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Sample records for accurate diagnostic assays

  1. Evaluation of a pan-serotype point-of-care rapid diagnostic assay for accurate detection of acute dengue infection.

    PubMed

    Vivek, Rosario; Ahamed, Syed Fazil; Kotabagi, Shalini; Chandele, Anmol; Khanna, Ira; Khanna, Navin; Nayak, Kaustuv; Dias, Mary; Kaja, Murali-Krishna; Shet, Anita

    2017-03-01

    The catastrophic rise in dengue infections in India and globally has created a need for an accurate, validated low-cost rapid diagnostic test (RDT) for dengue. We prospectively evaluated the diagnostic performance of NS1/IgM RDT (dengue day 1) using 211 samples from a pediatric dengue cohort representing all 4 serotypes in southern India. The dengue-positive panel consisted of 179 dengue real-time polymerase chain reaction (RT-PCR) positive samples from symptomatic children. The dengue-negative panel consisted of 32 samples from dengue-negative febrile children and asymptomatic individuals that were negative for dengue RT-PCR/NS1 enzyme-linked immunosorbent assay/IgM/IgG. NS1/IgM RDT sensitivity was 89.4% and specificity was 93.8%. The NS1/IgM RDT showed high sensitivity throughout the acute phase of illness, in primary and secondary infections, in different severity groups, and detected all 4 dengue serotypes, including coinfections. This NS1/IgM RDT is a useful point-of-care assay for rapid and reliable diagnosis of acute dengue and an excellent surveillance tool in our battle against dengue.

  2. Diagnostic limitations to accurate diagnosis of cholera.

    PubMed

    Alam, Munirul; Hasan, Nur A; Sultana, Marzia; Nair, G Balakrish; Sadique, A; Faruque, A S G; Endtz, Hubert P; Sack, R B; Huq, A; Colwell, R R; Izumiya, Hidemasa; Morita, Masatomo; Watanabe, Haruo; Cravioto, Alejandro

    2010-11-01

    The treatment regimen for diarrhea depends greatly on correct diagnosis of its etiology. Recent diarrhea outbreaks in Bangladesh showed Vibrio cholerae to be the predominant cause, although more than 40% of the suspected cases failed to show cholera etiology by conventional culture methods (CMs). In the present study, suspected cholera stools collected from every 50th patient during an acute diarrheal outbreak were analyzed extensively using different microbiological and molecular tools to determine their etiology. Of 135 stools tested, 86 (64%) produced V. cholerae O1 by CMs, while 119 (88%) tested positive for V. cholerae O1 by rapid cholera dipstick (DS) assay; all but three samples positive for V. cholerae O1 by CMs were also positive for V. cholerae O1 by DS assay. Of 49 stools that lacked CM-based cholera etiology despite most being positive for V. cholerae O1 by DS assay, 25 (51%) had coccoid V. cholerae O1 cells as confirmed by direct fluorescent antibody (DFA) assay, 36 (73%) amplified primers for the genes wbe O1 and ctxA by multiplex-PCR (M-PCR), and 31 (63%) showed El Tor-specific lytic phage on plaque assay (PA). Each of these methods allowed the cholera etiology to be confirmed for 97% of the stool samples. The results suggest that suspected cholera stools that fail to show etiology by CMs during acute diarrhea outbreaks may be due to the inactivation of V. cholerae by in vivo vibriolytic action of the phage and/or nonculturability induced as a host response.

  3. 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

  4. Acquisition of accurate data from intramolecular quenched fluorescence protease assays.

    PubMed

    Arachea, Buenafe T; Wiener, Michael C

    2017-04-01

    The Intramolecular Quenched Fluorescence (IQF) protease assay utilizes peptide substrates containing donor-quencher pairs that flank the scissile bond. Following protease cleavage, the dequenched donor emission of the product is subsequently measured. Inspection of the IQF literature indicates that rigorous treatment of systematic errors in observed fluorescence arising from inner-filter absorbance (IF) and non-specific intermolecular quenching (NSQ) is incompletely performed. As substrate and product concentrations vary during the time-course of enzyme activity, iterative solution of the kinetic rate equations is, generally, required to obtain the proper time-dependent correction to the initial velocity fluorescence data. Here, we demonstrate that, if the IQF assay is performed under conditions where IF and NSQ are approximately constant during the measurement of initial velocity for a given initial substrate concentration, then a simple correction as a function of initial substrate concentration can be derived and utilized to obtain accurate initial velocity data for analysis.

  5. Variola virus-specific diagnostic assays: characterization, sensitivity, and specificity.

    PubMed

    Kondas, Ashley V; Olson, Victoria A; Li, Yu; Abel, Jason; Laker, Miriam; Rose, Laura; Wilkins, Kimberly; Turner, Jonathan; Kline, Richard; Damon, Inger K

    2015-04-01

    A public health response relies upon rapid and reliable confirmation of disease by diagnostic assays. Here, we detail the design and validation of two variola virus-specific real-time PCR assays, since previous assays cross-reacted with newly identified cowpox viruses. The assay specificity must continually be reassessed as other closely related viruses are identified.

  6. 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.

  7. A lateral electrophoretic flow diagnostic assay.

    PubMed

    Lin, Robert; Skandarajah, Arunan; Gerver, Rachel E; Neira, Hector D; Fletcher, Daniel A; Herr, Amy E

    2015-03-21

    Immunochromatographic assays are a cornerstone tool in disease screening. To complement existing lateral flow assays (based on wicking flow) we introduce a lateral flow format that employs directed electrophoretic transport. The format is termed a "lateral e-flow assay" and is designed to support multiplexed detection using immobilized reaction volumes of capture antigen. To fabricate the lateral e-flow device, we employ mask-based UV photopatterning to selectively immobilize unmodified capture antigen along the microchannel in a barcode-like pattern. The channel-filling polyacrylamide hydrogel incorporates a photoactive moiety (benzophenone) to immobilize capture antigen to the hydrogel without a priori antigen modification. We report a heterogeneous sandwich assay using low-power electrophoresis to drive biospecimen through the capture antigen barcode. Fluorescence barcode readout is collected via a low-resource appropriate imaging system (CellScope). We characterize lateral e-flow assay performance and demonstrate a serum assay for antibodies to the hepatitis C virus (HCV). In a pilot study, the lateral e-flow assay positively identifies HCV+ human sera in 60 min. The lateral e-flow assay provides a flexible format for conducting multiplexed immunoassays relevant to confirmatory diagnosis in near-patient settings.

  8. Diagnostic assays used to control small ruminant lentiviruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The serological diagnostic tests such as the agar gel immunodiffusion (AGID) assay and various types of enzyme linked immunosorbent assays (ELISAs) have contributed to the reduction of small ruminant lentivirus infections worldwide. Since there are no treatments or efficacious vaccines, the serolog...

  9. Field-based multiplex and quantitative assay platforms for diagnostics

    NASA Astrophysics Data System (ADS)

    Venkatasubbarao, Srivatsa; Dixon, C. Edward; Chipman, Russell; Scherer, Axel; Beshay, Manal; Kempen, Lothar U.; Chandra Sekhar, Jai Ganesh; Yan, Hong; Puccio, Ava; Okonkwo, David; McClain, Stephen; Gilbert, Noah; Vyawahare, Saurabh

    2011-06-01

    The U.S. military has a continued interest in the development of handheld, field-usable sensors and test kits for a variety of diagnostic applications, such as traumatic brain injury (TBI) and infectious diseases. Field-use presents unique challenges for biosensor design, both for the readout unit and for the biological assay platform. We have developed robust biosensor devices that offer ultra-high sensitivity and also meet field-use needs. The systems under development include a multiplexed quantitative lateral flow test strip for TBI diagnostics, a field test kit for the diagnosis of pathogens endemic to the Middle East, and a microfluidic assay platform with a label-free reader for performing complex biological automated assays in the field.

  10. Bioengineering of Tobacco Mosaic Virus to Create a Non-Infectious Positive Control for Ebola Diagnostic Assays

    NASA Astrophysics Data System (ADS)

    Lam, Patricia; Gulati, Neetu M.; Stewart, Phoebe L.; Keri, Ruth A.; Steinmetz, Nicole F.

    2016-03-01

    The 2014 Ebola epidemic is the largest to date. There is no cure or treatment for this deadly disease; therefore there is an urgent need to develop new diagnostics to accurately detect Ebola. Current RT-PCR assays lack sensitive and reliable positive controls. To address this critical need, we devised a bio-inspired positive control for use in RT-PCR diagnostics: we encapsulated scrambled Ebola RNA sequences inside of tobacco mosaic virus to create a biomimicry that is non-infectious, but stable, and could therefore serve as a positive control in Ebola diagnostic assays. Here, we report the bioengineering and validation of this probe.

  11. Bioengineering of Tobacco Mosaic Virus to Create a Non-Infectious Positive Control for Ebola Diagnostic Assays

    PubMed Central

    Lam, Patricia; Gulati, Neetu M.; Stewart, Phoebe L.; Keri, Ruth A.; Steinmetz, Nicole F.

    2016-01-01

    The 2014 Ebola epidemic is the largest to date. There is no cure or treatment for this deadly disease; therefore there is an urgent need to develop new diagnostics to accurately detect Ebola. Current RT-PCR assays lack sensitive and reliable positive controls. To address this critical need, we devised a bio-inspired positive control for use in RT-PCR diagnostics: we encapsulated scrambled Ebola RNA sequences inside of tobacco mosaic virus to create a biomimicry that is non-infectious, but stable, and could therefore serve as a positive control in Ebola diagnostic assays. Here, we report the bioengineering and validation of this probe. PMID:27030058

  12. A Rapid Zika Diagnostic Assay to Measure Neutralizing Antibodies in Patients.

    PubMed

    Shan, Chao; Xie, Xuping; Ren, Ping; Loeffelholz, Michael J; Yang, Yujiao; Furuya, Andrea; Dupuis, Alan P; Kramer, Laura D; Wong, Susan J; Shi, Pei-Yong

    2017-03-01

    The potential association of microcephaly and other congenital abnormalities with Zika virus (ZIKV) infection during pregnancy underlines the critical need for a rapid and accurate diagnosis. Due to the short duration of ZIKV viremia in infected patients, a serologic assay that detects antibody responses to viral infection plays an essential role in diagnosing patient specimens. The current serologic diagnosis of ZIKV infection relies heavily on the labor-intensive Plaque Reduction Neutralization Test (PRNT) that requires more than one-week turnaround time and represents a major bottleneck for patient diagnosis. To overcome this limitation, we have developed a high-throughput assay for ZIKV and dengue virus (DENV) diagnosis that can attain the "gold standard" of the current PRNT assay. The new assay is homogeneous and utilizes luciferase viruses to quantify the neutralizing antibody titers in a 96-well format. Using 91 human specimens, we showed that the reporter diagnostic assay has a higher dynamic range and maintains the relative specificity of the traditional PRNT assay. Besides the improvement of assay throughput, the reporter virus technology has also shortened the turnaround time to less than two days. Collectively, our results suggest that, along with the viral RT-PCR assay, the reporter virus-based serologic assay could be potentially used as the first-line test for clinical diagnosis of ZIKV infection as well as for vaccine clinical trials.

  13. Redox-tagged peptide for capacitive diagnostic assays.

    PubMed

    Santos, Adriano; Piccoli, Julia P; Santos-Filho, Norival A; Cilli, Eduardo M; Bueno, Paulo R

    2015-06-15

    Early detection assays play a key role in the successful treatment of most diseases. Redox capacitive biosensors were recently introduced as a potential electroanalytical assay platform for point-of-care applications but alternative surfaces (besides a mixed layer containing ferrocene and antibody receptive component) for recruiting important clinical biomarkers are still needed. Aiming to develop alternative receptive surfaces for this novel electrochemical biosensing platform, we synthesized a ferrocene redox-tagged peptide capable of self-assembly into metallic interfaces, a potentially useful biological surface functionalization for bedside diagnostic assays. As a proof of concept we used C-reactive protein (CRP), as a model biomarker, and compared the obtained results to those of previously reported capacitive assays. The redox-tagged peptide approach shows a limit of detection of 0.8 nmol L(-1) (same as 94 ng mL(-1)) and a linear range (R(2)∼98%) with the logarithm of the concentration of the analyte comprising 0.5-10.0 nmol L(-1), within a clinical relevant range for CRP.

  14. A fluorescence-based quantitative real-time PCR assay for accurate Pocillopora damicornis species identification

    NASA Astrophysics Data System (ADS)

    Thomas, Luke; Stat, Michael; Evans, Richard D.; Kennington, W. Jason

    2016-09-01

    Pocillopora damicornis is one of the most extensively studied coral species globally, but high levels of phenotypic plasticity within the genus make species identification based on morphology alone unreliable. As a result, there is a compelling need to develop cheap and time-effective molecular techniques capable of accurately distinguishing P. damicornis from other congeneric species. Here, we develop a fluorescence-based quantitative real-time PCR (qPCR) assay to genotype a single nucleotide polymorphism that accurately distinguishes P. damicornis from other morphologically similar Pocillopora species. We trial the assay across colonies representing multiple Pocillopora species and then apply the assay to screen samples of Pocillopora spp. collected at regional scales along the coastline of Western Australia. This assay offers a cheap and time-effective alternative to Sanger sequencing and has broad applications including studies on gene flow, dispersal, recruitment and physiological thresholds of P. damicornis.

  15. Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection

    PubMed Central

    Abd El Wahed, Ahmed; Patel, Pranav; Faye, Oumar; Thaloengsok, Sasikanya; Heidenreich, Doris; Matangkasombut, Ponpan; Manopwisedjaroen, Khajohnpong; Sakuntabhai, Anavaj; Sall, Amadou A.; Hufert, Frank T.; Weidmann, Manfred

    2015-01-01

    Background Over 2.5 billion people are exposed to the risk of contracting dengue fever (DF). Early diagnosis of DF helps to diminish its burden on public health. Real-time reverse transcription polymerase amplification assays (RT-PCR) are the standard method for molecular detection of the dengue virus (DENV). Real-time RT-PCR analysis is not suitable for on-site screening since mobile devices are large, expensive, and complex. In this study, two RT-recombinase polymerase amplification (RT-RPA) assays were developed to detect DENV1-4. Methodology/Principal Findings Using two quantitative RNA molecular standards, the analytical sensitivity of a RT-RPA targeting the 3´non-translated region of DENV1-4 was found to range from 14 (DENV4) to 241 (DENV1-3) RNA molecules detected. The assay was specific and did not cross detect other Flaviviruses. The RT-RPA assay was tested in a mobile laboratory combining magnetic-bead based total nucleic acid extraction and a portable detection device in Kedougou (Senegal) and in Bangkok (Thailand). In Kedougou, the RT-RPA was operated at an ambient temperature of 38°C with auxiliary electricity tapped from a motor vehicle and yielded a clinical sensitivity and specificity of 98% (n=31) and 100% (n=23), respectively. While in the field trial in Bangkok, the clinical sensitivity and specificity were 72% (n=90) and 100%(n=41), respectively. Conclusions/Significance During the first 5 days of infection, the developed DENV1-4 RT-RPA assays constitute a suitable accurate and rapid assay for DENV diagnosis. Moreover, the use of a portable fluorescence-reading device broadens its application potential to the point-of-care for outbreak investigations. PMID:26075598

  16. Diagnostic accuracy and turnaround time of the Xpert MTB/RIF assay in routine clinical practice.

    PubMed

    Kwak, Nakwon; Choi, Sun Mi; Lee, Jinwoo; Park, Young Sik; Lee, Chang-Hoon; Lee, Sang-Min; Yoo, Chul-Gyu; Kim, Young Whan; Han, Sung Koo; Yim, Jae-Joon

    2013-01-01

    The Xpert MTB/RIF assay was introduced for timely and accurate detection of tuberculosis (TB). The aim of this study was to determine the diagnostic accuracy and turnaround time (TAT) of Xpert MTB/RIF assay in clinical practice in South Korea. We retrospectively reviewed the medical records of patients in whom Xpert MTB/RIF assay using sputum were requested. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for the diagnosis of pulmonary tuberculosis (PTB) and detection of rifampicin resistance were calculated. In addition, TAT of Xpert MTB/RIF assay was compared with those of other tests. Total 681 patients in whom Xpert MTB/RIF assay was requested were included in the analysis. The sensitivity, specificity, PPV and NPV of Xpert MTB/RIF assay for diagnosis of PTB were 79.5% (124/156), 100.0% (505/505), 100.0% (124/124) and 94.0% (505/537), respectively. Those for the detection of rifampicin resistance were 57.1% (8/14), 100.0% (113/113), 100.0% (8/8) and 94.9% (113/119), respectively. The median TAT of Xpert MTB/RIF assay to the report of results and results confirmed by physicians in outpatient settings were 0 (0-1) and 6 (3-7) days, respectively. Median time to treatment after initial evaluation was 7 (4-9) days in patients with Xpert MTB/RIF assay, but was 21 (7-33.5) days in patients without Xpert MTB/RIF assay. Xpert MTB/RIF assay showed acceptable sensitivity and excellent specificity for the diagnosis of PTB and detection of rifampicin resistance in areas with intermediate TB burden. Additionally, the assay decreased time to the initiation of anti-TB drugs through shorter TAT.

  17. Multicentre comparison of a diagnostic assay: aquaporin-4 antibodies in neuromyelitis optica

    PubMed Central

    Waters, Patrick; Reindl, Markus; Saiz, Albert; Schanda, Kathrin; Tuller, Friederike; Kral, Vlastimil; Nytrova, Petra; Sobek, Ondrej; Nielsen, Helle Hvilsted; Barington, Torben; Lillevang, Søren T; Illes, Zsolt; Rentzsch, Kristin; Berthele, Achim; Berki, Tímea; Granieri, Letizia; Bertolotto, Antonio; Giometto, Bruno; Zuliani, Luigi; Hamann, Dörte; van Pelt, E Daniëlle; Hintzen, Rogier; Höftberger, Romana; Costa, Carme; Comabella, Manuel; Montalban, Xavier; Tintoré, Mar; Siva, Aksel; Altintas, Ayse; Deniz, Günnur; Woodhall, Mark; Palace, Jacqueline; Paul, Friedemann; Hartung, Hans-Peter; Aktas, Orhan; Jarius, Sven; Wildemann, Brigitte; Vedeler, Christian; Ruiz, Anne; Leite, M Isabel; Trillenberg, Peter; Probst, Monika; Saschenbrecker, Sandra; Vincent, Angela; Marignier, Romain

    2016-01-01

    Objective Antibodies to cell surface central nervous system proteins help to diagnose conditions which often respond to immunotherapies. The assessment of antibody assays needs to reflect their clinical utility. We report the results of a multicentre study of aquaporin (AQP) 4 antibody (AQP4-Ab) assays in neuromyelitis optica spectrum disorders (NMOSD). Methods Coded samples from patients with neuromyelitis optica (NMO) or NMOSD (101) and controls (92) were tested at 15 European diagnostic centres using 21 assays including live (n=3) or fixed cell-based assays (n=10), flow cytometry (n=4), immunohistochemistry (n=3) and ELISA (n=1). Results Results of tests on 92 controls identified 12assays as highly specific (0–1 false-positive results). 32 samples from 50 (64%) NMO sera and 34 from 51 (67%) NMOSD sera were positive on at least two of the 12 highly specific assays, leaving 35 patients with seronegative NMO/spectrum disorder (SD). On the basis of a combination of clinical phenotype and the highly specific assays, 66 AQP4-Ab seropositive samples were used to establish the sensitivities (51.5–100%) of all 21 assays. The specificities (85.8–100%) were based on 92 control samples and 35 seronegative NMO/SD patient samples. Conclusions The cell-based assays were most sensitive and specific overall, but immunohistochemistry or flow cytometry could be equally accurate in specialist centres. Since patients with AQP4-Ab negative NMO/SD require different management, the use of both appropriate control samples and defined seronegative NMOSD samples is essential to evaluate these assays in a clinically meaningful way. The process described here can be applied to the evaluation of other antibody assays in the newly evolving field of autoimmune neurology. PMID:27113605

  18. An accurate assay for HCV based on real-time fluorescence detection of isothermal RNA amplification.

    PubMed

    Wu, Xuping; Wang, Jianfang; Song, Jinyun; Li, Jiayan; Yang, Yongfeng

    2016-09-01

    Hepatitis C virus (HCV) is one of the common reasons of liver fibrosis and hepatocellular carcinoma (HCC). Early, rapid and accurate HCV RNA detection is important to prevent and control liver disease. A simultaneous amplification and testing (SAT) assay, which is based on isothermal amplification of RNA and real-time fluorescence detection, was designed to optimize routine HCV RNA detection. In this study, HCV RNA and an internal control (IC) were amplified and analyzed simultaneously by SAT assay and detection of fluorescence using routine real-time PCR equipment. The assay detected as few as 10 copies of HCV RNA transcripts. We tested 705 serum samples with SAT, among which 96.4% (680/705) showed consistent results compared with routine real-time PCR. About 92% (23/25) discordant samples were confirmed to be same results as SAT-HCV by using a second real-time PCR. The sensitivity and specificity of SAT-HCV assay were 99.6% (461/463) and 100% (242/242), respectively. In conclusion, the SAT assay is an accurate test with a high specificity and sensitivity which may increase the detection rate of HCV. It is therefore a promising tool to diagnose HCV infection.

  19. A rapid, simple, and accurate plaque assay for human respiratory syncytial virus (HRSV).

    PubMed

    Kim, Kyung Sook; Kim, Ah Ra; Piao, Ying; Lee, Ju-Hie; Quan, Fu-Shi

    2017-03-31

    Plaque assays of human respiratory syncytial virus (HRSV) are time-consuming, requiring 4 to 7 days for plaque formation and several hours for dye staining. Here, we describe a simple method by which RSV plaques can be visualized and counted with the naked eye only 2 days after infection of HEp-2 cells. In this assay, the infected cells are stained with monoclonal antibodies and the plaques are developed using diaminobenzidine (DAB). We tested the accuracy of this new plaque assay by comparing the results obtained on days 1, 2, 3, 4, 5, and 6 post-infection. The whole procedure is significantly simpler than the traditional method, with an immunostaining process of around 1.5h. Our method is rapid, accurate, and simple; thus, it has the potential to significantly contribute to studies related to RSV disease.

  20. Diagnostic Assays for Polyomavirus JC and Progressive Multifocal Leukoencephalopathy

    PubMed Central

    White, Martyn K.; Sariyer, Ilker K.; Gordon, Jennifer; Delbue, Serena; Pietropaolo, Valeria; Berger, Joseph R.; Khalili, Kamel

    2016-01-01

    SUMMARY Progressive multifocal leukoencephalopathy (PML) is a devastating and often fatal demyelinating disease of the central nervous system (CNS) for which effective therapies are lacking. It is caused by the replication of polyomavirus JC (JCV) in the oligodendrocytes and astrocytes leading to their cytolytic death and loss of myelin from the subcortical white matter. While the virus is very common in human populations worldwide, the incidence of the disease is very low and confined almost exclusively to individuals with some form of immunological dysfunction. However, the number of people who constitute the at-risk population is growing larger and includes individuals with HIV-1/AIDS and patients receiving immunomodulatory therapies such as multiple sclerosis patients treated with natalizumab. Further adding to the public health significance of this disease are the difficulties encountered in the diagnosis of PML and the lack of useful biomarkers for PML progression. In this review, we examine the diagnostic assays that are available for different aspects of the JCV life cycle, their usefulness and drawbacks, and the prospects for improvements. PMID:26663440

  1. 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.

  2. 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

  3. Leveraging arthropod-borne disease surveillance assays for clinical diagnostic use.

    PubMed

    Melanson, Vanessa R; Scheirer, Jessica L; Van de Wyngaerde, Marshall T; Bourzac, Kevin; Wu, Shuenn-Jue; Kochel, Tadeusz; McAvin, James C

    2014-11-01

    Researchers at the Walter Reed Army Institute of Research have taken a joint service approach to filling an identified diagnostic capability gap by leveraging a vector surveillance assay. Specifically, the Army took a field-stable real-time polymerase chain reaction assay, developed by the Air Force, for dengue virus surveillance in arthropod vectors and collaborated with Navy researchers for utility in human diagnostics. As current Department of Defense diagnostic PCR assays employ the Joint Biological Agent Identification and Diagnostic System, the dengue assay was tested for use on this platform. The low rates of false negative and false positive dengue samples in clinical matrices demonstrate excellent utility as a human diagnostic assay. Overall, converting an arboviral vector surveillance assay to human diagnostic assay and potentially vice versa is both cost effective and labor reducing. Codevelopment with harmonization of vector surveillance and diagnostics offers monetary and resource advantages to the Department of Defense and should be considered as a path forward in times when downsizing threatens assay development and pathogen discovery.

  4. Accurate Quantification of Disease Markers in Human Serum Using Iron Oxide Nanoparticle-linked Immunosorbent Assay

    PubMed Central

    Zhang, Linlin; Tong, Sheng; Zhou, Jun; Bao, Gang

    2016-01-01

    Accurate and reliable quantification of biomarkers in the blood is essential in disease screening and diagnosis. Here we describe an iron oxide nanoparticle (IONP)-linked immunosorbent assay (ILISA) for detecting biomolecules in human serum. Sandwich ILISA was optimized for the detection of four important serological markers, IgA, IgG, IgM, and C-reactive protein (CRP), and assessed with normal sera, simulated disease-state sera and the serum samples from patients infected with West Nile virus (WNV) or human herpes virus (HHV). Our study shows that using the detection assay formulated with 18.8 nm wüstite nanocrystals, ILISA can achieve sub-picomolar detection sensitivity, and all four markers can be accurately quantified over a large dynamic range. In addition, ILISA is not susceptible to variations in operating procedures and shows better linearity and higher stability compared with ELISA, which facilitates its integration into detection methods suitable for point of care. Our results demonstrate that ILISA is a simple and versatile nanoplatform for highly sensitive and reliable detection of serological biomarkers in biomedical research and clinical applications. PMID:27375784

  5. Profile of Ventana ALK (D5F3) companion diagnostic assay for non-small-cell lung carcinomas.

    PubMed

    Conde, Esther; Hernandez, Susana; Prieto, Mario; Martinez, Rebeca; Lopez-Rios, Fernando

    2016-06-01

    The development of several ALK inhibitors means that the importance of accurately identifying ALK-positive lung cancer has never been greater. Therefore, it is crucial that ALK testing assays become more standardized. The aim of this review is to comment on the recently FDA-approved VENTANA ALK (D5F3) Companion Diagnostic (CDx) Assay. This kit provides high sensitivity and specificity for the detection of ALK rearrangements and seamless integration into the laboratory workflow, with a fully automated analytical phase and fast interpretation. The use of controls increases the sensitivity and specificity and a dichotomous scoring approach enhances reproducibility.

  6. A versatile assay for the accurate, time-resolved determination of cellular viability.

    PubMed

    Amano, Toyoki; Hirasawa, Ken ichi; O'Donohue, Michael J; Pernolle, Jean Claude; Shioi, Yuzo

    2003-03-01

    A convenient and versatile method for the accurate, time-resolved determination of cellular viability has been developed. The conventional viability indicator fluorescein diacetate (FDA), which is converted to the fluorescent compound fluorescein in living cells, was employed as a viability probe. Fluorescence emission from cells was measured using a spectrofluorimeter equipped with a magnetic stirrer. Using this assay cell suspensions exhibiting densities in the range 0.5 x 10(5) to 2.0 x 10(5) cells displayed a linear response when FDA concentrations less than 12 micro M were employed. To calibrate the method, viability standards were elaborated using different proportions of living and dead cells, and a correlation coefficient for the viability of tobacco BY-2 suspensions was calculated as 0.998. This viability assay was also found to be applicable to Chlamydomonas reinhardtii and Arabidopsis thaliana cultured cells. Using this cell viability assay, kinetic analyses of cell death could be performed. Using the proteinaceous elicitor from Phytophthora cryptogea, cryptogein, to induce cell death in tobacco cell suspensions, values for the maximum velocity of death induction rate (V(max)) and the LD50 (half-maximal velocity or k(1/2)) were calculated as 17.2 (% death/h) and 65 nM, respectively.

  7. The CSFV DNAChip: a novel diagnostic assay for classical swine fever virus.

    PubMed

    Kim, Yong Kwan; Lim, Seong-In; Cho, Yoon-Young; Song, Jae-Young; Kim, JoonBae; An, Dong-Jun

    2014-08-01

    A novel assay, the CSFV DNAChip, was developed to clearly and rapidly discriminate three genotypes of classical swine fever virus (CSFV). Total RNA was extracted from clinical samples and then subjected to a one-step reverse-transcription polymerase chain reaction (RT-PCR) using Cy3-labeled primers from the 5' non-coding region (NCR) of CSFV. Amplicons were hybridized to the CSFV DNAChip and fluorescence scanning was performed for detection of CSFV. A cut-off fluorescence intensity value of 5000 was determined by two-graph receiver operating curve (TG-ROC) analysis. The limit of detection values for the developed DNA chip assay were 0.313ng/μL for amplicon concentration and 1TCID50/100μL for virus titer. Using the developed DNA chip, 157 field samples (91 CSFV-positive and 66 CSFV-negative) were investigated. The genotypes determined by the CSFV DNAChip agreed completely with those determined by nucleotide sequence analysis of the viral genome. The developed CSFV DNAChip will be helpful in implementing a CSFV eradication strategy, as it provides a rapid and accurate diagnostic assay that can discriminate easily among CSFV genotypes.

  8. Diagnostic methodology is critical for accurately determining the prevalence of ichthyophonus infections in wild fish populations

    USGS Publications Warehouse

    Kocan, R.; Dolan, H.; Hershberger, P.

    2011-01-01

    Several different techniques have been employed to detect and identify Ichthyophonus spp. in infected fish hosts; these include macroscopic observation, microscopic examination of tissue squashes, histological evaluation, in vitro culture, and molecular techniques. Examination of the peer-reviewed literature revealed that when more than 1 diagnostic method is used, they often result in significantly different results; for example, when in vitro culture was used to identify infected trout in an experimentally exposed population, 98.7% of infected trout were detected, but when standard histology was used to confirm known infected tissues from wild salmon, it detected ~50% of low-intensity infections and ~85% of high-intensity infections. Other studies on different species reported similar differences. When we examined a possible mechanism to explain the disparity between different diagnostic techniques, we observed non-random distribution of the parasite in 3-dimensionally visualized tissue sections from infected hosts, thus providing a possible explanation for the different sensitivities of commonly used diagnostic techniques. Based on experimental evidence and a review of the peer-reviewed literature, we have concluded that in vitro culture is currently the most accurate diagnostic technique for determining infection prevalence of Ichthyophonus, particularly when the exposure history of the population is not known.

  9. Rapid clinical diagnostics assays using injection-molded planar waveguides

    NASA Astrophysics Data System (ADS)

    Herron, James N.; Wang, Hsu-Kun; Terry, Allen H.; Durtschi, Jacob D.; Tan, Lyndon; Astill, Mark E.; Smith, Richard S.; Christensen, Douglas A.

    1998-04-01

    The goal of our research program is to develop an evanescent wave immunoassay system that can be used in point-of-care and critical care settings. Several key attributes are required to accomplish this goal: (1) the assay system should be at least as sensitive as present day immunoassays; (2) assay time should be 5 minutes or less; (3) the assay protocol should be relatively simple; (4) the sensor should be capable of performing more than one assay on a single specimen; (5) the assay system should be able to accommodate specimens such as serum, plasma and whole blood; and (6) the sensor should be an inexpensive, disposable cartridge. Our laboratory has developed an injection-molded planar waveguide sensor that meets most, if not all, of these attributes. This sensor has been evaluated in a number of different immunoassays for analytes such as bovine serum albumin, human chorionic gonadotrophin, creatine phosphokinase MB and cardiac troponin I.

  10. Analytical applications of MIPs in diagnostic assays: future perspectives.

    PubMed

    Bedwell, Thomas S; Whitcombe, Michael J

    2016-03-01

    Many efforts have been made to produce artificial materials with biomimetic properties for applications in binding assays. Among these efforts, the technique of molecular imprinting has received much attention because of the high selectivity obtainable for molecules of interest, robustness of the produced polymers, simple and short synthesis, and excellent cost efficiency. In this review, progress in the field of molecularly imprinted sorbent assays is discussed-with a focus on work conducted from 2005 to date.

  11. Using Copula Distributions to Support More Accurate Imaging-Based Diagnostic Classifiers for Neuropsychiatric Disorders

    PubMed Central

    Bansal, Ravi; Hao, Xuejun; Liu, Jun; Peterson, Bradley S.

    2014-01-01

    Many investigators have tried to apply machine learning techniques to magnetic resonance images (MRIs) of the brain in order to diagnose neuropsychiatric disorders. Usually the number of brain imaging measures (such as measures of cortical thickness and measures of local surface morphology) derived from the MRIs (i.e., their dimensionality) has been large (e.g. >10) relative to the number of participants who provide the MRI data (<100). Sparse data in a high dimensional space increases the variability of the classification rules that machine learning algorithms generate, thereby limiting the validity, reproducibility, and generalizability of those classifiers. The accuracy and stability of the classifiers can improve significantly if the multivariate distributions of the imaging measures can be estimated accurately. To accurately estimate the multivariate distributions using sparse data, we propose to estimate first the univariate distributions of imaging data and then combine them using a Copula to generate more accurate estimates of their multivariate distributions. We then sample the estimated Copula distributions to generate dense sets of imaging measures and use those measures to train classifiers. We hypothesize that the dense sets of brain imaging measures will generate classifiers that are stable to variations in brain imaging measures, thereby improving the reproducibility, validity, and generalizability of diagnostic classification algorithms in imaging datasets from clinical populations. In our experiments, we used both computer-generated and real-world brain imaging datasets to assess the accuracy of multivariate Copula distributions in estimating the corresponding multivariate distributions of real-world imaging data. Our experiments showed that diagnostic classifiers generated using imaging measures sampled from the Copula were significantly more accurate and more reproducible than were the classifiers generated using either the real-world imaging

  12. Diagnostic yield of blood clot culture in the accurate diagnosis of enteric fever and human brucellosis.

    PubMed

    Mantur, Basappa G; Bidari, Laxman H; Akki, Aravind S; Mulimani, Mallanna S; Tikare, Nitin V

    2007-01-01

    Culture of blood is the most frequent, accurate means of diagnosing bacteremia in enteric fever and brucellosis. However, conventional blood culturing is slow in isolating bacteria causing these diseases. In this work, we evaluated the performance of blood clot culture and conventional whole blood cultures in the accurate diagnosis of enteric fever (253 cases) and human brucellosis (71cases). The blood clot culture was found to be much more sensitive for both Salmonella (more by 34.4%, P< 0.001) and Brucella (more by 22.6%, P<0.001) than whole blood culture. Bacterial growth was significantly faster in cultures of blood clot compared to whole blood (1.1 versus 2.6 days for Salmonella, 3.1 versus 8.2 days for Brucella melitensis, respectively). The rapid confirmation of the etiological agent would facilitate an early institution of appropriate antimicrobial therapy, thereby reducing clinical morbidity especially in an endemic population. It is worthwile practicing blood clot culture for the accurate diagnosis of enteric fever and brucellosis in developing countries where diagnostic facilities by advanced technologies like automated culture systems and PCR are not available.

  13. Limitations of automated remnant lipoprotein cholesterol assay for diagnostic use

    Technology Transfer Automated Retrieval System (TEKTRAN)

    I wish to comment on the limitations of automated remnant lipoprotein cholesterol (RemL-C) assay reported in Clinical Chemistry. Remnants are lipoprotein particles produced after newly formed triglyceride-rich lipoproteins (TRLs) of either hepatic or intestinal origin enter the plasma space and unde...

  14. Molecular diagnostic assays for infectious diseases in cats.

    PubMed

    Veir, Julia K; Lappin, Michael R

    2010-11-01

    With the advent of more accessible polymerase chain reaction panels, the use of molecular techniques for the detection of infectious organisms has become more routine in veterinary medicine. The use of molecular diagnostics is best reserved for the detection of organisms that are difficult to detect or identify expediently. In this article, the fundamentals of molecular techniques are reviewed along with an examination of specific feline infectious diseases in which diagnosis via molecular techniques is advantageous.

  15. Robust and accurate single nucleotide polymorphism genotyping by dynamic allele-specific hybridization (DASH): design criteria and assay validation.

