probe design and optimization is demonstrated using a dataset including the 16S rRNA gene of Rhodobacter sphaeroides as an additional target molecule. The LFEM and thermodynamic databases were incorporated into a computational tool (ProbeMelt) that is freely available at http://DECIPHER.cee.wisc.edu. PMID:22952791

  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. 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

  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. 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. PMID:26305321

  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. 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

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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

  8. PCR performance of the B-type DNA polymerase from the thermophilic euryarchaeon Thermococcus aggregans improved by mutations in the Y-GG/A motif

    PubMed Central

    Böhlke, Kristina; Pisani, Francesca M.; Vorgias, Constantinos E.; Frey, Bruno; Sobek, Harald; Rossi, Mosè; Antranikian, Garabed

    2000-01-01

    The effect of mutations in the highly conserved Y-GG/A motif of B-type DNA polymerases was studied in the DNA polymerase from the hyperthermophilic euryarchaeon Thermococcus aggregans. This motif plays a critical role in the balance between the synthesis and degradation of the DNA chain. Five different mutations of the tyrosine at position 387 (Tyr387→Phe, Tyr387→Trp, Tyr387→His, Tyr387→Asn and Tyr387→Ser) revealed that an aromatic ring system is crucial for the synthetic activity of the enzyme. Amino acids at this position lacking the ring system (Ser and Asn) led to a significant decrease in polymerase activity and to enhanced exonuclease activity, which resulted in improved enzyme fidelity. Exchange of tyrosine to phenylalanine, tryptophan or histidine led to phenotypes with wild-type-like fidelity but enhanced PCR performance that could be related to a higher velocity of polymerisation. With the help of a modelled structure of T.aggregans DNA polymerase, the biochemical data were interpreted proposing that the conformation of the flexible loop containing the Y-GG/A motif is an important factor for the equilibrium between DNA polymerisation and exonucleolysis. PMID:11024170

  9. 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. PMID:26971150

  10. Use of real-time PCR on faecal samples for detection of sub-clinical Salmonella infection in cattle did not improve the detection sensitivity compared to conventional bacteriology.

    PubMed

    Jensen, A N; Nielsen, L R; Baggesen, D L

    2013-05-01

    There is a need for more sensitive detection methods to improve effectiveness of control programmes of Salmonella enterica subsp. enterica serotypes (Salmonella) in cattle. We assessed the performance of a rapid, molecular-based, real-time PCR (rt-PCR) method against the conventional bacteriological culture-reference method (BCRM) on cattle faecal samples for detection of sub-clinical Salmonella infections in cattle. Thirty faecal samples were artificially contaminated with either 10 or 50 CFU of one of five strains of S. Dublin (SD) and S. Typhimurium (ST). The overall detection sensitivity of both rt-PCR and BCRM was 100% for ST and 78% for SD. Furthermore, 163 faecal samples from cattle herds with suspected Salmonella infection were tested to compare the relative performance of rt-PCR to BCRM on samples from naturally infected herds. The relative sensitivity of rt-PCR was 20% (3/15 BCRM positive samples) while the relative specificity and accuracy was 99% and 92%, respectively. Both methods had limitations for detecting low levels of SD (<1 CFU/g). Hence, the evaluated rt-PCR method did not provide a sensitive alternative to the BCRM for detection of bacteria in faecal samples of sub-clinically, Salmonella-infectious cattle. PMID:23391438

  11. 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

  12. Computational evaluation of exome sequence data using human and model organism phenotypes improves diagnostic efficiency

    PubMed Central

    Bone, William P.; Washington, Nicole L.; Buske, Orion J.; Adams, David R.; Davis, Joie; Draper, David; Flynn, Elise D.; Girdea, Marta; Godfrey, Rena; Golas, Gretchen; Groden, Catherine; Jacobsen, Julius; Köhler, Sebastian; Lee, Elizabeth M. J.; Links, Amanda E.; Markello, Thomas C.; Mungall, Christopher J.; Nehrebecky, Michele; Robinson, Peter N.; Sincan, Murat; Soldatos, Ariane G.; Tifft, Cynthia J.; Toro, Camilo; Trang, Heather; Valkanas, Elise; Vasilevsky, Nicole; Wahl, Colleen; Wolfe, Lynne A.; Boerkoel, Cornelius F.; Brudno, Michael; Haendel, Melissa A.; Gahl, William A.; Smedley, Damian

    2016-01-01

    Purpose: Medical diagnosis and molecular or biochemical confirmation typically rely on the knowledge of the clinician. Although this is very difficult in extremely rare diseases, we hypothesized that the recording of patient phenotypes in Human Phenotype Ontology (HPO) terms and computationally ranking putative disease-associated sequence variants improves diagnosis, particularly for patients with atypical clinical profiles. Genet Med 18 6, 608–617. Methods: Using simulated exomes and the National Institutes of Health Undiagnosed Diseases Program (UDP) patient cohort and associated exome sequence, we tested our hypothesis using Exomiser. Exomiser ranks candidate variants based on patient phenotype similarity to (i) known disease–gene phenotypes, (ii) model organism phenotypes of candidate orthologs, and (iii) phenotypes of protein–protein association neighbors. Genet Med 18 6, 608–617. Results: Benchmarking showed Exomiser ranked the causal variant as the top hit in 97% of known disease–gene associations and ranked the correct seeded variant in up to 87% when detectable disease–gene associations were unavailable. Using UDP data, Exomiser ranked the causative variant(s) within the top 10 variants for 11 previously diagnosed variants and achieved a diagnosis for 4 of 23 cases undiagnosed by clinical evaluation. Genet Med 18 6, 608–617. Conclusion: Structured phenotyping of patients and computational analysis are effective adjuncts for diagnosing patients with genetic disorders. Genet Med 18 6, 608–617. PMID:26562225

  13. 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

  14. The use of quantitative PCR for identification and quantification of Brachyspira pilosicoli, Lawsonia intracellularis and Escherichia coli fimbrial types F4 and F18 in pig feces.

    PubMed

    Ståhl, M; Kokotovic, B; Hjulsager, C K; Breum, S Ø; Angen, Ø

    2011-08-01

    Four quantitative PCR (qPCR) assays were evaluated for quantitative detection of Brachyspira pilosicoli, Lawsonia intracellularis, and E. coli fimbrial types F4 and F18 in pig feces. Standard curves were based on feces spiked with the respective reference strains. The detection limits from the spiking experiments were 10(2) bacteria/g feces for Bpilo-qPCR and Laws-qPCR, 10(3)CFU/g feces for F4-qPCR and F18-qPCR. The PCR efficiency for all four qPCR assays was between 0.91 and 1.01 with R(2) above 0.993. Standard curves, slopes and elevation, varied between assays and between measurements from pure DNA from reference strains and feces spiked with the respective strains. The linear ranges found for spiked fecal samples differed both from the linear ranges from pure culture of the reference strains and between the qPCR tests. The linear ranges were five log units for F4-qPCR, and Laws-qPCR, six log units for F18-qPCR and three log units for Bpilo-qPCR in spiked feces. When measured on pure DNA from the reference strains used in spiking experiments, the respective log ranges were: seven units for Bpilo-qPCR, Laws-qPCR and F18-qPCR and six log units for F4-qPCR. This shows the importance of using specific standard curves, where each pathogen is analysed in the same matrix as sample DNA. The qPCRs were compared to traditional bacteriological diagnostic methods and found to be more sensitive than cultivation for E. coli and B. pilosicoli. The qPCR assay for Lawsonia was also more sensitive than the earlier used method due to improvements in DNA extraction. In addition, as samples were not analysed for all four pathogen agents by traditional diagnostic methods, many samples were found positive for agents that were not expected on the basis of age and case history. The use of quantitative PCR tests for diagnosis of enteric diseases provides new possibilities for veterinary diagnostics. The parallel simultaneous analysis for several bacteria in multi-qPCR and the

  15. 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

  16. Strengths and limitations of microarray-based phenotype prediction: lessons learned from the IMPROVER Diagnostic Signature Challenge

    PubMed Central

    Tarca, Adi L.; Lauria, Mario; Unger, Michael; Bilal, Erhan; Boue, Stephanie; Kumar Dey, Kushal; Hoeng, Julia; Koeppl, Heinz; Martin, Florian; Meyer, Pablo; Nandy, Preetam; Norel, Raquel; Peitsch, Manuel; Rice, Jeremy J.; Romero, Roberto; Stolovitzky, Gustavo; Talikka, Marja; Xiang, Yang; Zechner, Christoph

    2013-01-01

    Motivation: After more than a decade since microarrays were used to predict phenotype of biological samples, real-life applications for disease screening and identification of patients who would best benefit from treatment are still emerging. The interest of the scientific community in identifying best approaches to develop such prediction models was reaffirmed in a competition style international collaboration called IMPROVER Diagnostic Signature Challenge whose results we describe herein. Results: Fifty-four teams used public data to develop prediction models in four disease areas including multiple sclerosis, lung cancer, psoriasis and chronic obstructive pulmonary disease, and made predictions on blinded new data that we generated. Teams were scored using three metrics that captured various aspects of the quality of predictions, and best performers were awarded. This article presents the challenge results and introduces to the community the approaches of the best overall three performers, as well as an R package that implements the approach of the best overall team. The analyses of model performance data submitted in the challenge as well as additional simulations that we have performed revealed that (i) the quality of predictions depends more on the disease endpoint than on the particular approaches used in the challenge; (ii) the most important modeling factor (e.g. data preprocessing, feature selection and classifier type) is problem dependent; and (iii) for optimal results datasets and methods have to be carefully matched. Biomedical factors such as the disease severity and confidence in diagnostic were found to be associated with the misclassification rates across the different teams. Availability: The lung cancer dataset is available from Gene Expression Omnibus (accession, GSE43580). The maPredictDSC R package implementing the approach of the best overall team is available at www.bioconductor.org or http://bioinformaticsprb.med.wayne.edu/. Contact

  17. 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. PMID:25835783

  18. 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. PMID:26412381

  19. Detection of Rare Drug Resistance Mutations by Digital PCR in a Human Influenza A Virus Model System and Clinical Samples

    PubMed Central

    Bushell, Claire A.; Grant, Paul R.; Cowen, Simon; Gutierrez-Aguirre, Ion; O'Sullivan, Denise M.; Žel, Jana; Milavec, Mojca; Foy, Carole A.; Nastouli, Eleni; Garson, Jeremy A.; Huggett, Jim F.

    2015-01-01

    Digital PCR (dPCR) is being increasingly used for the quantification of sequence variations, including single nucleotide polymorphisms (SNPs), due to its high accuracy and precision in comparison with techniques such as quantitative PCR (qPCR) and melt curve analysis. To develop and evaluate dPCR for SNP detection using DNA, RNA, and clinical samples, an influenza virus model of resistance to oseltamivir (Tamiflu) was used. First, this study was able to recognize and reduce off-target amplification in dPCR quantification, thereby enabling technical sensitivities down to 0.1% SNP abundance at a range of template concentrations, a 50-fold improvement on the qPCR assay used routinely in the clinic. Second, a method was developed for determining the false-positive rate (background) signal. Finally, comparison of dPCR with qPCR results on clinical samples demonstrated the potential impact dPCR could have on clinical research and patient management by earlier (trace) detection of rare drug-resistant sequence variants. Ultimately this could reduce the quantity of ineffective drugs taken and facilitate early switching to alternative medication when available. In the short term such methods could advance our understanding of microbial dynamics and therapeutic responses in a range of infectious diseases such as HIV, viral hepatitis, and tuberculosis. Furthermore, the findings presented here are directly relevant to other diagnostic areas, such as the detection of rare SNPs in malignancy, monitoring of graft rejection, and fetal screening. PMID:26659206

  20. Detection of Rare Drug Resistance Mutations by Digital PCR in a Human Influenza A Virus Model System and Clinical Samples.

