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Sample records for improved diagnostic pcr

  1. COLD-PCR: improving the sensitivity of molecular diagnostics assays

    PubMed Central

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

    2011-01-01

    The detection of low-abundance DNA variants or mutations is of particular interest to medical diagnostics, individualized patient treatment and cancer prognosis; however, detection sensitivity for low-abundance variants is a pronounced limitation of most currently available molecular assays. We have recently developed coamplification at lower denaturation temperature-PCR (COLD-PCR) to resolve this limitation. This novel form of PCR selectively amplifies low-abundance DNA variants from mixtures of wild-type and mutant-containing (or variant-containing) sequences, irrespective of the mutation type or position on the amplicon, by using a critical denaturation temperature. The use of a lower denaturation temperature in COLD-PCR results in selective denaturation of amplicons with mutation-containing molecules within wild-type mutant heteroduplexes or with a lower melting temperature. COLD-PCR can be used in lieu of conventional PCR in several molecular applications, thus enriching the mutant fraction and improving the sensitivity of downstream mutation detection by up to 100-fold. PMID:21405967

  2. 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.

  3. PCR Improves Diagnostic Yield from Lung Aspiration in Malawian Children with Radiologically Confirmed Pneumonia

    PubMed Central

    Carrol, Enitan D.; Mankhambo, Limangeni A.; Guiver, Malcolm; Banda, Daniel L.; Denis, Brigitte; Dove, Winifred; Jeffers, Graham; Molyneux, Elizabeth M.; Molyneux, Malcolm E.; Graham, Stephen M.

    2011-01-01

    Background Accurate data on childhood pneumonia aetiology are essential especially from regions where mortality is high, in order to inform case-management guidelines and the potential of prevention strategies such as bacterial conjugate vaccines. Yield from blood culture is low, but lung aspirate culture provides a higher diagnostic yield. We aimed to determine if diagnostic yield could be increased further by polymerase chain reaction (PCR) detection of bacteria (Streptococcus pneumoniae and Haemophilus influenzae b) and viruses in lung aspirate fluid. Methods A total of 95 children with radiological focal, lobar or segmental consolidation had lung aspirate performed and sent for bacterial culture and for PCR for detection of bacteria, viruses and Pneumocystis jirovecii. In children with a pneumococcal aetiology, pneumococcal bacterial loads were calculated in blood and lung aspirate fluid. Results Blood culture identified a bacterial pathogen in only 8 patients (8%). With the addition of PCR on lung aspirate samples, causative pathogens (bacterial, viral, pneumocystis) were identified singly or as co-infections in 59 children (62%). The commonest bacterial organism was S.pneumoniae (41%), followed by H. influenzae b (6%), and the commonest virus identified was adenovirus (16%), followed by human bocavirus (HBoV) (4%), either as single or co-infection. Conclusions In a select group of African children, lung aspirate PCR significantly improves diagnostic yield. Our study confirms a major role of S.pneumoniae and viruses in the aetiology of childhood pneumonia in Africa. PMID:21695128

  4. PCR assay for improved diagnostics of epitheliotropic disease virus (EEDV) in lake trout Salvelinus namaycush.

    PubMed

    Kurobe, T; Marcquenski, S; Hedrick, R P

    2009-03-09

    Epizootic epitheliotropic disease virus (EEDV) has caused catastrophic losses among hatchery-reared juvenile lake trout Salvelinus namaycush since the early 1980s and remains a major impediment to lake trout restoration in the Great Lakes basin of the USA. Although EEDV has been tentatively designated as a herpesvirus based upon morphological criteria, further characterization of the virus and development of improved detection methods have been hampered by the inability to propagate the virus in cell culture. Recently obtained sequence data for a region of the putative terminase gene from EEDV as well as the related Salmonid herpesvirus 1 and 2 have permitted the development of a polymerase chain reaction (PCR) assay for specific detection of EEDV. The new EEDV PCR demonstrated both an excellent analytic sensitivity and specificity and detected viral DNA as present in the skin of lake trout during periods of active viral outbreaks. In addition, EEDV DNA was detected among healthy appearing juveniles and in the ovarian fluids of spawning adults. Here we describe the development and initial validation steps of the EEDV PCR as a replacement for current diagnostic methods that require virus extraction from the skin, partial purification by isopycnic centrifugation, and visualization of negatively-stained virions by electron microscopy.

  5. The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis.

    PubMed

    Devonshire, Alison S; O'Sullivan, Denise M; Honeyborne, Isobella; Jones, Gerwyn; Karczmarczyk, Maria; Pavšič, Jernej; Gutteridge, Alice; Milavec, Mojca; Mendoza, Pablo; Schimmel, Heinz; Van Heuverswyn, Fran; Gorton, Rebecca; Cirillo, Daniela Maria; Borroni, Emanuele; Harris, Kathryn; Barnard, Marinus; Heydenrych, Anthenette; Ndusilo, Norah; Wallis, Carole L; Pillay, Keshree; Barry, Thomas; Reddington, Kate; Richter, Elvira; Mozioğlu, Erkan; Akyürek, Sema; Yalçınkaya, Burhanettin; Akgoz, Muslum; Žel, Jana; Foy, Carole A; McHugh, Timothy D; Huggett, Jim F

    2016-08-03

    Real-time PCR (qPCR) based methods, such as the Xpert MTB/RIF, are increasingly being used to diagnose tuberculosis (TB). While qualitative methods are adequate for diagnosis, the therapeutic monitoring of TB patients requires quantitative methods currently performed using smear microscopy. The potential use of quantitative molecular measurements for therapeutic monitoring has been investigated but findings have been variable and inconclusive. The lack of an adequate reference method and reference materials is a barrier to understanding the source of such disagreement. Digital PCR (dPCR) offers the potential for an accurate method for quantification of specific DNA sequences in reference materials which can be used to evaluate quantitative molecular methods for TB treatment monitoring. To assess a novel approach for the development of quality assurance materials we used dPCR to quantify specific DNA sequences in a range of prototype reference materials and evaluated accuracy between different laboratories and instruments. The materials were then also used to evaluate the quantitative performance of qPCR and Xpert MTB/RIF in eight clinical testing laboratories. dPCR was found to provide results in good agreement with the other methods tested and to be highly reproducible between laboratories without calibration even when using different instruments. When the reference materials were analysed with qPCR and Xpert MTB/RIF by clinical laboratories, all laboratories were able to correctly rank the reference materials according to concentration, however there was a marked difference in the measured magnitude. TB is a disease where the quantification of the pathogen could lead to better patient management and qPCR methods offer the potential to rapidly perform such analysis. However, our findings suggest that when precisely characterised materials are used to evaluate qPCR methods, the measurement result variation is too high to determine whether molecular quantification

  6. Improved detection of Burkholderia pseudomallei from non-blood clinical specimens using enrichment culture and PCR: narrowing diagnostic gap in resource-constrained settings.

    PubMed

    Tellapragada, Chaitanya; Shaw, Tushar; D'Souza, Annet; Eshwara, Vandana Kalwaje; Mukhopadhyay, Chiranjay

    2017-07-01

    To evaluate the diagnostic utility of enrichment culture and PCR for improved case detection rates of non-bacteraemic form of melioidosis in limited resource settings. Clinical specimens (n = 525) obtained from patients presenting at a tertiary care hospital of South India with clinical symptoms suggestive of community-acquired pneumonia, lower respiratory tract infections, superficial or internal abscesses, chronic skin ulcers and bone or joint infections were tested for the presence of Burkholderia pseudomallei using conventional culture (CC), enrichment culture (EC) and PCR. Sensitivity, specificity, positive and negative predictive values of CC and PCR were initially deduced using EC as the gold standard method. Further, diagnostic accuracies of all the three methods were analysed using Bayesian latent class modelling (BLCM). Detection rates of B. pseudomallei using CC, EC and PCR were 3.8%, 5.3% and 6%, respectively. Diagnostic sensitivities and specificities of CC and PCR were 71.4, 98.4% and 100 and 99.4%, respectively in comparison with EC as the gold standard test. With Bayesian latent class modelling, EC and PCR demonstrated sensitivities of 98.7 and 99.3%, respectively, while CC showed a sensitivity of 70.3% for detection of B. pseudomallei. An increase of 1.6% (95% CI: 1.08-4.32%) in the case detection rate of melioidosis was observed in the study population when EC and/or PCR were used in adjunct to the conventional culture technique. Our study findings underscore the diagnostic superiority of enrichment culture and/or PCR over conventional microbiological culture for improved case detection of melioidosis from non-blood clinical specimens. © 2017 John Wiley & Sons Ltd.

  7. Overcoming inhibition in real-time diagnostic PCR.

    PubMed

    Hedman, Johannes; Rådström, Peter

    2013-01-01

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

  8. Modification of two capripoxvirus quantitative real-time PCR assays to improve diagnostic sensitivity and include beta-actin as an internal positive control.

    PubMed

    Das, Amaresh; Deng, Ming Y; Babiuk, Shawn; McIntosh, Michael T

    2017-05-01

    Capripoxviruses (CaPVs), consisting of Sheeppox virus (SPV), Goatpox virus (GPV), and Lumpy skin disease virus (LSDV) species, cause economically significant diseases in sheep, goats, and cattle, respectively. Quantitative real-time polymerase chain reaction (qPCR) assays are routinely used for rapid detection of CaPVs in surveillance and outbreak management programs. We further modified and optimized 2 previously published CaPV qPCR assays, referred to as the Balinsky and Bowden assays, by changing commercial PCR reagents used in the tests. The modified assays displayed 100% analytical specificity and showed no apparent changes in analytical sensitivities for detection of CaPVs compared with the original assays. Diagnostic sensitivities, assessed using 50 clinical reference samples from experimentally infected sheep, goats, and cattle, improved from 82% to 92% for the modified Balinsky assay and from 58% to 82% for the modified Bowden assay. The modified qPCR assays were multiplexed for detection of beta-actin as an indicator for potential false-negative results. The multiplex modified qPCR assays exhibited the same diagnostic sensitivities as the singleplex assays suggesting their utility in the detection of CaPVs.

  9. The potential advantages of digital PCR for clinical virology diagnostics.

    PubMed

    Hall Sedlak, Ruth; Jerome, Keith R

    2014-05-01

    Digital PCR (dPCR), a new nucleic acid amplification technology, offers several potential advantages over real-time or quantitative PCR (qPCR), the current workhorse of clinical molecular virology diagnostics. Several studies have demonstrated dPCR assays for human cytomegalovirus or HIV, which give more precise and reproducible results than qPCR assays without sacrificing sensitivity. Here we review the literature comparing dPCR and qPCR performance in viral molecular diagnostic assays and offer perspective on the future of dPCR in clinical virology diagnostics.

  10. 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.

  11. 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

  12. 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

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

    PubMed

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

    2004-05-20

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

  14. Applications of competitor RNA in diagnostic reverse transcription-PCR.

    PubMed

    Kleiboeker, Steven B

    2003-05-01

    Detection of RNA viruses by reverse transcription (RT)-PCR has proven to be a useful approach for the diagnosis of infections caused by many viral pathogens. However, adequate controls are required for each step of the RT-PCR protocol to ensure the accuracies of diagnostic test results. Heterologous competitor RNA can be used as a control for a number of different aspects of diagnostic RT-PCR. Competitor RNA can be applied to assessments of the efficiency of RNA recovery during extraction procedures, detection of endogenous RT-PCR inhibitors that could lead to false-negative results, and quantification of viral template in samples used for diagnosis; competitor RNA can also be used as a positive control for the RT-PCR. In the present study, heterologous competitor RNA was synthesized by a method that uses two long oligonucleotide primers containing primer binding sites for RT-PCR amplification of porcine reproductive and respiratory syndrome virus or West Nile virus. Amplification of the competitor RNA by RT-PCR resulted in a product that was easily distinguished from the amplification product of viral RNA by agarose gel electrophoresis. Assessment of a variety of RNA samples prepared from routine submissions to a veterinary diagnostic laboratory found that either partial or complete inhibition of the RT-PCR could be demonstrated for approximately 20% of the samples. When inhibition was detected, either dilution of the sample or RNA extraction by an alternative protocol proved successful in eliminating the source of inhibition.

  15. Real time PCR in childhood tuberculosis: a valuable diagnostic tool.

    PubMed

    Dayal, Rajeshwar; Kashyap, Haripal; Pounikar, Gajanand; Kamal, Raj; Yadav, Neeraj Kumar; Singh, Manoj Kumar; Chauhan, Devendra Singh; Goyal, Ankur

    2015-02-01

    The present study was conducted to detect and quantitate Mycobacterium tuberculosis from various body fluid specimens of cases of tuberculosis by real time PCR technique and compare results with conventional PCR technique and culture. One hundred fifteen children (<18 y) with tuberculosis (diagnosed as per IAP guidelines) and 32 disease matched controls from the Department of Pediatrics, S.N. Medical College, Agra, were included in the study. Different body fluids (CSF, gastric aspirate, pleural fluid, ascitic fluid and lymph node aspirate) were subjected to culture, conventional PCR targeting insertion sequence 1S6110 and Real time PCR targeting 16srRNA of Mycobacterium tuberculosis. Real time PCR showed significantly better results than culture in all body fluids (p < 0.05). It was superior to conventional PCR in CSF (p < 0.05) but showed comparable results in gastric aspirate, pleural fluid, ascitic fluid and lymph node aspirate (p > 0.05). Hence, real time PCR is a promising diagnostic tool for childhood tuberculosis, particularly tubercular meningitis.

  16. Browsed twig environmental DNA: diagnostic PCR to identify ungulate species.

    PubMed

    Nichols, Ruth V; Königsson, Helena; Danell, Kjell; Spong, Göran

    2012-11-01

    Ungulate browsing can have a strong effect on ecological processes by affecting plant community structure and composition, with cascading effects on nutrient cycling and animal communities. However, in the absence of direct observations of foraging, species-specific foraging behaviours are difficult to quantify. We therefore know relatively little about foraging competition and species-specific browsing patterns in systems with several browsers. However, during browsing, a small amount of saliva containing buccal cells is deposited at the bite site, providing a source of environmental DNA (eDNA) that can be used for species identification. Here, we describe extraction and PCR protocols for a browser species diagnostic kit. Species-specific primers for mitochondrial DNA were optimized and validated using twigs browsed by captive animals. A time series showed that about 50% of the samples will amplify up to 12 weeks after the browsing event and that some samples amplify up to 24 weeks after browsing (12.5%). Applied to samples of natural browsing from an area where moose (Alces alces), roe deer (Capreolus capreolus), fallow deer (Cervus dama) and red deer (Cervus elaphus) are sympatric, amplification success reached 75%. This method promises to greatly improve our understanding of multispecies browsing systems without the need for direct observations. © 2012 Blackwell Publishing Ltd.

  17. 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

  18. 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.

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

    PubMed

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

    2017-03-15

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

  20. 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.

  1. 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

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

    PubMed

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

    2012-01-01

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

  3. Improved PCR performance using template DNA from formalin-fixed and paraffin-embedded tissues by overcoming PCR inhibition.

    PubMed

    Dietrich, Dimo; Uhl, Barbara; Sailer, Verena; Holmes, Emily Eva; Jung, Maria; Meller, Sebastian; Kristiansen, Glen

    2013-01-01

    Formalin-fixed and paraffin-embedded (FFPE) tissues represent a valuable source for biomarker studies and clinical routine diagnostics. However, they suffer from degradation of nucleic acids due to the fixation process. Since genetic and epigenetic studies usually require PCR amplification, this degradation hampers its use significantly, impairing PCR robustness or necessitating short amplicons. In routine laboratory medicine a highly robust PCR performance is mandatory for the clinical utility of genetic and epigenetic biomarkers. Therefore, methods to improve PCR performance using DNA from FFPE tissue are highly desired and of wider interest. The effect of template DNA derived from FFPE tissues on PCR performance was investigated by means of qPCR and conventional PCR using PCR fragments of different sizes. DNA fragmentation was analyzed via agarose gel electrophoresis. This study showed that poor PCR amplification was partly caused by inhibition of the DNA polymerase by fragmented DNA from FFPE tissue and not only due to the absence of intact template molecules of sufficient integrity. This PCR inhibition was successfully minimized by increasing the polymerase concentration, dNTP concentration and PCR elongation time thereby allowing for the robust amplification of larger amplicons. This was shown for genomic template DNA as well as for bisulfite-converted template DNA required for DNA methylation analyses. In conclusion, PCR using DNA from FFPE tissue suffers from inhibition which can be alleviated by adaptation of the PCR conditions, therefore allowing for a significant improvement of PCR performance with regard to variability and the generation of larger amplicons. The presented solutions to overcome this PCR inhibition are of tremendous value for clinical chemistry and laboratory medicine.

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

    PubMed

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

    2016-12-01

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

  5. Educational Improvement Act: Diagnostic Testing.

    ERIC Educational Resources Information Center

    Kentucky State Dept. of Education, Frankfort. Office of Research and Planning.

    The Kentucky Department of Education has a responsibility to provide technical assistance and consultative services to local school districts. Descriptions of the state selected diagnostic reading test, the Prescriptive Reading Inventory (PRI) and the diagnostic math test, the Diagnostic Math Inventory (DMI), are explained. Each school district in…

  6. A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything

    PubMed Central

    Kralik, Petr; Ricchi, Matteo

    2017-01-01

    Real time PCR (quantitative PCR, qPCR) is now a well-established method for the detection, quantification, and typing of different microbial agents in the areas of clinical and veterinary diagnostics and food safety. Although the concept of PCR is relatively simple, there are specific issues in qPCR that developers and users of this technology must bear in mind. These include the use of correct terminology and definitions, understanding of the principle of PCR, difficulties with interpretation and presentation of data, the limitations of qPCR in different areas of microbial diagnostics and parameters important for the description of qPCR performance. It is not our intention in this review to describe every single aspect of qPCR design, optimization, and validation; however, it is our hope that this basic guide will help to orient beginners and users of qPCR in the use of this powerful technique. PMID:28210243

  7. Statistical diagnostics emerging from external quality control of real-time PCR.

    PubMed

    Marubini, E; Verderio, P; Raggi, Casini C; Pazzagli, M; Orlando, C

    2004-01-01

    Besides the application of conventional qualitative PCR as a valuable tool to enrich or identify specific sequences of nucleic acids, a new revolutionary technique for quantitative PCR determination has been introduced recently. It is based on real-time detection of PCR products revealed as a homogeneous accumulating signal generated by specific dyes. However, as far as we know, the influence of the variability of this technique on the reliability of the quantitative assay has not been thoroughly investigated. A national program of external quality assurance (EQA) for real-time PCR determination involving 42 Italian laboratories has been developed to assess the analytical performance of real-time PCR procedures. Participants were asked to perform a conventional experiment based on the use of an external reference curve (standard curve) for real-time detection of three cDNA samples with different concentrations of a specific target. In this paper the main analytical features of the standard curve have been investigated in an attempt to produce statistical diagnostics emerging from external quality control. Specific control charts were drawn to help biochemists take technical decisions aimed at improving the performance of their laboratories. Overall, our results indicated a subset of seven laboratories whose performance appeared to be markedly outside the limits for at least one of the standard curve features investigated. Our findings suggest the usefulness of the approach presented here for monitoring the heterogeneity of results produced by different laboratories and for selecting those laboratories that need technical advice on their performance.

  8. 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

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

    PubMed

    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.

  10. RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers

    PubMed Central

    2011-01-01

    Background The polymerase chain reaction (PCR) is commonly used to detect the presence of nucleic acid sequences both in research and diagnostic settings. While high specificity is often achieved, biological requirements sometimes necessitate that primers are placed in suboptimal locations which lead to problems with the formation of primer dimers and/or misamplification of homologous sequences. Results Pyrococcus abyssi (P.a.) RNase H2 was used to enable PCR to be performed using blocked primers containing a single ribonucleotide residue which are activated via cleavage by the enzyme (rhPCR). Cleavage occurs 5'-to the RNA base following primer hybridization to the target DNA. The requirement of the primer to first hybridize with the target sequence to gain activity eliminates the formation of primer-dimers and greatly reduces misamplification of closely related sequences. Mismatches near the scissile linkage decrease the efficiency of cleavage by RNase H2, further increasing the specificity of the assay. When applied to the detection of single nucleotide polymorphisms (SNPs), rhPCR was found to be far more sensitive than standard allele-specific PCR. In general, the best discrimination occurs when the mismatch is placed at the RNA:DNA base pair. Conclusion rhPCR eliminates the formation of primer dimers and markedly improves the specificity of PCR with respect to off-target amplification. These advantages of the assay should find utility in challenging qPCR applications such as genotyping, high level multiplex assays and rare allele detection. PMID:21831278

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

    PubMed

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

    2013-02-01

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

  12. Diagnostic Approaches For Paediatric Tuberculosis By Use Of Different Specimen Types, Culture Methods, And Pcr

    PubMed Central

    Oberhelman, Richard A.; Soto-Castellares, Giselle; Gilman, Robert H.; Caviedes, Luz; Castillo, Maria E.; Kolevic, Lenka; Pino, Trinidad Del; Saito, Mayuko; Salazar-Lindo, Eduardo; Negron, Eduardo; Montenegro, Sonia; Laguna-Torres, V. Alberto; Moore, David A. J.; Evans, Carlton A.

    2010-01-01

    children treated for presumed PTB were culture-negative in all specimens, MODS culture of duplicate GAs considerably improved recovery by culture. PCR was insufficiently sensitive or specific for routine diagnostic use, but in high risk children duplicate GA PCR provided same-day identification of half of all culture-positive cases. Collection of duplicate GA specimens from high-risk children for MODS culture was the optimal diagnostic test. PMID:20656559

  13. 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

  14. 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.

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

    PubMed

    Purcell, Maureen K; Getchell, Rodman G; McClure, Carol A; Garver, Kyle A

    2011-09-01

    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.

  16. 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.

  17. A Pilot Study for the Evaluation of PCR as a Diagnostic Tool in Patients with Suspected Dermatophytoses

    PubMed Central

    Sharma, Robin; Gupta, Samiksha; Asati, Dinesh P.; Karuna, T.; Purwar, Shashank; Biswas, Debasis

    2017-01-01

    Context: The conventional diagnostic tools for dermatophytoses suffer from several limitations including low sensitivity, specificity, and long turn-around-time. Aims: The present study was, therefore, performed to evaluate the performance of a polymerase chain reaction (PCR)-based method for the diagnosis of this condition. Settings and Design: The study was conducted in the Dermatology outpatient department of a tertiary care teaching hospital in central India over a period of 3 months from July to September 2015. Materials and Methods: Forty participants, including 25 cases and 15 controls, were recruited in this observational study. Direct microscopy and fungal culture were performed from skin scrapings and nail clippings collected from the participants. PCR was also performed to amplify the chitin synthase 1 and internal transcribed spacer 2 genes from DNA samples extracted from the same clinical materials, using the method reported by Brillowska-Dabrowska et al. The diagnostic performance of fungal culture and PCR was compared using OpenEpi software. Results: We observed a significant male predominance among patients with dermatophytoses. The sensitivity of fungal culture and dermatophyte PCR to diagnose dermatophytoses was 24% and 48%, respectively, whereas the specificity of the two assays was 100% and 93.3%, respectively. The likelihood ratio of a positive PCR assay was 7.2 and the negative likelihood ratio was 0.5. PCR assay also delivered a significant shortening of the time-to-diagnosis, with the mean turn-around-time being 8 hours and 14 days for PCR and culture, respectively. Conclusion: This study, thus, highlights the potential merits of the dermatophyte PCR assay in achieving a rapid diagnosis of dermatophytoses and underscores its utility as a complementary test to improve the sensitivity of the conventional diagnostic tools for this condition, as well as to reliably differentiate this condition from other similar skin conditions. PMID:28584753

  18. A Pilot Study for the Evaluation of PCR as a Diagnostic Tool in Patients with Suspected Dermatophytoses.

    PubMed

    Sharma, Robin; Gupta, Samiksha; Asati, Dinesh P; Karuna, T; Purwar, Shashank; Biswas, Debasis

    2017-01-01

    The conventional diagnostic tools for dermatophytoses suffer from several limitations including low sensitivity, specificity, and long turn-around-time. The present study was, therefore, performed to evaluate the performance of a polymerase chain reaction (PCR)-based method for the diagnosis of this condition. The study was conducted in the Dermatology outpatient department of a tertiary care teaching hospital in central India over a period of 3 months from July to September 2015. Forty participants, including 25 cases and 15 controls, were recruited in this observational study. Direct microscopy and fungal culture were performed from skin scrapings and nail clippings collected from the participants. PCR was also performed to amplify the chitin synthase 1 and internal transcribed spacer 2 genes from DNA samples extracted from the same clinical materials, using the method reported by Brillowska-Dabrowska et al. The diagnostic performance of fungal culture and PCR was compared using OpenEpi software. We observed a significant male predominance among patients with dermatophytoses. The sensitivity of fungal culture and dermatophyte PCR to diagnose dermatophytoses was 24% and 48%, respectively, whereas the specificity of the two assays was 100% and 93.3%, respectively. The likelihood ratio of a positive PCR assay was 7.2 and the negative likelihood ratio was 0.5. PCR assay also delivered a significant shortening of the time-to-diagnosis, with the mean turn-around-time being 8 hours and 14 days for PCR and culture, respectively. This study, thus, highlights the potential merits of the dermatophyte PCR assay in achieving a rapid diagnosis of dermatophytoses and underscores its utility as a complementary test to improve the sensitivity of the conventional diagnostic tools for this condition, as well as to reliably differentiate this condition from other similar skin conditions.

  19. Schistosoma mansoni: a diagnostic approach to detect acute schistosomiasis infection in a murine model by PCR.

    PubMed

    Sandoval, Nidia; Siles-Lucas, Mar; Lopez Aban, Julio; Pérez-Arellano, José Luis; Gárate, Teresa; Muro, Antonio

    2006-10-01

    Schistosomiasis represents an increasing problem in non-endemic areas, due to the growing number of immigrants and to tourists contracting this disease in "off-the-beaten-track" tourism. Acute schistosomiasis is not diagnosed early due to the lack of diagnostic tools that are sufficiently sensitive enough to detect the parasite during the first weeks of infection. We have developed a diagnostic approach based on the detection of parasite DNA by polymerase chain reaction (PCR) in urine, comparing the performance of this new approach with the two currently used schistosomiasis diagnostic tools (Kato-Katz and ELISA) and the PCR in stool samples. This comparison was done in a Schistosoma mansoni murine experimental model, which permits follow up of the parasite from the acute to the chronic stage of infection. Our results suggest that this new PCR-based approach could be useful for the detection of acute schistosomiasis in easy-to-handle clinical samples such the urine.

  20. 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

  1. [Quality improvement of medical diagnostic laboratories].

    PubMed

    Horváth, Andrea Rita; Endröczi, Elemér; Mikó, Tivadar

    2003-07-13

    Service quality in medical laboratories is influenced by a number of variables. Medical laboratories have long recognized the need for total quality management that incorporates the continuous improvement of all stages, such as the pre-analytical, analytical and post-analytical phases, of the diagnostic process, in addition to the traditional internal and external quality control of analytical procedures. Based on national and international experience, continuous improvement of quality and its external assessment are of high priority in order to guarantee a reliable, effective and cost-effective diagnostic service. Certification of health care services, according to ISO 9001 standards in Hungarian hospitals, is not sufficient to prove professional competence of medical laboratories, which called for a system of laboratory accreditation. Accreditation is an external professional audit by which an independent accreditation body gives formal recognition that the medical laboratory is competent to provide high quality services that are compliant with rigorous professional standards of best practice. The primary aim of accreditation is the improvement of the quality of diagnostic services by voluntary participation, professional peer review, continuous training and education and compliance with professional standards. In vitro medical laboratories have pioneered quality control and quality assurance in health care. Based on these strengths and traditions, the introduction of the accreditation program of medical laboratories in Hungary is one of the key professional and ethical responsibilities of diagnostic professions, in order to improve the quality, efficiency and effectiveness of laboratory services during the course of Hungary's accession to the European Union.

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

    PubMed

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

    2015-04-01

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

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

    PubMed

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

    2016-01-01

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

  4. 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

  5. [Diagnostic value of R-banding Technique, Dual-color Fluore- scence In Situ Hybridization and Quantitative Real-time PCR in Acute Promyelocytic Leukemia].

    PubMed

    Peng, You-Fan; Liu, Yang; Zhang, Qiong; Zhang, Zhao-Xia

    2015-10-01

    To explore the diagnostic value of R-banding technique (RT), dual-color fluorescence in situ hybridization (D-FISH) and quantitative real-time PCR (RT-PCR) for acute promyelocytic leukemia. The cytogenetic characteristics and PML/RARα fusion gene in 340 patients with suspectable APL were analyzed by using 3 detection methods. MICM (morphology, immunology, cytogenetic and molecular biology) was used as diagnostic standard of APL, and the diagnostic value of RT, D-FISH and RT-PCR was evaluated by comparing the detection results of RT, D-FISH and RT-PCR as well as their combination. For the diagnosis of APL, the sensitivity of RT, D-FISH and RT-PCR was 81.3% (78/96), 95.0% (91/96) and 96.9% (93/96) respectively. RT failed to detect 18 cases, the results of D-FISH showed 5 cases with false positive and 2 cases with false negative, the RT-PCR showed 4 cases with false positive, 3 cases with false negative. The sensitivity and specificity of combined detection of 3 methods were 99.97% and 100% respectively. The 3 detection methods alone all have certain defects for diagnosis of APL, but their combined detection is helpful to improve the definitive diagnostic rate and can decrease misdiagnosis rate and missed diagnostic rate.

  6. PCR diagnostics underestimate the prevalence of avian malaria (Plasmodium relictum) in experimentally-infected passerines.

    PubMed

    Jarvi, Susan I; Schultz, Jeffrey J; Atkinson, Carter T

    2002-02-01

    Several polymerase chain reaction (PCR)-based methods have recently been developed for diagnosing malarial infections in both birds and reptiles, but a critical evaluation of their sensitivity in experimentally-infected hosts has not been done. This study compares the sensitivity of several PCR-based methods for diagnosing avian malaria (Plasmodium relictum) in captive Hawaiian honeycreepers using microscopy and a recently developed immunoblotting technique. Sequential blood samples were collected over periods of up to 4.4 yr after experimental infection and rechallenge to determine both the duration and detectability of chronic infections. Two new nested PCR approaches for detecting circulating parasites based on P. relictum 18S rRNA genes and the thrombospondin-related anonymous protein (TRAP) gene are described. The blood smear and the PCR tests were less sensitive than serological methods for detecting chronic malarial infections. Individually, none of the diagnostic methods was 100% accurate in detecting subpatent infections, although serological methods were significantly more sensitive (97%) than either nested PCR (61-84%) or microscopy (27%). Circulating parasites in chronically infected birds either disappear completely from circulation or to drop to intensities below detectability by nested PCR. Thus, the use of PCR as a sole means of detection of circulating parasites may significantly underestimate true prevalence.

  7. PCR diagnostics underestimate the prevalence of avian malaria (Plasmodium relictum) in experimentally-infected passerines

    USGS Publications Warehouse

    Jarvi, Susan I.; Schultz, Jeffrey J.; Atkinson, Carter T.

    2002-01-01

    Several polymerase chain reaction (PCR)-based methods have recently been developed for diagnosing malarial infections in both birds and reptiles, but a critical evaluation of their sensitivity in experimentally-infected hosts has not been done. This study compares the sensitivity of several PCR-based methods for diagnosing avian malaria (Plasmodium relictum) in captive Hawaiian honeycreepers using microscopy and a recently developed immunoblotting technique. Sequential blood samples were collected over periods of up to 4.4 yr after experimental infection and rechallenge to determine both the duration and detectability of chronic infections. Two new nested PCR approaches for detecting circulating parasites based on P. relictum 18S rRNA genes and the thrombospondin-related anonymous protein (TRAP) gene are described. The blood smear and the PCR tests were less sensitive than serological methods for detecting chronic malarial infections. Individually, none of the diagnostic methods was 100% accurate in detecting subpatent infections, although serological methods were significantly more sensitive (97%) than either nested PCR (61–84%) or microscopy (27%). Circulating parasites in chronically infected birds either disappear completely from circulation or to drop to intensities below detectability by nested PCR. Thus, the use of PCR as a sole means of detection of circulating parasites may significantly underestimate true prevalence.

  8. Improved case confirmation in meningococcal disease with whole blood Taqman PCR

    PubMed Central

    Hackett, S; Carrol, E; Guiver, M; Marsh, J; Sills, J; Thomson, A; Kaczmarski, E; Hart, C

    2002-01-01

    Background: The clinical diagnosis of meningococcal disease (MCD) can be difficult. Non-culture methods like the previous ELISA meningococcal PCR improved case confirmation rates, but were not ideal. A Taqman meningococcal PCR, using DNA extracted from serum (S-Taqman), which has an improved sensitivity compared to the ELISA method in vitro, was introduced into clinical practice in July 1997. A new whole blood DNA extraction method for Taqman (WB-Taqman) was introduced in September 1999. Aims: To determine the degree of improvement in the confirmation rate in clinically diagnosed MCD, following the introduction of WB-Taqman. Methods: A total of 192 patients (WB-Taqman) with possible or probable MCD, including those admitted to our paediatric intensive care unit, were studied. Admission EDTA samples obtained were sent for bacterial DNA detection at the Meningococcal Reference Unit (MRU), Manchester. These patients were compared to 319 patients with possible and probable MCD, seen at the same hospital prior to the introduction of WB-Taqman. Results: Following the introduction of WB-Taqman, 82 of the 95 probable cases (88%) had a positive meningococcal PCR result. This gives a diagnostic sensitivity and specificity for WB-Taqman of 87% and 100% respectively. Following WB-Taqman all blood culture positive patients were also PCR positive. Confirmation of cases by PCR rose from 47% (S-Taqman, n = 166) to 88% (WB-Taqman). When all confirmatory tests were included, case confirmation increased from 72% (S-Taqman) to 94% (WB-Taqman). Conclusion: The sensitivity of PCR in confirming clinical MCD has improved significantly with this new method. The gold standard for confirming cases of MCD is now the WB-Taqman PCR. PMID:12023187

  9. 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.

  10. Digital Mammography: Improvements in Breast Cancer Diagnostic

    NASA Astrophysics Data System (ADS)

    Montaño Zetina, Luis Manuel

    2006-01-01

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

  11. 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.

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2008-06-01

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

  14. 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.

  15. 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.

  16. Diagnostic Performance of Filter Paper Lesion Impression PCR for Secondarily Infected Ulcers and Nonulcerative Lesions Caused by Cutaneous Leishmaniasis▿

    PubMed Central

    Boggild, Andrea K.; Ramos, Ana Pilar; Valencia, Braulio Mark; Veland, Nicolas; Calderon, Flor; Arevalo, Jorge; Low, Donald E.; Llanos-Cuentas, Alejandro

    2011-01-01

    We compared traditional cutaneous leishmaniasis diagnostic methods to filter paper lesion impression (FPLI) PCR for secondarily infected ulcers and nonulcerative lesions. The sensitivity and specificity of FPLI PCR for secondarily infected lesions (n = 8) were 100%. In primarily nonulcerative lesions (n = 15), the sensitivity of FPLI PCR was inferior to that of pooled-invasive-specimen PCR (72.7% versus 100%) (P = 0.10). FPLI PCR is sensitive, specific, and unlike invasive procedures, can be used in secondarily infected ulcers. Invasive specimen collection is superior in nonulcerative lesions. PMID:21177908

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

    USDA-ARS?s Scientific Manuscript database

    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...

  18. Diagnostics based on nucleic acid sequence variant profiling: PCR, hybridization, and NGS approaches.

    PubMed

    Khodakov, Dmitriy; Wang, Chunyan; Zhang, David Yu

    2016-10-01

    Nucleic acid sequence variations have been implicated in many diseases, and reliable detection and quantitation of DNA/RNA biomarkers can inform effective therapeutic action, enabling precision medicine. Nucleic acid analysis technologies being translated into the clinic can broadly be classified into hybridization, PCR, and sequencing, as well as their combinations. Here we review the molecular mechanisms of popular commercial assays, and their progress in translation into in vitro diagnostics. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  19. Real Time Polymerase Chain Reaction (rt-PCR): A New Patent to Diagnostic Purposes for Paracoccidioidomycosis.

    PubMed

    Rocha-Silva, Fabiana; Gomes, Luciana I; Gracielle-Melo, Cidiane; Goes, Alfredo M; Caligiorne, Rachel B

    2017-01-01

    Paracoccidioidomycosis (PCM) is a systemic mycosis caused by dimorphic fungi Paracoccidioides brasiliensis and Paracoccidioides lutzii. It is prevalent in Latin American, mainly in Brazil. Therefore, PCM has fundamental impact on the Brazilian global economy, especially in public health system, since it is affecting economical active population in different country regions. The present study aimed to standardize the Real Time-Polymerase Chain Reaction (rt-PCR) for an efficient and safe PCM diagnosis amplifying the recombinant protein PB27 gene, only expressed by specimens of Paracoccidioides genus. To standardize a methodology of rt-PCR using species-specific primers and probe designed for annealing in this specific region of the fungi´s genome, amplifying the recombinant protein PB27 gene, only expressed by specimens of Paracoccidioides genus. Followed by design in silico, experiments were performed in vitro to determine rt-PCR specificity, efficiency and genome detection limit. The primers and probe sequences were deposited in Brazilian Coordination of Technological Innovation and Transfer (CTIT), under patent reference number BR1020160078830. The present study demonstrated the rt-PCR applicability for support on diagnosis of paracoccidioidomycosis, presenting low cost, which makes it affordable for public health services in developing countries as Brazil. It is noteworthy that it is necessary to validate this methodology using clinical samples before to use as a safe method of diagnosis. A review of all patents related to this topic was performed and it was shown that, to date, there are no records of patent on kits for paracoccidioidomycosis´s diagnostic. Indeed, there is still a lot to go to reach this goal. The reaction developed was standardized and patented, opening perspectives to molecular diagnosis development for paracoccidioidomycosis, since rt-PCR can be applied to a broad spectrum of infectious diseases. It would need to be tested in biological

  20. Comparison of PCR and other diagnostic techniques for detection of Helicobacter pylori infection in dyspeptic patients.

    PubMed Central

    Weiss, J; Mecca, J; da Silva, E; Gassner, D

    1994-01-01

    A sensitive and specific PCR-based assay to detect the Helicobacter pylori 16S rRNA gene present in formalin-fixed paraffin-embedded gastric biopsy specimens has been developed. A total of 95 patients with dyspepsia were evaluated for the presence of chronic active gastritis and an infection with H. pylori through the use of diagnostic assays based on biopsy specimens and serology. The "gold standard" for the presence of the bacteria was direct detection in histological sections of biopsy specimens by Giemsa stain. The results obtained with the PCR assay performed on the biopsy specimens (94% sensitivity and 100% specificity) were equivalent to the detection of H. pylori immunoglobulin G antibodies by the commercially available second-generation Cobas Core anti-H. pylori immunoglobulin G enzyme immunoassay (94% sensitivity and 98% specificity) for the diagnosis of H. pylori infection. Urease testing and bacterial culture of the biopsy specimens were inferior (88 and 70% sensitivity and 96% and 98% specificity, respectively). A Western blot (immunoblot) analysis had slightly greater sensitivity (96%), although specificity was reduced to 93%. This research prototype PCR assay was shown to be highly reliable for the detection of infection with H. pylori and the presence of chronic active gastritis in the population studied. PMID:7929755

  1. PCR Inhibition of a Quantitative PCR for Detection of Mycobacterium avium Subspecies Paratuberculosis DNA in Feces: Diagnostic Implications and Potential Solutions

    PubMed Central

    Acharya, Kamal R.; Dhand, Navneet K.; Whittington, Richard J.; Plain, Karren M.

    2017-01-01

    Molecular tests such as polymerase chain reaction (PCR) are increasingly being applied for the diagnosis of Johne’s disease, a chronic intestinal infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Feces, as the primary test sample, presents challenges in terms of effective DNA isolation, with potential for PCR inhibition and ultimately for reduced analytical and diagnostic sensitivity. However, limited evidence is available regarding the magnitude and diagnostic implications of PCR inhibition for the detection of MAP in feces. This study aimed to investigate the presence and diagnostic implications of PCR inhibition in a quantitative PCR assay for MAP (High-throughput Johne’s test) to investigate the characteristics of samples prone to inhibition and to identify measures that can be taken to overcome this. In a study of fecal samples derived from a high prevalence, endemically infected cattle herd, 19.94% of fecal DNA extracts showed some evidence of inhibition. Relief of inhibition by a five-fold dilution of the DNA extract led to an average increase in quantification of DNA by 3.3-fold that consequently increased test sensitivity of the qPCR from 55 to 80% compared to fecal culture. DNA extracts with higher DNA and protein content had 19.33 and 10.94 times higher odds of showing inhibition, respectively. The results suggest that the current test protocol is sensitive for herd level diagnosis of Johne’s disease but that test sensitivity and individual level diagnosis could be enhanced by relief of PCR inhibition, achieved by five-fold dilution of the DNA extract. Furthermore, qualitative and quantitative parameters derived from absorbance measures of DNA extracts could be useful for prediction of inhibitory fecal samples. PMID:28210245

  2. PCR Inhibition of a Quantitative PCR for Detection of Mycobacterium avium Subspecies Paratuberculosis DNA in Feces: Diagnostic Implications and Potential Solutions.