    PubMed

    Prince, J A; Feuk, L; Howell, W M; Jobs, M; Emahazion, T; Blennow, K; Brookes, A J

    2001-01-01

    We recently introduced a generic single nucleotide polymorphism (SNP) genotyping method, termed DASH (dynamic allele-specific hybridization), which entails dynamic tracking of probe (oligonucleotide) to target (PCR product) hybridization as reaction temperature is steadily increased. The reliability of DASH and optimal design rules have not been previously reported. We have now evaluated crudely designed DASH assays (sequences unmodified from genomic DNA) for 89 randomly selected and confirmed SNPs. Accurate genotype assignment was achieved for 89% of these worst-case-scenario assays. Failures were determined to be caused by secondary structures in the target molecule, which could be reliably predicted from thermodynamic theory. Improved design rules were thereby established, and these were tested by redesigning six of the failed DASH assays. This involved reengineering PCR primers to eliminate amplified target sequence secondary structures. This sophisticated design strategy led to complete functional recovery of all six assays, implying that SNPs in most if not all sequence contexts can be effectively scored by DASH. Subsequent empirical support for this inference has been evidenced by approximately 30 failure-free DASH assay designs implemented across a range of ongoing genotyping programs. Structured follow-on studies employed standardized assay conditions, and revealed that assay reproducibility (733 duplicated genotypes, six different assays) was as high as 100%, with an assay accuracy (1200 genotypes, three different assays) that exceeded 99.9%. No post-PCR assay failures were encountered. These findings, along with intrinsic low cost and high flexibility, validate DASH as an effective procedure for SNP genotyping.

  16. [Evaluation of the diagnostic power of 3 methods for assaying free T4. Results in the diagnostic strategy of hyperthyroidism].

    PubMed

    Fragu, P; Noel, M; Patois, E; Delarue, J C; Paugam-Capelle, J; Parmentier, C

    1985-01-01

    The free thyroxin (FT4) tests of Amersham, Clinical Assay and Corning Medical were evaluated in 240 patients who were suspected of hyperthyroidism. The diagnostic performances of the Corning method were of less value while those of the other methods were equivalent to that obtained with the free thyroxin index for an average cost reduced. Furthermore our results show that T3 determination is better than T4 determination in patients who remained doubtful after FT4. However the development of ultra-short methods of measurement of total thyroid hormone blood levels using fluorescence polarization could lead to reconsider the diagnostic strategy of hyperthyroidism.

  17. A diagnostic polymerase chain reaction assay for Zika virus.

    PubMed

    Balm, Michelle N D; Lee, Chun Kiat; Lee, Hong Kai; Chiu, Lily; Koay, Evelyn S C; Tang, Julian W

    2012-09-01

    Zika virus (ZIKV) is a mosquito-borne flavivirus. Infection results in a dengue-like illness with fever, headache, malaise, and a maculopapular rash. Nearly all cases are mild and self-limiting but in 2007, a large outbreak of ZIKV was reported from the island of Yap (in Micronesia, northwest of Indonesia). Singapore is already endemic for dengue, and its impact on public health and economic burden is significant. Other dengue-like infections (e.g., Chikungunya virus) are present. Yet only 10% of reported dengue cases have laboratory confirmation. The identification and control of other dengue-like, mosquito-transmitted infections is thus important for the health of Singapore's population, as well as its economy. Given that ZIKV shares the same Aedes mosquito vector with both dengue and Chikungunya, it is possible that this virus is present in Singapore and causing some of the mild dengue-like illness. A specific and sensitive one-step, reverse transcription polymerase chain reaction (RT-PCR) with an internal control (IC) was designed and tested on 88 archived samples of dengue-negative, Chikungunya-negative sera from patients presenting to our hospital with a dengue-like illness, to determine the presence of ZIKV in Singapore. The assay was specific for detection of ZIKV and displayed a lower limit of detection (LoD) of 140 copies viral RNA/reaction when tested on synthetic RNA standards prepared using pooled negative patient plasma. Of the 88 samples tested, none were positive for ZIKV RNA, however, the vast majority of these were from patients admitted to hospital and further study may be warranted in community-based environments.

  18. Stationary microfluidics: molecular diagnostic assays by moving magnetic beads through non-moving liquids

    NASA Astrophysics Data System (ADS)

    Becker, Holger; Carstens, Cornelia; Kuhlmeier, Dirk; Sandetskaya, Natalia; Schröter, Nicole; Zilch, Christian; Gärtner, Claudia

    2013-03-01

    Commonly, microfluidic devices are based on the movement of fluids. For molecular diagnostics assays which often include steps like PCR, this practically always involves a more or less complicated set of external pumps, valves and liquid controls. In the presented paper, we follow a different approach in which the fluid after sample introduction remains stationary and the main bioactive sample molecules are moved through a chain of reaction compartments which contain the different reagents necessary for the assay. The big advantage of this concept is the lack of any external fluid actuation/control. Results on sample carry-over experiments and complete assays will be given.

  19. Screening metagenomic data for viruses using the E-Probe Diagnostic Nucleic Acid Assay (EDNA)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There are many plant pathogen-specific diagnostic assays, based on PCR and immune-detection. However, the ability to test for large numbers of pathogens simultaneously is lacking. Next generation sequencing (NGS) allows one to detect all organisms within a given sample, but has computational limitat...

  20. An improved Bathocuproine assay for accurate valence identification and quantification of copper bound by biomolecules.

    PubMed

    Chen, Dinglong; Darabedian, Narek; Li, Zhiqiang; Kai, Tianhan; Jiang, Dianlu; Zhou, Feimeng

    2016-03-15

    Copper is an essential metal in all organisms. Reliably quantifying and identifying the copper content and oxidation state is crucial, since the information is essential to understanding protein structure and function. Chromophoric ligands, such as Bathocuproine (BC) and its water-soluble analog, Bathocuproinedisulfonic acid (BCS), preferentially bind Cu(I) over Cu(II), and therefore have been widely used as optical probes to determine the oxidation state of copper bound by biomolecules. However, the BCS assay is commonly misused, leading to erroneous conclusions regarding the role of copper in biological processes. By measuring the redox potential of Cu(II)-BCS2 and conducting UV-vis absorption measurements in the presence of oxidizable amino acids, the thermodynamic origin of the potential artifacts becomes evident. The BCS assay was improved by introducing a strong Cu(II) chelator EDTA prior to the addition of BCS to prevent interference that might arise from Cu(II) present in the sample. The strong Cu(II) chelator rids of all the potential errors inherent in the conventional BCS assay. Applications of the improved assay to peptides and protein containing oxidizable amino acid residues confirm that free Cu(II) no longer leads to artifacts, thereby resolving issues related to this persistently misused colorimetric assay of Cu(I) in biological systems.

  1. An accurate DNA marker assay for stem rust resistance gene Sr2 in wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The stem rust resistance gene Sr2 has provided broad-spectrum protection against stem rust (Puccinia graminis) since its wide spread deployment in wheat from the 1940s. Because Sr2 confers partial resistance which is difficult to select under field conditions, a DNA marker is desirable that accurate...

  2. Development and Validation of a Highly Accurate Quantitative Real-Time PCR Assay for Diagnosis of Bacterial Vaginosis

    PubMed Central

    Smith, William L.; Chadwick, Sean G.; Toner, Geoffrey; Mordechai, Eli; Adelson, Martin E.; Aguin, Tina J.; Sobel, Jack D.

    2016-01-01

    Bacterial vaginosis (BV) is the most common gynecological infection in the United States. Diagnosis based on Amsel's criteria can be challenging and can be aided by laboratory-based testing. A standard method for diagnosis in research studies is enumeration of bacterial morphotypes of a Gram-stained vaginal smear (i.e., Nugent scoring). However, this technique is subjective, requires specialized training, and is not widely available. Therefore, a highly accurate molecular assay for the diagnosis of BV would be of great utility. We analyzed 385 vaginal specimens collected prospectively from subjects who were evaluated for BV by clinical signs and Nugent scoring. We analyzed quantitative real-time PCR (qPCR) assays on DNA extracted from these specimens to quantify nine organisms associated with vaginal health or disease: Gardnerella vaginalis, Atopobium vaginae, BV-associated bacteria 2 (BVAB2, an uncultured member of the order Clostridiales), Megasphaera phylotype 1 or 2, Lactobacillus iners, Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus jensenii. We generated a logistic regression model that identified G. vaginalis, A. vaginae, and Megasphaera phylotypes 1 and 2 as the organisms for which quantification provided the most accurate diagnosis of symptomatic BV, as defined by Amsel's criteria and Nugent scoring, with 92% sensitivity, 95% specificity, 94% positive predictive value, and 94% negative predictive value. The inclusion of Lactobacillus spp. did not contribute sufficiently to the quantitative model for symptomatic BV detection. This molecular assay is a highly accurate laboratory tool to assist in the diagnosis of symptomatic BV. PMID:26818677

  3. An extended set of yeast-based functional assays accurately identifies human disease mutations

    PubMed Central

    Sun, Song; Yang, Fan; Tan, Guihong; Costanzo, Michael; Oughtred, Rose; Hirschman, Jodi; Theesfeld, Chandra L.; Bansal, Pritpal; Sahni, Nidhi; Yi, Song; Yu, Analyn; Tyagi, Tanya; Tie, Cathy; Hill, David E.; Vidal, Marc; Andrews, Brenda J.; Boone, Charles; Dolinski, Kara; Roth, Frederick P.

    2016-01-01

    We can now routinely identify coding variants within individual human genomes. A pressing challenge is to determine which variants disrupt the function of disease-associated genes. Both experimental and computational methods exist to predict pathogenicity of human genetic variation. However, a systematic performance comparison between them has been lacking. Therefore, we developed and exploited a panel of 26 yeast-based functional complementation assays to measure the impact of 179 variants (101 disease- and 78 non-disease-associated variants) from 22 human disease genes. Using the resulting reference standard, we show that experimental functional assays in a 1-billion-year diverged model organism can identify pathogenic alleles with significantly higher precision and specificity than current computational methods. PMID:26975778

  4. Design considerations for high-throughput screening and in vitro diagnostic assays.

    PubMed

    Achyuthan, Komandoor E; Whitten, David G

    2007-07-01

    This paper reviews the several different factors that must be considered during the development of assays for high throughput screening (HTS) or in vitro diagnostic (IVD) applications. The reader is introduced to the terminology used in assay development as well as the statistical approaches for evaluating the data. The review is intended to serve as a tutorial to biotechnology, pharmaceutical and clinical professionals, the academic researcher, as well as a guide for established investigators of HTS and IVD. This review is not a comprehensive treatise in its scope or content, but is meant to introduce the reader to key concepts of assay development. Elementary mathematical and statistical tools for designing robust assays and data management are described. While certain design concepts overlap HTS and IVDs, others are more pertinent to one or the other topic. An overview of the regulatory requirements for IVDs is included in the context of the United States Food and Drug Administration. Quality concepts and high content screening are also briefly described. The review does not focus upon any particular assay technology nor does it provide detailed laboratory procedures on specific assays. The references cited are not exhaustive, but meant to steer the reader toward a general status report of the various technologies discussed. The information presented in this review is not intended to replace the judgment of the experienced laboratory scientist. However, this review should assist the scientific professional in executing well designed assays and being aware of design considerations.

  5. Characterization of the Xanthomonas translucens Complex Using Draft Genomes, Comparative Genomics, Phylogenetic Analysis, and Diagnostic LAMP Assays.

    PubMed

    Langlois, Paul A; Snelling, Jacob; Hamilton, John P; Bragard, Claude; Koebnik, Ralf; Verdier, Valérie; Triplett, Lindsay R; Blom, Jochen; Tisserat, Ned A; Leach, Jan E

    2017-03-21

    Prevalence of Xanthomonas translucens, which causes cereal leaf streak (CLS) in cereal crops and bacterial wilt in forage and turfgrass species, has increased in many regions in recent years. Because the pathogen is seedborne in economically important cereals, it is a concern for international and interstate germplasm exchange and, thus, reliable and robust protocols for its detection in seed are needed. However, historical confusion surrounding the taxonomy within the species has complicated the development of accurate and reliable diagnostic tools for X. translucens. Therefore, we sequenced genomes of 15 X. translucens strains representing six different pathovars and compared them with additional publicly available X. translucens genome sequences to obtain a genome-based phylogeny for robust classification of this species. Our results reveal three main clusters: one consisting of pv. cerealis, one consisting of pvs. undulosa and translucens, and a third consisting of pvs. arrhenatheri, graminis, phlei, and poae. Based on genomic differences, diagnostic loop-mediated isothermal amplification (LAMP) primers were developed that clearly distinguish strains that cause disease on cereals, such as pvs. undulosa, translucens, hordei, and secalis, from strains that cause disease on noncereal hosts, such as pvs. arrhenatheri, cerealis, graminis, phlei, and poae. Additional LAMP assays were developed that selectively amplify strains belonging to pvs. cerealis and poae, distinguishing them from other pathovars. These primers will be instrumental in diagnostics when implementing quarantine regulations to limit further geographic spread of X. translucens pathovars.

  6. Optimization of a phage amplification assay to permit accurate enumeration of viable Mycobacterium avium subsp. paratuberculosis cells.

    PubMed

    Foddai, Antonio; Elliott, Christopher T; Grant, Irene R

    2009-06-01

    A commercially available phage amplification assay, FASTPlaqueTB (Biotec Laboratories, Ipswich, United Kingdom), when used according to the manufacturer's instructions, does not permit accurate enumeration of Mycobacterium avium subsp. paratuberculosis. The aim of this study was to optimize the phage amplification assay conditions to permit accurate quantification of viable M. avium subsp. paratuberculosis cells. The burst time for M. avium subsp. paratuberculosis was initially determined to inform decisions about optimal incubation time before plating, and then other test parameters were altered to evaluate how the correlation between plaque and colony counts was affected. The D29 mycobacteriophage replicates more slowly in M. avium subsp. paratuberculosis than in Mycobacterium smegmatis (used to optimize the commercial test originally), and the mean burst time for four M. avium subsp. paratuberulosis strains was 210 +/- 36.8 min at 37 degrees C compared to 63 +/- 17.5 min for M. smegmatis mc(2) 155. To achieve 100% correlation between plaque and colony counts, the optimized phage assay includes the following: (i) resuspension of the samples to be tested in Middlebrook 7H9 broth containing 10% oleic acid-albumin-dextrose-catalase and 2 mM calcium chloride, followed by overnight incubation at 37 degrees C before performance of the phage assay; (ii) a 2-h incubation of the sample with D29 mycobacteriophage before viricide treatment; and (iii) a further 90-min incubation after viricide treatment and neutralization up to the burst time (total incubation time, 210 min) before plating with M. smegmatis mc(2) 155 in 7H9 agar. The optimized phage amplification assay was able to detect 1 to 10 CFU/ml of M. avium subsp. paratuberculosis in spiked milk or broth within 48 h, as demonstrated by the results of several blind trials.

  7. Diagnostic accuracy of tumor necrosis factor-alpha assay for tuberculous pleurisy

    PubMed Central

    Li, Min; Luo, Zhuang; Zhu, Wenye; Khan, Rana Sami Ullah; Ummair, Saeed Ummai; Shi, Shaoqing

    2016-01-01

    Abstract Background: The diagnosis of tuberculous pleurisy is difficult and traditional methods are not always helpful. Many studies have focused on the tumor necrosis factor-alpha (TNF-α) assay in pleural effusion for the diagnosis of tuberculous pleurisy, but the results remain controversial. This meta-analysis was conducted to determine the overall diagnostic accuracy of TNF-α. Methods: Relevant studies were searched from PubMed, Embase, Web of Science, Chinese National Knowledge Infrastructure (CNKI), Wangfang, and Weipu. We pooled the published results and computed the accuracy measures, including sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR). Receiver operating characteristic curves (SROC) and the area under the curve (AUC) were used to summarize the overall test performance. Results: Twelve studies with 1022 patients met the inclusion criteria. The pooled sensitivity and specificity were 0.85 (95%CI, 0.81–0.89) and 0.80 (95% CI, 0.77–0.83) respectively. The area under the SROC curve was 0.89. Conclusions: The results of meta-analysis suggested that the TNF-α assay plays a vital role in the diagnosis of tuberculous pleurisy, whereas other test results or clinical findings should be interpreted together with the TNF-α assay to improve the overall diagnostic accuracy. PMID:27902616

  8. Impact of Clinical Symptoms on Interpretation of Diagnostic Assays for Clostridium difficile Infections▿

    PubMed Central

    Dubberke, Erik R.; Han, Zhuolin; Bobo, Linda; Hink, Tiffany; Lawrence, Brenda; Copper, Susan; Hoppe-Bauer, Joan; Burnham, Carey-Ann D.; Dunne, William Michael

    2011-01-01

    Asymptomatic Clostridium difficile colonization is common in hospitalized patients. Existing C. difficile assay comparisons lack data on severity of diarrhea or patient outcomes, limiting the ability to interpret their results in regard to the diagnosis of C. difficile infection (CDI). The objective of this study was to measure how including patient presentation with the C. difficile assay result impacted assay performance to diagnose CDI. Stool specimens from 150 patients that met inclusion and exclusion criteria were selected. Nine methods to detect C. difficile in stool were evaluated. All patients were interviewed prospectively to assess diarrhea severity. We then assessed how different reference standards, with and without the inclusion of patient presentation, impact the sensitivity, specificity, and positive and negative predictive values of the assays to diagnose CDI. There were minimal changes in sensitivity; however, specificity was significantly lower for the assays Tox A/B II, C. diff Chek-60, BD GeneOhm Cdiff, Xpert C. difficile, and Illumigene C. difficile and for toxigenic culture (P was <0.01 for all except Tox A/B II from fresh stool, for which the P value was 0.016) when the reference standard was recovery of toxigenic C. difficile from stool plus the presence of clinically significant diarrhea compared to when the reference standard was having at least four assays positive while ignoring diarrhea severity. There were 15 patients whose assay result was reported as negative but subsequently found to be positive by at least four assays in the comparison. None suffered from any CDI-related adverse events. In conclusion, clinical presentation is important when interpreting C. difficile diagnostic assays. PMID:21697328

  9. Diagnostic value of an enzyme-linked immunospot assay for interferon-γ in genitourinary tuberculosis.

    PubMed

    Lai, Chih-Cheng; Tan, Che-Kim; Lin, Sheng-Hsiang; Liao, Chun-Hsing; Huang, Yu-Tsung; Wang, Cheng-Yi; Wang, Jen-Yu; Lin, Hen-I; Hsueh, Po-Ren

    2010-11-01

    The aim of this study was to evaluate the diagnostic performance of an enzyme-linked immunospot (ELISPOT) assay for interferon-γ in patients with suspected genitourinary tuberculosis (TB). A total of 30 patients with suspected genitourinary TB at the National Taiwan University Hospital, Taipei, Taiwan, were prospectively enrolled from January 2007 to December 2009, and 12 of whom had positive urine culture for Mycobacterium tuberculosis. Frequency and dysuria were the most common symptoms noted in 6 (50.0%) and 4 (33.3%) patients, respectively. Pyuria was the most common finding of urinalysis noted in 11 (91.7%) patients. Six (50.0%) patients had positive acid-fast stain in urine. Among the 30 patients, 13 patients had positive ELISPOT assay. Eleven patients with positive ELISPOT assay had culture-confirmed TB, and the remaining 2 patients without evidence of active TB had positive ELISPOT assay. The overall sensitivity, specificity, positive predictive value, and negative predictive value for genitourinary TB diagnosis by the ELISPOT assay were 91.7% (95% confidence interval [CI], 59.8-99.6%), 88.9% (95% CI, 63.9-98.1%), 84.6% (95% CI, 53.7-97.3%), and 94.1% (95% CI, 69.2-96.7%), respectively. In conclusion, ELISPOT assay can provide useful support in diagnosing genitourinary TB.

  10. Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories.

    PubMed

    Christopher-Hennings, Jane; Araujo, Karla P C; Souza, Carlos J H; Fang, Ying; Lawson, Steven; Nelson, Eric A; Clement, Travis; Dunn, Michael; Lunney, Joan K

    2013-11-01

    Bead-based multiplex assays (BBMAs) are applicable for high throughput, simultaneous detection of multiple analytes in solution (from several to 50-500 analytes within a single, small sample volume). Currently, few assays are commercially available for veterinary applications, but they are available to identify and measure various cytokines, growth factors and their receptors, inflammatory proteins, kinases and inhibitors, neurobiology proteins, and pathogens and antibodies in human beings, nonhuman primates, and rodent species. In veterinary medicine, various nucleic acid and protein-coupled beads can be used in, or for the development of, antigen and antibody BBMAs, with the advantage that more data can be collected using approximately the same amount of labor as used for other antigen and antibody assays. Veterinary-related BBMAs could be used for detection of pathogens, genotyping, measurement of hormone levels, and in disease surveillance and vaccine assessment. It will be important to evaluate whether BBMAs are "fit for purpose," how costs and efficiencies compare between assays, which assays are published or commercially available for specific veterinary applications, and what procedures are involved in the development of the assays. It is expected that many veterinary-related BBMAs will be published and/or become commercially available in the next few years. The current review summarizes the BBMA technology and some of the currently available BBMAs developed for veterinary settings. Some of the human diagnostic BBMAs are also described, providing an example of possible templates for future development of new veterinary-related BBMAs.

  11. B-type natriuretic peptide rapid assay: a diagnostic test for heart failure.

    PubMed

    Ancheta, Irma B

    2006-01-01

    Hospitals are constantly besieged with congestive heart failure admissions. Current studies show that the advent of the B-type natriuretic peptide (BNP) rapid assay as a quick and easy blood test is beneficial to nurses in confirming the diagnosis of heart failure. B-type natriuretic peptide is a neurohormone produced by the failing heart in response to increased volume and cardiac overload. The BNP rapid assay measures the presence of BNP levels present in the circulating bloodstream to confirm the diagnosis of congestive heart failure. It is a simple blood test that can be done at the bedside or at the clinic so it is a valid point-of-care modality. Elevated levels suggest severity of heart failure and possibility of sudden death. This article focuses on the description of the diagnostic performance of the BNP rapid assay, its clinical dimensions, and its implications to nursing practice and collaborative practice models.

  12. Development and Validation of a Highly Accurate Quantitative Real-Time PCR Assay for Diagnosis of Bacterial Vaginosis.

    PubMed

    Hilbert, David W; Smith, William L; Chadwick, Sean G; Toner, Geoffrey; Mordechai, Eli; Adelson, Martin E; Aguin, Tina J; Sobel, Jack D; Gygax, Scott E

    2016-04-01

    Bacterial vaginosis (BV) is the most common gynecological infection in the United States. Diagnosis based on Amsel's criteria can be challenging and can be aided by laboratory-based testing. A standard method for diagnosis in research studies is enumeration of bacterial morphotypes of a Gram-stained vaginal smear (i.e., Nugent scoring). However, this technique is subjective, requires specialized training, and is not widely available. Therefore, a highly accurate molecular assay for the diagnosis of BV would be of great utility. We analyzed 385 vaginal specimens collected prospectively from subjects who were evaluated for BV by clinical signs and Nugent scoring. We analyzed quantitative real-time PCR (qPCR) assays on DNA extracted from these specimens to quantify nine organisms associated with vaginal health or disease:Gardnerella vaginalis,Atopobium vaginae, BV-associated bacteria 2 (BVAB2, an uncultured member of the orderClostridiales),Megasphaeraphylotype 1 or 2,Lactobacillus iners,Lactobacillus crispatus,Lactobacillus gasseri, andLactobacillus jensenii We generated a logistic regression model that identifiedG. vaginalis,A. vaginae, andMegasphaeraphylotypes 1 and 2 as the organisms for which quantification provided the most accurate diagnosis of symptomatic BV, as defined by Amsel's criteria and Nugent scoring, with 92% sensitivity, 95% specificity, 94% positive predictive value, and 94% negative predictive value. The inclusion ofLactobacillusspp. did not contribute sufficiently to the quantitative model for symptomatic BV detection. This molecular assay is a highly accurate laboratory tool to assist in the diagnosis of symptomatic BV.

  13. An accurate DNA marker assay for stem rust resistance gene Sr2 in wheat.

    PubMed

    Mago, R; Simkova, H; Brown-Guedira, G; Dreisigacker, S; Breen, J; Jin, Y; Singh, R; Appels, R; Lagudah, E S; Ellis, J; Dolezel, J; Spielmeyer, W

    2011-03-01

    The stem rust resistance gene Sr2 has provided broad-spectrum protection against stem rust (Puccinia graminis Pers. f. sp. tritici) since its wide spread deployment in wheat from the 1940s. Because Sr2 confers partial resistance which is difficult to select under field conditions, a DNA marker is desirable that accurately predicts Sr2 in diverse wheat germplasm. Using DNA sequence derived from the vicinity of the Sr2 locus, we developed a cleaved amplified polymorphic sequence (CAPS) marker that is associated with the presence or absence of the gene in 115 of 122 (95%) diverse wheat lines. The marker genotype predicted the absence of the gene in 100% of lines which were considered to lack Sr2. Discrepancies were observed in lines that were predicted to carry Sr2 but failed to show the CAPS marker. Given the high level of accuracy observed, the marker provides breeders with a selection tool for one of the most important disease resistance genes of wheat.

  14. Surface enhanced Raman spectroscopy based nanoparticle assays for rapid, point-of-care diagnostics

    NASA Astrophysics Data System (ADS)

    Driscoll, Ashley J.

    Nucleotide and immunoassays are important tools for disease diagnostics. Many of the current laboratory-based analytical diagnostic techniques require multiple assay steps and long incubation times before results are acquired. In the development of bioassays designed for detecting the emergence and spread of diseases in point-of-care (POC) and remote settings, more rapid and portable analytical methods are necessary. Nanoparticles provide simple and reproducible synthetic methods for the preparation of substrates that can be applied in colloidal assays, providing gains in kinetics due to miniaturization and plasmonic substrates for surface enhanced spectroscopies. Specifically, surface enhanced Raman spectroscopy (SERS) is finding broad application as a signal transduction method in immunological and nucleotide assays due to the production of narrow spectral peaks from the scattering molecules and the potential for simultaneous multiple analyte detection. The application of SERS to a no-wash, magnetic capture assay for the detection of West Nile Virus Envelope and Rift Valley Fever Virus N antigens is described. The platform utilizes colloid based capture of the target antigen in solution, magnetic collection of the immunocomplexes and acquisition of SERS spectra by a handheld Raman spectrometer. The reagents for a core-shell nanoparticle, SERS based assay designed for the capture of target microRNA implicated in acute myocardial infarction are also characterized. Several new, small molecule Raman scatterers are introduced and used to analyze the enhancing properties of the synthesized gold coated-magnetic nanoparticles. Nucleotide and immunoassay platforms have shown improvements in speed and analyte capture through the miniaturization of the capture surface and particle-based capture systems can provide a route to further surface miniaturization. A reaction-diffusion model of the colloidal assay platform is presented to understand the interplay of system

  15. Direct Reading of Bona Fide Barcode Assays for Diagnostics with Smartphone Apps

    PubMed Central

    Wong, Jessica X. H.; Li, Xiaochun; Liu, Frank S. F.; Yu, Hua-Zhong

    2015-01-01

    The desire to develop new point-of-care (POC) diagnostic tools has led to the adaptation of smartphones to tackle limitations in state-of-the-art instrumentation and centralized laboratory facilities. Today’s smartphones possess the computer-like ability to image and process data using mobile apps; barcode scanners are one such type of apps. We demonstrate herein that a diagnostic assay can be performed by patterning immunoassay strips in a bona fide barcode format such that after target binding and signal enhancement, the linear barcode can be read directly with a standard smartphone app. Quantitative analysis can then be performed based on the grayscale intensities with a customized mobile app. This novel diagnostic concept has been validated for a real-world application, i.e., the detection of human chorionic gonadotropin, a pregnancy hormone. With the possibility of multiplex detection, the barcode assay protocol promises to boost POC diagnosis research by the direct adaptation of mobile devices and apps. PMID:26122608

  16. Direct Reading of Bona Fide Barcode Assays for Diagnostics with Smartphone Apps

    NASA Astrophysics Data System (ADS)

    Wong, Jessica X. H.; Li, Xiaochun; Liu, Frank S. F.; Yu, Hua-Zhong

    2015-06-01

    The desire to develop new point-of-care (POC) diagnostic tools has led to the adaptation of smartphones to tackle limitations in state-of-the-art instrumentation and centralized laboratory facilities. Today’s smartphones possess the computer-like ability to image and process data using mobile apps; barcode scanners are one such type of apps. We demonstrate herein that a diagnostic assay can be performed by patterning immunoassay strips in a bona fide barcode format such that after target binding and signal enhancement, the linear barcode can be read directly with a standard smartphone app. Quantitative analysis can then be performed based on the grayscale intensities with a customized mobile app. This novel diagnostic concept has been validated for a real-world application, i.e., the detection of human chorionic gonadotropin, a pregnancy hormone. With the possibility of multiplex detection, the barcode assay protocol promises to boost POC diagnosis research by the direct adaptation of mobile devices and apps.

  17. Smartphone-Based Accurate Analysis of Retinal Vasculature towards Point-of-Care Diagnostics

    PubMed Central

    Xu, Xiayu; Ding, Wenxiang; Wang, Xuemin; Cao, Ruofan; Zhang, Maiye; Lv, Peilin; Xu, Feng

    2016-01-01

    Retinal vasculature analysis is important for the early diagnostics of various eye and systemic diseases, making it a potentially useful biomarker, especially for resource-limited regions and countries. Here we developed a smartphone-based retinal image analysis system for point-of-care diagnostics that is able to load a fundus image, segment retinal vessels, analyze individual vessel width, and store or uplink results. The proposed system was not only evaluated on widely used public databases and compared with the state-of-the-art methods, but also validated on clinical images directly acquired with a smartphone. An Android app is also developed to facilitate on-site application of the proposed methods. Both visual assessment and quantitative assessment showed that the proposed methods achieved comparable results to the state-of-the-art methods that require high-standard workstations. The proposed system holds great potential for the early diagnostics of various diseases, such as diabetic retinopathy, for resource-limited regions and countries. PMID:27698369

  18. High-Throughput Diagnostic Assay for a Highly Prevalent Cardiomyopathy-Associated MYBPC3 Variant

    PubMed Central

    Barefield, David Y; Lynch, Thomas L; Jagadeesan, Aravindakshan; Sanagala, Thriveni; Sadayappan, Sakthivel

    2016-01-01

    A 25-basepair deletion variant of MYBPC3 occurs at high frequency in individuals of South Asian descent and is estimated to affect 55 million people worldwide, carrying an increased likelihood of cardiomyopathy. Since this variant is prevalent and severe in this subpopulation, quick and affordable screening to provide risk-assessment to guide treatment for these patients is critical. An RNaseH qPCR assay was developed to quickly and specifically diagnose the presence of the 25-basepair deletion variant in MYBPC3. RNAseH-blocked nucleotide primers were designed to identify the presence or absence of the wild type MYBPC3 allele or the genomic sequence containing the 25-basepair deletion. Using this assay, three blinded operators were able to accurately determine the genotype from human genomic DNA samples from blood and saliva using a qPCR thermocycler. Furthermore, positive variant subjects were examined by both electrocardiography and echocardiography for the presence of cardiomyopathy. A simple, robust assay was established, verified and validated that can be automated to detect the presence of the highly prevalent 25-basepair deletion MYBPC3 variant using both blood and saliva samples. The assay will provide quick and accurate prescreening of individuals at high risk for cardiomyopathies and allow for better clinical identification of 25-basepair deletion MYBPC3 carriers in large cohort epidemiological studies. PMID:27990320

  19. 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

  20. A highly sensitive and selective diagnostic assay based on virus nanoparticles

    NASA Astrophysics Data System (ADS)

    Park, Jin-Seung; Cho, Moon Kyu; Lee, Eun Jung; Ahn, Keum-Young; Lee, Kyung Eun; Jung, Jae Hun; Cho, Yunjung; Han, Sung-Sik; Kim, Young Keun; Lee, Jeewon

    2009-04-01

    Early detection of the protein marker troponin I in patients with a higher risk of acute myocardial infarction can reduce the risk of death from heart attacks. Most troponin assays are currently based on the conventional enzyme linked immunosorbent assay and have detection limits in the nano- and picomolar range. Here, we show that by combining viral nanoparticles, which are engineered to have dual affinity for troponin antibodies and nickel, with three-dimensional nanostructures including nickel nanohairs, we can detect troponin levels in human serum samples that are six to seven orders of magnitude lower than those detectable using conventional enzyme linked immunosorbent assays. The viral nanoparticle helps to orient the antibodies for maximum capture of the troponin markers. High densities of antibodies on the surfaces of the nanoparticles and nanohairs lead to greater binding of the troponin markers, which significantly enhances detection sensitivities. The nickel nanohairs are re-useable and can reproducibly differentiate healthy serum from unhealthy ones. We expect other viral nanoparticles to form similar highly sensitive diagnostic assays for a variety of other protein markers.

  1. 78 FR 24425 - Assay Migration Studies for In Vitro Diagnostic Devices; Guidance for Industry and Food and Drug...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-25

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration Assay Migration Studies for In Vitro Diagnostic Devices...'s current thinking on ``migration studies'' for in vitro diagnostic device. It does not create...

  2. Optimizing odor identification testing as quick and accurate diagnostic tool for Parkinson's disease

    PubMed Central

    Mahlknecht, Philipp; Pechlaner, Raimund; Boesveldt, Sanne; Volc, Dieter; Pinter, Bernardette; Reiter, Eva; Müller, Christoph; Krismer, Florian; Berendse, Henk W.; van Hilten, Jacobus J.; Wuschitz, Albert; Schimetta, Wolfgang; Högl, Birgit; Djamshidian, Atbin; Nocker, Michael; Göbel, Georg; Gasperi, Arno; Kiechl, Stefan; Willeit, Johann; Poewe, Werner

    2016-01-01

    ABSTRACT Introduction The aim of this study was to evaluate odor identification testing as a quick, cheap, and reliable tool to identify PD. Methods Odor identification with the 16‐item Sniffin' Sticks test (SS‐16) was assessed in a total of 646 PD patients and 606 controls from three European centers (A, B, and C), as well as 75 patients with atypical parkinsonism or essential tremor and in a prospective cohort of 24 patients with idiopathic rapid eye movement sleep behavior disorder (center A). Reduced odor sets most discriminative for PD were determined in a discovery cohort derived from a random split of PD patients and controls from center A using L1‐regularized logistic regression. Diagnostic accuracy was assessed in the rest of the patients/controls as validation cohorts. Results Olfactory performance was lower in PD patients compared with controls and non‐PD patients in all cohorts (each P < 0.001). Both the full SS‐16 and a subscore of the top eight discriminating odors (SS‐8) were associated with an excellent discrimination of PD from controls (areas under the curve ≥0.90; sensitivities ≥83.3%; specificities ≥82.0%) and from non‐PD patients (areas under the curve ≥0.91; sensitivities ≥84.1%; specificities ≥84.0%) in all cohorts. This remained unchanged when patients with >3 years of disease duration were excluded from analysis. All 8 incident PD cases among patients with idiopathic rapid eye movement sleep behavior disorder were predicted with the SS‐16 and the SS‐8 (sensitivity, 100%; positive predictive value, 61.5%). Conclusions Odor identification testing provides excellent diagnostic accuracy in the distinction of PD patients from controls and diagnostic mimics. A reduced set of eight odors could be used as a quick tool in the workup of patients presenting with parkinsonism and for PD risk indication. © 2016 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and

  3. Bench-top validation testing of selected immunological and molecular Renibacterium salmoninarum diagnostic assays by comparison with quantitative bacteriological culture

    USGS Publications Warehouse

    Elliott, D.G.; Applegate, L.J.; Murray, A.L.; Purcell, M.K.; McKibben, C.L.