    PubMed

    Whale, Alexandra S; Bushell, Claire A; Grant, Paul R; Cowen, Simon; Gutierrez-Aguirre, Ion; O'Sullivan, Denise M; Žel, Jana; Milavec, Mojca; Foy, Carole A; Nastouli, Eleni; Garson, Jeremy A; Huggett, Jim F

    2016-02-01

    Digital PCR (dPCR) is being increasingly used for the quantification of sequence variations, including single nucleotide polymorphisms (SNPs), due to its high accuracy and precision in comparison with techniques such as quantitative PCR (qPCR) and melt curve analysis. To develop and evaluate dPCR for SNP detection using DNA, RNA, and clinical samples, an influenza virus model of resistance to oseltamivir (Tamiflu) was used. First, this study was able to recognize and reduce off-target amplification in dPCR quantification, thereby enabling technical sensitivities down to 0.1% SNP abundance at a range of template concentrations, a 50-fold improvement on the qPCR assay used routinely in the clinic. Second, a method was developed for determining the false-positive rate (background) signal. Finally, comparison of dPCR with qPCR results on clinical samples demonstrated the potential impact dPCR could have on clinical research and patient management by earlier (trace) detection of rare drug-resistant sequence variants. Ultimately this could reduce the quantity of ineffective drugs taken and facilitate early switching to alternative medication when available. In the short term such methods could advance our understanding of microbial dynamics and therapeutic responses in a range of infectious diseases such as HIV, viral hepatitis, and tuberculosis. Furthermore, the findings presented here are directly relevant to other diagnostic areas, such as the detection of rare SNPs in malignancy, monitoring of graft rejection, and fetal screening. PMID:26659206

  1. 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

  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. 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

  4. 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

  5. An Improved PCR-RFLP Assay for Detection and Genotyping of Asymptomatic Giardia lamblia Infection in a Resource-Poor Setting

    PubMed Central

    Hawash, Yoursry; Ghonaim, M. M.; Al-Shehri, S. S.

    2016-01-01

    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. PMID:26951972

  6. 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. PMID:26951972

  7. Effects of a Multidisciplinary Approach to Improve Volume of Diagnostic Material in CT-Guided Lung Biopsies

    PubMed Central

    Ferguson, Philip E.; Sales, Catherine M.; Hodges, Dalton C.; Sales, Elizabeth W.

    2015-01-01

    Background Recent publications have emphasized the importance of a multidisciplinary strategy for maximum conservation and utilization of lung biopsy material for advanced testing, which may determine therapy. This paper quantifies the effect of a multidisciplinary strategy implemented to optimize and increase tissue volume in CT-guided transthoracic needle core lung biopsies. The strategy was three-pronged: (1) once there was confidence diagnostic tissue had been obtained and if safe for the patient, additional biopsy passes were performed to further increase volume of biopsy material, (2) biopsy material was placed in multiple cassettes for processing, and (3) all tissue ribbons were conserved when cutting blocks in the histology laboratory. This study quantifies the effects of strategies #1 and #2. Design This retrospective analysis comparing CT-guided lung biopsies from 2007 and 2012 (before and after multidisciplinary approach implementation) was performed at a single institution. Patient medical records were reviewed and main variables analyzed include biopsy sample size, radiologist, number of blocks submitted, diagnosis, and complications. The biopsy sample size measured was considered to be directly proportional to tissue volume in the block. Results Biopsy sample size increased 2.5 fold with the average total biopsy sample size increasing from 1.0 cm (0.9–1.1 cm) in 2007 to 2.5 cm (2.3–2.8 cm) in 2012 (P<0.0001). The improvement was statistically significant for each individual radiologist. During the same time, the rate of pneumothorax requiring chest tube placement decreased from 15% to 7% (P = 0.065). No other major complications were identified. The proportion of tumor within the biopsy material was similar at 28% (23%–33%) and 35% (30%–40%) for 2007 and 2012, respectively. The number of cases with at least two blocks available for testing increased from 10.7% to 96.4% (P<0.0001). Conclusions The effect of this multidisciplinary strategy to CT

  8. 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

  9. 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 Central

    Evans, Jonathan; Swart, Marelize; Soko, Nyarai; Wonkam, Ambroise; Huzair, Farah

    2015-01-01

    Abstract 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

  10. 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…

  11. 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

  12. An Improved Real-Time PCR System for Broad-Spectrum Detection of Didymella bryoniae, the Causal Agent of Gummy Stem Blight of Cucurbits

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gummy stem blight (GSB) is a major disease of cucurbit crops (e.g., cantaloupe, cucumber and watermelon). Didymella bryoniae (anamorph Phoma cucurbitacearum), the causal agent of GSB, is often co-isolated from infected tissues with other Phoma spp. RAPD profiles and PCR primers have been used to de...

  13. 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

  14. 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

  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. 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.

  18. 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

  19. The Features of GGT in Patients with ATP8B1 or ABCB11 Deficiency Improve the Diagnostic Efficiency

    PubMed Central

    Wang, Neng-Li; Li, Li-Ting; Wu, Bing-Bing; Gong, Jing-Yu; Abuduxikuer, Kuerbanjiang; Li, Gang; Wang, Jian-She

    2016-01-01

    Background and Aims Genetic defects in ATP8B1 or ABCB11 account for the majority of cholestasis with low GGT. But the ranges for GGT in patients with ATP8B1 or ABCB11 deficiency are unclear. This study tried to unravel the features of GGT in these patients that improve diagnostic efficiency. Methods This study enrolled 207 patients with chronic cholestasis who were ordered to test for ATP8B1 and/or ABCB11 from January 2012 to December 2015. Additional 17 patients with ATPB81 or ABCB11 deficiency diagnosed between January 2004 and December 2011 were also enrolled in this study. 600 population-matched children served as controls. Clinical data were obtained by retrospectively reviewing medical records. Results A total of 26 patients were diagnosed with ATP8B1 deficiency and 30 patients were diagnosed with ABCB11 deficiency. GGT levels were similar between the two disorders at any observed month of age, but varied with age. The peak GGT value was <70U/L in the 2nd~6th month of life, <60U/L in the 7th~12th month and <50U/L beyond one year. GGT levels in patients with a genetic diagnosis were different from that in patients without a genetic diagnosis and controls. Larger ranges for GGT were found in patients without a genetic diagnosis. Some controls had GGT≥70U/L in the 2nd~6th month. Of the 207 patients, 39 (18.8%) obtained a genetic diagnosis. 111 patients met the ranges described above, including all the 39 patients with ATP8B1 or ABCB11 deficiency. The sensitivity was 100.0%. The rate of a positive molecular diagnosis increased to 35.1% (39/111 vs. 39/207, X2 = 10.363, P = 0.001). The remaining 96 patients exceeded the ranges described above and failed to receive a genetic diagnosis. These patients accounted for 43.8% of sequencing cost. Conclusions GGT levels in patients with ATP8B1 or ABCB11 deficiency varied with age. The peak GGT value was <70U/L in the 2nd~6th month of life, <60U/L in the 7th~12th month and <50U/L beyond one year. PMID:27050426

  20. SveDem, the Swedish Dementia Registry – A Tool for Improving the Quality of Diagnostics, Treatment and Care of Dementia Patients in Clinical Practice

    PubMed Central

    Religa, Dorota; Fereshtehnejad, Seyed-Mohammad; Cermakova, Pavla; Edlund, Ann-Katrin; Garcia-Ptacek, Sara; Granqvist, Nicklas; Hallbäck, Anne; Kåwe, Kerstin; Farahmand, Bahman; Kilander, Lena; Mattsson, Ulla-Britt; Nägga, Katarina; Nordström, Peter; Wijk, Helle; Wimo, Anders; Winblad, Bengt; Eriksdotter, Maria

    2015-01-01

    Background The Swedish Dementia Registry (SveDem) was developed with the aim to improve the quality of diagnostic work-up, treatment and care of patients with dementia disorders in Sweden. Methods SveDem is an internet based quality registry where several indicators can be followed over time. It includes information about the diagnostic work-up, medical treatment and community support (www.svedem.se). The patients are diagnosed and followed-up yearly in specialist units, primary care centres or in nursing homes. Results The database was initiated in May 2007 and covers almost all of Sweden. There were 28 722 patients registered with a mean age of 79.3 years during 2007–2012. Each participating unit obtains continuous online statistics from its own registrations and they can be compared with regional and national data. A report from SveDem is published yearly to inform medical and care professionals as well as political and administrative decision-makers about the current quality of diagnostics, treatment and care of patients with dementia disorders in Sweden. Conclusion SveDem provides knowledge about current dementia care in Sweden and serves as a framework for ensuring the quality of diagnostics, treatment and care across the country. It also reflects changes in quality dementia care over time. Data from SveDem can be used to further develop the national guidelines for dementia and to generate new research hypotheses. PMID:25695768

  1. 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.

  2. 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. PMID:26167220

  3. Autism according to diagnostic and statistical manual of mental disorders 5th edition: The need for further improvements

    PubMed Central

    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. PMID:26167220

  4. Evaluation of a commercial PCR test for the diagnosis of dermatophyte nail infections.

    PubMed

    Spiliopoulou, Anastasia; Bartzavali, Christina; Jelastopulu, Eleni; Anastassiou, Evangelos D; Christofidou, Myrto

    2015-01-01

    Tinea unguium, known as onychomycosis, is a dermatophyte infection of nails with worldwide distribution. Conventional methods for detecting fungi in nail specimens are either non-specific (microscopy) or insensitive (culture). PCR has been used to improve sensitivity in detecting the causative fungi in nail specimens from patients with suspected onychomycosis. Results of a commercial multiplex PCR for the detection of dermatophytes, especially Trichophyton rubrum (the main dermatophyte implicated), as compared to conventional methods are presented. A total of 418 nail scrapings obtained from dermatological outpatients were handled in the Laboratory of Microbiology between May 2010 and May 2013. Among them, multiplex PCR detected 126 (30.1 %) dermatophyte-positive samples, whereas culture revealed 44 (10.5 %). Direct microscopy revealed 63 (15.1 %) positive specimens. T. rubrum was identified in 116 out of 126 (92 %) positive PCR samples and 40 out of 44 (91 %) dermatophyte-positive cultures. Implementation of PCR increased species-specific detection of dermatophytes by 21.1 %, leading to a threefold increase as compared to culture alone. Multiplex PCR offers a time-saving diagnostic tool for tinea unguium and augments laboratory assistance to clinical evaluation for proper treatment. PMID:25418736

  5. 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 ...

  6. Advances in molecular diagnostics for Mycobacterium bovis.

    PubMed

    Collins, Desmond M

    2011-07-01

    The two most important molecular diagnostic techniques for bovine tuberculosis are the polymerase chain reaction (PCR) because of its rapid determination of infection, and DNA strain typing because of its ability to answer important epidemiological questions. PCR tests for Mycobacterium bovis have been improved through recent advances in PCR technology, but still lack the sensitivity of good culture methods, and in some situations are susceptible to giving both false negative and false positive results. Therefore, PCR does not usually replace the need for culture, but is used to provide fast preliminary results. DNA typing of M. bovis isolates by restriction endonuclease analysis (REA) was developed 25 years ago in New Zealand, and remains an important tool in the New Zealand control scheme, where the typing results are combined with other information to determine large and expensive possum poisoning operations. A range of other DNA typing systems developed for M. bovis in the 1990 s have assisted epidemiological investigations in some countries but are now less commonly used. Variable number tandem repeat (VNTR) typing and spoligotyping, either alone or together, have now become the preferred approaches as they are robust and amenable to electronic analysis and comparison. Spoligotyping gives only moderate discrimination but can be easily applied to large numbers of isolates, and VNTR typing provides better discrimination than all other methods except for REA. While the current typing techniques are sufficient for most epidemiological purposes, more discriminating methods are likely to become available in the near future. PMID:21420257

  7. 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

  8. Induced sputum deposition improves diagnostic yields of pulmonary alveolar proteinosis: A clinicopathological and methodological study of 17 cases.