    PubMed

    Acharya, Kamal R; Dhand, Navneet K; Whittington, Richard J; Plain, Karren M

    2017-01-01

    Molecular tests such as polymerase chain reaction (PCR) are increasingly being applied for the diagnosis of Johne's disease, a chronic intestinal infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Feces, as the primary test sample, presents challenges in terms of effective DNA isolation, with potential for PCR inhibition and ultimately for reduced analytical and diagnostic sensitivity. However, limited evidence is available regarding the magnitude and diagnostic implications of PCR inhibition for the detection of MAP in feces. This study aimed to investigate the presence and diagnostic implications of PCR inhibition in a quantitative PCR assay for MAP (High-throughput Johne's test) to investigate the characteristics of samples prone to inhibition and to identify measures that can be taken to overcome this. In a study of fecal samples derived from a high prevalence, endemically infected cattle herd, 19.94% of fecal DNA extracts showed some evidence of inhibition. Relief of inhibition by a five-fold dilution of the DNA extract led to an average increase in quantification of DNA by 3.3-fold that consequently increased test sensitivity of the qPCR from 55 to 80% compared to fecal culture. DNA extracts with higher DNA and protein content had 19.33 and 10.94 times higher odds of showing inhibition, respectively. The results suggest that the current test protocol is sensitive for herd level diagnosis of Johne's disease but that test sensitivity and individual level diagnosis could be enhanced by relief of PCR inhibition, achieved by five-fold dilution of the DNA extract. Furthermore, qualitative and quantitative parameters derived from absorbance measures of DNA extracts could be useful for prediction of inhibitory fecal samples.

  3. 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

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

    PubMed

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

    2017-10-01

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

  5. 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

  6. [An effective scheme to produce recombinant uracil-DNA glycosylase of Escherichia coli for PCR diagnostics].

    PubMed

    Dmitrochenko, A E; Turiianskaia, O M; Gilep, A A; Usanov, S A; Iantsevich, A V

    2014-01-01

    An effective scheme has been developed to produce recombinant uracil-DNA glycosylase of Escherichia coli K12 intended to be used for PCR diagnostics, making it possible to achieve a high yield of the end product using a two-stage purification. The gene encoding this enzyme was cloned into the pCWori vector within the same reading frame with six residues of histidine in the C-erminal sequence. Using this vector and the E. coli DH5alpha, a host-vector expression system has been developed and conditions for protein synthesis have been optimized. To purify the protein, metal affinity chromatography with further dialysis was used to remove imidazole. The enzyme yield was no less than 60 mg of the end protein per 1 L of the culture medium. The concordance between amino acid sequences of the recombinant and native enzymes was proved by peptide mass fingerprinting and mass spectrometry. A rapid test to determine the activity of the enzyme preparation was suggested. It was found that the activity of 1.0 mg of the recombinant protein is no less than 3 x 10(3) units. The recombinant enzyme was most stable at pH 8.0 and an ionic strength of the solution equal to 200 mM; it lost its activity completely for 10 min at 60 degrees C. Storage during 1 h at 20 degrees C resulted in the loss of no more than 30% of activity. In the enzyme preparation, the activity of DNase was absent. The free energy of the unfolding of the protein globule of the recombinant uracil-DNA glycosylase is 23.1 +/- 0.2 kJ/mol. The data obtained indicate that the recombinant enzyme may be recommended for use in PCR diagnostics to prevent the appearance of false positive results caused by pollution of the reaction mixture by products of the preceding reactions.

  7. Development of two quantitative real-time PCR diagnostic kits for HPV isolates from Korea.

    PubMed

    Jeeva, Subbiah; Kim, Nam-Il; Jang, In-Kwon; Choi, Tae-Jin

    2012-10-01

    Viral pathogens, alongside other pathogens, have major effects on crustacean aquaculture. Hepatopancreatic parvovirus (HPV) is an emerging virus in the shrimp industry and has been detected in shrimp farms worldwide. The HPV genome has greater diversity than other shrimp viruses owing to its wide host range and geographical distribution. Therefore, developing diagnostic tools is essential to detect even small copy numbers from the target region of native HPV isolates. We have developed two easy to use quantitative real-time PCR kits, called Green Star and Dual Star, which contain all of the necessary components for real-time PCR, including HPV primers, using the primers obtained from the sequences of HPV isolates from Korea, and analyzed their specificity, efficiency, and reproducibility. These two kits could detect from 1 to 1 × 10(9) copies of cloned HPV DNA. The minimum detection limits obtained from HPV-infected shrimp were 7.74 × 10(1) and 9.06 × 10(1) copies in the Green Star and Dual Star assay kits, respectively. These kits can be used for rapid, sensitive, and efficient screening for HPV isolates from Korea before the introduction of postlarval stages into culture ponds, thereby decreasing the incidence of early development of the disease.

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

    PubMed

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

    2014-06-01

    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.

  9. 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

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

    PubMed Central

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

    2013-01-01

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

  11. Better tests, better care: improved diagnostics for infectious diseases.

    PubMed

    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

    2013-12-01

    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.

  12. 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

  13. Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis

    PubMed Central

    Chong, Nikson Fatt-Ming

    2016-01-01

    The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40–45 PCR cycles. A total of eight mutagenesis experiments were successfully carried out, with each experiment mutating between two to six base pairs. Up to four adjacent codons were changed in a single experiment. This method is especially useful for codon optimization, where doublet or triplet rare codons can be changed using a single mutagenic primer set, in a single experiment. PMID:27995143

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

    PubMed

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

    2015-11-01

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

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

    PubMed

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

    2013-05-01

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

  16. 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.

  17. Rapid and sensitive detection of Yersinia pestis using amplification of plague diagnostic bacteriophages monitored by real-time PCR.

    PubMed

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

    2010-06-28

    Yersinia pestis, the agent of plague, has caused many millions of human deaths and still poses a serious threat to global public health. Timely and reliable detection of such a dangerous pathogen is of critical importance. Lysis by specific bacteriophages remains an essential method of Y. pestis detection and plague diagnostics. The objective of this work was to develop an alternative to conventional phage lysis tests--a rapid and highly sensitive method of indirect detection of live Y. pestis cells based on quantitative real-time PCR (qPCR) monitoring of amplification of reporter Y. pestis-specific bacteriophages. Plague diagnostic phages phiA1122 and L-413C were shown to be highly effective diagnostic tools for the detection and identification of Y. pestis by using qPCR with primers specific for phage DNA. The template DNA extraction step that usually precedes qPCR was omitted. phiA1122-specific qPCR enabled the detection of an initial bacterial concentration of 10(3) CFU/ml (equivalent to as few as one Y. pestis cell per 1-microl sample) in four hours. L-413C-mediated detection of Y. pestis was less sensitive (up to 100 bacteria per sample) but more specific, and thus we propose parallel qPCR for the two phages as a rapid and reliable method of Y. pestis identification. Importantly, phiA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR. Thus, we developed a novel assay for detection and identification of Y. pestis using amplification of specific phages monitored by qPCR. The method is simple, rapid, highly sensitive, and specific and allows the detection of only live bacteria.

  18. Upgrading Engine Test Cells for Improved Troubleshooting and Diagnostics

    DTIC Science & Technology

    2002-01-01

    being developed. To enable this, test cell fault detection and isolation capabilities will need to utilize all of the relevant engine and test...improved engine fault detection and isolation capabilities, various approaches for automated sensor validation, performance diagnostics and

  19. Development of a pan-Simbu real-time reverse transcriptase PCR for the detection of Simbu serogroup viruses and comparison with SBV diagnostic PCR systems

    PubMed Central

    2013-01-01

    Background Schmallenberg virus (SBV), a novel orthobunyavirus of the Simbu serogroup, was first identified in October 2011 in dairy cattle in Germany, where it caused fever, diarrhea and a drop in milk yield. Since then, SBV additionally has been detected in adult sheep and goats. Although symptoms of acute infection were not observed, infection during a vulnerable phase of pregnancy caused congenital malformations and stillbirths. In view of the current situation and the possible emergence of further Simbu serogroup members, a pan-Simbu real-time reverse transcriptase (RT) PCR system for the reliable detection of Simbu serogroup viruses should be developed. Methods In this study a pan-Simbu real-time RT-PCR system was established and compared to several SBV real-time RT-PCR assays. All PCR-systems were tested using a panel of different Simbu serogroup viruses as well as several field samples from diseased cattle, sheep and goats originating from all over Germany. Several pan-Simbu real-time RT-PCR products were sequenced via Sanger sequencing. Furthermore, in silico analyses were performed to investigate suitability for the detection of further orthobunyaviruses. Results All tested members of the Simbu serogroup (n = 14) as well as most of the field samples were successfully detected by the pan-Simbu real-time RT-PCR system. The comparison of this intercalating dye assay with different TaqMan probe-based assays developed for SBV diagnostics confirmed the functionality of the pan-Simbu assay for screening purposes. However, the SBV-TaqMan-assay SBV-S3 delivered the highest analytical sensitivity of less than ten copies per reaction for duplex systems including an internal control. In addition, for confirmation of SBV-genome detection the highly specific SBV-M1 assay was established. Conclusion The pan-Simbu real-time RT-PCR system was able to detect all tested members of the Simbu serogroup, most of the SBV field samples as well as three tested Bunyamwera

  20. 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

  1. 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

  2. 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.

  3. PCR-based retrospective evaluation of diagnostic samples for emergence of porcine deltacoronavirus in US swine.

    PubMed

    Sinha, Avanti; Gauger, Phillip; Zhang, Jianqiang; Yoon, Kyoung-Jin; Harmon, Karen

    2015-09-30

    Porcine deltacoronavirus (PDCoV) was first identified in Hong Kong in a regional surveillance study for Coronaviruses in 2012 and was detected for the first time in United States (US) swine in February 2014. However, it remains unknown if PDCoV had been introduced into the US prior to that time period. In the present study, 1734 clinical samples (903 cases) submitted to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) for enteric disease diagnosis between October 2012 and December 2013 were tested retrospectively for PDCoV using a virus-specific real-time reverse transcription (RT) PCR targeting conserved region of the membrane gene. PDCoV genome was first detected in a fecal sample collected on August 19th 2013 from Minnesota. Subsequently, PDCoV was observed in samples collected on August 20th and August 27th from Iowa and on August 29th from Illinois. Therefore, with available samples submitted to the ISU VDL, it can be inferred that PDCoV has been present in US swine at least since August 2013.

  4. Development and evaluation of a next-generation digital PCR diagnostic assay for ocular Chlamydia trachomatis infections.

    PubMed

    Roberts, Chrissy H; 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

    2013-07-01

    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 (r(2) = 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.

  5. 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

  6. Diagnostic Value of PCR Analysis of Bacteria and Fungi from Blood in Empiric-Therapy-Resistant Febrile Neutropenia ▿

    PubMed Central

    Nakamura, Akiko; Sugimoto, Yuka; Ohishi, Kohshi; Sugawara, Yumiko; Fujieda, Atsushi; Monma, Fumihiko; Suzuki, Kei; Masuya, Masahiro; Nakase, Kazunori; Matsushima, Yoshiko; Wada, Hideo; Katayama, Naoyuki; Nobori, Tsutomu

    2010-01-01

    This study aimed to assess the clinical utility of PCR for the analysis of bacteria and fungi from blood for the management of febrile neutropenic patients with hematologic malignancies. Using a PCR system able to detect a broad range of bacteria and fungi, we conducted a prospective pilot study of periodic analyses of blood from patients following intensive chemotherapy. When fever occurred, it was treated with empirical antibiotic therapy, basically without knowledge of the PCR results. In 23 febrile episodes during the neutropenic period, bacteria were detected by PCR in 11 cases, while the same species were identified by blood culture in 3 cases. In 10 out of 11 PCR-positive cases, fever could be managed by empirical therapy. In the empirical-therapy-resistant case, the identification of Stenotrophomonas maltophilia by PCR led to improvement of fever. No fungi were detected by PCR in febrile cases, while Aspergillus fumigatus was detected in one afebrile patient, several days before a clinical diagnosis was made. In subsequent sporadic PCR analyses in 15 cases of febrile neutropenia, bacteria were detected by both PCR and blood culture in 7 cases and by PCR alone in 6. Fungi were not detected. While fever was improved by empirical therapy in 12 out of the 13 PCR-positive cases, the identification of Pseudomonas aeruginosa by PCR in one therapy-resistant case contributed to the successful treatment of persistent fever. Our results indicate that PCR analysis of bacteria from blood provides essential information for managing empirical-therapy-resistant febrile neutropenia. PMID:20392911

  7. Diagnostic value of PCR analysis of bacteria and fungi from blood in empiric-therapy-resistant febrile neutropenia.

    PubMed

    Nakamura, Akiko; Sugimoto, Yuka; Ohishi, Kohshi; Sugawara, Yumiko; Fujieda, Atsushi; Monma, Fumihiko; Suzuki, Kei; Masuya, Masahiro; Nakase, Kazunori; Matsushima, Yoshiko; Wada, Hideo; Katayama, Naoyuki; Nobori, Tsutomu

    2010-06-01

    This study aimed to assess the clinical utility of PCR for the analysis of bacteria and fungi from blood for the management of febrile neutropenic patients with hematologic malignancies. Using a PCR system able to detect a broad range of bacteria and fungi, we conducted a prospective pilot study of periodic analyses of blood from patients following intensive chemotherapy. When fever occurred, it was treated with empirical antibiotic therapy, basically without knowledge of the PCR results. In 23 febrile episodes during the neutropenic period, bacteria were detected by PCR in 11 cases, while the same species were identified by blood culture in 3 cases. In 10 out of 11 PCR-positive cases, fever could be managed by empirical therapy. In the empirical-therapy-resistant case, the identification of Stenotrophomonas maltophilia by PCR led to improvement of fever. No fungi were detected by PCR in febrile cases, while Aspergillus fumigatus was detected in one afebrile patient, several days before a clinical diagnosis was made. In subsequent sporadic PCR analyses in 15 cases of febrile neutropenia, bacteria were detected by both PCR and blood culture in 7 cases and by PCR alone in 6. Fungi were not detected. While fever was improved by empirical therapy in 12 out of the 13 PCR-positive cases, the identification of Pseudomonas aeruginosa by PCR in one therapy-resistant case contributed to the successful treatment of persistent fever. Our results indicate that PCR analysis of bacteria from blood provides essential information for managing empirical-therapy-resistant febrile neutropenia.

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

    PubMed

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

    2014-12-01

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

  9. Rv1458c: a new diagnostic marker for identification of Mycobacterium tuberculosis complex in a novel duplex PCR assay.

    PubMed

    Shrivastava, Kamal; Garima, Kushal; Narang, Anshika; Bhattacharyya, Kausik; Vishnoi, Ekta; Singh, Roshan Kumar; Chaudhry, Anil; Prasad, Rajendra; Bose, Mridula; Varma-Basil, Mandira

    2017-03-01

    We explored the efficiency of Rv1458c, the gene encoding a putative ABC drug transporter specific for the Mycobacterium tuberculosis complex (MTBC), as a diagnostic marker. A 190 bp region of Rv1458c and a 300 bp region of hsp65 were targeted in a novel duplex PCR assay and the results were compared with those for PCR restriction analysis(PRA) using the restriction enzymes NruI and BamHI. Species identification of a subset of the isolates (n=50) was confirmed by sequencing. Clinical isolates of M. tuberculosis (n=426) obtained from clinically suspected patients of pulmonary tuberculosis and mycobacterial (n=13) and non-mycobacterial (n=8) reference strains were included in the study. The duplex PCR assay correctly identified 320/426 isolates as MTBC and 106/426 isolates as non-tuberculous mycobacteria(NTM). The test was 100 % specific and sensitive when compared with NruI/BamHI PCR restriction analysis and highlighted the use of Rv1458c as a diagnostic marker for MTBC. The duplex PCR assay could be developed for use as a screening test to identify MTBC in clinical specimens in peripheral laboratories with limited resources.

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

    PubMed Central

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

    2011-01-01

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

  11. O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis.

    PubMed

    Mekonnen, Solomon A; Beissner, Marcus; Saar, Malkin; Ali, Solomon; Zeynudin, Ahmed; Tesfaye, Kassahun; Adbaru, Mulatu G; Battke, Florian; Poppert, Sven; Hoelscher, Michael; Löscher, Thomas; Bretzel, Gisela; Herbinger, Karl-Heinz

    2017-10-02

    Onchocerciasis is a parasitic disease caused by the filarial nematode Onchocerca volvulus. In endemic areas, the diagnosis is commonly confirmed by microscopic examination of skin snip samples, though this technique is considered to have low sensitivity. The available melting-curve based quantitative real-time PCR (qPCR) using degenerated primers targeting the O-150 repeat of O. volvulus was considered insufficient for confirming the individual diagnosis, especially in elimination studies. This study aimed to improve detection of O. volvulus DNA in clinical samples through the development of a highly sensitive qPCR assay. A novel hydrolysis probe based qPCR assay was designed targeting the specific sequence of the O. volvulus O-5S rRNA gene. A total of 200 clinically suspected onchocerciasis cases were included from Goma district in South-west Ethiopia, from October 2012 through May 2013. Skin snip samples were collected and subjected to microscopy, O-150 qPCR, and the novel O-5S qPCR. Among the 200 individuals, 133 patients tested positive (positivity rate of 66.5%) and 67 negative by O-5S qPCR, 74 tested positive by microscopy (37.0%) and 78 tested positive by O-150 qPCR (39.0%). Among the 133 O-5S qPCR positive individuals, microscopy and O-150 qPCR detected 55.6 and 59.4% patients, respectively, implying a higher sensitivity of O-5S qPCR than microscopy and O-150 qPCR. None of the 67 individuals who tested negative by O-5S qPCR tested positive by microscopy or O-150 qPCR, implying 100% specificity of the newly designed O-5S qPCR assay. The novel O-5S qPCR assay is more sensitive than both microscopic examination and the existing O-150 qPCR for the detection of O. volvulus from skin snip samples. The newly designed assay is an important step towards appropriate individual diagnosis and control of onchocerciasis.

  12. PCR-electrospray ionization mass spectrometry: the potential to change infectious disease diagnostics in clinical and public health laboratories.

    PubMed

    Wolk, Donna M; Kaleta, Erin J; Wysocki, Vicki H

    2012-07-01

    During the past 20 years, microbial detection methods that are genetically based, such as real-time PCR and peptide nucleic acid fluorescent hybridization, coexisted with traditional microbiological methods and were typically based on the identification of individual genetic targets. For these methods to be successful, a potential cause of infection must be suspected. More recently, multiplex PCR and multiplex RT-PCR were used to enable more broad-range testing based on panels of suspected pathogens. PCR-electrospray ionization mass spectrometry (PCR-ESI/MS) has emerged as a technology that is capable of identifying nearly all known human pathogens either from microbial isolates or directly from clinical specimens. Assay primers are strategically designed to target one or more of the broad pathogen categories: bacterial, mycobacterial, fungal, or viral. With broad-range amplification followed by detection of mixed amplicons, the method can identify genetic evidence of known and unknown pathogens. This unique approach supports a higher form of inquiry, asking the following question: What is the genetic evidence of known or unknown pathogens in the patient sample? This approach has advantages over traditional assays that commonly target the presence or absence of one or more pathogens with known genetic composition. This review considers the breadth of the published literature and explores the possibilities, advantages, and limitations for implementation of PCR-ESI/MS in diagnostic laboratories.

  13. Use of universal 16S rRNA gene PCR as a diagnostic tool for venous access port-related bloodstream infections.

    PubMed

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

    2013-03-01

    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.

  14. 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

  15. Improving the PCR protocol to amplify a repetitive DNA sequence.

    PubMed

    Riet, J; Ramos, L R V; Lewis, R V; Marins, L F

    2017-09-21

    Although PCR-based techniques have become an essential tool in the field of molecular and genetic research, the amplification of repetitive DNA sequences is limited. This is due to the truncated nature of the amplified sequences, which are also prone to errors during DNA polymerase-based amplification. The complex structure of repetitive DNA can form hairpin loops, which promote dissociation of the polymerase from the template, impairing complete amplification, and leading to the formation of incomplete fragments that serve as megaprimers. These megaprimers anneal with other sequences, generating unexpected fragments in each PCR cycle. Our gene model, MaSp1, is 1037-bp long, with 68% GC content, and its amino acid sequence is characterized by poly-alanine-glycine motifs, which represent the repetitive codon consensus. We describe the amplification of the MaSp1 gene through minor changes in the PCR program. The results show that a denaturation temperature of 98°C is the key determinant in the amplification of the MaSp1 partial gene sequence.

  16. 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

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

    PubMed

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

    2014-01-01

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

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

    USDA-ARS?s Scientific Manuscript database

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

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

    PubMed Central

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

    1995-01-01

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

  20. A novel diagnostic platform based on multiplex ligase detection-PCR and microarray for simultaneous detection of swine viruses.

    PubMed

    Jiang, Yonghou; Guo, Yao; Wang, Ping; Dong, Qinfang; Opriessnig, Tanja; Cheng, Juhui; Xu, Hui; Ding, Xianfeng; Guo, Jiangfeng

    2011-12-01

    Simultaneous detection and identification of multiple pathogens is required in many diagnostic fields. In this study a novel method based on a multiplex ligase detection (LD)-polymerase chain reaction (PCR) and microarray (MLPM) is described to detect simultaneously several swine viruses involved in reproductive and/or respiratory problems. The multiplex diagnostic system was validated using standard plasmids, and clinical samples. Using this strategy as few as 10 copies of target plasmids were detected successfully. Each probe pair yielded specific positive signal only in its target site. In addition, when six target plasmids were present simultaneously sufficient robust signals were generated in their corresponding sites of six plasmid templates and no obvious signals were detected in non-target sites. Compared to real-time PCR, the MLPM showed specificities and sensitivities of 95.7-100% and 100% for 47 clinical samples tested, respectively. The results demonstrate that this novel assay is a specific, sensitive, and multiplex diagnostic method for detection of multiple pathogens and can also be adapted easily for diagnostic purposes.

  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.

  2. Simple Repeat-Primed PCR Analysis of the Myotonic Dystrophy Type 1 Gene in a Clinical Diagnostics Environment

    PubMed Central

    Dryland, Philippa A.; Doherty, Elaine; Love, Jennifer M.; Love, Donald R.

    2013-01-01

    Myotonic dystrophy type 1 is an autosomal dominant neuromuscular disorder that is caused by the expansion of a CTG trinucleotide repeat in the DMPK gene. The confirmation of a clinical diagnosis of DM-1 usually involves PCR amplification of the CTG repeat-containing region and subsequent sizing of the amplification products in order to deduce the number of CTG repeats. In the case of repeat hyperexpansions, Southern blotting is also used; however, the latter has largely been superseded by triplet repeat-primed PCR (TP-PCR), which does not yield a CTG repeat number but nevertheless provides a means of stratifying patients regarding their disease severity. We report here a combination of forward and reverse TP-PCR primers that allows for the simple and effective scoring of both the size of smaller alleles and the presence or absence of expanded repeat sequences. In addition, the CTG repeat-containing TP-PCR forward primer can target both the DM-1 and Huntington disease genes, thereby streamlining the work flow for confirmation of clinical diagnoses in a diagnostic laboratory. PMID:26317000

  3. Detection of Mixed-Species Infections of Plasmodium falciparum and Plasmodium vivax by Nested PCR and Rapid Diagnostic Tests in Southeastern Iran

    PubMed Central

    Ehtesham, Reyhaneh; Fazaeli, Asghar; Raeisi, Ahmad; Keshavarz, Hossein; Heidari, Aliehsan

    2015-01-01

    Coexistence of two species of Plasmodium in a single host has disrupted the diagnosis and treatment of malaria. This study was designed to evaluate the ability of rapid diagnostic test (RDT) kits for the diagnosis of mixed-species malaria infections in southeastern Iran. A total of 100 malaria patients were included in the study out of 164 randomly suspected symptomatic malaria patients from May to November 2012. Nested polymerase chain reaction (PCR) was also used to judge the ability of microscopy versus RDT kits for detecting mixed species. The sensitivity of light microscopy for the detection of mixed-species malaria infections was 16.6% (95% confidence interval [CI] = 3–49.1). Nested PCR revealed 12 patients with mixed-species infection. The CareStart Pv/Pf Combo kit detected 58% of the mixed-species infections, which were determined by nested PCR (sensitivity = 58.3%; 95% CI = 28.5–83.5). For identifying P. falciparum, P. vivax, and mixed-species infections, the concordance rates (kappa statistics) of microscopy and CareStart Pv/Pf Combo kit with nested PCR were 0.76 and 0.79, respectively (P = 0.001). This study underlines the effectiveness of RDT kits to improve the differentiation of mixed-species malaria infections in endemic areas where the prevalence of chloroquine resistance is high. PMID:25962771

  4. Polymorphism-specific PCR enhances the diagnostic performance of American tegumentary leishmaniasis and allows the rapid identification of Leishmania species from Argentina

    PubMed Central

    2012-01-01

    Background The diagnosis of the leishmaniases poses enormous challenges in Argentina. The Polymorphism-Specific PCR (PS-PCR) designed and validated in our laboratories has been proven effective for typifying the Leishmania genus from cultured material. Here we evaluated the performance of this method in the diagnosis of American tegumentary leishmaniasis (ATL) and the rapid identification of Leishmania spp. directly from clinical specimens. Methods A total of 63 patients from northwestern Argentina, with cutaneous or mucocutaneous lesions, underwent an ATL diagnosis protocol which included clinical examination, Leishmanin skin test, and microscopic examination of dermal smears. In addition, we performed PS-PCR on DNA directly extracted from the specimens scraped from the lesions. Results Out of the 63 patients, 44 were classified as ATL cases and 19 as non-ATL cases. The diagnostic sensitivity of the microscopic analysis of dermal smears and PS-PCR individually were 70.5% and 81%, respectively. When performing both tests in parallel, this parameter increased significantly to 97.6% (p = 0.0018). The specificities, on the other hand, were 100%, 84.2%, and 83.3% for the combination, respectively (p > 0.05). Using the PS-PCR analysis we successfully identified the Leishmania spp. in 31 out of the 44 ATL cases. Twenty-eight (90.3%) cases were caused by L. (V.) braziliensis, two (6.5%) by L. (V.) guyanensis, and one (3.2%) by L. (V.) panamensis. Conclusions The efficacy of the ATL diagnosis was significantly improved by combining the dermal smear examination with a PS-PCR analysis. Our strategy allowed us to reach the diagnosis of ATL with high accuracy regarding the species of the etiological agent in 70.5% of the cases. Moreover, we diagnosed two cases of the disseminated cutaneous form caused by L. (V.) braziliensis and a cutaneous case due to L. (V.) panamensis infection, both findings reported for the first time in Argentina. PMID:22894734

  5. 3D printing and milling a real-time PCR device for infectious disease diagnostics.

    PubMed

    Mulberry, Geoffrey; White, Kevin A; Vaidya, Manjusha; Sugaya, Kiminobu; Kim, Brian N

    2017-01-01

    Diagnosing infectious diseases using quantitative polymerase chain reaction (qPCR) offers a conclusive result in determining the infection, the strain or type of pathogen, and the level of infection. However, due to the high-cost instrumentation involved and the complexity in maintenance, it is rarely used in the field to make a quick turnaround diagnosis. In order to provide a higher level of accessibility than current qPCR devices, a set of 3D manufacturing methods is explored as a possible option to fabricate a low-cost and portable qPCR device. The key advantage of this approach is the ability to upload the digital format of the design files on the internet for wide distribution so that people at any location can simply download and feed into their 3D printers for quick manufacturing. The material and design are carefully selected to minimize the number of custom parts that depend on advanced manufacturing processes which lower accessibility. The presented 3D manufactured qPCR device is tested with 20-μL samples that contain various concentrations of lentivirus, the same type as HIV. A reverse-transcription step is a part of the device's operation, which takes place prior to the qPCR step to reverse transcribe the target RNA from the lentivirus into complementary DNA (cDNA). This is immediately followed by qPCR which quantifies the target sequence molecules in the sample during the PCR amplification process. The entire process of thermal control and time-coordinated fluorescence reading is automated by closed-loop feedback and a microcontroller. The resulting device is portable and battery-operated, with a size of 12 × 7 × 6 cm3 and mass of only 214 g. By uploading and sharing the design files online, the presented low-cost qPCR device may provide easier access to a robust diagnosis protocol for various infectious diseases, such as HIV and malaria.

  6. 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

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

    PubMed

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

    2014-01-01

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

  8. 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.

  9. Diagnostic Accuracy of Kato–Katz, FLOTAC, Baermann, and PCR Methods for the Detection of Light-Intensity Hookworm and Strongyloides stercoralis Infections in Tanzania

    PubMed Central

    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

    2014-01-01

    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

  10. Diagnostic value of a "wide-range" quantitative nested real-time PCR assay for varicella zoster virus myelitis.

    PubMed

    Takahashi, Teruyuki; Tamura, Masato; Takasu, Toshiaki

    2013-11-01

    Myelitis is one of the rarest neurological complications of varicella zoster virus (VZV) infection. In this study, the authors remodeled the "wide-range" quantitative nested real-time (QNRT) polymerase chain reaction (PCR) assay to quantitatively detect a small amount of VZV-DNA in cerebrospinal fluid (CSF). For use as a specific internal control "calibrator," an original mutation-VZV (MZ) plasmid was developed. The initial copy number of VZV-DNA in CSF specimens was measured by the amplification rate of the MZ-plasmid. For 17 consecutive CSF specimens collected from three elderly patients with VZV myelitis, the diagnostic value of the wide-range QNRT-PCR assay was evaluated and compared with other conventional PCR assays and enzyme immunoassay (EIA). The MZ-plasmid demonstrated statistically uniform amplifications (F=1.016) against a wide range (1-100,000) of copy numbers of mimic VZV-DNA. The wide-range QNRT-PCR assay quantitatively and rapidly (within 48 hr) detected 5,863, 3,052, 958, and 6,721 copies/ml of VZV-DNA in the CSF specimens collected from all patients in the acute phase. Additionally, there was a significant difference (*P=0.023) in the copy number of VZV-DNA between before and after acyclovir treatment. Other conventional single PCR assays all revealed negative results, but were nevertheless time-consuming (7 days). The IgG EIA-value for VZV was continually elevated throughout the clinical course of all patients. The MZ-plasmid was thus regarded as an appropriate "calibrator" in the wide-range QNRT-PCR assay. This assay is a novel, rapid, accurate, quantitative, and highly sensitive technique, and will contribute as a reliable and useful clinical examination for the rapid diagnosis of VZV infection to central nervous system. © 2013 Wiley Periodicals, Inc.

  11. The development of an improved streak tube for fusion diagnostics

    NASA Astrophysics Data System (ADS)

    Howorth, J. R.; Milnes, J. S.; Fisher, Y.; Jadwin, A.; Boni, R.; Jaanimagi, P. A.

    2016-11-01

    The fusion diagnostic community, including the National Ignition Facility, the Laboratory for Laser Energetics, Megajoule in France, and others require optical recording instruments with precise time resolution covering a dynamic range of many orders of magnitude. In 2012, LLE, Photek, and Sydor Instruments embarked on the re-design of an improved streak tube for fusion diagnostics. As a baseline we started with the Photek ST-Y streak tube which is a member of the RCA design dating back to 1957, because the tube body can accommodate a 35 mm long photocathode, and consequently more fibre coupled diagnostic channels than smaller designs. Electron optical modelling was carried out by both Paul Jaanimagi in the US and by Photek with different software packages in a parallel exercise. Our goal was to address some of the short-comings of this tube, the initial approach being to increase the field between the photocathode and extractor electrode from 700 to 1300 V/mm to reduce space charge effects and to improve time resolution. Many changes and modifications were made: the time resolution was improved to 5 ps, the usable cathode length was increased from 20 mm to 32 mm under high extraction field operation, and the off-axis spatial resolution was substantially improved compared to other tubes of this format. Several tubes have been built and tested in Sydor ROSS-5800 streak cameras.

  12. 3D printing and milling a real-time PCR device for infectious disease diagnostics

    PubMed Central

    Mulberry, Geoffrey; White, Kevin A.; Vaidya, Manjusha; Sugaya, Kiminobu

    2017-01-01

    Diagnosing infectious diseases using quantitative polymerase chain reaction (qPCR) offers a conclusive result in determining the infection, the strain or type of pathogen, and the level of infection. However, due to the high-cost instrumentation involved and the complexity in maintenance, it is rarely used in the field to make a quick turnaround diagnosis. In order to provide a higher level of accessibility than current qPCR devices, a set of 3D manufacturing methods is explored as a possible option to fabricate a low-cost and portable qPCR device. The key advantage of this approach is the ability to upload the digital format of the design files on the internet for wide distribution so that people at any location can simply download and feed into their 3D printers for quick manufacturing. The material and design are carefully selected to minimize the number of custom parts that depend on advanced manufacturing processes which lower accessibility. The presented 3D manufactured qPCR device is tested with 20-μL samples that contain various concentrations of lentivirus, the same type as HIV. A reverse-transcription step is a part of the device’s operation, which takes place prior to the qPCR step to reverse transcribe the target RNA from the lentivirus into complementary DNA (cDNA). This is immediately followed by qPCR which quantifies the target sequence molecules in the sample during the PCR amplification process. The entire process of thermal control and time-coordinated fluorescence reading is automated by closed-loop feedback and a microcontroller. The resulting device is portable and battery-operated, with a size of 12 × 7 × 6 cm3 and mass of only 214 g. By uploading and sharing the design files online, the presented low-cost qPCR device may provide easier access to a robust diagnosis protocol for various infectious diseases, such as HIV and malaria. PMID:28586401

  13. 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

  14. Diagnostic PCR assays to unravel food web interactions in cereal crops with focus on biological control of aphids.

    PubMed

    Staudacher, Karin; Jonsson, Mattias; Traugott, Michael

    Successful biological control of agricultural pests is dependent on a thorough understanding of the underlying trophic interactions between predators and prey. Studying trophic interactions can be challenging, particularly when generalist predators that frequently use multiple prey and interact with both pest and alternative prey are considered. In this context, diagnostic PCR proved to be a suitable approach, however at present, prey-specific PCR primers necessary for assessing such interactions across trophic levels are missing. Here we present a new set of 45 primers designed to target a wide range of invertebrate taxa common to temperate cereal crops: cereal aphids, their natural enemies such as carabid beetles, ladybeetles, lacewings, and spiders, and potential alternative prey groups (earthworms, springtails, and dipterans). These primers were combined in three 'ready to use' multiplex PCR assays for quick and cost-effective analyses of large numbers of predator samples. The assays were tested on 560 carabids collected in barley fields in Sweden. Results from this screening suggest that aphids constitute a major food source for carabids in cereal crops (overall DNA detection rate: 51 %), whereas alternative extraguild and intraguild prey appear to be less frequently preyed upon when aphids are present (11 % for springtails and 12 % for earthworms; 1 % for spiders and 4 % for carabids). In summary, the newly developed molecular assays proved reliable and effective in assessing previously cryptic predator-prey trophic interactions, specifically with focus on biological control of aphids. The diagnostic PCR assays will be applicable manifold as the targeted invertebrates are common to many agricultural systems of the temperate region.

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

    PubMed

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

    2009-01-01

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

  16. Targeted sequencing with enrichment PCR: a novel diagnostic method for the detection of EGFR mutations

    PubMed Central

    Kang, Suki; Kim, Baek Gil; Han, Hyun Ho; Lee, Joo Hyun; Kim, Ji Eun; Shim, Hyo Sup; Cho, Nam Hoon

    2015-01-01

    Epidermal growth factor receptor (EGFR) is an important mediator of tumor cell survival and proliferation. The detection of EGFR mutations can predict prognoses and indicate when treatment with EGFR tyrosine kinase inhibitors should be used. As such, the development of highly sensitive methods for detecting EGFR mutations is important. Targeted next-generation sequencing is an effective method for diagnosing mutations. We compared the abilities of enrichment PCR followed by ultra-deep pyrosequencing (UDP), UDP alone, and PNA-mediated RT-PCR clamping to detect low-frequency EGFR mutations in tumor cell lines and tissue samples. Using enrichment PCR-UDP, we were able to detect the E19del and L858R mutations at minimum frequencies of 0.01% and 0.05%, respectively, in the PC-9 and H197 tumor cell lines. We also confirmed the sensitivity of detecting the E19del mutation by performing a titration analysis in FFPE tumor samples. The lowest mutation frequency detected was 0.0692% in tissue samples. EGFR mutations with frequencies as low as 0.01% were detected using enrichment PCR-UDP, suggesting that this method is a valuable tool for detecting rare mutations, especially in scarce tissue samples or those with small quantities of DNA. PMID:25915533

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

    PubMed

    Li, Jin; Makrigiorgos, G Mike

    2009-04-01

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

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  3. 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

  4. 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. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Improved PCR Methods for Detection of African Rabies and Rabies-Related Lyssaviruses ▿

    PubMed Central

    Coertse, Jessica; Weyer, Jacqueline; Nel, Louis H.; Markotter, Wanda

    2010-01-01

    Eleven different lyssavirus species, four of which occur on the African continent, are presently recognized. These viruses cause rabies, the burden of which is highest in the developing world, where routine laboratory diagnosis is often not available. From an epidemiological and control perspective, it is necessary that diagnostic methods detect the diversity of lyssaviruses present in different regions of the world. A published and widely used heminested reverse transcription-PCR (hnRT-PCR) was evaluated for its ability to detect a panel of diverse African lyssaviruses. Due to the limitations experienced for this assay, an alternative hnRT-PCR was developed. The new assay was found to be accurate and sensitive in the detection of African lyssavirus RNA in a variety of clinical specimens. The assay was further adapted to a real-time PCR platform to allow rapid, one-step, quantitative, and single-probe detection, and an internal control for the verification of sample preparation was included. The limit of detection of the real-time PCR assay was 10 RNA copies per reaction, with inter- and intra-assay variability below 4%. Subsequently, in demonstrating utility, both assays were successfully applied to antemortem rabies diagnosis in humans. We believe that the quantitative real-time PCR assay could find application as a routine confirmatory test for rabies diagnosis in the future and that it will serve as a valuable research tool in the biology of African lyssaviruses. Alternatively, the hnRT-PCR assay can be used in laboratories that do not have access to expensive real-time PCR equipment for sensitive diagnosis of lyssaviruses. PMID:20810772

  6. [Evaluation of COBAS TaqMan: a real-time PCR-based diagnostic kit for mycobacteria].

    PubMed

    Yonemaru, Makoto; Horiba, Masahide; Tada, Atsuhiko; Nagai, Takayuki

    2009-12-01

    The real-time PCR-based diagnostic kits, COBAS TaqMan MTB and COBAS TaqMan MAI (Roche Diagnostics, Tokyo, Japan), were developed to detect Mycobacterium tuberculosis (MTB) and M. avium (MAV)/M. intracellulare (MIN), respectively. The TaqMan kits simultaneously perform amplification and detection of mycobacterial DNA to reduce assay time. We evaluated the diagnostic accuracy of both TaqMan kits in 781 clinical specimens, and compared the results with those obtained from the AMPLICOR MTB and MAI kits. With smear-positive specimens, the TaqMan kits showed 100% concordance with AMPLICOR in MTB, MAV and MIN. With all specimens, the concordances of TaqMan with AMPLICOR were 99.1%, 99.0%, and 99.7% in MTB, MAV and MIN, respectively. Four specimens for MTB and one for MAV were AMPLICOR positive/TaqMan negative. Among them, two specimens were culture-positive for MTB and one for MAV. Three specimens for MTB, seven for MAV, and two for MIN were AMPLICOR negative/TaqMan positive. Among them, two specimens were culture-positive for MTB, seven for MAV, and one for MIN. In twelve out of 21 specimens in which AMPLICOR failed to activate PCR, TaqMan successfully determined the results which were in concordance with those of mycobacterial culture. Thus, our data suggest that the accuracy of TaqMan in detecting mycobacterial DNA is superior to that of AMPLICOR. We conclude that TaqMan, which is an easy and rapid DNA amplification test, is useful for detecting MTB, MAV and MIN.

  7. Improving detection of avian malaria from host blood: a step towards a standardised protocol for diagnostics.

    PubMed

    Niebuhr, Chris N; Blasco-Costa, Isabel

    2016-10-01

    Avian malaria, caused by Plasmodium spp., has been linked to the mortality and population-level declines in native birds in some regions. While molecular diagnostic methods have greatly improved our ability to detect infections of both human and bird malaria, failing to identify false negatives remains an important handicap, particularly for avian malaria due to host DNA presence in the bird blood cells. In an attempt to improve the accuracy of diagnostics by PCR, we evaluated the performance of a commercial silica-membrane-based DNA extraction kit by modifying the protocol with four unpooled elution volume alternatives. Our results suggest that the best template is the DNA extract obtained from the second eluate of a first 50 μL elution step. In one case, the only band visible was from this second eluate and, thus, may not have been identified as positive for Plasmodium spp. if a different elution protocol had been followed. Our results are likely explained by the concept of size exclusion chromatography by which particles of different sizes will elute at different rates. Overall, first elution templates may consist of a lower ratio of parasite to host DNA, while second eluates may contain a higher parasite to host DNA ratio. A low ratio of parasite to host DNA is a concern in detecting chronic infections, in which birds typically carry low levels of parasitemia, making accurate diagnostics imperative when identifying reservoirs of disease that could lead to spillback events.