    2013-01-01

    No gold standard assay exhibiting error-free classification of results has been identified for detection of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Validation of diagnostic assays for R. salmoninarum has been hindered by its unique characteristics and biology, and difficulties in locating suitable populations of reference test animals. Infection status of fish in test populations is often unknown, and it is commonly assumed that the assay yielding the most positive results has the highest diagnostic accuracy, without consideration of misclassification of results. In this research, quantification of R. salmoninarum in samples by bacteriological culture provided a standardized measure of viable bacteria to evaluate analytical performance characteristics (sensitivity, specificity and repeatability) of non-culture assays in three matrices (phosphate-buffered saline, ovarian fluid and kidney tissue). Non-culture assays included polyclonal enzyme-linked immunosorbent assay (ELISA), direct smear fluorescent antibody technique (FAT), membrane-filtration FAT, nested polymerase chain reaction (nested PCR) and three real-time quantitative PCR assays. Injection challenge of specific pathogen-free Chinook salmon, Oncorhynchus tshawytscha (Walbaum), with R. salmoninarum was used to estimate diagnostic sensitivity and specificity. Results did not identify a single assay demonstrating the highest analytical and diagnostic performance characteristics, but revealed strengths and weaknesses of each test.

  4. The cryptococcal antigen lateral flow assay: A point-of-care diagnostic at an opportune time.

    PubMed

    Tang, Michele W; Clemons, Karl V; Katzenstein, David A; Stevens, David A

    2016-08-01

    Cryptococcal meningitis is a devastating HIV-related opportunistic infection, affecting nearly 1 million individuals and causing over 500 000 deaths each year. The burden of disease is greatest in sub-Saharan Africa and Southeast Asia, where cryptococcal disease is the most common cause of meningitis. Rapid, accurate and affordable diagnosis of cryptococcal disease has been lacking in many of the most heavily affected areas. Here, we review a point-of-care assay for cryptococcal disease, the dipstick-formatted cryptococcal antigen lateral flow assay (LFA) (IMMY, Norman, OK). In comparison to culture, the assay is 99.5% sensitive and 98% specific. In comparison to other commercially available tests for cryptococcal antigen, the LFA has equal or superior sensitivity and specificity in CSF, plasma and serum samples. We discuss potential applications for the use of the assay in resource-limited settings, including what is likely to be an important role of the LFA in screening for early cryptococcal infection before clinical disease and in evaluating pre-emptive treatment.

  5. 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.

  6. 4D laser camera for accurate patient positioning, collision avoidance, image fusion and adaptive approaches during diagnostic and therapeutic procedures.

    PubMed

    Brahme, Anders; Nyman, Peter; Skatt, Björn

    2008-05-01

    A four-dimensional (4D) laser camera (LC) has been developed for accurate patient imaging in diagnostic and therapeutic radiology. A complementary metal-oxide semiconductor camera images the intersection of a scanned fan shaped laser beam with the surface of the patient and allows real time recording of movements in a three-dimensional (3D) or four-dimensional (4D) format (3D +time). The LC system was first designed as an accurate patient setup tool during diagnostic and therapeutic applications but was found to be of much wider applicability as a general 4D photon "tag" for the surface of the patient in different clinical procedures. It is presently used as a 3D or 4D optical benchmark or tag for accurate delineation of the patient surface as demonstrated for patient auto setup, breathing and heart motion detection. Furthermore, its future potential applications in gating, adaptive therapy, 3D or 4D image fusion between most imaging modalities and image processing are discussed. It is shown that the LC system has a geometrical resolution of about 0, 1 mm and that the rigid body repositioning accuracy is about 0, 5 mm below 20 mm displacements, 1 mm below 40 mm and better than 2 mm at 70 mm. This indicates a slight need for repeated repositioning when the initial error is larger than about 50 mm. The positioning accuracy with standard patient setup procedures for prostate cancer at Karolinska was found to be about 5-6 mm when independently measured using the LC system. The system was found valuable for positron emission tomography-computed tomography (PET-CT) in vivo tumor and dose delivery imaging where it potentially may allow effective correction for breathing artifacts in 4D PET-CT and image fusion with lymph node atlases for accurate target volume definition in oncology. With a LC system in all imaging and radiation therapy rooms, auto setup during repeated diagnostic and therapeutic procedures may save around 5 min per session, increase accuracy and allow

  7. Raman Spectroscopy Provides a Powerful Diagnostic Tool for Accurate Determination of Albumin Glycation

    PubMed Central

    Dingari, Narahara Chari; Horowitz, Gary L.; Kang, Jeon Woong; Dasari, Ramachandra R.; Barman, Ishan

    2012-01-01

    We present the first demonstration of glycated albumin detection and quantification using Raman spectroscopy without the addition of reagents. Glycated albumin is an important marker for monitoring the long-term glycemic history of diabetics, especially as its concentrations, in contrast to glycated hemoglobin levels, are unaffected by changes in erythrocyte life times. Clinically, glycated albumin concentrations show a strong correlation with the development of serious diabetes complications including nephropathy and retinopathy. In this article, we propose and evaluate the efficacy of Raman spectroscopy for determination of this important analyte. By utilizing the pre-concentration obtained through drop-coating deposition, we show that glycation of albumin leads to subtle, but consistent, changes in vibrational features, which with the help of multivariate classification techniques can be used to discriminate glycated albumin from the unglycated variant with 100% accuracy. Moreover, we demonstrate that the calibration model developed on the glycated albumin spectral dataset shows high predictive power, even at substantially lower concentrations than those typically encountered in clinical practice. In fact, the limit of detection for glycated albumin measurements is calculated to be approximately four times lower than its minimum physiological concentration. Importantly, in relation to the existing detection methods for glycated albumin, the proposed method is also completely reagent-free, requires barely any sample preparation and has the potential for simultaneous determination of glycated hemoglobin levels as well. Given these key advantages, we believe that the proposed approach can provide a uniquely powerful tool for quantification of glycation status of proteins in biopharmaceutical development as well as for glycemic marker determination in routine clinical diagnostics in the future. PMID:22393405

  8. Isothermal Diagnostic Assays for Monitoring Single Nucleotide Polymorphisms in Necator americanus Associated with Benzimidazole Drug Resistance

    PubMed Central

    Rashwan, Nour; Bourguinat, Catherine; Keller, Kathy; Gunawardena, Nipul Kithsiri; de Silva, Nilanthi; Prichard, Roger

    2016-01-01

    Background Soil-transmitted helminths (STHs) are the most prevalent intestinal helminths of humans, and a major cause of morbidity in tropical and subtropical countries. The benzimidazole (BZ) drugs albendazole (ABZ) and mebendazole (MBZ) are used for treatment of human STH infections and this use is increasing dramatically with massive drug donations. Frequent and prolonged use of these drugs could lead to the emergence of anthelmintic resistance as has occurred in nematodes of livestock. Previous molecular assays for putative resistance mutations have been based mainly on PCR amplification and sequencing. However, these techniques are complicated and time consuming and not suitable for resource-constrained situations. A simple, rapid and sensitive genotyping method is required to monitor for possible developing resistance to BZ drugs. Methods To address this problem, single nucleotide polymorphism (SNP) detection assays were developed based on the Smart amplification method (SmartAmp2) to target codons 167, 198, and 200 in the β-tubulin isotype 1 gene for the hookworm Necator americanus. Findings Diagnostic assays were developed and applied to analyze hookworm samples by both SmartAmp2 and conventional sequencing methods and the results showed high concordance. Additionally, fecal samples spiked with N. americanus larvae were assessed and the results showed that the Aac polymerase used has high tolerance to inhibitors in fecal samples. Conclusion The N. americanus SmartAmp2 SNP detection assay is a new genotyping tool that is rapid, sensitive, highly specific and efficient with the potential to be used as a field tool for monitoring SNPs associated with BZ resistance. However, further validation on large numbers of field samples is required. PMID:27930648

  9. Technical and diagnostic performance of five commercial anti-diphtheria toxoid IgG enzyme-linked immunosorbent assay kits.

    PubMed

    Faruq, A; Dadson, L; Cox, H; Alcock, F; Parker, A R

    2010-10-01

    The technical and diagnostic performances of five commercially available enzyme-linked immunosorbent assays for the measurement of anti-diphtheria toxoid IgG antibodies were evaluated. There was good agreement between the relative sensitivities of the five assays, but the relative specificity of one of the assays differed from that of the other four assays. Three of the five assays possessed recoveries of the international reference material NIBSC 00/496 within the range of 90% to 110% at antibody levels >0.1 IU/ml. The data suggest that there are manufacture-dependent differences in relative sensitivity, specificity, and accuracy for the determination of anti-diphtheria toxoid IgG antibodies that could result in different diagnostic interpretations.

  10. Development of a simple, accurate SPME-based method for assay of VOCs in column breakthrough experiments.

    PubMed

    Salaices Avila, Manuel Alejandro; Breiter, Roman; Mott, Henry

    2007-01-01

    Solid-phase microextraction (SPME) with gas chromatography is to be used for assay of effluent liquid samples from soil column experiments associated with VOC fate/transport studies. One goal of the fate/transport studies is to develop accurate, highly reproducible column breakthrough curves for 1,2-cis-dichloroethylene (cis-DCE) and trichloroethylene (TCE) to better understand interactions with selected natural solid phases. For SPME, the influences of the sample equilibration time, extraction temperature and the ratio of volume of sample bottle to that of the liquid sample (V(T)/V(w)) are the critical factors that could influence accuracy and precision of the measured results. Equilibrium between the gas phase and liquid phase was attained after 200 min of equilibration time. The temperature must be carefully controlled due to variation of both the Henry's constant (K(h)) and the fibre/gas phase distribution coefficient (K(fg)). K(h) decreases with decreasing temperature while K(fg) increases. Low V(T)/V(w) yields better sensitivity but results in analyte losses and negative bias of the resultant assay. High V(T)/V(w) ratio yields reduced sensitivity but analyte losses were found to be minimal, leading to better accuracy and reproducibility. A fast SPME method was achieved, 5 min for SPME extraction and 3.10 min for GC analysis. A linear calibration function in the gas phase was developed to analyse the breakthrough curve data, linear between a range of 0.9-236 microgl(-1), and a detection limit lower than 5 microgl(-1).

  11. Comparative Evaluation of the Diagnostic Performance of the Prototype Cepheid GeneXpert Ebola Assay

    PubMed Central

    Jansen van Vuren, Petrus; Grobbelaar, Antoinette; Storm, Nadia; Conteh, Ousman; Konneh, Kelfala; Kamara, Abdul; Sanne, Ian

    2015-01-01

    The Ebola virus disease (EVD) outbreak in West Africa has highlighted an urgent need for point-of-care (POC) assays for the diagnosis of this devastating disease in resource-limited African countries. The diagnostic performance characteristics of a prototype Cepheid GeneXpert Ebola POC used to detect Ebola virus (EBOV) in stored serum and plasma samples collected from suspected EVD cases in Sierra Leone in 2014 and 2015 was evaluated. The GeneXpert Ebola POC is a self-contained single-cartridge automated system that targets the glycoprotein (GP) and nucleoprotein (NP) genes of EBOV and yields results within 90 min. Results from 281 patient samples were compared to the results of a TaqMan real-time reverse transcription-PCR (RT-PCR) targeting the polymerase gene and performed on two real-time PCR machines. Agreement between the three platforms was 100% at cycle threshold (CT) values of ≤34.99, but discordant results were noted between CT values of 35 and 45.The diagnostic sensitivity of the three platforms was 100% in 91 patient samples that were confirmed to be infectious by virus isolation. All three molecular platforms detected viral EBOV RNA in additional samples that did not contain viable EBOV. The analytical sensitivity of the GeneXpert Ebola POC for the detection of NP was higher, and comparable to that of polymerase gene detection, than that for the detection of GP when using a titrated laboratory stock of EBOV. There was no detectable cross-reactivity with other hemorrhagic fever viruses or arboviruses. The GeneXpert Ebola POC offers an easy to operate and sensitive diagnostic tool that can be used for the rapid screening of suspected EVD cases in treatment or in holding centers during EVD outbreaks. PMID:26637383

  12. Comparative Evaluation of the Diagnostic Performance of the Prototype Cepheid GeneXpert Ebola Assay.

    PubMed

    Jansen van Vuren, Petrus; Grobbelaar, Antoinette; Storm, Nadia; Conteh, Ousman; Konneh, Kelfala; Kamara, Abdul; Sanne, Ian; Paweska, Janusz T

    2016-02-01

    The Ebola virus disease (EVD) outbreak in West Africa has highlighted an urgent need for point-of-care (POC) assays for the diagnosis of this devastating disease in resource-limited African countries. The diagnostic performance characteristics of a prototype Cepheid GeneXpert Ebola POC used to detect Ebola virus (EBOV) in stored serum and plasma samples collected from suspected EVD cases in Sierra Leone in 2014 and 2015 was evaluated. The GeneXpert Ebola POC is a self-contained single-cartridge automated system that targets the glycoprotein (GP) and nucleoprotein (NP) genes of EBOV and yields results within 90 min. Results from 281 patient samples were compared to the results of a TaqMan real-time reverse transcription-PCR (RT-PCR) targeting the polymerase gene and performed on two real-time PCR machines. Agreement between the three platforms was 100% at cycle threshold (CT) values of ≤34.99, but discordant results were noted between CT values of 35 and 45.The diagnostic sensitivity of the three platforms was 100% in 91 patient samples that were confirmed to be infectious by virus isolation. All three molecular platforms detected viral EBOV RNA in additional samples that did not contain viable EBOV. The analytical sensitivity of the GeneXpert Ebola POC for the detection of NP was higher, and comparable to that of polymerase gene detection, than that for the detection of GP when using a titrated laboratory stock of EBOV. There was no detectable cross-reactivity with other hemorrhagic fever viruses or arboviruses. The GeneXpert Ebola POC offers an easy to operate and sensitive diagnostic tool that can be used for the rapid screening of suspected EVD cases in treatment or in holding centers during EVD outbreaks.

  13. 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.

  14. Diagnostic accuracy of two multiplex real-time polymerase chain reaction assays for the diagnosis of meningitis in children in a resource-limited setting

    PubMed Central

    Khumalo, Jermaine; Nicol, Mark; Hardie, Diana; Muloiwa, Rudzani; Mteshana, Phindile

    2017-01-01

    Introduction Accurate etiological diagnosis of meningitis is important, but difficult in resource-limited settings due to prior administration of antibiotics and lack of viral diagnostics. We aimed to develop and validate 2 real-time multiplex PCR (RT-PCR) assays for the detection of common causes of community-acquired bacterial and viral meningitis in South African children. Methods We developed 2 multiplex RT- PCRs for detection of S. pneumoniae, N. meningitidis, H. influenzae, enteroviruses, mumps virus and herpes simplex virus. We tested residual CSF samples from children presenting to a local paediatric hospital over a one-year period, whose CSF showed an abnormal cell count. Results were compared with routine diagnostic tests and the final discharge diagnosis. We calculated accuracy of the bacterial RT-PCR assay compared to CSF culture and using World Health Organisation definitions of laboratory-confirmed bacterial meningitis. Results From 292 samples, bacterial DNA was detected in 12 (4.1%) and viral nucleic acids in 94 (32%). Compared to CSF culture, the sensitivity and specificity of the bacterial RT-PCR was 100% and 97.2% with complete agreement in organism identification. None of the cases positive by viral RT-PCR had a bacterial cause confirmed on CSF culture. Only 9/90 (10%) of patients diagnosed clinically as bacterial meningitis or partially treated bacterial meningitis tested positive with the bacterial RT-PCR. Discussion In this population the use of 2 multiplex RT-PCRs targeting 6 common pathogens gave promising results. If introduced into routine diagnostic testing, these multiplex RT-PCR assays would supplement other diagnostic tests, and have the potential to limit unnecessary antibiotic therapy and hospitalisation. PMID:28346504

  15. Detection of ESKAPE Bacterial Pathogens at the Point of Care Using Isothermal DNA-Based Assays in a Portable Degas-Actuated Microfluidic Diagnostic Assay Platform.

    PubMed

    Renner, Lars D; Zan, Jindong; Hu, Linda I; Martinez, Manuel; Resto, Pedro J; Siegel, Adam C; Torres, Clint; Hall, Sara B; Slezak, Tom R; Nguyen, Tuan H; Weibel, Douglas B

    2017-02-15

    An estimated 1.5 billion microbial infections occur globally each year and result in ∼4.6 million deaths. A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients. The result is a trial-and-error approach that is limited in effectiveness, increases risk for patients while contributing to antimicrobial drug resistance, and reduces the lifetime of antibiotics. This paper addresses this important diagnostic technology gap by describing a low-cost, portable, rapid, and easy-to-use microfluidic cartridge-based system for detecting the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacterial pathogens that are most commonly associated with antibiotic resistance. The point-of-care molecular diagnostic system consists of a vacuum-degassed microfluidic cartridge preloaded with lyophilized recombinase polymerase amplification (RPA) assays and a small portable battery-powered electronic incubator/reader. The isothermal RPA assays detect the targeted ESKAPE pathogens with high sensitivity (e.g., a limit of detection of ∼10 nucleic acid molecules) that is comparable to that of current PCR-based assays, and they offer advantages in power consumption, engineering, and robustness, which are three critical elements required for the point-of-care setting.

  16. Validation of a TaqMan diagnostic assay for the systematic development of Phytophthora genus and species specific markers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genus Phytophthora contains many species that are not native to the USA and have the potential to cause significant damage to agriculture and native ecosystems. A genus and species-specific diagnostic assay was developed based on mitochondrial gene order differences that allowed for the systemat...

  17. Development of internally controlled duplex real-time NASBA diagnostics assays for the detection of microorganisms associated with bacterial meningitis.

    PubMed

    Clancy, Eoin; Coughlan, Helena; Higgins, Owen; Boo, Teck Wee; Cormican, Martin; Barrett, Louise; Smith, Terry J; Reddington, Kate; Barry, Thomas

    2016-08-01

    Three duplex molecular beacon based real-time Nucleic Acid Sequence Based Amplification (NASBA) assays have been designed and experimentally validated targeting RNA transcripts for the detection and identification of Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae respectively. Each real-time NASBA diagnostics assay includes an endogenous non-competitive Internal Amplification Control (IAC) to amplify the splice variant 1 mRNA of the Homo sapiens TBP gene from human total RNA. All three duplex real-time NASBA diagnostics assays were determined to be 100% specific for the target species tested for. Also the Limits of Detection (LODs) for the H. influenzae, N. meningitidis and S. pneumoniae duplex real-time NASBA assays were 55.36, 0.99, and 57.24 Cell Equivalents (CE) respectively. These robust duplex real-time NASBA diagnostics assays have the potential to be used in a clinical setting for the rapid (<60min) specific detection and identification of the most prominent microorganisms associated with bacterial meningitis in humans.

  18. Diagnostic difficulties of factor XI deficiencies: interferences' assay or real deficit?

    PubMed

    Gaymard, Alexandre; Nougier, Christophe

    2016-06-01

    Madam P, 77 years old, consulted in the hemostasis department after a coagulation anomaly was discovered during her preoperative test for a total hip prosthesis. After confirmation of a persistent and increased aPTT, additional tests were performed and showed the presence of antiphospholipid antibodies. Factor VIII level could be corrected after the plasma dilution to 1/40(th). But successive dilutions were not enough to obtain a correct factor IX (FIX) and factor XI (FXI) level. FIX level was obtained by chromogenic method in order to avoid the interferences caused by the antibodies. Finally, despite the change of reagents and dilutions up to 1/160(th), the FXI level couldn't be determined. Despite these results and those of the thrombin generation assay, the surgery was successfully done without specific treatment thanks to the absence of hemorrhagic history. This observation highlights the diagnostic and monitoring difficulties for uncommon clotting factor deficit. The development of interference free test could increase the support for these patients.

  19. Evaluation of Signature Erosion in Ebola Virus Due to Genomic Drift and Its Impact on the Performance of Diagnostic Assays

    PubMed Central

    Sozhamannan, Shanmuga; Holland, Mitchell Y.; Hall, Adrienne T.; Negrón, Daniel A.; Ivancich, Mychal; Koehler, Jeffrey W.; Minogue, Timothy D.; Campbell, Catherine E.; Berger, Walter J.; Christopher, George W.; Goodwin, Bruce G.; Smith, Michael A.

    2015-01-01

    Genome sequence analyses of the 2014 Ebola Virus (EBOV) isolates revealed a potential problem with the diagnostic assays currently in use; i.e., drifting genomic profiles of the virus may affect the sensitivity or even produce false-negative results. We evaluated signature erosion in ebolavirus molecular assays using an in silico approach and found frequent potential false-negative and false-positive results. We further empirically evaluated many EBOV assays, under real time PCR conditions using EBOV Kikwit (1995) and Makona (2014) RNA templates. These results revealed differences in performance between assays but were comparable between the old and new EBOV templates. Using a whole genome approach and a novel algorithm, termed BioVelocity, we identified new signatures that are unique to each of EBOV, Sudan virus (SUDV), and Reston virus (RESTV). Interestingly, many of the current assay signatures do not fall within these regions, indicating a potential drawback in the past assay design strategies. The new signatures identified in this study may be evaluated with real-time reverse transcription PCR (rRT-PCR) assay development and validation. In addition, we discuss regulatory implications and timely availability to impact a rapidly evolving outbreak using existing but perhaps less than optimal assays versus redesign these assays for addressing genomic changes. PMID:26090727

  20. Fungal disease detection in plants: Traditional assays, novel diagnostic techniques and biosensors.

    PubMed

    Ray, Monalisa; Ray, Asit; Dash, Swagatika; Mishra, Abtar; Achary, K Gopinath; Nayak, Sanghamitra; Singh, Shikha

    2017-01-15

    Fungal diseases in commercially important plants results in a significant reduction in both quality and yield, often leading to the loss of an entire plant. In order to minimize the losses, it is essential to detect and identify the pathogens at an early stage. Early detection and accurate identification of pathogens can control the spread of infection. The present article provides a comprehensive overview of conventional methods, current trends and advances in fungal pathogen detection with an emphasis on biosensors. Traditional techniques are the "gold standard" in fungal detection which relies on symptoms, culture-based, morphological observation and biochemical identifications. In recent times, with the advancement of biotechnology, molecular and immunological approaches have revolutionized fungal disease detection. But the drawback lies in the fact that these methods require specific and expensive equipments. Thus, there is an urgent need for rapid, reliable, sensitive, cost effective and easy to use diagnostic methods for fungal pathogen detection. Biosensors would become a promising and attractive alternative, but they still have to be subjected to some modifications, improvements and proper validation for on-field use.

  1. Development of silicon photonic microring resonator biosensors for multiplexed cytokine assays and in vitro diagnostics

    NASA Astrophysics Data System (ADS)

    Luchansky, Matthew Sam

    In order to guide critical care therapies that are personalized to a patient's unique disease state, a diagnostic or theranostic medical device must quickly provide a detailed biomolecular understanding of disease onset and progression. This detailed molecular understanding of cellular processes and pathways requires the ability to measure multiple analytes in parallel. Though many traditional sensing technologies for biomarker analysis and fundamental biological studies (i.e. enzyme-linked immunosorbent assays, real-time polymerase chain reaction, etc.) rely on single-parameter measurements, it has become increasingly clear that the inherent complexity of many human illnesses and pathways necessitates quantitative and multiparameter analysis of biological samples. Currently used analytical methods are deficient in that they often provide either highly quantitative data for a single biomarker or qualitative data for many targets, but methods that simultaneously provide highly quantitative analysis of many targets have yet to be adequately developed. Fields such as medical diagnostics and cellular biology would benefit greatly from a technology that enables rapid, quantitative and reproducible assays for many targets within a single sample. In an effort to fill this unmet need, this doctoral dissertation describes the development of a clinically translational biosensing technology based on silicon photonics and developed in the chemistry research laboratory of Ryan C. Bailey. Silicon photonic microring resonators, a class of high-Q optical sensors, represent a promising platform for rapid, multiparameter in vitro measurements. The original device design utilizes 32-ring arrays for real-time biomolecular sensing without fluorescent labels, and these optical biosensors display great potential for more highly multiplexed (100s-1000s) measurements based on the impressive scalability of silicon device fabrication. Though this technology can be used to detect a variety of

  2. Hyperthermostable binding molecules on phage: Assay components for point-of-care diagnostics for active tuberculosis infection.

    PubMed

    Zhao, Ning; Spencer, John; Schmitt, Margaret A; Fisk, John D

    2017-03-15

    Tuberculosis is the leading cause of death from infectious disease worldwide. The low sensitivity, extended processing time, and high expense of current diagnostics are major challenges to the detection and treatment of tuberculosis. Mycobacterium tuberculosis ornithine transcarbamylase (Mtb OTC, Rv1656) has been identified in the urine of patients with active TB infection and is a promising target for point-of-care diagnostics. Specific binding proteins with low nanomolar affinities for Mtb OTC were selected from a phage display library built upon a hyperthermostable Sso7d scaffold. Phage particles displaying Sso7d variants were utilized to generate a sandwich ELISA-based assay for Mtb OTC. The assay response is linear between 2 ng/mL and 125 ng/mL recombinant Mtb OTC and has a limit of detection of 400 pg/mL recombinant Mtb OTC. The assay employing a phage-based detection reagent is comparable to commercially-available antibody-based biosensors. Importantly, the assay maintains functionality at both neutral and basic pH in presence of salt and urea over the range of concentrations typical for human urine. Phage-based diagnostic systems may feature improved physical stability and cost of production relative to traditional antibody-based reagents, without sacrificing specificity and sensitivity.

  3. From SOMAmer-Based Biomarker Discovery to Diagnostic and Clinical Applications: A SOMAmer-Based, Streamlined Multiplex Proteomic Assay

    PubMed Central

    Kraemer, Stephan; Vaught, Jonathan D.; Bock, Christopher; Gold, Larry; Katilius, Evaldas; Keeney, Tracy R.; Kim, Nancy; Saccomano, Nicholas A.; Wilcox, Sheri K.; Zichi, Dom; Sanders, Glenn M.

    2011-01-01

    Recently, we reported a SOMAmer-based, highly multiplexed assay for the purpose of biomarker identification. To enable seamless transition from highly multiplexed biomarker discovery assays to a format suitable and convenient for diagnostic and life-science applications, we developed a streamlined, plate-based version of the assay. The plate-based version of the assay is robust, sensitive (sub-picomolar), rapid, can be highly multiplexed (upwards of 60 analytes), and fully automated. We demonstrate that quantification by microarray-based hybridization, Luminex bead-based methods, and qPCR are each compatible with our platform, further expanding the breadth of proteomic applications for a wide user community. PMID:22022604

  4. Diagnostic Accuracy of a New d-Dimer Assay (Sclavo Auto d-Dimer) for Exclusion of Deep Vein Thrombosis in Symptomatic Outpatients.

    PubMed

    Legnani, Cristina; Cini, Michela; Frascaro, Mirella; Rodorigo, Giuseppina; Sartori, Michelangelo; Cosmi, Benilde

    2017-04-01

    In patients presenting non-high clinical pretest probability (PTP), a negative d-dimer can exclude venous thromboembolism without imaging tests. However, each d-dimer assay should be validated in prospective studies. We evaluated an automated d-dimer immunoassay using the Sclavo Auto d-dimer (Sclavo Diagnostics Int, Sovicille, Italy) provided by Dasit Diagnostica (Cornaredo, Milan, Italy). Three hundred two consecutive outpatients suspected of leg deep vein thrombosis (DVT) with non-high PTP were included. The Sclavo Auto d-dimer assay was evaluated on 2 analyzers (Sysmex CA-7000 and Sysmex CS-2100; Sysmex Corporation, Kobe, Japan, provided by Dasit). The cutoff value (200 ng/mL) was established a priori. Prevalence of DVT was 11.9%. Since no false-negative patients were detected, the sensitivity and negative predictive values (NPVs) were 100% (sensitivity = CA-7000: 100% [95% confidence interval, CI: 93.3-100], CS-2100: 100% [95% CI: 93.3-100]; NPV = CA-7000: 100% [95% CI: 97.9-100], CS-2100: 100% [95% CI: 98.0-100]). Specificity was 65.4% (95% CI: 59.4-71.1) and 69.2% (95% CI: 63.3-74.7) for CA-7000 and CS-2100, respectively. Specificity increased when a higher cutoff value (234 ng/mL) was used for patients aged ≥60 years without compromising the safety. Assay reproducibility was satisfactory at concentrations near the cutoff value (total coefficient of variations <10%). In conclusion, the Sclavo Auto d-dimer assay was accurate when used for DVT diagnostic workup in outpatients with non-high PTP. Based on its high sensitivity and NPV, it can be used as a stand-alone test in outpatients with non-high PTP. Given its high specificity, the number of patients in whom further imaging techniques can be avoided increased, improving the yield of the test.

  5. Diagnostic test accuracy of loop-mediated isothermal amplification assay for Mycobacterium tuberculosis: systematic review and meta-analysis

    PubMed Central

    Nagai, Kenjiro; Horita, Nobuyuki; Yamamoto, Masaki; Tsukahara, Toshinori; Nagakura, Hideyuki; Tashiro, Ken; Shibata, Yuji; Watanabe, Hiroki; Nakashima, Kentaro; Ushio, Ryota; Ikeda, Misako; Narita, Atsuya; Kanai, Akinori; Sato, Takashi; Kaneko, Takeshi

    2016-01-01

    Diagnostic test accuracy of the loop-mediated isothermal amplification (LAMP) assay for culture proven tuberculosis is unclear. We searched electronic databases for both cohort and case-control studies that provided data to calculate sensitivity and specificity. The index test was any LAMP assay including both commercialized kits and in-house assays. Culture-proven M. tuberculosis was considered a positive reference test. We included 26 studies on 9330 sputum samples and one study on 315 extra-pulmonary specimens. For sputum samples, 26 studies yielded the summary estimates of sensitivity of 89.6% (95% CI 85.6–92.6%), specificity of 94.0% (95% CI 91.0–96.1%), and a diagnostic odds ratio of 145 (95% CI 93–226). Nine studies focusing on Loopamp MTBC yielded the summary estimates of sensitivity of 80.9% (95% CI 76.0–85.1%) and specificity of 96.5% (95% CI 94.7–97.7%). Loopamp MTBC had higher sensitivity and lower specificity for smear-positive sputa compared to smear-negative sputa. In-house assays showed higher sensitivity and lower specificity compared to Loopamp MTBC. LAMP promises to be a useful test for the diagnosis of TB, however there is still need to improve the assay to make it simpler, cheaper and more efficient to make it competitive against other PCR methods already available. PMID:27958360

  6. Exploiting Bacterial Whole-Genome Sequencing Data for Evaluation of Diagnostic Assays: Campylobacter Species Identification as a Case Study.

    PubMed

    Jansen van Rensburg, Melissa J; Swift, Craig; Cody, Alison J; Jenkins, Claire; Maiden, Martin C J

    2016-12-01

    The application of whole-genome sequencing (WGS) to problems in clinical microbiology has had a major impact on the field. Clinical laboratories are now using WGS for pathogen identification, antimicrobial susceptibility testing, and epidemiological typing. WGS data also represent a valuable resource for the development and evaluation of molecular diagnostic assays, which continue to play an important role in clinical microbiology. To demonstrate this application of WGS, this study used publicly available genomic data to evaluate a duplex real-time PCR (RT-PCR) assay that targets mapA and ceuE for the detection of Campylobacter jejuni and Campylobacter coli, leading global causes of bacterial gastroenteritis. In silico analyses of mapA and ceuE primer and probe sequences from 1,713 genetically diverse C. jejuni and C. coli genomes, supported by RT-PCR testing, indicated that the assay was robust, with 1,707 (99.7%) isolates correctly identified. The high specificity of the mapA-ceuE assay was the result of interspecies diversity and intraspecies conservation of the target genes in C. jejuni and C. coli Rare instances of a lack of specificity among C. coli isolates were due to introgression in mapA or sequence diversity in ceuE The results of this study illustrate how WGS can be exploited to evaluate molecular diagnostic assays by using publicly available data, online databases, and open-source software.

  7. Exploiting Bacterial Whole-Genome Sequencing Data for Evaluation of Diagnostic Assays: Campylobacter Species Identification as a Case Study

    PubMed Central

    Jansen van Rensburg, Melissa J.; Swift, Craig; Cody, Alison J.; Jenkins, Claire

    2016-01-01

    The application of whole-genome sequencing (WGS) to problems in clinical microbiology has had a major impact on the field. Clinical laboratories are now using WGS for pathogen identification, antimicrobial susceptibility testing, and epidemiological typing. WGS data also represent a valuable resource for the development and evaluation of molecular diagnostic assays, which continue to play an important role in clinical microbiology. To demonstrate this application of WGS, this study used publicly available genomic data to evaluate a duplex real-time PCR (RT-PCR) assay that targets mapA and ceuE for the detection of Campylobacter jejuni and Campylobacter coli, leading global causes of bacterial gastroenteritis. In silico analyses of mapA and ceuE primer and probe sequences from 1,713 genetically diverse C. jejuni and C. coli genomes, supported by RT-PCR testing, indicated that the assay was robust, with 1,707 (99.7%) isolates correctly identified. The high specificity of the mapA-ceuE assay was the result of interspecies diversity and intraspecies conservation of the target genes in C. jejuni and C. coli. Rare instances of a lack of specificity among C. coli isolates were due to introgression in mapA or sequence diversity in ceuE. The results of this study illustrate how WGS can be exploited to evaluate molecular diagnostic assays by using publicly available data, online databases, and open-source software. PMID:27733632

  8. The Diagnostic Performance of a Single GeneXpert MTB/RIF Assay in an Intensified Tuberculosis Case Finding Survey among HIV-Infected Prisoners in Malaysia

    PubMed Central

    Al-Darraji, Haider Abdulrazzaq Abed; Razak, Humaira Abd; Ng, Kee Peng; Altice, Frederick L.; Kamarulzaman, Adeeba

    2013-01-01

    Background Delays in tuberculosis (TB) diagnosis, particularly in prisons, is associated with detrimental outcomes. The new GeneXpert MTB/RIF assay (Xpert) offers accurate and rapid diagnosis of active TB, but its performance in improving case detection in high-transmission congregate settings has yet to be evaluated. We assessed the diagnostic accuracy of a single Xpert assay in an intensified case finding survey among HIV-infected prisoners in Malaysia. Methods HIV-infected prisoners at a single site provided two early-morning sputum specimens to be examined using fluorescence smear microscopy, BACTEC MGIT 960 liquid culture and a single Xpert. The sensitivity, specificity, negative and positive predictive values of Xpert were calculated relative to gold-standard results using MGIT 960 liquid culture. Relevant clinical and demographic data were used to examine correlates of active TB disease. Results The majority of enrolled subjects with complete data (N=125) were men (90.4%), age <40 years (61.6%) and had injected drugs (75.2%). Median CD4 lymphocyte count was 337 cells/µL (IQR 149-492); only 19 (15.2%) were receiving antiretroviral therapy. Of 15 culture-positive TB cases, single Xpert assay accurately detected only eight previously undiagnosed TB cases, resulting in a sensitivity, specificity, positive predictive value and negative predictive value of 53.3% (95% CI 30.12-75.2%), 100% (95% CI 96.6-100%), 100% (95% CI 67.56-100%) and 94.0% (95% CI 88.2-97.1%), respectively. Only 1 of 15 (6.7%) active TB cases was smear-positive. The prevalence (12%) of undiagnosed active pulmonary TB (15 of 125 prisoners) was high and associated with longer duration of drug use (AOR 1.14, 95% CI 1.03-1.26, for each year of drug use). Conclusions Single Xpert assay improved TB case detection and outperformed AFB smear microscopy, but yielded low screening sensitivity. Further examination of the impact of HIV infection on the diagnostic performance of the new assay alongside

  9. Detection of ESKAPE Bacterial Pathogens at the Point of Care Using Isothermal DNA-Based Assays in a Portable Degas-Actuated Microfluidic Diagnostic Assay Platform

    PubMed Central

    Renner, Lars D.; Zan, Jindong; Hu, Linda I.; Martinez, Manuel; Resto, Pedro J.; Siegel, Adam C.; Torres, Clint; Hall, Sara B.; Slezak, Tom R.