    PubMed

    Huang, Ziling; Yi, Xianghua; Luo, Benfang; Zhu, Jian; Wu, Yunjin; Jiang, Wenxia; Chu, Haiqing; Yang, Zhongmin; Li, Shuai; Zhu, Hailong; Zhang, Suxia; Zhang, Lanjing; Zeng, Yu

    2016-01-01

    Pulmonary alveolar proteinosis (PAP) is a rare diffuse lung disease characterized by the accumulation of intra-alveolar lipoprotein-like surfactants. Lung core biopsy and bronchoalveolar lavage (BAL) fluid are currently the two major sources of sampling for diagnosis. In the present study, we assessed the value of induced sputum in diagnosing PAP by transmission electron microscopy and examined the PAP 2-year death rate in Asians. Transmission electron microscopy was performed on the samples from 17 patients with PAP, 13 patients with inflammatory lung diseases, and 13 healthy adults. The PAP patients were followed up for 3-156 months, and inflammatory lung diseases patients or healthy adults for 12-36 months. The ultrastructural features including diagnostic lamellar bodies of induced sputum deposition (ISD) samples were similar to that of the BAL fluid sediment. However, the rates of lamellar bodies were 73.7% in the ISD group, significantly higher than the spontaneous sputum deposition (SSD) group (42.1%, P < .0487) and similar to the BAL sediment (76.2%) and the lung biopsy (54.5%) groups. The overall 2-year death rate of our PAP patients was 17.6% (3/17), not statistically different from the healthy adults and patients with inflammatory diseases (0/13, P = .237 for both). ISD may be the preferred non-invasive sampling method for diagnosing PAP by electronic microscopy because of the higher diagnostic yield than SSD. The diagnostic yields of this noninvasive method were similar to that of lung core biopsy and BAL. PMID:26583435

  9. 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

  10. 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. PMID:25173868

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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 Central

    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

    2015-01-01

    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. PMID:26085614

  16. Oxygen-atom concentrations measured in flames: a method to improve the accuracy of laser-induced fluorescence diagnostics.

    PubMed

    Myhr, F H; Driscoll, J F

    2001-10-20

    A procedure is proposed, denoted as the corrected laser-induced fluorescence (LIF) method, that reduces the error associated with the unavoidable photodissociation of O(2) molecules that has limited the measurement of oxygen-atom concentrations in the past. Two different laser intensities are employed, and the two signals that are obtained with two-photon LIF diagnostics are used to correct for the photolysis error. We measured oxygen-atom concentrations using this method at 33 locations in lean and rich flames. Results are compared with values determined by use of two independent techniques: the partial equilibrium method and equilibrium calculations. The measurements also quantify the shot noise, the photolysis errors, and the critical laser intensity required to avoid photolysis errors. PMID:18364818

  17. Improving patient flow and timeliness in the diagnosis and management of breast abnormalities: the impact of a rapid diagnostic unit

    PubMed Central

    Racz, J.M.; Holloway, C.M.B.; Huang, W.; Hong, N.J. Look

    2016-01-01

    Background Efforts to streamline the diagnosis and treatment of breast abnormalities are necessary to limit patient anxiety and expedite care. In the present study, we examined the effect of a rapid diagnostic unit (rdu) on wait times to clinical investigations and definitive treatment. Methods A retrospective before–after series, each considering a 1-year period, examined consecutive patients with suspicious breast lesions before and after initiation of the rdu. Patient consultations, clinical investigations, and lesion characteristics were captured from time of patient referral to initiation of definitive treatment. Outcomes included time (days) to clinical investigations, to delivery of diagnosis, and to management. Groups were compared using the Fisher exact test or Student t-test. Results The non-rdu group included 287 patients with 164 invasive breast carcinomas. The rdu group included 260 patients with 154 invasive carcinomas. The rdu patients had more single visits for biopsy (92% rdu vs. 78% non-rdu, p < 0.0001). The rdu group also had a significantly shorter wait time from initial consultation to delivery of diagnosis (mean: 2.1 days vs. 16.7 days, p = 0.0001) and a greater chance of receiving neoadjuvant chemotherapy (37% vs. 24%, p = 0.0106). Overall time from referral to management remained statistically unchanged (mean: 53 days with the rdu vs. 50 days without the rdu, p = 0.3806). Conclusions Introduction of a rdu appears to reduce wait times to definitive diagnosis, but not to treatment initiation, suggesting that obstacles to care delivery can occur at several points along the diagnostic trajectory. Multipronged efforts to reduce system-related delays to definitive treatment are needed. PMID:27330363

  18. 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

  19. 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.

  20. 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. PMID:27393622

  1. 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. PMID:25673634

  2. [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

  3. Intestinal parasitic infections in an industrialized country; a new focus on children with better DNA-based diagnostics.

    PubMed

    Verweij, Jaco J; van Lieshout, Lisette

    2011-10-01

    In recent years, the isolation of parasitic DNA from faecal samples and PCR techniques, have been improved and simplified. Moreover, the introduction of real-time PCR has made it possible to multiplex different targets into one reaction. These new technical possibilities make it feasible to introduce PCR with its unsurpassed sensitivity and specificity in a routine laboratory setting for the diagnosis of intestinal parasites. Detection rates of the parasitic infections included in the PCR are increased significantly compared with microscopy. Molecular diagnostics, especially in children, reveal a possible cause of the gastrointestinal complaints in many more cases compared with conventional methods. Usually in GP patients no other pathogenic parasites are detected using microscopy and in the returning travellers additional parasites are found with microscopy in a minority of cases only. Multiplex real-time PCR offers a highly sensitive and specific diagnostic alternative for labour intensive microscopy in clinical laboratory practice. Additional diagnostic methods for the detection of parasitic infections that are not included as PCR target can be limited to a selected group of patients. PMID:21859503

  4. 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...

  5. 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.

  6. Underdiagnosing of Mycoplasma pneumoniae infections as revealed by use of a respiratory multiplex PCR panel.

    PubMed

    Dalpke, Alexander; Zimmermann, Stefan; Schnitzler, Paul

    2016-09-01

    We compared a multiplex PCR diagnostic approach against specific PCR diagnosis for detection of Mycoplasma pneumoniae infection. Seventy-five percent of all M. pneumoniae infections were only detected "unintentionally" by the use of the multiplex PCR indicating underdiagnosing of M. pneumoniae due to absence of clinical suspicion. PMID:27377674

  7. 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. PMID:26911230

  8. 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

  9. 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

  10. 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…

  11. Integrated diagnostics

    NASA Technical Reports Server (NTRS)

    Hunthausen, Roger J.

    1988-01-01

    Recently completed projects in which advanced diagnostic concepts were explored and/or demonstrated are summarized. The projects begin with the design of integrated diagnostics for the Army's new gas turbine engines, and advance to the application of integrated diagnostics to other aircraft subsystems. Finally, a recent project is discussed which ties together subsystem fault monitoring and diagnostics with a more complete picture of flight domain knowledge.

  12. 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

  13. 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

  14. Surveillance for Malaria Elimination in Swaziland: A National Cross-Sectional Study Using Pooled PCR and Serology

    PubMed Central

    Hsiang, Michelle S.; Hwang, Jimee; Kunene, Simon; Drakeley, Chris; Kandula, Deepika; Novotny, Joseph; Parizo, Justin; Jensen, Trevor; Tong, Marcus; Kemere, Jordan; Dlamini, Sabelo; Moonen, Bruno; Angov, Evelina; Dutta, Sheetij; Ockenhouse, Christian; Dorsey, Grant; Greenhouse, Bryan

    2012-01-01

    Background To guide malaria elimination efforts in Swaziland and other countries, accurate assessments of transmission are critical. Pooled-PCR has potential to efficiently improve sensitivity to detect infections; serology may clarify temporal and spatial trends in exposure. Methodology/Principal Findings Using a stratified two-stage cluster, cross-sectional design, subjects were recruited from the malaria endemic region of Swaziland. Blood was collected for rapid diagnostic testing (RDT), pooled PCR, and ELISA detecting antibodies to Plasmodium falciparum surface antigens. Of 4330 participants tested, three were RDT-positive yet false positives by PCR. Pooled PCR led to the identification of one P. falciparum and one P. malariae infection among RDT-negative participants. The P. falciparum-infected participant reported recent travel to Mozambique. Compared to performing individual testing on thousands of samples, PCR pooling reduced labor and consumable costs by 95.5%. Seropositivity was associated with age ≥20 years (11·7% vs 1·9%, P<0.001), recent travel to Mozambique (OR 4.4 [95% CI 1.0–19.0]) and residence in southeast Swaziland (RR 3.78, P<0.001). Conclusions The prevalence of malaria infection and recent exposure in Swaziland are extremely low, suggesting elimination is feasible. Future efforts should address imported malaria and target remaining foci of transmission. Pooled PCR and ELISA are valuable surveillance tools for guiding elimination efforts. PMID:22238621

  15. 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

  16. 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

  17. Sonication cultures of explanted components as an add-on test to routinely conducted microbiological diagnostics improve pathogen detection.

    PubMed

    Holinka, Johannes; Bauer, Leonhard; Hirschl, Alexander M; Graninger, Wolfgang; Windhager, Reinhard; Presterl, Elisabeth

    2011-04-01

    The purpose of this study was to improve the pathogen detection in prosthetic joint infections, particularly to evaluate the feasibility of the sonication culture method in the clinical routine. Explanted components of all patients with presumptive prosthetic or implant infection were sonicated separately in sterile containers to dislodge the adherent bacteria from the surfaces and cultured. The results of sonication culture were compared to the conventional tissue culture. We investigated 60 consecutive patients with loosening of the prostheses or implants Forty patients had septic and 20 aseptic loosening (24 knee prostheses, 21 hip prostheses, 6 mega-prostheses, 2 shoulder prostheses, 6 osteosynthesis, 1 spinal instrumentation). The sensitivity of sonication fluid culture was 83.3%, of single positive tissue culture was 72.2% and 61.1% when two or more cultures yielded the same microorganism. In patients receiving antibiotic therapy the sensitivity was 65.9%, 57.5%, and 42.5%, respectively. Pathogens detected in a single tissue culture as well as in sonication culture yielded a significantly higher rate of prosthetic infection than conventional tissue culture alone (p = 0.008), even in patients receiving continuous antibiotic therapy before explantation (p = 0.016). The sonication method represents an essential add-on in pathogen detection compared to conventional tissue culture. PMID:21337398

  18. Improving on-wafer CD correlation analysis using advanced diagnostics and across-wafer light-source monitoring

    NASA Astrophysics Data System (ADS)

    Alagna, Paolo; Zurita, Omar; Rechtsteiner, Gregory; Lalovic, Ivan; Bekaert, Joost

    2014-04-01

    With the implementation of multi-patterning ArF-immersion for sub 20nm integrated circuits (IC), advances in equipment monitoring and control are needed to support on-wafer yield performance. These in-situ equipment monitoring improvements, along with advanced litho-cell corrections based on on-wafer measurements, enable meeting stringent overlay and CD control requirements for advanced lithography patterning. The importance of light-source performance on lithography pattering (CD and overlay) has been discussed in previous publications.[1-3] Recent developments of Cymer ArF light-source metrology and on-board monitoring enable end-users to detect, for each exposed wafer, changes in the near-field and far-field spatial profiles and polarization performance, [4-6] in addition to the key `optical' scalar parameters, such as bandwidth, wavelength and energy. The major advantage of this capability is that the key performance metrics are sampled at rates matched to wafer performance, e.g. every exposure field across the wafer, which is critical for direct correlation with on-wafer performance for process control and excursion detection.

  19. Inclusion of Neuropsychological Scores in Atrophy Models Improves Diagnostic Classification of Alzheimer's Disease and Mild Cognitive Impairment

    PubMed Central

    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. PMID:26101520

  20. A flu optical immunoassay (ThermoBioStar's FLU OIA): a diagnostic tool for improved influenza management.

    PubMed Central

    Tucker, S P; Cox, C; Steaffens, J

    2001-01-01

    ThermoBioStar's and Biota's flu optical immunoassay (FLU OIA) is a rapid test designed to diagnose influenza A and B infection using a variety of specimen types. The assay uses highly sensitive thin-film detection methods, coupled with specific monoclonal antibodies to the nucleoprotein. The test is simple to perform, requires no instrumentation and is intended to provide a result within 15 min of test initiation in the 'point-of-care' environment. In initial clinical studies, the assay was demonstrated to be equivalent to culture in identifying infected individuals. Subsequent independent studies using a variety of sample types have demonstrated sensitivity ranging from 48 to 100% and specificities ranging from 93 to 97%. In addition to detecting human strains, this assay has been demonstrated to be capable of detecting a variety of avian and non-human mammalian influenza viruses. The FLU OIA test has been used in large-scale surveillance schemes intended to provide rapid epidemiological data during normal influenza seasons and has demonstrated the potential for fulfilling a similar role for multispecies surveillance in, for example, conditions that offer challenges for conventional virus isolation methods. Conceivably, such use should facilitate the timely recognition of influenza outbreaks and prioritization of positive specimens for more conventional, laboratory characterization, leading to improved interpandemic surveillance and rapid reaction in the face of the next pandemic. PMID:11779392

  1. 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

  2. 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

  3. 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. PMID:25450004

  4. 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

  5. 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...

  6. 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...