  8. 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.

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

    PubMed Central

    2014-01-01

    PCR detection of intestinal protozoa is often restrained by a poor DNA recovery or by inhibitors present in feces. The need for an extraction protocol that can overcome these obstacles is therefore clear. QIAamp® DNA Stool Mini Kit (Qiagen) was evaluated for its ability to recover DNA from oocysts/cysts directly from feces. Twenty-five Giardia-positive, 15 Cryptosporidium-positive, 15 Entamoeba histolytica-positive, and 45 protozoa-free samples were processed as control by microscopy and immunoassay tests. DNA extracts were amplified using 3 sets of published primers. Following the manufacturer's protocol, the kit showed sensitivity and specificity of 100% towards Giardia and Entamoeba. However, for Cryptosporidium, the sensitivity and specificity were 60% (9/15) and 100%, respectively. A series of optimization experiments involving various steps of the kit's protocol were conducted using Cryptosporidium-positive samples. The best DNA recoveries were gained by raising the lysis temperature to the boiling point for 10 min and the incubation time of the InhibitEX tablet to 5 min. Also, using a pre-cooled ethanol for nucleic acid precipitation and small elution volume (50-100 µl) were valuable. The sensitivity of the amended protocol to Cryptosporidium was raised to 100%. Cryptosporidium DNA was successfully amplified by either the first or the second primer set. When applied on parasite-free feces spiked with variable oocysts/cysts counts, ≈ 2 oocysts/cysts were theoretically enough for detection by PCR. To conclude, the Qiagen kit with the amended protocol was proved to be suitable for protozoan DNA extraction directly from feces and support PCR diagnosis. PMID:25031466

  10. 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.

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

    PubMed Central

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

    2014-01-01

    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

  12. 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

  13. Improved timestep-size diagnostic edits for TRAC-P

    SciTech Connect

    Giguere, P.T.

    1996-04-01

    Improvements have been made to the timestep-size selection logic diagnostic edits of the Transient Reactor Analysis Code (TRAC), specifically to the TRAC-P version. These include both a precise account of the reason for the selection for individual timesteps and thermal-hydraulic information on mesh cells that control the timestep size. The new edits can be specified by user input as a range of timestep numbers, problem time, or both. A description of the current timestep controls in effect in TRAC-P is also given.

  14. Improving Model Performance through Process-Based Diagnostics

    NASA Astrophysics Data System (ADS)

    Clune, T.; Kuo, K.; Schmidt, G. A.; Bauer, M. P.; Oloso, A. O.

    2013-12-01

    Many of the aspects of the climate system that are of the greatest interest (e.g., the sensitivity of the system to external forcings) are emergent properties that arise via the complex interplay between disparate processes. This is also true for climate models -- most diagnostics are not a function of an isolated portion of source code, but rather are affected by multiple components and procedures. Thus any model-observation mismatch is hard to attribute to any specific piece of code or imperfection in a specific model assumption. An alternative approach is to identify diagnostics that are more closely tied to specific processes -- implying that if a mismatch is found, it should be much easier to identify and address specific algorithmic choices that will improve the simulation. However, this approach requires looking at model output and observational data in a more sophisticated way than the more traditional production of monthly or annual mean quantities. The data must instead be filtered in time and space for examples of the specific process being targeted. We are developing a data analysis environment called PROcess-Based Explorer (PROBE) that seeks to enable efficient and systematic computation of process-based diagnostics on very large sets of data. In this environment, investigators can define arbitrarily complex filters and then seamlessly perform computations in parallel on the filtered output from their model. The same analysis can be performed on additional related data sets (e.g., reanalyses) thereby enabling routine comparisons between model and observational data. PROBE also incorporates workflow technology to automatically update computed diagnostics for subsequent executions of a model. In this presentation, we will discuss the design and current status of PROBE as well as share results from some preliminary use cases.

  15. Comparing the Performance of Hybrid Capture II and Polymerase Chain Reaction (PCR) for the Identification of Cervical Dysplasia in the Screening and Diagnostic Settings.

    PubMed

    Luu, Hung N; Adler-Storthz, Karen; Dillon, Laura M; Follen, Michele; Scheurer, Michael E

    2013-01-01

    Both PCR and Hybrid Capture II (HCII) have been used for identifying cervical dysplasia; however, comparisons on the performance between these two tests show inconsistent results. We evaluated the performance of HCII and PCR MY09/11 in both screening and diagnostic populations in sub-sample of 1,675 non-pregnant women from a cohort in three clinical centers in the United States and Canada. Sensitivity, specificity, positive predictive value, negative predictive value, and concordance between the two tests were calculated. Specificity of HCII in detecting low-grade squamous intraepithelial lesion (LSIL) was higher in the screening group (88.7%; 95% CI: 86.2%-90.8%) compared to the diagnostic group (46.3%; 95% CI: 42.1%-50.6%); however, specificity of PCR was low in both the screening (32.8%; 95% CI: 29.6%-36.2%) and diagnostic (14.4%; 95% CI: 11.6%-17.6%) groups. There was comparable sensitivity by both tests in both groups to detect high-grade squamous intraepithelial lesion (HSIL); however, HCII was more specific (89.1%; 95% CI: 86.8%-91.0%; 66.2%; 95% CI: 62.0%-70.1%) than PCR (33.3%; 95% CI: 30.2%-36.5%; 17.9%; 95% CI: 14.8%-21.6%) in the screening and diagnostic groups, respectively. Overall agreement for HPV positivity was approximately 50% between HCII and PCR MY09/11; with more positive results coming from the PCR MY09/11. In the current study, PCR MY09/11 was more sensitive but less specific than HCII in detecting LSIL, and HCII was more sensitive and specific in detecting HSIL than PCR in both screening and diagnostic groups.

  16. 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.

  17. 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

  18. A Unique Capsule Locus in the Newly Designated Actinobacillus pleuropneumoniae Serovar 16 and Development of a Diagnostic PCR Assay

    PubMed Central

    Li, Yanwen; Sárközi, Rita; Gottschalk, Marcelo; Angen, Øystein; Nedbalcova, Katerina; Rycroft, Andrew N.; Fodor, László

    2017-01-01

    ABSTRACT Actinobacillus pleuropneumoniae causes pleuropneumonia, an economically significant lung disease of pigs. Recently, isolates of A. pleuropneumoniae that were serologically distinct from the previously characterized 15 serovars were described, and a proposal was put forward that they comprised a new serovar, serovar 16. Here we used whole-genome sequencing of the proposed serovar 16 reference strain A-85/14 to confirm the presence of a unique capsular polysaccharide biosynthetic locus. For molecular diagnostics, primers were designed from the capsule locus of strain A-85/14, and a PCR was formulated that differentiated serovar 16 isolates from all 15 known serovars and other common respiratory pathogenic/commensal bacteria of pigs. Analysis of the capsule locus of strain A-85/14 combined with the previous serological data show the existence of a sixteenth serovar—designated serovar 16—of A. pleuropneumoniae. PMID:28053219

  19. A Unique Capsule Locus in the Newly Designated Actinobacillus pleuropneumoniae Serovar 16 and Development of a Diagnostic PCR Assay.

    PubMed

    Bossé, Janine T; Li, Yanwen; Sárközi, Rita; Gottschalk, Marcelo; Angen, Øystein; Nedbalcova, Katerina; Rycroft, Andrew N; Fodor, László; Langford, Paul R

    2017-03-01

    Actinobacillus pleuropneumoniae causes pleuropneumonia, an economically significant lung disease of pigs. Recently, isolates of A. pleuropneumoniae that were serologically distinct from the previously characterized 15 serovars were described, and a proposal was put forward that they comprised a new serovar, serovar 16. Here we used whole-genome sequencing of the proposed serovar 16 reference strain A-85/14 to confirm the presence of a unique capsular polysaccharide biosynthetic locus. For molecular diagnostics, primers were designed from the capsule locus of strain A-85/14, and a PCR was formulated that differentiated serovar 16 isolates from all 15 known serovars and other common respiratory pathogenic/commensal bacteria of pigs. Analysis of the capsule locus of strain A-85/14 combined with the previous serological data show the existence of a sixteenth serovar-designated serovar 16-of A. pleuropneumoniae.

  20. Orthogonal amplification of nanoparticles for improved diagnostic sensing.

    PubMed

    Peterson, Vanessa M; Castro, Cesar M; Lee, Hakho; Weissleder, Ralph

    2012-04-24

    There remains an ongoing need for fast, highly sensitive, and quantitative technologies that can detect and profile rare cells in freshly harvested samples. Recent developments in nanomaterial-based detection platforms provide advantages over traditional approaches in terms of signal sensitivity, stability, and the possibility for performing multiplexed measurements. Here, we describe a bioorthogonal, nanoparticle amplification technique capable of rapid augmentation of detection sensitivities by up to 1-2 orders of magnitude over current methods. This improvement in sensitivity was achieved by (i) significantly reducing background noise arising from nonspecific nanoparticle binding, (ii) increasing nanomaterial binding through orthogonal rounds of amplification, and (iii) implementing a cleavage step to improve assay robustness. The developed method allowed sensitive detection and molecular profiling of scant tumor cells directly in unpurified human clinical samples such as ascites. With its high sensitivity and simplified assay steps, this technique will likely have broad utility in nanomaterial-based diagnostics.

  1. 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

  2. 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.

  3. Diagnostic Evaluation of Multiplexed Reverse Transcription-PCR Microsphere Array Assay for Detection of Foot-and-Mouth and Look-Alike Disease Viruses▿

    PubMed Central

    Hindson, Benjamin J.; Reid, Scott M.; Baker, Brian R.; Ebert, Katja; Ferris, Nigel P.; Tammero, Lance F. Bentley; Lenhoff, Raymond J.; Naraghi-Arani, Pejman; Vitalis, Elizabeth A.; Slezak, Thomas R.; Hullinger, Pamela J.; King, Donald P.

    2008-01-01

    A high-throughput multiplexed assay was developed for the differential laboratory detection of foot-and-mouth disease virus (FMDV) from viruses that cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses by using multiplexed reverse transcription-PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the 17 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 assay was evaluated using 287 field samples, including 247 samples (213 true-positive samples and 35 true-negative samples) from suspected 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 to those of two singleplex rRT-PCR assays, using virus isolation with antigen enzyme-linked immunosorbent assays as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% (95% confidence interval [CI], 89.8 to 96.4%), and the sensitivity was 98.1% (95% CI, 95.3 to 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 by using focused single-target rRT-PCR assays. PMID:18216216

  4. 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.

  5. [PCR: basics and new developments].

    PubMed

    Berchtold, M W; Hübscher, U

    1996-01-01

    Since its discovery 10 years ago PCR has been introduced for a variety of practical applications. PCR has opened new dimensions particularly in laboratory diagnostics because of its sensitivity, accuracy and speed. In spite of the availability of user friendly kits, basic knowledge is of great importance for the user especially if PCR has to be optimized for special needs or when specific problems arise. The general mechanism of the reaction and the significance of the reaction components and the PCR conditions are discussed initially. Several recent developments in PCR (new enzymes, RNA-PCR, improvements of the specificity, prevention of contamination and development of new equipment) that are critical for the user are shortly introduced. Finally, "long PCR" is discussed in order to demonstrate that even 10 years after the invention of PCR significant new breakthroughs in the PCR technology are still possible.

  6. PCR screening for 22q11.2 microdeletion: development of a new cost-effective diagnostic tool.

    PubMed

    Gioli-Pereira, L; Pereira, A C; Mesquita, S M; Lopes, A A; Krieger, J E

    2006-07-15

    Del22q11.2 syndrome is the most frequent known chromosomal microdeletion syndrome. Previous studies suggest that a substantial number of patients with congenital heart disease have a 22q11 deletion. The molecular diagnosis of Del22q11.2 is usually made by fluorescence in situ hybridization, an expensive and not widely available technique. We developed an efficient and cost-effective PCR SNP assay designed for the screening of 22q11.2 deletion through consecutive homozygosity. Through the screening of dbSNP we have selected SNP markers located in the 22q11.2 microdeleted region. Population heterozygosities were determined in 213 normal individuals. Designed assays consisted of PCR amplification followed by restriction enzyme digestion. Fragments generated were visualized on agarose gel and genotyped. Selected markers were: rs5748411, rs2238778, rs4819523 and rs4680. All selected markers were localized in the 22q11.2 deleted region. Allele and genotype frequencies of all selected markers were under Hardy-Weinberg equilibrium. Selected SNPs were not in linkage disequilibrium. Predicted assay specificity was estimated to be 92.86% in the Brazilian population. The use of consecutive homozygosity in this SNP-based diagnostic test may be used as a cost-effective tool in reference molecular genetics laboratories.

  7. A newly developed BVDV-1 RT-qPCR Taqman assay based on Italian isolates: evaluation as a diagnostic tool.

    PubMed

    Zoccola, Roberto; Mazzei, Maurizio; Carrozza, Maria Luisa; Ricci, Emanuele; Forzan, Mario; Pizzurro, Federica; Giammarioli, Monica; Bandecchi, Patrizia; Tolari, Francesco

    2017-01-26

    A single-step TaqMan® RT-qPCR was developed for the detection of bovine viral diarrhea virus type 1 (BVDV-1), an important pathogen of cattle worldwide. The assay was based on conserved 5'UTR sequences of Italian BVDV-1 isolates. In order to establish a diagnostic protocol which simplifies sample collection and processing, the assay was tested on a variety of biological specimens collected from persistently infected calves. The samples analyzed included PBMCs, plasma, dry blood, ear notch and hair bulb. Time and costs required for the analysis of each type of specimen were compared. The RT-qPCR, whose lower limit of detection was 100 copies of viral RNA (1 TCID50), correctly identified all PI animals, irrespective of the type of specimen. The highest copy numbers were obtained from the RNAs extracted from PBMCs, ear notches and hair bulbs. Hair bulb-supernatants directly used as a template allowed identification of all PI animals. In conclusion, based on time and cost evaluation, the most effective and efficient protocol was the one based on the direct analysis of hair bulb-supernatants, avoiding the RNA extraction step.

  8. Diagnostic accuracy of fungal PCR and β-D-glucan for detection of candidaemia: a preliminary evaluation.

    PubMed

    McKeating, Cara; White, P Lewis; Posso, Raquel; Palmer, Michael; Johnson, Elizabeth; McMullan, Ronan

    2017-09-28

    Although treatment for candidaemia is time critical, culture-based tests prolong turnaround times and may promote underdiagnosis. Non-culture-based tests have the potential to overcome these difficulties but are in limited clinical use. The aim of this work was to undertake an initial evaluation of two non-culture-based tests for diagnosis of candidaemia. Patients with candidaemia were identified prospectively over a 4-month period. Sera drawn from case (candidaemic) and control (non-candidaemic) patients on the same day as the positive blood culture were tested with both the Renishaw RenDx Fungiplex test and a commercial β-D-glucan (BDG) assay (Fungitell, Associates of Cape Cod). Sensitivity and specificity were calculated independently and in combination, using paired blood culture as the reference standard. There were 10 eligible case patients and 39 negative controls. PCR sensitivity and specificity were found to be 44.4% (95% CI 18.9% to 73.3%) and 87.2% (72.8% to 94.8%), respectively. BDG sensitivity and specificity were 80% (47.9% to 95.4%) and 89.7% (75.9% to 96.5%), respectively. When combining PCR and BDG, sensitivity was 90% (95% CI 57.4% to 100%) and specificity was 79.5% (64.2% to 89.5%). When two sequential specimens were tested, PCR sensitivity increased to 60% (95% CI 31.2% to 83.3%) and BDG sensitivity to 90% (54.7% to 100%). A combination of tests, or a single test at multiple time points, may be preferable to relying on one test at a single time point. This should be accounted for in design of future diagnostic accuracy studies of tests for invasive candidosis. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  9. Diagnostic needle arthroscopy and the economics of improved diagnostic accuracy: a cost analysis.

    PubMed

    Voigt, Jeffrey D; Mosier, Michael; Huber, Bryan

    2014-10-01

    Hundreds of thousands of surgical arthroscopy procedures are performed annually in the United States (US) based on MRI findings. There are situations where these MRI findings are equivocal or indeterminate and because of this clinicians commonly perform the arthroscopy in order not to miss pathology. Recently, a less invasive needle arthroscopy system has been introduced that is commonly performed in the physician office setting and that may help improve the accuracy of diagnostic findings. This in turn may prevent unnecessary follow-on arthroscopy procedures from being performed. The purpose of this analysis is to determine whether the in-office diagnostic needle arthroscopy system can provide cost savings by reducing unnecessary follow on arthroscopy procedures. Data obtained from a recent trial and from a systematic review were used in comparing the accuracy of MRI and VisionScope needle arthroscopy (VSI) with standard arthroscopy (gold standard). The resultant false positive and false negative findings were then used to evaluate the costs of follow-on procedures. These differences were then modeled for the US patient population diagnosed and treated for meniscal knee pathology (most common disorder) to determine if a technology such as VSI could save the US healthcare system money. Data on surgical arthroscopy procedures in the US for meniscal knee pathology were used (calendar year [CY] 2010). The costs of performing diagnostic and surgical arthroscopy procedures (using CY 2013 Medicare reimbursement amounts), costs associated with false negative findings, and the costs for treating associated complications arising from diagnostic and therapeutic arthroscopy procedures were assessed. In patients presenting with medial meniscal pathology (International Classification of Diseases, 9th edition, Clinical Modification [ICD9CM] diagnosis 836.0), VSI in place of MRI (standard of care) resulted in a net cost savings to the US system of US$115-US$177 million (CY 2013

  10. Improved FLT3/ITD PCR assay predicts outcome following allogeneic transplant for AML

    PubMed Central

    Grunwald, Michael R.; Tseng, Li-Hui; Lin, Ming-Tseh; Pratz, Keith W.; Eshleman, James R.; Levis, Mark J.; Gocke, Christopher D.

    2014-01-01

    Acute myeloid leukemia (AML) patients harboring internal tandem duplication (ITD) mutations of the FMS-like tyrosine kinase 3 (FLT3) gene carry a poor prognosis. While allogeneic transplantation may improve outcomes, relapse occurs frequently. The FLT3/ITD mutation has been deemed an unsuitable minimal residual disease (MRD) marker because it is unstable and because the standard assay for the mutation is relatively insensitive. The FLT3 mutation is undetectable by polymerase chain reaction (PCR) at pre- or post-transplant time points in many FLT3/ITD AML patients who subsequently relapse following transplant. We report the application of a new technique, tandem duplication PCR (TD-PCR), for detecting MRD in FLT3/ITD AML patients. Between October 2004 and January 2012, 54 FLT3/ITD AML patients in remission underwent transplantation at our institution. Of 37 patients with available Day 60 marrow samples, 28 (76%) were evaluable for MRD detection. In seven (25%) of the 28 patients, the FLT3/ITD mutation was detectable by TD-PCR, but not by standard PCR, on day 60. Six out of the seven patients (86%) with MRD by TD-PCR have relapsed to date compared with only 2 of 21 (10%) patients who were negative for MRD (p = 0.0003). The ability to detect MRD by this sensitive technique may provide an opportunity for early clinical intervention. PMID:25240816

  11. PCR-hybridization after sonication improves diagnosis of implant-related infection.

    PubMed

    Esteban, Jaime; Alonso-Rodriguez, Noelia; del-Prado, Gema; Ortiz-Pérez, Alberto; Molina-Manso, Diana; Cordero-Ampuero, Jose; Sandoval, Enrique; Fernández-Roblas, Ricardo; Gómez-Barrena, Enrique

    2012-06-01

    We wanted to improve the diagnosis of implant-related infection using molecular biological techniques after sonication. We studied 258 retrieved implant components (185 prosthetic implants and 73 osteosynthesis implants) from 126 patients. 47 patients had a clinical diagnosis of infection (108 components) and 79 patients did not (150 components). The fluids from sonication of retrieved implants were tested in culture and were also analyzed using a modified commercial PCR kit for detection of Gram-positive and Gram-negative bacteria (GenoType BC; Hain Lifescience) after extraction of the DNA. 38 of 47 patients with a clinical diagnosis of infection were also diagnosed as being infected using culture and/or PCR (35 by culture alone). Also, 24 patients of the 79 cases with no clinical diagnosis of infection were identified microbiologically as being infected (4 by culture, 16 by PCR, and 4 by both culture and PCR). Comparing culture and PCR, positive culture results were obtained in 28 of the 79 patients and positive PCR results were obtained in 35. There were 21 discordant results in patients who were originally clinically diagnosed as being infected and 28 discordant results in patients who had no clinical diagnosis of infection. For prosthetic joint infections and relative to culture, molecular detection can increase (by one tenth) the number of patients diagnosed as having an infection. Positive results from patients who have no clinical diagnosis of infection must be interpreted carefully.

  12. 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.

  13. Improving on the diagnostic characteristics of echocardiography for pulmonary hypertension.

    PubMed

    Broderick-Forsgren, Kathleen; Davenport, Clemontina A; Sivak, Joseph A; Hargett, Charles William; Foster, Michael C; Monteagudo, Andrew; Armour, Alicia; Rajagopal, Sudarshan; Arges, Kristine; Velazquez, Eric J; Samad, Zainab

    2017-03-24

    This retrospective study evaluated the diagnostic characteristics of a combination of echocardiographic parameters for pulmonary hypertension (PH). Right ventricular systolic pressure (RVSP) estimation by echocardiography (echo) is used to screen for PH. However, the sensitivity of this method is suboptimal. We hypothesized that RVSP estimation in conjunction with other echo parameters would improve the value of echo for PH. The Duke Echo database was queried for adult patients with known or suspected PH who had undergone both echo and right heart catheterization (RHC) within a 24 h period between 1/1/2008 and 12/31/2013. Patients with complex congenital heart disease, heart transplantation, ventricular assist device, or on mechanical ventilation at time of study were excluded. Diagnostic characteristics of several echo parameters (right atrial enlargement, pulmonary artery (PA) enlargement, RV enlargement, RV dysfunction, and RVSP) for PH (mean PA pressure 25 mmHg on RHC) were evaluated among 1007 patients. RVSP ≥40 had a sensitivity of 77% (accuracy 77), while RVSP ≥35 had the highest sensitivity at 88% (81% accuracy). PA enlargement had the lowest sensitivity at 17%. The area under the curve (AUC) for RVSP was 0.844. A model including RVSP, RA, PA, RV enlargement and RV dysfunction had a higher AUC (AUC = 0.87) than RVSP alone. The value of echo as a screening test for PH is improved by a model incorporating a lower RVSP in addition to other right heart parameters. These findings need to be validated in prospective cohorts.

  14. 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

  15. 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.

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

    PubMed Central

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

    2012-01-01

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

  17. Improved Efficiency and Robustness in qPCR and Multiplex End-Point PCR by Twisted Intercalating Nucleic Acid Modified Primers

    PubMed Central

    Schneider, Uffe Vest; Mikkelsen, Nikolaj Dam; Lindqvist, Anja; Okkels, Limei Meng; Jøhnk, Nina; Lisby, Gorm

    2012-01-01

    We introduce quantitative polymerase chain reaction (qPCR) primers and multiplex end-point PCR primers modified by the addition of a single ortho-Twisted Intercalating Nucleic Acid (o-TINA) molecule at the 5′-end. In qPCR, the 5′-o-TINA modified primers allow for a qPCR efficiency of 100% at significantly stressed reaction conditions, increasing the robustness of qPCR assays compared to unmodified primers. In samples spiked with genomic DNA, 5′-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers. In unspiked samples, replacement of unmodified DNA primers with 5′-o-TINA modified primers permits an increased qPCR stringency. Compared to unmodified DNA primers, this allows for a qPCR efficiency of 100% at lowered primer concentrations and at increased annealing temperatures with unaltered cross-reactivity for primers with single nucleobase mismatches. In a previously published octaplex end-point PCR targeting diarrheagenic Escherichia coli, application of 5′-o-TINA modified primers allows for a further reduction (>45% or approximately one hour) in overall PCR program length, while sustaining the amplification and analytical sensitivity for all targets in crude bacterial lysates. For all crude bacterial lysates, 5′-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures for all eight targets. Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness. Thus, 5′-o-TINA modified primers are advantageous in PCR assays, where one or more primer pairs are required to perform at stressed reaction conditions. PMID:22701644

  18. Improved efficiency and robustness in qPCR and multiplex end-point PCR by twisted intercalating nucleic acid modified primers.

    PubMed

    Schneider, Uffe Vest; Mikkelsen, Nikolaj Dam; Lindqvist, Anja; Okkels, Limei Meng; Jøhnk, Nina; Lisby, Gorm

    2012-01-01

    We introduce quantitative polymerase chain reaction (qPCR) primers and multiplex end-point PCR primers modified by the addition of a single ortho-Twisted Intercalating Nucleic Acid (o-TINA) molecule at the 5'-end. In qPCR, the 5'-o-TINA modified primers allow for a qPCR efficiency of 100% at significantly stressed reaction conditions, increasing the robustness of qPCR assays compared to unmodified primers. In samples spiked with genomic DNA, 5'-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers. In unspiked samples, replacement of unmodified DNA primers with 5'-o-TINA modified primers permits an increased qPCR stringency. Compared to unmodified DNA primers, this allows for a qPCR efficiency of 100% at lowered primer concentrations and at increased annealing temperatures with unaltered cross-reactivity for primers with single nucleobase mismatches. In a previously published octaplex end-point PCR targeting diarrheagenic Escherichia coli, application of 5'-o-TINA modified primers allows for a further reduction (>45% or approximately one hour) in overall PCR program length, while sustaining the amplification and analytical sensitivity for all targets in crude bacterial lysates. For all crude bacterial lysates, 5'-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures for all eight targets. Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness. Thus, 5'-o-TINA modified primers are advantageous in PCR assays, where one or more primer pairs are required to perform at stressed reaction conditions.

  19. 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.

  20. Strategies to Improve Efficiency and Specificity of Degenerate Primers in PCR.

    PubMed

    Campos, Maria Jorge; Quesada, Alberto

    2017-01-01

    PCR with degenerate primers can be used to identify the coding sequence of an unknown protein or to detect a genetic variant within a gene family. These primers, which are complex mixtures of slightly different oligonucleotide sequences, can be optimized to increase the efficiency and/or specificity of PCR in the amplification of a sequence of interest by the introduction of mismatches with the target sequence and balancing their position toward the primers 5'- or 3'-ends. In this work, we explain in detail examples of rational design of primers in two different applications, including the use of specific determinants at the 3'-end, to: (1) improve PCR efficiency with coding sequences for members of a protein family by fully degeneration at a core box of conserved genetic information, with the reduction of degeneration at the 5'-end, and (2) optimize specificity of allelic discrimination of closely related orthologous by 5'-end degenerate primers.

  1. Diagnostic accuracy of quantitative real-time PCR assay versus clinical and Gram stain identification of bacterial vaginosis.

    PubMed

    Menard, J-P; Mazouni, C; Fenollar, F; Raoult, D; Boubli, L; Bretelle, F

    2010-12-01

    The purpose of this investigation was to determine the diagnostic accuracy of quantitative real-time polymerase chain reaction (PCR) assay in diagnosing bacterial vaginosis versus the standard methods, the Amsel criteria and the Nugent score. The Amsel criteria, the Nugent score, and results from the molecular tool were obtained independently from vaginal samples of 163 pregnant women who reported abnormal vaginal symptoms before 20 weeks gestation. To determine the performance of the molecular tool, we calculated the kappa value, sensitivity, specificity, and positive and negative predictive values. Either or both of the Amsel criteria (≥3 criteria) and the Nugent score (score ≥7) indicated that 25 women (15%) had bacterial vaginosis, and the remaining 138 women did not. DNA levels of Gardnerella vaginalis or Atopobium vaginae exceeded 10(9) copies/mL or 10(8) copies/mL, respectively, in 34 (21%) of the 163 samples. Complete agreement between both reference methods and high concentrations of G. vaginalis and A. vaginae was found in 94.5% of women (154/163 samples, kappa value = 0.81, 95% confidence interval 0.70-0.81). The nine samples with discordant results were categorized as intermediate flora by the Nugent score. The molecular tool predicted bacterial vaginosis with a sensitivity of 100%, a specificity of 93%, a positive predictive value of 73%, and a negative predictive value of 100%. The quantitative real-time PCR assay shows excellent agreement with the results of both reference methods for the diagnosis of bacterial vaginosis.

  2. Stratified computed tomography findings improve diagnostic accuracy for appendicitis

    PubMed Central

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

    2014-01-01

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

  3. Balanitis xerotica obliterans: has its diagnostic accuracy improved with time?

    PubMed Central

    Patwardhan, Nitin

    2017-01-01

    Objectives We observed whether general practitioners are referring more appropriately for balanitis xerotica obliterans in regards to circumcision, especially at a time of clinical concern, and whether their discriminative abilities were affected by age. We also aimed to explore if balanitis xerotica obliterans was over-diagnosed by surgeons potentially leading to unnecessary circumcisions of healthy foreskins. Design Cross-sectional descriptive study. Setting Leicester Royal Infirmary. Participants All children less than 16 years of age were included and were subsequently split into two categories: those less than or equal to five years and those above five years. Circumcision was justified if surgeon found pathology under foreskin commissioning guidelines set by the Royal College of Surgeons of England. After clinical diagnosis of balanitis xerotica obliterans, the pathological database was searched for histological confirmation. Main outcome measures Has diagnostic accuracy improved amongst general practitioners for balanitis xerotica obliterans and is there a high clinical to histological confirmation. Results Of the total patients, 14.5% were diagnosed clinically with balanitis xerotica obliterans. Only 66.7% of cases were histologically confirmed with chronic inflammation found in the rest; 5.5% of all boys referred had balanitis xerotica obliterans on histology; and 8.2% of children <5 had clinical balanitis xerotica obliterans with 1.7% confirmed histologically. This was in contrast with 18.1% and 9.2% found in the older cohort. Conclusion There remains a high diagnostic inaccuracy amongst general practitioners when referring for balanitis xerotica obliterans. This is greatest in those under five years. Although balanitis xerotica obliterans was over-diagnosed, no healthy foreskin underwent unnecessary circumcision. PMID:28620502

  4. Quantification of serum SOX2 DNA with FQ-PCR potentially provides a diagnostic biomarker for lung cancer.

    PubMed

    Wu, Yanfeng; Du, Xiao; Xue, Chengjun; Li, Detao; Zheng, Qian; Li, Xue; Chen, Hui

    2013-12-01

    Sex-determining region Y-box 2 (SOX2), as a subunit of transcription and reprogramming factor, plays a critical role in the development and progression of many malignancies, including lung cancer through gene amplification. In the present study, we aimed to quantify the levels of serum SOX2 DNA, analyze its diagnostic value and compare it with existing clinical parameters in lung cancer, and purpose to provide a novel tumor marker for lung cancer. Serum DNA was extracted from 94 lung cancer patients, 10 benign lung diseases, and 30 healthy volunteers, and then the levels of SOX2 DNA were quantified using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR). The data were analyzed by statistical software SPSS14.0. The present results show that serum SOX2 DNA level in lung cancer group was higher compared to the levels in benign lung diseases group (u = 102.0, p < 0.001) or healthy group (u = 140.0, p < 0.001), and it was closely associated with TNM stage, histopathological type, and tumor size (p = 0.031, p = 0.012, and p = 0.010, respectively). However, serum SOX2 DNA levels of lung cancer patients were not associated with age, gender, smoking status, lymph node metastasis, or tumor differentiation (p > 0.05). ROC curve showed a sensitivity of 78.9% and a specificity of 82.5% for the ability of serum SOX2 DNA to detect lung cancer at the cutoff value of 1,078.3 copies/ul. Furthermore, we assessed the associations of serum SOX2 levels with clinical existing lung tumor markers, such as squamous cell carcinoma antigen, cytokeratin fragment 21-1, and neuron-specific enolase. The sensitivity was increased from 24.9, 66.1, and 39.1 to 84.2, 92.8, and 87.5%, respectively, by the combination of serum SOX2 DNA. Taken together, quantification of serum SOX2 DNA by FQ-PCR may serve as a novel accessory diagnostic tool for the clinical screening and detection of lung cancer.

  5. Improved Serotype-Specific Dengue Virus Detection in Trinidad and Tobago using a Multiplex, Real-Time RT-PCR

    PubMed Central

    Waggoner, Jesse J.; Sahadeo, Nikita S. D.; Brown, Arianne; Mohamed-Hadley, Alisha; Hadley, Dexter; Carrington, Leslie; Carrington, Christine V. F.; Pinsky, Benjamin A.

    2014-01-01

    Dengue virus (DENV) transmission occurs throughout the Caribbean, though laboratory confirmation and epidemiologic surveillance is limited by the availability of serotype-specific molecular diagnostics. In this study, we show that a serotype-specific DENV multiplex, real-time RT-PCR detected DENV RNA in significantly more samples (82/182) than a reference hemi-nested RT-PCR (57/182; p=0.01). PMID:25533614

  6. Diagnostic value of conjunctival swab sampling associated with nested PCR for different categories of dogs naturally exposed to Leishmania infantum infection.

    PubMed

    Di Muccio, Trentina; Veronesi, Fabrizia; Antognoni, Maria Teresa; Onofri, Andrea; Piergili Fioretti, Daniela; Gramiccia, Marina

    2012-08-01

    The objective of the present study was to evaluate the diagnostic performance of a noninvasive assay, conjunctival swab (CS) nested-PCR (n-PCR), for diagnosing canine leishmaniasis (CanL) in different stages of infection in comparison to the performance of the indirect immunofluorescence antibody test (IFAT), lymph node microscopy, and buffy coat n-PCR. To this end, we performed a cross-sectional survey among 253 nonselected dogs in areas of endemicity in central Italy. We also performed a longitudinal study of CS n-PCR among 20 sick dogs undergoing antileishmanial treatment. In the first study, among the 72 animals that were positive by at least one test (28.45%), CS n-PCR showed the best relative performance (76.38%), with a high concordance in comparison to standard IFAT serology (κ = 0.75). The highest positivity rates using CS n-PCR were found in asymptomatic infected dogs (84.2%) and sick dogs (77.8%); however, the sensitivity of the assay was not associated with the presence of clinical signs. In the follow-up study on treated sick dogs, CS n-PCR was the most sensitive assay, with promising prognostic value for relapses. The univariate analysis of risk factors for CanL based on CS n-PCR findings showed a significant correlation with age (P = 0.012), breed size (P = 0.026), habitat (P = 4.9 × 10(-4)), and previous therapy (P = 0.014). Overall, the results indicated that CS n-PCR was the most sensitive assay of the less invasive diagnostic methods and could represent a good option for the early and simple diagnosis of CanL infection in asymptomatic animals and for monitoring relapses in drug-treated dogs.

  7. Improvement in Laboratory Diagnosis of Wound Botulism and Tetanus among Injecting Illicit-Drug Users by Use of Real-Time PCR Assays for Neurotoxin Gene Fragments

    PubMed Central

    Akbulut, D.; Grant, K. A.; McLauchlin, J.

    2005-01-01

    An upsurge in wound infections due to Clostridium botulinum and Clostridium tetani among users of illegal injected drugs (IDUs) occurred in the United Kingdom during 2003 and 2004. A real-time PCR assay was developed to detect a fragment of the neurotoxin gene of C. tetani (TeNT) and was used in conjunction with previously described assays for C. botulinum neurotoxin types A, B, and E (BoNTA, -B, and -E). The assays were sensitive, specific, rapid to perform, and applicable to investigating infections among IDUs using DNA extracted directly from wound tissue, as well as bacteria growing among mixed microflora in enrichment cultures and in pure culture on solid media. A combination of bioassay and PCR test results confirmed the clinical diagnosis in 10 of 25 cases of suspected botulism and two of five suspected cases of tetanus among IDUs. The PCR assays were in almost complete agreement with the conventional bioassays when considering results from different samples collected from the same patient. The replacement of bioassays by real-time PCR for the isolation and identification of both C. botulinum and C. tetani demonstrates a sensitivity and specificity similar to those of conventional approaches. However, the real-time PCR assays substantially improves the diagnostic process in terms of the speed of results and by the replacement of experimental animals. Recommendations are given for an improved strategy for the laboratory investigation of suspected wound botulism and tetanus among IDUs. PMID:16145075

  8. Improvement in laboratory diagnosis of wound botulism and tetanus among injecting illicit-drug users by use of real-time PCR assays for neurotoxin gene fragments.

    PubMed

    Akbulut, D; Grant, K A; McLauchlin, J

    2005-09-01

    An upsurge in wound infections due to Clostridium botulinum and Clostridium tetani among users of illegal injected drugs (IDUs) occurred in the United Kingdom during 2003 and 2004. A real-time PCR assay was developed to detect a fragment of the neurotoxin gene of C. tetani (TeNT) and was used in conjunction with previously described assays for C. botulinum neurotoxin types A, B, and E (BoNTA, -B, and -E). The assays were sensitive, specific, rapid to perform, and applicable to investigating infections among IDUs using DNA extracted directly from wound tissue, as well as bacteria growing among mixed microflora in enrichment cultures and in pure culture on solid media. A combination of bioassay and PCR test results confirmed the clinical diagnosis in 10 of 25 cases of suspected botulism and two of five suspected cases of tetanus among IDUs. The PCR assays were in almost complete agreement with the conventional bioassays when considering results from different samples collected from the same patient. The replacement of bioassays by real-time PCR for the isolation and identification of both C. botulinum and C. tetani demonstrates a sensitivity and specificity similar to those of conventional approaches. However, the real-time PCR assays substantially improves the diagnostic process in terms of the speed of results and by the replacement of experimental animals. Recommendations are given for an improved strategy for the laboratory investigation of suspected wound botulism and tetanus among IDUs.

  9. Diagnostic performance of a multiple real-time PCR assay in patients with suspected sepsis hospitalized in an internal medicine ward.

    PubMed

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

    2012-04-01

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

  10. 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

  11. Improving the safety of room air pneumoperitoneum for diagnostic laparoscopy.

    PubMed

    Ikechebelu, J I; Okeke, C A F

    2008-06-01

    Laparoscopic examination is a useful investigation in the evaluation of infertile women. To perform this test, pneumoperitoneum is required to distend the abdomen, improve visibility and displace the intestines out of the pelvis. Several gases have been used to achieve this purpose including Nitrous Oxide (N2O), Carbondioxide (CO2), Helium, Xenon andAir. This was a prospective study in a private fertility centre in Nnewi, Nigeria aimed at reducing the morbidities inherent in the use Room Air pneumoperitoneum for diagnostic laparoscopy. This was sequel to an earlier study, which revealed that women who had Room Air pneumoperitoneum had a higher port wound infection rate, abdominal discomfort (feeling of retained gas in the abdomen) and shoulder pain with resultant delayed return to normal activity than women who had Co2 pneumoperitoneum. This study demonstrated that the use of soda lime to purify the Room Air and a low pressure suction pump to evacuate the air after the procedure significantly reduced the wound infection rate and virtually eliminated the abdominal discomfort and shoulder pain associated with Room Air pneumoperitoneum. This was followed by early return to normal activity. Therefore, use of Room Air for pneumoperitoneum is safe and affordable. It is recommended for low resource settings.

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

    PubMed

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

    2015-01-01

    Campylobacter jejuni is responsible for the majority of Campylobacter infections. As the molecular epidemiological study of outbreaks, pulsed-field gel electrophoresis (PFGE) is performed in general. But PFGE has several problems. PCR binary typing (P-BIT) method is a typing method for Campylobacter spp. that was recently developed, and was reported to have a similar discriminatory power and stability to those of PFGE. We modified the P-BIT method from 18 monoplex PCRs to two multiplex PCR systems (mP-BIT). The same results were obtained from monoplex PCRs using original primers and multiplex PCR in the representative isolates. The mP-BIT can analyze 48 strains at a time by using 96-well PCR systems and can identify C. jejuni because mP-BIT includes C. jejuni marker. The typing of the isolates by the mP-BIT and PFGE demonstrated generally concordant results and the mP-BIT method (D = 0.980) has a similar discriminatory power to that of PFGE with SmaI digest (D = 0.975) or KpnI digest (D = 0.987) as with original article. The mP-BIT method is quick, simple and easy, and comes to be able to perform it at low cost by having become a multiplex PCR system. Therefore, the mP-BIT method with two multiplex PCR systems has high potential for a rapid first-line surveillance typing assay of C. jejuni and can be used for routine surveillance and outbreak investigations of C. jejuni in the future. Copyright © 2014 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  13. 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…

  14. Simultaneous detection of five different DNA targets by real-time Taqman PCR using the Roche LightCycler480: Application in viral molecular diagnostics.