    2016-01-01

    ABSTRACT An estimated 1.5 billion microbial infections occur globally each year and result in ∼4.6 million deaths. A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients. The result is a trial-and-error approach that is limited in effectiveness, increases risk for patients while contributing to antimicrobial drug resistance, and reduces the lifetime of antibiotics. This paper addresses this important diagnostic technology gap by describing a low-cost, portable, rapid, and easy-to-use microfluidic cartridge-based system for detecting the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacterial pathogens that are most commonly associated with antibiotic resistance. The point-of-care molecular diagnostic system consists of a vacuum-degassed microfluidic cartridge preloaded with lyophilized recombinase polymerase amplification (RPA) assays and a small portable battery-powered electronic incubator/reader. The isothermal RPA assays detect the targeted ESKAPE pathogens with high sensitivity (e.g., a limit of detection of ∼10 nucleic acid molecules) that is comparable to that of current PCR-based assays, and they offer advantages in power consumption, engineering, and robustness, which are three critical elements required for the point-of-care setting. IMPORTANCE This paper describes a portable system for rapidly identifying bacteria in resource-limited environments; we highlight the capabilities of the technology by detecting different pathogens within the ESKAPE collection, which cause nosocomial infections. The system is designed around isothermal DNA-based assays housed within an autonomous plastic cartridge that are designed with the end user in mind, who may have limited technological training. Displaying

  10. Kininogen Cleavage Assay: Diagnostic Assistance for Kinin-Mediated Angioedema Conditions

    PubMed Central

    Defendi, Federica; Charignon, Delphine; Ghannam, Arije; Habib, Mohammed; Drouet, Christian

    2016-01-01

    Background Angioedema without wheals (AE) is a symptom characterised by localised episodes of oedema presumably caused by kinin release from kininogen cleavage. It can result from a hereditary deficiency in C1 Inhibitor (C1Inh), but it can present with normal level of C1Inh. These forms are typically difficult to diagnose although enhanced kinin production is suspected or demonstrated in some cases. Objectives We wanted to investigate bradykinin overproduction in all AE condition with normal C1Inh, excluding cases with enhanced kinin catabolism, and to propose this parameter as a disease biomarker. Methods We retrospectively investigated high molecular weight kininogen (HK) cleavage pattern, using gel electrophoresis and immunorevelation. Plasma samples were drawn using the same standardised procedure from blood donors or AE patients with normal C1Inh conditions, normal kinin catabolism, and without prophylaxis. Results Circulating native HK plasma concentrations were similar in the healthy men (interquartile range: 98–175μg/mL, n = 51) and in healthy women (90–176μg/mL, n = 74), while HK cleavage was lower (p<0.001) in men (0–5%) than women (3–9%). Patients exhibited lower native HK concentration (p<10−4; 21–117μg/mL, n = 31 for men; 0–84μg/mL, n = 41 for women) and higher HK cleavage (p<10−4; 10–30% and 14–89%, respectively) than healthy donors. Pathological thresholds were set at: <72μg/mL native HK, >14.4% HK cleavage for men; <38μg/mL; native HK, >33.0% HK cleavage for women, with >98% specificity achieved for all parameters. In plasma from patients undergoing recovery two months after oestrogen/progestin combination withdrawal (n = 13) or two weeks after AE attack (n = 2), HK cleavage was not fully restored, suggesting its use as a post-attack assay. Conclusion As a diagnostic tool, HK cleavage can offer physicians supportive arguments for kinin production in suspected AE cases and improve patient follow-up in clinical trials or

  11. Time-resolved immunofluorimetic assay (TRIFMA): diagnostic method of the future

    NASA Astrophysics Data System (ADS)

    Missler, U.; Gaida, U.; Li, Hong; Wood, W. G.

    1993-05-01

    This article describes the development and clinical evaluation of two-site immunometric assays for ferritin, thyrotropin (TSH), and tumor necrosis factor alpha (TNF(alpha) ) using time resolved fluorescent measurement with streptavidin-europium (STAV-Eu+3) as a label. The liquid phase antibodies were labeled with amidocaproylbiotin-N- hydroxysuccinimide ester. All three assays were based on microtiterplate technology and could be completed within a working day (incubation times less than 4 h). The ferritin assay was compared with luminescent and enzyme labeled assays using identical components. The TSH assay was compared with a commercial immunoluminometric assay whereas the TNF assay was unable to be compared with another method, only with standards from an independent source. The performance data was excellent with lower detection limits for TSH from < 0.003 mU/l and for TNF(alpha) under 10 ng/l. Intra-assay precision was acceptable within the range of interest with TSH < 4% (0.2 - 50 mU/l), TNF(alpha) < 15% (70 - 8000 ng/l and ferritin < 8% (10 - 500 g/l). Inter-assay precision was < 6% for TSH, < 16% for TNF(alpha) and < 8.5% for ferritin. All assays were performed using commercially available components and proved suitable for routine use.

  12. Validation of Three Early Ejaculation Diagnostic Tools: A Composite Measure Is Accurate and More Adequate for Diagnosis by Updated Diagnostic Criteria

    PubMed Central

    Jern, Patrick; Piha, Juhana; Santtila, Pekka

    2013-01-01

    Purpose To validate three early ejaculation diagnostic tools, and propose a new tool for diagnosis in line with proposed changes to diagnostic criteria. Significant changes to diagnostic criteria are expected in the near future. Available screening tools do not necessarily reflect proposed changes. Materials and Methods Data from 148 diagnosed early ejaculation patients (Mage = 42.8) and 892 controls (Mage = 33.1 years) from a population-based sample were used. Participants responded to three different questionnaires (Premature Ejaculation Profile; Premature Ejaculation Diagnostic Tool; Multiple Indicators of Premature Ejaculation). Stopwatch measured ejaculation latency times were collected from a subsample of early ejaculation patients. We used two types of responses to the questionnaires depending on the treatment status of the patients 1) responses regarding the situation before starting pharmacological treatment and 2) responses regarding current situation. Logistic regressions and Receiver Operating Characteristics were used to assess ability of both the instruments and individual items to differentiate between patients and controls. Results All instruments had very good precision (Areas under the Curve ranging from .93-.98). A new five-item instrument (named CHecklist for Early Ejaculation Symptoms – CHEES) consisting of high-performance variables selected from the three instruments had validity (Nagelkerke R2 range .51-.79 for backwards/forwards logistic regression) equal to or slightly better than any individual instrument (i.e., had slightly higher validity statistics, but these differences did not achieve statistical significance). Importantly, however, this instrument was more in line with proposed changes to diagnostic criteria. Conclusions All three screening tools had good validity. A new 5-item diagnostic tool (CHEES) based on the three instruments had equal or somewhat more favorable validity statistics compared to the other three tools, but is

  13. Simulated Respiratory Secretion for Use in the Development of Influenza Diagnostic Assays

    PubMed Central

    McCaul, Kate C.; Mei, Hong; Sasman, Amy; He, Jie; Kramp, William; Shively, Roxanne; Yan, Ke; Henrickson, Kelly J.

    2016-01-01

    Many assays have been developed for the detection of influenza virus which is an important respiratory pathogen. Development of these assays commonly involves the use of human clinical samples for validation of their performance. However, clinical samples can be difficult to obtain, deteriorate over time, and be inconsistent in composition. The goal of this study was to develop a simulated respiratory secretion (SRS) that could act as a surrogate for clinical samples. To this end, we determined the effects major respiratory secretion components (Na+, K+, Ca2+, cells, albumin IgG, IgM, and mucin) have on the performance of influenza assays including both nucleic acid amplification and rapid antigen assays. Minimal effects on the molecular assays were observed for all of the components tested, except for serum derived human IgG, which suppressed the signal of the rapid antigen assays. Using dot blots we were able to show anti-influenza nucleoprotein IgG antibodies are common in human respiratory samples. We composed a SRS that contained mid-point levels of human respiratory sample components and studied its effect compared to phosphate buffered saline and virus negative clinical sample matrix on the Veritor, Sofia, CDC RT-PCR, Simplexa, cobas Liat, and Alere i influenza assays. Our results demonstrated that a SRS can interact with a variety of test methods in a similar manner to clinical samples with a similar impact on test performance. PMID:27870895

  14. Accurate detection and quantification of the fish viral hemorrhagic Septicemia virus (VHSv) with a two-color fluorometric real-time PCR assay.

    PubMed

    Pierce, Lindsey R; Willey, James C; Palsule, Vrushalee V; Yeo, Jiyoun; Shepherd, Brian S; Crawford, Erin L; Stepien, Carol A

    2013-01-01

    Viral Hemorrhagic Septicemia virus (VHSv) is one of the world's most serious fish pathogens, infecting >80 marine, freshwater, and estuarine fish species from Eurasia and North America. A novel and especially virulent strain - IVb - appeared in the Great Lakes in 2003, has killed many game fish species in a series of outbreaks in subsequent years, and shut down interstate transport of baitfish. Cell culture is the diagnostic method approved by the USDA-APHIS, which takes a month or longer, lacks sensitivity, and does not quantify the amount of virus. We thus present a novel, easy, rapid, and highly sensitive real-time quantitative reverse transcription PCR (qRT-PCR) assay that incorporates synthetic competitive template internal standards for quality control to circumvent false negative results. Results demonstrate high signal-to-analyte response (slope = 1.00±0.02) and a linear dynamic range that spans seven orders of magnitude (R(2) = 0.99), ranging from 6 to 6,000,000 molecules. Infected fishes are found to harbor levels of virus that range to 1,200,000 VHSv molecules/10(6) actb1 molecules with 1,000 being a rough cut-off for clinical signs of disease. This new assay is rapid, inexpensive, and has significantly greater accuracy than other published qRT-PCR tests and traditional cell culture diagnostics.

  15. Accurate Detection and Quantification of the Fish Viral Hemorrhagic Septicemia virus (VHSv) with a Two-Color Fluorometric Real-Time PCR Assay

    PubMed Central

    Palsule, Vrushalee V.; Yeo, Jiyoun; Shepherd, Brian S.; Crawford, Erin L.; Stepien, Carol A.

    2013-01-01

    Viral Hemorrhagic Septicemia virus (VHSv) is one of the world's most serious fish pathogens, infecting >80 marine, freshwater, and estuarine fish species from Eurasia and North America. A novel and especially virulent strain – IVb – appeared in the Great Lakes in 2003, has killed many game fish species in a series of outbreaks in subsequent years, and shut down interstate transport of baitfish. Cell culture is the diagnostic method approved by the USDA-APHIS, which takes a month or longer, lacks sensitivity, and does not quantify the amount of virus. We thus present a novel, easy, rapid, and highly sensitive real-time quantitative reverse transcription PCR (qRT-PCR) assay that incorporates synthetic competitive template internal standards for quality control to circumvent false negative results. Results demonstrate high signal-to-analyte response (slope = 1.00±0.02) and a linear dynamic range that spans seven orders of magnitude (R2 = 0.99), ranging from 6 to 6,000,000 molecules. Infected fishes are found to harbor levels of virus that range to 1,200,000 VHSv molecules/106 actb1 molecules with 1,000 being a rough cut-off for clinical signs of disease. This new assay is rapid, inexpensive, and has significantly greater accuracy than other published qRT-PCR tests and traditional cell culture diagnostics. PMID:23977162

  16. Accurate, quantitative assays for the hydrolysis of soluble type I, II, and III /sup 3/H-acetylated collagens by bacterial and tissue collagenases

    SciTech Connect

    Mallya, S.K.; Mookhtiar, K.A.; Van Wart, H.E.

    1986-11-01

    Accurate and quantitative assays for the hydrolysis of soluble /sup 3/H-acetylated rat tendon type I, bovine cartilage type II, and human amnion type III collagens by both bacterial and tissue collagenases have been developed. The assays are carried out at any temperature in the 1-30/sup 0/C range in a single reaction tube and the progress of the reaction is monitored by withdrawing aliquots as a function of time, quenching with 1,10-phenanthroline, and quantitation of the concentration of hydrolysis fragments. The latter is achieved by selective denaturation of these fragments by incubation under conditions described in the previous paper of this issue. The assays give percentages of hydrolysis of all three collagen types by neutrophil collagenase that agree well with the results of gel electrophoresis experiments. The initial rates of hydrolysis of all three collagens are proportional to the concentration of both neutrophil or Clostridial collagenases over a 10-fold range of enzyme concentrations. All three assays can be carried out at collagen concentrations that range from 0.06 to 2 mg/ml and give linear double reciprocal plots for both tissue and bacterial collagenases that can be used to evaluate the kinetic parameters K/sub m/ and k/sub cat/ or V/sub max/. The assay developed for the hydrolysis of rat type I collagen by neutrophil collagenase is shown to be more sensitive by at least one order of magnitude than comparable assays that use rat type I collagen fibrils or gels as substrate.

  17. [Protein biomarker measurement and simple/rapid diagnostics with supersensitive and multiplex assay, MUSTag technology].

    PubMed

    Shibasaki, Futoshi; Morizane, Yoshihito; Makisaka, Noriko

    2009-11-01

    Recently, we face the rapid progression of an aging population, and so the importance of preventive medicine is growing. We would all like to pursue a healthy life during old age through effective treatment on the basis of the early detection of diseases. In this situation, we have developed MUSTag (Multiple Simultaneous Tag) assay technology through an innovative modification of the immuno-PCR method for the super-sensitive and multiplex detection of target biomarkers. In MUSTag technology, each different oligo-tag simultaneously detects multiplex protein targets with extremely high-level sensitivity (more than 10 fg(10(-15) g)/ml) in a dose-dependent manner by qRT-PCR (maximum: 3 plexes). Herein we report our recent results of multiple cytokine assays or disease-specific biomarker assays using MUSTag technology, and, further, clinical results from patients with cancer, ischemic brain, or heart attack, who need a prompt and predictive diagnosis for adequate treatment.

  18. 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.

  19. Stable isotope dilution assay for the accurate determination of mycotoxins in maize by UHPLC-MS/MS.

    PubMed

    Varga, Elisabeth; Glauner, Thomas; Köppen, Robert; Mayer, Katharina; Sulyok, Michael; Schuhmacher, Rainer; Krska, Rudolf; Berthiller, Franz

    2012-03-01

    A fast, easy-to-handle and cost-effective analytical method for 11 mycotoxins currently regulated in maize and other cereal-based food products in Europe was developed and validated for maize. The method is based on two extraction steps using different acidified acetonitrile–water mixtures. Separation is achieved using ultrahigh-performance liquid chromatography (UHPLC) by a linear water–methanol gradient. After electrospray ionisation, tandem mass spectrometric detection is performed in dynamic multiple reaction monitoring mode. Since accurate mass spectrometric quantification is hampered by matrix effects, uniformly [(13)C]-labelled mycotoxins for each of the 11 compounds were added to the sample extracts prior to UHPLC-MS/MS analysis. Method performance parameters were obtained by spiking blank maize samples with mycotoxins before as well as after extraction on six levels in triplicates. The twofold extraction led to total recoveries of the extraction steps between 97% and 111% for all target analytes, including fumonisins. The [(13)C]-labelled internal standards efficiently compensated all matrix effects in electrospray ionisation, leading to apparent recoveries between 88% and 105% with reasonable additional costs. The relative standard deviations of the whole method were between 4% and 11% for all analytes. The trueness of the method was verified by the measurement of several maize test materials with well-characterized concentrations. In conclusion, the developed method is capable of determining all regulated mycotoxins in maize and presuming similar matrix effects and extraction recovery also in other cereal-based foods.

  20. Development of a diagnostic gene expression assay for tuberculosis and its use under field conditions in African buffaloes (Syncerus caffer).

    PubMed

    Parsons, Sven D C; Menezes, Angela M; Cooper, David; Walzl, Gerhard; Warren, Robin M; van Helden, Paul D

    2012-08-15

    The development of diagnostic tests for tuberculosis (TB) in exotic species is constrained by host biology and the limited availability of suitable assay reagents. As such, we evaluated a gene expression assay (GEA) which is easily modified for novel species and allows for initial sample processing under field conditions. African buffaloes (Syncerus caffer) were categorized using the single comparative intradermal tuberculin test, and blood from test-positive and test-negative animals was incubated for 20 h in "Nil" tubes (containing saline) and "TB Antigen" tubes (containing Mycobacterium tuberculosis complex (MTC)-specific antigens) of a commercial human TB test, the QuantiFERON(®)-TB Gold (In-Tube) (QFT) assay. Blood samples were then stabilized in RNAlater(®) and transported to the laboratory for RNA extraction. A Custom TaqMan GEA was used to calculate the relative abundance of interferon-gamma (IFN-γ) mRNA in the TB Antigen tube compared to that in the Nil tube as a marker of immune activation in response to MTC antigen recognition. The GEA results from the two buffalo groups were compared and a cutoff value of 2.85 was calculated to differentiate between animals from these groups with a sensitivity of 80% (95% C.I.: 56-94%) and a specificity of 95% (95% C.I.: 75-100%). Further optimization of this assay could provide a highly useful tool for the diagnosis of MTC infection in exotic species.

  1. Avian eyelid assay, a new diagnostic method for detecting botulinum neurotoxin serotypes A, B and E.

    PubMed

    Wang, Jinglin; Gao, Shan; Zhang, Qien; Kang, Lin; Liu, Yanhua

    2007-06-01

    Based upon botulinum neurotoxins' (BoNT) mechanism of action, a novel, rapid, and sensitive avian eyelid assay was developed to detect Clostridium botulinum neurotoxin serotypes A, B and E in assay buffer and mimic samples. It showed that chick was the most optimal model of 20-selected laboratory, non-laboratory animals. The eyelid closure of chick was the indicator symptom for positive results. The detection limits achieved range from 5 to 250 mouse LD(50) for toxin types A, B, and E in a buffer system and mimic samples. No cross reactivity occurred when using staphylococcal enterotoxin B, diphtheria toxin and nerve agent sarin, but cross reactivity was obtained in more than 6h for using high dose of tetanus toxin. This cross reactivity can be differentiated by BoNT neutralization tests with a serotype-specific antiserum in parallel. The avian eyelid assay can be performed within as short a time as 0.4-6 h. We report here the development of avian eyelid assay is the second animal bioassay for the detection of toxin types A, B, and E which approaches the sensitivity of the mouse bioassay, and is simple to perform as well as rapid to yield results.

  2. Molecular diagnostic assays for cervical neoplasia: emerging markers for the detection of high-grade cervical disease.

    PubMed

    Malinowski, Douglas P

    2005-04-01

    The accurate detection and diagnosis of cervical carcinoma and its malignant precursors (collectively referred to as high-grade cervical disease) represents one of the current challenges in clinical medicine and cytopathology. The advent of molecular diagnostics and the use of whole-genome profiling using DNA microarrays promises to yield improved understanding of the disease process with the subsequent development of more accurate diagnostic procedures based upon these discoveries. Recent reports describing a variety of experimental approaches have identified a series of candidate genes that are overexpressed in cervical carcinoma. In this article, representative examples of these markers and the resulting translational research will be reviewed within the context of improved cervical disease detection. An emerging class of markers, the minichromosome maintenance protein family of DNA licensing factors (MCM-2, MCM-6, MCM-7), shows promise for the specific detection of high-grade cervical disease using simple antibody-based immunochemistry formats. These proteins are overexpressed in cervical disease as a result of infection by oncogenic strains of human papillomavirus (HPV) and subsequent uncontrolled activation of gene transcription and aberrant S-phase induction, mediated through the E2F transcription factor pathway. This behavior appears to be a hallmark of high-grade cervical disease and provides the link between oncogenic HPV infections and the molecular behavior of cervical neoplasia (CN). The use of these molecular descriptors of CN in simple immunochemistry formats compatible with conventional cytology preparations is anticipated to improve the screening and detection of cervical disease within the healthcare system.

  3. Panel-based Genetic Diagnostic Testing for Inherited Eye Diseases is Highly Accurate and Reproducible and More Sensitive for Variant Detection Than Exome Sequencing

    PubMed Central

    Bujakowska, Kinga M.; Sousa, Maria E.; Fonseca-Kelly, Zoë D.; Taub, Daniel G.; Janessian, Maria; Wang, Dan Yi; Au, Elizabeth D.; Sims, Katherine B.; Sweetser, David A.; Fulton, Anne B.; Liu, Qin; Wiggs, Janey L.; Gai, Xiaowu; Pierce, Eric A.

    2015-01-01

    Purpose Next-generation sequencing (NGS) based methods are being adopted broadly for genetic diagnostic testing, but the performance characteristics of these techniques have not been fully defined with regard to test accuracy and reproducibility. Methods We developed a targeted enrichment and NGS approach for genetic diagnostic testing of patients with inherited eye disorders, including inherited retinal degenerations, optic atrophy and glaucoma. In preparation for providing this Genetic Eye Disease (GEDi) test on a CLIA-certified basis, we performed experiments to measure the sensitivity, specificity, reproducibility as well as the clinical sensitivity of the test. Results The GEDi test is highly reproducible and accurate, with sensitivity and specificity for single nucleotide variant detection of 97.9% and 100%, respectively. The sensitivity for variant detection was notably better than the 88.3% achieved by whole exome sequencing (WES) using the same metrics, due to better coverage of targeted genes in the GEDi test compared to commercially available exome capture sets. Prospective testing of 192 patients with IRDs indicated that the clinical sensitivity of the GEDi test is high, with a diagnostic rate of 51%. Conclusion The data suggest that based on quantified performance metrics, selective targeted enrichment is preferable to WES for genetic diagnostic testing. PMID:25412400

  4. Allele Specific Locked Nucleic Acid Quantitative PCR (ASLNAqPCR): An Accurate and Cost-Effective Assay to Diagnose and Quantify KRAS and BRAF Mutation

    PubMed Central

    Morandi, Luca; de Biase, Dario; Visani, Michela; Cesari, Valentina; De Maglio, Giovanna; Pizzolitto, Stefano; Pession, Annalisa; Tallini, Giovanni

    2012-01-01

    The use of tyrosine kinase inhibitors (TKIs) requires the testing for hot spot mutations of the molecular effectors downstream the membrane-bound tyrosine kinases since their wild type status is expected for response to TKI therapy. We report a novel assay that we have called Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR). The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. We designed primers specific for codon 12/13 KRAS mutations and BRAF V600E, and validated the assay with 300 routine samples from a variety of sources, including cytology specimens. All were analyzed by ASLNAqPCR and Sanger sequencing. Discordant cases were pyrosequenced. ASLNAqPCR correctly identified BRAF and KRAS mutations in all discordant cases and all had a mutated/wild type DNA ratio below the analytical sensitivity of the Sanger method. ASLNAqPCR was 100% specific with greater accuracy, positive and negative predictive values compared with Sanger sequencing. The analytical sensitivity of ASLNAqPCR is 0.1%, allowing quantification of mutated DNA in small neoplastic cell clones. ASLNAqPCR can be performed in any laboratory with real-time PCR equipment, is very cost-effective and can easily be adapted to detect hot spot mutations in other oncogenes. PMID:22558339

  5. Navigating the Rapids: The Development of Regulated Next-Generation Sequencing-Based Clinical Trial Assays and Companion Diagnostics

    PubMed Central

    Pant, Saumya; Weiner, Russell; Marton, Matthew J.

    2014-01-01

    Over the past decade, next-generation sequencing (NGS) technology has experienced meteoric growth in the aspects of platform, technology, and supporting bioinformatics development allowing its widespread and rapid uptake in research settings. More recently, NGS-based genomic data have been exploited to better understand disease development and patient characteristics that influence response to a given therapeutic intervention. Cancer, as a disease characterized by and driven by the tumor genetic landscape, is particularly amenable to NGS-based diagnostic (Dx) approaches. NGS-based technologies are particularly well suited to studying cancer disease development, progression and emergence of resistance, all key factors in the development of next-generation cancer Dxs. Yet, to achieve the promise of NGS-based patient treatment, drug developers will need to overcome a number of operational, technical, regulatory, and strategic challenges. Here, we provide a succinct overview of the state of the clinical NGS field in terms of the available clinically targeted platforms and sequencing technologies. We discuss the various operational and practical aspects of clinical NGS testing that will facilitate or limit the uptake of such assays in routine clinical care. We examine the current strategies for analytical validation and Food and Drug Administration (FDA)-approval of NGS-based assays and ongoing efforts to standardize clinical NGS and build quality control standards for the same. The rapidly evolving companion diagnostic (CDx) landscape for NGS-based assays will be reviewed, highlighting the key areas of concern and suggesting strategies to mitigate risk. The review will conclude with a series of strategic questions that face drug developers and a discussion of the likely future course of NGS-based CDx development efforts. PMID:24860780

  6. Performance and Diagnostic Accuracy of a Urine-Based Human Papillomavirus Assay in a Referral Population.

    PubMed

    Cuzick, Jack M; Cadman, Louise; Ahmad, Amar Sabri; Ho, Linda; Terry, George; Kleeman, Michelle; Lyons, Deirdre; Austin, Janet; Stoler, Mark H; Vibat, Cecile Rose T; Dockter, Janel; Robbins, David; Billings, Paul R; Erlander, Mark G

    2017-02-21

    Background HPV testing from clinician-collected cervical and self-collected cervico-vaginal samples is more sensitive for detecting CIN2+/CIN3+ than cytology-based screening, stimulating interest in HPV testing from urine. The objective was to determine the performance of the Trovagene HPV test for the detection of CIN2+ from urine and PreservCyt cervical samples. Methods Women referred for colposcopy at St Mary's Hospital London, following abnormal cytology, were recruited to this diagnostic accuracy study by convenience sampling (September 2011 and April 2013). 501 paired urine and cervical samples were collected.

  7. Diagnostic Value of T-cell Interferon-γ Release Assays on Synovial Fluid for Articular Tuberculosis: A Pilot Study

    PubMed Central

    Cheng, Xin-He; Bian, Sai-Nan; Zhang, Yue-Qiu; Zhang, Li-Fan; Shi, Xiao-Chun; Yang, Bo; Zhang, Feng-Chun; Liu, Xiao-Qing

    2016-01-01

    Background: Tuberculosis (TB) remains a major global public health challenge. Articular TB is an important form of extrapulmonary tuberculosis, and its diagnosis is difficult because of the low sensitivity of traditional methods. The aim of this study was to analyze the diagnostic value of T-SPOT.TB on synovial fluid for the diagnosis of articular TB. Methods: Patients with suspected articular TB were enrolled consecutively between August 2011 and December 2015. T-SPOT.TB was performed on both synovial fluid mononuclear cells (SFMCs) and peripheral blood mononuclear cells (PBMCs). The final diagnosis of articular TB was independent of the T-SPOT.TB result. The diagnostic sensitivity, specificity, predictive value, and likelihood ratio of T-SPOT.TB on SFMCs and PBMCs were analyzed. Results: Twenty patients with suspected articular TB were enrolled. Six were diagnosed with articular TB, and 14 patients were diagnosed with other diseases. Sensitivity and specificity were 83% and 86% for T-SPOT.TB on SFMCs, and 67% and 69% for T-SPOT.TB on PBMCs, respectively. The positive predictive value (PPV) and negative predictive value (NPV) of T-SPOT.TB on SFMCs were 71% and 92%, respectively. The PPV and NPV were 50% and 82% for T-SPOT.TB on PBMCs. Conclusion: Sensitivity, specificity, and NPV of T-SPOT.TB on SFMCs appeared higher than that on PBMCs, indicating that T-SPOT.TB on SFMCs might be a rapid and accurate diagnostic test for articular TB. PMID:27174325

  8. A diagnostic one-step real-time reverse transcription polymerase chain reaction method for accurate detection of influenza virus type A

    PubMed Central

    Behzadi, Mohammad Amin; Alborzi, Abdolvahab

    2016-01-01

    Introduction Influenza A is known as a public health concern worldwide. In this study, a novel one-step real-time reverse transcription polymerase chain reaction (rtRT-PCR) assay was designed and optimized for the detection of influenza A viruses. Material and methods The primers and probe were designed based on the analysis of 90 matrix nucleotide sequence data of influenza type A subtypes from the GenBank database of the National Center for Biotechnology Information (NCBI). The influenza virus A/Tehran/5652/2010 (H1N1 pdm09) was used as a reference. The rtRT-PCR assay was optimized, compared with that of the World Health Organization (WHO), and its analytical sensitivity, specificity and reproducibility were evaluated. In total, 64 nasopharyngeal swabs from patients with influenza-like illness (ILI) and 41 samples without ILI symptoms were tested for the virus, using conventional cell culture, direct immunofluorescence antibody (DFA) methods, and one-step rtRT-PCR with the designed primer set and probe and the WHO’s. Results The optimized assay results were similar to the WHO’s. The optimized assay results were similar to WHO’s, with non-significant differences for 10–103 copies of viral RNA/reaction (p > 0.05). It detected 10 copies of viral RNA/reaction with high reproducibility and no cross reactivity with other respiratory viruses. A specific cytopathic effect was observed in 6/64 (9.37%) of the ILI group using conventional culture and DFA staining methods; however, it was not seen in non-ILI. Also, the results of our assay and the WHO’s were similar to those of viral isolation and DFA staining. Conclusions Given the high specificity, sensitivity and reproducibility of this novel assay, it can serve as a reliable diagnostic tool for the detection of influenza A viruses in clinical specimens and lab experiments. PMID:27904520

  9. RAD sequencing yields a high success rate for westslope cutthroat and rainbow trout species-diagnostic SNP assays

    USGS Publications Warehouse

    Stephen J. Amish,; Paul A. Hohenlohe,; Sally Painter,; Robb F. Leary,; Muhlfeld, Clint C.; Fred W. Allendorf,; Luikart, Gordon

    2012-01-01

    Hybridization with introduced rainbow trout threatens most native westslope cutthroat trout populations. Understanding the genetic effects of hybridization and introgression requires a large set of high-throughput, diagnostic genetic markers to inform conservation and management. Recently, we identified several thousand candidate single-nucleotide polymorphism (SNP) markers based on RAD sequencing of 11 westslope cutthroat trout and 13 rainbow trout individuals. Here, we used flanking sequence for 56 of these candidate SNP markers to design high-throughput genotyping assays. We validated the assays on a total of 92 individuals from 22 populations and seven hatchery strains. Forty-six assays (82%) amplified consistently and allowed easy identification of westslope cutthroat and rainbow trout alleles as well as heterozygote controls. The 46 SNPs will provide high power for early detection of population admixture and improved identification of hybrid and nonhybridized individuals. This technique shows promise as a very low-cost, reliable and relatively rapid method for developing and testing SNP markers for nonmodel organisms with limited genomic resources.

  10. RAD sequencing yields a high success rate for westslope cutthroat and rainbow trout species-diagnostic SNP assays.

    PubMed

    Amish, Stephen J; Hohenlohe, Paul A; Painter, Sally; Leary, Robb F; Muhlfeld, Clint; Allendorf, Fred W; Luikart, Gordon

    2012-07-01

    Hybridization with introduced rainbow trout threatens most native westslope cutthroat trout populations. Understanding the genetic effects of hybridization and introgression requires a large set of high-throughput, diagnostic genetic markers to inform conservation and management. Recently, we identified several thousand candidate single-nucleotide polymorphism (SNP) markers based on RAD sequencing of 11 westslope cutthroat trout and 13 rainbow trout individuals. Here, we used flanking sequence for 56 of these candidate SNP markers to design high-throughput genotyping assays. We validated the assays on a total of 92 individuals from 22 populations and seven hatchery strains. Forty-six assays (82%) amplified consistently and allowed easy identification of westslope cutthroat and rainbow trout alleles as well as heterozygote controls. The 46 SNPs will provide high power for early detection of population admixture and improved identification of hybrid and nonhybridized individuals. This technique shows promise as a very low-cost, reliable and relatively rapid method for developing and testing SNP markers for nonmodel organisms with limited genomic resources.

  11. ADIBO-based "click" chemistry for diagnostic peptide micro-array fabrication: physicochemical and assay characteristics.

    PubMed

    Prim, Denis; Rebeaud, Fabien; Cosandey, Vincent; Marti, Roger; Passeraub, Philippe; Pfeifer, Marc E

    2013-08-16

    Several azide-derivatized and fluorescently-labeled peptides were immobilized on azadibenzocyclooctyne (ADIBO)-activated slide surfaces via a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction revealing excellent immobilization kinetics, good spot homogeneities and reproducible fluorescence signal intensities. A myc-peptide micro-array immunoassay showed an antibody limit-of-detection (LOD) superior to a microtiter plate-based ELISA. Bovine serum albumin (BSA) and dextran covalently attached via "click" chemistry more efficiently reduced non-specific binding (NSB) of fluorescently-labeled IgG to the microarray surface in comparison to immobilized hexanoic acid and various types of polyethylene glycol (PEG) derivatives. Confirmation of these findings via further studies with other proteins and serum components could open up new possibilities for human sample and microarray platform-based molecular diagnostic tests.

  12. Quantitative molecular diagnostic assays of grain washes for Claviceps purpurea are correlated with visual determinations of ergot contamination

    PubMed Central

    Comte, Alexia; Gräfenhan, Tom; Links, Matthew G.; Hemmingsen, Sean M.

    2017-01-01

    We examined the epiphytic microbiome of cereal grain using the universal barcode chaperonin-60 (cpn60). Microbial community profiling of seed washes containing DNA extracts prepared from field-grown cereal grain detected sequences from a fungus identified only to Class Sordariomycetes. To identify the fungal sequence and to improve the reference database, we determined cpn60 sequences from field-collected and reference strains of the ergot fungus, Claviceps purpurea. These data allowed us to identify this fungal sequence as deriving from C. purpurea, and suggested that C. purpurea DNA is readily detectable on agricultural commodities, including those for which ergot was not identified as a grading factor. To get a sense of the prevalence and level of C. purpurea DNA in cereal grains, we developed a quantitative PCR assay based on the fungal internal transcribed spacer (ITS) and applied it to 137 samples from the 2014 crop year. The amount of Claviceps DNA quantified correlated strongly with the proportion of ergot sclerotia identified in each grain lot, although there was evidence that non-target organisms were responsible for some false positives with the ITS-based assay. We therefore developed a cpn60-targeted loop-mediated isothermal amplification assay and applied it to the same grain wash samples. The time to positive displayed a significant, inverse correlation to ergot levels determined by visual ratings. These results indicate that both laboratory-based and field-adaptable molecular diagnostic assays can be used to detect and quantify pathogen load in bulk commodities using cereal grain washes. PMID:28257512

  13. Diagnostic Values and Limitations of (1,3)-β-D-Glucans and Galactomannan Assays for Invasive Fungal Infection in Patients Admitted to Pediatric Intensive Care Unit.

    PubMed

    Zheng, Fang; Zha, Hui; Yang, Dandan; Deng, Jun; Zhang, Zhiquan

    2017-04-01

    The relationship among (1,3)-β-D-glucans (BG), galactomannan (GM), and the risk of developing invasive fungal infections (IFI) has been observed in adult ICU and in children with hematological malignancies. Only scant data evaluated the value of BG/GM assays for diagnosis of IFI in patients with nonhematological diseases in pediatric intensive care unit (PICU). In this study, we assessed the diagnostic value of these markers for IFI in PICU. The records of 230 patients were retrospectively evaluated. Out of 117 patients (7 proven, 23 probable, and 87 cases without evidence of IFI) performed GM and BG assays. The results showed many factors were associated with false-positive test results. Patients who aged over 3 years had higher levels of GM and BG than younger infants. The levels of BG were higher in subjects with dairy, human blood products, antibiotics, and corticosteroids therapy than in cases without these treatments. Unlike BG assay, GM assay was less susceptible to above-mentioned factors expect blood products. The levels of BG and GM in IFI cases were dramatically higher than in controls. The diagnostic performance of these assays showed that GM assay had better results when compared with BG assay. On the whole, negative predictive value in both GM and BG assays was dramatically higher than other diagnostic parameters. In conclusion, BG assay was highly susceptible to many factors, and GM assay could be useful for diagnosis of IFI for its high sensitivity, but the over benefit of this assay limited in its inadequate specificity. The comparative advantage of BG and BG assays lied in excluding IFI in non-hematological PICU patients.

  14. Diagnostic significance and clinical impact of quantitative assays for diagnosis of human cytomegalovirus infection/disease in immunocompromised patients.