  7. Application of 5′-Nuclease PCR for Quantitative Detection of Listeria monocytogenes in Pure Cultures, Water, Skim Milk, and Unpasteurized Whole Milk

    PubMed Central

    Nogva, Hege Karin; Rudi, Knut; Naterstad, Kristine; Holck, Askild; Lillehaug, Dag

    2000-01-01

    PCR techniques have significantly improved the detection and identification of bacterial pathogens. Countless adaptations and applications have been described, including quantitative PCR and the latest innovation, real-time PCR. In real-time PCR, e.g., the 5′-nuclease chemistry renders the automated and direct detection and quantification of PCR products possible (P. M. Holland et al., Proc. Natl. Acad. Sci. USA 88:7276–7280, 1991). We present an assay for the quantitative detection of Listeria monocytogenes based on the 5′-nuclease PCR using a 113-bp amplicon from the listeriolysin O gene (hlyA) as the target. The assay was positive for all isolates of L. monocytogenes tested (65 isolates including the type strain) and negative for all other Listeria strains (16 isolates from five species tested) and several other bacteria (18 species tested). The application of 5′-nuclease PCR in diagnostics requires a quantitative sample preparation step. Several magnetic bead-based strategies were evaluated, since these systems are simple and relatively easy to automate. The combination of nonspecific binding of bacteria to paramagnetic beads, with subsequent DNA purification by use of the same beads, gave the most satisfactory result. The detection limit was approximately 6 to 60 CFU, quantification was linear over at least 7 log units, and the method could be completed within 3 h. In conclusion, a complete quantitative method for L. monocytogenes in water and in skimmed and raw milk was developed. PMID:11010869

  8. International collaborative study to compare reverse transcriptase PCR assays for detection and genotyping of noroviruses.

    PubMed

    Vinjé, Jan; Vennema, Harry; Maunula, Leena; von Bonsdorff, Carl-Henrik; Hoehne, Marina; Schreier, Eckart; Richards, Alison; Green, Jon; Brown, David; Beard, Suzanne S; Monroe, Stephan S; de Bruin, Erwin; Svensson, Lennart; Koopmans, Marion P G

    2003-04-01

    To allow more rapid and internationally standardized assessment of the spread of noroviruses (previously called Norwalk-like viruses [NLVs]) as important food-borne pathogens, harmonization of methods for their detection is needed. Diagnosis of NLVs in clinical diagnostic laboratories is usually performed by reverse transciptase PCR (RT-PCR) assays. In the present study, the performance of five different RT-PCR assays for the detection of NLVs was evaluated in an international collaborative study by five laboratories in five countries with a coded panel of 91 fecal specimens. The assays were tested for their sensitivity, detection limit, and ease of standardization. In total, NLVs could be detected by at least one RT-PCR assay in 69 (84%) of the samples that originally tested positive. Sensitivity ranged from 52 to 73% overall and from 54 to 100% and 58 to 85% for genogroup I and II viruses, respectively. In all, 64% of the false-negative results were obtained with a set of diluted stools (n = 20) that may have lost quality upon storage. Sensitivity was improved when these samples were excluded from analysis. No one single assay stood out as the best, although the p1 assay demonstrated the most satisfactory overall performance. To promote comparability of data, this assay will be recommended for newly starting groups in future collaborative studies. PMID:12682125

  9. A thermally baffled device for highly stabilized convective PCR.

    PubMed

    Chang, Hsiao-Fen Grace; Tsai, Yun-Long; Tsai, Chuan-Fu; Lin, Ching-Ko; Lee, Pei-Yu; Teng, Ping-Hua; Su, Chen; Jeng, Chien-Chung

    2012-05-01

    Rayleigh-Bénard convective PCR is a simple and effective design for amplification of DNA. Convective PCR is, however, extremely sensitive to environmental temperature fluctuations, especially when using small- diameter test tubes. Therefore, this method is inherently unstable with limited applications. Here, we present a convective PCR device that has been modified by adding thermal baffles. With this thermally baffled device the influence from fluctuations in environmental temperature were significantly reduced, even in a wind tunnel (1 m/s). The thermally baffled PCR instrument described here has the potential to be used as a low-cost, point-of-care device for PCR-based molecular diagnostics in the field. PMID:22241586

  10. An improved molecular assay for Tritrichomonas foetus.

    PubMed

    Grahn, R A; BonDurant, R H; van Hoosear, K A; Walker, R L; Lyons, L A

    2005-01-01

    Tritrichomonas foetus (T. foetus) is the causative agent of bovine trichomonosis, a sexually transmitted disease leading to abortion (from 1 to 8 months gestation), infertility, and occasional pyometra. The annual losses to the U.S. beef industry are estimated to be in the hundreds of millions of dollars. Currently, the "gold standard" diagnostic test for trichomonosis in most countries is the cultivation of live organisms from reproductive secretions. The cultured organisms can then be followed by PCR assays with primers that amplify T. foetus to the exclusion of all other trichomonad species. Thus, negative results present as null data, indistinguishable from failed PCR amplification during T. foetus specific amplification. Our newly developed assay improves previously developed PCR based techniques by using diagnostic size variants from within the internal transcribed spacer 1 (ITS1) region that is between the 18S rRNA and 5.8S rRNA subunits. This new PCR assay amplifies trichomonad DNA from a variety of genera and positively identifies the causative agent in the bovine trichomonad infection. This approach eliminates false negatives found in some current assays as well as identifying the causative agent of trichomonad infection. Additionally, our assay incorporates a fluorescently labeled primer enabling high sensitivity and rapid assessment of the specific trichomonad species. Moreover, electrophoretic separation of amplified samples can be outsourced, thus eliminating the need for diagnostic laboratories to purchase expensive analysis equipment. PMID:15619373

  11. Evaluation of PCR based coprodiagnosis of human opisthorchiasis.

    PubMed

    Stensvold, C R; Saijuntha, W; Sithithaworn, P; Wongratanacheewin, S; Strandgaard, H; Ornbjerg, N; Johansen, M V

    2006-01-01

    In this study, a recently developed PCR test for the detection of Opisthorchis viverrini in human faecal samples was evaluated using two parasitological methods as references. During a survey of foodborne trematodes (FBT) in the Vientiane Province, Lao PDR, 85 samples were collected and evaluated for FBT eggs by the Kato Katz (KK) technique, the formalin ethyl acetate concentration technique (FECT) and a PCR analysis for the distinction between O. viverrini and other FBT. The two parasitological methods did not differ in the ability of detecting FBT eggs, and a single KK reading was characterized by a sensitivity of 85% when compared to two FECT readings. The PCR tested positive only in cases where eggs had been demonstrated by parasitological examination. However, the PCR tested negative in some samples with very high egg counts. Demonstrating a PCR sensitivity of approximately 50% in samples with faecal egg counts>1000, the previously reported PCR sensitivity based on in vitro studies was not supported. It is believed that technical problems rather than diagnostic reference related issues were responsible for the relatively low PCR performance. Further studies should aim at optimizing DNA extraction and amplification, and future PCR evaluation should include specificity control such as the scanning electron microscopy of eggs in test samples or the expulsion of adult trematodes from PCR tested individuals. PMID:16253202

  12. Single-step PCR in molecular diagnosis of hepatitis C virus infection.

    PubMed Central

    Farma, E; Boeri, E; Bettini, P; Repetto, C M; McDermott, J; Lillo, F B; Varnier, O E

    1996-01-01

    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. PMID:8940466

  13. 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

  14. Transgene Detection by Digital Droplet PCR

    PubMed Central

    Moser, Dirk A.; Braga, Luca; Raso, Andrea; Zacchigna, Serena; Giacca, Mauro; Simon, Perikles

    2014-01-01

    Somatic gene therapy is a promising tool for the treatment of severe diseases. Because of its abuse potential for performance enhancement in sports, the World Anti-Doping Agency (WADA) included the term ‘gene doping’ in the official list of banned substances and methods in 2004. Several nested PCR or qPCR-based strategies have been proposed that aim at detecting long-term presence of transgene in blood, but these strategies are hampered by technical limitations. We developed a digital droplet PCR (ddPCR) protocol for Insulin-Like Growth Factor 1 (IGF1) detection and demonstrated its applicability monitoring 6 mice injected into skeletal muscle with AAV9-IGF1 elements and 2 controls over a 33-day period. A duplex ddPCR protocol for simultaneous detection of Insulin-Like Growth Factor 1 (IGF1) and Erythropoietin (EPO) transgenic elements was created. A new DNA extraction procedure with target-orientated usage of restriction enzymes including on-column DNA-digestion was established. In vivo data revealed that IGF1 transgenic elements could be reliably detected for a 33-day period in DNA extracted from whole blood. In vitro data indicated feasibility of IGF1 and EPO detection by duplex ddPCR with high reliability and sensitivity. On-column DNA-digestion allowed for significantly improved target detection in downstream PCR-based approaches. As ddPCR provides absolute quantification, it ensures excellent day-to-day reproducibility. Therefore, we expect this technique to be used in diagnosing and monitoring of viral and bacterial infection, in detecting mutated DNA sequences as well as profiling for the presence of foreign genetic material in elite athletes in the future. PMID:25375130

  15. [Comparative attempts for the establishment and optimisation of a PCR on Leishmania for the purpose of diagnosis].

    PubMed

    Fürnkranz, Ursula; Walochnik, Julia; Grimm, Felix; Deplazes, Peter; Aspöck, Horst

    2004-01-01

    Leishmania spp. are the causative agents of visceral, cutaneous and mucocutaneous leishmaniosis with several taxa ("species") of the genus Leishmania being involved in human disease. As diagnostics based on microscopical detection of the parasites or on serological tests are often unsatisfactory, also molecular biological methods, particularly the polymerase chain reaction (PCR), have been employed for the detection of Leishmania spp. in the past years. The aim of the present study was to compare different PCR-protocols and optimise them for our needs, placing emphasis on the improvement of DNA isolation. PCR was performed with whole cell DNA isolated from cultures, as well as from simulated blood samples and clinical samples. Three different methods for the isolation of DNA from blood samples and two different PCR-protocols, one amplifying a fragment of the 18S rDNA and one for the amplification of the whole kDNA-circle, were applied and compared. No significant difference in sensitivity was detected between the different PCR-protocols, however, it was shown that the highest yield of DNA was achieved with a DNA isolation protocol based on urea. PMID:15683040

  16. 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

  17. 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.

  18. PCR microfluidic devices for DNA amplification.

    PubMed

    Zhang, Chunsun; Xu, Jinliang; Ma, Wenli; Zheng, Wenling

    2006-01-01

    The miniaturization of biological and chemical analytical devices by micro-electro-mechanical-systems (MEMS) technology has posed a vital influence on such fields as medical diagnostics, microbial detection and other bio-analysis. Among many miniaturized analytical devices, the polymerase chain reaction (PCR) microchip/microdevices are studied extensively, and thus great progress has been made on aspects of on-chip micromachining (fabrication, bonding and sealing), choice of substrate materials, surface chemistry and architecture of reaction vessel, handling of necessary sample fluid, controlling of three or two-step temperature thermocycling, detection of amplified nucleic acid products, integration with other analytical functional units such as sample preparation, capillary electrophoresis (CE), DNA microarray hybridization, etc. However, little has been done on the review of above-mentioned facets of the PCR microchips/microdevices including the two formats of flow-through and stationary chamber in spite of several earlier reviews [Zorbas, H. Miniature continuous-flow polymerase chain reaction: a breakthrough? Angew Chem Int Ed 1999; 38 (8):1055-1058; Krishnan, M., Namasivayam, V., Lin, R., Pal, R., Burns, M.A. Microfabricated reaction and separation systems. Curr Opin Biotechnol 2001; 12:92-98; Schneegabeta, I., Köhler, J.M. Flow-through polymerase chain reactions in chip themocyclers. Rev Mol Biotechnol 2001; 82:101-121; deMello, A.J. DNA amplification: does 'small' really mean 'efficient'? Lab Chip 2001; 1: 24N-29N; Mariella, Jr. R. MEMS for bio-assays. Biomed Microdevices 2002; 4 (2):77-87; deMello AJ. Microfluidics: DNA amplification moves on. Nature 2003; 422:28-29; Kricka, L.J., Wilding, P. Microchip PCR. Anal BioAnal Chem 2003; 377:820-825]. In this review, we survey the advances of the above aspects among the PCR microfluidic devices in detail. Finally, we also illuminate the potential and practical applications of PCR microfluidics to some fields such

  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. Diagnostic Imaging

    MedlinePlus

    Diagnostic imaging lets doctors look inside your body for clues about a medical condition. A variety of machines and ... and activities inside your body. The type of imaging your doctor uses depends on your symptoms and ...