    PubMed

    Molenkamp, Richard; van der Ham, Alwin; Schinkel, Janke; Beld, Marcel

    2007-05-01

    One of the most interesting aspects of real-time PCR based on the detection of fluorophoric labeled oligonucleotides is the possibility of being able to detect conveniently multiple targets in the same PCR reaction. Recently, Roche Diagnostics launched a real-time PCR platform, the LightCycler480 (LC480), which should be well suited for multiplex real-time PCR analysis. In this paper the performance of the LC480 and accompanying software for the detection of five different targets was analyzed. Target DNAs mixed at equimolar concentrations were detected reproducibly and quantitatively. In addition, mixing different concentrations of the five targets demonstrated that the LC480 is capable of providing quantitative results for a mixture of DNA sequences without losing sensitivity. When applied to the practice of molecular diagnosis of four respiratory viral infections the multiplex assay showed almost complete concordance with corresponding single-target PCRs. The application of multiplex PCR for the detection of multiple pathogens within the same sample will provide a major contribution to the efficiency, logistics and cost-effectiveness of molecular diagnostics.

  15. 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).

  16. Towards Accreditation of Diagnostic Models for Improved Performance

    DTIC Science & Technology

    2004-10-02

    analysis. Secondly, while performing testability, the diagnostic algorithm is not included to assess Anuradha Kodali et al. This is an open-access...to assess the diagnosis (Sheppard, & Simpson, 1998). Considering these factors, Interactive Diagnostic Modeling Evaluator (i-DME) ( Kodali , Robinson...requirements set before to suit practical compulsions. This may lead to changing the basic principles and to refine the existing methods continuously

  17. 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

  18. Refined NrfA phylogeny improves PCR-based nrfA gene detection.

    PubMed

    Welsh, Allana; Chee-Sanford, Joanne C; Connor, Lynn M; Löffler, Frank E; Sanford, Robert A

    2014-04-01

    Dissimilatory nitrate reduction to ammonium (DNRA) and denitrification are contrasting microbial processes in the terrestrial nitrogen (N) cycle, in that the former promotes N retention and the latter leads to N loss (i.e., the formation of gaseous products). The nitrite reductase NrfA catalyzes nitrite reduction to ammonium, the enzyme associated with respiratory nitrite ammonification and the key step in DNRA. Although well studied biochemically, the diversity and phylogeny of this enzyme had not been rigorously analyzed. A phylogenetic analysis of 272 full-length NrfA protein sequences distinguished 18 NrfA clades with robust statistical support (>90% Bayesian posterior probabilities). Three clades possessed a CXXCH motif in the first heme-binding domain, whereas all other clades had a CXXCK motif in this location. The analysis further identified a KXRH or KXQH motif between the third and fourth heme-binding motifs as a conserved and diagnostic feature of all pentaheme NrfA proteins. PCR primers targeting a portion of the heme-binding motifs that flank this diagnostic region yielded the expected 250-bp-long amplicons with template DNA from eight pure cultures and 16 new nrfA-containing isolates. nrfA amplicons obtained with template DNA from two geomorphically distinct agricultural soils could be assigned to one of the 18 NrfA clades, providing support for this expanded classification. The extended NrfA phylogeny revealed novel diagnostic features of DNRA populations and will be useful to assess nitrate/nitrite fate in natural and engineered ecosystems.

  19. A computerized methodology for improved virus typing by PCR-RFLP gel electrophoresis.

    PubMed

    Maramis, Christos F; Delopoulos, Anastasios N; Lambropoulos, Alexandros F

    2011-08-01

    The analysis of digitized images from polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP)gel electrophoresis examinations is a popular method for virus typing, i.e., for identifying the virus type(s) that have infected an investigated biological sample. However, being mostly manual, the conventional virus typing protocol remains laborious, time consuming, and error prone. In order to overcome these shortcomings,we propose a computerized methodology for improving virus typing via PCR-RFLP gel electrophoresis. A novel realistic observation model of the viral DNA motion on the gel matrix is employed to assist in exploiting additional virus-related information in comparison to the conventional approaches. The extracted rich information is fed to a novel typing algorithm, resulting in faster and more accurate decisions. The proposed methodology is evaluated for the case of the human papillomavirus typing on a dataset of 80 real and 1500 simulated samples, producing very satisfactory results.Ind

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

    PubMed

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

    2013-11-01

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

  1. 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.

  2. Companion diagnostics-a tool to improve pharmacotherapy.

    PubMed

    Jørgensen, Jan Trøst; Hersom, Maria

    2016-12-01

    The variability of pharmacotherapy can be of a significant magnitude, and the main reason for this is often diseases heterogeneity. Patients who have similar diagnoses very often respond differently to the same pharmacological intervention, with great variability in both efficacy and safety outcome. Despite having discussed personalized medicine for more than a decade, we still see that most drug prescriptions for severe chronic diseases are largely based on 'trial and error' and not on solid biomarker data. However, with the advance of molecular diagnostics and a subsequent increased understanding of disease mechanisms, things are slowly changing. Within the last few years, we have seen an increasing number of predictive biomarker assays being developed to guide the use of targeted cancer drugs. This type of assay is called companion diagnostics and is developed in parallel to the drug using the drug-diagnostic co-development model. The development of companion diagnostics is a relatively new discipline and in this review, different aspects will be discussed including clinical and regulatory issues. Furthermore, examples of drugs, such as the ALK and PD-1/PD-L1 inhibitors, that have been approved recently together with a companion or complimentary diagnostic will be given.

  3. Comparative study of diagnostic accuracy of established PCR assays and in-house developed sdaA PCR method for detection of Mycobacterium tuberculosis in symptomatic patients with pulmonary tuberculosis.

    PubMed

    Nimesh, Manoj; Joon, Deepali; Pathak, Anil Kumar; Saluja, Daman

    2013-11-01

    Indian contribution to global burden of tuberculosis is about 26%. In the present study we have developed an in-house PCR assay using primers for sdaA gene of Mycobacterium tuberculosis and evaluated against already established primers devR, IS6110, MPB64, rpoB primers for diagnosis of pulmonary tuberculosis. Using universal sample preparation (USP) method, DNA was extracted from sputum specimens of 412 symptomatic patients from Delhi, India. The DNA so extracted was used as template for PCR amplification using primers targeting sdaA, devR, IS6110, MPB64 and rpoB genes. Out of 412, 149 specimens were considered positive based on composite reference standard (CRS) criteria. The in-house designed sdaA PCR showed high specificity (96.5%), the high positive likelihood ratio (28), the high sensitivity (95.9%), and the very low negative likelihood ratio (0.04) in comparison to CRS. Based on our results, the sdaA PCR assay can be considered as one of the most reliable diagnostic tests in comparison to other PCR based detection methods. Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

  4. 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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  5. Evaluation of an Improved U.S. Food and Drug Administration Method for the Detection of Cyclospora cayetanensis in Produce Using Real-Time PCR.

    PubMed

    Murphy, Helen R; Lee, Seulgi; da Silva, Alexandre J

    2017-07-01

    Cyclospora cayetanensis is a protozoan parasite that causes human diarrheal disease associated with the consumption of fresh produce or water contaminated with C. cayetanensis oocysts. In the United States, foodborne outbreaks of cyclosporiasis have been linked to various types of imported fresh produce, including cilantro and raspberries. An improved method was developed for identification of C. cayetanensis in produce at the U.S. Food and Drug Administration. The method relies on a 0.1% Alconox produce wash solution for efficient recovery of oocysts, a commercial kit for DNA template preparation, and an optimized TaqMan real-time PCR assay with an internal amplification control for molecular detection of the parasite. A single laboratory validation study was performed to assess the method's performance and compare the optimized TaqMan real-time PCR assay and a reference nested PCR assay by examining 128 samples. The samples consisted of 25 g of cilantro or 50 g of raspberries seeded with 0, 5, 10, or 200 C. cayetanensis oocysts. Detection rates for cilantro seeded with 5 and 10 oocysts were 50.0 and 87.5%, respectively, with the real-time PCR assay and 43.7 and 94.8%, respectively, with the nested PCR assay. Detection rates for raspberries seeded with 5 and 10 oocysts were 25.0 and 75.0%, respectively, with the real-time PCR assay and 18.8 and 68.8%, respectively, with the nested PCR assay. All unseeded samples were negative, and all samples seeded with 200 oocysts were positive. Detection rates using the two PCR methods were statistically similar, but the real-time PCR assay is less laborious and less prone to amplicon contamination and allows monitoring of amplification and analysis of results, making it more attractive to diagnostic testing laboratories. The improved sample preparation steps and the TaqMan real-time PCR assay provide a robust, streamlined, and rapid analytical procedure for surveillance, outbreak response, and regulatory testing of foods for

  6. 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…

  7. 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…

  8. 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…

  9. 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…

  10. 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…

  11. 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…

  12. 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.

  13. 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.

  14. 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

  15. Multiplex blood PCR in combination with blood cultures for improvement of microbiological documentation of infection in febrile neutropenia.

    PubMed

    Lamoth, F; Jaton, K; Prod'hom, G; Senn, L; Bille, J; Calandra, T; Marchetti, O

    2010-10-01

    The frequent lack of microbiological documentation of infection by blood cultures (BC) has a major impact on clinical management of febrile neutropenic patients, especially in cases of unexplained persistent fever. We assessed the diagnostic utility of the LightCycler SeptiFast test (SF), a multiplex blood PCR, in febrile neutropenia. Blood for BC and SF was drawn at the onset of fever and every 3 days of persistent fever. SF results were compared with those of BC, clinical documentation of infection, and standard clinical, radiological, and microbiological criteria for invasive fungal infections (IFI). A total of 141 febrile neutropenic episodes in 86 hematological patients were studied: 44 (31%) microbiologically and 49 (35%) clinically documented infections and 48 (34%) unexplained fevers. At the onset of fever, BC detected 44 microorganisms in 35/141 (25%) episodes. Together, BC and SF identified 78 microorganisms in 61/141 (43%) episodes (P = 0.002 versus BC or SF alone): 12 were detected by BC and SF, 32 by BC only, and 34 by SF only. In 19/52 (37%) episodes of persistent fever, SF detected 28 new microorganisms (7 Gram-positive bacterial species, 15 Gram-negative bacterial species, and 6 fungal species [89% with a clinically documented site of infection]) whereas BC detected only 4 pathogens (8%) (P = 0.001). While BC did not detect fungi, SF identified 5 Candida spp. and 1 Aspergillus sp. in 5/7 probable or possible cases of IFI. Using SeptiFast PCR combined with blood cultures improves microbiological documentation in febrile neutropenia, especially when fever persists and invasive fungal infection is suspected. Technical adjustments may enhance the efficiency of this new molecular tool in this specific setting.

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

    PubMed

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

    2013-02-20

    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. Copyright © 2012 John Wiley & Sons, Ltd.

  17. 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

  18. Absolute quantification by droplet digital PCR versus analog real-time PCR

    PubMed Central

    Hindson, Christopher M; Chevillet, John R; Briggs, Hilary A; Gallichotte, Emily N; Ruf, Ingrid K; Hindson, Benjamin J; Vessella, Robert L; Tewari, Muneesh

    2014-01-01

    Nanoliter-sized droplet technology paired with digital PCR (ddPCR) holds promise for highly precise, absolute nucleic acid quantification. Our comparison of microRNA quantification by ddPCR and real-time PCR revealed greater precision (coefficients of variation decreased by 37–86%) and improved day-to-day reproducibility (by a factor of seven) of ddPCR but with comparable sensitivity. When we applied ddPCR to serum microRNA biomarker analysis, this translated to superior diagnostic performance for identifying individuals with cancer. PMID:23995387

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

    PubMed Central

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

    2016-01-01

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

  20. 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.

  1. 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

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

    PubMed

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

    2008-10-01

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

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

    PubMed

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

    2008-10-01

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

  4. MT-PCR panel detection of canine parvovirus (CPV-2): Vaccine and wild-type CPV-2 can be difficult to differentiate in canine diagnostic fecal samples.

    PubMed

    Meggiolaro, Maira N; Ly, Anna; Rysnik-Steck, Benjamin; Silva, Carolina; Zhang, Joshua; Higgins, Damien P; Muscatello, Gary; Norris, Jacqueline M; Krockenberger, Mark; Šlapeta, Jan

    2017-03-01

    Canine parvovirus (CPV-2) remains an important cause of devastating enteritis in young dogs. It can be successfully prevented with live attenuated CPV-2 vaccines when given at the appropriate age and in the absence of maternal antibody interference. Rapid diagnosis of parvoviral enteritis in young dogs is essential to ensuring suitable barrier nursing protocols within veterinary hospitals. The current diagnostic trend is to use multiplexed PCR panels to detect an array of pathogens commonly responsible for diarrhea in dogs. The multiplexed PCR assays do not distinguish wild from vaccine CPV-2. They are highly sensitive and detect even a low level of virus shedding, such as those caused by the CPV-2 vaccine. The aim of this study was to identify the CPV-2 subtypes detected in diagnostic specimens and rule out occult shedding of CPV-2 vaccine strains. For a total of 21 samples that tested positive for CPV-2 in a small animal fecal pathogens diagnostic multiplexed tandem PCR (MT-PCR) panel during 2014-2016 we partially characterized the VP2 gene of CPV-2. Vaccine CPV-2 strain, wild type CPV-2a subtypes and vaccine-like CPV-2b subtypes were detected. High copy number was indicative of wild-type CPV-2a presence, but presence of vaccine-like CPV-2b had a variable copy number in fecal samples. A yardstick approach to a copy number or Ct-value to discriminate vaccine strain from a wild type virus of CPV-2 can be, in some cases, potentially misleading. Therefore, discriminating vaccine strain from a wild type subtype of CPV-2 remains ambitious.

  5. When do individuals help close others improve? The role of information diagnosticity.

    PubMed

    Pemberton, M; Sedikides, C

    2001-08-01

    On the basis of the self-evaluation maintenance model (SEM; Tesser, 1988), it was hypothesized that individuals give less improving information to relationally close (rather than distant) others, out of concern for being outperformed by close others in the future. Further, this effect only occurs if diagnostic and valid criteria for success are present. Three studies confirmed the hypotheses. In Studies 1 and 2, participants gave less improving information to familiar than to unfamiliar others in a domain (academics) in which diagnostic assessment criteria (grades) were available. This pattern was not found in a domain (social life) without diagnostic criteria. These results were replicated in Study 3, in which relative performance and diagnosticity of assessment criteria were manipulated and amount of improving information given to friends and strangers was measured. Diagnosticity of comparison information is an important addition to the SEM model.

  6. 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.

  7. 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.

  8. Improving the treatment of musculoskeletal infections with molecular diagnostics.

    PubMed

    Tarkin, Ivan S; Dunman, Paul M; Garvin, Kevin L

    2005-08-01

    Molecular diagnostic strategies have been implemented to enhance the treatment of musculoskeletal infections. Once primarily a research tool, molecular-based assays, have become accepted clinical tests for the genomic detection of certain pathogens involved in bone and joint infections. Currently, culture remains the gold standard for identifying most organisms causing infection. However, molecular assays are beneficial in clinical cases in which standard culture-based tests are unreliable or untimely. We will review current clinical utility of this emerging technology and roles for assays in the future.

  9. 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.

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

    ERIC Educational Resources Information Center

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

    2010-01-01

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

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

    ERIC Educational Resources Information Center

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

    2010-01-01

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

  12. Molecular diagnostic toolkit for Rhizophagus irregularis isolate DAOM-197198 using quantitative PCR assay targeting the mitochondrial genome.

    PubMed

    Badri, Amine; Stefani, Franck O P; Lachance, Geneviève; Roy-Arcand, Line; Beaudet, Denis; Vialle, Agathe; Hijri, Mohamed

    2016-10-01

    Rhizophagus irregularis (previously named Glomus irregulare) is one of the most widespread and common arbuscular mycorrhizal fungal (AMF) species. It has been recovered worldwide in agricultural and natural soils, and the isolate DAOM-197198 has been utilized as a commercial inoculant for two decades. Despite the ecological and economical importance of this taxon, specific markers for quantification of propagules by quantitative real-time PCR (qPCR) are extremely limited and none have been rigorously validated for quality control of manufactured products such as biofertilizers. From the sequencing of 14 complete AMF mitochondrial (mt) genomes, a qPCR assay using a hydrolysis probe designed in the single copy cox3-rnl intergenic region was tested and validated to specifically and accurately quantify the spores of R. irregularis isolate DAOM-197198. Specificity tests were performed using standard PCR and qPCR, and results clearly showed that the primers specifically amplified the isolate DAOM-197198, yielding a PCR product of 106 bp. According to the qPCR analyses on spores produced in vitro, the average copy number of mt genomes per spore was 3172 ± 304 SE (n = 6). Quantification assays were successfully undertaken on known and unknown samples in liquid suspensions and commercial dry formulations to show the accuracy, precision, robustness, and reproducibility of the qPCR assay. This study provides a powerful molecular toolkit specifically designed to quantify spores of the model AMF isolate DAOM-197198. The approach of molecular toolkit used in our study could be applied to other AMF taxa and will be useful to research institutions and governmental and industrial laboratories running routine quality control of AMF-based products.

  13. Development and evaluation of one-step rRT-PCR and immunohistochemical methods for detection of Rift Valley fever virus in biosafety level 2 diagnostic laboratories.

    PubMed

    Drolet, Barbara S; Weingartl, Hana M; Jiang, Jieyuan; Neufeld, James; Marszal, Peter; Lindsay, Robbin; Miller, Myrna M; Czub, Markus; Wilson, William C

    2012-02-01

    Rift Valley fever virus (RVFV) is a zoonotic insect transmitted virus endemic to Africa and the Arabian Peninsula. Infection causes abortions and high mortality in newborn ruminants. The overall human infection rate is <1%; however, fatality rates in those with severe clinical disease have been reported as high as 29%. The potential of RVFV as a bioterrorism agent and/or being accidentally introduced into North America is widely recognized. Currently, regional veterinary biosafety level 2 (BSL-2) diagnostic laboratories lack safe, modern, validated diagnostic tests to detect RVFV. An existing one-step real-time RT-PCR (rRT-PCR) assay was modified for quick virus inactivation for use in BSL-2 laboratories, evaluated on serum and tissue samples from experimentally infected lambs and calves, and compared to virus isolation. Viremia was detected in all inoculated sheep with titers reaching 10(6.5) plaque forming units/ml, or up to 10(10) viral RNA copies/ml. Viremia in calves was lower and not detected in all inoculated animals; however, all animals became transiently febrile and were infected as determined by rRT-PCR of tissues. Virus was isolated from rRT-PCR-positive liver and/or spleen in 33% of lamb and 41% of calf samples between 2 and 7 days post inoculation. For RVFV antigen detection, reagents are typically produced at BSL-3Ag or BSL-4 conditions and require inactivation and safety testing for use outside of containment. In this study, antiserum against recombinant RVFV-nucleocapsid (N) was produced to develop an immunohistochemical (IHC) assay which was subsequently evaluated on formalin fixed lamb and calf tissues at BSL-2 laboratory conditions. Antigen was detected by IHC in 79% of rRT-PCR-positive sheep and 70% of rRT-PCR-positive calf tissues tested. Once validated and approved by national regulatory agencies, these assays can be safely produced and distributed to regional diagnostic laboratories, providing capacity for early detection of RVFV in

  14. Comparison of bacterial culture and qPCR testing of rectal and pen floor samples as diagnostic approaches to detect enterotoxic Escherichia coli in nursery pigs.

    PubMed

    Weber, N R; Nielsen, J P; Hjulsager, C K; Jorsal, S E; Haugegaard, S; Hansen, C F; Pedersen, K S

    2017-08-01

    Enterotoxigenic E. coli (ETEC) are a major cause of diarrhoea in weaned pigs. The objective of this study was to evaluate the agreement at pen level among three different diagnostic approaches for the detection of ETEC in groups of nursery pigs with diarrhoea. The diagnostic approaches used were: bacterial culturing of faecal samples from three pigs (per pen) with clinical diarrhoea and subsequent testing for virulence genes in E. coli isolates; bacterial culturing of pen floor samples and subsequent testing for virulence genes in E. coli isolates; qPCR testing of pen floor samples in order to determine the quantity of F18 and F4 genes. The study was carried out in three Danish pig herds and included 31 pens with a pen-level diarrhoea prevalence of > 25%, as well as samples from 93 diarrhoeic nursery pigs from these pens. All E. coli isolates were analysed by PCR and classified as ETEC when genes for one or more adhesin factors and one or more enterotoxins were detected. A total of 208 E. coli colonies from pig samples and 172 E. coli colonies from pen floor samples were isolated. Haemolytic activity was detected on blood agar plates in 111 (29.2%) of the 380 colonies that were isolated. The only adhesin factor detected in this study was F18. When comparing bacterial culture or qPCR testing of pen floor samples with detection of ETEC-positive diarrhoeic pigs by culture, agreement was found in 26 (83.9%, Kappa = 0.665) and 23 (74.2%, Kappa = 0.488) of the pens, respectively. Agreement was observed between the detection of ETEC by bacterial culture and qPCR in the same pen floor sample in 26 (83.9%, Kappa = 0.679) pens. We observed an acceptable agreement for the detection of ETEC-positive diarrhoeic nursery pigs in pen samples for both bacterial culture of pen floor samples and qPCR. This study showed that both bacterial culture and qPCR testing of pen floor samples can be used as a diagnostic approach for detecting groups of ETEC-positive diarrhoeic nursery pigs

  15. Assessment of the Usefulness of Multiplex Real-Time PCR Tests in the Diagnostic and Therapeutic Process of Pneumonia in Hospitalized Children: A Single-Center Experience

    PubMed Central

    Bartkowska-Śniatkowska, Alicja; Jończyk-Potoczna, Katarzyna; Wysocka-Leszczyńska, Joanna; Bobkowski, Waldemar; Fichna, Piotr; Sobkowiak, Paulina; Mazur-Melewska, Katarzyna; Bręborowicz, Anna; Wysocki, Jacek; Januszkiewicz-Lewandowska, Danuta

    2017-01-01

    The aim of the study was assessment of the usefulness of multiplex real-time PCR tests in the diagnostic and therapeutic process in children hospitalized due to pneumonia and burdened with comorbidities. Methods. The study group included 97 children hospitalized due to pneumonia at the Karol Jonscher Teaching Hospital in Poznań, in whom multiplex real-time PCR tests (FTD respiratory pathogens 33; fast-track diagnostics) were used. Results. Positive test results of the test were achieved in 74 patients (76.3%). The average age in the group was 56 months. Viruses were detected in 61 samples (82% of all positive results); bacterial factors were found in 29 samples (39% of all positive results). The presence of comorbidities was established in 90 children (92.78%). On the basis of the obtained results, 5 groups of patients were established: viral etiology of infection, 34 patients; bacterial etiology, 7 patients; mixed etiology, 23 patients; pneumocystis, 9 patients; and no etiology diagnosed, 24 patients. Conclusions. Our analysis demonstrated that the participation of viruses in causing severe lung infections is significant in children with comorbidities. Multiplex real-time PCR tests proved to be more useful in establishing the etiology of pneumonia in hospitalized children than the traditional microbiological examinations. PMID:28182108

  16. Assessment of the Usefulness of Multiplex Real-Time PCR Tests in the Diagnostic and Therapeutic Process of Pneumonia in Hospitalized Children: A Single-Center Experience.

    PubMed

    Gowin, Ewelina; Bartkowska-Śniatkowska, Alicja; Jończyk-Potoczna, Katarzyna; Wysocka-Leszczyńska, Joanna; Bobkowski, Waldemar; Fichna, Piotr; Sobkowiak, Paulina; Mazur-Melewska, Katarzyna; Bręborowicz, Anna; Wysocki, Jacek; Januszkiewicz-Lewandowska, Danuta

    2017-01-01

    The aim of the study was assessment of the usefulness of multiplex real-time PCR tests in the diagnostic and therapeutic process in children hospitalized due to pneumonia and burdened with comorbidities. Methods. The study group included 97 children hospitalized due to pneumonia at the Karol Jonscher Teaching Hospital in Poznań, in whom multiplex real-time PCR tests (FTD respiratory pathogens 33; fast-track diagnostics) were used. Results. Positive test results of the test were achieved in 74 patients (76.3%). The average age in the group was 56 months. Viruses were detected in 61 samples (82% of all positive results); bacterial factors were found in 29 samples (39% of all positive results). The presence of comorbidities was established in 90 children (92.78%). On the basis of the obtained results, 5 groups of patients were established: viral etiology of infection, 34 patients; bacterial etiology, 7 patients; mixed etiology, 23 patients; pneumocystis, 9 patients; and no etiology diagnosed, 24 patients. Conclusions. Our analysis demonstrated that the participation of viruses in causing severe lung infections is significant in children with comorbidities. Multiplex real-time PCR tests proved to be more useful in establishing the etiology of pneumonia in hospitalized children than the traditional microbiological examinations.

  17. Development of diagnostic SCAR markers for genomic DNA amplifications in breast carcinoma by DNA cloning of high-GC RAMP-PCR fragments.

    PubMed

    Fu, Shangyi; Cheng, Jingliang; Wei, Chunli; Yang, Luquan; Xiao, Xiuli; Zhang, Dianzheng; Stewart, M David; Fu, Junjiang

    2017-07-04

    Cancer is genetically heterogeneous regarding to molecular genetic characteristics and pathogenic pathways. A wide spectrum of biomarkers, including DNA markers, is used in determining genomic instability, molecular subtype determination and disease prognosis, and estimating sensitivity to different drugs in clinical practice. In a previous study, we developed highly effective DNA markers using improved random amplified polymorphic DNA (RAPD) with high-GC primers, which is a valuable approach for the genetic authentication of medicinal plants. In this study, we applied this effective DNA marker technique to generate genetic fingerprints that detect genomic alterations in human breast cancer tissues and then developed sequence-characterized amplified region (SCAR) markers. Three SCAR markers (BC10-1, BC13-4 and BC31-2) had high levels of genomic DNA amplification in breast cancer. The PHKG2 and RNF40 genes are either overlapping or close to the sequences of SCAR marker BC13-4, while SCAR marker BC10-1 is in the intron and overlap the DPEP1 gene, suggesting that alterations in the expression of these genes could contribute to cancer progression. Screening of breast cancer cell lines showed that the mRNA expression levels for the PHKG2 and DPEP1 were lower in non-tumorigenic mammary epithelial cell MCF10A, but elevated in other cell lines. The DPEP1 mRNA level in invasive ductal carcinoma specimens was significantly higher than that of the adjacent normal tissues in women. Taken together, high-GC RAMP-PCR provides greater efficacy in measuring genomic DNA amplifications, deletion or copy number variations. Furthermore, SCAR markers BC10-1 and BC13-4 might be useful diagnostic markers for breast cancer carcinomas.

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

    PubMed

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

    2017-07-01

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

  19. Improving Inspection and Maintenance Performance and On-board Diagnostics Monitor Readiness Memo

    EPA Pesticide Factsheets

    This EPA memorandum transmits an updated list of vehicles that exhibit issues related to OBD (on board diagnostics) monitor readiness and makes suggestions for how Inspection/Maintenance (I/M) programs can improve operational performance by addressing

  20. 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

  1. 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.

  2. Ages of celiac disease: from changing environment to improved diagnostics.

    PubMed

    Tommasini, Alberto; Not, Tarcisio; Ventura, Alessandro

    2011-08-28

    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.

  3. Brief communication: multiplex X/Y-PCR improves sex identification in aDNA analysis.

    PubMed

    Schmidt, Diane; Hummel, Susanne; Herrmann, Bernd

    2003-08-01

    This study introduces a polymerase chain reaction (PCR)-based multiplex approach to improve the certainty of molecular sex identification on archaeological skeletal material. We coamplified amelogenin, two X-chromosomal short tandem repeats (STRs) (DXS6789 and DXS9898), and two Y-specific STRs (DYS391 and DYS392). The amplification results of this multiplex approach back each other up, and enable a reliable sex identification. This coamplification of X- and Y-specific markers in a multiplex assay combines the added advantage of positive identification of both female and male individuals with raising the validity of the diagnosis by obtaining multiple data simultaneously. This multiplex system was successfully applied to 3,000-year-old bone material.

  4. Improved extraction of PCR-quality community DNA from digesta and fecal samples.

    PubMed

    Yu, Zhongtang; Morrison, Mark

    2004-05-01

    Several DNA extraction methods have been reported for use with digesta or fecal samples, but problems are often encountered in terms of relatively low DNA yields and/or recovering DNA free of inhibitory substances. Here we report a modified method to extract PCR-quality microbial community DNA from these types of samples, which employs bead beating in the presence of high concentrations of sodium dodecyl sulfate (SDS), salt, and EDTA, and with subsequent DNA purification by QIAamp columns [referred to as repeated bead beating plus column (RBB + C) method]. The RBB + C method resulted in a 1.5- to 6-fold increase in DNA yield when compared to three other widely used methods. The community DNA prepared with the RBB + C method was also free of inhibitory substances and resulted in improved denaturing gradient gel electrophoresis (DGGE) profiles, which is indicative of a more complete lysis and representation of microbial diversity present in such samples.

  5. Improved RT-PCR Assay to Quantitate the Pri-, Pre-, and Mature microRNAs with Higher Efficiency and Accuracy.

    PubMed

    Tong, Li; Xue, Huihui; Xiong, Li; Xiao, Junhua; Zhou, Yuxun

    2015-10-01

    Understanding of the functional significance of microRNAs (miRNAs) requires efficient and accurate detection method. In this study, we developed an improved miRNAs quantification system based on quantitative real-time polymerase chain reaction (qRT-PCR). This method showed higher efficiency and accuracy to survey the expression of primary miRNAs (pri-miRNAs), precursor miRNAs (pre-miRNAs), and mature miRNAs. Instead of relative quantification method, we quantified the pri-miRNAs and pre-miRNAs with absolute qRT-PCR based on SYBR Green I fluorescence. This improvement corrected for the inaccuracy caused by the differences in amplicon length and PCR efficiency. We also used SYBR Green method to quantify mature miRNAs based on the stem-loop qRT-PCR method. We extended the pairing part of the stem-loop reverse transcript (RT) primer from 6 to 11 bp, which greatly increased the efficiency of reverse transcription PCR (RT-PCR). The performance of the improved RT primer was tested using synthetic mature miRNAs and tissue RNA samples. Results showed that the improved RT primer demonstrated dynamic range of seven orders of magnitude and sensitivity of detection of hundreds of copies of miRNA molecules.

  6. 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.

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

    PubMed Central

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

    2014-01-01

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

  8. Detection of malaria infection in blood transfusion: a comparative study among real-time PCR, rapid diagnostic test and microscopy: sensitivity of Malaria detection methods in blood transfusion.

    PubMed

    Hassanpour, Gholamreza; Mohebali, Mehdi; Raeisi, Ahmad; Abolghasemi, Hassan; Zeraati, Hojjat; Alipour, Mohsen; Azizi, Ebrahim; Keshavarz, Hossein

    2011-06-01

    The transmission of malaria by blood transfusion was one of the first transfusion-transmitted infections recorded in the world. Transfusion-transmitted malaria may lead to serious problems because infection with Plasmodium falciparum may cause rapidly fatal death. This study aimed to compare real-time polymerase chain reaction (real-time PCR) with rapid diagnostic test (RDT) and light microscopy for the detection of Plasmodium spp. in blood transfusion, both in endemic and non-endemic areas of malaria disease in Iran. Two sets of 50 blood samples were randomly collected. One set was taken from blood samples donated in blood bank of Bandar Abbas, a city located in a malarious-endemic area, and the other set from Tehran, a non-endemic one. Light microscopic examination on both thin and thick smears, RDTs, and real-time PCR were performed on the blood samples and the results were compared. Thin and thick light microscopic examinations of all samples as well as RDT results were negative for Plasmodium spp. Two blood samples from endemic area were positive only with real-time PCR. It seems that real-time PCR as a highly sensitive method can be helpful for the confirmation of malaria infection in different units of blood transfusion organization especially in malaria-endemic areas where the majority of donors may be potentially infected with malaria parasites.

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

    PubMed

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

    2009-05-01

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

  10. An improved DNA marker technique for genetic characterization using RAMP-PCR with high-GC primers.

    PubMed

    Wei, C L; Cheng, J L; Khan, M A; Yang, L Q; Imani, S; Chen, H C; Fu, J J

    2016-09-16

    Random amplified polymorphic DNA (RAPD) is a widely used molecular marker technique. As traditional RAPD has poor reproducibility and productivity, we previously developed an improved RAPD method (termed RAMP-PCR), which increased the reproducibility, number of bands, and efficiency of studies on polymorphism. To further develop the efficiency of this method, we used high-GC content primers for improved RAMP-PCR with DNA samples from Lonicera japonica. Comparison of amplification profiles obtained by standard RAPD primers with those obtained by regular PCR and RAMP-PCR, and high-GC primers with regular PCR and RAMP-PCR showed that the average number of bands and polymorphisms per primer gradually and significantly increased (from 6.4 to 15.0 and from 4.6 to 10.2, respectively). Cluster dendrograms showed similar results, indicating that this new method is consistent and reproducible. A total of 22 samples from different species, including plants, animals, and humans, were used for RAMP-PCR with high-GC primers. Multiple bands were successfully amplified from all samples, demonstrating that this method is a reliable technique with consistent results and may be of general interest in studies on different genera and species. We developed highly effective DNA markers, which can provide a more effective and potentially valuable approach than traditional RAPD for the genetic identification of various organisms, particularly of medicinal plants.

  11. Diagnostic Accuracy of Real-Time PCR Assays Targeting 16S rRNA and lipl32 Genes for Human Leptospirosis in Thailand: A Case-Control Study

    PubMed Central

    Thaipadunpanit, Janjira; Chierakul, Wirongrong; Wuthiekanun, Vanaporn; Limmathurotsakul, Direk; Amornchai, Premjit; Boonslip, Siriphan; Smythe, Lee D.; Limpaiboon, Roongrueng; Hoffmaster, Alex R.; Day, Nicholas P. J.; Peacock, Sharon J.

    2011-01-01

    Background Rapid PCR-based tests for the diagnosis of leptospirosis can provide information that contributes towards early patient management, but these have not been adopted in Thailand. Here, we compare the diagnostic sensitivity and specificity of two real-time PCR assays targeting rrs or lipL32 for the diagnosis of leptospirosis in northeast Thailand. Methods/Principal Findings A case-control study of 266 patients (133 cases of leptospirosis and 133 controls) was constructed to evaluate the diagnostic sensitivity and specificity (DSe & DSp) of both PCR assays. The median duration of illness prior to admission of cases was 4 days (IQR 2–5 days; range 1–12 days). DSe and DSp were determined using positive culture and/or microscopic agglutination test (MAT) as the gold standard. The DSe was higher for the rrs assay than the lipL32 assay (56%, (95% CI 47–64%) versus 43%, (95% CI 34–52%), p<0.001). No cases were positive for the lipL32 assay alone. There was borderline evidence to suggest that the DSp of the rrs assay was lower than the lipL32 assay (90% (95% CI 83–94%) versus 93%, (95%CI 88–97%), p = 0.06). Nine controls gave positive reactions for both assays and 5 controls gave a positive reaction for the rrs assay alone. The DSe of the rrs and lipL32 assays were high in the subgroup of 39 patients who were culture positive for Leptospira spp. (95% and 87%, respectively, p = 0.25). Conclusions/Significance Early detection of Leptospira using PCR is possible for more than half of patients presenting with leptospirosis and could contribute to individual patient care. PMID:21283633

  12. [Improvement of laboratory diagnostics of cholera due to genetically altered (hybrid) variants of cholera Vibrio biovar El Tor].

    PubMed

    Savel'eva, I V; Khatsukov, K X; Savel'eva, E I; Moskvitina, S I; Kovalev, D A; Savel'ev, V N; Kulichenko, A N; Antonenko, A D; Babenyshev, B V

    2015-01-01

    Improvement of laboratory diagnostics of cholera taking into the account appearance of hybrid variants of cholera vibrio El Tor biovar in the 1990s. Phenotypic and molecular-genetic properties of typical toxigenic (151 strains) and hybrid (102 strains) variants of El Tor biovar cholera vibrios, isolated in the Caucuses in 1970-1990 and 1993-1998, respectively, were studied. Toxigenicity gene DNA fragments, inherent to El Tor biovars or classic, were detected by using a reagent kit "Genes of Vibrio cholerae variant ctxB-rstR-rstC, REF" developed by us. Reagent kit "Genes of V. cholerae variant ctxB-rstR-rstC, REF" is proposed to be used for laboratory diagnostics of cholera during study of material from humans or environmental objects and for identification of V. cholerae 01 on genome level in PCR-analysis as a necessary addition to the classic scheme of bacteriological analysis. Laboratory diagnostics of cholera due to genetically altered (hybrid) variants of cholera vibrio El Tor biovar is based on a complex study of material from humans and environmental objects by routine bacteriologic and PCR-analysis methods with the aim of detection of gene DNA fragments in the studied material, that determine biovar (classic or El Tor), identification of V. cholerae O1 strains with differentiation of El Tor vibrios into typical and altered, as well as determination of enterotoxin, produced by the specific cholera vibrio strain (by the presence ctxB(El) or ctxB(Cl) gene DNA fragment, coding biosynthesis of CT-2 or CT-1, respectively).

  13. Trichomonas vaginalis: investigation of a novel diagnostic method in urine samples using cysteine proteinase 4 gene and PCR technique.

    PubMed

    Vatanshenassan, Mansoureh; Rezaie, Sassan; Mohebali, Mehdi; Niromand, Nasrin; Kazemi, Bahram; Babaei, Zahra; Rezaeian, Mostafa

    2010-10-01

    Trichomonas vaginalis is the agent of a highly prevalent sexually transmitted disease that leads to vaginitis, urethritis, ectocervicitis and has been associated with human immunodeficiency virus (HIV). Detection of T. vaginalis based on wet-mount microscopy and culture methods is insensitive and time consuming, respectively. Thus the quest for reliable PCR techniques of T. vaginalis in vaginal discharge and urine sample is more importance. In this study, 500 urine and vaginal-discharge samples were collected from women referred to Sexual Transmitted Disease Clinic of Mirzakuchakkhan Hospital in Tehran, Iran between May 2008 and March 2009. Wet-mount and culture methods were done on the vaginal discharges, and PCR assay targeting cysteine proteinase 4 (CP4) was performed on the urine samples. The present study demonstrated 16 (3.2%) of patients were infected with T. vaginalis using culture and wet-mount, whereas PCR assay using CP4 could detect 12 (2.4%) positivity. Sensitivity and specificity of urine PCR assay compared to culture were 80% (95% CI, 54-96) and 99.6% (95% CI, 98.96-100), respectively. These results indicate that using urine-based detection method for T. vaginalis may not be appropriate in women.

  14. Higher blood volumes improve the sensitivity of direct PCR diagnosis of blood stream tuberculosis among HIV-positive patients: an observation study.

    PubMed

    Bwanga, Freddie; Disqué, Claudia; Lorenz, Michael G; Allerheiligen, Vera; Worodria, William; Luyombya, Allan; Najjingo, Irene; Weizenegger, Michael

    2015-02-06

    Blood stream tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB) is common among HIV-positive patients, turning rapidly fatal unless detected and treated promptly. Blood culture is currently the standard test for the detection of MTB in whole blood but results take weeks; patients deteriorate markedly and often die before a diagnosis of blood stream TB is made. Rapid molecular tests on whole blood, with potential for same day diagnosis of blood stream TB usually show low sensitivity due to the problem of insufficient MTB DNA template when extraction is performed directly on low blood volumes. This study assessed the influence of blood volume on the sensitivity of a HyBeacon PCR assay-the FluoroType MTB (Hain Lifescience, Nehren, Germany) on direct detection of MTB in whole blood. Prospective recruitment of HIV-positive patients with clinical suspicion of blood stream TB but not on anti-TB or HIV drug treatment was done. Venous blood samples were collected and DNA extracted using the MolYsis (Molzym, Bremen, Germany) methods; for study A, from duplicate 1 ml (42 patients) and for study B (31 patients) from 9 ml EDTA blood samples. The FluoroType MTB PCR assay targeting an IS6110 sequence was performed and results compared with blood culture. The diagnostic sensitivity and specificity of the FluoroType MTB PCR in study A was 33% and 97%, respectively. Corresponding values in study B were 71% and 96%, respectively. In both studies, one case each of blood culture-negative blood stream TB was detected with the FluoroType MTB PCR assay. The median time to positivity of blood culture was 20.1 (range 12-32) for study A and 19.9 days (range 15-30) for study B. Larger blood volumes (9 ml) improved and gave acceptable sensitivity of direct PCR diagnosis of blood stream TB.

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

    PubMed

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

    2015-06-15

    A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed to detect and type bovine papillomaviruses (BPVs) from tumors in cattle. Two degenerate primer sets targeting the BPV L1 gene, subAup/subAdw and subBup/subBdw, and one restriction enzyme RsaI were used in this assay. In silico analyses of the restriction enzyme sites in the PCR fragments of 13 BPV sequences (BPV-1 to -13) revealed that all known BPVs are differentiated by the PCR-RFLP assay. Analyses of 63 previously typed clinical samples, that included teat papillomas and both esophageal and urinary bladder cancer biopsies, show that the assay clearly differentiates between eight clinically important BPV types (BPV-1 to -6, -9, -10), and discriminates between single and multiple infections. To further assess the reliability of the PCR-RFLP method amplified fragments were sequenced. A high correlation (95%) was observed when the results of the PCR-RFLP method were compared with PCR-sequencing. Differences in typing occurred for 3 of 63 specimens; PCR-RFLP identified additional BPV types in these specimens, while the PCR-sequencing identified only one. These results indicate that the PCR-RFLP method reported here is simpler and more reliable in the detection and typing of BPVs from bovine tumor samples than PCR-sequencing. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Comparison of the GenMark Diagnostics eSensor respiratory viral panel to real-time PCR for detection of respiratory viruses in children.