    PubMed

    Gerna, G; Percivalle, E; Baldanti, F; Sarasini, A; Zavattoni, M; Furione, M; Torsellini, M; Revello, M G

    1998-07-01

    In recent years several assays have been developed for quantitation of human cytomegalovirus (HCMV) in blood of immunocompromised (transplanted and AIDS) patients. It is currently agreed that the only reliable indication of the degree of dissemination of HCMV infection/disease is the measurement of HCMV in blood. Diagnosis of HCMV end-organ disease (organ localizations) often does not benefit from quantitation of virus in blood, but requires detection and quantification of virus in samples taken locally. The most important and clinically useful diagnostic assays for HCMV quantitation in blood are: i) viremia, quantifying infectious HCMV carried by peripheral blood leukocytes (PBL); ii) pp65-antigenemia, quantifying the number of PBL positive for HCMV pp65 in the nucleus; iii) circulating cytomegalic endothelial cell (CEC) viremia (CEC-viremia) measuring the number of circulating CEC carrying infectious HCMV (during the antigenemia assay); iv) leuko- and plasma-DNAemia, quantifying the number of HCMV genome equivalents present in PBL or plasma, respectively, by quantitative polymerase chain reaction (Q-PCR). Other less widely used assays are: i) determination of immediate early and late gene transcripts (mRNA) to detect active viral infection; ii) in situ hybridization to detect viral nucleic acid (DNA or RNA) in tissue sections or cell smears; iii) in situ PCR to detect a low DNA copy number in single cells. Monitoring of HCMV infection/disease in transplant recipients and AIDS patients has established threshold values for different assays above which HCMV-related clinical symptoms are likely to appear. These values are approximately 10 for viremia, 100 for antigenemia and 1,000 GE for leukoDNAemia, and are valid for both solid organ and bone marrow transplant recipients as well as AIDS patients, whereas presence of even a single circulating CEC is sufficient to suggest the presence of a disseminated HCMV infection with potential organ involvement. Monitoring of

  15. A support vector machine model provides an accurate transcript-level-based diagnostic for major depressive disorder

    PubMed Central

    Yu, J S; Xue, A Y; Redei, E E; Bagheri, N

    2016-01-01

    Major depressive disorder (MDD) is a critical cause of morbidity and disability with an economic cost of hundreds of billions of dollars each year, necessitating more effective treatment strategies and novel approaches to translational research. A notable barrier in addressing this public health threat involves reliable identification of the disorder, as many affected individuals remain undiagnosed or misdiagnosed. An objective blood-based diagnostic test using transcript levels of a panel of markers would provide an invaluable tool for MDD as the infrastructure—including equipment, trained personnel, billing, and governmental approval—for similar tests is well established in clinics worldwide. Here we present a supervised classification model utilizing support vector machines (SVMs) for the analysis of transcriptomic data readily obtained from a peripheral blood specimen. The model was trained on data from subjects with MDD (n=32) and age- and gender-matched controls (n=32). This SVM model provides a cross-validated sensitivity and specificity of 90.6% for the diagnosis of MDD using a panel of 10 transcripts. We applied a logistic equation on the SVM model and quantified a likelihood of depression score. This score gives the probability of a MDD diagnosis and allows the tuning of specificity and sensitivity for individual patients to bring personalized medicine closer in psychiatry. PMID:27779627

  16. An Efficient Multiplex PCR-Based Assay as a Novel Tool for Accurate Inter-Serovar Discrimination of Salmonella Enteritidis, S. Pullorum/Gallinarum and S. Dublin

    PubMed Central

    Xiong, Dan; Song, Li; Tao, Jing; Zheng, Huijuan; Zhou, Zihao; Geng, Shizhong; Pan, Zhiming; Jiao, Xinan

    2017-01-01

    Salmonella enterica serovars Enteritidis, Pullorum/Gallinarum, and Dublin are infectious pathogens causing serious problems for pig, chicken, and cattle production, respectively. Traditional serotyping for Salmonella is costly and labor-intensive. Here, we established a rapid multiplex PCR method to simultaneously identify three prevalent Salmonella serovars Enteritidis, Pullorum/Gallinarum, and Dublin individually for the first time. The multiplex PCR-based assay focuses on three genes tcpS, lygD, and flhB. Gene tcpS exists only in the three Salmonella serovars, and lygD exists only in S. Enteritidis, while a truncated region of flhB gene is only found in S. Pullorum/Gallinarum. The sensitivity and specificity of the multiplex PCR assay using three pairs of specific primers for these genes were evaluated. The results showed that this multiplex PCR method could accurately identify Salmonella Enteritidis, Pullorum/Gallinarum, and Dublin from eight non-Salmonella species and 27 Salmonella serovars. The least concentration of genomic DNA that could be detected was 58.5 pg/μL and the least number of cells was 100 CFU. Subsequently, this developed method was used to analyze clinical Salmonella isolates from one pig farm, one chicken farm, and one cattle farm. The results showed that blinded PCR testing of Salmonella isolates from the three farms were in concordance with the traditional serotyping tests, indicating the newly developed multiplex PCR system could be used as a novel tool to accurately distinguish the three specific Salmonella serovars individually, which is useful, especially in high-throughput screening. PMID:28360901

  17. 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

  18. A real-time PCR assay for accurate quantification of the individual members of the Altered Schaedler Flora microbiota in gnotobiotic mice.

    PubMed

    Gomes-Neto, João Carlos; Mantz, Sara; Held, Kyler; Sinha, Rohita; Segura Munoz, Rafael R; Schmaltz, Robert; Benson, Andrew K; Walter, Jens; Ramer-Tait, Amanda E

    2017-04-01

    Changes in the gastrointestinal microbial community are frequently associated with chronic diseases such as Inflammatory Bowel Diseases. However, understanding the relationship of any individual taxon within the community to host physiology is made complex due to the diversity and individuality of the gut microbiota. Defined microbial communities such as the Altered Schaedler Flora (ASF) help alleviate the challenges of a diverse microbiota by allowing one to interrogate the relationship between individual bacterial species and host responses. An important aspect of studying these relationships with defined microbial communities is the ability to measure the population abundance and dynamics of each member. Herein, we describe the development of an improved ASF species-specific and sensitive real-time quantitative polymerase chain reaction (qPCR) for use with SYBR Green chemistry to accurately assess individual ASF member abundance. This approach targets hypervariable regions V1 through V3 of the 16S rRNA gene of each ASF taxon to enhance assay specificity. We demonstrate the reproducibility, sensitivity and application of this new method by quantifying each ASF bacterium in two inbred mouse lines. We also used it to assess changes in ASF member abundance before and after acute antibiotic perturbation of the community as well as in mice fed two different diets. Additionally, we describe a nested PCR assay for the detection of lowly abundant ASF members. Altogether, this improved qPCR method will facilitate gnotobiotic research involving the ASF community by allowing for reproducible quantification of its members under various physiological conditions.

  19. Accurate and sensitive real-time PCR assays using intergenic spacer 1 region to differentiate Cryptococcus gattii sensu lato and Cryptococcus neoformans sensu lato.

    PubMed

    Tavares, Eliandro Reis; Azevedo, Caroline Souza; Panagio, Luciano Aparecido; Pelisson, Marsileni; Pinge-Filho, Phileno; Venancio, Emerson José; Barros, Tânia Fraga; Yamada-Ogatta, Sueli Fumie; Yamauchi, Lucy Megumi

    2016-01-01

    In this work, two accurate and sensitive real-time polymerase chain reaction (PCR) assays to differentiate pathogenic Cryptococcus gattii sensu lato (s.l.) and C. neoformans sensu lato (s.l.) targeting the intergenic spacer 1 (IGS1) region from rDNA locus were developed. Specific primers were designed based on their IGS1 sequence analyses and the optimal real-time PCR assays showed that the dissociation curves generated two different melting peaks, at 82.8 and 84.2ºC for C. gattii s.l. and C. neoformans s.l., respectively. No amplifications were observed in the negative template control. The minimum limit of detection of both primers was 100 plasmid copies per reaction, and they were highly specific when tested with a range of fungal DNAs. Overall, the results showed that the designed primers completely differentiated C. gattii s.l. and C. neoformans s.l. from clinical and environmental sources with great accuracy when compared to phenotypic identification, with no cross-reactivity to other fungal DNA.

  20. 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.

  1. 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

  2. Compact, accurate description of diagnostic neutral beam propagation and attenuation in a high temperature plasma for charge exchange recombination spectroscopy analysis.

    PubMed

    Bespamyatnov, Igor O; Rowan, William L; Granetz, Robert S

    2008-10-01

    Charge exchange recombination spectroscopy on Alcator C-Mod relies on the use of the diagnostic neutral beam injector as a source of neutral particles which penetrate deep into the plasma. It employs the emission resulting from the interaction of the beam atoms with fully ionized impurity ions. To interpret the emission from a given point in the plasma as the density of emitting impurity ions, the density of beam atoms must be known. Here, an analysis of beam propagation is described which yields the beam density profile throughout the beam trajectory from the neutral beam injector to the core of the plasma. The analysis includes the effects of beam formation, attenuation in the neutral gas surrounding the plasma, and attenuation in the plasma. In the course of this work, a numerical simulation and an analytical approximation for beam divergence are developed. The description is made sufficiently compact to yield accurate results in a time consistent with between-shot analysis.

  3. Retrospective screening of relevant pesticide metabolites in food using liquid chromatography high resolution mass spectrometry and accurate-mass databases of parent molecules and diagnostic fragment ions.

    PubMed

    Polgár, László; García-Reyes, Juan F; Fodor, Péter; Gyepes, Attila; Dernovics, Mihály; Abrankó, László; Gilbert-López, Bienvenida; Molina-Díaz, Antonio

    2012-08-03

    In recent years, the detection and characterization of relevant pesticide metabolites in food is an important task in order to evaluate their formation, kinetics, stability, and toxicity. In this article, a methodology for the systematic screening of pesticides and their main metabolites in fruit and vegetable samples is described, using LC-HRMS and accurate-mass database search of parent compounds and their diagnostic fragment ions. The approach is based on (i) search for parent pesticide molecules; (ii) search for their metabolites in the positive samples, assuming common fragmentation pathways between the metabolites and parent pesticide molecules; and (iii) search for pesticide conjugates using the data from both parent species and diagnostic fragment ions. An accurate-mass database was constructed consisting of 1396 compounds (850 parent compounds, 447 fragment ions and 99 metabolites). The screening process was performed by the software in an automated fashion. The proposed methodology was evaluated with 29 incurred samples and the output obtained was compared to standard pesticide testing methods (targeted LC-MS/MS). Examples on the application of the proposed approach are shown, including the detection of several pesticide glycosides derivatives, which were found with significantly relevant intensities. Glucose-conjugated forms of parent compounds (e.g., fenhexamid-O-glucoside) and those of metabolites (e.g., despropyl-iprodione-N-glycoside) were detected. Facing the lack of standards for glycosylated pesticides, the study was completed with the synthesis of fenhexamid-O-glucoside for quantification purposes. In some cases the pesticide derivatives were found in a relatively high ratio, drawing the attention to these kinds of metabolites and showing that they should not be neglected in multi-residue methods. The global coverage obtained on the 29 analyzed samples showed the usefulness and benefits of the proposed approach and highlights the practical

  4. 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.

  5. 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.

  6. Sporadic Creutzfeldt-Jakob disease diagnostic accuracy is improved by a new CSF ELISA 14-3-3γ assay.

    PubMed

    Leitão, M J; Baldeiras, I; Almeida, M R; Ribeiro, M H; Santos, A C; Ribeiro, M; Tomás, J; Rocha, S; Santana, I; Oliveira, C R

    2016-05-13

    Protein 14-3-3 is a reliable marker of rapid neuronal damage, specifically increased in cerebrospinal fluid (CSF) of sporadic Creutzfeldt-Jakob disease (sCJD) patients. Its detection is usually performed by Western Blot (WB), prone to methodological issues. Our aim was to evaluate the diagnostic performance of a recently developed quantitative enzyme-linked immunosorbent (ELISA) assay for 14-3-3γ, in comparison with WB and other neurodegeneration markers. CSF samples from 145 patients with suspicion of prion disease, later classified as definite sCJD (n=72) or Non-prion diseases (Non-CJD; n=73) comprised our population. 14-3-3 protein was determined by WB and ELISA. Total Tau (t-Tau) and phosphorylated Tau (p-Tau) were also evaluated. Apolipoprotein E gene (ApoE) and prionic protein gene (PRNP) genotyping was assessed. ELISA 14-3-3γ levels were significantly increased in sCJD compared to Non-CJD patients (p<0.001), showing very good accuracy (AUC=0.982; sensitivity=97%; specificity=94%), and matching WB results in 81% of all cases. It strongly correlated with t-Tau and p-Tau (p<0.0001), showing slightly higher specificity (14-3-3 WB - 63%; Tau - 90%; p-Tau/t-Tau ratio - 88%). From WB inconclusive results (n=44), ELISA 14-3-3γ correctly classified 41 patients. Additionally, logistic regression analysis selected ELISA 14-3-3γ as the best single predictive marker for sCJD (overall accuracy=93%). ApoE and PRNP genotypes did not influence ELISA 14-3-3γ levels. Despite specificity for 14-3-3γ isoform, ELISA results not only match WB evaluation but also help discrimination of inconclusive results. Our results therefore reinforce this assay as a single screening test, allowing higher sample throughput and unequivocal results.

  7. Profile of the MP Diagnostics HTLV Blot 2.4 test: a supplemental assay for the confirmation and differentiation of antibodies to HTLV-1 and HTLV-2.

    PubMed

    Miller, Liane

    2016-01-01

    As the first US FDA-approved assay for supplemental HTLV testing, the MP Diagnostics HTLV Blot 2.4 is an effective and efficient method for confirming and differentiating HTLV type infection in repeatedly reactive samples. Novel and patented antigens added increased sensitivity in identifying specimens from infected individuals while differentiating those from uninfected individuals with false reactivity.

  8. Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A molecular diagnostic assay utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 39 °C and target-specific primers and probe were developed for the rapid, sensitive, and specific detection of tomato chlorotic dwarf viroid (TCDVd) in ...

  9. 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.

  10. Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification.

    PubMed

    Hammond, Rosemarie W; Zhang, Shulu

    2016-10-01

    A molecular diagnostic assay utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 39°C and target-specific primers and probe were developed for the rapid, sensitive, and specific detection of tomato chlorotic dwarf viroid (TCDVd) in infected leaf and seed tissues. The performance of the AmplifyRP(®) Acceler8™ RT-RPA diagnostic assay, utilizing a lateral flow strip contained within an amplicon detection chamber, was evaluated and the results were compared with a standard RT-PCR assay. The AmplifyRP(®) Acceler8™ assay was specific for TCDVd in leaf and seed tissues, its sensitivity was comparable to conventional RT-PCR in leaf tissues, and it does not require extensive sample purification, specialized equipment, or technical expertise. This is the first report utilizing an RT-RPA assay to detect viroids and the assay can be used both in the laboratory and in the field for TCDVd detection.

  11. Multicountry prospective clinical evaluation of two enzyme-linked immunosorbent assays and two rapid diagnostic tests for diagnosing dengue fever.

    PubMed

    Pal, Subhamoy; Dauner, Allison L; Valks, Andrea; Forshey, Brett M; Long, Kanya C; Thaisomboonsuk, Butsaya; Sierra, Gloria; Picos, Victor; Talmage, Sara; Morrison, Amy C; Halsey, Eric S; Comach, Guillermo; Yasuda, Chadwick; Loeffelholz, Michael; Jarman, Richard G; Fernandez, Stefan; An, Ung Sam; Kochel, Tadeusz J; Jasper, Louis E; Wu, Shuenn-Jue L

    2015-04-01

    We evaluated four dengue diagnostic devices from Alere, including the SD Bioline Dengue Duo (nonstructural [NS] 1 Ag and IgG/IgM), the Panbio Dengue Duo Cassette (IgM/IgG) rapid diagnostic tests (RDTs), and the Panbio dengue IgM and IgG capture enzyme-linked immunosorbent assays (ELISAs) in a prospective, controlled, multicenter study in Peru, Venezuela, Cambodia, and the United States, using samples from 1,021 febrile individuals. Archived, well-characterized samples from an additional 135 febrile individuals from Thailand were also used. Reference testing was performed on all samples using an algorithm involving virus isolation, in-house IgM and IgG capture ELISAs, and plaque reduction neutralization tests (PRNT) to determine the infection status of the individual. The primary endpoints were the clinical sensitivities and specificities of these devices. The SD Bioline Dengue Duo had an overall sensitivity of 87.3% (95% confidence interval [CI], 84.1 to 90.2%) and specificity of 86.8% (95% CI, 83.9 to 89.3%) during the first 14 days post-symptom onset (p.s.o.). The Panbio Dengue Duo Cassette demonstrated a sensitivity of 92.1% (87.8 to 95.2%) and specificity of 62.2% (54.5 to 69.5%) during days 4 to 14 p.s.o. The Panbio IgM capture ELISA had a sensitivity of 87.6% (82.7 to 91.4%) and specificity of 88.1% (82.2 to 92.6%) during days 4 to 14 p.s.o. Finally, the Panbio IgG capture ELISA had a sensitivity of 69.6% (62.1 to 76.4%) and a specificity of 88.4% (82.6 to 92.8%) during days 4 to 14 p.s.o. for identification of secondary dengue infections. This multicountry prospective study resulted in reliable real-world performance data that will facilitate data-driven laboratory test choices for managing patient care during dengue outbreaks.

  12. Multicountry Prospective Clinical Evaluation of Two Enzyme-Linked Immunosorbent Assays and Two Rapid Diagnostic Tests for Diagnosing Dengue Fever

    PubMed Central

    Dauner, Allison L.; Valks, Andrea; Forshey, Brett M.; Long, Kanya C.; Thaisomboonsuk, Butsaya; Sierra, Gloria; Picos, Victor; Talmage, Sara; Morrison, Amy C.; Halsey, Eric S.; Comach, Guillermo; Yasuda, Chadwick; Loeffelholz, Michael; Jarman, Richard G.; Fernandez, Stefan; An, Ung Sam; Kochel, Tadeusz J.; Jasper, Louis E.; Wu, Shuenn-Jue L.

    2015-01-01

    We evaluated four dengue diagnostic devices from Alere, including the SD Bioline Dengue Duo (nonstructural [NS] 1 Ag and IgG/IgM), the Panbio Dengue Duo Cassette (IgM/IgG) rapid diagnostic tests (RDTs), and the Panbio dengue IgM and IgG capture enzyme-linked immunosorbent assays (ELISAs) in a prospective, controlled, multicenter study in Peru, Venezuela, Cambodia, and the United States, using samples from 1,021 febrile individuals. Archived, well-characterized samples from an additional 135 febrile individuals from Thailand were also used. Reference testing was performed on all samples using an algorithm involving virus isolation, in-house IgM and IgG capture ELISAs, and plaque reduction neutralization tests (PRNT) to determine the infection status of the individual. The primary endpoints were the clinical sensitivities and specificities of these devices. The SD Bioline Dengue Duo had an overall sensitivity of 87.3% (95% confidence interval [CI], 84.1 to 90.2%) and specificity of 86.8% (95% CI, 83.9 to 89.3%) during the first 14 days post-symptom onset (p.s.o.). The Panbio Dengue Duo Cassette demonstrated a sensitivity of 92.1% (87.8 to 95.2%) and specificity of 62.2% (54.5 to 69.5%) during days 4 to 14 p.s.o. The Panbio IgM capture ELISA had a sensitivity of 87.6% (82.7 to 91.4%) and specificity of 88.1% (82.2 to 92.6%) during days 4 to 14 p.s.o. Finally, the Panbio IgG capture ELISA had a sensitivity of 69.6% (62.1 to 76.4%) and a specificity of 88.4% (82.6 to 92.8%) during days 4 to 14 p.s.o. for identification of secondary dengue infections. This multicountry prospective study resulted in reliable real-world performance data that will facilitate data-driven laboratory test choices for managing patient care during dengue outbreaks. PMID:25588659

  13. Application of dosimetry systems and cytogenetic status of the child population exposed to diagnostic X-rays by use of the cytokinesis-block micronucleus cytome assay.

    PubMed

    Gajski, Goran; Milković, Durđica; Ranogajec-Komor, Mária; Miljanić, Saveta; Garaj-Vrhovac, Vera

    2011-10-01

    Low-dose ionizing radiation used for medical purposes is one of the definite risk factors for cancer development, and children exposed to ionizing radiation are at a relatively greater cancer risk as they have more rapidly dividing cells than adults and have longer life expectancy. Since cytokinesis-block micronucleus cytome (CBMN Cyt) assay has become one of the standard endpoints for radiation biological dosimetry, we used that assay in the present work for the assessment of different types of chromosomal damage in children exposed to diagnostic X-ray procedures. Twenty children all with pulmonary diseases between the ages of 4 and 14 years (11.30 ± 2.74) were evaluated. Absorbed dose measurements were conducted for posterior-anterior projection on the forehead, thyroid gland, gonads, chest and back. Doses were measured using thermoluminescence and radiophotoluminescent dosimetry systems. It was shown that, after diagnostic X-rays, the mean total number of CBMN Cyt assay parameters (micronucleus, nucleoplasmic bridges and nuclear buds) was significantly higher than prior to diagnostic procedure and that interindividual differences existed for each monitored child. For the nuclear division index counted prior and after examination, no significant differences were noted among mean group values. These data suggest that even low-dose diagnostic X-ray exposure may induce damaging effect in the somatic DNA of exposed children, indicating that immense care should be given in both minimizing and optimizing radiation exposure to diminish the radiation burden, especially in the youngest population.

  14. Diagnostic and genetic studies on fibrin-stabilizing factor with a new assay based on amine incorporation.

    PubMed

    Lorand, L; Urayama, T; De Kiewiet, J W; Nossel, H L

    1969-06-01

    -stabilizing factor, though not quite as low as the other two relatives. Two infusions (1 liter each) of fresh normal plasma, administered about 26 hr apart, brought levels in the patient's plasma close to those found in the mother and brother. The corrective power of the transfusions, however, rapidly declined within 5-6 days. Futility of the last transfusion could be ascribed to the appearance of a neutralizing antibody directed against the precursor stabilizing factor, a serious complication. General diagnostic versatility and potential of the quantitative amine incorporation assay with plasma is discussed.

  15. DNA Detection of Schistosoma japonicum: Diagnostic Validity of a LAMP Assay for Low-Intensity Infection and Effects of Chemotherapy in Humans

    PubMed Central

    Zhao, Bo; Wang, Yan-Yan; Cao, Yun; Zhang, Hui-Qin; Zhu, Xing-Quan; He, Yong-Kang; Xia, Chao-Ming

    2015-01-01

    Background Schistosomiasis has decreased significantly in prevalence and intensity of infection in China, thus more accurate and sensitive methods are desperately needed for the further control of schistosomiasis. The present work aimed to assess the utility of the loop-mediated isothermal amplification (LAMP) for detection of light intensity infection or false-negative patients and patients post-treatment, targeting the highly repetitive retrotransposon SjR2 of Schistosoma japonicum. Methodology/ Principal Findings LAMP was first assessed in rabbits with low intensity infection (EPG<10). Then 110 patient sera from Hunan Province, China, and 47 sera after treatment by praziquantel were used to evaluate the diagnostic validity of LAMP. Meanwhile, 42 sera from healthy individuals in a non-endemic area, and 60 sera from "healthy” residents who were identified as being negative for feces examination and immuno-methods in an endemic area were also examined. The results showed that LAMP could detect S. japonicum DNA in sera from rabbits at 3rd day post-infection. Following administration of praziquantel, the S. japonicum DNA in rabbit sera became negative at 10 weeks post-treatment. Of 110 sera from patients, LAMP showed 95.5% sensitivity, and even for 41 patients with less than 10 EPG, the sensitivity of LAMP still reached to 95.1%. For 47 patients after treatment, the negative conversion rate of S. japonicum DNA in patient sera increased from 23.4%, 61.7% to 83.0% at 3 months, 6 months and 9 months post-treatment, respectively. No false-positive result was obtained for 42 human sera from non-endemic area, while for the 60 “healthy” individuals from endemic area, 10 (16.7%) individuals were positive by LAMP, which suggested that these individuals might be false-negative patients. Conclusions/ Significance The present study demonstrated that the LAMP assay is sensitive, specific, and affordable, which would help reduce schistosomiasis transmission through targeted

  16. Authentication of animal signatures in traditional Chinese medicine of Lingyang Qingfei Wan using routine molecular diagnostic assays.

    PubMed

    Cao, Meng; Wang, Jikun; Yao, Lu; Xie, Suhua; Du, Jing; Zhao, Xingbo

    2014-01-01

    Lingyang Qingfei Wan produced by Beijing TongRenTang is a long-standing and popular medicine in China and international pharmaceutical markets. Concerns continue to be raised about the legality of usage of saiga antelope, which was defined as endangered species by Convention on International Trade in Endangered Species of Wild Fauna and Flora legislation and internal legislation in China. Therefore, the alternative pill in which substitutes saiga antelope with goat in the formula of Lingyang Qingfei Wan was developed. In order to authenticate the origin of animal contents in Lingyang Qingfei Wan and its alternative pill, molecular diagnostic assay was utilized by mtDNA polymorphism analysis. Four universal primer pairs containing mtDNA 12SrRNA, 16SrRNA, cytochrome b gene and cytochrome oxidase I were employed to obtain species-specific sequences of saiga antelope and goat, and multiple species-specific primer pairs for saiga antelope and goat were used to identify the animal origin in patent pills according to nucleotide polymorphisms between the two species. In additions, alternative techniques were attempted surrounding dilemmas of low concentration of target DNAs and presence of PCR-inhibitory substances in organic ingredients within complex pill. Results revealed that all species-specific primers could be successfully used for authentication of animal origin within complex pill, and sample preprocessing was critical during experimental manipulation. Internal positive control was an efficient and cost-effective way to assist in monitoring the potential interference from inhibitory substances which existed in the highly processed pills.

  17. Test and Evaluation of Field-Deployable Infectious Disease Diagnostic Assays in Support of the Joint Biological Agent Identification and Diagnosis System (JBAIDS): Malaria (Plasmodium/JBAIDS)

    DTIC Science & Technology

    2012-05-31

    Disease Diagnostic Assays in Support of the Joint Biological Agent Identification and Diagnosis System 5b. GRANT NUMBER (JBAIDS): Malaria (Plasmodium...JBAIDS) 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER James C. McAvin (Designated PI July 2011), Clinical Research Division/59th...ORGANIZATION NAME(S) AND ADDRESS(ESI 8. PERFORMING ORGANIZATION Clinical Research Division, CSPG/SGVUL, 59th MDW, 2200 Bergquist Drive, REPORT NUMBER

  18. Inventions leading to the development of the diagnostic test kit industry--from the modern pregnancy test to the sandwich assays.

    PubMed

    Wide, Leif

    2005-01-01

    The universities are encouraged by the government nowadays to stimulate innovations and also to provide the proper machinery for assisting the protection and commercialisation of innovations. A better understanding of the innovation process may help to create an atmosphere suitable for inventions at the university. Examples can be taken from successful innovations previously made at the university. During the 1960's I made a series of inventions, which ultimately led to the development of the diagnostic test kit industry. The first, which I made as an undergraduate, was a simple and reliable test kit for diagnosis of pregnancy. This was followed by the solid phase radioimmunoassay and a solid phase assay for vitamin B12; next, the dual specific non-competitive sandwich assay and the in-vitro test for diagnosis of allergy, called RAST (Radioallergosorbent test). Organon in Holland with the pregnancy test kit, and Pharmacia in Sweden with test kits for radioimmunoassay, became pioneers among the diagnostic test kit industries. Pharmacia Diagnostics later became one of the leading diagnostic test kit companies in the world and has continued to be so in the field of allergy diagnosis. Each one of these inventions started with a few unique observations leading to a technical development. The pregnancy test as well as the allergy test emerged from the development of assay methods with unique qualities with the subsequent search for appropriate applications. The foreseeing of a commercial value on a future market was a very important step. This was followed by the search for a suitable industry interested to exploit the invention with its new business opportunity i.e. apply for a patent, produce and market the products, which in my case consisted of the necessary reagents and equipments for particular diagnostic tests. Finally, an agreement had to be settled between the entrepreneur and the inventors. This report describes these inventions and particularly discusses some

  19. Development of a diagnostic real-time polymerase chain reaction assay for the detection of invasive Haemophilus influenzae in clinical samples.

    PubMed

    Meyler, Kenneth L; Meehan, Mary; Bennett, Desiree; Cunney, Robert; Cafferkey, Mary

    2012-12-01

    Since the introduction of the Haemophilus influenzae serotype b vaccine, invasive H. influenzae disease has become dominated by nontypeable (NT) strains. Several widely used molecular diagnostic methods have been shown to lack sensitivity or specificity in the detection of some of these strains. Novel real-time assays targeting the fucK, licA, and ompP2 genes were developed and evaluated. The fucK assay detected all strains of H. influenzae tested (n = 116) and had an analytical sensitivity of 10 genome copies/polymerase chain reaction (PCR). This assay detected both serotype b and NT H. influenzae in 12 previously positive specimens (culture and/or bexA PCR) and also detected H. influenzae in a further 5 of 883 culture-negative blood and cerebrospinal fluid (CSF) samples. The fucK assay has excellent potential as a diagnostic test for detection of typeable and nontypeable strains of invasive H. influenzae in clinical samples of blood and CSF.

  20. Population-based Tay-Sachs screening among Ashkenazi Jewish young adults in the 21st century: Hexosaminidase A enzyme assay is essential for accurate testing.

    PubMed

    Schneider, Adele; Nakagawa, Sachiko; Keep, Rosanne; Dorsainville, Darnelle; Charrow, Joel; Aleck, Kirk; Hoffman, Jodi; Minkoff, Sherman; Finegold, David; Sun, Wei; Spencer, Andrew; Lebow, Johannah; Zhan, Jie; Apfelroth, Stephen; Schreiber-Agus, Nicole; Gross, Susan

    2009-11-01

    Tay-Sachs disease (TSD) carrier screening, initiated in the 1970s, has reduced the birth-rate of Ashkenazi Jews with TSD worldwide by 90%. Recently, several nationwide programs have been established that provide carrier screening for the updated panel of Jewish genetic diseases on college campuses and in Jewish community settings. The goals of this study were to determine the performance characteristics of clinical TSD testing in college- and community-based screening programs and to determine if molecular testing alone is adequate in those settings. Clinical data for TSD testing were retrospectively anonymized and subsequently analyzed for 1,036 individuals who participated in these programs. The performance characteristics of the serum and the platelet Hexosaminidase assays were compared, and also correlated with the results of targeted DNA analysis. The serum assay identified 29 carriers and the platelet assay identified 35 carriers for carrier rates of 1/36 and 1/29, respectively. One hundred sixty-nine samples (16.3%) were inconclusive by serum assay in marked contrast to four inconclusive samples (0.4%) by the platelet assay. Molecular analysis alone would have missed four of the 35 carriers detected by the platelet assay, yielding a false negative rate of 11.4% with a sensitivity of 88.6%. Based on the results of this study, platelet assay was superior to serum with a minimal inconclusive rate. Due to changing demographics of the Ashkenazi Jewish population, molecular testing alone in the setting of broad-based population screening programs is not sufficient, and biochemical analysis should be the assay of choice.

  1. Clinical and diagnostic developments of a gamma interferon release assay for use in bovine tuberculosis control programs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Currently the Bovigam assay is used as an official supplemental test within the bovine tuberculosis eradication program. This assay measures interferon-gamma (IFN-gamma) produced by lymphocytes in response to specific antigens. The objectives of the present study were to evaluate two Mycobacterium ...

  2. 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.

  3. Performance of the Elecsys Rubella IgG Assay in the Diagnostic Laboratory Setting for Assessment of Immune Status

    PubMed Central

    Bartelt, Uwe; Knotek, Frank; Bunn, Kristina; Strobel, Sirpa; Dietz, Klaus; Enders, Gisela

    2013-01-01

    Rubella in early pregnancy bears a high risk for congenital defects (e.g., cataracts, hearing loss, and heart disease) and for long-term sequelae in the newborn. Despite implementation of vaccination programs in many regions, the threat of devastating consequences from congenital rubella virus infection remains and careful screening of maternal immune status before and during pregnancy helps to reduce the risk. This study compared the performance of the Elecsys Rubella IgG assay with that of other assays routinely used for screening. Samples from 1,090 women undergoing routine antenatal care were tested using the Elecsys and Enzygnost Rubella IgG assays and the hemagglutination inhibition test. Samples with hemagglutination inhibition titers of <32 (n = 148) were additionally tested using the Vidas, AxSYM, Liaison, and Architect Rubella IgG assays. Agreement of qualitative results from the Elecsys, Enzygnost, and hemagglutination inhibition assays was good in all samples. All assays showed 100.0% specificity. In samples with hemagglutination inhibition titers of <32, the Elecsys, AxSYM, and Enzygnost assays showed higher sensitivity (>90.0%) than the other immunoassays (78.6 to 82.4%). The Elecsys assay reported significantly higher rubella virus IgG levels than the other immunoassays across the whole set of 1,090 samples, with the largest bias and deviation from limits of agreement in Bland-Altman analysis. In conclusion, the Elecsys assay is highly sensitive and specific with regard to qualitative results and suitable for routine automated screening. However, given the considerable variation between quantitative results from different immunoassays, testing methods should be documented and the same assay used throughout an individual's antenatal follow-up wherever possible. PMID:23345585

  4. 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

  5. An Automated High-Throughput Metabolic Stability Assay Using an Integrated High-Resolution Accurate Mass Method and Automated Data Analysis Software

    PubMed Central

    Shah, Pranav; Kerns, Edward; Nguyen, Dac-Trung; Obach, R. Scott; Wang, Amy Q.; Zakharov, Alexey; McKew, John; Simeonov, Anton; Hop, Cornelis E. C. A.

    2016-01-01

    Advancement of in silico tools would be enabled by the availability of data for metabolic reaction rates and intrinsic clearance (CLint) of a diverse compound structure data set by specific metabolic enzymes. Our goal is to measure CLint for a large set of compounds with each major human cytochrome P450 (P450) isozyme. To achieve our goal, it is of utmost importance to develop an automated, robust, sensitive, high-throughput metabolic stability assay that can efficiently handle a large volume of compound sets. The substrate depletion method [in vitro half-life (t1/2) method] was chosen to determine CLint. The assay (384-well format) consisted of three parts: 1) a robotic system for incubation and sample cleanup; 2) two different integrated, ultraperformance liquid chromatography/mass spectrometry (UPLC/MS) platforms to determine the percent remaining of parent compound, and 3) an automated data analysis system. The CYP3A4 assay was evaluated using two long t1/2 compounds, carbamazepine and antipyrine (t1/2 > 30 minutes); one moderate t1/2 compound, ketoconazole (10 < t1/2 < 30 minutes); and two short t1/2 compounds, loperamide and buspirone (t½ < 10 minutes). Interday and intraday precision and accuracy of the assay were within acceptable range (∼12%) for the linear range observed. Using this assay, CYP3A4 CLint and t1/2 values for more than 3000 compounds were measured. This high-throughput, automated, and robust assay allows for rapid metabolic stability screening of large compound sets and enables advanced computational modeling for individual human P450 isozymes. PMID:27417180

  6. Diagnostic value of animal-side antibody assays for rapid detection of Mycobacterium bovis or Mycobacterium microti infection in South American camelids.

    PubMed

    Lyashchenko, Konstantin P; Greenwald, Rena; Esfandiari, Javan; Rhodes, Shelley; Dean, Gillian; de la Rua-Domenech, Ricardo; Meylan, Mireille; Vordermeier, H Martin; Zanolari, Patrik

    2011-12-01

    Tuberculosis (TB) in South American camelids (SAC) is caused by Mycobacterium bovis or Mycobacterium microti. Two serological methods, rapid testing (RT) and the dual-path platform (DPP) assay, were evaluated using naturally infected SAC. The study population included 156 alpacas and 175 llamas in Great Britain, Switzerland, and the United States. TB due to M. bovis (n = 44) or M. microti (n = 8) in 35 alpacas and 17 llamas was diagnosed by gross pathology examination and culture. Control animals were from herds with no TB history. The RT and the DPP assay showed sensitivities of 71% and 74%, respectively, for alpacas, while the sensitivity for llamas was 77% for both assays. The specificity of the DPP assay (98%) was higher than that of RT (94%) for llamas; the specificities of the two assays were identical (98%) for alpacas. When the two antibody tests were combined, the parallel-testing interpretation (applied when either assay produced a positive result) enhanced the sensitivities of antibody detection to 89% for alpacas and 88% for llamas but at the cost of lower specificities (97% and 93%, respectively), whereas the serial-testing interpretation (applied when both assays produced a positive result) maximized the specificity to 100% for both SAC species, although the sensitivities were 57% for alpacas and 65% for llamas. Over 95% of the animals with evidence of TB failed to produce skin test reactions, thus confirming concerns about the validity of this method for testing SAC. The findings suggest that serological assays may offer a more accurate and practical alternative for antemortem detection of camelid TB.