  1. A web server for performing electronic PCR

    PubMed Central

    Rotmistrovsky, Kirill; Jang, Wonhee; Schuler, Gregory D.

    2004-01-01

    ‘Electronic PCR’ (e-PCR) refers to a computational procedure that is used to search DNA sequences for sequence tagged sites (STSs), each of which is defined by a pair of primer sequences and an expected PCR product size. To gain speed, our implementation extracts short ‘words’ from the 3′ end of each primer and stores them in a sorted hash table that can be accessed efficiently during the search. One recent improvement is the use of overlapping discontinuous words to allow matches to be found despite the presence of a mismatch. Moreover, it is possible to allow gaps in the alignment between the primer and the sequence. The effect of these changes is to improve sensitivity without significantly affecting specificity. The new software provides a search mode using a query STS against a sequence database to augment the previously available mode using a query sequence against an STS database. Finally, e-PCR may now be used through a web service, with search results linked to other web resources such as the UniSTS database and the MapViewer genome browser. The e-PCR web server may be found at www.ncbi.nlm.nih.gov/sutils/e-pcr. PMID:15215361

  2. Diagnostic radiology

    SciTech Connect

    Leeds, N.E.; Jacobson, H.G.

    1986-10-17

    Developments in the burgeoning field of diagnostic radiology have continued apace. Four areas that represent either subspecialities or technological advances in diagnostic radiology will be considered in this report: ultrasonography, interventional radiology, nuclear radiology, and magnetic resonance. In no sense is the exclusion of other subdisciplines and modalities (eg, pediatric radiology, computed tomography) and indication of their of importance or their failure to include innovative concepts.

  3. A proline racemase based PCR for identification of Trypanosoma vivax in cattle blood.

    PubMed

    Fikru, Regassa; Hagos, Ashenafi; Rogé, Stijn; Reyna-Bello, Armando; Gonzatti, Mary Isabel; Merga, Bekana; Goddeeris, Bruno Maria; Büscher, Philippe

    2014-01-01

    A study was conducted to develop a Trypanosoma vivax (T. vivax) specific PCR based on the T. vivax proline racemase (TvPRAC) gene. Forward and reverse primers were designed that bind at 764-783 bp and 983-1002 bp of the gene. To assess its specificity, TvPRAC PCR was conducted on DNA extracted from different haemotropic pathogens: T. vivax from Nigeria, Ethiopia and Venezuela, T. congolense Savannah type, T. brucei brucei, T. evansi, T. equiperdum, T. theileri, Theileria parva, Anaplasma marginale, Babesia bovis and Babesia bigemina and from bovine, goat, mouse, camel and human blood. The analytical sensitivity of the TvPRAC PCR was compared with that of the ITS-1 PCR and the 18S PCR-RFLP on a dilution series of T. vivax DNA in water. The diagnostic performance of the three PCRs was compared on 411 Ethiopian bovine blood specimens collected in a former study. TvPRAC PCR proved to be fully specific for T. vivax, irrespective of its geographical origin. Its analytical sensitivity was lower than that of ITS-1 PCR. On these bovine specimens, TvPRAC PCR detected 8.3% T. vivax infections while ITS-1 PCR and 18S PCR-RFLP detected respectively 22.6 and 6.1% T. vivax infections. The study demonstrates that a proline racemase based PCR could be used, preferably in combination with ITS-1 PCR, as a species-specific diagnostic test for T. vivax infections worldwide. PMID:24416292

  4. 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...

  5. 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

  6. 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. PMID:26497302

  7. 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...

  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. Methods for producing partially digested restriction DNA fragments and for producing a partially modified PCR product

    DOEpatents

    Wong, Kwong-Kwok

    2000-01-01

    The present invention is an improved method of making a partially modified PCR product from a DNA fragment with a polymerase chain reaction (PCR). In a standard PCR process, the DNA fragment is combined with starting deoxynucleoside triphosphates, a primer, a buffer and a DNA polymerase in a PCR mixture. The PCR mixture is then reacted in the PCR producing copies of the DNA fragment. The improvement of the present invention is adding an amount of a modifier at any step prior to completion of the PCR process thereby randomly and partially modifying the copies of the DNA fragment as a partially modified PCR product. The partially modified PCR product may then be digested with an enzyme that cuts the partially modified PCR product at unmodified sites thereby producing an array of DNA restriction fragments.

  10. 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

  11. Discrimination of gastrointestinal nematode eggs from crude fecal egg preparations by inhibitor-resistant conventional and real-time PCR.

    PubMed

    Demeler, Janina; Ramünke, Sabrina; Wolken, Sonja; Ianiello, Davide; Rinaldi, Laura; Gahutu, Jean Bosco; Cringoli, Giuseppe; von Samson-Himmelstjerna, Georg; Krücken, Jürgen

    2013-01-01

    Diagnosis of gastrointestinal nematodes relies predominantly on coproscopic methods such as flotation, Kato-Katz, McMaster or FLOTAC. Although FLOTAC allows accurate quantification, many nematode eggs can only be differentiated to genus or family level. Several molecular diagnostic tools discriminating closely related species suffer from high costs for DNA isolation from feces and limited sensitivity since most kits use only small amounts of feces (<1 g). A direct PCR from crude egg preparations was designed for full compatibility with FLOTAC to accurately quantify eggs per gram feces (epg) and determine species composition. Eggs were recovered from the flotation solution and concentrated by sieving. Lysis was achieved by repeated boiling and freezing cycles - only Trichuris eggs required additional mechanic disruption. Egg lysates were directly used as template for PCR with Phusion DNA polymerase which is particularly resistant to PCR inhibitors. Qualitative results were obtained with feces of goats, cattle, horses, swine, cats, dogs and mice. The finally established protocol was also compatible with quantitative real-time PCR in the presence of EvaGreen and no PCR inhibition was detectable when extracts were diluted at least fourfold. Sensitivity was comparable to DNA isolation protocols and spiked samples with five epg were reliably detected. For Toxocara cati a detection limit below one epg was demonstrated. It was possible to distinguish T. cati and Toxocara canis using high resolution melt (HRM) analysis, a rapid tool for species identification. In human samples, restriction fragment length polymorphism (RFLP) and HRM analysis were used to discriminate Necator americanus and Ancylostoma duodenale. The method is able to significantly improve molecular diagnosis of gastrointestinal nematodes by increasing speed and sensitivity while decreasing overall costs. For identification of species or resistance alleles, analysis of PCR products with many different post

  12. Comparative Assessment of a Commercial Kit and Two Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis

    PubMed Central

    Morelle, Christelle; Varlet-Marie, Emmanuelle; Brenier-Pinchart, Marie-Pierre; Cassaing, Sophie; Pelloux, Hervé; Bastien, Patrick

    2012-01-01

    Toxoplasmosis is a worldwide infection that may cause severe disease and is regarded as a serious health problem in France. Detection of the parasite by molecular methods is crucial for diagnosing the disease. The extreme diversity of methods and performances of Toxoplasma PCR assays makes the use of commercial PCR kits an attractive alternative, as they offer a chance for standardization. We compared the performances of three molecular methods for the detection of Toxoplasma gondii DNA in amniotic fluid: a commercial method using nested PCR and two laboratory-developed methods, one using conventional PCR and the other one real-time PCR. This evaluation was based upon a T. gondii DNA serial dilution assay, three amniotic fluid samples spiked with T. gondii at different concentrations, and a clinical cohort of 33 amniotic fluid samples. The T. gondii DNA serial dilution assay showed a much lower sensitivity for the commercial kit than for the laboratory-developed methods. Moreover, out of 12 proven congenital toxoplasmosis cases, 91.7% were detected by the laboratory-developed assays, whereas only 50% were detected by the commercial kit. A lack of sensitivity of the method, partly due to the presence of PCR inhibitors, was the main drawback of the commercial method. This study emphasizes that commercial PCR diagnostic kits do not systematically perform better than carefully optimized laboratory-developed methods. There is a need for thorough evaluation of such kits by proficient groups, as well as for performance standards that commercial kits can be tested against to improve confidence in those selected by health care providers. PMID:23035184

  13. Simultaneous differential detection of Chlamydophila abortus, Chlamydophila pecorum and Coxiella burnetii from aborted ruminant's clinical samples using multiplex PCR

    PubMed Central

    2009-01-01

    Background Chlamydiosis and Q fever, two zoonosis, are important causes of ruminants' abortion around the world. They are caused respectively by strictly intracellular and Gram negative bacterium Chlamydophila abortus (Cp. abortus) and Coxiella burnetii (C. burnetii). Chlamydophila pecorum (Cp. pecorum) is commonly isolated from the digestive tract of clinically inconspicuous ruminants but the abortive and zoonotic impact of this bacterium is still unknown because Cp. pecorum is rarely suspected in abortion cases of small ruminants. We have developed a multiplex PCR (m-PCR) for rapid simultaneous differential detection of Cp. abortus, Cp. pecorum and C. burnetii in clinical samples taken from infected animals. Results Specific PCR primers were designed and a sensitive and specific m-PCR was developed to detect simultaneously, in one tube reaction, three specific fragments of 821, 526 and 687-bp long for Cp. abortus, Cp. pecorum and C. burnetii respectively. This m-PCR assay was performed on 253 clinical samples taken from infected ruminant's flocks that have showed problems of abortion diseases. Thus, 67 samples were infected by either one of the three pathogens: 16 (13 vaginal swabs and 3 placentas) were positive for Cp. abortus, 2 were positive for Cp. pecorum (1 vaginal swab and 1 placenta) and 49 samples (33 vaginal swabs, 11 raw milks, 4 faeces and 1 placenta) were positive for C. burnetii. Two vaginal swabs were m-PCR positive of both Cp. abortus and C. burnetii and none of the tested samples was shown to be infected simultaneously with the three pathogens. Conclusion We have successfully developed a rapid multiplex PCR that can detect and differentiate Cp. abortus, Cp. pecorum and C. burnetii; with a good sensitivity and specificity. The diagnosis of chlamydiosis and Q fever may be greatly simplified and performed at low cost. In addition, the improvement in diagnostic techniques will enhance our knowledge regarding the prevalence and the pathogenetic

  14. Discrimination of Gastrointestinal Nematode Eggs from Crude Fecal Egg Preparations by Inhibitor-Resistant Conventional and Real-Time PCR

    PubMed Central

    Demeler, Janina; Ramünke, Sabrina; Wolken, Sonja; Ianiello, Davide; Rinaldi, Laura; Gahutu, Jean Bosco; Cringoli, Giuseppe; von Samson-Himmelstjerna, Georg; Krücken, Jürgen

    2013-01-01

    Diagnosis of gastrointestinal nematodes relies predominantly on coproscopic methods such as flotation, Kato-Katz, McMaster or FLOTAC. Although FLOTAC allows accurate quantification, many nematode eggs can only be differentiated to genus or family level. Several molecular diagnostic tools discriminating closely related species suffer from high costs for DNA isolation from feces and limited sensitivity since most kits use only small amounts of feces (<1 g). A direct PCR from crude egg preparations was designed for full compatibility with FLOTAC to accurately quantify eggs per gram feces (epg) and determine species composition. Eggs were recovered from the flotation solution and concentrated by sieving. Lysis was achieved by repeated boiling and freezing cycles – only Trichuris eggs required additional mechanic disruption. Egg lysates were directly used as template for PCR with Phusion DNA polymerase which is particularly resistant to PCR inhibitors. Qualitative results were obtained with feces of goats, cattle, horses, swine, cats, dogs and mice. The finally established protocol was also compatible with quantitative real-time PCR in the presence of EvaGreen and no PCR inhibition was detectable when extracts were diluted at least fourfold. Sensitivity was comparable to DNA isolation protocols and spiked samples with five epg were reliably detected. For Toxocara cati a detection limit below one epg was demonstrated. It was possible to distinguish T. cati and Toxocara canis using high resolution melt (HRM) analysis, a rapid tool for species identification. In human samples, restriction fragment length polymorphism (RFLP) and HRM analysis were used to discriminate Necator americanus and Ancylostoma duodenale. The method is able to significantly improve molecular diagnosis of gastrointestinal nematodes by increasing speed and sensitivity while decreasing overall costs. For identification of species or resistance alleles, analysis of PCR products with many different post

  15. Development, application and validation of a Taqman real-time RT-PCR assay for the detection of infectious salmon anaemia virus (ISAV) in Atlantic salmon (Salmo salar).