    PubMed

    Pierce, Virginia M; Hodinka, Richard L

    2012-11-01

    A novel eSensor respiratory viral panel (eSensor RVP) multiplexed nucleic acid amplification test (GenMark Diagnostics, Inc., Carlsbad, CA) was compared to laboratory-developed real-time PCR assays for the detection of various respiratory viruses. A total of 250 frozen archived pediatric respiratory specimens previously characterized as either negative or positive for one or more viruses by real-time PCR were examined using the eSensor RVP. Overall agreement between the eSensor RVP and corresponding real-time PCR assays for shared analytes was 99.2% (kappa = 0.96 [95% confidence interval {CI}, 0.94 to 0.98]). The combined positive percent agreement was 95.4% (95% CI, 92.5 to 97.3); the negative percent agreement was 99.7% (95% CI, 99.4 to 99.8). The mean real-time PCR threshold cycle (C(T)) value for specimens with discordant results was 39.73 (95% CI, 38.03 to 41.43). Detection of coinfections and correct identification of influenza A virus subtypes were comparable between methods. Of note, the eSensor RVP rhinovirus assay was found to be more sensitive and specific than the corresponding rhinovirus real-time PCR. In contrast, the eSensor RVP adenovirus B, C, and E assays demonstrated some cross-reactivity when tested against known adenovirus serotypes representing groups A through F. The eSensor RVP is robust and relatively easy to perform, it involves a unique biosensor technology for target detection, and its multiplexed design allows for efficient and simultaneous interrogation of a single specimen for multiple viruses. Potential drawbacks include a slower turnaround time and the need to manipulate amplified product during the protocol, increasing the possibility of contamination.

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

    PubMed

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

    2015-09-01

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

  18. High-resolution melting PCR assay, applicable for diagnostics and screening studies, allowing detection and differentiation of several Babesia spp. infecting humans and animals.

    PubMed

    Rozej-Bielicka, Wioletta; Masny, Aleksander; Golab, Elzbieta

    2017-08-10

    The goal of the study was to design a single tube PCR test for detection and differentiation of Babesia species in DNA samples obtained from diverse biological materials. A multiplex, single tube PCR test was designed for amplification of approximately 400 bp region of the Babesia 18S rRNA gene. Universal primers were designed to match DNA of multiple Babesia spp. and to have low levels of similarity to DNA sequences of other intracellular protozoa and Babesia hosts. The PCR products amplified from Babesia DNA isolated from human, dog, rodent, deer, and tick samples were subjected to high-resolution melting analysis for Babesia species identification. The designed test allowed detection and differentiation of four Babesia species, three zoonotic (B. microti, B. divergens, B. venatorum) and one that is generally not considered zoonotic-Babesia canis. Both detection and identification of all four species were possible based on the HRM curves of the PCR products in samples obtained from the following: humans, dogs, rodents, and ticks. No cross-reactivity with DNA of Babesia hosts or Plasmodium falciparum and Toxoplasma gondii was observed. The lack of cross-reactivity with P. falciparum DNA might allow using the assay in endemic malaria areas. The designed assay is the first PCR-based test for detection and differentiation of several Babesia spp. of medical and veterinary importance, in a single tube reaction. The results of the study show that the designed assay for Babesia detection and identification could be a practical and inexpensive tool for diagnostics and screening studies of diverse biological materials.

  19. 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.

  20. 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

  1. Improved spectral analysis for the motional Stark effect diagnostic

    SciTech Connect

    Ko, J.; Klabacha, J.

    2012-10-15

    The magnetic pitch angle and the magnitude from reversed field pinch plasmas in the Madison symmetric torus (MST) have been routinely obtained from fully resolved motional Stark effect (MSE) spectrum analyses. Recently, the spectrum fit procedure has been improved by initializing and constraining the fit parameters based on the MSE model in the atomic data and analysis structure. A collisional-radiative model with level populations nlm-resolved up to n= 4 and a simple Born approximation for ion-impact cross sections is used for this analysis. Measurement uncertainty is quantified by making MSE measurements with multiple views of a single spatial location, ranging 5%-15% for typical MST operation conditions. A multi-view fit improves the goodness of fit of MSE spectral features and background.

  2. Molecular phylogenetic analysis of Enterobius vermicularis and development of an 18S ribosomal DNA-targeted diagnostic PCR.

    PubMed

    Zelck, Ulrike E; Bialek, Ralf; Weiss, Michael

    2011-04-01

    We genetically characterized pinworms obtained from 37 children from different regions of Germany and established new species-specific molecular diagnostic tools. No ribosomal DNA diversity was found; the phylogenetic position of Enterobius vermicularis within the Oxyurida order and its close relationship to the Ascaridida and Spirurida orders was confirmed.

  3. Use of chimeric influenza viruses as a novel internal control for diagnostic rRT-PCR assays.

    PubMed

    Wang, Xueliang; Liu, Fen; Jiang, Lingli; Bao, Yun; Xiao, Yanqun; Wang, Hualiang

    2016-02-01

    Real-time quantitative reverse transcriptase polymerase chain reaction (rRT-PCR) is now widely used to detect viral pathogens in various human specimens. The application of internal controls to validate the entire process of these assays is necessary to prevent false-negative results caused by unexpected inhibition or inefficient extraction. In the present study, we describe a strategy to produce a stable internal control for rRT-PCR by packaging foreign RNA into influenza virions using plasmid-based reverse genetics technology. The envelope structure of influenza virus can effectively protect RNA segments from RNase digestion, which provides an advantage for its routine use as an internal control. Utilizing this approach, we successfully generated a recombinant influenza virus (rPR8-HCV) containing the 5′ untranslated region (5′UTR) of the hepatitis C virus (HCV) RNA genome. After inactivation and purification, the rPR8-HCV particles were demonstrated to be RNase resistant and stable at 4 °C for at least 252 days in human plasma, with no degradation even after being frozen and thawed multiple times. These results were reproducible in the COBAS TaqMan HCV test for 164 days. Moreover, the chimeric influenza virus particles could be easily produced in embryonated eggs and were noninfectious after inactivation treatment. Additionally, this strategy could also be adapted for real-time clinical applications of other RNA targets, providing a universal approach with broad clinical applications in rRT-PCR assays.

  4. Formal art observation training improves medical students' visual diagnostic skills.

    PubMed

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

    2008-07-01

    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. To improve students' visual acumen through structured observation of artworks, understanding of fine arts concepts and applying these skills to patient care. Prospective, partially randomized pre- vs. post-course evaluation using mixed-methods data analysis. Twenty-four pre-clinical student participants were compared to 34 classmates at a similar stage of training. 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. 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. 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). This interdisciplinary course improved participants' capacity to make accurate observations of art and physical findings.

  5. The investigation of the truncated mbtA gene within the mycobactin cluster of Mycobacterium avium subspecies paratuberculosis as a novel diagnostic marker for real-time PCR.

    PubMed

    de Kruijf, Marcel; Coffey, Aidan; O'Mahony, Jim

    2017-05-01

    The inability of Mycobacterium avium subspecies paratuberculosis (MAP) to produce endogenous mycobactin in-vitro is most likely due to the presence of a truncated mbtA gene within the mycobactin cluster of MAP. The main goal of this study was to investigate this unique mbtA truncation as a potential novel PCR diagnostic marker for MAP. Novel primers were designed that were located within the truncated region and the contiguous MAP2179 gene. Primers were evaluated against non-MAP isolates and no amplicons were generated. The detection limit of this mbtA-MAP2179 target was evaluated using a range of MAP DNA concentrations, MAP inoculated faecal material and 20 MAP isolates. The performance of mbtA-MAP2179 was compared to the established f57 target. The detection limits recorded for MAP K-10 DNA and from MAP K-10 inoculated faecal samples were 0.34pg and 10(4)CFU/g respectively for both f57 and mbtA-MAP2179. A detection limit of 10(3)CFU/g was recorded for both targets, but not achieved consistently. The detection limit of MAP from inoculated faecal material was successful at 10(3)CFU/g for mbtA-MAP2179 when FAM probe real-time PCR was used. A MAP cell concentration of 10(2)CFU/g was detected successfully, but again not consistently achieved. All 20 mycobacterial isolates were successfully identified as MAP by f57 and mbtA-MAP2179. Interestingly, the mbtA-MAP2179 real-time PCR assay resulted in the formation of a unique melting curve profile that contained two melting curve peaks rather than one single peak. This melting curve phenomenon was attributed towards the asymmetrical GC% distribution within the mbtA-MAP2179 amplicon. This study investigated the implementation of the mbtA-MAP2179 target as a novel diagnostic marker and the detection limits obtained with mbtA-MAP2179 were comparable to the established f57 target, making the mbtA-MAP2179 an adequate confirmatory target. Moreover, the mbtA-MAP2179 target could be implemented in multiplex real-time PCR assays and

  6. 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

  7. The theoretical reliability of PCR-based fish viral diagnostic methods is critically affected when they are applied to fish populations with low prevalence and virus loads.

    PubMed

    Dopazo, C P; Moreno, P; Olveira, J G; Borrego, J J

    2017-09-15

    The reliability of PCR techniques is an important issue in viral diagnosis, and it is even crucial when they must be applied for detection of viruses in asymptomatic carriers. The problems will arise when the aim is to study fish wild populations, where the viral loads and prevalence values are extremely low. We have evaluated several PCR procedures employed by two laboratories for monitoring fish captured in several oceanographic campaigns performed in the Gulf of Cádiz. To evaluate the reliability of different diagnostics test used, we have re-analyzed fish samples that had been previously subjected to diagnosis for a surveillance of viruses performed in 2010-2011 in wild fish populations. The following parameters were employed: the clinical sensitivity (Ss), the clinical specificity (Sp), the predictive positive value (PPV), the predictive negative value (PNV), and the positive and negative likelihood ratio (LR(+) and LR(-) ). For viral nervious necrosis virus (VNNV), a RT-PCR procedure supplemented by nested PCR showed the highest values (100%) for all the parameters. For viral hemorrhagic septicemia virus (VHSV), the highest values were provided by RT-PCR supplemented by dot blot hybridization. In the case of Infectious pancreatic necrosis virus (IPNV), none of the procedures yielded 100% for any parameter. The results obtained for viral prevalence indicate: (i) that the conservation of the samples at -80 °C did not affect to the capacity of detection of the virus in the tissues, and (ii) that the reproducibility of the diagnosis can be affected by factors including the staff experience and/or the materials employed. Finally, the use of a combination of procedures in advised to ensure the maximum reliability of the diagnosis when it is applied to asymptomatic fish populations. This paper describes a strategy of combining diagnostic tests for the surveillance and monitoring of wild fish populations to reduce underestimation of the prevalence of viruses this

  8. 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. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  9. 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

  10. 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.

  11. Multisegment one-step RT-PCR fluorescent labeling of influenza A virus genome for use in diagnostic microarray applications

    NASA Astrophysics Data System (ADS)

    Vasin, A. V.; Sandybaev, N. T.; Plotnikova, M. A.; Klotchenko, S. A.; Chervyakova, O. V.; Strochkov, V. M.; Taylakova, E. T.; Elpaeva, E. A.; Komissarov, A. B.; Egorov, V. V.; Koshemetov, J. K.; Kiselev, O. I.; Mamadaliev, S. M.

    2011-04-01

    Microarray technology is one of the most challenging methods of influenza A virus subtyping, which is based on the antigenic properties of viral surface glycoproteins - hemagglutinin and neuraminidase. On the example of biochip for detection of influenza A/H5N1 virus we showed the possibility of using multisegment RTPCR method for amplification of fluorescently labeled cDNA of all possible influenza A virus subtypes with a single pair of primers in influenza diagnostic microarrays.

  12. Real-time qPCR improves meningitis pathogen detection in invasive bacterial-vaccine preventable disease surveillance in Fiji.

    PubMed

    Dunne, Eileen M; Mantanitobua, Silivia; Singh, Shalini P; Reyburn, Rita; Tuivaga, Evelyn; Rafai, Eric; Tikoduadua, Lisi; Porter, Barbara; Satzke, Catherine; Strachan, Janet E; Fox, Kimberly K; Jenkins, Kylie M; Jenney, Adam; Baro, Silo; Mulholland, E Kim; Kama, Mike; Russell, Fiona M

    2016-12-23

    As part of the World Health Organization Invasive Bacterial-Vaccine Preventable Diseases (IB-VPD) surveillance in Suva, Fiji, cerebrospinal fluid (CSF) samples from suspected meningitis patients of all ages were examined by traditional methods (culture, Gram stain, and latex agglutination for bacterial antigen) and qPCR for Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae. Of 266 samples tested, pathogens were identified in 47 (17.7%). S. pneumoniae was the most common pathogen detected (n = 17) followed by N. meningitidis (n = 13). The use of qPCR significantly increased detection of IB-VPD pathogens (P = 0.0001): of 35 samples that were qPCR positive for S. pneumoniae, N. meningitidis, and H. influenzae, only 10 were culture positive. This was particularly relevant for N. meningitidis, as only 1/13 cases was culture positive. Molecular serotyping by microarray was used to determine pneumococcal serotypes from 9 of 16 (56%) of samples using DNA directly extracted from CSF specimens. Results indicate that qPCR significantly increases detection of S. pneumoniae, N. meningitidis, and H. influenzae in CSF, and that application of molecular diagnostics is a feasible way to enhance local and global surveillance for IB-VPD.

  13. Real-time qPCR improves meningitis pathogen detection in invasive bacterial-vaccine preventable disease surveillance in Fiji

    PubMed Central

    Dunne, Eileen M.; Mantanitobua, Silivia; Singh, Shalini P.; Reyburn, Rita; Tuivaga, Evelyn; Rafai, Eric; Tikoduadua, Lisi; Porter, Barbara; Satzke, Catherine; Strachan, Janet E.; Fox, Kimberly K.; Jenkins, Kylie M.; Jenney, Adam; Baro, Silo; Mulholland, E. Kim; Kama, Mike; Russell, Fiona M.

    2016-01-01

    As part of the World Health Organization Invasive Bacterial-Vaccine Preventable Diseases (IB-VPD) surveillance in Suva, Fiji, cerebrospinal fluid (CSF) samples from suspected meningitis patients of all ages were examined by traditional methods (culture, Gram stain, and latex agglutination for bacterial antigen) and qPCR for Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae. Of 266 samples tested, pathogens were identified in 47 (17.7%). S. pneumoniae was the most common pathogen detected (n = 17) followed by N. meningitidis (n = 13). The use of qPCR significantly increased detection of IB-VPD pathogens (P = 0.0001): of 35 samples that were qPCR positive for S. pneumoniae, N. meningitidis, and H. influenzae, only 10 were culture positive. This was particularly relevant for N. meningitidis, as only 1/13 cases was culture positive. Molecular serotyping by microarray was used to determine pneumococcal serotypes from 9 of 16 (56%) of samples using DNA directly extracted from CSF specimens. Results indicate that qPCR significantly increases detection of S. pneumoniae, N. meningitidis, and H. influenzae in CSF, and that application of molecular diagnostics is a feasible way to enhance local and global surveillance for IB-VPD. PMID:28009001

  14. Digital PCR Improves Mutation Analysis in Pancreas Fine Needle Aspiration Biopsy Specimens

    PubMed Central

    Court, Colin M.; Kim, Stephen; Braxton, David R.; Hou, Shuang; Muthusamy, V. Raman; Watson, Rabindra R.; Sedarat, Alireza; Tseng, Hsian-Rong; Tomlinson, James S.

    2017-01-01

    Applications of precision oncology strategies rely on accurate tumor genotyping from clinically available specimens. Fine needle aspirations (FNA) are frequently obtained in cancer management and often represent the only source of tumor tissues for patients with metastatic or locally advanced diseases. However, FNAs obtained from pancreas ductal adenocarcinoma (PDAC) are often limited in cellularity and/or tumor cell purity, precluding accurate tumor genotyping in many cases. Digital PCR (dPCR) is a technology with exceptional sensitivity and low DNA template requirement, characteristics that are necessary for analyzing PDAC FNA samples. In the current study, we sought to evaluate dPCR as a mutation analysis tool for pancreas FNA specimens. To this end, we analyzed alterations in the KRAS gene in pancreas FNAs using dPCR. The sensitivity of dPCR mutation analysis was first determined using serial dilution cell spiking studies. Single-cell laser-microdissection (LMD) was then utilized to identify the minimal number of tumor cells needed for mutation detection. Lastly, dPCR mutation analysis was performed on 44 pancreas FNAs (34 formalin-fixed paraffin-embedded (FFPE) and 10 fresh (non-fixed)), including samples highly limited in cellularity (100 cells) and tumor cell purity (1%). We found dPCR to detect mutations with allele frequencies as low as 0.17%. Additionally, a single tumor cell could be detected within an abundance of normal cells. Using clinical FNA samples, dPCR mutation analysis was successful in all preoperative FNA biopsies tested, and its accuracy was confirmed via comparison with resected tumor specimens. Moreover, dPCR revealed additional KRAS mutations representing minor subclones within a tumor that were not detected by the current clinical gold standard method of Sanger sequencing. In conclusion, dPCR performs sensitive and accurate mutation analysis in pancreas FNAs, detecting not only the dominant mutation subtype, but also the additional rare

  15. Digital PCR Improves Mutation Analysis in Pancreas Fine Needle Aspiration Biopsy Specimens.

    PubMed

    Sho, Shonan; Court, Colin M; Kim, Stephen; Braxton, David R; Hou, Shuang; Muthusamy, V Raman; Watson, Rabindra R; Sedarat, Alireza; Tseng, Hsian-Rong; Tomlinson, James S

    2017-01-01

    Applications of precision oncology strategies rely on accurate tumor genotyping from clinically available specimens. Fine needle aspirations (FNA) are frequently obtained in cancer management and often represent the only source of tumor tissues for patients with metastatic or locally advanced diseases. However, FNAs obtained from pancreas ductal adenocarcinoma (PDAC) are often limited in cellularity and/or tumor cell purity, precluding accurate tumor genotyping in many cases. Digital PCR (dPCR) is a technology with exceptional sensitivity and low DNA template requirement, characteristics that are necessary for analyzing PDAC FNA samples. In the current study, we sought to evaluate dPCR as a mutation analysis tool for pancreas FNA specimens. To this end, we analyzed alterations in the KRAS gene in pancreas FNAs using dPCR. The sensitivity of dPCR mutation analysis was first determined using serial dilution cell spiking studies. Single-cell laser-microdissection (LMD) was then utilized to identify the minimal number of tumor cells needed for mutation detection. Lastly, dPCR mutation analysis was performed on 44 pancreas FNAs (34 formalin-fixed paraffin-embedded (FFPE) and 10 fresh (non-fixed)), including samples highly limited in cellularity (100 cells) and tumor cell purity (1%). We found dPCR to detect mutations with allele frequencies as low as 0.17%. Additionally, a single tumor cell could be detected within an abundance of normal cells. Using clinical FNA samples, dPCR mutation analysis was successful in all preoperative FNA biopsies tested, and its accuracy was confirmed via comparison with resected tumor specimens. Moreover, dPCR revealed additional KRAS mutations representing minor subclones within a tumor that were not detected by the current clinical gold standard method of Sanger sequencing. In conclusion, dPCR performs sensitive and accurate mutation analysis in pancreas FNAs, detecting not only the dominant mutation subtype, but also the additional rare

  16. Improving diagnostic recognition of primary hyperparathyroidism with machine learning.

    PubMed

    Somnay, Yash R; Craven, Mark; McCoy, Kelly L; Carty, Sally E; Wang, Tracy S; Greenberg, Caprice C; Schneider, David F

    2017-04-01

    decrease the accuracy significantly (area under receiver operating characteristic = 0.985). In mild disease cases, however, the Bayesian network model classified correctly 71.1% of patients with normal calcium and 92.1% with normal parathyroid hormone levels preoperatively. Bayesian networking and AdaBoost improved the accuracy of all parathyroid hormone patients to 97.2% cases (area under receiver operating characteristic = 0.994), and 91.9% of primary hyperparathyroidism patients with mild disease. This was significantly improved relative to Bayesian networking alone (P < .0001). Machine learning can diagnose accurately primary hyperparathyroidism without human input even in mild disease. Incorporation of this tool into electronic medical record systems may aid in recognition of this under-diagnosed disorder. Copyright © 2016 Elsevier Inc. All rights reserved.

  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. Improving communication of diagnostic radiology findings through structured reporting.

    PubMed

    Schwartz, Lawrence H; Panicek, David M; Berk, Alexandra R; Li, Yuelin; Hricak, Hedvig

    2011-07-01

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

  19. Improving Communication of Diagnostic Radiology Findings through Structured Reporting

    PubMed Central

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

    2011-01-01

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

  20. Improved adalimumab dose decision with comprehensive diagnostics data.

    PubMed

    Zänker, Michael; Becher, Gunther; Arbach, Olga; Maurer, Marcus; Stuhlmüller, Bruno; Schäfer, Astrid; Strohner, Pavel; Brand, Janko

    2017-08-28

    Monoclonal antibodies are important in the treatment of rheumatoid arthritis (RA). This is the first trial to monitor the effect of adalimumab dose escalation in persistently active RA. The aim of this study was to identify the response to adalimumab to improve the basis for making decision in relation to actual drug capacity in serum. The disease activity of RA patients was assessed with CDAI and DAS28 before administration of additional 40 mg adalimumab one week after standard injection. Serum samples were analysed using the recoveryELISA technology, a combination of sandwich ELISA and competitive assay. The recoveryELISA measure the concentrations of free TNF-α, drug level, and the remaining active adalimumab in the patients' sera. An adalimumab concentration of 5.0-10.0 g/mL was defined as the targeted therapeutic window. Five of 8 patients achieved moderate EULAR response by dose escalation. The results of the free adalimumab and TNF-α neutralisation measurements allowed a separation of the cohort (n=17) into three groups. Group 1 represents 18% of the patients with free adalimumab level higher 30.0 μg/mL and TNF-α neutralisation above 95%. Group 2 (47%) consists of patients within the therapeutic window with balanced free adalimumab and TNF-α neutralisation values. Group 3 contains 35% of the cohort with low concentrations of free adalimumab and lowest remaining TNF-α-neutralisation capacity. Anti-drug antibodies were detected in four patients but did not prevent response to treatment. Drug and antigen monitoring using recoveryELISA may support dose decision to avoid unnecessary switch in medication or possible overtreatment.

  1. Development of a semi-nested PCR for the improved detection of Mycoplasma bovis from bovine milk and mucosal samples.

    PubMed

    Hayman, Bernadette; Hirst, Robert

    2003-02-02

    A new forward primer, Mb-F, was designed to improve the sensitivity and reproducibility of the Mycoplasma bovis-specific PCR developed by Ghadersohi et al. [Vet. Microbiol. 56 (1997) 87] for testing clinical samples. A semi-nested (SN) PCR configuration was developed and this provided enhanced sensitivity and reproducibility. The detection limit of the SN PCR was in the range of 10-100cfu/ml and the correct amplicon was amplified from 9.15pg/microliter of total extracted DNA (mixture of M. bovis and bovine cellular DNA). A dot blot assay was also developed and compared with the SN PCR on a number of randomly selected milk and mucosal samples. The dot blot had the same level of detection as the SN PCR. The specificity of the SN configuration was confirmed by Southern blot analysis and automated sequencing of the PCR product. The results from the tests on the samples from cattle, together with those from sheep, provided evidence that M. bovis is host-specific and that most cattle are colonised. The assay was shown to be specific, sensitive and reproducible and could be used successfully to detect M. bovis directly from clinical material without pre-enrichment.

  2. 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. Published by Elsevier Ireland Ltd.

  3. Novel cystatin B mutation and diagnostic PCR assay in an Unverricht-Lundborg progressive myoclonus epilepsy patient.

    PubMed

    Bespalova, I N; Adkins, S; Pranzatelli, M; Burmeister, M

    1997-09-19

    Two mutations in the cystatin B gene, a 3' splice mutation and a stop codon mutation, were previously found in patients with progressive myoclonus epilepsy of Unverricht-Lundborg type [Pennacchio et al. (1996): Science 271:1731-1734]. We present here a new mutation 2404deltaTC: a 2-bp deletion within the third exon of the cystatin B gene in an Unverricht-Lundborg patient. This mutation results in a frameshift and consequently premature termination of protein synthesis. Complete sequencing of the coding region and splice junctions of the cystatin B gene showed that neither of the two previously known mutations was present in this patient. The level of cystatin B mRNA in an immortalized cell line was found to be decreased, as had been reported for other Unverricht-Lundborg patients. The new mutation further supports the argument that defects in the cystatin B gene cause the Unverricht-Lundborg form of progressive myoclonus epilepsy. We describe a simple PCR method which can detect the 2404deltaTC deletion. This assay, together with previously described PCR assays for the other two known mutations, should prove useful in confirming clinically difficult diagnoses of Unverricht-Lundborg disease.

  4. Improved high sensitivity screen for Huntington disease using a one-step triplet-primed PCR and melting curve assay

    PubMed Central

    Zhao, Mingjue; Cheah, Felicia S. H.; Chen, Min; Lee, Caroline G.; Law, Hai-Yang

    2017-01-01

    Molecular diagnosis of Huntington disease (HD) is currently performed by fluorescent repeat-flanking or triplet-primed PCR (TP-PCR) with capillary electrophoresis (CE). However, CE requires multiple post-PCR steps and may result in high cost in high-throughput settings. We previously described a cost-effective single-step molecular screening strategy employing the use of melting curve analysis (MCA). However, because it relies on repeat-flanking PCR, its efficiency in detecting expansion mutations decreases with increasing size of the repeat, which could lead to false-negative results. To address this pitfall, we have developed an improved screening assay coupling TP-PCR, which has been shown in CE-based assays to detect all expanded alleles regardless of size, with MCA in a rapid one-step assay. A companion protocol for rapid size confirmation of expansion-positive samples is also described. The assay was optimized on 30 genotype-known DNAs, and two plasmids pHTT(CAG)26 and pHTT(CAG)33 were used to establish the threshold temperatures (TTs) distinguishing normal from expansion-positive samples. In contrast to repeat-flanking PCR MCA, TP-PCR MCA displayed much higher sensitivity for detecting large expansions. All 30 DNAs generated distinct melt peak Tms which correlated well with each sample’s larger allele. Normal samples were clearly distinguished from affected samples. The companion sizing protocol accurately sized even the largest expanded allele of ~180 CAGs. Blinded analysis of 69 clinical samples enriched for HD demonstrated 100% assay sensitivity and specificity in sample segregation. The assay targets the HTT CAG repeat specifically, tolerates a wide range of input DNA, and works well using DNA from saliva and buccal swab in addition to blood. Therefore, rapid, accurate, reliable, and high-throughput detection/exclusion of HD can be achieved using this one-step screening assay, at less than half the cost of fluorescent PCR with CE. PMID:28700716

  5. Improved detection of Lassa virus by reverse transcription-PCR targeting the 5' region of S RNA.

    PubMed

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

    2010-06-01

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

  6. Combined assessment of midbrain hyperechogenicity, hyposmia and motor asymmetry improves diagnostic accuracy in early Parkinson's disease.

    PubMed

    Poewe, Werner; Mahlknecht, Philipp

    2012-08-01

    The differential diagnosis of Parkinsonian syndromes can be challenging, particularly in early disease stages, when overlapping clinical signs and symptoms may lead to erroneous classification. However, an early differentiation between Parkinson's disease (PD) and other diseases causing Parkinsonism is crucial for prognostic and therapeutic reasons and is essential for clinical research. In a recent study, Busse et al. investigated the diagnostic utility of a set of tests to improve diagnostic differentiation between PD, essential tremor and other Parkinsonian disorders. The authors studied a total of 632 patients divided into a retrospective (n = 517) and a prospective (n = 115) group. Diagnostic anchors were based on clinical criteria. Combining midbrain hyperechogenicity, hyposmia and motor asymmetry increased specificity and positive predictive value for diagnosis of PD up to 98% at the expense of sensitivity, whereas two features provided 91% sensitivity with 77% specificity. The results of this study further support the diagnostic utility of transcranial sonography in diagnosing PD.

  7. Real-time PCR-based analysis of the human bile microRNAome identifies miR-9 as a potential diagnostic biomarker for biliary tract cancer.

    PubMed

    Shigehara, Kengo; Yokomuro, Shigeki; Ishibashi, Osamu; Mizuguchi, Yoshiaki; Arima, Yasuo; Kawahigashi, Yutaka; Kanda, Tomohiro; Akagi, Ichiro; Tajiri, Takashi; Yoshida, Hiroshi; Takizawa, Toshihiro; Uchida, Eiji

    2011-01-01

    Biliary tract cancer (BTC) is often difficult to diagnose definitively, even through histological examination. MicroRNAs (miRNAs) regulate a variety of physiological processes. In recent years, it has been suggested that profiles for circulating miRNAs, as well as those for tissue miRNAs, have the potential to be used as diagnostic biomarkers for cancer. The aim of this study was to confirm the existence of miRNAs in human bile and to assess their potential as clinical biomarkers for BTC. We sampled bile from patients who underwent biliary drainage for biliary diseases such as BTC and choledocholithiasis. PCR-based miRNA detection and miRNA cloning were performed to identify bile miRNAs. Using high-throughput real-time PCR-based miRNA microarrays, the expression profiles of 667 miRNAs were compared in patients with malignant disease (n = 9) and age-matched patients with the benign disease choledocholithiasis (n = 9). We subsequently characterized bile miRNAs in terms of stability and localization. Through cloning and using PCR methods, we confirmed that miRNAs exist in bile. Differential analysis of bile miRNAs demonstrated that 10 of the 667 miRNAs were significantly more highly expressed in the malignant group than in the benign group at P<0.0005. Setting the specificity threshold to 100% showed that some miRNAs (miR-9, miR-302c*, miR-199a-3p and miR-222*) had a sensitivity level of 88.9%, and receiver-operating characteristic analysis demonstrated that miR-9 and miR-145* could be useful diagnostic markers for BTC. Moreover, we verified the long-term stability of miRNAs in bile, a characteristic that makes them suitable for diagnostic use in clinical settings. We also confirmed that bile miRNAs are localized to the malignant/benign biliary epithelia. These findings suggest that bile miRNAs could be informative biomarkers for hepatobiliary disease and that some miRNAs, particularly miR-9, may be helpful in the diagnosis and clinical management of BTC.

  8. Real-Time PCR-Based Analysis of the Human Bile MicroRNAome Identifies miR-9 as a Potential Diagnostic Biomarker for Biliary Tract Cancer

    PubMed Central

    Shigehara, Kengo; Yokomuro, Shigeki; Ishibashi, Osamu; Mizuguchi, Yoshiaki; Arima, Yasuo; Kawahigashi, Yutaka; Kanda, Tomohiro; Akagi, Ichiro; Tajiri, Takashi; Yoshida, Hiroshi; Takizawa, Toshihiro; Uchida, Eiji

    2011-01-01

    Biliary tract cancer (BTC) is often difficult to diagnose definitively, even through histological examination. MicroRNAs (miRNAs) regulate a variety of physiological processes. In recent years, it has been suggested that profiles for circulating miRNAs, as well as those for tissue miRNAs, have the potential to be used as diagnostic biomarkers for cancer. The aim of this study was to confirm the existence of miRNAs in human bile and to assess their potential as clinical biomarkers for BTC. We sampled bile from patients who underwent biliary drainage for biliary diseases such as BTC and choledocholithiasis. PCR-based miRNA detection and miRNA cloning were performed to identify bile miRNAs. Using high-throughput real-time PCR-based miRNA microarrays, the expression profiles of 667 miRNAs were compared in patients with malignant disease (n = 9) and age-matched patients with the benign disease choledocholithiasis (n = 9). We subsequently characterized bile miRNAs in terms of stability and localization. Through cloning and using PCR methods, we confirmed that miRNAs exist in bile. Differential analysis of bile miRNAs demonstrated that 10 of the 667 miRNAs were significantly more highly expressed in the malignant group than in the benign group at P<0.0005. Setting the specificity threshold to 100% showed that some miRNAs (miR-9, miR-302c*, miR-199a-3p and miR-222*) had a sensitivity level of 88.9%, and receiver-operating characteristic analysis demonstrated that miR-9 and miR-145* could be useful diagnostic markers for BTC. Moreover, we verified the long-term stability of miRNAs in bile, a characteristic that makes them suitable for diagnostic use in clinical settings. We also confirmed that bile miRNAs are localized to the malignant/benign biliary epithelia. These findings suggest that bile miRNAs could be informative biomarkers for hepatobiliary disease and that some miRNAs, particularly miR-9, may be helpful in the diagnosis and clinical management of BTC. PMID

  9. An improved arbitrary primed PCR method for rapid characterization of transposon insertion sites.

    PubMed

    Das, Sankar; Noe, Jody C; Paik, Sehmi; Kitten, Todd

    2005-10-01

    Modifications were made to published arbitrary primed polymerase chain reaction (AP-PCR) procedures that resulted in increased specificity and sensitivity. Several arbitrary primer sequences were also evaluated, resulting in recommendations for primer design.

  10. Improvement of a PCR method for the detection of necrotizing hepatopancreatitis in shrimp.

    PubMed

    Nunan, Linda M; Pantoja, Carlos; Lightner, Donald V

    2008-06-19

    Necrotizing hepatopancreatitis (NHP) is considered to be one of the most important bacterial diseases affecting penaeid shrimp culture and is caused by an unclassified Gram-negative, pleomorphic, intracellular Alphaproteobacterium. Due to the enteric nature of the bacteria, PCR is the one non-lethal method available for detection of the pathogen. Over a decade ago, a PCR protocol was developed for detection of NHP, which over the subsequent years was shown to occasionally generate false positive reactions. The University of Arizona Aquaculture Pathology Laboratory has developed a set of primers and PCR cycling parameters that have been tested on a variety of DNA templates, using 2 types of PCR reagent systems, which eliminated the generation of false positive amplicons.

  11. 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...

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-08-31

    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.

  14. Automated degenerate PCR primer design for high-throughput sequencing improves efficiency of viral sequencing.

    PubMed

    Li, Kelvin; Shrivastava, Susmita; Brownley, Anushka; Katzel, Dan; Bera, Jayati; Nguyen, Anh Thu; Thovarai, Vishal; Halpin, Rebecca; Stockwell, Timothy B

    2012-11-06

    In a high-throughput environment, to PCR amplify and sequence a large set of viral isolates from populations that are potentially heterogeneous and continuously evolving, the use of degenerate PCR primers is an important strategy. Degenerate primers allow for the PCR amplification of a wider range of viral isolates with only one set of pre-mixed primers, thus increasing amplification success rates and minimizing the necessity for genome finishing activities. To successfully select a large set of degenerate PCR primers necessary to tile across an entire viral genome and maximize their success, this process is best performed computationally. We have developed a fully automated degenerate PCR primer design system that plays a key role in the J. Craig Venter Institute's (JCVI) high-throughput viral sequencing pipeline. A consensus viral genome, or a set of consensus segment sequences in the case of a segmented virus, is specified using IUPAC ambiguity codes in the consensus template sequence to represent the allelic diversity of the target population. PCR primer pairs are then selected computationally to produce a minimal amplicon set capable of tiling across the full length of the specified target region. As part of the tiling process, primer pairs are computationally screened to meet the criteria for successful PCR with one of two described amplification protocols. The actual sequencing success rates for designed primers for measles virus, mumps virus, human parainfluenza virus 1 and 3, human respiratory syncytial virus A and B and human metapneumovirus are described, where >90% of designed primer pairs were able to consistently successfully amplify >75% of the isolates. Augmenting our previously developed and published JCVI Primer Design Pipeline, we achieved similarly high sequencing success rates with only minor software modifications. The recommended methodology for the construction of the consensus sequence that encapsulates the allelic variation of the targeted

  15. Application of Routine Diagnostic Procedure, VITEK 2 Compact, MALDI-TOF MS, and PCR Assays in Identification Procedure of Bacterial Strain with Ambiguous Phenotype.

    PubMed

    Książczyk, Marta; Kuczkowski, Maciej; Dudek, Bartłomiej; Korzekwa, Kamila; Tobiasz, Anna; Korzeniowska-Kowal, Agnieszka; Paluch, Emil; Wieliczko, Alina; Bugla-Płoskońska, Gabriela

    2016-05-01

    In diagnostic microbiology as well as in microbiological research, the identification of a microorganism is a crucial and decisive stage. A broad choice of methods is available, based on both phenotypic and molecular properties of microbes. The aim of this study was to compare the application of phenotypic and molecular tools in bacterial identification on the example of Gram-negative intestine rod with an ambiguous phenotype. Different methods of identification procedure, which based on various properties of bacteria, were applied, e.g., microscopic observation of single-bacterial cells, macroscopic observation of bacterial colonies morphology, the automated system of microorganism identification (biochemical tests), the mass spectrometry method (analysis of bacterial proteome), and genetic analysis with PCR reactions. The obtained results revealed discrepancies in the identification of the tested bacterial strain with an atypical phenotype: mucous morphology of colonies, not characteristic for either E. coli and Citrobacter spp., mass spectrometry analysis of proteome initially assigned the tested strain to Citrobacter genus (C. freundii) and biochemical profiles pointed to Escherichia coli. A decisive method in the current study was genetic analysis with PCR reactions which identified conserved genetic sequences highly specific to E. coli species in the genome of the tested strain.

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

    PubMed Central

    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; Meya, David B; Boulware, David R

    2015-01-01

    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: CMV (n=2), VZV (n=2), HHV-6 (n=1), and Streptococcus pneumoniae (n=1). The FilmArray Meningitis/Encephalitis panel offers a promising platform for rapid meningitis diagnosis. PMID:26711635

  17. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Rendering of mycobacteria safe for molecular diagnostic studies and development of a lysis method for strand displacement amplification and PCR.

    PubMed Central

    Zwadyk, P; Down, J A; Myers, N; Dey, M S

    1994-01-01

    Two criteria must be met before mycobacterial specimens can be tested by DNA amplification methods: (i) the sample must be rendered noninfectious, and (ii) the organisms must be lysed to free the DNA. Previous publications reporting DNA amplification of mycobacteria have concentrated on lysis and amplification procedures and have not addressed the issue of sample safety. We have shown that heating of samples below 100 degrees C may not consistently kill mycobacteria; however, heating at 100 degrees C in a boiling-water bath or a forced-air oven for a minimum of 5 min kills mycobacteria, including Mycobacterium thermoresistibile. Furthermore, heating at 100 degrees C for 30 min consistently lyses mycobacteria to produce short fragments of DNA that are suitable for amplification by PCR and strand displacement amplification. This procedure works with clinical samples digested by the n-acetyl cysteine-NaOH method as well as with suspensions of organisms in phosphate buffer. This paper also demonstrates the feasibility of using strand displacement amplification with clinical specimens. Images PMID:7814537

  19. Diagnostic Utility of the Social Skills Improvement System Performance Screening Guide

    ERIC Educational Resources Information Center

    Krach, S. Kathleen; McCreery, Michael P.; Wang, Ye; Mohammadiamin, Houra; Cirks, Christen K.

    2017-01-01

    Researchers investigated the diagnostic utility of the Social Skills Improvement System: Performance Screening Guide (SSIS-PSG). Correlational, regression, receiver operating characteristic (ROC), and conditional probability analyses were run to compare ratings on the SSIS-PSG subscales of Prosocial Behavior, Reading Skills, and Math Skills, to…

  20. Rapid diagnostic PCR assays for members of the Culicoides obsoletus and Culicoides pulicaris species complexes, implicated vectors of bluetongue virus in Europe.

    PubMed

    Nolan, Damien V; Carpenter, Simon; Barber, James; Mellor, Philip S; Dallas, John F; Mordue Luntz, A Jennifer; Piertney, Stuart B

    2007-09-20

    Biting midges of the Culicoides obsoletus Meigen and Culicoides pulicaris L. species complexes (Diptera: Ceratopogonidae) are increasingly implicated as vectors of bluetongue virus in Palearctic regions. However, predicting epidemiological risk and the spread of disease is hampered because whilst vector competence of Culicoides is expressed only in adult females, morphological identification of constituent species is only readily applicable to adult males and some species distinguishing traits have overlapping character states. Furthermore, adult males are typically rare in field collections, making characterisation of Culicoides communities impossible. Here we highlight the utility of mitochondrial cytochrome oxidase subunit I (COI) DNA sequences for taxonomic resolution and species identification of all species within C. obsoletus and C. pulicarus complexes. Culicoides were collected from 18 sites in the UK and Continental Europe, and identified to species level, or species complex level, based on morphological characters. The sample comprised four species from the C. obsoletus complex (n = 88) and five species from the C. pulicaris complex (n = 39). The DNA sequence of the 5' end of the COI gene was obtained from all individuals. Each member species formed a well-supported reciprocally monophyletic clade in a maximum likelihood phylogeny. Levels of DNA sequence divergence were sufficiently high between species to allow the design of species-specific PCR primers that can be used in PCR for identification of members of the C. pulicaris complex or in a multiplex PCR to identify members of the C. obsoletus complex. This approach provides a valuable diagnostic tool for monitoring species composition in mixed field collections of Culicoides.