  7. Ferric plasmonic nanoparticles, aptamers, and magnetofluidic chips: toward the development of diagnostic surface-enhanced Raman spectroscopy assays

    NASA Astrophysics Data System (ADS)

    Marks, Haley; Huang, Po-Jung; Mabbott, Samuel; Graham, Duncan; Kameoka, Jun; Coté, Gerard

    2016-12-01

    Conjugation of aptamers and their corresponding analytes onto plasmonic nanoparticles mediates the formation of nanoparticle assemblies: molecularly bound nanoclusters that cause a measurable change in the colloid's optical properties. The optimization of a surface-enhanced Raman spectroscopy (SERS) competitive binding assay utilizing plasmonic "target" and magnetic "probe" nanoparticles for the detection of the toxin bisphenol-A (BPA) is presented. These assay nanoclusters were housed inside three types of optofluidic chips patterned with magnetically activated nickel pads, in either a straight or array pattern. Both Fe2O3 and Fe2CoO4 were compared as potential magnetic cores for the silver-coated probe nanoparticles. We found that the Ag@Fe2O3 particles were, on average, more uniform in size and more stable than Ag@Fe2CoO4, whereas the addition of cobalt significantly improved the collection time of particles. Using Raman mapping of the assay housed within the magnetofluidic chips, it was determined that a 1×5 array of 50 μm square nickel pads provided the most uniform SERS enhancement of the assay (coefficient of variation ˜25%) within the magnetofluidic chip. Additionally, the packaged assay demonstrated the desired response to BPA, verifying the technology's potential to translate magnetic nanoparticle assays into a user-free optical analysis platform.

  8. Accuracy of molecular diagnostics in pemphigus and bullous pemphigoid: comparison of commercial and modified mosaic indirect immunofluorescence tests as well as enzyme-linked immunosorbent assays

    PubMed Central

    Seraszek-Jaros, Agnieszka; Bowszyc-Dmochowska, Monika; Kaczmarek, Elżbieta; Pietkiewicz, Paweł; Bartkiewicz, Paweł; Dmochowski, Marian

    2017-01-01

    Introduction Pemphigus and bullous pemphigoid (BP) are identified by autoantibodies (abs) against desmoglein 1, 3 (DSG1/3) and BP180/BP230, respectively. A novel mosaic to indirect immunofluorescence (IIF) using purified BP180 recombinant proteins spotted on slide and transfected cells expressing BP230, DSG1, DSG3 is available. The commercial (IgG detection) and modified (IgG4 detection) mosaic for indirect immunofluorescence (IIFc – IIF commercial, IIFm – IIF modified) and IgG ELISAs were evaluated in pemphigus and bullous pemphigoid (BP) molecular diagnostics. Aim To compare diagnostic accuracy of commercial (IgG detection) and modified (IgG4 detection) mosaic IIF assay and to examine the diagnostic value of ELISAs in relation to mosaic IIF in routine laboratory diagnostics of pemphigus and BP. Material and methods Sera from 37 BP and 19 pemphigus patients were studied. Associations between tests were assessed using Fisher’s exact test. Results There are associations between the positive/negative samples detected by IIFc with desmoglein1 (DSG1)/desmoglein3 (DSG3)/BP230 transfected cells and ELISAs and no association between anti-BP180 IgG detection by IIFc and ELISA. IIFm with DSG1 and DSG3 showed both 100% sensitivity and 100% and 78% specificity, respectively, and 100% and 83% positive predictive value in relation to IIFc. IIFm with BP230 had 87% specificity, 55% sensitivity, whereas IIFm with BP180 had a 100% sensitivity and 13% specificity in relation to IIFc. Conclusions The IIFc with DSG1/DSG3/BP230 transfected cells, excluding BP180 spots, is an alternative method to ELISA in pemphigus/BP diagnostics. IgG4 antibodies, both pathogenically and diagnostically important, are inconsistently detectable with IIFm. PMID:28261028

  9. Reprint of 'Draw your assay: Fabrication of low-cost paper-based diagnostic and multi-well test zones by drawing on a paper'.

    PubMed

    Oyola-Reynoso, Stephanie; Heim, Andrew P; Halbertsma-Black, Julian; Zhao, C; Tevis, Ian D; Çınar, Simge; Cademartiri, Rebecca; Liu, Xinyu; Bloch, Jean-Francis; Thuo, Martin M

    2015-12-01

    Interest in low-cost diagnostic devices has recently gained attention, in part due to the rising cost of healthcare and the need to serve populations in resource-limited settings. A major challenge in the development of such devices is the need for hydrophobic barriers to contain polar bio-fluid analytes. Key approaches in lowering the cost in diagnostics have centered on (i) development of low-cost fabrication techniques/processes, (ii) use of affordable materials, or, (iii) minimizing the need for high-tech tools. This communication describes a simple, low-cost, adaptable, and portable method for patterning paper and subsequent use of the patterned paper in diagnostic tests. Our approach generates hydrophobic regions using a ball-point pen filled with a hydrophobizing molecule suspended in a solvent carrier. An empty ball-point pen was filled with a solution of trichloro perfluoroalkyl silane in hexanes (or hexadecane), and the pen used to draw lines on Whatman® chromatography 1 paper. The drawn regions defined the test zones since the trichloro silane reacts with the paper to give a hydrophobic barrier. The formation of the hydrophobic barriers is reaction kinetic and diffusion-limited, ensuring well defined narrow barriers. We performed colorimetric glucose assays and enzyme-linked immuno-sorbent assay (ELISA) using the created test zones. To demonstrate the versatility of this approach, we fabricated multiple devices on a single piece of paper and demonstrated the reproducibility of assays on these devices. The overall cost of devices fabricated by drawing are relatively lower (

  10. Draw your assay: Fabrication of low-cost paper-based diagnostic and multi-well test zones by drawing on a paper.

    PubMed

    Oyola-Reynoso, Stephanie; Heim, Andrew P; Halbertsma-Black, Julian; Zhao, C; Tevis, Ian D; Çınar, Simge; Cademartiri, Rebecca; Liu, Xinyu; Bloch, Jean-Francis; Thuo, Martin M

    2015-11-01

    Interest in low-cost diagnostic devices has recently gained attention, in part due to the rising cost of healthcare and the need to serve populations in resource-limited settings. A major challenge in the development of such devices is the need for hydrophobic barriers to contain polar bio-fluid analytes. Key approaches in lowering the cost in diagnostics have centered on (i) development of low-cost fabrication techniques/processes, (ii) use of affordable materials, or, (iii) minimizing the need for high-tech tools. This communication describes a simple, low-cost, adaptable, and portable method for patterning paper and subsequent use of the patterned paper in diagnostic tests. Our approach generates hydrophobic regions using a ball-point pen filled with a hydrophobizing molecule suspended in a solvent carrier. An empty ball-point pen was filled with a solution of trichloro perfluoroalkyl silane in hexanes (or hexadecane), and the pen used to draw lines on Whatman® chromatography 1 paper. The drawn regions defined the test zones since the trichloro silane reacts with the paper to give a hydrophobic barrier. The formation of the hydrophobic barriers is reaction kinetic and diffusion-limited, ensuring well defined narrow barriers. We performed colorimetric glucose assays and enzyme-linked immuno-sorbent assay (ELISA) using the created test zones. To demonstrate the versatility of this approach, we fabricated multiple devices on a single piece of paper and demonstrated the reproducibility of assays on these devices. The overall cost of devices fabricated by drawing are relatively lower (

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

    PubMed 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

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

    PubMed

    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.

  13. Driving efficiency in a high-throughput metabolic stability assay through a generic high-resolution accurate mass method and automated data mining.

    PubMed

    Shui, Wenqing; Lin, Song; Zhang, Allen; Chen, Yan; Huang, Yingying; Sanders, Mark

    2011-08-01

    Improving analytical throughput is the focus of many quantitative workflows being developed for early drug discovery. For drug candidate screening, it is common practice to use ultra-high performance liquid chromatography (U-HPLC) coupled with triple quadrupole mass spectrometry. This approach certainly results in short analytical run time; however, in assessing the true throughput, all aspects of the workflow needs to be considered, including instrument optimization and the necessity to re-run samples when information is missed. Here we describe a high-throughput metabolic stability assay with a simplified instrument set-up which significantly improves the overall assay efficiency. In addition, as the data is acquired in a non-biased manner, high information content of both the parent compound and metabolites is gathered at the same time to facilitate the decision of which compounds to proceed through the drug discovery pipeline.

  14. Total Vitamin D Assay Comparison of the Roche Diagnostics “Vitamin D Total” Electrochemiluminescence Protein Binding Assay with the Chromsystems HPLC Method in a Population with both D2 and D3 forms of Vitamin D

    PubMed Central

    Abdel-Wareth, Laila; Haq, Afrozul; Turner, Andrew; Khan, Shoukat; Salem, Arwa; Mustafa, Faten; Hussein, Nafiz; Pallinalakam, Fasila; Grundy, Louisa; Patras, Gemma; Rajah, Jaishen

    2013-01-01

    This study compared two methods of assaying the 25-hydroxylated metabolites of cholecalciferol (vitamin D3) and ergocalciferol (vitamin D2). A fully automated electrochemiluminescence assay from Roche Diagnostics and an HPLC based method from Chromsystems were used to measure vitamin D levels in surplus sera from 96 individuals, where the majority has the D2 form of the vitamin. Deming regression, concordance rate, correlation and Altman Bland agreement were performed. Seventy two subjects (75%) had a D2 concentration >10 nmol/L while the remaining twenty four subjects had vitamin D2 concentration of less than 10 nmol/L by HPLC. Overall, the Roche Diagnostics method showed a negative bias of −2.59 ± 4.11 nmol/L on the e602 as compared to the HPLC with a concordance rate of 84%. The concordance rate was 91% in samples with D2 of less than 10 nmol/L and 82% in those with D2 concentration >10 nmol/L. The overall correlation had an r value of 0.77. The r value was higher in samples with D2 levels of less than 10 nmol/L, r = 0.96, as compared to those with D2 values of greater than 10 nmol/L, r = 0.74. The observed bias had little impact on clinical decision and therefore is clinically acceptable. PMID:23525081

  15. Total vitamin D assay comparison of the Roche Diagnostics "Vitamin D total" electrochemiluminescence protein binding assay with the Chromsystems HPLC method in a population with both D2 and D3 forms of vitamin D.

    PubMed

    Abdel-Wareth, Laila; Haq, Afrozul; Turner, Andrew; Khan, Shoukat; Salem, Arwa; Mustafa, Faten; Hussein, Nafiz; Pallinalakam, Fasila; Grundy, Louisa; Patras, Gemma; Rajah, Jaishen

    2013-03-22

    This study compared two methods of assaying the 25-hydroxylated metabolites of cholecalciferol (vitamin D3) and ergocalciferol (vitamin D2). A fully automated electrochemiluminescence assay from Roche Diagnostics and an HPLC based method from Chromsystems were used to measure vitamin D levels in surplus sera from 96 individuals, where the majority has the D2 form of the vitamin. Deming regression, concordance rate, correlation and Altman Bland agreement were performed. Seventy two subjects (75%) had a D2 concentration >10 nmol/L while the remaining twenty four subjects had vitamin D2 concentration of less than 10 nmol/L by HPLC. Overall, the Roche Diagnostics method showed a negative bias of -2.59 ± 4.11 nmol/L on the e602 as compared to the HPLC with a concordance rate of 84%. The concordance rate was 91% in samples with D2 of less than 10 nmol/L and 82% in those with D2 concentration >10 nmol/L. The overall correlation had an r value of 0.77. The r value was higher in samples with D2 levels of less than 10 nmol/L, r = 0.96, as compared to those with D2 values of greater than 10 nmol/L, r = 0.74. The observed bias had little impact on clinical decision and therefore is clinically acceptable.

  16. Target Product Profile for a Diagnostic Assay to Differentiate between Bacterial and Non-Bacterial Infections and Reduce Antimicrobial Overuse in Resource-Limited Settings: An Expert Consensus

    PubMed Central

    Dittrich, Sabine; Tadesse, Birkneh Tilahun; Moussy, Francis; Chua, Arlene; Zorzet, Anna; Tängdén, Thomas; Dolinger, David L.; Page, Anne-Laure; Crump, John A.; D’Acremont, Valerie; Bassat, Quique; Lubell, Yoel; Newton, Paul N.; Heinrich, Norbert; Rodwell, Timothy J.; González, Iveth J.

    2016-01-01

    Acute fever is one of the most common presenting symptoms globally. In order to reduce the empiric use of antimicrobial drugs and improve outcomes, it is essential to improve diagnostic capabilities. In the absence of microbiology facilities in low-income settings, an assay to distinguish bacterial from non-bacterial causes would be a critical first step. To ensure that patient and market needs are met, the requirements of such a test should be specified in a target product profile (TPP). To identify minimal/optimal characteristics for a bacterial vs. non-bacterial fever test, experts from academia and international organizations with expertise in infectious diseases, diagnostic test development, laboratory medicine, global health, and health economics were convened. Proposed TPPs were reviewed by this working group, and consensus characteristics were defined. The working group defined non-severely ill, non-malaria infected children as the target population for the desired assay. To provide access to the most patients, the test should be deployable to community health centers and informal health settings, and staff should require <2 days of training to perform the assay. Further, given that the aim is to reduce inappropriate antimicrobial use as well as to deliver appropriate treatment for patients with bacterial infections, the group agreed on minimal diagnostic performance requirements of >90% and >80% for sensitivity and specificity, respectively. Other key characteristics, to account for the challenging environment at which the test is targeted, included: i) time-to-result <10 min (but maximally <2 hrs); ii) storage conditions at 0–40°C, ≤90% non-condensing humidity with a minimal shelf life of 12 months; iii) operational conditions of 5–40°C, ≤90% non-condensing humidity; and iv) minimal sample collection needs (50–100μL, capillary blood). This expert approach to define assay requirements for a bacterial vs. non-bacterial assay should guide

  17. Target Product Profile for a Diagnostic Assay to Differentiate between Bacterial and Non-Bacterial Infections and Reduce Antimicrobial Overuse in Resource-Limited Settings: An Expert Consensus.

    PubMed

    Dittrich, Sabine; Tadesse, Birkneh Tilahun; Moussy, Francis; Chua, Arlene; Zorzet, Anna; Tängdén, Thomas; Dolinger, David L; Page, Anne-Laure; Crump, John A; D'Acremont, Valerie; Bassat, Quique; Lubell, Yoel; Newton, Paul N; Heinrich, Norbert; Rodwell, Timothy J; González, Iveth J

    2016-01-01

    Acute fever is one of the most common presenting symptoms globally. In order to reduce the empiric use of antimicrobial drugs and improve outcomes, it is essential to improve diagnostic capabilities. In the absence of microbiology facilities in low-income settings, an assay to distinguish bacterial from non-bacterial causes would be a critical first step. To ensure that patient and market needs are met, the requirements of such a test should be specified in a target product profile (TPP). To identify minimal/optimal characteristics for a bacterial vs. non-bacterial fever test, experts from academia and international organizations with expertise in infectious diseases, diagnostic test development, laboratory medicine, global health, and health economics were convened. Proposed TPPs were reviewed by this working group, and consensus characteristics were defined. The working group defined non-severely ill, non-malaria infected children as the target population for the desired assay. To provide access to the most patients, the test should be deployable to community health centers and informal health settings, and staff should require <2 days of training to perform the assay. Further, given that the aim is to reduce inappropriate antimicrobial use as well as to deliver appropriate treatment for patients with bacterial infections, the group agreed on minimal diagnostic performance requirements of >90% and >80% for sensitivity and specificity, respectively. Other key characteristics, to account for the challenging environment at which the test is targeted, included: i) time-to-result <10 min (but maximally <2 hrs); ii) storage conditions at 0-40°C, ≤90% non-condensing humidity with a minimal shelf life of 12 months; iii) operational conditions of 5-40°C, ≤90% non-condensing humidity; and iv) minimal sample collection needs (50-100μL, capillary blood). This expert approach to define assay requirements for a bacterial vs. non-bacterial assay should guide product

  18. Rapid Diagnosis of Pulmonary Tuberculosis with the LCx Mycobacterium tuberculosis Assay and Comparison with Conventional Diagnostic Techniques

    PubMed Central

    Rohner, Peter; Jahn, Esther I. M.; Ninet, Beatrice; Ionati, Concetta; Weber, Rainer; Auckenthaler, Raymond; Pfyffer, Gaby E.

    1998-01-01

    The LCx MTB amplification assay is a nucleic acid amplification test intended for the direct detection of Mycobacterium tuberculosis complex in respiratory specimens. We evaluated its performance on 2,001 consecutive respiratory specimens; 78 were culture positive for M. tuberculosis. Sensitivity, specificity, and positive and negative predictive values of this assay for all specimens compared to culture results were 88.5, 97.7, 60.5, and 99.5%, respectively. When referred to resolved clinical diagnosis of active tuberculosis, these values improved to 90.2, 98.4, 72.8, and 99.5%, respectively. PMID:9738065

  19. Sensitive and accurate identification of protein–DNA binding events in ChIP-chip assays using higher order derivative analysis

    PubMed Central

    Barrett, Christian L.; Cho, Byung-Kwan

    2011-01-01

    Immuno-precipitation of protein–DNA complexes followed by microarray hybridization is a powerful and cost-effective technology for discovering protein–DNA binding events at the genome scale. It is still an unresolved challenge to comprehensively, accurately and sensitively extract binding event information from the produced data. We have developed a novel strategy composed of an information-preserving signal-smoothing procedure, higher order derivative analysis and application of the principle of maximum entropy to address this challenge. Importantly, our method does not require any input parameters to be specified by the user. Using genome-scale binding data of two Escherichia coli global transcription regulators for which a relatively large number of experimentally supported sites are known, we show that ∼90% of known sites were resolved to within four probes, or ∼88 bp. Over half of the sites were resolved to within two probes, or ∼38 bp. Furthermore, we demonstrate that our strategy delivers significant quantitative and qualitative performance gains over available methods. Such accurate and sensitive binding site resolution has important consequences for accurately reconstructing transcriptional regulatory networks, for motif discovery, for furthering our understanding of local and non-local factors in protein–DNA interactions and for extending the usefulness horizon of the ChIP-chip platform. PMID:21051353

  20. Application of 3D Printing Technology in Increasing the Diagnostic Performance of Enzyme-Linked Immunosorbent Assay (ELISA) for Infectious Diseases.

    PubMed

    Singh, Harpal; Shimojima, Masayuki; Shiratori, Tomomi; An, Le Van; Sugamata, Masami; Yang, Ming

    2015-07-08

    Enzyme-linked Immunosorbent Assay (ELISA)-based diagnosis is the mainstay for measuring antibody response in infectious diseases and to support pathogen identification of potential use in infectious disease outbreaks and clinical care of individual patients. The development of laboratory diagnostics using readily available 3D printing technologies provides a timely opportunity for further expansion of this technology into immunodetection systems. Utilizing available 3D printing platforms, a '3D well' was designed and developed to have an increased surface area compared to those of 96-well plates. The ease and rapidity of the development of the 3D well prototype provided an opportunity for its rapid validation through the diagnostic performance of ELISA in infectious disease without modifying current laboratory practices for ELISA. The improved sensitivity of the 3D well of up to 2.25-fold higher compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization and Lab-On-a-Chip platforms to reduce time, volume of reagents and samples needed for such assays in the laboratory diagnosis of infectious and other diseases including applications in other disciplines.

  1. Diagnostic Accuracy of GeneXpert MTB/RIF Assay in Comparison to Conventional Drug Susceptibility Testing Method for the Diagnosis of Multidrug-Resistant Tuberculosis.

    PubMed

    Pandey, Pratikshya; Pant, Narayan Dutt; Rijal, Komal Raj; Shrestha, Bhawana; Kattel, Sirita; Banjara, Megha Raj; Maharjan, Bhagwan; Kc, Rajendra

    2017-01-01

    Xpert MTB/RIF assay is regarded as a great achievement of modern medicine for the rapid diagnosis of multidrug-resistant tuberculosis (MDR-TB). The main purpose of this study was to determine the performance of Xpert MTB/RIF assay compared to conventional drug susceptibility testing (DST) method for the diagnosis of MDR-TB. A comparative cross sectional study was carried out at German-Nepal Tuberculosis Project, Kathmandu, Nepal, from April 2014 to September 2014. A total of 88 culture positive clinical samples (83 pulmonary and 5 extra-pulmonary) received during the study period were analyzed for detection of multidrug-resistant tuberculosis by both GeneXpert MTB/RIF assay and conventional DST method. McNemar chi square test was used to compare the performance of Xpert with that of DST method. A p-value of less than 0.05 was considered as statistically significant. Of total 88 culture positive samples, one was reported as invalid while 2 were found to contain nontuberculous Mycobacteria (NTM). Among remaining 85 Mycobacterium tuberculosis culture positive samples, 69 were found to be MDR-TB positive by both methods. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of GeneXpert MTB/RIF assay were found to be 98.6%, 100%, 100% and 93.8% respectively. Statistically, there was no significant difference between the diagnostic performance of Xpert and conventional DST method for detection of MDR-TB. GeneXpert MTB/RIF assay was found to be highly sensitive, specific and comparable to gold standard conventional DST method for the diagnosis of MDR-TB.

  2. Diagnostic Accuracy of GeneXpert MTB/RIF Assay in Comparison to Conventional Drug Susceptibility Testing Method for the Diagnosis of Multidrug-Resistant Tuberculosis

    PubMed Central

    Pandey, Pratikshya; Rijal, Komal Raj; Shrestha, Bhawana; Kattel, Sirita; Banjara, Megha Raj; Maharjan, Bhagwan; KC, Rajendra

    2017-01-01

    Xpert MTB/RIF assay is regarded as a great achievement of modern medicine for the rapid diagnosis of multidrug-resistant tuberculosis (MDR-TB). The main purpose of this study was to determine the performance of Xpert MTB/RIF assay compared to conventional drug susceptibility testing (DST) method for the diagnosis of MDR-TB. A comparative cross sectional study was carried out at German-Nepal Tuberculosis Project, Kathmandu, Nepal, from April 2014 to September 2014. A total of 88 culture positive clinical samples (83 pulmonary and 5 extra-pulmonary) received during the study period were analyzed for detection of multidrug-resistant tuberculosis by both GeneXpert MTB/RIF assay and conventional DST method. McNemar chi square test was used to compare the performance of Xpert with that of DST method. A p-value of less than 0.05 was considered as statistically significant. Of total 88 culture positive samples, one was reported as invalid while 2 were found to contain nontuberculous Mycobacteria (NTM). Among remaining 85 Mycobacterium tuberculosis culture positive samples, 69 were found to be MDR-TB positive by both methods. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of GeneXpert MTB/RIF assay were found to be 98.6%, 100%, 100% and 93.8% respectively. Statistically, there was no significant difference between the diagnostic performance of Xpert and conventional DST method for detection of MDR-TB. GeneXpert MTB/RIF assay was found to be highly sensitive, specific and comparable to gold standard conventional DST method for the diagnosis of MDR-TB. PMID:28081227

  3. Hemoglobin precipitation greatly improves 4-methylumbelliferone-based diagnostic assays for lysosomal storage diseases in dried blood spots.

    PubMed

    Oemardien, L F; Boer, A M; Ruijter, G J G; van der Ploeg, A T; de Klerk, J B C; Reuser, A J J; Verheijen, F W

    2011-01-01

    Derivatives of 4-methylumbelliferone (4MU) are favorite substrates for the measurement of lysosomal enzyme activities in a wide variety of cell and tissue specimens. Hydrolysis of these artificial substrates at acidic pH leads to the formation of 4-methylumbelliferone, which is highly fluorescent at a pH above 10. When used for the assay of enzyme activities in dried blood spots the light emission signal can be very low due to the small sample size so that the patient and control ranges are not widely separated. We have investigated the hypothesis that quenching of the fluorescence by hemoglobin leads to appreciable loss of signal and we show that the precipitation of hemoglobin with trichloroacetic acid prior to the measurement of 4-methylumbelliferone increases the height of the output signal up to eight fold. The modified method provides a clear separation of patients' and controls' ranges for ten different lysosomal enzyme assays in dried blood spots, and approaches the conventional leukocyte assays in outcome quality.

  4. Raman spectroscopy for medical diagnostics--From in-vitro biofluid assays to in-vivo cancer detection.

    PubMed

    Kong, Kenny; Kendall, Catherine; Stone, Nicholas; Notingher, Ioan

    2015-07-15

    Raman spectroscopy is an optical technique based on inelastic scattering of light by vibrating molecules and can provide chemical fingerprints of cells, tissues or biofluids. The high chemical specificity, minimal or lack of sample preparation and the ability to use advanced optical technologies in the visible or near-infrared spectral range (lasers, microscopes, fibre-optics) have recently led to an increase in medical diagnostic applications of Raman spectroscopy. The key hypothesis underpinning this field is that molecular changes in cells, tissues or biofluids, that are either the cause or the effect of diseases, can be detected and quantified by Raman spectroscopy. Furthermore, multivariate calibration and classification models based on Raman spectra can be developed on large "training" datasets and used subsequently on samples from new patients to obtain quantitative and objective diagnosis. Historically, spontaneous Raman spectroscopy has been known as a low signal technique requiring relatively long acquisition times. Nevertheless, new strategies have been developed recently to overcome these issues: non-linear optical effects and metallic nanoparticles can be used to enhance the Raman signals, optimised fibre-optic Raman probes can be used for real-time in-vivo single-point measurements, while multimodal integration with other optical techniques can guide the Raman measurements to increase the acquisition speed and spatial accuracy of diagnosis. These recent efforts have advanced Raman spectroscopy to the point where the diagnostic accuracy and speed are compatible with clinical use. This paper reviews the main Raman spectroscopy techniques used in medical diagnostics and provides an overview of various applications.

  5. Analytical Validation of a Highly Quantitative, Sensitive, Accurate, and Reproducible Assay (HERmark®) for the Measurement of HER2 Total Protein and HER2 Homodimers in FFPE Breast Cancer Tumor Specimens

    PubMed Central

    Larson, Jeffrey S.; Goodman, Laurie J.; Tan, Yuping; Defazio-Eli, Lisa; Paquet, Agnes C.; Cook, Jennifer W.; Rivera, Amber; Frankson, Kristi; Bose, Jolly; Chen, Lili; Cheung, Judy; Shi, Yining; Irwin, Sarah; Kiss, Linda D. B.; Huang, Weidong; Utter, Shannon; Sherwood, Thomas; Bates, Michael; Weidler, Jodi; Parry, Gordon; Winslow, John; Petropoulos, Christos J.; Whitcomb, Jeannette M.

    2010-01-01

    We report here the results of the analytical validation of assays that measure HER2 total protein (H2T) and HER2 homodimer (H2D) expression in Formalin Fixed Paraffin Embedded (FFPE) breast cancer tumors as well as cell line controls. The assays are based on the VeraTag technology platform and are commercially available through a central CAP-accredited clinical reference laboratory. The accuracy of H2T measurements spans a broad dynamic range (2-3 logs) as evaluated by comparison with cross-validating technologies. The measurement of H2T expression demonstrates a sensitivity that is approximately 7–10 times greater than conventional immunohistochemistry (IHC) (HercepTest). The HERmark assay is a quantitative assay that sensitively and reproducibly measures continuous H2T and H2D protein expression levels and therefore may have the potential to stratify patients more accurately with respect to response to HER2-targeted therapies than current methods which rely on semiquantitative protein measurements (IHC) or on indirect assessments of gene amplification (FISH). PMID:21151530

  6. β-human chorionic gonadotropin assay in vaginal washing fluid for the accurate diagnosis of premature rupture of membranes during late pregnancy

    PubMed Central

    Temel, Orhan; Çöğendez, Ebru; Selçuk, Selçuk; Asoğlu, Mehmet Reşit; Kaya, Erdal

    2013-01-01

    Objective To determine whether the measurement of beta-human chorionic gonadotropin (β-hCG) levels in vaginal fluid is useful for the diagnosis of premature rupture of membranes (PROM). Material and Methods A total of 92 pregnant women between 24 and 40 weeks gestation participated in this study. The patients with fluid leaking from the vagina were designated Group 1, the patients with no fluid leaking from the vagina were Group 2, and those with a suspicion of fluid leaking from the vagina were classified as Group 3. Irrigating the posterior vaginal fornix with 5 mL sterile saline was used to measure β-hCG levels of the patients. Receiver operator curve (ROC) analysis was used to determine the cut-off value for a positive diagnosis. Results The β-hCG levels of vaginal fluid were measured as 20.5±25.0 mIU/mL, 254.6±346.8 mIU/mL, and 74.3±100.8 mIU/mL in Group 1, Group 2, and Group 3, respectively. Vaginal β-hCG level was higher statistically significantly in Group 2 than Group 1 and 3 (p<0.001). 100 mIU/mL was accepted as a cut-off value by using the receiver operating characteristic curve. According to 100 mIU/mL, sensitivity, specificity, positive predictive and negative predictive values were calculated as 71.2, 100, 100, and 65.1%, respectively. Conclusion The study showed that the measurement of β-hCG level in vaginal washing fluid is an efficient and easy diagnostic test for predicting the amount of fluid leaking from the vagina. However, due to the low negative predictive value of the test, it would not be convenient in daily practice. PMID:24592106

  7. Cerebrospinal Fluid IL-10 and IL-10/IL-6 as Accurate Diagnostic Biomarkers for Primary Central Nervous System Large B-cell Lymphoma

    PubMed Central

    Song, Yang; Zhang, Wei; Zhang, Li; Wu, Wei; Zhang, Yan; Han, Xiao; Yang, Chen; Zhang, Lu; Zhou, Daobin

    2016-01-01

    Early diagnosis of primary central nervous system lymphoma (PCNSL) represents a challenge, and cerebrospinal fluid (CSF) cytokines may be diagnostic biomarkers for PCNSL. We used an electrochemiluminescence immunoassay to measure interleukin (IL)-10, IL-6, IL-8 and tumor necrosis factor α (TNF-α) in the CSF of 22 B cell PCNSL patients and 80 patients with other CNS diseases. CSF IL-10 was significantly higher in PCNSL patients than in the control group (median 74.7 pg/ml vs < 5.0 pg/ml, P < 0.000). Using a CSF IL-10 cutoff value of 8.2 pg/ml, the diagnostic sensitivity and specificity were 95.5% and 96.1%, respectively (AUC, 0.957; 95% CI, 0.901–1.000). For a CSF IL-10/IL-6 cutoff value of 0.72, the sensitivity was 95.5%, and the specificity was 100.0% (AUC, 0.976; 95% CI, 0.929–1.000). An increased CSF IL-10 level at diagnosis and post-treatment was associated with poor Progression free survival (PFS) for patients with PCNSL (P = 0.0181 and P = 0.0002, respectively). A low diagnostic value for PCNSL was found with CSF IL-8 or TNF-α. In conclusion, increased CSF IL-10 was a reliable diagnostic biomarker for large B cell PCNSL, and an IL-10/IL-6 ratio facilitates differentiation from other conditions, especially a CNS infection. PMID:27924864

  8. 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.

  9. 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.

  10. Nucleic Acid Amplification Based Diagnostic of Lyme (Neuro-)borreliosis - Lost in the Jungle of Methods, Targets, and Assays?

    PubMed

    Nolte, Oliver

    2012-01-01

    Laboratory based diagnosis of infectious diseases usually relies on culture of the disease causing micro-organism, followed by identification and susceptibility testing. Since Borrelia burgdorferi sensu lato, the etiologic agent of Lyme disease or Lyme borreliosis, requires very specific culture conditions (e.g. specific liquid media, long term cul-ture) traditional bacteriology is often not done on a routine basis. Instead, confirmation of the clinical diagnosis needs ei-ther indirect techniques (like serology or measurement of cellular activity in the presence of antigens) or direct but culture independent techniques, like microscopy or nucleic acid amplification techniques (NAT), with polymerase chain reaction (PCR) being the most frequently applied NAT method in routine laboratories. NAT uses nucleic acids of the disease causing micro-organism as template for amplification, isolated from various sources of clinical specimens. Although the underlying principle, adoption of the enzymatic process running during DNA duplication prior to prokaryotic cell division, is comparatively easy, a couple of 'pitfalls' is associated with the technique itself as well as with interpretation of the results. At present, no commercial, CE-marked and sufficiently validated PCR assay is available. A number of homebrew assays have been published, which are different in terms of target (i.e. the gene targeted by the amplification primers), method (nested PCR, PCR followed by hybridization, real-time PCR) and validation criteria. Inhibitory compounds may lead to false negative results, if no appropriate internal control is included. Carry-over of amplicons, insufficient handling and workflow and/or insufficiently validated targets/primers may result in false positive results. Different targets may yield different analytical sensitivity, depending, among other factors, of the redundancy of a target gene in the genome. Per-formance characteristics (e.g. analytical sensitivity and

  11. Nucleic Acid Amplification Based Diagnostic of Lyme (Neuro-)borreliosis – Lost in the Jungle of Methods, Targets, and Assays?

    PubMed Central

    Nolte, Oliver

    2012-01-01

    Laboratory based diagnosis of infectious diseases usually relies on culture of the disease causing micro-organism, followed by identification and susceptibility testing. Since Borrelia burgdorferi sensu lato, the etiologic agent of Lyme disease or Lyme borreliosis, requires very specific culture conditions (e.g. specific liquid media, long term cul-ture) traditional bacteriology is often not done on a routine basis. Instead, confirmation of the clinical diagnosis needs ei-ther indirect techniques (like serology or measurement of cellular activity in the presence of antigens) or direct but culture independent techniques, like microscopy or nucleic acid amplification techniques (NAT), with polymerase chain reaction (PCR) being the most frequently applied NAT method in routine laboratories. NAT uses nucleic acids of the disease causing micro-organism as template for amplification, isolated from various sources of clinical specimens. Although the underlying principle, adoption of the enzymatic process running during DNA duplication prior to prokaryotic cell division, is comparatively easy, a couple of ‘pitfalls’ is associated with the technique itself as well as with interpretation of the results. At present, no commercial, CE-marked and sufficiently validated PCR assay is available. A number of homebrew assays have been published, which are different in terms of target (i.e. the gene targeted by the amplification primers), method (nested PCR, PCR followed by hybridization, real-time PCR) and validation criteria. Inhibitory compounds may lead to false negative results, if no appropriate internal control is included. Carry-over of amplicons, insufficient handling and workflow and/or insufficiently validated targets/primers may result in false positive results. Different targets may yield different analytical sensitivity, depending, among other factors, of the redundancy of a target gene in the genome. Per-formance characteristics (e.g. analytical sensitivity and

  12. 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

  13. 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.