    PubMed

    Snow, M; McKay, P; McBeath, A J A; Black, J; Doig, F; Kerr, R; Cunningham, C O; Nylund, A; Devold, M

    2006-01-01

    Infectious salmon anaemia (ISA) is a disease of cultured Atlantic salmon (Salmo salar) which was successfully eradicated from Scotland following its emergence in 1998. The rapid deployment of sensitive diagnostic methods for the detection of ISA virus (ISAV) was fundamental to the swift eradication of ISA disease in Scotland and continues to be of crucial importance to surveillance of the aquaculture industry. This study reports the development, validation, application and interpretation of two independent, highly sensitive and specific semi-quantitative Taqman real-time RT-PCR (qRT-PCR) methods for the detection of ISAV. Such technology offers considerable advantages over conventional RT-PCR methods in current routine use for ISAV surveillance. These include an increased sensitivity, enhanced specificity, semi-quantification using endogenous controls, a lack of subjectivity in results interpretation, speed of processing and improved contamination control. PMID:17058489

  16. Reducing Diagnostic Bias.

    ERIC Educational Resources Information Center

    McLaughlin, Jerry E.

    2002-01-01

    Skill using the American Psychiatric Association's 2000 "Diagnostic and Statistical Manual of Mental Disorders, Text Revision" is essential for increased professional credibility, career marketability, and third-party reimbursement of professional counselors. This article focuses on how to improve counselors' skill with the manual, by providing…

  17. 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

  18. Sequence polymorphism can produce serious artifacts in real-time PCR assays: lessons from Pacific oysters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Since it was first described in the mid-1990s, quantitative real time PCR (Q-PCR) has been widely used in many fields of biomedical research and molecular diagnostics. This method is routinely used to validate whole transcriptome analyses such as DNA microarrays, suppressive subtractive hybridizati...

  19. PCR+ In Diesel Fuels and Emissions Research

    SciTech Connect

    McAdams, H.T.

    2002-04-15

    In past work for the U.S. Department of Energy (DOE) and Oak Ridge National Laboratory (ORNL), PCR+ was developed as an alternative methodology for building statistical models. PCR+ is an extension of Principal Components Regression (PCR), in which the eigenvectors resulting from Principal Components Analysis (PCA) are used as predictor variables in regression analysis. The work was motivated by the observation that most heavy-duty diesel (HDD) engine research was conducted with test fuels that had been ''concocted'' in the laboratory to vary selected fuel properties in isolation from each other. This approach departs markedly from the real world, where the reformulation of diesel fuels for almost any purpose leads to changes in a number of interrelated properties. In this work, we present new information regarding the problems encountered in the conventional approach to model-building and how the PCR+ method can be used to improve research on the relationship between fuel characteristics and engine emissions. We also discuss how PCR+ can be applied to a variety of other research problems related to diesel fuels.

  20. Highly precise measurement of HIV DNA by droplet digital PCR.

    PubMed

    Strain, Matthew C; Lada, Steven M; Luong, Tiffany; Rought, Steffney E; Gianella, Sara; Terry, Valeri H; Spina, Celsa A; Woelk, Christopher H; Richman, Douglas D

    2013-01-01

    Deoxyribonucleic acid (DNA) of the human immunodeficiency virus (HIV) provides the most sensitive measurement of residual infection in patients on effective combination antiretroviral therapy (cART). Droplet digital PCR (ddPCR) has recently been shown to provide highly accurate quantification of DNA copy number, but its application to quantification of HIV DNA, or other equally rare targets, has not been reported. This paper demonstrates and analyzes the application of ddPCR to measure the frequency of total HIV DNA (pol copies per million cells), and episomal 2-LTR (long terminal repeat) circles in cells isolated from infected patients. Analysis of over 300 clinical samples, including over 150 clinical samples assayed in triplicate by ddPCR and by real-time PCR (qPCR), demonstrates a significant increase in precision, with an average 5-fold decrease in the coefficient of variation of pol copy numbers and a >20-fold accuracy improvement for 2-LTR circles. Additional benefits of the ddPCR assay over qPCR include absolute quantification without reliance on an external standard and relative insensitivity to mismatches in primer and probe sequences. These features make digital PCR an attractive alternative for measurement of HIV DNA in clinical specimens. The improved sensitivity and precision of measurement of these rare events should facilitate measurements to characterize the latent HIV reservoir and interventions to eradicate it. PMID:23573183

  1. Highly Precise Measurement of HIV DNA by Droplet Digital PCR

    PubMed Central

    Strain, Matthew C.; Lada, Steven M.; Luong, Tiffany; Rought, Steffney E.; Gianella, Sara; Terry, Valeri H.; Spina, Celsa A.; Woelk, Christopher H.; Richman, Douglas D.

    2013-01-01

    Deoxyribonucleic acid (DNA) of the human immunodeficiency virus (HIV) provides the most sensitive measurement of residual infection in patients on effective combination antiretroviral therapy (cART). Droplet digital PCR (ddPCR) has recently been shown to provide highly accurate quantification of DNA copy number, but its application to quantification of HIV DNA, or other equally rare targets, has not been reported. This paper demonstrates and analyzes the application of ddPCR to measure the frequency of total HIV DNA (pol copies per million cells), and episomal 2-LTR (long terminal repeat) circles in cells isolated from infected patients. Analysis of over 300 clinical samples, including over 150 clinical samples assayed in triplicate by ddPCR and by real-time PCR (qPCR), demonstrates a significant increase in precision, with an average 5-fold decrease in the coefficient of variation of pol copy numbers and a >20-fold accuracy improvement for 2-LTR circles. Additional benefits of the ddPCR assay over qPCR include absolute quantification without reliance on an external standard and relative insensitivity to mismatches in primer and probe sequences. These features make digital PCR an attractive alternative for measurement of HIV DNA in clinical specimens. The improved sensitivity and precision of measurement of these rare events should facilitate measurements to characterize the latent HIV reservoir and interventions to eradicate it. PMID:23573183

  2. 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

  3. 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.

  4. PCR in laboratory diagnosis of human Borrelia burgdorferi infections.

    PubMed Central

    Schmidt, B L

    1997-01-01

    The laboratory diagnosis of Lyme borreliosis, the most prevalent vector-borne disease in the United States and endemic in parts of Europe and Asia, is currently based on serology with known limitations. Direct demonstration of Borrelia burgdorferi by culture may require weeks, while enzyme-linked immunosorbent assays for antigen detection often lack sensitivity. The development of the PCR has offered a new dimension in the diagnosis. Capable of amplifying minute amounts of DNA into billions of copies in just a few hours, PCR facilitates the sensitive and specific detection of DNA or RNA of pathogenic organisms. This review is restricted to applications of PCR methods in the diagnosis of human B. burgdorferi infections. In the first section, methodological aspects, e.g., sample preparation, target selection, primers and PCR methods, and detection and control of inhibition and contamination, are highlighted. In the second part, emphasis is placed on diagnostic aspects, where PCR results in patients with dermatological, neurological, joint, and ocular manifestations of the disease are discussed. Here, special attention is given to monitoring treatment efficacy by PCR tests. Last, specific guidelines on how to interpret PCR results, together with the advantages and limitations of these new techniques, are presented. PMID:8993863

  5. 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. PMID:27143822

  6. 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

  7. PCR and real-time PCR assays to detect fungi of Alternaria alternata species.

    PubMed

    Kordalewska, Milena; Brillowska-Dąbrowska, Anna; Jagielski, Tomasz; Dworecka-Kaszak, Bożena

    2015-01-01

    Fungi of the Alternaria genus are mostly associated with allergic diseases. However, with a growing number of immunocompromised patients, these fungi, with A. alternata being the most prevalent one, are increasingly recognized as etiological agents of infections (phaeohyphomycoses) in humans. Nowadays, identification of Alternaria spp. requires their pure culture and is solely based on morphological criteria. Clinically, Alternaria infections may be indistinguishable from other fungal diseases. Therefore, a diagnostic result is often delayed or even not achieved at all. In this paper we present easy to perform and interpret PCR and real-time PCR assays enabling detection of A. alternata species. On the basis of alignment of β-tubulin gene sequences, A. alternata-specific primers were designed. DNA from fungal isolates, extracted in a two-step procedure, were used in PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The assays specificity was confirmed, since positive results were obtained for all A. alternata isolates, and no positive results were obtained neither for other molds, dermatophytes, yeast-like fungi, nor human DNA. The assays developed here enable fast and unambiguous identification of A. alternata pathogens. PMID:26610309

  8. Development and Validation of an Improved PCR Method Using the 23S-5S Intergenic Spacer for Detection of Rickettsiae in Dermacentor variabilis Ticks and Tissue Samples from Humans and Laboratory Animals.

    PubMed

    Kakumanu, Madhavi L; Ponnusamy, Loganathan; Sutton, Haley T; Meshnick, Steven R; Nicholson, William L; Apperson, Charles S

    2016-04-01

    A novel nested PCR assay was developed to detectRickettsiaspp. in ticks and tissue samples from humans and laboratory animals. Primers were designed for the nested run to amplify a variable region of the 23S-5S intergenic spacer (IGS) ofRickettsiaspp. The newly designed primers were evaluated using genomic DNA from 11Rickettsiaspecies belonging to the spotted fever, typhus, and ancestral groups and, in parallel, compared to otherRickettsia-specific PCR targets (ompA,gltA, and the 17-kDa protein gene). The new 23S-5S IGS nested PCR assay amplified all 11Rickettsiaspp., but the assays employing other PCR targets did not. The novel nested assay was sensitive enough to detect one copy of a cloned 23S-5S IGS fragment from "CandidatusRickettsia amblyommii." Subsequently, the detection efficiency of the 23S-5S IGS nested assay was compared to those of the other three assays using genomic DNA extracted from 40 adultDermacentor variabilisticks. The nested 23S-5S IGS assay detectedRickettsiaDNA in 45% of the ticks, while the amplification rates of the other three assays ranged between 5 and 20%. The novel PCR assay was validated using clinical samples from humans and laboratory animals that were known to be infected with pathogenic species ofRickettsia The nested 23S-5S IGS PCR assay was coupled with reverse line blot hybridization with species-specific probes for high-throughput detection and simultaneous identification of the species ofRickettsiain the ticks. "CandidatusRickettsia amblyommii,"R. montanensis,R. felis, andR. belliiwere frequently identified species, along with some potentially novelRickettsiastrains that were closely related toR. belliiandR. conorii. PMID:26818674

  9. Development and Validation of an Improved PCR Method Using the 23S-5S Intergenic Spacer for Detection of Rickettsiae in Dermacentor variabilis Ticks and Tissue Samples from Humans and Laboratory Animals

    PubMed Central

    Kakumanu, Madhavi L.; Ponnusamy, Loganathan; Sutton, Haley T.; Meshnick, Steven R.; Nicholson, William L.

    2016-01-01

    A novel nested PCR assay was developed to detect Rickettsia spp. in ticks and tissue samples from humans and laboratory animals. Primers were designed for the nested run to amplify a variable region of the 23S-5S intergenic spacer (IGS) of Rickettsia spp. The newly designed primers were evaluated using genomic DNA from 11 Rickettsia species belonging to the spotted fever, typhus, and ancestral groups and, in parallel, compared to other Rickettsia-specific PCR targets (ompA, gltA, and the 17-kDa protein gene). The new 23S-5S IGS nested PCR assay amplified all 11 Rickettsia spp., but the assays employing other PCR targets did not. The novel nested assay was sensitive enough to detect one copy of a cloned 23S-5S IGS fragment from “Candidatus Rickettsia amblyommii.” Subsequently, the detection efficiency of the 23S-5S IGS nested assay was compared to those of the other three assays using genomic DNA extracted from 40 adult Dermacentor variabilis ticks. The nested 23S-5S IGS assay detected Rickettsia DNA in 45% of the ticks, while the amplification rates of the other three assays ranged between 5 and 20%. The novel PCR assay was validated using clinical samples from humans and laboratory animals that were known to be infected with pathogenic species of Rickettsia. The nested 23S-5S IGS PCR assay was coupled with reverse line blot hybridization with species-specific probes for high-throughput detection and simultaneous identification of the species of Rickettsia in the ticks. “Candidatus Rickettsia amblyommii,” R. montanensis, R. felis, and R. bellii were frequently identified species, along with some potentially novel Rickettsia strains that were closely related to R. bellii and R. conorii. PMID:26818674

  10. 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.