  1. Improving the Specificity of Plasmodium falciparum Malaria Diagnosis in High-Transmission Settings with a Two-Step Rapid Diagnostic Test and Microscopy Algorithm.

    PubMed

    Murungi, Moses; Fulton, Travis; Reyes, Raquel; Matte, Michael; Ntaro, Moses; Mulogo, Edgar; Nyehangane, Dan; Juliano, Jonathan J; Siedner, Mark J; Boum, Yap; Boyce, Ross M

    2017-05-01

    Poor specificity may negatively impact rapid diagnostic test (RDT)-based diagnostic strategies for malaria. We performed real-time PCR on a subset of subjects who had undergone diagnostic testing with a multiple-antigen (histidine-rich protein 2 and pan-lactate dehydrogenase pLDH [HRP2/pLDH]) RDT and microscopy. We determined the sensitivity and specificity of the RDT in comparison to results of PCR for the detection of Plasmodium falciparum malaria. We developed and evaluated a two-step algorithm utilizing the multiple-antigen RDT to screen patients, followed by confirmatory microscopy for those individuals with HRP2-positive (HRP2(+))/pLDH-negative (pLDH(-)) results. In total, dried blood spots (DBS) were collected from 276 individuals. There were 124 (44.9%) individuals with an HRP2(+)/pLDH(+) result, 94 (34.1%) with an HRP2(+)/pLDH(-) result, and 58 (21%) with a negative RDT result. The sensitivity and specificity of the RDT compared to results with real-time PCR were 99.4% (95% confidence interval [CI], 95.9 to 100.0%) and 46.7% (95% CI, 37.7 to 55.9%), respectively. Of the 94 HRP2(+)/pLDH(-) results, only 32 (34.0%) and 35 (37.2%) were positive by microscopy and PCR, respectively. The sensitivity and specificity of the two-step algorithm compared to results with real-time PCR were 95.5% (95% CI, 90.5 to 98.0%) and 91.0% (95% CI, 84.1 to 95.2), respectively. HRP2 antigen bands demonstrated poor specificity for the diagnosis of malaria compared to that of real-time PCR in a high-transmission setting. The most likely explanation for this finding is the persistence of HRP2 antigenemia following treatment of an acute infection. The two-step diagnostic algorithm utilizing microscopy as a confirmatory test for indeterminate HRP2(+)/pLDH(-) results showed significantly improved specificity with little loss of sensitivity in a high-transmission setting. Copyright © 2017 American Society for Microbiology.

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

    PubMed Central

    Sint, Daniela; Raso, Lorna; Traugott, Michael

    2012-01-01

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

  3. 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

  4. Linear amplification of target prior to PCR for improved low template DNA results.

    PubMed

    Grisedale, Kelly; van Daal, Angela

    2014-01-01

    Forensic analysis of genetic material is often limited by the quantity and quality of DNA available for examination. Stochastic effects associated with low amounts of starting template can lead to a reduction in the quality of the result, making interpretation difficult. This paper presents an amplification method to copy target DNA in a linear fashion prior to short tandem repeat (STR) analysis to increase the available starting template without introducing the amplification bias seen in other methods used to increase the sensitivity of PCR. Results show that implementing the pre- PCR procedure allows for greater allele recovery in multiplex STR analysis compared with samples that were not subjected to prior processing.

  5. An improved qPCR protocol for rapid detection and quantification of Clostridium difficile in cattle feces.

    PubMed

    Bandelj, Petra; Logar, Katarina; Usenik, Alenka M; Vengust, Modest; Ocepek, Matjaz

    2013-04-01

    Clostridium difficile (CD) can cause a significant and transmissible disease in animals and humans, with poorly understood epidemiology. Animals have been suggested as a possible source of infection and environment contamination. It is necessary that a precise and rapid diagnostic tool is available for the detection of CD from clinical and/or environmental samples. A quantitative real-time PCR (qPCR) protocol for CD detection defined by Penders et al. (FEMS Microbiol Lett, 243, 2005, 141-147) was modified. The modified protocol, supported by a novel extraction method, was tested on CD-spiked cattle feces and clinical fecal samples from calves. Quantification was performed targeting CD 16S rRNA gene. Three different commonly used TaqMan universal PCR master mixes were also compared. Results indicate that the modified protocol is very sensitive with an LOD of 7.72 CD cells per g CD-spiked feces. The protocol is capable of precise quantification with an LOQ of 77.2 CD cells per g CD-spiked feces, R(2) between 0.9957 and 0.9968, isolation efficiency from 87.89% to 90.96%, and an interassay CV ranging from 3.71% to 9.57%. The qPCR protocol for the detection and quantification of CD from animal feces investigated and described in this article using MIQE guidelines has the lowest detection and quantification limits published to date. Therefore, it can be implemented for precise epidemiological investigations of CD infections in animals and humans. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  6. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  8. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  10. 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

  11. 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.

  12. 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

  13. External quality assessment study for ebolavirus PCR-diagnostic promotes international preparedness during the 2014 – 2016 Ebola outbreak in West Africa

    PubMed Central

    Jacobsen, Sonja; Patel, Pranav; Rieger, Toni; Eickmann, Markus; Becker, Stephan; Günther, Stephan; Naidoo, Dhamari; Schrick, Livia; Keeren, Kathrin; Targosz, Angelina; Teichmann, Anette; Formenty, Pierre; Niedrig, Matthias

    2017-01-01

    During the recent Ebola outbreak in West Africa several international mobile laboratories were deployed to the mainly affected countries Guinea, Sierra Leone and Liberia to provide ebolavirus diagnostic capacity. Additionally, imported cases and small outbreaks in other countries required global preparedness for Ebola diagnostics. Detection of viral RNA by reverse transcription polymerase chain reaction has proven effective for diagnosis of ebolavirus disease and several assays are available. However, reliability of these assays is largely unknown and requires serious evaluation. Therefore, a proficiency test panel of 11 samples was generated and distributed on a global scale. Panels were analyzed by 83 expert laboratories and 106 data sets were returned. From these 78 results were rated optimal and 3 acceptable, 25 indicated need for improvement. While performance of the laboratories deployed to West Africa was superior to the overall performance there was no significant difference between the different assays applied. PMID:28459810

  14. External quality assessment study for ebolavirus PCR-diagnostic promotes international preparedness during the 2014 - 2016 Ebola outbreak in West Africa.

    PubMed

    Ellerbrok, Heinz; Jacobsen, Sonja; Patel, Pranav; Rieger, Toni; Eickmann, Markus; Becker, Stephan; Günther, Stephan; Naidoo, Dhamari; Schrick, Livia; Keeren, Kathrin; Targosz, Angelina; Teichmann, Anette; Formenty, Pierre; Niedrig, Matthias

    2017-05-01

    During the recent Ebola outbreak in West Africa several international mobile laboratories were deployed to the mainly affected countries Guinea, Sierra Leone and Liberia to provide ebolavirus diagnostic capacity. Additionally, imported cases and small outbreaks in other countries required global preparedness for Ebola diagnostics. Detection of viral RNA by reverse transcription polymerase chain reaction has proven effective for diagnosis of ebolavirus disease and several assays are available. However, reliability of these assays is largely unknown and requires serious evaluation. Therefore, a proficiency test panel of 11 samples was generated and distributed on a global scale. Panels were analyzed by 83 expert laboratories and 106 data sets were returned. From these 78 results were rated optimal and 3 acceptable, 25 indicated need for improvement. While performance of the laboratories deployed to West Africa was superior to the overall performance there was no significant difference between the different assays applied.

  15. Controlled hot start and improved specificity in carrying out PCR utilizing touch-up and loop incorporated primers (TULIPS).

    PubMed

    Ailenberg, M; Silverman, M

    2000-11-01

    The PCR technique often yields nonspecific products. To overcome this problem, a simple, specific and efficient method was designed: touch-up and loop incorporated primers (TULIPS)-PCR. This approach utilizes loop primers (i.e., additional nontemplate 5' sequence that self-anneals to the 3' region and inhibits initiation of polymerization). Upon heating of the reaction, the primers melt, initiating hot start. The reaction also uses touch-up pre-cycling with gradual elevation in annealing temperatures to ensure correct pairing. The method has been validated with glyceraldehyde-3-phosphate dehydrogenase (GAPD) primers, and its general applicability is demonstrated by specific amplification of the human gelatinase A transgene from genomic DNA extracted from transgenic mice tails. The TULIPS-PCR protocol is a novel method. The self-annealing primers utilized in this method offer improved specificity and more robust synthesis compared with touch-down and manual hot start PCR. It is performed without the need to open, pause or add to the reaction mixture any nonrectant components, such as wax, antibody or nonspecific dsDNA.

  16. Improved detection of Escherichia coli and coliform bacteria by multiplex PCR.

    PubMed

    Molina, Felipe; López-Acedo, Elena; Tabla, Rafael; Roa, Isidro; Gómez, Antonia; Rebollo, José E

    2015-06-04

    The presence of coliform bacteria is routinely assessed to establish the microbiological safety of water supplies and raw or processed foods. Coliforms are a group of lactose-fermenting Enterobacteriaceae, which most likely acquired the lacZ gene by horizontal transfer and therefore constitute a polyphyletic group. Among this group of bacteria is Escherichia coli, the pathogen that is most frequently associated with foodborne disease outbreaks and is often identified by β-glucuronidase enzymatic activity or by the redundant detection of uidA by PCR. Because a significant fraction of essential E. coli genes are preserved throughout the bacterial kingdom, alternative oligonucleotide primers for specific E. coli detection are not easily identified. In this manuscript, two strategies were used to design oligonucleotide primers with differing levels of specificity for the simultaneous detection of total coliforms and E. coli by multiplex PCR. A consensus sequence of lacZ and the orphan gene yaiO were chosen as targets for amplification, yielding 234 bp and 115 bp PCR products, respectively. The assay designed in this work demonstrated superior detection ability when tested with lab collection and dairy isolated lactose-fermenting strains. While lacZ amplicons were found in a wide range of coliforms, yaiO amplification was highly specific for E. coli. Additionally, yaiO detection is non-redundant with enzymatic methods.

  17. [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".

  18. TCRβ clonality improves diagnostic yield of TCRγ clonality in refractory celiac disease.

    PubMed

    Perfetti, Vittorio; Brunetti, Laura; Biagi, Federico; Ciccocioppo, Rachele; Bianchi, Paola I; Corazza, Gino R

    2012-09-01

    Refractory celiac disease (RCD) is a preneoplastic condition as many patients develop an enteropathy-type T-cell lymphoma, a mature T-cell receptor α-β lymphoma arising in the gut with an ominous outcome. Recently, research focused on a population of intraepithelial intestinal lymphocytes expressing the same lymphoma T-cell receptor variable region (V)γ, as shown by polymerase chain reaction (PCR) analysis and sequencing. Meanwhile, the Biomedicine and Health-2 Concerted Action has made available standardized, highly specific, and sensitive PCR assays not only for Vγ but also for Vβ. We verified whether analyzing both rearrangements in duodenal biopsies from RCD patients increases the diagnostic accuracy of this method. Duodenal biopsies were analyzed from 15 RCD patients, 21 negative controls, and 2 positive controls (enteropathy-type T-cell lymphoma complicating celiac disease). Multiplex clonality analyses were performed according to the Biomedicine and Health-2 protocols. PCR products were cloned and sequenced. Monoclonal rearrangements were found in 5/15 samples from patients with RCD (both rearrangements in 2 cases, Vβ only in 2, and only 1 solitary Vγ clonality). Monoclonality was found in 4/8 of the RCD patients who subsequently died, whereas only 1/7 of the patients still alive presented a monoclonal rearrangement. Positive controls revealed both monoclonal rearrangements; rearrangements were not detected in 20 of 21 negative controls. Sequencing of the amplified fragments confirmed the results. The combined analysis of both rearrangements allowed recognition of monoclonal populations in otherwise negative patients, with detection rates from 20% (Vγ only) to 33% (Vγ and Vβ), thus raising the likelihood of early identification of RCD patients at high risk of death.

  19. Comparison of four rapid diagnostic tests, ELISA, microscopy and PCR for the detection of Giardia lamblia, Cryptosporidium spp. and Entamoeba histolytica in feces.

    PubMed

    Van den Bossche, Dorien; Cnops, Lieselotte; Verschueren, Jacob; Van Esbroeck, Marjan

    2015-03-01

    Microscopy is the diagnostic reference standard for the detection of parasites, but it is labor-intensive and requires experience. Rapid diagnostic tests (RDTs) can provide an alternative to microscopy. RDTs from four different manufacturers were compared to enzyme-linked immunosorbent assay (ELISA), microscopy and/or parasite-specific real-time PCR: ImmunoCardSTAT!®CGE (Meridian Bioscience Inc., Cincinnati, Ohio, USA) (A), Crypto/Giardia Duo-Strip (Coris Bioconcepts, Gembloux, Belgium) (B), RIDA®QUICK Cryptosporidium/Giardia/Entamoeba Combi (R-BioPharm, Darmstadt, Germany) (C) and Giardia/Cryptosporidium Quik Chek (Techlab Inc., Blacksburg, Virginia, USA) (D). Thirty frozen samples were analyzed retrospectively. For Giardia lamblia (n=12) and Cryptosporidium (n=12) sensitivities ranged from 58% (B), over 83% (A, C) to 100% (D) and from 92% (B) to 100% (A, C, D), respectively. Specificity for both G. lamblia and Cryptosporidium was 100% for all RDT brands. Sensitivity for Entamoeba histolytica (n=5) was 100%, while specificity reached 80% (A) to 88% (C). In a prospective study, fresh samples were tested. For G. lamblia (n=30), sensitivity ranged from 66% (B), over 79% (A) and 83% (C) to 100% (D) and specificity varied between 94% (D) and 100% (A, B, C). For Cryptosporidium (n=3), sensitivity was 100% for all brands except (B) (67%) and specificities were 95% (A, B), 98% (C) and 100% (D). E. histolytica (n=1) was detected by both (A) and (C), while specificity was 81% and 87% respectively. RDTs can be a valuable tool when microscopic expertise is poor and in remote and outbreak settings where other techniques are often not available and rapid diagnosis is required. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. A Novel Teaching-Learning-Based Optimization for Improved Mutagenic Primer Design in Mismatch PCR-RFLP SNP Genotyping.

    PubMed

    Cheng, Yu-Huei

    2016-01-01

    Many single nucleotide polymorphisms (SNPs) for complex genetic diseases are genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in small-scale basic research studies. It is an essential work to design feasible PCR-RFLP primer pair and find out available restriction enzymes to recognize the target SNP for PCR experiments. However, many SNPs are incapable of performing PCR-RFLP makes SNP genotyping become unpractical. A genetic algorithm (GA) had been proposed for designing mutagenic primer and get available restriction enzymes, but it gives an unrefined solution in mutagenic primers. In order to improve the mutagenic primer design, we propose TLBOMPD (TLBO-based Mutagenic Primer Design) a novel computational intelligence-based method that uses the notion of "teaching and learning" to search for more feasible mutagenic primers and provide the latest available restriction enzymes. The original Wallace's formula for the calculation of melting temperature is maintained, and more accurate calculation formulas of GC-based melting temperature and thermodynamic melting temperature are introduced into the proposed method. Mutagenic matrix is also reserved to increase the efficiency of judging a hypothetical mutagenic primer if involve available restriction enzymes for recognizing the target SNP. Furthermore, the core of SNP-RFLPing version 2 is used to enhance the mining work for restriction enzymes based on the latest REBASE. Twenty-five SNPs with mismatch PCR-RFLP screened from 288 SNPs in human SLC6A4 gene are used to appraise the TLBOMPD. Also, the computational results are compared with those of the GAMPD. In the future, the usage of the mutagenic primers in the wet lab needs to been validated carefully to increase the reliability of the method. The TLBOMPD is implemented in JAVA and it is freely available at http://tlbompd.googlecode.com/.

  1. 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

  2. Intramuscular compartment pressure measurement in chronic exertional compartment syndrome: new and improved diagnostic criteria.

    PubMed

    Roscoe, David; Roberts, Andrew J; Hulse, David

    2015-02-01

    Patients with chronic exertional compartment syndrome (CECS) have pain during exercise that subsides with rest. Diagnosis is usually confirmed by intramuscular compartment pressure (IMCP) measurement. Controversy exists regarding the accuracy of existing diagnostic criteria. (1) To compare dynamic IMCP measurement and anthropometric factors between patients with CECS and asymptomatic controls and (2) to establish the diagnostic utility of dynamic IMCP measurement. Cohort study (diagnosis); Level of evidence, 2. A total of 40 men aged 21 to 40 years were included in the study: 20 with symptoms of CECS of the anterior compartment and 20 asymptomatic controls. Diagnoses other than CECS were excluded with rigorous inclusion criteria and magnetic resonance imaging. The IMCP was measured continuously before, during, and after participants exercised on a treadmill, wearing identical footwear and carrying a 15-kg load. Pain experienced by study subjects increased incrementally as the study progressed (P < .001). Pain levels experienced by the case group during each phase of the exercise were significantly different (P = .021). Subjects had higher IMCP immediately upon standing at rest compared with controls (23.8 mm Hg [controls] vs 35.5 mm Hg [subjects]; P = .006). This relationship persisted throughout the exercise protocol, with the greatest difference corresponding to the period of maximal tolerable pain (68.7 mm Hg [controls] vs 114 mm Hg [subjects]; P < .001). Sensitivity and specificity were consistently higher than the existing criteria with improved diagnostic value (sensitivity = 63%, specificity = 95%; likelihood ratio = 12.5 [95% CI, 3.2-49]). Anterior compartment IMCP is elevated immediately upon standing at rest in subjects with CECS. In patients with symptoms consistent with CECS, diagnostic utility of IMCP measurement is improved when measured continuously during exercise. A cutoff of 105 mm Hg in phase 2 provides better diagnostic accuracy than do the

  3. Sequence Depth, Not PCR Replication, Improves Ecological Inference from Next Generation DNA Sequencing

    PubMed Central

    Smith, Dylan P.; Peay, Kabir G.

    2014-01-01

    Recent advances in molecular approaches and DNA sequencing have greatly progressed the field of ecology and allowed for the study of complex communities in unprecedented detail. Next generation sequencing (NGS) can reveal powerful insights into the diversity, composition, and dynamics of cryptic organisms, but results may be sensitive to a number of technical factors, including molecular practices used to generate amplicons, sequencing technology, and data processing. Despite the popularity of some techniques over others, explicit tests of the relative benefits they convey in molecular ecology studies remain scarce. Here we tested the effects of PCR replication, sequencing depth, and sequencing platform on ecological inference drawn from environmental samples of soil fungi. We sequenced replicates of three soil samples taken from pine biomes in North America represented by pools of either one, two, four, eight, or sixteen PCR replicates with both 454 pyrosequencing and Illumina MiSeq. Increasing the number of pooled PCR replicates had no detectable effect on measures of α- and β-diversity. Pseudo-β-diversity – which we define as dissimilarity between re-sequenced replicates of the same sample – decreased markedly with increasing sampling depth. The total richness recovered with Illumina was significantly higher than with 454, but measures of α- and β-diversity between a larger set of fungal samples sequenced on both platforms were highly correlated. Our results suggest that molecular ecology studies will benefit more from investing in robust sequencing technologies than from replicating PCRs. This study also demonstrates the potential for continuous integration of older datasets with newer technology. PMID:24587293

  4. Improved strategies and optimization of calibration models for real-time PCR absolute quantification.

    PubMed

    Sivaganesan, Mano; Haugland, Richard A; Chern, Eunice C; Shanks, Orin C

    2010-09-01

    Real-time PCR absolute quantification applications are becoming more common in the recreational and drinking water quality industries. Many methods 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, the generation of a standard curve for each qPCR experiment set-up can be expensive and time consuming, especially for studies with large numbers of unknown samples. As a result, many researchers have adopted a master calibration strategy where a single curve is derived from DNA standard measurements generated from multiple instrument runs. However, a master curve can inflate uncertainty associated with intercept and slope parameters and decrease the accuracy of unknown sample DNA target concentration estimates. Here we report two alternative strategies termed 'pooled' and 'mixed' for the generation of calibration equations from absolute standard curves which can help reduce the cost and time of laboratory testing, as well as the uncertainty in calibration model parameter estimates. In this study, four different strategies for generating calibration models were compared based on a series of repeated experiments for two different qPCR assays using a Monte Carlo Markov Chain method. The hierarchical Bayesian approach allowed for the comparison of uncertainty in intercept and slope model parameters and the optimization of experiment design. Data suggests that the 'pooled' model can reduce uncertainty in both slope and intercept parameter estimates compared to the traditional single curve approach. In addition, the 'mixed' model achieved uncertainty estimates similar to the 'single' model while increasing the number of available reaction wells per instrument run. Published by Elsevier Ltd.

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

    PubMed

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

    2008-11-01

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

  6. 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.

  7. Microfluidic Purification and Concentration of Malignant Pleural Effusions for Improved Molecular and Cytomorphological Diagnostics

    PubMed Central

    Go, Derek E.; Talati, Ish; Ying, Yong; Rao, Jianyu; Kulkarni, Rajan P.; Di Carlo, Dino

    2013-01-01

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

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

    SciTech Connect

    Shepard, W.S.

    1994-12-01

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

  9. Added diagnostic value and impact on antimicrobial therapy of 16S rRNA PCR and amplicon sequencing on resected heart valves in infective endocarditis: a prospective cohort study.

    PubMed

    Peeters, B; Herijgers, P; Beuselinck, K; Verhaegen, J; Peetermans, W E; Herregods, M-C; Desmet, S; Lagrou, K

    2017-06-19

    For adequate management and therapy of infective endocarditis (IE), identification of the causative pathogen is crucial but molecular testing results are not currently included in diagnostic criteria. The added diagnostic value and impact on antimicrobial therapy of 16S rRNA PCR and amplicon sequencing (16S rRNA PCR) performed on excised heart valves from patients with IE was evaluated alongside the effect of pre-operative antibiotics on the performance of blood culture (BC), valve culture (VC) and 16S rRNA PCR. All patients undergoing valve surgery for definite or possible IE, according to modified Duke Criteria, were prospectively included from July 2013 up to and including June 2016. In all, 127 patients were included. Sensitivity for detecting the causative micro-organism in 120 post-operative definite IE patients was 26% for VC and 87% for BC and 16S rRNA PCR. 16S rRNA PCR, VC and BC were equally sensitive for different valve types and causative pathogens. In 27 (21%) definite IE patients, 16S rRNA PCR clarified discrepant culture results or was the only method identifying the causative pathogen. In 12 (10%) post-operative definite IE cases, molecular testing results influenced antimicrobial therapy. The very good performance characteristics, added diagnostic value and impact on antimicrobial therapy of molecular testing of heart valves should support the incorporation of molecular testing in diagnostic criteria and guidelines for IE. Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  10. Molecular diagnostic of cytomegalovirus, Epstein Barr virus and Herpes virus 6 infections among blood donors by multiplex real-time PCR in Ouagadougou, Burkina Faso.

    PubMed

    Traore, Lassina; Tao, Issoufou; Bisseye, Cyrille; Diarra, Birama; Compaore, Tegwindé Rebeca; Nebie, Yacouba; Assih, Maleki; Ouedraogo, Alice; Zohoncon, Theodora; Djigma, Florencia; Ouermi, Djénéba; Barro, Nicolas; Sanou, Mahamoudou; Ouedraogo, Rasmata Traore; Simpore, Jacques

    2016-01-01

    In most developing countries, Cytomegalovirus (CMV), Epstein Barr virus (EBV) and Herpes virus 6 (HHV-6) are not diagnosed in blood donors. The aim of this study is to determine the prevalence of these viruses in blood donors from the city of Ouagadougou, Burkina Faso. The study included 198 blood donors of the Regional Blood Transfusion Centre of Ouagadougou. Multiplex real time PCR was used to diagnose the three viruses. Statistical analysis was performed with the software EpiInfo version 6 and SPSS version 17. P values ≤ 0.05 were considered significant. Of 198 samples tested, 18 (9.1%) were positive to at least one of the three viruses. In fact, 10 (5.1%) were positive for EBV, 10 (5.1%) positive for CMV and 12 (6.1%) positive for HHV-6. Viral infections were higher in women than in men, EBV (8,6% versus 4.3%), CMV (8.6% versus 3.7%) and HHV-6 (11.4% versus 4.9%). EBV / CMV / HHV-6 co-infection was found in 3.5% (7/198) of blood donors. The prevalence recorded in this study is low compared to those found in previous studies from the sub-region among blood donors. The molecular diagnostic test used in our study could explain the differences with previous studies.

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

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

  12. Best diagnostic approach for the genetic evaluation of fetuses after intrauterine death in first, second or third trimester: QF-PCR, karyotyping and/or genome wide SNP array analysis

    PubMed Central

    2014-01-01

    Background The aim of this study was to evaluate the best diagnostic approach for the genetic analysis of samples from first, second and third trimester intrauterine fetal deaths (IUFDs). We examined a total of 417 IUFD samples from fetuses with and without congenital anomalies. On 414 samples, karyotyping (N = 46) and/or rapid aneuploidy testing by QF-PCR (N = 371) was performed). One hundred sixty eight samples with a normal test result were subsequently tested by genome wide Single Nucleotide Polymorphism (SNP) array analysis. Three samples were only analyzed by array. Results In 50 (12.0%) samples an aneuploidy was detected by QF-PCR and/or karyotyping, representing 47.1% of first, 13.2% of second and 3.4% of third trimester pregnancies. Karyotyping and QF-PCR failed in 4 (8.7%) and 7 (1.9%) samples, respectively, concerning mostly contaminated amniotic fluid samples from third trimester pregnancies. Clinically relevant aberrations were identified in 4.2% (all fetuses with malformations) of the 168 samples tested by SNP array. Inherited copy number variants (CNVs) were detected in 5.4% and 8.9% showed CNVs of unknown clinical relevance as parental inheritance could not be studied yet. In a sample from a fetus suspect for Meckel-Grüber syndrome, the genotype information from the SNP array revealed various stretches of homozygosity, including one stretch encompassing the CEP290 gene. Subsequent CEP290 mutation analysis revealed a homozygous, pathogenic mutation in this gene. Conclusions Based on our experience we recommend QF-PCR as the first-line test in IUFD samples of first and second trimester pregnancies to exclude aneuploidy before performing array analysis. The chance to detect aneuploidy in third trimester pregnancies is relatively low and therefore array analysis can be performed as a first-tier test. A tissue sample, instead of amniotic fluid, is preferred because of a higher success rate in testing. We emphasize the need for analysis of parental

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

    PubMed

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

    2015-10-01

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

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

    PubMed Central

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

    2016-01-01

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

  15. Improving diagnostic accuracy using agent-based distributed data mining system.

    PubMed

    Sridhar, S

    2013-09-01

    The use of data mining techniques to improve the diagnostic system accuracy is investigated in this paper. The data mining algorithms aim to discover patterns and extract useful knowledge from facts recorded in databases. Generally, the expert systems are constructed for automating diagnostic procedures. The learning component uses the data mining algorithms to extract the expert system rules from the database automatically. Learning algorithms can assist the clinicians in extracting knowledge automatically. As the number and variety of data sources is dramatically increasing, another way to acquire knowledge from databases is to apply various data mining algorithms that extract knowledge from data. As data sets are inherently distributed, the distributed system uses agents to transport the trained classifiers and uses meta learning to combine the knowledge. Commonsense reasoning is also used in association with distributed data mining to obtain better results. Combining human expert knowledge and data mining knowledge improves the performance of the diagnostic system. This work suggests a framework of combining the human knowledge and knowledge gained by better data mining algorithms on a renal and gallstone data set.

  16. Wavenumber selection based analysis in Raman spectroscopy improves skin cancer diagnostic specificity.

    PubMed

    Zhao, Jianhua; Zeng, Haishan; Kalia, Sunil; Lui, Harvey

    2016-02-07

    Real-time Raman spectroscopy can be used to assist in assessing skin lesions suspicious for cancer. Most of the diagnostic algorithms are based on full band of the Raman spectra, either in the fingerprint region or the high wavenumber region. In this paper we explored wavenumber selection based analysis in Raman spectroscopy for skin cancer diagnosis. Wavenumber selection was implemented using windows of wavenumber and leave-one-out cross-validated stepwise regression or least and shrinkage selection operator (LASSO). The diagnostic algorithms were then generated from the selected windows of wavenumber using multivariate statistical analyses, including principal component and general discriminate analysis (PC-GDA) and partial least squares (PLS). In total a combined cohort of 645 confirmed lesions from 573 patients encompassing skin cancers, precancers and benign skin lesions were included, which were divided into training cohort (n = 518) and testing cohort (n = 127) according to the measurement time. It was found that the area under the receiver operating characteristic curve (ROC) was improved from 0.861-0.891 to 0.891-0.911 and the diagnostic specificity for fixed sensitivity 0.99-0.90 was improved from 0.17-0.65 to 0.20-0.75 with wavenumber selection based analysis.

  17. Improved Spectral Fitting Models for the B-Stark Diagnostic at DIII-D

    NASA Astrophysics Data System (ADS)

    Pablant, N. A.; Grierson, B. A.; Burrell, K. H.; Groebner, R. J.; Kaplan, D. H.; Holcomb, C. T.

    2010-11-01

    Recent results are presented from the B-Stark diagnostic installed on the DIII-D tokamak. This diagnostic provides measurements of the magnitude and direction of the internal magnetic field. The B-Stark system is a version of a motional Stark effect (MSE) diagnostic based on the relative line intensities and spacing of the Stark split Dα emission from injected neutral beams. Improvements to the spectral fitting model are presented, including the addition of an analytical model for Dα emission from the fast-ion distribution. We discuss the accuracy of using in-situ beam-into-gas calibrations to find the beam emission line profiles, the viewing direction and the transmission properties of the collection optics. We also present results of efforts to improve the determination of the beam emission line profiles. Finally, the magnetic field measured with the B-Stark system is compared to values found from plasma equilibrium reconstructions (EFIT) and the MSE polarimetry system on DIII-D.

  18. Directed evolution of lectins by an improved error-prone PCR and ribosome display method.

    PubMed

    Hu, Dan; Tateno, Hiroaki; Hirabayashi, Jun

    2014-01-01

    Lectins are useful reagents for the structural characterization of glycans. However, currently available lectins have an apparent drawback in their "repertoire," lacking some critical probes, such as those for sulfated glycans. Thus, engineering lectins with novel specificity would be of great practical value. Here, we describe a directed evolution strategy to tailor novel lectins for novel specificity or biological functions. Our strategy uses a reinforced ribosome display-based selection combined with error-prone PCR to isolate mutants with target specificity and an evanescent-field fluorescence-assisted glycoconjugate microarray to rapidly evaluate the specificity of selected mutants. A successful case of screening a lectin, which has acquired an ability to recognize 6-sulfo-galactose-terminated glycans, is described.

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

    PubMed Central

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

    2007-01-01

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

  20. 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.

  1. Point-of-care diagnostics to improve maternal and neonatal health in low-resource settings.

    PubMed

    Majors, Catherine E; Smith, Chelsey A; Natoli, Mary E; Kundrod, Kathryn A; Richards-Kortum, Rebecca

    2017-08-23

    Each day, approximately 830 women and 7400 newborns die from complications during pregnancy and childbirth. Improving maternal and neonatal health will require bringing rapid diagnosis and treatment to the point of care in low-resource settings. However, to date there are few diagnostic tools available that can be used at the point of care to detect the leading causes of maternal and neonatal mortality in low-resource settings. Here we review both commercially available diagnostics and technologies that are currently in development to detect the leading causes of maternal and neonatal mortality, highlighting key gaps in development where innovative design could increase access to technology and enable rapid diagnosis at the bedside.

  2. Linear combination methods to improve diagnostic/prognostic accuracy on future observations

    PubMed Central

    Kang, Le; Liu, Aiyi; Tian, Lili

    2014-01-01

    Multiple diagnostic tests or biomarkers can be combined to improve diagnostic accuracy. The problem of finding the optimal linear combinations of biomarkers to maximise the area under the receiver operating characteristic curve has been extensively addressed in the literature. The purpose of this article is threefold: (1) to provide an extensive review of the existing methods for biomarker combination; (2) to propose a new combination method, namely, the nonparametric stepwise approach; (3) to use leave-one-pair-out cross-validation method, instead of re-substitution method, which is overoptimistic and hence might lead to wrong conclusion, to empirically evaluate and compare the performance of different linear combination methods in yielding the largest area under receiver operating characteristic curve. A data set of Duchenne muscular dystrophy was analysed to illustrate the applications of the discussed combination methods. PMID:23592714

  3. Improved microbiological diagnostic due to utilization of a high-throughput homogenizer for routine tissue processing.

    PubMed

    Redanz, Sylvio; Podbielski, Andreas; Warnke, Philipp

    2015-07-01

    Tissue specimens are valuable materials for microbiological diagnostics and require swift and accurate processing. Established processing methods are complex, labor intensive, hardly if at all standardizable, and prone to incorporate contaminants. To improve analyses from tissue samples in routine microbiological diagnostics, by facilitating, fastening, and standardizing processing as well as increasing the microbial yield, performance of Precellys 24 high-throughput tissue homogenizer was evaluated. Therefore, tissue samples were artificially inoculated with Staphylococcus aureus, Escherichia coli, and Candida albicans in 3 different ways on the surface and within the material. Microbial yield from homogenized samples was compared to direct plating method. Further, as proof of principle, routine tissue samples from knee and hip endoprosthesis infections were analyzed. The process of tissue homogenization with Precellys 24 homogenizer is easy and fast to perform and allows for a high degree of standardization. Microbial yield after homogenization was significantly higher as compared to conventional plating technique.

  4. Improved diagnostic accuracy of malignant neck lumps by a simple BMVC staining assay.

    PubMed

    Liao, Li-Jen; Kang, Chi-Chih; Jan, I-Shiow; Chen, Huei-Chin; Wang, Chiung-Lin; Lou, Pei-Jen; Chang, Ta-Chau

    2009-04-01

    A handheld device based on fluorescence of 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC) staining was established for the rapid, point-of-care screening of cancer cells (see Chang and co-workers, Analyst, 2007, 132, 745). Offering instant screening of cancer at low cost, here we apply this simple assay in clinical tests on fine needle aspirates of neck masses from 114 outpatients (115 specimens). The diagnostic accuracy of this simple method alone is ca. 80% (80/99). The combination of the BMVC test and the fine needle aspiration (FNA) cytology reduced the non-diagnosis from 17 cases in FNA cytology to 6 cases in the combined method. Moreover, an algorithm is proposed to improve the diagnostic accuracy of malignant neck lumps up to nearly 100%.

  5. 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

  6. Improving the diagnostic performance of lung scintigraphy in suspected pulmonary embolic disease.

    PubMed

    Gleeson, F V; Turner, S; Scarsbrook, A F

    2006-12-01

    to determine the effectiveness of a new imaging algorithm in the investigation of suspected pulmonary embolism (PE). A new imaging algorithm for suspected PE was introduced following the installation of a multisection computed tomography (CT) machine at our institution. Before its installation, patients with suspected PE were evaluated with ventilation/perfusion (V/Q) scintigraphy. Subsequently, patients were triaged according to chest radiography (CR) and respiratory history to either lung scintigraphy or CT pulmonary angiography (CTPA). Patients with a normal CR and no history of lung disease were evaluated using perfusion (Q) scintigraphy [ventilation (V) scintigraphy was no longer performed]. Patients with an abnormal CR, asthma or chronic lung disease were evaluated using CTPA. All V/Q images in a continuous 3-year period before the introduction of the new imaging algorithm and all Q images performed in a 3-year period after its introduction were retrospectively reviewed. Imaging reports were categorized into normal, non-diagnostic (low or intermediate probability) or high probability for PE. Patients in the later group who subsequently underwent CTPA, were also reviewed. After the policy change the percentage of normal scintigrams significantly increased (39 to 60%; p<0.001). There was a non-significant increase in the percentage of high probability scintigrams (15 to 18%; p=0.716). Overall the diagnostic yield of lung scintigraphy improved significantly (54 to 78%; p<0.001). the diagnostic performance of lung scintigraphy can be improved by careful triage of patients to either Q scintigraphy or CTPA based on clinical history and CR findings. Q scintigraphy remains a valuable diagnostic test in the investigation of suspected PE in carefully selected patients.

  7. A Traditionally Administered Short Course Failed to Improve Medical Students’ Diagnostic Performance

    PubMed Central

    Noguchi, Yoshinori; Matsui, Kunihiko; Imura, Hiroshi; Kiyota, Masatomo; Fukui, Tsuguya

    2004-01-01

    BACKGROUND Quite often medical students or novice residents have difficulty in ruling out diseases even though they are quite unlikely and, due to this difficulty, such students and novice residents unnecessarily repeat laboratory or imaging tests. OBJECTIVE To explore whether or not a carefully designed short training course teaching Bayesian probabilistic thinking improves the diagnostic ability of medical students. PARTICIPANTS AND METHODS Ninety students at 2 medical schools were presented with clinical scenarios of coronary artery disease corresponding to high, low, and intermediate pretest probabilities. The students’ estimates of test characteristics of exercise stress test, and pretest and posttest probability for each scenario were evaluated before and after the short course. RESULTS The pretest probability estimates by the students, as well as their proficiency in applying Bayes's theorem, were improved in the high pretest probability scenario after the short course. However, estimates of pretest probability in the low pretest probability scenario, and their proficiency in applying Bayes's theorem in the intermediate and low pretest probability scenarios, showed essentially no improvement. CONCLUSION A carefully designed, but traditionally administered, short course could not improve the students’ abilities in estimating pretest probability in a low pretest probability setting, and subsequently students remained incompetent in ruling out disease. We need to develop educational methods that cultivate a well-balanced clinical sense to enable students to choose a suitable diagnostic strategy as needed in a clinical setting without being one-sided to the “rule-in conscious paradigm.” PMID:15109340

  8. An Improved PCR-Restriction Fragment Length Polymorphism (RFLP) Method for the Identification of cry1-Type Genes

    PubMed Central

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

    2013-01-01

    The cry1-type genes of Bacillus thuringiensis represent the largest cry gene family, which contains 50 distinct holotypes. It is becoming more and more difficult to identify cry1-type genes using current methods because of the increasing number of cry1-type genes. In the present study, an improved PCR-restriction fragment length polymorphism (PCR-RFLP) method which can distinguish 41 holotypes of cry1-type genes was developed. This improved method was used to identify cry1-type genes in 20 B. thuringiensis strains that are toxic to lepidoptera. The results showed that the improved method can efficiently identify single and clustered cry1-type genes and can be used to evaluate cry1-type genes in novel strain collections of B. thuringiensis. Among the detected cry1-type genes, we identified four novel genes, cry1Ai, cry1Bb, cry1Ja, and cry1La. The bioassay results from the expressed products of the four novel cry genes showed that Cry1Ai2, Cry1Bb2, and Cry1Ja2 were highly toxic against Plutella xylostella, whereas Cry1La2 exhibited no activity. Moreover, Cry1Ai2 had good lethal activity against Ostrinia furnacalis, Hyphantria cunea, Chilo suppressalis, and Bombyx mori larvae and considerable weight loss activity against Helicoverpa armigera. PMID:23995930

  9. Early diagnostic suggestions improve accuracy of GPs: a randomised controlled trial using computer-simulated patients

    PubMed Central

    Kostopoulou, Olga; Rosen, Andrea; Round, Thomas; Wright, Ellen; Douiri, Abdel; Delaney, Brendan

    2015-01-01

    Background Designers of computerised diagnostic support systems (CDSSs) expect physicians to notice when they need advice and enter into the CDSS all information that they have gathered about the patient. The poor use of CDSSs and the tendency not to follow advice once a leading diagnosis emerges would question this expectation. Aim To determine whether providing GPs with diagnoses to consider before they start testing hypotheses improves accuracy. Design and setting Mixed factorial design, where 297 GPs diagnosed nine patient cases, differing in difficulty, in one of three experimental conditions: control, early support, or late support. Method Data were collected over the internet. After reading some initial information about the patient and the reason for encounter, GPs requested further information for diagnosis and management. Those receiving early support were shown a list of possible diagnoses before gathering further information. In late support, GPs first gave a diagnosis and were then shown which other diagnoses they could still not discount. Results Early support significantly improved diagnostic accuracy over control (odds ratio [OR] 1.31; 95% confidence interval [95%CI] = 1.03 to 1.66, P = 0.027), while late support did not (OR 1.10; 95% CI = 0.88 to 1.37). An absolute improvement of 6% with early support was obtained. There was no significant interaction with case difficulty and no effect of GP experience on accuracy. No differences in information search were detected between experimental conditions. Conclusion Reminding GPs of diagnoses to consider before they start testing hypotheses can improve diagnostic accuracy irrespective of case difficulty, without lengthening information search. PMID:25548316

  10. 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.

  11. 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.

  12. Molecular diagnosis of bacterial vaginosis: Does adjustment for total bacterial load or human cellular content improve diagnostic performance?