  14. Rapid infectious diseases diagnostics using Smartphones

    PubMed Central

    Bates, Matthew

    2015-01-01

    The “Smartphone” is an almost universal possession in high-income populations, and is rapidly becoming so in lower-income regions, particularly among urban populations, and serves social networking and a quest for information and knowledge. The field of infectious disease diagnostics is at a potential watershed moment, with the essential building blocks for the development of diagnostic assays being ever more available and affordable, which is leading to creative innovative approaches to developing much-needed accurate and simple point-of-care (POC) diagnostic tools for high disease burden, low-income settings. We review the importance and implications of a paper published in Science Translational Medicine on the development of a smartphone-powered and -controlled multiplex immunological assay that tests for HIV and syphilis simultaneously. This is reviewed in the context of other prototype smartphone-enabled/assisted diagnostic devices, and how such developments might shape the future of the POC diagnostics field. PMID:26488011

  15. Application of cancer‐associated glycoforms and glycan‐binding probes to an in vitro diagnostic multivariate index assay for precise diagnoses of cancer

    PubMed Central

    Kang, Jeong Gu

    2016-01-01

    Personalized medicine has emerged as a widely accepted trend in medicine for the efficacious and safe treatment of various diseases. It covers every medical treatment tailored according to various properties of individuals. Cancer‐associated glycosylation mirrors cancer states more precisely, and this “sweet side of cancer” is thus intended to spur the development of an advanced in vitro diagnostic system. The changes of glyco‐codes are often subtle and thus not easy to trace, thereby making it difficult to discriminate changes from various compounding factors. Special glycan‐binding probes, often lectins, can be paired with aglycosylated antibodies to enable quantitative and qualitative measurements of glycoforms. With the in vitro diagnosis multivariate index assay (IVDMIA) considered to be capable of yielding patient‐specific results, the combinatorial use of multiple glycoproteins may be a good modality to ensure disease‐specific, personalized diagnoses. PMID:27005968

  16. Cotton-based Diagnostic Devices

    PubMed Central

    Lin, Shang-Chi; Hsu, Min-Yen; Kuan, Chen-Meng; Wang, Hsi-Kai; Chang, Chia-Ling; Tseng, Fan-Gang; Cheng, Chao-Min

    2014-01-01

    A good diagnostic procedure avoids wasting medical resources, is easy to use, resists contamination, and provides accurate information quickly to allow for rapid follow-up therapies. We developed a novel diagnostic procedure using a “cotton-based diagnostic device” capable of real-time detection, i.e., in vitro diagnostics (IVD), which avoids reagent contamination problems common to existing biomedical devices and achieves the abovementioned goals of economy, efficiency, ease of use, and speed. Our research reinforces the advantages of an easy-to-use, highly accurate diagnostic device created from an inexpensive and readily available U.S. FDA-approved material (i.e., cotton as flow channel and chromatography paper as reaction zone) that adopts a standard calibration curve method in a buffer system (i.e., nitrite, BSA, urobilinogen and uric acid assays) to accurately obtain semi-quantitative information and limit the cross-contamination common to multiple-use tools. Our system, which specifically targets urinalysis diagnostics and employs a multiple biomarker approach, requires no electricity, no professional training, and is exceptionally portable for use in remote or home settings. This could be particularly useful in less industrialized areas. PMID:25393975

  17. Sensitive Detection and Simultaneous Discrimination of Influenza A and B Viruses in Nasopharyngeal Swabs in a Single Assay Using Next-Generation Sequencing-Based Diagnostics

    PubMed Central

    Liu, Jikun; Vemula, Sai Vikram; Lin, Corinna; Tan, Jiying; Ragupathy, Viswanath; Wang, Xue; Mbondji-wonje, Christelle; Ye, Zhiping; Landry, Marie L.; Hewlett, Indira

    2016-01-01

    Reassortment of 2009 (H1N1) pandemic influenza virus (pdH1N1) with other strains may produce more virulent and pathogenic forms, detection and their rapid characterization is critical. In this study, we reported a “one-size-fits-all” approach using a next-generation sequencing (NGS) detection platform to extensively identify influenza viral genomes for diagnosis and determination of novel virulence and drug resistance markers. A de novo module and other bioinformatics tools were used to generate contiguous sequence and identify influenza types/subtypes. Of 162 archived influenza-positive patient specimens, 161(99.4%) were positive for either influenza A or B viruses determined using the NGS assay. Among these, 135(83.3%) were A(H3N2), 14(8.6%) were A(pdH1N1), 2(1.2%) were A(H3N2) and A(pdH1N1) virus co-infections and 10(6.2%) were influenza B viruses. Of the influenza A viruses, 66.7% of A(H3N2) viruses tested had a E627K mutation in the PB2 protein, and 87.8% of the influenza A viruses contained the S31N mutation in the M2 protein. Further studies demonstrated that the NGS assay could achieve a high level of sensitivity and reveal adequate genetic information for final laboratory confirmation. The current diagnostic platform allows for simultaneous identification of a broad range of influenza viruses, monitoring emerging influenza strains with pandemic potential that facilitating diagnostics and antiviral treatment in the clinical setting and protection of the public health. PMID:27658193

  18. Diagnostic tests for amoebic liver abscess: comparison of enzyme-linked immunosorbent assay (ELISA) and counterimmunoelectrophoresis (CIE).

    PubMed

    Restrepo, M I; Restrepo, Z; Elsa Villareal, C L; Aguirre, A; Restrepo, M

    1996-01-01

    The liver abscess is the most frequent extraintestinal complication of intestinal amoebiasis: its diagnosis is suggested by the clinical picture but it must be confirmed by paraclinic tests. Themost stringent diagnosis requires identification of E. histolytica. But this is possible only in a few cases. Serological tests greatly improve the diagnosis of this severe complication of amoebiasis. We compared the Enzyme Linfed Immunosorbent Assay and the Counterimmunoelectrophoresis techniques. Both techniques were used to detect amoebic antibodies in 50 control patients, 30 patients with liver abscess and 30 patients with intestinal amoebiasis. All the sera from control patients gave negative results in both techniques. When analysing the sera from patients with intestinal amoebiasis, 10% of them were positive by ELISA but non by CIE. The sera of patients with liver abscess, we found that 90% were positive by the ELISA method and 66.6% by the CIE technique. In patients with amoebic liver abscess, the results showed that the ELISA was more sensitive than the CIE, as it presented a higher sensitivity (100%) than that of the CIE technique (66%).

  19. Osteopenic disease in growing pigs: diagnostic methods using serum and urine calcium and phosphorus values, parathoromone assay, and bone analysis.

    PubMed

    Hagemoser, W A; Goff, J P; Sanderson, T P; Haynes, J S

    2000-11-01

    This research was performed to evaluate the utility of several serum and urine parameters as well as bone ash and plasma parathormone assay to diagnose and monitor diet-related osteopenia in growing pigs. Five diets were tested as follows: calcium-deficient, phosphorus-replete; moderate-deficiency of calcium and phosphorus; marked deficiency of calcium and phosphorus; calcium replete, phosphorus deficient; and vitamin D deficient. Parameters monitored included serum calcium and phosphorus as well as ratios of urine calcium to creatinine, phosphorus to creatinine, calcium to phosphorus, and percent fractional excretions of calcium and phosphorus. Plasma parathormone (PTH) levels were monitored in 2 of 3 experiments. Osteopenic bone differences at necropsy were evaluated by bone density, percent ash, ash per milliliter bone, calcium per milliliter bone, and phosphorus per milliliter bone. Marked change in urine mineral parameters, especially the calcium-to-phosphorus ratio, typically occurred within 1 to 2 days of treatment and preceded significant change in serum mineral or plasma PTH by 2 to 3 weeks. When monitored, plasma PTH levels were elevated following treatment, which confirms the hyperparathyroid state induced by the test diets. Significant differences in bone mineralization between control and treatment diets at necropsy were generally observed. The results of this study indicate that the analysis of urine minerals offers an early, noninvasive technique to investigate diet-associated osteopenic disease in growing pigs, which can be supported further by bone mineral analysis at postmortem using techniques herein described. Several urine mineral reference intervals for application to field investigations are included. Research into application of similar techniques to evaluate calcium and phosphorus homeostasis in pigs of all ages, including gestating and lactating gilts and sows, appears warranted.

  20. The Impact of a Line Probe Assay Based Diagnostic Algorithm on Time to Treatment Initiation and Treatment Outcomes for Multidrug Resistant TB Patients in Arkhangelsk Region, Russia

    PubMed Central

    Eliseev, Platon; Balantcev, Grigory; Nikishova, Elena; Gaida, Anastasia; Bogdanova, Elena; Enarson, Donald; Ornstein, Tara; Detjen, Anne; Dacombe, Russell; Gospodarevskaya, Elena; Phillips, Patrick P. J.; Mann, Gillian; Squire, Stephen Bertel; Mariandyshev, Andrei

    2016-01-01

    Background In the Arkhangelsk region of Northern Russia, multidrug-resistant (MDR) tuberculosis (TB) rates in new cases are amongst the highest in the world. In 2014, MDR-TB rates reached 31.7% among new cases and 56.9% among retreatment cases. The development of new diagnostic tools allows for faster detection of both TB and MDR-TB and should lead to reduced transmission by earlier initiation of anti-TB therapy. Study Aim The PROVE-IT (Policy Relevant Outcomes from Validating Evidence on Impact) Russia study aimed to assess the impact of the implementation of line probe assay (LPA) as part of an LPA-based diagnostic algorithm for patients with presumptive MDR-TB focusing on time to treatment initiation with time from first-care seeking visit to the initiation of MDR-TB treatment rather than diagnostic accuracy as the primary outcome, and to assess treatment outcomes. We hypothesized that the implementation of LPA would result in faster time to treatment initiation and better treatment outcomes. Methods A culture-based diagnostic algorithm used prior to LPA implementation was compared to an LPA-based algorithm that replaced BacTAlert and Löwenstein Jensen (LJ) for drug sensitivity testing. A total of 295 MDR-TB patients were included in the study, 163 diagnosed with the culture-based algorithm, 132 with the LPA-based algorithm. Results Among smear positive patients, the implementation of the LPA-based algorithm was associated with a median decrease in time to MDR-TB treatment initiation of 50 and 66 days compared to the culture-based algorithm (BacTAlert and LJ respectively, p<0.001). In smear negative patients, the LPA-based algorithm was associated with a median decrease in time to MDR-TB treatment initiation of 78 days when compared to the culture-based algorithm (LJ, p<0.001). However, several weeks were still needed for treatment initiation in LPA-based algorithm, 24 days in smear positive, and 62 days in smear negative patients. Overall treatment outcomes

  1. Point of care diagnostics for sexually transmitted infections: perspectives and advances

    PubMed Central

    Gaydos, Charlotte; Hardick, Justin

    2014-01-01

    Accurate and inexpensive point-of-care (POC) tests are urgently needed to control sexually transmitted infection (STI) epidemics, so that patients can receive immediate diagnoses and treatment. Current POC assays for Chlamydia trachomatis and Neisseria gonorrhoeae perform inadequately and require better assays. Diagnostics for Trichomonas vaginalis rely on wet preparation, with some notable advances. Serological POC assays for syphilis can impact resource-poor settings, with many assays available, but only one available in the U.S. HIV POC diagnostics demonstrate the best performance, with excellent assays available. There is a rapid assay for HSV lesion detection; but no POC serological assays are available. Despite the inadequacy of POC assays for treatable bacterial infections, application of technological advances offers the promise of advancing POC diagnostics for all STIs. PMID:24484215

  2. Identification and Expression of Babesia ovis Secreted Antigen 1 and Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Cao, Shinuo; Xuan, Xuenan; Sevinc, Mutlu; Ceylan, Onur

    2015-01-01

    In order to identify immunoreactive proteins that are usable for the immunological diagnosis of Babesia ovis infections, a phage lambda cDNA expression library was constructed and screened using parasite-specific immune serum. Immunoscreening resulted in the identification of a full-length cDNA clone encoding a secreted protein designated Babesia ovis secreted antigen 1 (BoSA1). The full-length BoSA1 cDNA contained a 1,137-bp open reading frame that encoded a protein of 378 amino acids, with a signal peptide and 2 internal repeat domains. The theoretical molecular mass of the mature protein was 42.5 kDa. Recombinant BoSA1 (rBoSA1) protein was expressed in Escherichia coli strain DH5α cells as a glutathione S-transferase (GST) fusion protein and was purified by affinity chromatography. Purified rBoSA1 was tested for reactivity with sera from animals experimentally or naturally infected with B. ovis, in an indirect enzyme-linked immunosorbent assay (ELISA). The results showed that specific antibodies against rBoSA1 were detectable on days 7 and 8 of the experimental infection and were maintained during the sampling period. Additionally, 38 field sera taken from sheep naturally infected with B. ovis gave strong positive reactions in the ELISA between day 20 and day 30 of treatment. As a result, the identified recombinant BoSA1 protein seems to be a promising diagnostic antigen that is usable for the development of serological assays for the diagnosis of ovine babesiosis. This is the first report on the molecular cloning, expression, and potential use of a recombinant antigen for the diagnosis of ovine babesiosis. PMID:25694531

  3. Expression of the Lassa virus nucleocapsid protein in insect cells infected with a recombinant baculovirus: application to diagnostic assays for Lassa virus infection.

    PubMed

    Barber, G N; Clegg, J C; Lloyd, G

    1990-01-01

    The coding region of the gene for the nucleocapsid protein of Lassa virus has been inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) using the transfer vector pAcYM1, so that expression of the foreign DNA is under the control of the promoter of the AcNPV polyhedrin gene. Infection of cultured Spodoptera frugiperda cells with recombinant virus resulted in the synthesis of high levels of a protein that was indistinguishable from the authentic Lassa virus protein by SDS gel electrophoresis and immunoblotting with a variety of specific immune sera and monoclonal antibodies (MAbs). The kinetics of appearance of the protein were comparable to those of polyhedrin production in wild-type AcNPV-infected cells. The recombinant material was antigenic when used in ELISA for Lassa virus-specific antibodies, reacting well with MAbs specific for the nucleocapsid protein and with sera from experimentally infected guinea-pigs. The recombinant ELISA was able to clearly distinguish sera from human cases of Lassa fever against a panel of known negative sera of African origin. Recombinant-infected insect cells were an effective substitute for mammalian cells infected with Lassa virus itself in the immunofluorescence assay for Lassa virus-specific antibodies. This system offers attractive alternatives to the use of Lassa virus-infected materials as reagents in diagnostic procedures.

  4. Incremental Yield of Including Determine-TB LAM Assay in Diagnostic Algorithms for Hospitalized and Ambulatory HIV-Positive Patients in Kenya

    PubMed Central

    Ferlazzo, Gabriella; Bevilacqua, Paolo; Kirubi, Beatrice; Ardizzoni, Elisa; Wanjala, Stephen; Sitienei, Joseph; Bonnet, Maryline

    2017-01-01

    Background Determine-TB LAM assay is a urine point-of-care test useful for TB diagnosis in HIV-positive patients. We assessed the incremental diagnostic yield of adding LAM to algorithms based on clinical signs, sputum smear-microscopy, chest X-ray and Xpert MTB/RIF in HIV-positive patients with symptoms of pulmonary TB (PTB). Methods Prospective observational cohort of ambulatory (either severely ill or CD4<200cells/μl or with Body Mass Index<17Kg/m2) and hospitalized symptomatic HIV-positive adults in Kenya. Incremental diagnostic yield of adding LAM was the difference in the proportion of confirmed TB patients (positive Xpert or MTB culture) diagnosed by the algorithm with LAM compared to the algorithm without LAM. The multivariable mortality model was adjusted for age, sex, clinical severity, BMI, CD4, ART initiation, LAM result and TB confirmation. Results Among 474 patients included, 44.1% were severely ill, 69.6% had CD4<200cells/μl, 59.9% had initiated ART, 23.2% could not produce sputum. LAM, smear-microscopy, Xpert and culture in sputum were positive in 39.0% (185/474), 21.6% (76/352), 29.1% (102/350) and 39.7% (92/232) of the patients tested, respectively. Of 156 patients with confirmed TB, 65.4% were LAM positive. Of those classified as non-TB, 84.0% were LAM negative. Adding LAM increased the diagnostic yield of the algorithms by 36.6%, from 47.4% (95%CI:39.4–55.6) to 84.0% (95%CI:77.3–89.4%), when using clinical signs and X-ray; by 19.9%, from 62.2% (95%CI:54.1–69.8) to 82.1% (95%CI:75.1–87.7), when using clinical signs and microscopy; and by 13.4%, from 74.4% (95%CI:66.8–81.0) to 87.8% (95%CI:81.6–92.5), when using clinical signs and Xpert. LAM positive patients had an increased risk of 2-months mortality (aOR:2.7; 95%CI:1.5–4.9). Conclusion LAM should be included in TB diagnostic algorithms in parallel to microscopy or Xpert request for HIV-positive patients either ambulatory (severely ill or CD4<200cells/μl) or hospitalized. LAM

  5. New diagnostic tools in schistosomiasis.

    PubMed

    Utzinger, J; Becker, S L; van Lieshout, L; van Dam, G J; Knopp, S

    2015-06-01

    Schistosomiasis is a water-based parasitic disease that affects over 250 million people. Control efforts have long been in vain, which is one reason why schistosomiasis is considered a neglected tropical disease. However, since the new millennium, interventions against schistosomiasis are escalating. The initial impetus stems from a 2001 World Health Assembly resolution, urging member states to scale-up deworming of school-aged children with the anthelminthic drug praziquantel. Because praziquantel is safe, efficacious and inexpensive when delivered through the school platform, diagnosis before drug intervention was deemed unnecessary and not cost-effective. Hence, there was little interest in research and development of novel diagnostic tools. With the recent publication of the World Health Organization (WHO) Roadmap to overcome the impact of neglected tropical diseases in 2020, we have entered a new era. Elimination of schistosomiasis has become the buzzword and this has important ramifications for diagnostic tools. Indeed, measuring progress towards the WHO Roadmap and whether local elimination has been achieved requires highly accurate diagnostic assays. Here, we introduce target product profiles for diagnostic tools that are required for different stages of a schistosomiasis control programme. We provide an update of the latest developments in schistosomiasis diagnosis, including microscopic techniques, rapid diagnostic tests for antigen detection, polymerase chain reaction (PCR) assays and proxy markers for morbidity assessments. Particular emphasis is placed on challenges and solutions for new technologies to enter clinical practice.

  6. Identification of Borrelia burgdorferi ospC genotypes in canine tissue following tick infestation: Implications for Lyme disease vaccine and diagnostic assay design

    PubMed Central

    Rhodes, D.V.L.; Earnhart, C.G.; Mather, T.N.; Meeus, P.F.M; Marconi, R.T.

    2013-01-01

    In endemic regions, Lyme disease is a potential health threat to dogs. Canine Lyme disease manifests with arthritis-induced lameness, anorexia, fever, lethargy, lymphadenopathy and, in some cases, fatal glomerulonephritis. A recent study revealed that the regional mean for the percentage of seropositive dogs in the Northeast of the USA is 11.6%. The outer surface protein C (OspC) of Lyme disease spirochetes is an important virulence factor required for the establishment of infection in mammals. It is a leading candidate in human and canine Lyme disease vaccine development efforts. Over 30 distinct ospC phyletic types have been defined. It has been hypothesized that ospC genotype may influence mammalian host range. In this study, Ixodes scapularis ticks collected from the field in Rhode Island were assessed for infection with B. burgdorferi. Ticks were fed on purpose bred beagles to repletion and infection of the dogs was assessed through serology and PCR. Tissue biopsies (n = 2) were collected from each dog 49 days post-tick infestation (dpi) and the ospC genotype of the infecting strains determined by direct PCR of DNA extracted from tissue or by PCR after cultivation of spirochetes from biopsy samples. The dominant ospC types associated with B. burgdorferi canine infections differed from those associated with human infection, indicating a relationship between ospC sequence and preferred host range. Knowledge of the most common ospC genotypes associated specifically with infection of dogs will facilitate the rational design of OspC-based canine Lyme disease vaccines and diagnostic assays. PMID:23962611

  7. Identification of Borrelia burgdorferi ospC genotypes in canine tissue following tick infestation: implications for Lyme disease vaccine and diagnostic assay design.

    PubMed

    Rhodes, D V L; Earnhart, C G; Mather, T N; Meeus, P F M; Marconi, R T

    2013-11-01

    In endemic regions, Lyme disease is a potential health threat to dogs. Canine Lyme disease manifests with arthritis-induced lameness, anorexia, fever, lethargy, lymphadenopathy and, in some cases, fatal glomerulonephritis. A recent study revealed that the regional mean for the percentage of seropositive dogs in the north-east of the USA is 11.6%. The outer surface protein C (OspC) of Lyme disease spirochetes is an important virulence factor required for the establishment of infection in mammals. It is a leading candidate in human and canine Lyme disease vaccine development efforts. Over 30 distinct ospC phyletic types have been defined. It has been hypothesized that ospC genotype may influence mammalian host range. In this study, Ixodes scapularis ticks collected from the field in Rhode Island were assessed for infection with B. burgdorferi. Ticks were fed on purpose bred beagles to repletion and infection of the dogs was assessed through serology and PCR. Tissue biopsies (n=2) were collected from each dog 49 days post-tick infestation (dpi) and the ospC genotype of the infecting strains determined by direct PCR of DNA extracted from tissue or by PCR after cultivation of spirochetes from biopsy samples. The dominant ospC types associated with B. burgdorferi canine infections differed from those associated with human infection, indicating a relationship between ospC sequence and preferred host range. Knowledge of the most common ospC genotypes associated specifically with infection of dogs will facilitate the rational design of OspC-based canine Lyme disease vaccines and diagnostic assays.

  8. Point-of-care diagnosis and prognostication of cryptococcal meningitis with the cryptococcal antigen lateral flow assay on cerebrospinal fluid.

    PubMed

    Kabanda, Taseera; Siedner, Mark J; Klausner, Jeffrey D; Muzoora, Conrad; Boulware, David R

    2014-01-01

    The cryptococcal antigen (CRAG) lateral flow assay (LFA) had 100% sensitivity and specificity on cerebrospinal fluid samples. Pretreatment LFA titers correlated with quantitative cultures (R(2) = 0.7) and predicted 2- and 10-week mortality. The CRAG LFA is an accurate diagnostic assay for CSF and should be considered for point-of-care diagnosis of cryptococcal meningitis.

  9. Integrating the Xpert MTB/RIF Assay into a Diagnostic Workflow for Rapid Detection of Mycobacterium tuberculosis in a Low-Prevalence Area

    PubMed Central

    Deggim, Vanessa; Somoskovi, Akos; Voit, Antje; Böttger, Erik C.

    2013-01-01

    The Xpert MTB/RIF assay is a rapid and fully automated real-time PCR assay. The performance of the Xpert MTB/RIF assay as a primary screening test for urgent clinical specimens was evaluated during a 2-year period. The results showed that replacing smear microscopy with the Xpert MTB/RIF assay facilitates laboratory handling and improves the sensitivity and specificity of Mycobacterium tuberculosis detection. PMID:23616455

  10. Diagnostic Values of the QuantiFERON-TB Gold In-Tube Assay Carried out in China for Diagnosing Pulmonary Tuberculosis

    PubMed Central

    Li, Fabin; Longuet, Christophe; Vernet, Guy; Goletti, Delia; Zhao, Yanlin; Lagrange, Philippe H.

    2015-01-01

    Background Interferon-release assays (IGRAs) for diagnosing active pulmonary tuberculosis (PTB) are not yet fully validated, particularly in high TB-endemic areas as the People's Republic of China (PRC). The aim of this report was to assess the performance of the QuantiFERON-TB Gold In-tube (QFT-GIT) and tuberculin skin test (TST), in addition to microbiological results, as contributors for diagnosing active PTB in the PRC. Methods/Principal Findings A total of 300 PTB patients, 41 disease controls (DC) and 59 healthy community controls (HCC) were included prospectively between May 2010 and April 2011 from two provinces of the PRC (Heilongjiang and Zhejiang). The QFT-GIT and TST yielded an overall sensitivity for active TB of 80.9% and 86.2%, and a specificity of 36.6% and 26.8%, respectively. The province of origin and smear microscopy status did not significantly impact the diagnostic values for PTB. However, using the TST with a 10 mm cut-off point, a significantly higher proportion of LTBI was observed in the DC than the HCC (p=0.01). Discordant results between the QFT-GIT and TST were found among 1/3 of the PTB, HCC and DC. Two-thirds of the individuals presented TST-positive/QFT-GIT-negative discordant results. The TST-negative/QFT-GIT-positive result was not associated with age or bacillary load. Cumulative QFT-GIT and TST positive results increased the overall sensitivity (95.9%), but it was associated with a dramatic decrease of the overall specificity (24.8%) leading to a suboptimal PPV (80.1%) and a low NPV (61.1%). Conclusions/Significance The usefulness of the QFT-GIT to diagnose active TB in high TB-endemic countries remains doubtful because like the TST, the QFT-GIT cannot distinguish between LTBI and active TB. Used as single stand-alone tests, both the QFT-GIT and TST have very limited roles in the diagnosis of active PTB. However, the combined use of SM, the TST and QFT-GIT may allow for the exclusion of ATB. PMID:25867946

  11. The diagnostic accuracy of the GenoType® Mtbdrsl assay for the detection of resistance to second-line anti-tuberculosis drugs

    PubMed Central

    Theron, Grant; Peter, Jonny; Richardson, Marty; Barnard, Marinus; Donegan, Sarah; Warren, Rob; Steingart, Karen R; Dheda, Keertan

    2014-01-01

    Background Accurate and rapid tests for tuberculosis (TB) drug resistance are critical for improving patient care and decreasing the transmission of drug-resistant TB. Genotype®MTBDRsl (MTBDRsl) is the only commercially-available molecular test for detecting resistance in TB to the fluoroquinolones (FQs; ofloxacin, moxifloxacin and levofloxacin) and the second-line injectable drugs (SLIDs; amikacin, kanamycin and capreomycin), which are used to treat patients with multidrug-resistant (MDR-)TB. Objectives To obtain summary estimates of the diagnostic accuracy ofMTBDRsl for FQ resistance, SLID resistance and extensively drug-resistant TB (XDR-TB; defined asMDR-TB plus resistance to a FQand a SLID) when performed (1) indirectly (ie on culture isolates confirmed as TB positive) and (2) directly (ie on smear-positive sputum specimens). To compare summary estimates of the diagnostic accuracy of MTBDRsl for FQ resistance, SLID resistance and XDR-TB by type of testing (indirect versus direct testing). The populations of interest were adults with drug-susceptible TB or drug-resistant TB. The settings of interest were intermediate and central laboratories. Search methods We searched the following databases without any language restriction up to 30 January 2014: Cochrane Infectious Diseases Group Specialized Register; MEDLINE; EMBASE; ISI Web of Knowledge; MEDION; LILACS; BIOSIS; SCOPUS; the metaRegister of Controlled Trials; the search portal of the World Health Organization International Clinical Trials Registry Platform; and ProQuest Dissertations & Theses A&I. Selection criteria We included all studies that determined MTBDRsl accuracy against a defined reference standard (culture-based drug susceptibility testing (DST), genetic testing or both).We included cross-sectional and diagnostic case-control studies.We excluded unpublished data and conference proceedings. Data collection and analysis For each study, two review authors independently extracted data using a

  12. A real time polymerase chain reaction assay for quantification of Edwardsiella ictaluri in catfish pond water and genetic homogeneity of diagnostic case isolates from Mississippi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A quantitative polymerase chain reaction (qPCR) assay was developed for the detection and quantification of Edwardsiella ictaluri in channel catfish Ictalurus punctatus pond water using modifications to a published E. ictaluri–specific qPCR assay and previously established protocols for the molecula...

  13. Evaluation of Commercial Diagnostic Assays for the Specific Detection of Avian Influenza A (H7N9) Virus RNA Using a Quality-Control Panel and Clinical Specimens in China

    PubMed Central

    Chen, Suhong; Wang, Dayan; Li, Changgui; Wu, Xing; Li, Lili; Bai, Dongting; Zhang, Chuntao; Wang, Junzhi

    2015-01-01

    A novel avian influenza A H7N9-subtype virus emerged in China in 2013 and threatened global public health. Commercial kits that specifically detect avian influenza A (H7N9) virus RNA are urgently required to prepare for the emergence and potential pandemic of this novel influenza virus. The safety and effectiveness of three commercial molecular diagnostic assays were evaluated using a quality-control panel and clinical specimens collected from over 90 patients with confirmed avian influenza A (H7N9) virus infections. The analytical performance evaluation showed that diverse influenza H7N9 viruses can be detected with high within- and between-lot reproducibility and without cross-reactivity to other influenza viruses (H1N1 pdm09, seasonal H1N1, H3N2, H5N1 and influenza B). The detection limit of all the commercial assays was 2.83 Log10 copies/μl [0.7 Log10TCID50/mL of avian influenza A (H7N9) virus strain A/Zhejiang/DTID-ZJU01/2013], which is comparable to the method recommended by the World Health Organization (WHO). In addition, using a WHO-Chinese National Influenza Center (CNIC) method as a reference for clinical evaluation, positive agreement of more than 98% was determined for all of the commercial kits, while negative agreement of more than 99% was observed. In conclusion, our findings provide comprehensive evidence for the high performance of three commercial diagnostic assays and suggest the application of these assays as rapid and effective diagnostic tools for avian influenza A (H7N9) virus in the routine clinical practice of medical laboratories. PMID:26361351

  14. A review of Theileria diagnostics and epidemiology

    PubMed Central

    Mans, Ben J.; Pienaar, Ronel; Latif, Abdalla A.

    2015-01-01

    An extensive range of serological and molecular diagnostic assays exist for most of the economically important Theileira species such as T. annulata, T. equi, T. lestoquardi, T. parva, T. uilenbergi and other more benign species. Diagnostics of Theileria is considered with regard to sensitivity and specificity of current molecular and serological assays and their use in epidemiology. In the case of serological assays, cross-reactivity of genetically closely related species reduces the use of the gold standard indirect fluorescent antibody test (IFAT). Development of antigen-specific assays does not necessarily address this problem, since closely related species will potentially have similar antigens. Even so, serological assays remain an important line of enquiry in epidemiological surveys. Molecular based assays have exploded in the last decade with significant improvements in sensitivity and specificity. In this review, the current interpretation of what constitute a species in Theileria and its impact on accurate molecular diagnostics is considered. Most molecular assays based on conventional or real-time PCR technology have proven to be on standard with regard to analytical sensitivity. However, consideration of the limits of detection in regard to total blood volume of an animal indicates that most assays may only detect >400,000 parasites/L blood. Even so, natural parasitaemia distribution in carrier-state animals seems to be above this limit of detection, suggesting that most molecular assays should be able to detect the majority of infected individuals under endemic conditions. The potential for false-negative results can, however, only be assessed within the biological context of the parasite within its vertebrate host, i.e. parasitaemia range in the carrier-state that will support infection of the vector and subsequent transmission. PMID:25830110

  15. Diagnostic Performance of Five Assays for Anti-Hepatitis E Virus IgG and IgM in a Large Cohort Study

    PubMed Central

    Karlsson, Marie; Mellgren, Åsa; Konar, Jan; Sandberg, Elisabeth; Lasson, Anders; Castedal, Maria; Magnius, Lars; Lagging, Martin

    2015-01-01

    Determination of anti-hepatitis E virus (anti-HEV) antibodies is still enigmatic. There is no gold standard, and results obtained with different assays often diverge. Herein, five assays were compared for detection of anti-HEV IgM and IgG. Serum samples from 500 Swedish blood donors and 316 patients, of whom 136 had suspected HEV infection, were analyzed. Concordant results for IgM and IgG with all assays were obtained only for 71% and 70% of patients with suspected hepatitis E, respectively. The range of sensitivity for anti-HEV detection was broad (42% to 96%); this was reflected in the detection limit, which varied up to 19-fold for IgM and 17-fold for IgG between assays. HEV RNA was analyzed in all patients and in those blood donors reactive for anti-HEV in any assay, and it was found in 26 individuals. Among all of the assays, both anti-HEV IgG and IgM were detected in 10 of those individuals. Twelve had only IgG and, in 7 of those 12, IgG was only detected with the two most sensitive assays. Three of the HEV-RNA-positive samples were negative for anti-HEV IgM and IgG in all assays. With the two most sensitive assays, anti-HEV IgG was identified in 16% of the blood donor samples and in 66% of patients with suspected HEV infection. Because several HEV-RNA-positive samples had only anti-HEV IgG without anti-HEV IgM or lacked anti-HEV antibodies, analysis for HEV RNA may be warranted as a complement in the laboratory diagnosis of ongoing HEV infection. PMID:26659210

  16. Comparison of Diagnostic Accuracy of Microscopy and Flow Cytometry in Evaluating N-Methyl-D-Aspartate Receptor Antibodies in Serum Using a Live Cell-Based Assay

    PubMed Central

    Ramberger, Melanie; Peschl, Patrick; Schanda, Kathrin; Irschick, Regina; Höftberger, Romana; Deisenhammer, Florian; Rostásy, Kevin; Berger, Thomas; Dalmau, Josep; Reindl, Markus

    2015-01-01

    N-methyl-D-aspartate receptor (NMDAR) encephalitis is an autoimmune neurological disease, diagnosed by a specific autoantibody against NMDAR. Antibody testing using commercially available cell-based assays (CBA) or immunohistochemistry on rat brain tissue has proven high specificity and sensitivity. Here we compare an immunofluorescence live CBA to a flow cytometry (FACS) based assay to detect NMDAR antibodies by their binding to the surface of HEK293A cells functionally expressing NMDAR. Both assays were first established using a discovery group of 76 individuals and then validated in a group of 32 patients in a blinded manner. In the CBA, 23 of 23 patients with NMDAR encephalitis were positive for NMDAR antibodies and 0 of 85 controls (32 healthy controls and 53 patients with other neurological diseases), resulting in a sensitivity and specificity of 100% (95% confidence intervals (CI) 85.1–100.0 and 95.7–100.0, respectively). The FACS based assay detected NMDAR antibodies in 20 of 23 patients and in 0 of 85 controls. Therefore, with an equally high specificity (95% CI 95.7–100.0) the sensitivity of the FACS based assay was 87% (95% CI 66.4–97.2). Comparing antibody titers from CBA with delta median fluorescence intensities from FACS showed a high concordance (kappa = 0.943, p<0.0001) and correlation (r = 0.697, p<0.0001). In conclusion, evaluation of the FACS based assay revealed a lower sensitivity and high inter-assay variation, making the CBA a more reliable detection method. PMID:25815887

  17. Comparison of diagnostic accuracy of microscopy and flow cytometry in evaluating N-methyl-D-aspartate receptor antibodies in serum using a live cell-based assay.

    PubMed

    Ramberger, Melanie; Peschl, Patrick; Schanda, Kathrin; Irschick, Regina; Höftberger, Romana; Deisenhammer, Florian; Rostásy, Kevin; Berger, Thomas; Dalmau, Josep; Reindl, Markus

    2015-01-01

    N-methyl-D-aspartate receptor (NMDAR) encephalitis is an autoimmune neurological disease, diagnosed by a specific autoantibody against NMDAR. Antibody testing using commercially available cell-based assays (CBA) or immunohistochemistry on rat brain tissue has proven high specificity and sensitivity. Here we compare an immunofluorescence live CBA to a flow cytometry (FACS) based assay to detect NMDAR antibodies by their binding to the surface of HEK293A cells functionally expressing NMDAR. Both assays were first established using a discovery group of 76 individuals and then validated in a group of 32 patients in a blinded manner. In the CBA, 23 of 23 patients with NMDAR encephalitis were positive for NMDAR antibodies and 0 of 85 controls (32 healthy controls and 53 patients with other neurological diseases), resulting in a sensitivity and specificity of 100% (95% confidence intervals (CI) 85.1-100.0 and 95.7-100.0, respectively). The FACS based assay detected NMDAR antibodies in 20 of 23 patients and in 0 of 85 controls. Therefore, with an equally high specificity (95% CI 95.7-100.0) the sensitivity of the FACS based assay was 87% (95% CI 66.4-97.2). Comparing antibody titers from CBA with delta median fluorescence intensities from FACS showed a high concordance (kappa = 0.943, p<0.0001) and correlation (r = 0.697, p<0.0001). In conclusion, evaluation of the FACS based assay revealed a lower sensitivity and high inter-assay variation, making the CBA a more reliable detection method.

  18. The various assays for measuring activity states of factor VIIa in plasma and therapeutic products: Diagnostic value and analytical usefulness in various pathophysiological states.

    PubMed

    Amiral, Jean; Dunois, Claire; Amiral, Cédric; Seghatchian, Jerard

    2016-12-29

    The key coagulation factor FVII, and its activated form FVIIa, present a major interest for their role at the initiation phase of blood coagulation, and because they can activate all blood coagulation cascade, through the extrinsic, but also the intrinsic pathway. Blood activation initiated through FVII is first presented, as it is understood nowadays. Measurement of FVII and FVIIa were of main interest for epidemiological studies, but FVIIa contribution to assay results was only deduced. The introduction of specific FVIIa assays, functional or immunoassays, allowed measuring directly FVIIa without any interference of non-activated FVII, or other coagulation factors or their activated forms. The various methods available, and their characteristics are presented, with a special focus on two assays developed by our group for FVIIa (a clotting one and a chromogenic one). The FVIIa clotting assay shows evident superiority for measuring its activity in plasma, in pathophysiological conditions. The normal range is <2.5ng/ml, which represents less than 0.5% of the FVII protein. FVIIa is elevated in some pathological states. The chromogenic assay is of interest for assigning the potency of FVIIa concentrates, as it has a higher dynamic range. Both assays are fully automatable on laboratory instruments, and standardized in a satisfactory manner thanks to the use of the FVIIa concentrate WHO International Standard (NIBSC). The various applications and usefulness of FVIIa laboratory assays are discussed, for the measurement of therapeutic products, or for following recoveries in treated patients, including hemophiliacs with inhibitors, patients with severe bleeding risk (liver diseases, surgery, trauma, …), and lastly for measurement of its activity in therapeutic products.

  19. Expression of Epstein-Barr virus antibodies EA-IgG, Rta-IgG, and VCA-IgA in nasopharyngeal carcinoma and their use in a combined diagnostic assay.