  11. A Pentaplex PCR Assay for the Detection and Differentiation of Shigella Species

    PubMed Central

    Ojha, Suvash Chandra; Yean Yean, Chan; Ismail, Asma; Banga Singh, Kirnpal-Kaur

    2013-01-01

    The magnitude of shigellosis in developing countries is largely unknown because an affordable detection method is not available. Current laboratory diagnosis of Shigella spp. is laborious and time consuming and has low sensitivity. Hence, in the present study, a molecular-based diagnostic assay which amplifies simultaneously four specific genes to identify invC for Shigella genus, rfc for S. flexneri, wbgZ for S. sonnei, and rfpB for S. dysenteriae, as well as one internal control (ompA) gene, was developed in a single reaction to detect and differentiate Shigella spp. Validation with 120 Shigella strains and 37 non-Shigella strains yielded 100% specificity. The sensitivity of the PCR was 100 pg of genomic DNA, 5.4 × 104 CFU/ml, or approximately 120 CFU per reaction mixture of bacteria. The sensitivity of the pentaplex PCR assay was further improved following preincubation of the stool samples in Gram-negative broth. A preliminary study with 30 diarrhoeal specimens resulted in no cross-reaction with other non-Shigella strains tested. We conclude that the developed pentaplex PCR assay is robust and can provide information about the four target genes that are essential for the identification of the Shigella genus and the three Shigella species responsible for the majority of shigellosis cases. PMID:23509722

  12. Optimization of LipL32 PCR assay for increased sensitivity in diagnosing leptospirosis.

    PubMed

    Galloway, Renee L; Hoffmaster, Alex R

    2015-07-01

    Early diagnosis of leptospirosis in humans is critical with regard to initiation of appropriate treatment; however, the gold standard serological test cannot detect antibodies until nearly a week after symptom onset. PCR has been shown to be sensitive and specific in the early phase of leptospirosis. Previously, we developed and validated a TaqMan PCR assay targeting lipL32. We reoptimized and validated this assay using PerfeCTa® qPCR ToughMix®, Low ROX™ (Quanta Biosciences, Gaithersburg, MD, USA). For optimization with the new mix, the final primer concentrations were increased from 0.5 μmol/L to 0.9 μmol/L compared to our previous assay, and the probe concentration increased from 0.1 μmol/L to 0.125 μmol/L. This newly optimized assay resulted in a lower limit of detection and increased diagnostic sensitivity. Here, we present the performance data of the improved assay and describe several clinical cases that were initially negative but tested positive using the optimized assay. PMID:25912810

  13. 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. PMID:25850696

  14. 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

  15. Identification of Actinobacillus actinomycetemcomitans in subgingival plaque by PCR.

    PubMed Central

    Flemmig, T F; Rüdiger, S; Hofmann, U; Schmidt, H; Plaschke, B; Strätz, A; Klaiber, B; Karch, H

    1995-01-01

    The purpose of this study was to assess the sensitivity and specificity of the PCR in detecting Actinobacillus actinomycetemcomitans. The PCR's detection capability was compared with those of three other methods: culture-enhanced PCR (CE-PCR), colony hybridization (CH), and conventional culture with presumptive biochemical identification. A 285-bp stretch of the leukotoxin gene lktA of A. actinomycetemcomitans was amplified by PCR with primers TT-15 and TT-16. For CH, the PCR product was labeled with digoxigenin and used as a hybridization probe. Nucleotide sequence analysis of the PCR product of A. actinomycetemcomitans 1D4 and 1664 and three clinical isolates revealed complete homology among the tested strains, with only one base substitution (at position 1344) in comparison with the published sequence. With artificially infected subgingival plaque, the detection limit of PCR for A. actinomycetemcomitans was 10(3) CFU/ml of plaque suspension. Culturing subgingival plaque on tryptic soy-serum-bacitracin-vancomycin agar prior to PCR (CE-PCR) improved the limit of detection to 10(2) CFU/ml. Analysis of subgingival plaque samples from 35 patients with periodontal disease and 10 periodontally healthy subjects revealed that CE-PCR and CH had the highest overall rate of A. actinomycetemcomitans detection (both 58%), followed by PCR and culture (both 42%). With CH as the "gold standard", the sensitivities of CE-PCR, PCR, and culture were 88, 65, and 58%, respectively; the specificities were 84, 89, and 79%, respectively. The CE-PCR provided acceptable positive and negative predictive values (> or = 70%) when the prevalence of A. actinomycetemcomitans varied between 30 and 70%. PCR alone provided comparable predictive values over a narrower range of prevalence rates (30 to 50%), while culture did not afford acceptable predictive values at any prevalence rate. PCR and CE-PCR were found to be superior to culture with presumptive biochemical identification and should be the

  16. New Diagnostic Aides for Melanoma

    PubMed Central

    Ferris, Laura K.; Harris, Ryan J.

    2012-01-01

    Synopsis Detection of melanoma at an early stage is crucial to improving survival rates in melanoma. Accurate diagnosis by current techniques including dermatoscopy remains difficult, and new tools are needed to improve our diagnostic abilities. This article discusses recent advances in diagnostic techniques including confocal scanning laser microscopy, MelaFind, Siascopy, noninvasive genomic detection, as well as other future possibilities to aid in diagnosing melanoma. Advantages and barriers to implementation of the various technologies are discussed as well. PMID:22800557

  17. Molecular diagnostics in tuberculosis: basis and implications for therapy.

    PubMed

    Balasingham, Seetha V; Davidsen, Tonje; Szpinda, Irena; Frye, Stephan A; Tønjum, Tone

    2009-01-01

    The processing of clinical specimens in the mycobacterial diagnostic laboratory has undergone remarkable improvements during the last decade. While microscopy and culture are still the major backbone for laboratory diagnosis of tuberculosis on a worldwide basis, new methods including molecular diagnostic tests have evolved over the last two decades. The majority of molecular tests have been focused on (i) detection of nucleic acids, both DNA and RNA, that are specific to Mycobacterium tuberculosis, by amplification techniques such as polymerase chain reaction (PCR); and (ii) detection of mutations in the genes that are associated with resistance to antituberculosis drugs by sequencing or nucleic acid hybridization. Recent developments in direct and rapid detection of mycobacteria, with emphasis on M. tuberculosis species identification by 16S rRNA gene sequence analysis or oligohybridization and strain typing, as well as detection of drug susceptibility patterns, all contribute to these advances. Generally, the balance between genome instability and genome maintenance as the basis for evolutionary development, strain diversification and resistance development is important, because it cradles the resulting M. tuberculosis phenotype. At the same time, semi-automated culture systems have contributed greatly to the increased sensitivity and reduced turnaround time in the mycobacterial analysis of clinical specimens. Collectively, these advances are particularly important for establishing the diagnosis of tuberculosis in children. More basic and operational research to appraise the impact and cost effectiveness of new diagnostic technologies must, however, be carried out. Furthermore, the design and quality of clinical trials evaluating new diagnostics must be improved to allow clinical and laboratory services that would provide rapid response to test results. Thus, important work remains before the new diagnostic tools can be meaningfully integrated into national

  18. Utility of PCR in diagnosing pulmonary tuberculosis.

    PubMed Central

    Bennedsen, J; Thomsen, V O; Pfyffer, G E; Funke, G; Feldmann, K; Beneke, A; Jenkins, P A; Hegginbothom, M; Fahr, A; Hengstler, M; Cleator, G; Klapper, P; Wilkins, E G

    1996-01-01

    At present, the rapid diagnosis of pulmonary tuberculosis rests with microscopy. However, this technique is insensitive and many cases of pulmonary tuberculosis cannot be initially confirmed. Nucleic acid amplification techniques are extremely sensitive, but when they are applied to tuberculosis diagnosis, they have given variable results. Investigators at six centers in Europe compared a standardized PCR system (Amplicor; Roche) against conventional culture methods. Defined clinical information was collected. Discrepant samples were retested, and inhibition assays and backup amplification with a separate primer pair were performed. Mycobacterium tuberculosis complex organisms were recovered from 654 (9.1%) of 7,194 samples and 293 (7.8%) of 3,738 patients. Four hundred fifty-two of the M. tuberculosis isolates from 204 patients were smear positive and culture positive. Among the culture-positive specimens, PCR had a sensitivity of 91.4% for smear-positive specimens and 60.9% for smear-negative specimens, with a specificity of 96.1%. Analysis of 254 PCR-positive, culture-negative specimens with discrepant results revealed that 130 were from patients with recently diagnosed tuberculosis and 94 represented a presumed laboratory error. Similar analysis of 118 PCR-negative, culture-positive specimens demonstrated that 27 discrepancies were due to presumed uneven aliquot distribution and 11 were due to presumed laboratory error; PCR inhibitors were detected in 8 specimens. Amplicor enables laboratories with little previous experience with nucleic acid amplification to perform PCR. Disease in more than 60% of the patients with tuberculosis with smear-negative, culture-positive specimens can be diagnosed at the time of admission, and potentially all patients with smear-positive specimens can immediately be confirmed as being infected with M. tuberculosis, leading to improved clinical management. PMID:8735089

  19. Digital droplet PCR on disk.

    PubMed

    Schuler, Friedrich; Trotter, Martin; Geltman, Marcel; Schwemmer, Frank; Wadle, Simon; Domínguez-Garrido, Elena; López, María; Cervera-Acedo, Cristina; Santibáñez, Paula; von Stetten, Felix; Zengerle, Roland; Paust, Nils

    2016-01-01

    Existing systems for digital droplet PCR (ddPCR) either suffer from low integration or are difficult to introduce to mass fabrication. Here we present an integrated system that is compatible to mass fabrication and combines emulsification, PCR, and fluorescence readout in a single chamber within a disposable cartridge (disk). Droplets are generated by injecting the sample into fluorinated oil via centrifugal step emulsification. The resulting emulsion is aligned in the PCR and readout zone by capillary action. During thermocycling, gas bubbles generated by degassing are removed by capillary driven transport through tapered regions in the PCR chamber. Thereby, the positioning of the emulsion within the readout zone of the PCR chamber is ensured at any time and no bubbles are present during readout. Manual handling of the disk solely requires pipetting of oil and PCR mix into the inlet structures, placing the disk into the thermocycler and subsequently into a microarray scanner. The functionality of the ddPCR process chain is demonstrated by quantitative detection of the cystic fibrosis causing mutation p.Phe508del, which is of interest for non-invasive prenatal testing (NIPT). The mutation was detected in a concentration range spanning four orders of magnitude. We envision that this work will lay the base for the development of highly integrated sample-to-digital-answer PCR systems that can be employed in routine clinical diagnosis. PMID:26610263

  20. 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

  1. High-throughput real-time PCR-based genotyping without DNA purification

    PubMed Central

    2012-01-01

    Background While improvements in genotyping technology have allowed for increased throughput and reduced time and expense, protocols remain hindered by the slow upstream steps of isolating, purifying, and normalizing DNA. Various methods exist for genotyping samples directly through blood, without having to purify the DNA first. These procedures were designed to be used on smaller throughput systems, however, and have not yet been tested for use on current high-throughput real-time (q)PCR based genotyping platforms. In this paper, a method of quantitative qPCR-based genotyping on blood without DNA purification was developed using a high-throughput qPCR platform. Findings The performances of either DNA purified from blood or the same blood samples without DNA purification were evaluated through qPCR-based genotyping. First, 60 different mutations prevalent in the Ashkenazi Jewish population were genotyped in 12 Ashkenazi Jewish individuals using the QuantStudio™12K Flex Real-Time PCR System. Genotyping directly from blood gave a call rate of 99.21%, and an accuracy of 100%, while the purified DNA gave a call rate of 92.49%, and an accuracy of 99.74%. Although no statistical difference was found for these parameters, an F test comparing the standard deviations of the wild type clusters for the two different methods indicated significantly less variation when genotyping directly from blood instead of after DNA purification. To further establish the ability to perform high-throughput qPCR based genotyping directly from blood, 96 individuals of Ashkenazi Jewish decent were genotyped for the same 60 mutations (5,760 genotypes in 5 hours) and resulted in a call rate of 98.38% and a diagnostic accuracy of 99.77%. Conclusion This study shows that accurate qPCR-based high-throughput genotyping can be performed without DNA purification. The direct use of blood may further expedite the entire genotyping process, reduce costs, and avoid tracking errors which can occur during

  2. Predicting Gene Structures from Multiple RT-PCR Tests

    NASA Astrophysics Data System (ADS)

    Kováč, Jakub; Vinař, Tomáš; Brejová, Broňa

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

  3. Quantitative PCR analysis of salivary pathogen burden in periodontitis

    PubMed Central

    Salminen, Aino; Kopra, K. A. Elisa; Hyvärinen, Kati; Paju, Susanna; Mäntylä, Päivi; Buhlin, Kåre; Nieminen, Markku S.; Sinisalo, Juha; Pussinen, Pirkko J.