    PubMed

    Plummer, E L; Garland, S M; Bradshaw, C S; Law, M G; Vodstrcil, L A; Hocking, J S; Fairley, C K; Tabrizi, S N

    2017-02-01

    We investigated the utility of quantitative PCR assays for diagnosis of bacterial vaginosis and found that while the best model utilized bacterial copy number adjusted for total bacterial load (sensitivity=98%, specificity=93%, AUC=0.95[95%CI=0.93,0.97]), adjusting for total bacterial or human cell load did not consistently increase the diagnostic performance of the assays. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2015-11-01

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

  14. A two-photon laser induced fluorescence diagnostic with improved sensitivity, localization, and measurement rate

    NASA Astrophysics Data System (ADS)

    Elliott, Drew; Scime, Earl; Short, Zachary

    2016-10-01

    A two-photon absorption laser induced fluorescence diagnostic has been developed for measuring neutrals in fusion plasmas. Implementation of this diagnostic on the HIT-SI3 spheromak has demonstrated the sensitivity of the diagnostic and shown that measurements taken over several plasma pulses are possible. These measurements yielded an unexpected loss of signal when complex collection optics were utilized. Simulations show that this loss of signal can be explained by chromatic aberrations caused by the disparate Kr and D emission. This loss of signal has been addressed with the development of a new calibration scheme involving xenon gas. The Xe calibration scheme emission occurs at 656.00 nm while the deuterium emission is 656.09 nm. This nearly identical emission allows for advanced optical techniques such as confocal collection/injection and spatial filtering to be employed without loss of signal. Spatial filtering has been demonstrated to decrease noise while improving measurement localization, while confocal collection/injection allows for probing and measuring to occur through one viewport. The Xe scheme also allows for a Doppler-free hydrogen measurement. Doppler-free measurements eliminate the need to scan the laser spectrally thus greatly increasing the rate of measurement.

  15. Visual representation of statistical information improves diagnostic inferences in doctors and their patients.

    PubMed

    Garcia-Retamero, Rocio; Hoffrage, Ulrich

    2013-04-01

    Doctors and patients have difficulty inferring the predictive value of a medical test from information about the prevalence of a disease and the sensitivity and false-positive rate of the test. Previous research has established that communicating such information in a format the human mind is adapted to-namely natural frequencies-as compared to probabilities, boosts accuracy of diagnostic inferences. In a study, we investigated to what extent these inferences can be improved-beyond the effect of natural frequencies-by providing visual aids. Participants were 81 doctors and 81 patients who made diagnostic inferences about three medical tests on the basis of information about prevalence of a disease, and the sensitivity and false-positive rate of the tests. Half of the participants received the information in natural frequencies, while the other half received the information in probabilities. Half of the participants only received numerical information, while the other half additionally received a visual aid representing the numerical information. In addition, participants completed a numeracy scale. Our study showed three important findings: (1) doctors and patients made more accurate inferences when information was communicated in natural frequencies as compared to probabilities; (2) visual aids boosted accuracy even when the information was provided in natural frequencies; and (3) doctors were more accurate in their diagnostic inferences than patients, though differences in accuracy disappeared when differences in numerical skills were controlled for. Our findings have important implications for medical practice as they suggest suitable ways to communicate quantitative medical data.

  16. Diagnostic delays in children with early-onset epilepsy: impact, reasons, and opportunities to improve care

    PubMed Central

    Berg, Anne T.; Loddenkemper, Tobias; Baca, Christine B.

    2014-01-01

    in childhood. Several factors influence diagnostic delays and may represent opportunities for intervention and improved care. PMID:24313635

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

    NASA Astrophysics Data System (ADS)

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

    2016-11-01

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

  18. Optimization of Classification Strategies of Acetowhite Temporal Patterns towards Improving Diagnostic Performance of Colposcopy

    PubMed Central

    Acosta-Mesa, Héctor Gabriel; Cruz-Ramírez, Nicandro; Hernández-Jiménez, Rodolfo

    2017-01-01

    Efforts have been being made to improve the diagnostic performance of colposcopy, trying to help better diagnose cervical cancer, particularly in developing countries. However, improvements in a number of areas are still necessary, such as the time it takes to process the full digital image of the cervix, the performance of the computing systems used to identify different kinds of tissues, and biopsy sampling. In this paper, we explore three different, well-known automatic classification methods (k-Nearest Neighbors, Naïve Bayes, and C4.5), in addition to different data models that take full advantage of this information and improve the diagnostic performance of colposcopy based on acetowhite temporal patterns. Based on the ROC and PRC area scores, the k-Nearest Neighbors and discrete PLA representation performed better than other methods. The values of sensitivity, specificity, and accuracy reached using this method were 60% (95% CI 50–70), 79% (95% CI 71–86), and 70% (95% CI 60–80), respectively. The acetowhitening phenomenon is not exclusive to high-grade lesions, and we have found acetowhite temporal patterns of epithelial changes that are not precancerous lesions but that are similar to positive ones. These findings need to be considered when developing more robust computing systems in the future. PMID:28744318

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

    PubMed

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

    2016-11-01

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

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

    SciTech Connect

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

    2004-10-31

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

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

    PubMed

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

    2014-03-01

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

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

    NASA Astrophysics Data System (ADS)

    Lopresto, Michael C.

    The results of administering the Astronomy Diagnostic Test (ADT) to introductory astronomy students at Henry Ford Community College over three years have shown gains comparable with national averages. Results have also accurately corresponded to course goals, showing greater gains in topics covered in more detail, and lower gains in topics covered in less detail. Also evident in the results were topics for which improvement of instruction is needed. These factors and the ease with which the ADT can be administered constitute evidence of the usefulness of the ADT as an assessment instrument for introductory astronomy.

  3. Extravascular lung water index improves the diagnostic accuracy of lung injury in patients with shock.

    PubMed

    Chew, Michelle S; Ihrman, Lilian; During, Joachim; Bergenzaun, Lill; Ersson, Anders; Undén, Johan; Ryden, Jörgen; Åkerman, Eva; Larsson, Marina

    2012-01-03

    The diagnosis of acute lung injury (ALI) may be more robust if more accurate physiological markers can be identified. Extravascular lung water (EVLW) is one possible marker, and it has been shown to correlate with respiratory function and mortality in patients with sepsis. Whether EVLW confers diagnostic value in a general population with shock, as well as which index performs best, is unclear. We investigated the diagnostic accuracy of various EVLW indices in patients with shock. We studied a prospective, observational cohort of 51 patients with shock admitted to a tertiary ICU. EVLW was measured within 6 hours of ICU admission and indexed to actual body weight (EVLW/ABW), predicted body weight (EVLW/PBW) and pulmonary blood volume (EVLW/PBV). The relationship of these indices to the diagnosis and severity of lung injury and ICU mortality were studied. Positive and negative likelihood ratios, pre- and posttest odds for diagnosis of lung injury and mortality were calculated. All EVLW indices were higher among patients with lung injury and significantly correlated with respiratory parameters. Furthermore, all EVLW indices were significantly higher in nonsurvivors. The use of EVLW improves the posttest OR for the diagnosis of ALI, acute respiratory distress syndrome (ARDS) and severe lung injury (sLI) by up to eightfold. Combining increased EVLW and a diagnosis of ALI, ARDS or sLI increases the posttest odds of ICU mortality. EVLW/ABW and EVLW/PBV demonstrated the best diagnostic performance in this population. EVLW was associated with degree of lung injury and mortality, regardless of the index used, confirming that it may be used as a bedside indicator of disease severity. The use of EVLW as a bedside test conferred added diagnostic value for the identification of patients with lung injury.

  4. Extravascular lung water index improves the diagnostic accuracy of lung injury in patients with shock

    PubMed Central

    2012-01-01

    Introduction The diagnosis of acute lung injury (ALI) may be more robust if more accurate physiological markers can be identified. Extravascular lung water (EVLW) is one possible marker, and it has been shown to correlate with respiratory function and mortality in patients with sepsis. Whether EVLW confers diagnostic value in a general population with shock, as well as which index performs best, is unclear. We investigated the diagnostic accuracy of various EVLW indices in patients with shock. Methods We studied a prospective, observational cohort of 51 patients with shock admitted to a tertiary ICU. EVLW was measured within 6 hours of ICU admission and indexed to actual body weight (EVLW/ABW), predicted body weight (EVLW/PBW) and pulmonary blood volume (EVLW/PBV). The relationship of these indices to the diagnosis and severity of lung injury and ICU mortality were studied. Positive and negative likelihood ratios, pre- and posttest odds for diagnosis of lung injury and mortality were calculated. Results All EVLW indices were higher among patients with lung injury and significantly correlated with respiratory parameters. Furthermore, all EVLW indices were significantly higher in nonsurvivors. The use of EVLW improves the posttest OR for the diagnosis of ALI, acute respiratory distress syndrome (ARDS) and severe lung injury (sLI) by up to eightfold. Combining increased EVLW and a diagnosis of ALI, ARDS or sLI increases the posttest odds of ICU mortality. EVLW/ABW and EVLW/PBV demonstrated the best diagnostic performance in this population. Conclusions EVLW was associated with degree of lung injury and mortality, regardless of the index used, confirming that it may be used as a bedside indicator of disease severity. The use of EVLW as a bedside test conferred added diagnostic value for the identification of patients with lung injury. PMID:22214612

  5. 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

  6. 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...

  7. 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...

  8. Design and application of an internal amplification control to improve Dehalococcoides mccartyi 16S rRNA gene enumeration by qPCR.

    PubMed

    Hatt, Janet K; Ritalahti, Kirsti M; Ogles, Dora M; Lebrón, Carmen A; Löffler, Frank E

    2013-10-01

    Dehalococcoides mccartyi (Dhc) strains are keystone bacteria for reductive dechlorination of chlorinated ethenes to nontoxic ethene in contaminated aquifers. Enumeration of Dhc biomarker genes using quantitative real-time PCR (qPCR) in groundwater is a key component of site assessment and bioremediation monitoring. Unfortunately, standardized qPCR procedures that recognize impaired measurements due to PCR inhibition, low template DNA concentrations, or analytical error are not available, thus limiting confidence in qPCR data. To improve contemporary approaches for enumerating Dhc in environmental samples, multiplex qPCR assays were designed to quantify the Dhc 16S rRNA gene and one of two different internal amplification controls (IACs): a modified Dhc 16S rRNA gene fragment (Dhc*) and the firefly luciferase gene luc. The Dhc* IAC exhibited competitive inhibition in qPCR with the Dhc 16S rRNA gene template when the ratio of either target was 100-fold greater than the other target. A multiplex qPCR assay with the luc IAC avoided competitive inhibition and accurately quantified Dhc abundances ranging from ∼10 to 10(7) 16S rRNA gene copies per reaction. The addition of ∼10(6) E. coli luc IAC to simulated groundwater amended with the Dhc-containing consortium KB-1 yielded reproducible luc counts after DNA extraction and multiplex qPCR enumeration. The application of the luc IAC assay improved Dhc biomarker gene quantification from simulated groundwater samples and is a valuable approach for "ground truthing" qPCR data obtained in different laboratories, thus reducing ambiguity associated with qPCR enumeration and reproducibility.

  9. Odor Identification Screening Improves Diagnostic Classification in Incipient Alzheimer’s Disease

    PubMed Central

    Quarmley, Megan; Moberg, Paul J.; Mechanic-Hamilton, Dawn; Kabadi, Sushila; Arnold, Steven E.; Wolk, David A.; Roalf, David R.

    2017-01-01

    Background Measurements of olfaction may serve as useful biomarkers of incipient dementia. Here we examine the improvement in diagnostic accuracy of Alzheimer’s disease (AD) and mild cognitive impairment (MCI) when assessing both cognitive functioning and odor identification. Objective To determine the utility of odor identification as a supplementary screening test in incipient AD. Methods Sniffin’ Sticks Odor Identification Test (SS-OIT) and the Montreal Cognitive Assessment (MoCA) were administered in 262 AD, 174 MCI [150 amnestic (aMCI), and 24 non-amnestic (naMCI)], and 292 healthy older adults (HOA). Results Odor identification scores were higher in HOA relative to MCI or AD groups, and MCI outperformed AD. Odor identification scores were higher in aMCI single domain than aMCI multiple domain. Complementing MoCA scores with the SS-OIT significantly improved diagnostic accuracy of individuals with AD and MCI, including within MCI subgroups. Discussion Odor identification is a useful supplementary screening tool that provides additional information relevant for clinical categorization of AD and MCI, including those who are at highest risk to convert to AD. PMID:27886011

  10. Improving the diagnostic accuracy of dysplastic and melanoma lesions using the decision template combination method.

    PubMed

    Faal, Maryam; Miran Baygi, Mohammad Hossein; Kabir, Ehsanollah

    2013-02-01

    Melanoma is the most dangerous type of skin cancer, and early detection of suspicious lesions can decrease the mortality rate of this cancer. In this article, we present a multi-classifier system for improving the diagnostic accuracy of melanoma and dysplastic lesions based on the decision template combination rule. First, the lesion is differentiated from the surrounding healthy skin in an image. Next, shape, colour and texture features are extracted from the lesion image. Different subsets of these features are fed to three different classifiers: k-nearest neighbour (k-NN), support vector machine (SVM) and linear discriminant analysis (LDA). The decision template method is used to combine the outputs of these classifiers. The proposed method has been evaluated on a set of 436 dermatoscopic images of benign, dysplastic and melanoma lesions. The final classifier ensemble delivers a total classification accuracy of 80.46%, with 67.73% of dysplastic lesions correctly classified and 83.53% of melanoma lesions correctly classified. The results show that the proposed method significantly increases the diagnostic accuracy of dysplastic and melanoma lesions compared with a single classifier. The total classification rate is also improved. © 2012 John Wiley & Sons A/S.

  11. Improvements To A High-Frequency Fiber-Optic System For Plasma Diagnostics

    NASA Astrophysics Data System (ADS)

    Ogle, J. W.; Lyons, P. B.; Looney, L.; Hocker, L.; Nelson, M. A.; Zagarino, P. A.; Davies, T. J.; Simmons, R. D.; Selk, R.; Hopkins, B.

    1982-01-01

    A system for high-frequency recording of plasma diagnostics has previously been reported. Substantial improvements have been made in the system response, dynamic range, and calibration of the system. Plastic-clad silica fiber is used as a radiation-to-light converter using the Cerenkov process. A spectral equalizer device is used to compensate for the material dispersion in the fiber, increasing the frequency response (,1 GHz-km) and the dynamic range (a factor of >20 over a FWHM 1 nm, 50% transmitting interference filter). The calibration system uses a pulsed injection laser diode (<100 ps FWHM) injected into the fiber at the radiation end of the fiber and detected by a microchannel plate photomultiplier tube on the recording end. The injection laser diode is triggered by a synchronous trigger delay unit, which also triggers a sampling or real time scope after as much as 10 μs delay with <50 ps jitter. The system improvements will be desribed in more detail and the utility of these components in other plasma diagnostic systems will be discussed.

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

    PubMed

    Peeling, Rosanna W

    2015-11-01

    Diagnostics play a critical role in clinical decision making, and in disease control and prevention. Rapid point-of-care (POC) tests for infectious diseases can improve access to diagnosis and patient management, but the quality of these tests vary, quality of testing is often not assured and there are few mechanisms to capture test results for surveillance when the testing is so decentralised. A new generation of POC molecular tests that are highly sensitive and specific, robust and easy to use are now available for deployment in low resource settings. Decentralisation of testing outside of the laboratory can put tremendous stress on the healthcare system and presents challenges for training and quality assurance. A feature of many of these POC molecular devices is that they are equipped with data transmission capacities. In a digital age, it is possible to link data from diagnostic laboratories and POC test readers and devices to provide data on testing coverage, disease trends and timely information for early warning of infectious disease outbreaks to inform design or optimisation of disease control and elimination programmes. Data connectivity also allows control programmes to monitor the quality of tests and testing, and optimise supply chain management; thus, increasing the efficiency of healthcare systems and improving patient outcomes. © The Author 2015. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. 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.

  14. Improvement of sampling plans for Salmonella detection in pooled table eggs by use of real-time PCR.

    PubMed

    Pasquali, Frédérique; De Cesare, Alessandra; Valero, Antonio; Olsen, John Emerdhal; Manfreda, Gerardo

    2014-08-01

    Eggs and egg products have been described as the most critical food vehicles of salmonellosis. The prevalence and level of contamination of Salmonella on table eggs are low, which severely affects the sensitivity of sampling plans applied voluntarily in some European countries, where one to five pools of 10 eggs are tested by the culture based reference method ISO 6579:2004. In the current study we have compared the testing-sensitivity of the reference culture method ISO 6579:2004 and an alternative real-time PCR method on Salmonella contaminated egg-pool of different sizes (4-9 uninfected eggs mixed with one contaminated egg) and contamination levels (10°-10(1), 10(1)-10(2), 10(2)-10(3)CFU/eggshell). Two hundred and seventy samples corresponding to 15 replicates per pool size and inoculum level were tested. At the lowest contamination level real-time PCR detected Salmonella in 40% of contaminated pools vs 12% using ISO 6579. The results were used to estimate the lowest number of sample units needed to be tested in order to have a 95% certainty not falsely to accept a contaminated lot by Monte Carlo simulation. According to this simulation, at least 16 pools of 10 eggs each are needed to be tested by ISO 6579 in order to obtain this confidence level, while the minimum number of pools to be tested was reduced to 8 pools of 9 eggs each, when real-time PCR was applied as analytical method. This result underlines the importance of including analytical methods with higher sensitivity in order to improve the efficiency of sampling and reduce the number of samples to be tested. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. 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.].

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

    PubMed

    Besseris, George J

    2014-01-01

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

  17. 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.

  18. Combination of immunohistochemistry, FISH and RT-PCR shows high incidence of Xp11 translocation RCC: comparison of three different diagnostic methods.

    PubMed

    Lee, Hyun Jung; Shin, Dong Hoon; Noh, Gyu You; Kim, Young Keum; Kim, Ahrong; Shin, Nari; Lee, Jung Hee; Choi, Kyung Un; Kim, Jee Yeon; Lee, Chang Hun; Sol, Mee Young; Rha, Seo Hee; Park, Sung Woo

    2017-05-09

    We evaluated the frequency of translocation renal cell carcinoma (RCC) by reverse transcription polymerase chain reaction (RT-PCR) and how well the TFE3 immunoreactivity is concordant with TFE3 gene translocation status proved by fluorescence in situ hybridization (FISH) assay and RT-PCR. TFE3 and Cathepsin K expression was analyzed by immunohistochemistry in 185 RCC cases, and 48 cases either of more than weak expression of TFE3 or of positivity for Cathepsin K were done for FISH analysis and RT-PCR. All the RT-PCR positive cases were confirmed by cloning and sequencing. Of the 14 cases with strong nuclear TFE3 expression, 12 showed a break-apart signal by FISH. ASPL- and PRCC-TFE3 translocations were detected in 13 and one case, respectively, by RT-PCR. Of 21 cases with weak TFE3 expression, five were translocation-positive by FISH. ASPL-, PRCC-, and PSF-TFE3 translocations were detected by RT-PCR (n=3, 3, and 1, respectively). All 13 TFE3-negative/cathepsin K-positive cases were negative by FISH and two each harbored ASPL- and PRCC-TFE3 translocations that were detected by RT-PCR. A high rate of TFE3 immunoreactivity (8.6%) was confirmed by RT-PCR (13.5%) and FISH (9.7%). Higher translocation rate of RT-PCR means RT-PCR detected translocation in TFE3 weak expression group and only cathepsin K positive group more specifically than FISH. Thus, RT-PCR would complement FISH analysis for detecting translocation RCC with fusion partners.

  19. The Iron Distribution and Magnetic Properties of Schistosome Eggshells: Implications for Improved Diagnostics

    PubMed Central

    Lucyk-Maurer, Rafael; Kerr, Roland; Candido, Renata R. F.; Toh, Shu Q.; Saunders, Martin; Shaw, Jeremy A.; Suvorova, Alexandra; Hofmann, Andreas; House, Michael J.; Woodward, Robert C.; Graeff-Teixera, Carlos; St. Pierre, Timothy G.; Jones, Malcolm K.

    2013-01-01

    Background Schistosoma mansoni and Schistosoma japonicum are the most frequent causative agents of human intestinal schistosomiasis. Approximately 200 million people in the world are infected with schistosomes. Diagnosis of schistosomiasis is often difficult. High percentages of low level infections are missed in routine fecal smear analysis and current diagnostic methodologies are inadequate to monitor the progress of parasite control, especially in areas with low transmission. Improved diagnostic methods are urgently needed to evaluate the success of elimination programs. Recently, a magnetic fractionation method for isolation of parasite eggs from feces was described, which uses magnetic microspheres to form parasite egg – magnetic microsphere conjugates. This approach enables screening of larger sample volumes and thus increased diagnostic sensitivity. The mechanism of formation of the conjugates remains unexplained and may either be related to specific surface characteristics of eggs and microspheres or to their magnetic properties. Methods/Principal Findings Here, we investigated iron localization in parasite eggs, specifically in the eggshells. We determined the magnetic properties of the eggs, studied the motion of eggs and egg-microsphere conjugates in magnetic fields and determined species specific affinity of parasite eggs to magnetic microspheres. Our study shows that iron is predominantly localized in pores in the eggshell. Parasite eggs showed distinct paramagnetic behaviour but they did not move in a magnetic field. Magnetic microspheres spontaneously bound to parasite eggs without the presence of a magnetic field. S. japonicum eggs had a significantly higher affinity to bind microspheres than S. mansoni eggs. Conclusions/Significance Our results suggest that the interaction of magnetic microspheres and parasite eggs is unlikely to be magnetic in origin. Instead, the filamentous surface of the eggshells may be important in facilitating the binding

  20. A simple bioscore improves diagnostic accuracy of sepsis after surgery.

    PubMed

    Liu, Zimeng; Chen, Juan; Liu, Yongjun; Si, Xiang; Jiang, Zhiyi; Zhang, Xuyu; Guan, Xiangdong

    2016-01-01

    Rapid and accurate prediction for sepsis remains a challenge in surgical intensive care units. Detection of individual biomarkers is often of marginal usefulness, and several biomarkers are difficult to measure in the clinical setting. The aim of this study was to evaluate the diagnostic and prognostic performance of three routine biomarkers, procalcitonin (PCT), B-type natriuretic peptide (BNP), and lymphocyte percentage, as individual or in combination for sepsis in surgical critically ill patients. Circulating PCT, BNP, and lymphocyte percentage were measured in surgical patients on admission to the intensive care unit. A bioscore system combining these biomarkers was constructed. All studied variables were analyzed according to the diagnosis and clinical outcomes of sepsis. A total of 320 consecutive patients were included in the analysis. One hundred fifty-six patients presented with sepsis. In the patients with sepsis, levels of PCT and BNP increased and lymphocyte percentage decreased. For individual biomarkers, PCT achieved the best area under the curve for the diagnosis of sepsis, whereas the diagnostic performance of the bioscore was better than that of each individual biomarker (area under the curve, 0.914 [95% confidence interval, 0.862-0.951]). Levels of BNP and bioscore increased in nonsurvivors in the entire cohort, but the accuracy of these two variables for mortality prediction was lower than that shown by Acute Physiology and Chronic Health Evaluation II score. Furthermore, bioscore failed to predict outcomes in septic patients. A simple bioscore combining PCT together with BNP and lymphocyte percentage improves the diagnostic accuracy for sepsis in surgical critically ill patients but fails to predict outcomes in surgical patients with sepsis. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. 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

  2. Neuropsychological Criteria for Mild Cognitive Impairment Improves Diagnostic Precision, Biomarker Associations, and Progression Rates

    PubMed Central

    Bondi, Mark W.; Edmonds, Emily C.; Jak, Amy J.; Clark, Lindsay R.; Delano-Wood, Lisa; McDonald, Carrie R.; Nation, Daniel A.; Libon, David J.; Au, Rhoda; Galasko, Douglas; Salmon, David P.

    2014-01-01

    progression. Refinement of MCI diagnostic methods may also yield gains in biomarker and clinical trial study findings because of improvements in sample compositions of ‘true positive’ cases and removal of ‘false positive’ cases. PMID:24844687

  3. Use of Bayesian Networks to Probabilistically Model and Improve the Likelihood of Validation of Microarray Findings by RT-PCR

    PubMed Central

    English, Sangeeta B.; Shih, Shou-Ching; Ramoni, Marco F.; Smith, Lois E.; Butte, Atul J.

    2014-01-01

    Though genome-wide technologies, such as microarrays, are widely used, data from these methods are considered noisy; there is still varied success in downstream biological validation. We report a method that increases the likelihood of successfully validating microarray findings using real time RT-PCR, including genes at low expression levels and with small differences. We use a Bayesian network to identify the most relevant sources of noise based on the successes and failures in validation for an initial set of selected genes, and then improve our subsequent selection of genes for validation based on eliminating these sources of noise. The network displays the significant sources of noise in an experiment, and scores the likelihood of validation for every gene. We show how the method can significantly increase validation success rates. In conclusion, in this study, we have successfully added a new automated step to determine the contributory sources of noise that determine successful or unsuccessful downstream biological validation. PMID:18790084

  4. Secondary signs may improve the diagnostic accuracy of equivocal ultrasounds for suspected appendicitis in children.

    PubMed

    Partain, Kristin N; Patel, Adarsh; Travers, Curtis; McCracken, Courtney E; Loewen, Jonathan; Braithwaite, Kiery; Heiss, Kurt F; Raval, Mehul V

    2016-10-01

    Ultrasound (US) is the preferred imaging modality for evaluating appendicitis. Our purpose was to determine if including secondary signs (SS) improve diagnostic accuracy in equivocal US studies. Retrospective review identified 825 children presenting with concern for appendicitis and with a right lower quadrant (RLQ) US. Regression models identified which SS were associated with appendicitis. Test characteristics were demonstrated. 530 patients (64%) had equivocal US reports. Of 114 (22%) patients with equivocal US undergoing CT, those with SS were more likely to have appendicitis (48.6% vs 14.6%, p<0.001). Of 172 (32%) patients with equivocal US admitted for observation, those with SS were more likely to have appendicitis (61.0% vs 33.6%, p<0.001). SS associated with appendicitis included fluid collection (adjusted odds ratio (OR) 13.3, 95% confidence interval (CI) 2.1-82.8), hyperemia (OR=2.0, 95%CI 1.5-95.5), free fluid (OR=9.8, 95%CI 3.8-25.4), and appendicolith (OR=7.9, 95%CI 1.7-37.2). Wall thickness, bowel peristalsis, and echogenic fat were not associated with appendicitis. Equivocal US that included hyperemia, a fluid collection, or an appendicolith had 96% specificity and 88% accuracy. Use of SS in RLQ US assists in the diagnostic accuracy of appendicitis. SS may guide clinicians and reduce unnecessary CT and admissions. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2011-02-01

    The diagnosis of cattle-related sensitization is complicated by the variability and complexity of cattle allergen extracts. To evaluate a modified diagnostic procedure leading to more accurate results especially in the early phase of sensitization. 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. 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. 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.

  6. 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

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

    PubMed Central

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

    2012-01-01

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

  8. 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.

  9. 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.

  10. 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.

  11. Integration of transcriptomics and metabonomics: improving diagnostics, biomarker identification and phenotyping in ulcerative colitis.

    PubMed

    Bjerrum, Jacob Tveiten; Rantalainen, Mattias; Wang, Yulan; Olsen, Jørgen; Nielsen, Ole Haagen

    2014-01-01

    A systems biology approach to multi-faceted diseases has provided an opportunity to establish a holistic understanding of the processes at play. Thus, the current study merges transcriptomics and metabonomics data in order to improve diagnostics, biomarker identification and to explore the possibilities of a molecular phenotyping of ulcerative colitis (UC) patients. Biopsies were obtained from the descending colon of 43 UC patients (22 active UC and 21 quiescent UC) and 15 controls. Genome-wide gene expression analyses were performed using Affymetrix GeneChip Human Genome U133 Plus 2.0. Metabolic profiles were generated using (1)H Nuclear magnetic resonance spectroscopy (Bruker 600 MHz, Bruker BioSpin, Rheinstetten, Germany). Data were analyzed with the use of orthogonal-projection to latent structure-discriminant analysis and a multivariate logistic regression model fitted by lasso. Prediction performance was evaluated using nested Monte Carlo cross-validation. The prediction performance of the merged data sets and that of relative small (<20 variables) multivariate biomarker panels suggest that it is possible to discriminate between active UC, quiescent UC, and controls; between patients with or without steroid dependency, as well as between early or late disease onset. Consequently, this study demonstrates that the novel approach of integrating metabonomics and transcriptomics combines the better of the two worlds, and provides us with clinical applicable candidate biomarker panels. These combined panels improve diagnostics and more importantly also the molecular phenotyping in UC and provide insight into the pathophysiological processes at play, making optimized and personalized medication a possibility.

  12. Patient navigation improves cancer diagnostic resolution: an individually randomized clinical trial in an underserved population.

    PubMed

    Raich, Peter C; Whitley, Elizabeth M; Thorland, William; Valverde, Patricia; Fairclough, Diane

    2012-10-01

    Barriers to timely resolution of abnormal cancer screening tests add to cancer health disparities among low-income, uninsured, and minority populations. We conducted a randomized trial to evaluate the impact of lay patient navigators on time to resolution and completion of follow-up testing among patients with abnormal screening tests in a medically underserved patient population. Denver Health, the safety-net health care system serving Denver, is one of 10 performance sites participating in the Patient Navigation Research Program. Of 993 eligible subjects with abnormal screening tests randomized to navigation and no-navigation (control) arms and analyzed, 628 had abnormal breast screens (66 abnormal clinical breast examinations, 304 BIRADS 0, 200 BIRADS 3, 58 BIRADS 4 or 5) whereas 235 had abnormal colorectal and 130 had abnormal prostate screens. Time to resolution was significantly shorter in the navigated group (stratified log rank test, P < 0.001). Patient navigation improved diagnostic resolution for patients presenting with mammographic BIRADS 3 (P = 0.0003) and BIRADS 0 (P = 0.09), but not BIRADS 4/5 or abnormal breast examinations. Navigation shortened the time for both colorectal (P = 0.0017) and prostate screening resolution (P = 0.06). Participant demographics included 72% minority, 49% with annual household income less than $10,000, and 36% uninsured. Patient navigation positively impacts time to resolution of abnormal screening tests for breast, colorectal, and prostate cancers in a medically underserved population. By shortening the time to and increasing the proportion of patients with diagnostic resolution patient navigation could reduce disparities in stage at diagnosis and improve cancer outcomes. 2012 AACR

  13. Patient Navigation Improves Cancer Diagnostic Resolution: An Individually Randomized Clinical Trial in an Underserved Population

    PubMed Central

    Raich, Peter C.; Whitley, Elizabeth M.; Thorland, William; Valverde, Patricia; Fairclough, Diane

    2012-01-01

    Background Barriers to timely resolution of abnormal cancer screening tests add to cancer health disparities among low income, uninsured and minority populations. We conducted a randomized trial to evaluate the impact of lay patient navigators on time to resolution and completion of follow-up testing among patients with abnormal screening tests in a medically underserved patient population. Methods Denver Health (DH), the safety-net healthcare system serving Denver, is one of ten performance sites participating in the Patient Navigation Research Program (PNRP). Of 993 eligible subjects with abnormal screening tests randomized to navigation and no-navigation (control) arms and analyzed, 628 had abnormal breast screens (66 abnormal clinical breast examinations, 304 BIRADS 0, 200 BIRADS 3, 58 BIRADS 4 or 5) while 235 had abnormal colorectal and 130 had abnormal prostate screens. Results Time to resolution was significantly shorter in the navigated group (stratified log rank test, p<0.001). Patient navigation improved diagnostic resolution for patients presenting with mammographic BIRADS 3 (p=0.0003) and BIRADS 0 (p=0.09), but not BIRADS 4/5 or abnormal breast exams. Navigation shortened the time for both colorectal (p=0.0017) and prostate screening resolution (p=0.06). Participant demographics included 72% minority, 49% with annual household income less than $10,000, and 36% uninsured. Conclusions Patient navigation positively impacts time to resolution of abnormal screening tests for breast, colorectal and prostate cancers in a medically underserved population. Impact By shortening the time to and increasing the proportion of patients with diagnostic resolution patient navigation could reduce disparities in stage at diagnosis and improve cancer outcomes. PMID:23045537

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

    PubMed

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

    2011-11-01

    Using the classical process improvement framework of Plan-Do-Study-Act (PDSA), the diagnostic radiology department of a tertiary hospital identified several patient cycle time reduction strategies. Experimentation of these strategies (which included procurement of new machines, hiring of new staff, redesign of queue system, etc.) through pilot scale implementation was impractical because it might incur substantial expenditure or be operationally disruptive. With this in mind, simulation modeling was used to test these strategies via performance of "what if" analyses. Using the output generated by the simulation model, the team was able to identify a cost-free cycle time reduction strategy, which subsequently led to a reduction of patient cycle time and achievement of a management-defined performance target. As healthcare professionals work continually to improve healthcare operational efficiency in response to rising healthcare costs and patient expectation, simulation modeling offers an effective scientific framework that can complement established process improvement framework like PDSA to realize healthcare process enhancement. © 2011 National Association for Healthcare Quality.

  15. 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…

  16. 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

  17. 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.

  18. Diagnostic Ultrasound Impulses Improve Microvascular Flow in Patients With STEMI Receiving Intravenous Microbubbles.

    PubMed

    Mathias, Wilson; Tsutsui, Jeane M; Tavares, Bruno G; Xie, Feng; Aguiar, Miguel O D; Garcia, Diego R; Oliveira, Mucio T; Soeiro, Alexandre; Nicolau, Jose C; Lemos, Pedro A; Rochitte, Carlos E; Ramires, José A F; Kalil, Roberto; Porter, Thomas R

    2016-05-31

    Pre-clinical trials have demonstrated that, during intravenous microbubble infusion, high mechanical index (HMI) impulses from a diagnostic ultrasound (DUS) transducer might restore epicardial and microvascular flow in acute ST-segment elevation myocardial infarction (STEMI). The purpose of this study was to test the safety and efficacy of this adjunctive approach in humans. From May 2014 through September 2015, patients arriving with their first STEMI were randomized to either DUS intermittent HMI impulses (n = 20) just prior to emergent percutaneous coronary intervention (PCI) and for an additional 30 min post-PCI (HMI + PCI), or low mechanical index (LMI) imaging only (n = 10) for perfusion assessments before and after PCI (LMI + PCI). All studies were conducted during an intravenous perflutren lipid microsphere infusion. A control reference group (n = 70) arrived outside of the time window of ultrasound availability and received emergent PCI alone (PCI only). Initial epicardial recanalization rates prior to emergent PCI and improvements in microvascular flow were compared between ultrasound-treated groups. Median door-to-dilation times were 82 ± 26 min in the LMI + PCI group, 72 ± 15 min in the HMI + PCI group, and 103 ± 42 min in the PCI-only group (p = NS). Angiographic recanalization prior to PCI was seen in 12 of 20 HMI + PCI patients (60%) compared with 10% of LMI + PCI and 23% of PCI-only patients (p = 0.002). There were no differences in microvascular obstructed segments prior to treatment, but there were significantly smaller proportions of obstructed segments in the HMI + PCI group at 1 month (p = 0.001) and significant improvements in left ventricular ejection fraction (p < 0.005). HMI impulses from a diagnostic transducer, combined with a commercial microbubble infusion, can prevent microvascular obstruction and improve functional outcome when added to the contemporary PCI management of acute STEMI. (Therapeutic Use of Ultrasound in

  19. [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.

  20. 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%).

  1. Improvements in a Tracer-Encapsulated Solid Pellet and Its Injector for More Advanced Plasma Diagnostics

    NASA Astrophysics Data System (ADS)

    Tamura, Naoki; Sudo, Shigeru; Suzuki, Chihiro; Funaba, Hisamichi; Takagi, Masaru; Satoh, Nakahiro; Hayashi, Hiromi; Maeno, Hiroya; Yokota, Mitsuhiro; Ogawa, Hideki

    2017-04-01

    A Tracer-Encapsulated Solid Pellet (TESPEL) has been developed for promoting a precise study of the impurity transport in a magnetically-confined high-temperature plasma. This paper gives a brief report of the recent improvements in the TESPEL and its injector for more advanced plasma diagnostics. The TESPEL can be considered as a double-layered impurity pellet. This structure enables us to produce a both poloidally and toroidally localized “tracer” impurity source in the plasma, and to specify the total amount of the tracer impurity deposited in the plasma precisely. Recent experiments on the Large Helical Device by using the TESPEL suggest that the importance of the impurity source location in the impurity transport study. Thus we have developed new-type TESPELs, which are greatly improved in regard to the above-mentioned unique features. In addition, we also developed a new TESPEL injector, which enables us to inject the TESPEL obliquely into the plasma. This injector can also contribute to a further shallower penetration of the TESPEL into the plasma.

  2. Vaginal examination does not improve diagnostic accuracy in early pregnancy bleeding.

    PubMed

    Johnstone, Chris

    2013-06-01

    The study aims to determine if a vaginal examination improves diagnostic accuracy when assessing women who present to the ED with vaginal bleeding in the first trimester of pregnancy. One hundred and thirty-five women with first trimester bleeding were randomised to have a vaginal examination (n = 61) or not (n = 74). They were given a provisional diagnosis, and then a final diagnosis after ultrasound, beta-human chorionic gonadotropin and gynaecological follow up. The provisional diagnosis was considered accurate if it matched the final diagnosis. The provisional and final diagnoses matched in a little over half of the cases, and there was no statistical difference between the two groups (χ(2) = 0.005, P = 0.94). In a stable patient presenting to the ED with first trimester bleeding, clinical diagnosis is highly inaccurate and is not improved by vaginal examination. Routine vaginal examination is not necessary as part of the initial patient assessment. © 2013 The Author. EMA © 2013 Australasian College for Emergency Medicine and Australasian Society for Emergency Medicine.

  3. Using the Astronomy Diagnostic Test to Identify Teaching Strategies that Improve Conceptual Understanding

    NASA Astrophysics Data System (ADS)

    Deming, G. L.; Hufnagel, B.

    2002-05-01

    The Astronomy Diagnostic Test (ADT) was developed in order to assess learning in undergraduate introductory astronomy classes, but an underlying goal was to use the information supplied by the ADT to improve student learning. The ADT National Project collected pre-course (5346 students) and post-course (3842 students) test results from 97 classes at a variety of institutions in 31 states. These results have been compiled in an extensive database. The overall gain between pre-course and post-course average scores amounts to a disappointing 15%, but significant gains are identifiable for specific questions in individual classes. Results from the ADT National Project database will be presented for specific questions with minimal gains. Astronomy education researchers in Maryland are beginning to use ADT results to identify minimal gain concepts and then to modify and assess instructional strategies with the goal of improving student learning. A comparison will be made between ADT pre-course and post-course responses for several classes in which different teaching methods were used. Successful teaching strategies applicable in a variety of class settings will be offered and instructors are encouraged to become involved in assessing results in their own introductory astronomy classes. This research was supported by the National Science Foundation through grant REC-0089239.

  4. A quadruplex real-time qPCR assay for the simultaneous assessment of total human DNA, human male DNA, DNA degradation and the presence of PCR inhibitors in forensic samples: a diagnostic tool for STR typing.

    PubMed

    Hudlow, William R; Chong, Mavis Date; Swango, Katie L; Timken, Mark D; Buoncristiani, Martin R

    2008-03-01

    A quadruplex real-time qPCR assay was developed to simultaneously assess total human DNA, human male DNA, DNA degradation and PCR inhibitors in forensic samples. Specifically, the assay utilizes a approximately 170-190bp target sequence that spans the TH01 STR locus to quantify total human DNA (nuTH01), a 137 bp target sequence directly adjacent to the SRY gene to quantify human male DNA (nuSRY), a 67 bp target sequence flanking the CSF1PO STR locus (nuCSF) to assess degradation (nuCSF:nuTH01 ratio) and a 77 bp synthetic DNA template used as an internal PCR control target sequence (IPC) for the assessment of PCR inhibition. Validation studies, performed on an ABI 7500 SDS instrument using TaqMan and TaqManMGB detection, indicate each of the targets in the quadruplex assay performs effectively and is informative even when challenged with DNase-degraded and hematin-inhibited samples. The nuTH01-nuSRY-nuCSF-IPC quadruplex qPCR assay is envisioned to assist in the choice of the most informative DNA typing system available, which may include standard autosomal STR typing when the results indicate the presence of non-degraded, single gender DNA or non-degraded, male:female mixtures at ratios expected to yield probative alleles; Y STR typing in samples containing a male component that is overwhelmed by the presence of an excess of female DNA; reduced amplicon size STR typing ("MiniSTRs") where the nuCSF:nuTH01 ratio indicates the sample is highly degraded; enhanced STR amplification with additional AmpliTaq Gold/BSA and/or sample clean-up when the presence of PCR inhibitors is suggested by a delayed IPC C(T) value or mitochondrial DNA typing in samples where little to no nuclear DNA is detected. The present study includes evaluations of species specificity, sensitivity, precision, reproducibility, male-female mixtures, population samples and applications to various casework-type samples as indicated by the Scientific Working Group on DNA Analysis Methods (SWGDAM

  5. 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

  6. Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human Breast Cancer Diagnostics and Therapeutic Decision Making

    DTIC Science & Technology

    2014-10-01

    sorting data coming from human and mouse adult mammary gland , and coming from the fetal mammary rudiment, to define gene expression profiles of...AD_____________ Award Number: W81XWH-12-1-0106 TITLE: Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human...SUBTITLE Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human Breast Cancer Diagnostics and Therapeutic Decision Making Improve

  7. 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.