    2015-01-01

    Our aim was to investigate the value of salivary concentrations of four major periodontal pathogens and their combination in diagnostics of periodontitis. The Parogene study included 462 dentate subjects (mean age 62.9 ± 9.2 years) with coronary artery disease (CAD) diagnosis who underwent an extensive clinical and radiographic oral examination. Salivary levels of four major periodontal bacteria were measured by quantitative real-time PCR (qPCR). Median salivary concentrations of Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia, as well as the sum of the concentrations of the four bacteria, were higher in subjects with moderate to severe periodontitis compared to subjects with no to mild periodontitis. Median salivary Aggregatibacter actinomycetemcomitans concentrations did not differ significantly between the subjects with no to mild periodontitis and subjects with moderate to severe periodontitis. In logistic regression analysis adjusted for age, gender, diabetes, and the number of teeth and implants, high salivary concentrations of P. gingivalis, T. forsythia, and P. intermedia were significantly associated with moderate to severe periodontitis. When looking at different clinical and radiographic parameters of periodontitis, high concentrations of P. gingivalis and T. forsythia were significantly associated with the number of 4–5 mm periodontal pockets, ≥6 mm pockets, and alveolar bone loss (ABL). High level of T. forsythia was associated also with bleeding on probing (BOP). The combination of the four bacteria, i.e., the bacterial burden index, was associated with moderate to severe periodontitis with an odds ratio (OR) of 2.40 (95% CI 1.39–4.13). When A. actinomycetemcomitans was excluded from the combination of the bacteria, the OR was improved to 2.61 (95% CI 1.51–4.52). The highest OR 3.59 (95% CI 1.94–6.63) was achieved when P. intermedia was further excluded from the combination and only the levels of P. gingivalis and

  4. A High Malaria Prevalence Identified by PCR among Patients with Acute Undifferentiated Fever in India

    PubMed Central

    Haanshuus, Christel Gill; Chandy, Sara; Manoharan, Anand; Vivek, Rosario; Mathai, Dilip; Xena, Deepika; Singh, Ashita; Langeland, Nina; Blomberg, Bjørn; Vasanthan, George; Sitaram, Usha; Appasamy, Jonathan; Nesaraj, Joel; Henry, Anil; Patil, Suvarna; Alvarez-Uria, Gerardo; Armstrong, Lois; Mørch, Kristine

    2016-01-01

    Background Approximately one million malaria cases were reported in India in 2015, based on microscopy. This study aims to assess the malaria prevalence among hospitalised fever patients in India identified by PCR, and to evaluate the performance of routine diagnostic methods. Methods During June 2011-December 2012, patients admitted with acute undifferentiated fever to seven secondary level community hospitals in Assam (Tezpur), Bihar (Raxaul), Chhattisgarh (Mungeli), Maharashtra (Ratnagiri), Andhra Pradesh (Anantapur) and Tamil Nadu (Oddanchatram and Ambur) were included. The malaria prevalence was assessed by polymerase chain reaction (PCR), routine microscopy, and a rapid diagnostic test (RDT) with PCR as a reference method. Results The malaria prevalence by PCR was 19% (268/1412) ranging from 6% (Oddanchatram, South India) to 35% (Ratnagiri, West India). Among malaria positive patients P. falciparum single infection was detected in 46%, while 38% had P. vivax, 11% mixed infections with P. falciparum and P. vivax, and 5% P. malariae. Compared to PCR, microscopy had sensitivity of 29% and specificity of 98%, while the RDT had sensitivity of 24% and specificity of 99%. Conclusions High malaria prevalence was identified by PCR in this cohort. Routine diagnostic methods had low sensitivity compared to PCR. The results suggest that malaria is underdiagnosed in rural India. However, low parasitaemia controlled by immunity may constitute a proportion of PCR positive cases, which calls for awareness of the fact that other pathogens could be responsible for the febrile disease in submicroscopic malaria. PMID:27389396

  5. Universal reverse-transcriptase real-time PCR for infectious hematopoietic necrosis virus (IHNV)

    USGS Publications Warehouse

    Purcell, Maureen K.; Thompson, Rachel L.; Garver, Kyle A.; Hawley, Laura M.; Batts, William N.; Sprague, Laura; Sampson, Corie; Winton, James R.

    2013-01-01

    Infectious hematopoietic necrosis virus (IHNV) is an acute pathogen of salmonid fishes in North America, Europe and Asia and is reportable to the World Organization for Animal Health (OIE). Phylogenetic analysis has identified 5 major virus genogroups of IHNV worldwide, designated U, M, L, E and J; multiple subtypes also exist within those genogroups. Here, we report the development and validation of a universal IHNV reverse-transcriptase real-time PCR (RT-rPCR) assay targeting the IHNV nucleocapsid (N) gene. Properties of diagnostic sensitivity (DSe) and specificity (DSp) were defined using laboratory-challenged steelhead trout Oncorhynchus mykiss, and the new assay was compared to the OIE-accepted conventional PCR test and virus isolation in cell culture. The IHNV N gene RT-rPCR had 100% DSp and DSe and a higher estimated diagnostic odds ratio (DOR) than virus culture or conventional PCR. The RT-rPCR assay was highly repeatable within a laboratory and highly reproducible between laboratories. Field testing of the assay was conducted on a random sample of juvenile steelhead collected from a hatchery raceway experiencing an IHN epizootic. The RT-rPCR detected a greater number of positive samples than cell culture and there was 40% agreement between the 2 tests. Overall, the RT-rPCR assay was highly sensitive, specific, repeatable and reproducible and is suitable for use in a diagnostic setting.

  6. A method for the simulation of normal, carrier and affected controls for PCR-RFLP screening of a genetic disease in dairy cattle.

    PubMed

    Mukhopadhyaya, P N; Mehta, H H; Rathod, R N

    2000-12-01

    A technique is described that may be used to create in vitro mutations in PCR templates to generate affected and carrier controls for diagnostic testing when DNA from such individuals is not easily obtained. The method is demonstrated for a PCR-RFLP diagnostic test of the genetic disorder BLAD (Bovine Leukocyte Adhesion Deficiency). PMID:11090268

  7. Gas Research Institute improved fracturing. Unconventional natural gas program, eastern devonian shales diagnostic program: Black No. 1 well experiment results. Third quarterly report, October 1979-December 1979

    SciTech Connect

    Schuster, C.L.

    1980-02-01

    During the last quarter of 1979, Sandia National Laboratories participated in an experiment with Thurlow Weed and Associates and the Morgantown Energy Technology Center. This Devonian Shale gas stimulation experiment was conducted in an area north of Columbus, Ohio. One purpose of the experiment was to apply the diagnostic instrumentation that is available for fracture mapping and characterization to increase our understanding of the stimulation technique. The induced fracture apparently followed a pre-existing fracture vertically from the borehole with an orientation of the N 62/sup 0/ E and in the latter stages of the stimulation turned into a shallower horizontal fracture. This fracture behavior was confirmed by several diagnostic analyses and demonstrates the insight that can be gained by fully instrumented stimulation experiments.

  8. Magnetic separation techniques in diagnostic microbiology.

    PubMed Central

    Olsvik, O; Popovic, T; Skjerve, E; Cudjoe, K S; Hornes, E; Ugelstad, J; Uhlén, M

    1994-01-01

    The principles of magnetic separation aided by antibodies or other specific binding molecules have been used for isolation of specific viable whole organisms, antigens, or nucleic acids. Whereas growth on selective media may be helpful in isolation of a certain bacterial species, immunomagnetic separation (IMS) technology can isolate strains possessing specific and characteristic surface antigens. Further separation, cultivation, and identification of the isolate can be performed by traditional biochemical, immunologic, or molecular methods. PCR can be used for amplification and identification of genes of diagnostic importance for a target organism. The combination of IMS and PCR reduces the assay time to several hours while increasing both specificity and sensitivity. Use of streptavidin-coated magnetic beads for separation of amplified DNA fragments, containing both biotin and a signal molecule, has allowed for the conversion of the traditional PCR into an easy-to-read microtiter plate format. The bead-bound PCR amplicons can also easily be sequenced in an automated DNA sequencer. The latter technique makes it possible to obtain sequence data of 300 to 600 bases from 20 to 30 strains, starting with clinical samples, within 12 to 24 h. Sequence data can be used for both diagnostic and epidemiologic purposes. IMS has been demonstrated to be a useful method in diagnostic microbiology. Most recent publications describe IMS as a method for enhancing the specificity and sensitivity of other detection systems, such as PCR, and providing considerable savings in time compared with traditional diagnostic systems. The relevance to clinical diagnosis has, however, not yet been fully established for all of these new test principles. In the case of PCR, for example, the presence of specific DNA in a food sample does not demonstrate the presence of a live organism capable of inducing a disease. However, all tests offering increased sensitivity and specificity of detection

  9. The Orthology Clause in the Next Generation Sequencing Era: Novel Reference Genes Identified by RNA-seq in Humans Improve Normalization of Neonatal Equine Ovary RT-qPCR Data

    PubMed Central

    Scarlet, Dragos; Ertl, Reinhard; Aurich, Christine; Steinborn, Ralf

    2015-01-01

    Background Vertebrate evolution is accompanied by a substantial conservation of transcriptional programs with more than a third of unique orthologous genes showing constrained levels of expression. Moreover, there are genes and exons exhibiting excellent expression stability according to RNA-seq data across a panel of eighteen tissues including the ovary (Human Body Map 2.0). Results We hypothesized that orthologs of these exons would also be highly uniformly expressed across neonatal ovaries of the horse, which would render them appropriate reference genes (RGs) for normalization of reverse transcription quantitative PCR (RT-qPCR) data in this context. The expression stability of eleven novel RGs (C1orf43, CHMP2A, EMC7, GPI, PSMB2, PSMB4, RAB7A, REEP5, SNRPD3, VCP and VPS29) was assessed by RT-qPCR in ovaries of seven neonatal fillies and compared to that of the expressed repetitive element ERE-B, two universal (OAZ1 and RPS29) and four traditional RGs (ACTB, GAPDH, UBB and B2M). Expression stability analyzed with the software tool RefFinder top ranked the normalization factor constituted of the genes SNRPD3 and VCP, a gene pair that is not co-expressed according to COEXPRESdb and GeneMANIA. The traditional RGs GAPDH, B2M, ACTB and UBB were only ranked 3rd and 12th to 14th, respectively. Conclusions The functional diversity of the novel RGs likely facilitates expression studies over a wide range of physiological and pathological contexts related to the neonatal equine ovary. In addition, this study augments the potential for RT-qPCR-based profiling of human samples by introducing seven new human RG assays (C1orf43, CHMP2A, EMC7, GPI, RAB7A, VPS29 and UBB). PMID:26536597

  10. Comparative analysis of human papillomavirus detection by PCR and non-isotopic in situ hybridisation.

    PubMed Central

    Herrington, C S; Anderson, S M; Bauer, H M; Troncone, B; de Angelis, M L; Noell, H; Chimera, J A; Van Eyck, S L; McGee, J O

    1995-01-01

    AIMS--To assess the relative diagnostic performance of the polymerase chain reaction (PCR) and non-isotopic in situ hybridisation (NISH) and to correlate these data with cytopathological assessment. METHODS--Paired analysis of human papillomavirus (HPV) detection was performed by PCR and NISH on exfoliated cervical cells from 122 women attending a routine gynaecological examination. PCR amplification followed by generic and HPV type specific hybridisation was compared with NISH on a parallel cervical smear. RESULTS--Overall, 32 cases were positive by NISH and 61 positive by PCR. Of the 105 cases in which both PCR and NISH were interpretable, 76 (26%) were normal smears, 20 of which were HPV positive by NISH and 37 (49%) by PCR. Of 17 borderline smears, two were NISH positive and 12 PCR positive. Eight