  8. Fast molecular diagnostics of canine T-cell lymphoma by PCR and capillary gel electrophoresis with laser-induced fluorescence detector.

    PubMed

    Jeon, Seonsook; Lee, Mi-Jin; Park, Jinho; Kang, Seong Ho

    2007-07-01

    Lymphoma is the most common hematopoietic tumor in dogs and manifests as a proliferation of malignant lymphoid cells primarily affecting the lymph nodes or solid visceral organs. We describe the use of capillary gel electrophoresis (CGE) with a laser-induced fluorescence (LIF) detector based on polymerase chain reaction (PCR) to rapidly detect a disorder of the canine T-cell receptor gamma (TCRgamma) gene. After the PCR amplification of the specific TCR( gene in dogs, the 90-bp DNA fragment amplified was separated in a fused-silica capillary by CGE-LIF. Under an electric field of 375 V/cm and with a sieving matrix of 1.5% poly (ethyleneoxide) (M(r) 600,000), the amplified PCR products were analyzed within 4 min by CGE separation. When the CGE-LIF method was applied to real clinical samples of the specific DNA fragment of the TCR( gene, the migration time and the corrected peak area showed relative standard deviations (n=5) of 0.29% and 0.58%, respectively. Both methods of CGE-LIF and slab gel electrophoresis showed same results for nine clinical samples. This PCR/CGE-LIF technique may prove to be a new fast and simple tool for the rapid diagnosis of the PCR-amplified DNA of canine T-cell lymphoma.

  9. An improved electronic microarray-based diagnostic assay for identification of MEFV mutations.

    PubMed

    Moutereau, Stéphane; Narwa, Rémy; Matheron, Catherine; Vongmany, Natalie; Simon, Emmanuelle; Goossens, Michel

    2004-06-01

    Recent technological advances, such as DNA chip devices that allow automated, high-throughput genotyping, promise to considerably improve the detection capability of single-nucleotide polymorphisms (SNPs) in clinically relevant genes. We used the NanoChip(R) Molecular Biology Workstation (Nanogen, www.nanogen.com) and recently introduced microelectronic array technology to develop a detection method for the more frequent mutations involved in familial Mediterranean fever (FMF), an autosomal recessive disease that affects several ethnic groups in the Mediterranean population, whose early diagnosis is crucial if severe complications are to be prevented. We adapted the previously described Nanogen procedures to FMF mutation analysis, introducing modifications that notably improve the technique. First, as the original procedure makes use of costly dye-tagged reporter sequences, we devised a universal reporter strategy, which was first evaluated and validated on the robust, previously established factor V Leiden and factor II (prothrombin) NanoChip diagnostic assays. FMF (MEFV), factor V (F5), and factor II (F2) genotypes identified using this improved system were totally concordant with results of other genotyping methods (denaturing gradient gel electrophoresis [DGGE], SSCP, and RFLP analysis). Second, we showed that the target sequences loaded on the NanoChip cartridges can be rehybridized several times in a highly reproducible manner, allowing sequential analysis of mutations. Thus, we devised a strategy that allows us to monitor the possible interference of additional mutations or SNPs at probe or stabilizer sequences. Finally, a comparative cost per sample analysis demonstrates that the accurate and reproducible FMF mutation detection assay we developed can be readily implemented in the clinical laboratory setting at reasonable expense. Copyright 2004 Wiley-Liss, Inc.

  10. Analysis of prostate cancer localization toward improved diagnostic accuracy of transperineal prostate biopsy.

    PubMed

    Sakamoto, Yoshiro; Fukaya, Kaori; Haraoka, Masaki; Kitamura, Kosuke; Toyonaga, Yoichiro; Tanaka, Michio; Horie, Shigeo

    2014-09-01

    Delineating the precise localization of prostate cancer is important in improving the diagnostic accuracy of prostate biopsy. In Juntendo University Nerima Hospital, initial 12-core or repeat 16-core biopsies were performed using a transrectal ultrasound guided transperineal prostate biopsy method. We step-sectioned prostates from radical prostatectomy specimens at 5-mm intervals from the urethra to the urinary bladder and designated five regions: the (1) Apex, (2) Apex-Mid, (3) Mid, (4) Mid-Base, and (5) Base. We then mapped prostate cancer localization on eight zones around the urethra for each of those regions. Prostate cancer was detected in 93 cases of 121 cases (76.9%) in the Apex, in 115 cases (95.0%) in the Apex-Mid, in 101 cases (83.5%) in the Mid, in 71 cases (58.7%) in the Mid-Base, and in 23 cases (19.0%) in the Base. In 99.2% of all cases, prostate cancers were detected from the Apex to Mid regions. For this reason, transperineal prostate biopsies have routinely been prioritized in the Apex, Apex-Mid, and Mid regions, while the Base region of the prostate was considered to be of lesser importance. Our analyses of prostate cancer localization revealed a higher rate of cancer in the posterior portion of the Apex, antero-medial and postero-medial portion of the Apex-Mid and antero-medial and postero-lateral portion of the Mid. The transperineal prostate biopsies in our institute performed had a sensitivity of 70.9%, a specificity of 96.6%, a positive predictive value (PPV) of 92.2% and a negative predictive value (NPV) of 85.5%. The concordance of prostate cancer between prostatectomy specimens and biopsies is comparatively favorable. According to our study, the diagnostic accuracy of transperineal prostate biopsy can be improved in our institute by including the anterior portion of the Apex-Mid and Mid regions in the 12-core biopsy or 16-core biopsy, such that a 4-core biopsy of the anterior portion is included.

  11. A comprehensive custom panel design for routine hereditary cancer testing: preserving control, improving diagnostics and revealing a complex variation landscape

    PubMed Central

    Castellanos, Elisabeth; Gel, Bernat; Rosas, Inma; Tornero, Eva; Santín, Sheila; Pluvinet, Raquel; Velasco, Juan; Sumoy, Lauro; del Valle, Jesús; Perucho, Manuel; Blanco, Ignacio; Navarro, Matilde; Brunet, Joan; Pineda, Marta; Feliubadaló, Lidia; Capellá, Gabi; Lázaro, Conxi; Serra, Eduard

    2017-01-01

    We wanted to implement an NGS strategy to globally analyze hereditary cancer with diagnostic quality while retaining the same degree of understanding and control we had in pre-NGS strategies. To do this, we developed the I2HCP panel, a custom bait library covering 122 hereditary cancer genes. We improved bait design, tested different NGS platforms and created a clinically driven custom data analysis pipeline. The I2HCP panel was developed using a training set of hereditary colorectal cancer, hereditary breast and ovarian cancer and neurofibromatosis patients and reached an accuracy, analytical sensitivity and specificity greater than 99%, which was maintained in a validation set. I2HCP changed our diagnostic approach, involving clinicians and a genetic diagnostics team from panel design to reporting. The new strategy improved diagnostic sensitivity, solved uncertain clinical diagnoses and identified mutations in new genes. We assessed the genetic variation in the complete set of hereditary cancer genes, revealing a complex variation landscape that coexists with the disease-causing mutation. We developed, validated and implemented a custom NGS-based strategy for hereditary cancer diagnostics that improved our previous workflows. Additionally, the existence of a rich genetic variation in hereditary cancer genes favors the use of this panel to investigate their role in cancer risk. PMID:28051113

  12. A comprehensive custom panel design for routine hereditary cancer testing: preserving control, improving diagnostics and revealing a complex variation landscape.

    PubMed

    Castellanos, Elisabeth; Gel, Bernat; Rosas, Inma; Tornero, Eva; Santín, Sheila; Pluvinet, Raquel; Velasco, Juan; Sumoy, Lauro; Del Valle, Jesús; Perucho, Manuel; Blanco, Ignacio; Navarro, Matilde; Brunet, Joan; Pineda, Marta; Feliubadaló, Lidia; Capellá, Gabi; Lázaro, Conxi; Serra, Eduard

    2017-01-04

    We wanted to implement an NGS strategy to globally analyze hereditary cancer with diagnostic quality while retaining the same degree of understanding and control we had in pre-NGS strategies. To do this, we developed the I2HCP panel, a custom bait library covering 122 hereditary cancer genes. We improved bait design, tested different NGS platforms and created a clinically driven custom data analysis pipeline. The I2HCP panel was developed using a training set of hereditary colorectal cancer, hereditary breast and ovarian cancer and neurofibromatosis patients and reached an accuracy, analytical sensitivity and specificity greater than 99%, which was maintained in a validation set. I2HCP changed our diagnostic approach, involving clinicians and a genetic diagnostics team from panel design to reporting. The new strategy improved diagnostic sensitivity, solved uncertain clinical diagnoses and identified mutations in new genes. We assessed the genetic variation in the complete set of hereditary cancer genes, revealing a complex variation landscape that coexists with the disease-causing mutation. We developed, validated and implemented a custom NGS-based strategy for hereditary cancer diagnostics that improved our previous workflows. Additionally, the existence of a rich genetic variation in hereditary cancer genes favors the use of this panel to investigate their role in cancer risk.

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

    PubMed

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

    2009-02-28

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

  14. Panel of 23S rRNA Gene-Based Real-Time PCR Assays for Improved Universal and Group-Specific Detection of Phytoplasmas▿ †

    PubMed Central

    Hodgetts, Jennifer; Boonham, Neil; Mumford, Rick; Dickinson, Matthew

    2009-01-01

    Primers and probes based on the 23S rRNA gene have been utilized to design a range of real-time PCR assays for routine phytoplasma diagnostics. These assays have been authenticated as phytoplasma specific and shown to be at least as sensitive as nested PCR. A universal assay to detect all phytoplasmas has been developed, along with a multiplex assay to discriminate 16SrI group phytoplasmas from members of all of the other 16Sr groups. Assays for the 16SrII, 16SrIV, and 16SrXII groups have also been developed to confirm that the 23S rRNA gene can be used to design group-specific assays. PMID:19270148

  15. Improved performance with saliva and urine as alternative DNA sources for malaria diagnosis by mitochondrial DNA-based PCR assays.

    PubMed

    Putaporntip, C; Buppan, P; Jongwutiwes, S

    2011-10-01

    Saliva and urine from malaria-infected individuals contain trace amounts of Plasmodium DNA, and therefore, could be used as alternative specimens for diagnosis. A nested PCR targeting the mitochondrial cytochrome b gene (Cytb-PCR) of four human malaria species and Plasmodium knowlesi was developed and tested with 693 blood samples from febrile patients living in diverse malaria-endemic areas of Thailand, and compared with microscopy and nested PCR targeting small-subunit rRNA (18S-PCR). Cytb-PCR was 16% and 39.8% more sensitive than 18S-PCR and microscopy, respectively, in detecting all of these malarial species in blood samples. Importantly, 34% and 17% of Plasmodium falciparum and Plasmodium vivax mono-infections, respectively, detected by microscopy were, in fact, mixed P. falciparum and P. non-falciparum infections. Analysis of matched blood, saliva and urine from 157 individuals showed that microscopy and Cytb-PCR of saliva yielded no significant difference in detecting P. falciparum and P. vivax. However, Cytb-PCR of saliva was more sensitive than microscopy for diagnosis of mixed-species infections. A combination of Cytb-PCR of saliva and of urine significantly outperformed microscopy (p 0.0098 for P. falciparum, p 0.006 for P. vivax, and p 0.0002 for mixed infections). Furthermore, Plasmodium malariae and P. knowlesi could also be identified in saliva and urine with this method. Therefore, the Cytb-PCR developed herein offers a high potential for the use of both saliva and urine for malaria diagnosis, with a sensitivity comparable with or superior to that of microscopy.

  16. Post-mortem computed tomography coaxial cutting needle biopsy to facilitate the detection of bacterioplankton using PCR probes as a diagnostic indicator for drowning.

    PubMed

    Rutty, Guy N; Johnson, Christopher; Amoroso, Jasmin; Robinson, Claire; Bradley, Carina J; Morgan, Bruno

    2017-01-01

    We report for the first time the use of coaxial cutting needle biopsy, guided by post-mortem computed tomography (PMCT), to sample internal body tissues for bacterioplankton PCR analysis to investigate drowning. This technical report describes the biopsy technique, the comparison of the needle biopsy and the invasive autopsy sampling results, as well as the PMCT and autopsy findings. By using this new biopsy sampling approach for bacterioplankton PCR, we have developed on previous papers describing the minimally invasive PMCT approach for the diagnosis of drowning. When such a system is used, the operator must take all precautions to avoid contamination of the core biopsy samples due to the sensitivity of PCR-based analytic systems.

  17. 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. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

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

    PubMed

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

    2010-08-27

    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 degrees C or 65 degrees 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. Copyright 2010 Elsevier Inc. All rights reserved.

  19. Utilisation of multi-frequency VEMPs improves diagnostic accuracy for Meniere's disease.

    PubMed

    Maxwell, Rebecca; Jerin, Claudia; Gürkov, Robert

    2017-01-01

    To determine whether vestibular evoked myogenic potential (VEMP) measurements that combine the VEMP 500/1000 Hz frequency tuning ratio and the inter-aural asymmetry ratio can reliably detect unilateral Meniere's disease ears as compared to healthy controls. Forty-two consecutive patients with certain unilateral Meniere's disease (as confirmed using a locally enhanced inner ear MRI (LEIM)) were assessed. Cervical vestibular evoked myogenic potentials (cVEMP) and ocular vestibular evoked myogenic potentials (oVEMP) were recorded at 500 and 1000 Hz. The VEMP amplitudes, asymmetry ratios, and the 500/1000 Hz amplitude ratios were compared with those of 21 age-matched healthy controls. A multi-frequency VEMPs score that combined: (1) the cVEMP 500/1000 Hz amplitude ratio, (2) the oVEMP 500/1000 Hz amplitude ratio, (3) the 500 Hz cVEMP asymmetry ratio, (4) the 1000 Hz cVEMP asymmetry ratio, produced a ROC curve with an area under the curve (AUC) of 0.814. The inclusion of audiology data further improved the result to 0.906. This score can be used to discriminate with a good degree of clinical accuracy between Meniere's ears (unilateral) and those of healthy controls. Multi-frequency VEMP analysis offers a simple, cost-effective solution to the diagnostic difficulties presented by Meniere's disease.

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

    PubMed

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

    2016-01-01

    Currently, Western blot is used to confirm the initial serodiagnosis of HIV infection by antibody detection. However, a major deficiency of the Western blot relates to a lack of sufficient sensitivity in detecting HIV antibodies. This report describes a simple, sensitive and inexpensive bead-based assay for detection of early HIV infection. A panel of 138 positive specimens including 105 blood donors and 33 MSM with known Western blot results were evaluated using Luminex xMAP at Tianjin Centers for Disease Control and Prevention (CDC). We demonstrate a superior sensitivity of Luminex xMAP compared with Western blot. Of the 87 confirmed HIV positive blood donors, Western blot only confirmed 65 cases with 74.7% (65/87) sensitivity while Luminex xMAP identified 72 cases with 82.8% (72/87) sensitivity (p<0.05). Western blot and Luminex xMAP verified 13 and 19 of 33 MSM specimens, respectively. The sensitivity was 39.4% (13/33) for Western blot and 57.6% (19/33) for Luminex xMAP (p<0.1). Luminex xMAP combined with Western blot improves the diagnostic sensitivity of HIV infection at an early stage, and reduces the chances of missed diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Improved diagnostic accuracy of lung perfusion imaging using Tc-99m MAA SPECT

    SciTech Connect

    O'Donnell, J.K.; Golish, J.A.; Go, R.T.; Risius, B.; Graor, R.A.; MacIntyre, W.J.; Feiglin, D.H.

    1984-01-01

    The addition of emission tomography (SPECT) to pulmonary perfusion imaging should improve diagnostic accuracy by detecting perfusion defects otherwise masked by superimposition of normal lung activity and by reducing problems with interpretation of defects that result from overlying soft tissue or pleural effusions. In order to examine the contribution of SPECT in the scintigraphic evaluation for pulmonary embolus (PE), the authors have obtained both planar and SPECT studies in 94 cases of suspected PE. All studies employed 3-4 mCi of Tc-99m MAA and standard six-view planar image acquisition. SPECT raw data of 64 images were then acquired over a 360 degree transaxial rotation with subsequent computer reconstruction. Xe-133 ventilation studies were performed when clinically indicated and tolerated by the patient. For 19 studies angiographic (AN) correlation was obtained within 24 hours. In 16/19 planar and SPECT both gave a high probability of PE but SPECT gave better segmental localization and showed better agreement with the number of defects seen at AN. In 3 indeterminate planar scans, 2 were low probability with SPECT and had negative AN. The third, a patient with Wegener's vasculitis, remained indeterminate with SPECT and had negative AN. Five patients with PE had repeat planar/SPECT/AN studies to evaluate response to treatment. SPECT correlated better with AN findings in each case. The authors conclude that SPECT perfusion imaging provides better anatomic accuracy for defects representing PE and is the non-invasive technique of choice for documenting response to therapy.

  2. [The implementation of diagnostic and curative technologies of improvement of population health in ambulatory polyclinic conditions].

    PubMed

    Murakhovskiĭ, A G; Babenko, A I; Bravve, Iu I

    2012-01-01

    The article deals with the main groups of diseases as a reason for adult population to consult a municipal polyclinics for diagnostics and ambulatory treatment. These diseases are arterial hypertension, diabetes mellitus, gastritis, duodenitis, influenza and acute respiratory disease. It is established that the leading groups of technologies on the implementation of diagnostic process are the laboratory clinical analyses and common diagnostics measures which comprise 72.7% of all diagnostic technologies. In polyclinics, the foundation of treatment process is the common treatment measures which comprise 68.6% of all treatment technologies. This fact testifies the non-significant percentage of implementation of high-technology diagnostics technique and treatment in the ambulatory polyclinic conditions.

  3. 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.

  4. 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

  5. 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…

  6. Can Latent Class Analysis Be Used to Improve the Diagnostic Process in Pediatric Patients with Chronic Ataxia?

    PubMed

    Klassen, Samantha; Dufault, Brenden; Salman, Michael S

    2017-04-01

    Chronic ataxia is a relatively common symptom in children. There are numerous causes of chronic ataxia, making it difficult to derive a diagnosis in a timely manner. We hypothesized that the efficiency of the diagnostic process can be improved with systematic analysis of clinical features in pediatric patients with chronic ataxia. Our aim was to improve the efficiency of the diagnostic process in pediatric patients with chronic ataxia. A cohort of 184 patients, aged 0-16 years with chronic ataxia who received medical care at Winnipeg Children's Hospital during 1991-2008, was ascertained retrospectively from several hospital databases. Clinical details were extracted from hospital charts. The data were compared among the more common diseases using univariate analysis to identify pertinent clinical features that could potentially improve the efficiency of the diagnostic process. Latent class analysis was then conducted to detect unique patterns of clinical features and to determine whether these patterns could be associated with chronic ataxia diagnoses. Two models each with three classes were chosen based on statistical criteria and clinical knowledge for best fit. Each class represented a specific pattern of presenting symptoms or other clinical features. The three classes corresponded to a plausible and shorter list of possible diagnoses. For example, developmental delay and hypotonia correlated best with Angelman syndrome. Specific patterns of presenting symptoms or other clinical features can potentially aid in the initial assessment and diagnosis of pediatric patients with chronic ataxia. This will likely improve the efficiency of the diagnostic process.

  7. 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…

  8. 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

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

    PubMed

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

    2014-07-01

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

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

    PubMed

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

    2015-06-01

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

  11. Adding a selective enrichment step to the iQ-Check real-time PCR improves the detection of Salmonella in naturally contaminated retail turkey meat products.

    PubMed

    Fakhr, M K; McEvoy, J M; Sherwood, J S; Logue, C M

    2006-07-01

    The aim of this study was to compare the real-time iQ-Check Salmonella kit (Bio-Rad) with the immunocapture assay RapidCheck Salmonella method, and a conventional culture method (FSIS, USDA) in detecting Salmonella in naturally contaminated turkey meat products. This study was also designed to determine if a selective enrichment step might improve the real-time detection of Salmonella. Using the culture method, Salmonella was recovered from 49 out of 99 retail turkey meat samples collected. RapidCheck failed to detect 11 Salmonella samples that were positive by the culture method. The iQ-Check real-time PCR also failed to detect three samples that were positive by the culture method. However, when carried out after a selective enrichment step, the iQ-Check real-time PCR detected all 49 Salmonella samples recovered by the culture method. The iQ-Check real-time PCR detected the presence of Salmonella in some samples that were not recovered by the culture method. Adding a selective enrichment step to the iQ-Check real-time PCR improves the detection of Salmonella in naturally contaminated turkey meat samples. The iQ-Check Salmonella real-time PCR can be used as a rapid method to monitor Salmonella in turkey meat, together with conventional culture methodology.

  12. Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human Breast Cancer Diagnostics and Therapeutic Decision Making

    DTIC Science & Technology

    2014-10-01

    Conclusion. We have used FAC sorting data coming from human and mouse adult mammary gland , and coming from the fetal mammary rudiment, to define gene...AD_____________ Award Number: W81XWH-12-1-0107 TITLE: Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human...4. TITLE AND SUBTITLE Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human Breast Cancer Diagnostics and Therapeutic

  13. Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human Breast Cancer Diagnostics and Therapeutic Decision Making

    DTIC Science & Technology

    2015-12-01

    satellites ” resulted in reversion of the cells to an epithelial state, re-entry into the cell cycle, and restoration of their ability to generate both...1 AWARD NUMBER: W81XWH-12-1-0107 TITLE: Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human Breast Cancer...W81XWH-12-1-0107 Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human Breast Cancer Diagnostics and Therapeutic Decision Making

  14. Specific detection of enteroaggregative hemorrhagic Escherichia coli O104:H4 strains by use of the CRISPR locus as a target for a diagnostic real-time PCR.

    PubMed

    Delannoy, Sabine; Beutin, Lothar; Burgos, Ylanna; Fach, Patrick

    2012-11-01

    In 2011, a large outbreak of an unusual bacterial strain occurred in Europe. This strain was characterized as a hybrid of an enteroaggregative Escherichia coli (EAEC) and a Shiga toxin-producing E. coli (STEC) strain of the serotype O104:H4. Here, we present a single PCR targeting the clustered regularly interspaced short palindromic repeats locus of E. coli O104:H4 (CRISPR(O104:H4)) for specific detection of EAEC STEC O104:H4 strains from different geographical locations and time periods. The specificity of the CRISPR(O104:H4) PCR was investigated using 1,321 E. coli strains, including reference strains for E. coli O serogroups O1 to O186 and flagellar (H) types H1 to H56. The assay was compared for specificity using PCR assays targeting different O104 antigen-encoding genes (wbwC(O104), wzx(O104), and wzy(O104)). The PCR assays reacted with all types of E. coli O104 strains (O104:H2, O104:H4, O104:H7, and O104:H21) and with E. coli O8 and O9 strains carrying the K9 capsular antigen and were therefore not specific for detection of the EAEC STEC O104:H4 type. A single PCR developed for the CRISPR(O104:H4) target was sufficient for specific identification and detection of the 48 tested EAEC STEC O104:H4 strains. The 35 E. coli O104 strains expressing H types other than H4 as well as 8 E. coli strains carrying a K9 capsular antigen tested all negative for the CRISPR(O104:H4) locus. Only 12 (0.94%) of the 1,273 non-O104:H4 E. coli strains (serotypes Ont:H2, O43:H2, O141:H2, and O174:H2) reacted positive in the CRISPR(O104:H4) PCR (99.06% specificity).

  15. Specific Detection of Enteroaggregative Hemorrhagic Escherichia coli O104:H4 Strains by Use of the CRISPR Locus as a Target for a Diagnostic Real-Time PCR

    PubMed Central

    Delannoy, Sabine; Beutin, Lothar; Burgos, Ylanna

    2012-01-01

    In 2011, a large outbreak of an unusual bacterial strain occurred in Europe. This strain was characterized as a hybrid of an enteroaggregative Escherichia coli (EAEC) and a Shiga toxin-producing E. coli (STEC) strain of the serotype O104:H4. Here, we present a single PCR targeting the clustered regularly interspaced short palindromic repeats locus of E. coli O104:H4 (CRISPRO104:H4) for specific detection of EAEC STEC O104:H4 strains from different geographical locations and time periods. The specificity of the CRISPRO104:H4 PCR was investigated using 1,321 E. coli strains, including reference strains for E. coli O serogroups O1 to O186 and flagellar (H) types H1 to H56. The assay was compared for specificity using PCR assays targeting different O104 antigen-encoding genes (wbwCO104, wzxO104, and wzyO104). The PCR assays reacted with all types of E. coli O104 strains (O104:H2, O104:H4, O104:H7, and O104:H21) and with E. coli O8 and O9 strains carrying the K9 capsular antigen and were therefore not specific for detection of the EAEC STEC O104:H4 type. A single PCR developed for the CRISPRO104:H4 target was sufficient for specific identification and detection of the 48 tested EAEC STEC O104:H4 strains. The 35 E. coli O104 strains expressing H types other than H4 as well as 8 E. coli strains carrying a K9 capsular antigen tested all negative for the CRISPRO104:H4 locus. Only 12 (0.94%) of the 1,273 non-O104:H4 E. coli strains (serotypes Ont:H2, O43:H2, O141:H2, and O174:H2) reacted positive in the CRISPRO104:H4 PCR (99.06% specificity). PMID:22895033

  16. 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. Copyright © 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  17. 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.

  18. 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

  19. Code-based Diagnostic Algorithms for Idiopathic Pulmonary Fibrosis. Case Validation and Improvement.

    PubMed

    Ley, Brett; Urbania, Thomas; Husson, Gail; Vittinghoff, Eric; Brush, David R; Eisner, Mark D; Iribarren, Carlos; Collard, Harold R

    2017-06-01

    Population-based studies of idiopathic pulmonary fibrosis (IPF) in the United States have been limited by reliance on diagnostic code-based algorithms that lack clinical validation. To validate a well-accepted International Classification of Diseases, Ninth Revision, code-based algorithm for IPF using patient-level information and to develop a modified algorithm for IPF with enhanced predictive value. The traditional IPF algorithm was used to identify potential cases of IPF in the Kaiser Permanente Northern California adult population from 2000 to 2014. Incidence and prevalence were determined overall and by age, sex, and race/ethnicity. A validation subset of cases (n = 150) underwent expert medical record and chest computed tomography review. A modified IPF algorithm was then derived and validated to optimize positive predictive value. From 2000 to 2014, the traditional IPF algorithm identified 2,608 cases among 5,389,627 at-risk adults in the Kaiser Permanente Northern California population. Annual incidence was 6.8/100,000 person-years (95% confidence interval [CI], 6.1-7.7) and was higher in patients with older age, male sex, and white race. The positive predictive value of the IPF algorithm was only 42.2% (95% CI, 30.6 to 54.6%); sensitivity was 55.6% (95% CI, 21.2 to 86.3%). The corrected incidence was estimated at 5.6/100,000 person-years (95% CI, 2.6-10.3). A modified IPF algorithm had improved positive predictive value but reduced sensitivity compared with the traditional algorithm. A well-accepted International Classification of Diseases, Ninth Revision, code-based IPF algorithm performs poorly, falsely classifying many non-IPF cases as IPF and missing a substantial proportion of IPF cases. A modification of the IPF algorithm may be useful for future population-based studies of IPF.

  20. 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.

  1. Health-based risk adjustment: improving the pharmacy-based cost group model by adding diagnostic cost groups.

    PubMed

    Prinsze, Femmeke J; van Vliet, René C J A

    Since 1991, risk-adjusted premium subsidies have existed in the Dutch social health insurance sector, which covered about two-thirds of the population until 2006. In 2002, pharmacy-based cost groups (PCGs) were included in the demographic risk adjustment model, which improved the goodness-of-fit, as measured by the R2, to 11.5%. The model's R2 reached 22.8% in 2004, when inpatient diagnostic information was added in the form of diagnostic cost groups (DCGs). PCGs and DCGs appear to be complementary in their ability to predict future costs. PCGs particularly improve the R2 for outpatient expenses, whereas DCGs improve the R2 for inpatient expenses. In 2006, this system of risk-adjusted premium subsidies was extended to cover the entire population.

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

    PubMed

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

    2011-01-01

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

  3. 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

  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. 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

    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.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2013-12-01

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

  8. Production of mono- and polyclonal antibodies to Citrus leprosis virus C2 and their application in triple antibody sandwich ELISA and immunocapture RT-PCR diagnostic assays.

    PubMed

    Choudhary, Nandlal; Roy, Avijit; Leon, M G; Wei, G; Nakhla, M K; Levy, L; Brlansky, R H

    2017-05-01

    The newly discovered Citrus leprosis virus cytoplasmic type 2 (CiLV-C2) is one of the causal virus of citrus leprosis disease complex; which leads to substantial loss of citrus production in the states of Meta and Casanare of Colombia. Specific and sensitive detection methods are needed to monitor the dissemination of CiLV-C2 in Colombia, and to prevent introduction of CiLV-C2 to other citrus growing countries. Toward this end, putative coat protein gene (CPG) of CiLV-C2 was amplified from CiLV-C2 infected citrus tissues. The CPG was cloned, expressed and purified a recombinant coat protein of ∼31kDa which used to generate monoclonal antibodies and polyclonal antisera. Four monoclonal antibodies and two polyclonal antisera were selected as being specific following Western blotting. The monoclonal antibody MAb E5 and polyclonal antiserum PAb UF715 were selected testing with an extract of CiLV-C2 infected leaves using triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). In addition, an immunocapture RT-PCR was standardized using MAb E5 for specific and sensitive detection of CiLV-C2. The standardized TAS-ELISA and IC-RT-PCR were able to detect CiLV-C2 in the extracts of symptomatic citrus leprosis tissues up to the dilutions of 1:160 and 1:2580, respectively. Result demonstrated that CiLV-C2 is present in citrus orchards in Meta and Casanare citrus growing areas of Colombia. TAS-ELISA could be used for routine detection of CiLV-C2, epidemiological studies, and for border inspections for quarantine purposes. IC-RT-PCR could be valuable for CiLV-C2 validation and viral genome analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. 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

  10. [Molecular diagnostics of infectious diseases for the ambulatory practice].

    PubMed

    Dumoulin, A

    2014-10-08

    Molecular diagnostics methods are not limited to specialized centers anymore. They play an important role for the diagnostic of infections commonly encountered in the clinical practice. Especially the detection of pathogens difficult to cultivate, such as viruses, has been greatly improved by these methods. Often, PCR has become the gold standard for the diagnostics of these pathogens. However, PCR cannot be used in any case, and it is not fail proof. Therefore, it is important to know when to use molecular methods and what are their strengths and weaknesses, in order to prescribe them rationally. This article reviews the characteristics of molecular tests and their main indications in the ambulatory setting.

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

    PubMed

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

    2016-10-01

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

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

    PubMed Central

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

    2012-01-01

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

  13. Microradiography of microcalcifications in breast specimen: a new histological correlation procedure and the effect of improved resolution on diagnostic validity.

    PubMed

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

    2012-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

  15. 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.

  16. Quantitative PCR for Detection of Shigella Improves Ascertainment of Shigella Burden in Children with Moderate-to-Severe Diarrhea in Low-Income Countries

    PubMed Central

    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

    2013-01-01

    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 × 104 ipaH copies per 100 ng of stool DNA for set 1 (n = 877). One hundred fifty-eight (18%) specimens yielded >2.9 × 104 ipaH copies. Ninety (10%) specimens were positive by traditional culture for Shigella. Individuals with ≥2.9 × 104 ipaH copies have 5.6-times-higher odds of having diarrhea than those with <2.9 × 104 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 × 104 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

  17. 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...

  18. 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...

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  2. Astrovirus Diagnostics

    PubMed Central

    Pérot, Philippe; Lecuit, Marc; Eloit, Marc

    2017-01-01

    Various methods exist to detect an astrovirus infection. Current methods include electron microscopy (EM), cell culture, immunoassays, polymerase chain reaction (PCR) and various other molecular approaches that can be applied in the context of diagnostic or in surveillance studies. With the advent of metagenomics, novel human astrovirus (HAstV) strains have been found in immunocompromised individuals in association with central nervous system (CNS) infections. This work reviews the past and current methods for astrovirus detection and their uses in both research laboratories and for medical diagnostic purposes. PMID:28085120

  3. Wavenumber selection based analysis in Raman spectroscopy improves skin cancer diagnostic specificity at high sensitivity levels (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Zhao, Jianhua; Zeng, Haishan; Kalia, Sunil; Lui, Harvey

    2017-02-01

    Background: Raman spectroscopy is a non-invasive optical technique which can measure molecular vibrational modes within tissue. A large-scale clinical study (n = 518) has demonstrated that real-time Raman spectroscopy could distinguish malignant from benign skin lesions with good diagnostic accuracy; this was validated by a follow-up independent study (n = 127). Objective: Most of the previous diagnostic algorithms have typically been based on analyzing the full band of the Raman spectra, either in the fingerprint or high wavenumber regions. Our objective in this presentation is to explore wavenumber selection based analysis in Raman spectroscopy for skin cancer diagnosis. Methods: A wavenumber selection algorithm was implemented using variably-sized wavenumber windows, which were determined by the correlation coefficient between wavenumbers. Wavenumber windows were chosen based on accumulated frequency from leave-one-out cross-validated stepwise regression or least and shrinkage selection operator (LASSO). The diagnostic algorithms were then generated from the selected wavenumber windows using multivariate statistical analyses, including principal component and general discriminant analysis (PC-GDA) and partial least squares (PLS). A total cohort of 645 confirmed lesions from 573 patients encompassing skin cancers, precancers and benign skin lesions were included. Lesion measurements were divided into training cohort (n = 518) and testing cohort (n = 127) according to the measurement time. Result: The area under the receiver operating characteristic curve (ROC) improved from 0.861-0.891 to 0.891-0.911 and the diagnostic specificity for sensitivity levels of 0.99-0.90 increased respectively from 0.17-0.65 to 0.20-0.75 by selecting specific wavenumber windows for analysis. Conclusion: Wavenumber selection based analysis in Raman spectroscopy improves skin cancer diagnostic specificity at high sensitivity levels.

  4. Development of Dual TaqMan Based One-Step rRT-PCR Assay Panel for Rapid and Accurate Diagnostic Test of MERS-CoV: A Novel Human Coronavirus, Ahead of Hajj Pilgrimage

    PubMed Central

    Hashemzadeh, Mohammad Sadegh; Rasouli, Rahimeh; Zahraei, Bentolhoda; Izadi, Morteza; Tat, Mahdi; Saadat, Seyed Hassan; Najarasl, Mohammad; Khansari Nejad, Behzad; Dorostkar, Ruhollah

    2016-01-01

    Background Coronaviruses (CoVs) are large ribonucleic acid (RNA) viruses causing primarily respiratory disease in humans. A novel human coronavirus, subsequently named middle east respiratory syndrome coronavirus (MERS-CoV), was first reported in Saudi Arabia in September of 2012. With increasing numbers of infections and deaths from MERS-CoV, development of a rapid and reliable kit was crucial to prevent further spread of MERS-CoV. Objectives In this study, we present two real-time reverse-transcription polymerase chain reaction (rRT-PCR) assays for in-house rapid and sensitive diagnostic testing of MERS-CoV, detecting the regions upstream of the envelope gene (upE) and open reading frame (ORF) 1b, respectively, for initial screening and final confirmation of MERS-CoV infection, as recommended by the world health organization (WHO). Materials and Methods In this experimental study, acquiring patient samples was difficult; thus, according to WHO recommendations and standard protocols, we synthesized RNA sequences of upE and ORF1b genes as the template signatures and TaqMan based-diagnostic rRT-PCR assays were carried out using these synthetic genes for detection of MERS-CoV. In this research, we also inaugurated a cell-free system to transcribe these RNA sequences using the DNA templates synthesized. Results The upE and ORF1b based one-step rRT-PCR assays were optimized by testing several times via different synthetic RNAs, and validation results were highly successful. The sensitivity obtained for upE was fewer than ten copies of RNA template per reaction and for ORF1b was 50 or fewer copies per reaction. Conclusions This study showed that the developed rRT-PCR assays are rapid, reliable, reproducible, specific, sensitive, and simple tools for detection of MERS-CoV. Finally, a kit consisting of two assay signatures and controls was assembled, which can be distributed to public health laboratories in Iran to support international MERS-CoV surveillance and public

  5. 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)

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

    ERIC Educational Resources Information Center

    LoPresto, Michael C.

    2007-01-01

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

  7. Getting Inside the Instructional Process: A Collaborative Diagnostic Process for Improving College Teaching.

    ERIC Educational Resources Information Center

    Cooper, Colleen R.

    1982-01-01

    Discusses a diagnostic technique called the Collaborative Analysis and Action Planning Process (CAP), which focuses on patterns of behaviors that are produced, interpreted, and responded to by college teachers and students during a lesson. The steps involved in the CAP process are described, and a 16-item reference list is included. (Author/JL)

  8. 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)

  9. Whole Genome Sequencing Expands Diagnostic Utility and Improves Clinical Management in Pediatric Medicine.

    PubMed

    Stavropoulos, Dimitri J; Merico, Daniele; Jobling, Rebekah; Bowdin, Sarah; Monfared, Nasim; Thiruvahindrapuram, Bhooma; Nalpathamkalam, Thomas; Pellecchia, Giovanna; Yuen, Ryan K C; Szego, Michael J; Hayeems, Robin Z; Shaul, Randi Zlotnik; Brudno, Michael; Girdea, Marta; Frey, Brendan; Alipanahi, Babak; Ahmed, Sohnee; Babul-Hirji, Riyana; Porras, Ramses Badilla; Carter, Melissa T; Chad, Lauren; Chaudhry, Ayeshah; Chitayat, David; Doust, Soghra Jougheh; Cytrynbaum, Cheryl; Dupuis, Lucie; Ejaz, Resham; Fishman, Leona; Guerin, Andrea; Hashemi, Bita; Helal, Mayada; Hewson, Stacy; Inbar-Feigenberg, Michal; Kannu, Peter; Karp, Natalya; Kim, Raymond; Kronick, Jonathan; Liston, Eriskay; MacDonald, Heather; Mercimek-Mahmutoglu, Saadet; Mendoza-Londono, Roberto; Nasr, Enas; Nimmo, Graeme; Parkinson, Nicole; Quercia, Nada; Raiman, Julian; Roifman, Maian; Schulze, Andreas; Shugar, Andrea; Shuman, Cheryl; Sinajon, Pierre; Siriwardena, Komudi; Weksberg, Rosanna; Yoon, Grace; Carew, Chris; Erickson, Raith; Leach, Richard A; Klein, Robert; Ray, Peter N; Meyn, M Stephen; Scherer, Stephen W; Cohn, Ronald D; Marshall, Christian R

    2016-01-13

    The standard of care for first-tier clinical investigation of the etiology of congenital malformations and neurodevelopmental disorders is chromosome microarray analysis (CMA) for copy number variations (CNVs), often followed by gene(s)-specific sequencing searching for smaller insertion-deletions (indels) and single nucleotide variant (SNV) mutations. Whole genome sequencing (WGS) has the potential to capture all classes of genetic variation in one experiment; however, the diagnostic yield for mutation detection of WGS compared to CMA, and other tests, needs to be established. In a prospective study we utilized WGS and comprehensive medical annotation to assess 100 patients referred to a paediatric genetics service and compared the diagnostic yield versus standard genetic testing. WGS identified genetic variants meeting clinical diagnostic criteria in 34% of cases, representing a 4-fold increase in diagnostic rate over CMA (8%) (p-value = 1.42e-05) alone and >2-fold increase in CMA plus targeted gene sequencing (13%) (p-value = 0.0009). WGS identified all rare clinically significant CNVs that were detected by CMA. In 26 patients, WGS revealed indel and missense mutations presenting in a dominant (63%) or a recessive (37%) manner. We found four subjects with mutations in at least two genes associated with distinct genetic disorders, including two cases harboring a pathogenic CNV and SNV. When considering medically actionable secondary findings in addition to primary WGS findings, 38% of patients would benefit from genetic counseling. Clinical implementation of WGS as a primary test will provide a higher diagnostic yield than conventional genetic testing and potentially reduce the time required to reach a genetic diagnosis.

  10. Polymerase chain reaction (PCR) detection of B cell clonality in Sjögren's syndrome patients: a diagnostic tool of clonal expansion

    PubMed Central

    Guzmán, L M; Castillo, D; Aguilera, S O

    2010-01-01

    Sjögren's syndrome (SS) is an autoimmune disease characterized by clonal B cell attack of the exocrine glands and dysregulated expression of B cell-activating factor (BAFF). Based upon the current data of increased rates of lymphoid malignancy, as non-Hodgkin's lymphoma (NHL) is associated with SS, we propose the detection of clonal rearrangements of immunoglobulin heavy chain (IgH) gene in those patients as a predictor of malignant clonal expansion. To test our proposal, we examined the IgH clonal rearrangements in SS patients (60) and healthy control subjects (42) having chronic non-specific sialadenitis, to deter