    PubMed

    Li, Y; Wang, K; Yin, S K; Zheng, H L; Min, D L

    2016-03-18

    Epstein-Barr virus (EBV) infection is closely associated with nasopharyngeal carcinoma, which can be monitored by the levels of Rta protein antibody IgG (Rta-IgG), early antigen antibody (EA-IgG), and viral capsid antibody (VCA-IgA). In the present study, we investigated the serum levels of Rta-IgG, EA-IgG, and VCA-IgA in nasopharyngeal cancer patients, and the diagnostic value of a combined assay that includes these antibodies in addition to the EBV-DNA. A total of 56 nasopharyngeal cancer patients were recruited as the study population, along with 48 benign rhinitis patients and 42 healthy individuals. Serum EA-IgG, Rta-IgG, and VCA-IgA levels were measured by enzyme-linked immunosorbent assay, and EBV-DNA was quantified with PCR. The diagnostic value of these indices was further evaluated by ROC curve analysis. The expression levels of EA-IgG, Rta-IgG, VCA-IgA, and EBV-DNA were elevated in the nasopharyngeal cancer patients, who had higher levels of these antibodies than those in the rhinitis patients, followed by the healthy individuals. These indices were also increased with advanced TNM stage. The overall diagnostic efficacy was ranked as follows: VCA-IgA, Rta-IgA, EA-IgA, and EBV-DNA. The combined diagnosis using these four indices increased the sensitivity to 98.21% and the negative predictive value to 98.61%, without any significant compromise on the test specificity. In conclusion, EA-IgG, Rta-IgG, VCA-IgA, and EBV-DNA expression levels were elevated in nasopharyngeal patients. The combined diagnostic value of these serum indices has important implications in nasopharyngeal carcinoma.

  20. Evaluation of an Automated Rapid Diagnostic Assay for Detection of Gram-Negative Bacteria and Their Drug-Resistance Genes in Positive Blood Cultures

    PubMed Central

    Tojo, Masayoshi; Fujita, Takahiro; Ainoda, Yusuke; Nagamatsu, Maki; Hayakawa, Kayoko; Mezaki, Kazuhisa; Sakurai, Aki; Masui, Yoshinori; Yazaki, Hirohisa; Takahashi, Hiroshi; Miyoshi-Akiyama, Tohru; Totsuka, Kyoichi; Kirikae, Teruo; Ohmagari, Norio

    2014-01-01

    We evaluated the performance of the Verigene Gram-Negative Blood Culture Nucleic Acid Test (BC-GN; Nanosphere, Northbrook, IL, USA), an automated multiplex assay for rapid identification of positive blood cultures caused by 9 Gram-negative bacteria (GNB) and for detection of 9 genes associated with β-lactam resistance. The BC-GN assay can be performed directly from positive blood cultures with 5 minutes of hands-on and 2 hours of run time per sample. A total of 397 GNB positive blood cultures were analyzed using the BC-GN assay. Of the 397 samples, 295 were simulated samples prepared by inoculating GNB into blood culture bottles, and the remaining were clinical samples from 102 patients with positive blood cultures. Aliquots of the positive blood cultures were tested by the BC-GN assay. The results of bacterial identification between the BC-GN assay and standard laboratory methods were as follows: Acinetobacter spp. (39 isolates for the BC-GN assay/39 for the standard methods), Citrobacter spp. (7/7), Escherichia coli (87/87), Klebsiella oxytoca (13/13), and Proteus spp. (11/11); Enterobacter spp. (29/30); Klebsiella pneumoniae (62/72); Pseudomonas aeruginosa (124/125); and Serratia marcescens (18/21); respectively. From the 102 clinical samples, 104 bacterial species were identified with the BC-GN assay, whereas 110 were identified with the standard methods. The BC-GN assay also detected all β-lactam resistance genes tested (233 genes), including 54 blaCTX-M, 119 blaIMP, 8 blaKPC, 16 blaNDM, 24 blaOXA-23, 1 blaOXA-24/40, 1 blaOXA-48, 4 blaOXA-58, and 6 blaVIM. The data shows that the BC-GN assay provides rapid detection of GNB and β-lactam resistance genes in positive blood cultures and has the potential to contributing to optimal patient management by earlier detection of major antimicrobial resistance genes. PMID:24705449

  1. Evaluation of an automated rapid diagnostic assay for detection of Gram-negative bacteria and their drug-resistance genes in positive blood cultures.

    PubMed

    Tojo, Masayoshi; Fujita, Takahiro; Ainoda, Yusuke; Nagamatsu, Maki; Hayakawa, Kayoko; Mezaki, Kazuhisa; Sakurai, Aki; Masui, Yoshinori; Yazaki, Hirohisa; Takahashi, Hiroshi; Miyoshi-Akiyama, Tohru; Totsuka, Kyoichi; Kirikae, Teruo; Ohmagari, Norio

    2014-01-01

    We evaluated the performance of the Verigene Gram-Negative Blood Culture Nucleic Acid Test (BC-GN; Nanosphere, Northbrook, IL, USA), an automated multiplex assay for rapid identification of positive blood cultures caused by 9 Gram-negative bacteria (GNB) and for detection of 9 genes associated with β-lactam resistance. The BC-GN assay can be performed directly from positive blood cultures with 5 minutes of hands-on and 2 hours of run time per sample. A total of 397 GNB positive blood cultures were analyzed using the BC-GN assay. Of the 397 samples, 295 were simulated samples prepared by inoculating GNB into blood culture bottles, and the remaining were clinical samples from 102 patients with positive blood cultures. Aliquots of the positive blood cultures were tested by the BC-GN assay. The results of bacterial identification between the BC-GN assay and standard laboratory methods were as follows: Acinetobacter spp. (39 isolates for the BC-GN assay/39 for the standard methods), Citrobacter spp. (7/7), Escherichia coli (87/87), Klebsiella oxytoca (13/13), and Proteus spp. (11/11); Enterobacter spp. (29/30); Klebsiella pneumoniae (62/72); Pseudomonas aeruginosa (124/125); and Serratia marcescens (18/21); respectively. From the 102 clinical samples, 104 bacterial species were identified with the BC-GN assay, whereas 110 were identified with the standard methods. The BC-GN assay also detected all β-lactam resistance genes tested (233 genes), including 54 bla(CTX-M), 119 bla(IMP), 8 bla(KPC), 16 bla(NDM), 24 bla(OXA-23), 1 bla(OXA-24/40), 1 bla(OXA-48), 4 bla(OXA-58), and 6 blaVIM. The data shows that the BC-GN assay provides rapid detection of GNB and β-lactam resistance genes in positive blood cultures and has the potential to contributing to optimal patient management by earlier detection of major antimicrobial resistance genes.

  2. Enzyme-linked PNA lectin binding assay compared with CA19-9 and CEA radioimmunoassay as a diagnostic blood test for pancreatic cancer.

    PubMed Central

    Ching, C. K.; Rhodes, J. M.

    1989-01-01

    Previous studies have shown that sera from patients with pancreatic cancer often contain a mucus glycoprotein that expresses the oncofetal antigen galactose 1-3, N-acetyl galactosamine, which is the T blood group antigen and the binding site for the lectin peanut agglutinin (PNA). An enzyme-linked lectin assay has been developed to quantify PNA-binding glycoproteins in serum and has been evaluated as a serological test for pancreatic cancer. Sera were studied from 53 patients with pancreatic cancer and 154 controls, including benign obstructive jaundice, acute and chronic pancreatitis, chronic liver disease and inflammatory bowel disease. The enzyme-linked peanut lectin assay proved highly reproducible and has 77% sensitivity and 83% specificity for pancreatic cancer, results that are very similar to those achieved in the same sera by CA19-9 radioimmunoassay (75% sensitivity, 82% specificity with the upper limit of normal set at 37 u ml-1). CEA assay proved less useful (60% sensitivity, 47% specificity). In this study better results were obtained if an upper limit of normal of 50 u ml-1 was used for CA19-9 (75% sensitivity, 92% specificity). Combination of CA19-9 assay with the upper limit set at 50 u ml-1 and the peanut lectin assay improved the sensitivity to 85% with only a slight fall in specificity (85%). These results compare well with published results for ultrasound and CT scanning. PMID:2736232

  3. Diagnostic performance of an indirect enzyme-linked immunosorbent assay (ELISA) to detect bovine leukemia virus antibodies in bulk-tank milk samples

    PubMed Central

    Nekouei, Omid; Durocher, Jean; Keefe, Greg

    2016-01-01

    This study assessed the diagnostic performance of a commercial ELISA for detecting bovine leukemia virus antibodies in bulk-tank milk samples from eastern Canada. Sensitivity and specificity of the test were estimated at 97.2% and 100%, respectively. The test was recommended as a cost-efficient tool for large-scale screening programs. PMID:27429469

  4. Design and validation of a qPCR assay for accurate detection and initial serogrouping of Legionella pneumophila in clinical specimens by the ESCMID Study Group for Legionella Infections (ESGLI).

    PubMed

    Mentasti, M; Kese, D; Echahidi, F; Uldum, S A; Afshar, B; David, S; Mrazek, J; De Mendonça, R; Harrison, T G; Chalker, V J

    2015-07-01

    Prompt detection of Legionella pneumophila is essential for rapid investigation of legionellosis. Furthermore, as the majority of L. pneumophila infections are caused by serogroup 1 (sg1) strains, rapid identification of such strains can be critical in both routine and outbreak scenarios. The ESCMID Study Group for Legionella Infections (ESGLI) was established in 2012 and immediately identified as a priority the validation of a reliable, easy to perform and interpret, cost-effective qPCR assay to standardise the detection of L. pneumophila DNA amongst members. A novel L. pneumophila assay targeting the mip gene was designed and combined with previously published methodologies amplifying the sg1 marker (wzm) and the green fluorescent protein gene (gfp) internal process control. The resulting triplex assay was validated internationally on the three qPCR platforms used by the majority of European Legionella reference laboratories: ABI 7500 (Life Technologies), LightCycler 480 Instrument II (Roche) and Rotor-Gene Q (Qiagen). Clinical and EQA specimens were tested together with a large panel of strains (251 in total) to validate the assay. The assay proved to be 100% specific for L. pneumophila and sg1 DNA both in silico and in vitro. Efficiency values for mip and wzm assays ranged between 91.97 and 97.69%. Limit of detection values estimated with 95% confidence were adopted for mip and wzm assays on all three qPCR platforms. Inhibition was not observed. This study describes a robust assay that could be widely implemented to standardise the molecular detection of L. pneumophila among ESGLI laboratories and beyond.

  5. 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.

  6. Drug-specific in vitro secretion of IFNγ in the diagnosis of drug-induced exanthemas: electrochemiluminescence assay versus previously used diagnostic methods.

    PubMed

    Porebski, Grzegorz; Czarnobilska, Ewa

    2015-01-01

    The in vitro diagnosis of delayed drug hypersensitivity reactions remains a research problem. We measured drug-specific IFNγ release in peripheral blood mononuclear cells sampled from patients with drug-induced maculopapular exanthema and the age- and sex-matched control group. This is the first study to directly cross-compare an ultrasensitive assay based on an emerging electrochemiluminescence technology (ECL), the standard lymphocyte proliferation assay and three following tests detecting IFNγ at different steps of its production: intracellular in CD3+CD4+ cells (flow cytometry), secretion at the single cell level (enzyme-linked immunospot assay), bulk content in cell culture supernatant (enzyme-linked immunosorbent assay, ELISA). The highest rate of drug-positive responses were recorded for ELISA and ECL tests (56.25%). No false-positive responses were observed--all tests were negative in the control group. We demonstrated that IFNγ-detecting ELISA is not less efficient than ECL test, however, it is easily available and cheap, which makes it a potential method of choice in the future.

  7. A Loop-Mediated Isothermal Amplification (LAMP) Assay for Early Detection of Schistosoma mansoni in Stool Samples: A Diagnostic Approach in a Murine Model

    PubMed Central

    Fernández-Soto, Pedro; Gandasegui Arahuetes, Javier; Sánchez Hernández, Alicia; López Abán, Julio; Vicente Santiago, Belén; Muro, Antonio

    2014-01-01

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

  8. Comparison of high-resolution melting analysis, TaqMan Allelic discrimination assay, and sanger sequencing for Clopidogrel efficacy genotyping in routine molecular diagnostics.

    PubMed

    Zhang, Lina; Cui, Guanglin; Li, Zongzhe; Wang, Haoran; Ding, Hu; Wang, Dao Wen

    2013-09-01

    Clopidogrel, as a routine antiplatelet drug, is widely used in patients to reduce cardiovascular events following percutaneous coronary intervention. Because of genetic variation, patients undergoing percutaneous coronary intervention show differing responses to clopidogrel therapy. Recently, five single nucleotide polymorphisms (SNPs) within CYP2C19 (rs4244285, rs4986893, rs12248560), ABCB1 (rs1045642), and ITGB3 (rs5918) were identified that contribute prominently to variability in response to clopidogrel. Given that Sanger sequencing is labor intensive and time consuming, rapid genotyping methods for SNP detection are urgently required before clopidogrel therapy. Accordingly, we developed a high-resolution melting analysis (HRMA) and TaqMan allelic discrimination assay (TaqMan) to genotype those five SNPs, and compared these two assays with Sanger sequencing on accuracy of genotyping as well as operational characteristics. These two assays showed high accuracy (0.995, 95% CI 0.991 to 0.998 for HRMA; 0.997, 95% CI 0.994 to 0.999 for TaqMan, respectively), sensitivity (0.996, 95% CI 0.989 to 1.002 for HRMA; 0.998, 95% CI 0.993 to 1.002 for TaqMan, respectively), and specificity (0.995, 95% CI 0.991 to 0.999 for HRMA; 0.996, 95% CI 0.993 to 1.000 for TaqMan, respectively). Our study indicates that HRMA and TaqMan are easier to operate and obviously faster than Sanger sequencing. In conclusion, HRMA and TaqMan are rapid, convenient, and reliable assays for clopidogrel efficacy genotyping.

  9. A rapid dipstick antigen capture assay for the diagnosis of falciparum malaria. WHO Informal Consultation on Recent Advances in Diagnostic Techniques and Vaccines for Malaria.

    PubMed Central

    1996-01-01

    Recent advances in the diagnosis of Plasmodium falciparum infections have made it possible to consider supplementing light microscopy with a standardized dipstick antigen capture assay based on the detection of a parasite-specific protein, which is secreted by the asexual blood stages and immature gametocytes but not by the other stages. Field trials indicate that this dipstick assay provides consistently reproducible results, with a threshold of detection of P. falciparum parasitaemia similar to that obtained by high quality routine malaria microscopy and a specificity and sensitivity of around 90% compared with standard thick blood film microscopy. The stability, reproducibility, and ease of use of the assay clearly indicate that it has potential for application in the management of malaria, particularly at the peripheral health care level, provided its accuracy can be assured and that it can be made affordable. Consideration should be given to its wider use where operational requirements and resources so justify, and where decisions are based on adequate evaluation of the existing health delivery systems. PMID:8653815

  10. Rapid and simple polymerase chain reaction-based diagnostic assays for GM2 gangliosidosis variant 0 (Sandhoff-like disease) in Japanese domestic cats.

    PubMed

    Rahman, Mohammad M; Shoubudani, Tomoaki; Mizukami, Keijiro; Chang, Hye-Sook; Hossain, Mohammad A; Yabuki, Akira; Mitani, Sawane; Higo, Takashi; Arai, Toshiro; Yamato, Osamu

    2011-03-01

    Polymerase chain reaction (PCR)-based assays combined with microchip electrophoresis were developed and evaluated for diagnosis and genotyping of GM2 gangliosidosis variant 0 (Sandhoff-like disease) in Japanese domestic cats. A preliminary genotyping survey was carried out in the population of Japanese domestic cats (1,015 cats in total) in southern Japan. Three kinds of assays including PCR primer-induced restriction analysis (PIRA) and mutagenically separated (MS)-PCR were carried out using blood-stained Flinders Technology Associates filter papers (FTA cards) as templates. The PCR products were analyzed by both agarose gel and microchip electrophoreses. All assays were sufficient to determine the genotypes of this disease, but MS-PCR offered the most rapid and simplest test, as it does not need the restriction enzyme step required in PCR-PIRA. The use of microchip electrophoresis in combination with FTA cards for sampling could shorten the time required for genotyping and simplify the procedure as well. The genotyping survey in the current study did not find any cats that possessed the mutant allele, suggesting that the prevalence of this allele is low (<0.1%) in southern Japan.

  11. Development of a fluorescence resonance energy transfer peptide library technology for detection of protease contaminants in protein-based raw materials used in diagnostic assays.

    PubMed

    Kapprell, Hans-Peter; Maurer, Andreas; Kramer, Florian; Heinrich, Boris; Buenning, Carsten; Narvaez, Alfredo; Kalbacher, Hubert; Flad, Thomas

    2011-10-01

    Protease impurities in raw materials used in enzyme immunoassays can impair assay performance. This risk may be greatly decreased if incoming protein-based raw materials are controlled for protease impurities or if protease inhibitors are used in the assay formulations. As many different proteases might occur in protein raw materials, it is desirable to have a general test for protease contamination. With the help of a fluorescence resonance energy transfer peptide library containing about 2.5 million peptides, we have succeeded in establishing such a system, with sensitivity in the nanogram range for known proteases. Protease contamination was found to differ between different raw materials and was correlated with assay performance. Protease activity in contaminated raw materials could be suppressed to various degrees with different chemical inhibitors or by thermal treatment. This technology is suited for the control of incoming protein-based raw materials used for enzyme immunoassays, as well as for the optimization of the use of protein inhibitors or thermal treatment of protein-based raw materials for the inactivation of proteases.

  12. Lyophilisation of influenza, rabies and Marburg lentiviral pseudotype viruses for the development and distribution of a neutralisation -assay-based diagnostic kit.

    PubMed

    Mather, Stuart T; Wright, Edward; Scott, Simon D; Temperton, Nigel J

    2014-12-15

    Pseudotype viruses (PVs) are chimeric, replication-deficient virions that mimic wild-type virus entry mechanisms and can be safely employed in neutralisation assays, bypassing the need for high biosafety requirements and performing comparably to established serological assays. However, PV supernatant necessitates -80°C long-term storage and cold-chain maintenance during transport, which limits the scope of dissemination and application throughout resource-limited laboratories. We therefore investigated the effects of lyophilisation on influenza, rabies and Marburg PV stability, with a view to developing a pseudotype virus neutralisation assay (PVNA) based kit suitable for affordable global distribution. Infectivity of each PV was calculated after lyophilisation and immediate reconstitution, as well as subsequent to incubation of freeze-dried pellets at varying temperatures, humidities and timepoints. Integrity of glycoprotein structure following treatment was also assessed by employing lyophilised PVs in downstream PVNAs. In the presence of 0.5M sucrose-PBS cryoprotectant, each freeze-dried pseudotype was stably stored for 4 weeks at up to 37°C and could be neutralised to the same potency as unlyophilised PVs when employed in PVNAs. These results confirm the viability of a freeze-dried PVNA-based kit, which could significantly facilitate low-cost serology for a wide portfolio of emerging infectious viruses.

  13. Interpreting Assays for the Detection of Streptococcus pneumoniae

    PubMed Central

    2011-01-01

    Streptococcus pneumoniae is both an aggressive pathogen and a normal part of the human respiratory microbiome. Clinicians and microbiologists have struggled to develop tests that can identify pneumococcal respiratory infection and accurately distinguish colonization from invasive disease. Molecular methods hold the promise of an improved ability to rapidly detect microorganisms in respiratory secretions and to make an accurate diagnosis; however, interpretation of diagnostic testing for S. pneumoniae remains problematic. Molecular assays, such as those targeting the pneumolysin gene, may cross-react with other streptococcal species, confounding detection and quantification. Assays that target the autolysin gene appear to be more specific. Even when accurately identified, however, the significance of S. pneumoniae DNA detected in clinical samples is difficult to determine. Here we will discuss the challenges faced in the interpretation of molecular testing for S. pneumoniae, and some strategies that might be used to improve our ability to diagnose pneumococcal respiratory infection. PMID:21460292

  14. Characterization of SNP and Structural Variations in the Mitochondrial Genomes of Tilletia indica and Its Closely Related Species Formed Basis for a Simple Diagnostic Assay

    PubMed Central

    Tan, Mui-Keng; Raman, Harsh; Chambers, Grant; Sharma, Indu; Chen, Zhiliang; Deshpande, Nandan; Wilkins, Marc R.

    2016-01-01

    Tilletia indica causes the disease Karnal bunt in wheat. The disease is under international quarantine regulations. Comparative mitochondrial (mt) genome analysis of T. indica (KX394364 and DQ993184) and T. walkeri (EF536375) has found 325 to 328 SNPs, 57 to 60 short InDels (from 1 to 13 nt), two InDels (30 and 61 nt) and five (>200 nt) presence/absence variations (PAVs) between the two species. The mt genomes of both species have identical gene order. The numbers of SNPs and InDels between the mt genomes of the two species are approximately nine times of the corresponding numbers between the two T. indica isolates. There are eight SNPs between T. indica and T. walkeri that resulted in amino acid substitutions in the mt genes of cob, nad2 and nad5. In contrast, there is no amino acid substitution in the mt genes of the T. indica isolates from the SNPs found. The five PAVs present in T. indica (DQ993184) are absent in T. walkeri. Four PAVs are more than 1 kb and are not present in every T. indica isolate. Analysis of their presence and absence separates a collection of T. indica isolates into 11 subgroups. Two PAVs have ORFs for the LAGLIDAG endonuclease and two have ORFs for the GIY-YIG endonuclease family, which are representatives of homing endonuclease genes (HEGs). These intron- encoded HEGs confer intron mobility and account for their fluid distribution in T. indica isolates. The small PAV of 221 bp, present in every T. indica isolate and unique to the species, was used as the genetic fingerprint for the successful development of a rapid, highly sensitive and specific loop mediated isothermal amplification (LAMP) assay. The simple procedure of the LAMP assay and the easy detection formats will enable the assay to be automated for high throughput diagnosis. PMID:27814391

  15. Use of qPCR and genomic sequencing to diagnose Plasmodium ovale wallikeri malaria in a returned soldier in the setting of a negative rapid diagnostic assay.

    PubMed

    Cohen, Robert; Feghali, Karla; Alemayehu, Saba; Komisar, Jack; Hang, Jun; Weina, Peter J; Coggeshall, Patricia; Kamau, Edwin; Zapor, Michael

    2013-09-01

    Plasmodium ovale is one of several clinically relevant malaria species known to cause disease in humans. However, in contrast to Plasmodium falciparum and Plasmodium vivax, which are responsible for most cases of human malaria, P. ovale has a wide distribution but low prevalence in tropical regions. Here, we report the case of a soldier returning from Liberia with P. ovale wallikeri malaria. This case highlights the limitations of both microscopy and the malaria rapid diagnostic test for diagnosing infection with P. ovale and for distinguishing P. ovale wallikeri from P. ovale curtisi. To our knowledge, this is the first case report in which quantitative real-time polymerase chain reaction amplification using the Cytochrome B gene, coupled with genomic sequencing of the potra locus, was used for definitive diagnosis of P. ovale wallikeri malaria.

  16. 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.

  17. Development of fluorescent methods for DNA methyltransferase assay

    NASA Astrophysics Data System (ADS)

    Li, Yueying; Zou, Xiaoran; Ma, Fei; Tang, Bo; Zhang, Chun-yang

    2017-03-01

    DNA methylation modified by DNA methyltransferase (MTase) plays an important role in regulating gene transcription, cell growth and proliferation. The aberrant DNA MTase activity may lead to a variety of human diseases including cancers. Therefore, accurate and sensitive detection of DNA MTase activity is crucial to biomedical research, clinical diagnostics and therapy. However, conventional DNA MTase assays often suffer from labor-intensive operations and time-consuming procedures. Alternatively, fluorescent methods have significant advantages of simplicity and high sensitivity, and have been widely applied for DNA MTase assay. In this review, we summarize the recent advances in the development of fluorescent methods for DNA MTase assay. These emerging methods include amplification-free and the amplification-assisted assays. Moreover, we discuss the challenges and future directions of this area.

  18. Diagnostic criteria for an enzyme-linked immunosorbent assay for occult heartworm disease: standardization of the test system in naturally exposed dogs.

    PubMed

    Gillis, J M; Smith, R D; Todd, K S

    1984-11-01

    The development of criteria for interpreting and reporting the results of an occult heartworm enzyme-linked immunosorbent assay to practitioners is described. The antigen is a saline extract of adult female Dirofilaria immitis. The cutoff absorbance A400 nm values were estimated, using 106 dogs free of infection. Any A400 nm value less than 0.526 is considered negative and values greater than or equal to 0.784 are positive. Intermediate A400 nm values are interpreted as suspect. Absorbance values for serum samples from 13 client-owned amicrofilaremic dogs revealed a bimodal distribution consistent with presumptive diagnosis based on clinical signs, which indicates that the test may be used to support a diagnosis of occult heartworm disease. The present serotest, however, is unable to distinguish microfilaremic dogs from noninfected dogs. Serum from dogs infected with other common helminths failed to crossreact with the D immitis antigen, with the exception of Dipetalonema reconditum.

  19. A solid phase enzyme-linked immunosorbent assay for the antigenic detection of Legionella pneumophila (serogroup 1): A compliment for the space station diagnostic capability

    NASA Technical Reports Server (NTRS)

    Hejtmancik, Kelly E.

    1987-01-01

    It is necessary that an adequate microbiology capability be provided as part of the Health Maintenance Facility (HMF) to support expected microbial disease events and environmental monitoring during long periods of space flight. The application of morphological and biochemical studies to confirm the presence of certain bacterial and fungal disease agents are currently available and under consideration. This confirmation would be facilitated through employment of serological methods to aid in the identification of bacterial, fungal, and viral agents. A number of serological approaches are currently being considered, including the use of Enzyme Linked Immunosorbent Assay (ELISA) technology, which could be utilized during microgravity conditions. A solid phase, membrane supported ELISA for the detection of Legionella pneumophila, an expected disease agent, was developed to show a potential model system that would meet the HMF requirements and specifications for the future space station. These studies demonstrate the capability of membrane supported ELISA systems for identification of expected microbial disease agents as part of the HMF.

  20. Diagnostic accuracy of the Cepheid GeneXpert vanA/vanB assay ver. 1.0 to detect the vanA and vanB vancomycin resistance genes in Enterococcus from perianal specimens.

    PubMed

    Marner, Erin S; Wolk, Donna M; Carr, Jeanne; Hewitt, Carolyn; Dominguez, Lorraine L; Kovacs, Thomas; Johnson, Desiree R; Hayden, Randall T

    2011-04-01

    Rapid detection of vancomycin-resistant enterococci (VRE) carriers could be useful to health care facilities to minimize transmission. To that end, we compared the performance of the Cepheid GeneXpert vanA/vanB assay with that of direct and broth-enriched culture methods for detection of VRE from perianal swabs. Enterococci were cultivated on Enterococcosel™ agar with 8 μg/mL vancomycin, Bile Esculin Azide Agar with 6 μg/mL vancomycin, and Bile Esculin Azide Enterococcosel Broth. Compared to the reference standard (combination of direct agar plating, broth-enriched culture, and clinical chart review), the sensitivity, specificity, positive predictive value, and negative predictive value of the vanA/vanB assay were 96.4%, 93.0%, 92.0%, and 96.9%, respectively (n=184). The 95% limit of detection was 100 colony-forming units (CFU)/mL for vanA and 114 CFU/mL for vanB. In summary, the GeneXpert vanA/vanB assay is a rapid and accurate method to identify vanA/vanB-colonized patients for VRE screening programs that use perianal swab specimens.

  1. Simultaneous identification of three highly pathogenic Eimeria species in rabbits using a multiplex PCR diagnostic assay based on ITS1-5.8S rRNA-ITS2 fragments.

    PubMed

    Yan, Wenchao; Wang, Wenlong; Wang, Tianqi; Suo, Xun; Qian, Weifeng; Wang, Shuai; Fan, Di

    2013-03-31

    Eimeria stiedai, E. intestinalis, and E. flavescens are highly pathogenic in rabbits, especially rabbits younger than 3 months. In this study, the complete ITS1-5.8S rRNA-ITS2 sequences of six rabbit Eimeria species, E. stiedai, E. intestinalis, E. flavescens, E. media, E. magna, and E. irresidua, were cloned with universal primers for the genus Eimeria and genomic DNA of LY and KF isolates as templates. These results revealed that both ITS1 and ITS2 sequences were specific to each Eimeria species in rabbits. A specific and sensitive multiplex PCR diagnostic assay based on polymorphic sites of ITS1 and ITS2 was developed and used to identify the three highly pathogenic species from rabbits, E. stiedai, E. intestinalis, and E. flavescens. Our findings provide a powerful tool for the clinical differentiation of highly pathogenic Eimeria species in rabbits and the study of the population genetics of rabbit coccidia.

  2. Combined assays for serum carcinoembryonic antigen and microRNA-17-3p offer improved diagnostic potential for stage I/II colon cancer

    PubMed Central

    ZHU, JINHAI; DONG, HUIMING; ZHANG, QIONG; ZHANG, SHANGWU

    2015-01-01

    Colorectal cancer is among the leading causes of cancer-related mortality, one of the main reasons for which is the lack of an effective screening method for early-stage disease. The levels of carcinoembryonic antigen (CEA) and microRNA (miR)-17-3p in the serum of 70 patients with stage I/II colon cancer and 70 healthy volunteers were determined, and the diagnostic value of CEA plus miR-17-3p detection for colon cancer was assessed. The levels of CEA were measured by a radioimmunoassay method, and those of miR-17-3p using the reverse transcription-quantitative polymerase chain reaction method. miR-16 was used as the endogenous control, as it displayed high stability, high abundance and low variability in the analyzed serum samples. The receiver operating characteristic (ROC) curve analysis indicated the potential diagnostic value of the two markers and the area under the ROC curve (AUC) for CEA and miR-17-3p was 0.719 (95% CI: 0.658–0.843) and 0.807 (95% CI: 0.748–0.906), respectively. At a threshold of 9.6 ng/ml for CEA, the optimal sensitivity and specificity were 74.6 and 84.3%, respectively, in discriminating colon cancer patients from healthy controls. At a threshold of 2.98 for miR-17-3p, the sensitivity and the specificity were 83.6 and 72.9%, respectively. A combined ROC analysis using CEA and miR-17-3p revealed an AUC of 0.929 (95% CI: 0.834–0.978) with a sensitivity of 96.4% and a specificity of 95.7% in discriminating colon cancer patients from healthy controls. In conclusion, both CEA and miR-17-3p were highly expressed in the serum of our series of colon cancer patients. CEA plus miR-17-3p detection significantly increased the sensitivity and specificity in discriminating stage I/II colon cancer patients from healthy controls. Therefore, combined detection of serum CEA and miR-17-3p levels may have the potential to become a new laboratory method for the early clinical diagnosis of colon cancer. PMID:26807240

  3. 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.

  4. DNA-based diagnostics for genetically modified cotton: decaplex PCR assay to differentiate MON531 and MON15985 Bt cotton events.

    PubMed

    Randhawa, Gurinder Jit; Singh, Monika; Chhabra, Rashmi

    2013-01-01

    The adoption rate and global area under cultivation of genetically modified (GM) crops is dramatically increasing in recent past. GM cotton has occupied 25.0 million hectares (mha) comprising 15.6% of the global area under GM cultivation. Bt cotton, expressing delta-endotoxins from Bacillus thuringiensis (Bt), is the only commercialized crop in India that is planted on an area of 10.6 mha. With the increase in development and commercialization of GM crops, it is necessary to develop appropriate qualitative and quantitative methods for detection of different GM events. Robust diagnostics for GM detection need to be developed and implemented to monitor and detect different events of GM cotton in India. This chapter summarizes the methods based on polymerase chain reaction (PCR) being employed for detection of different GM events of cotton. We describe a decaplex PCR method for identification and differentiation of two major commercialized events of Bt cotton, i.e., MON531 and MON15985, in India.

  5. A two-step approach for sequencing spliceosome-related genes as a complementary diagnostic assay in MDS patients with ringed sideroblasts.

    PubMed

    Janusz, Kamila; Del Rey, Mónica; Abáigar, María; Collado, Rosa; Ivars, David; Hernández-Sánchez, María; Valiente, Alberto; Robledo, Cristina; Benito, Rocío; Díez-Campelo, María; Ramos, Fernando; Kohlmann, Alexander; Cañizo, Consuelo Del; Hernández-Rivas, Jesús María

    2017-02-04

    Our study aimed to analyze the presence of mutations in SF3B1 and other spliceosome-related genes in myelodysplastic syndromes with ringed sideroblasts (MDS-RS) by combining conventional Sanger and next-generation sequencing (NGS) methods, and to determine the feasibility of this approach in a clinical setting. 122 bone marrow samples from MDS-RS patients were studied. Initially, exons 14 and 15 of the SF3B1 gene were analyzed by Sanger sequencing. Secondly, they were studied by NGS covering besides SF3B1, SRSF2, U2AF1 and ZRSR2 genes. An 86% of all patients showed mutations in the SF3B1 gene. Six of them, which were not identifiable by conventional sequencing in the first diagnostic step, were revealed by NGS. In addition, 19.5% of cases showed mutations in other splicing genes: SRSF2, U2AF1, and ZRSR2. Furthermore, 8.7% of patients had two mutations in SF3B1, SF3B1 and SRSF2, and SF3B1 and U2AF1, while 5.7% showed no mutations in the four spliceosome-related genes analyzed. The combined use of conventional Sanger and NGS allows the identification of mutations in spliceosome-related genes in almost all MDS patients with RS. This two-step approach is affordable and could be useful as a complementary technique in cases with an unclear diagnosis.

  6. 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.

  7. Accurate Classification of Germinal Center B-Cell-Like/Activated B-Cell-Like Diffuse Large B-Cell Lymphoma Using a Simple and Rapid Reverse Transcriptase-Multiplex Ligation-Dependent Probe Amplification Assay: A CALYM Study.

    PubMed

    Mareschal, Sylvain; Ruminy, Philippe; Bagacean, Cristina; Marchand, Vinciane; Cornic, Marie; Jais, Jean-Philippe; Figeac, Martin; Picquenot, Jean-Michel; Molina, Thierry Jo; Fest, Thierry; Salles, Gilles; Haioun, Corinne; Leroy, Karen; Tilly, Hervé; Jardin, Fabrice

    2015-04-09

    Diffuse large B-cell lymphoma, the most common non-Hodgkin lymphoma, is subdivided into germinal center B-cell-like and activated B-cell-like subtypes. Unfortunately, these lymphomas are difficult to differentiate in routine diagnosis, impeding the development of treatments. Patients with these lymphomas can benefit from specific therapies. We therefore developed a simple and rapid classifier based on a reverse transcriptase multiplex ligation-dependent probe amplification assay and 14 gene signatures. Compared with the Affymetrix U133+2 gold standard, all 46 samples (95% CI, 92%-100%) of a validation cohort classified by both techniques were attributed to the expected subtype. Similarly, 93% of the 55 samples (95% CI, 82%-98%) of a second independent series characterized with a mid-throughput gene expression profiling method were classified correctly. Unclassifiable sample proportions reached 13.2% and 13.8% in these cohorts, comparable with the frequency originally reported. The developed assay was also sensitive enough to obtain reliable results from formalin-fixed, paraffin-embedded samples and flexible enough to include prognostic factors such as MYC/BCL2 co-expression. Finally, in a series of 135 patients, both overall (P = 0.01) and progression-free (P = 0.004) survival differences between the two subtypes were confirmed. Because the multiplex ligation-dependent probe amplification method is already in use and requires only common instruments and reagents, it could easily be applied to clinical trial patient stratification to help in treatment decisions.

  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.

  9. Positive skin and serologic test results of diagnostic assays for bovine tuberculosis and subsequent isolation of Mycobacterium interjectum in a pygmy hippopotamus (Hexaprotodon liberiensis).

    PubMed

    Bouts, Tim; Vordermeier, Martin; Flach, Edmund; Routh, Andrew

    2009-09-01

    A 20-yr-old male pygmy hippopotamus (Hexaprotodon liberiensis), weighing 250 kg, arrived at Zoological Society London Whipsnade Zoo (United Kingdom) from a captive collection in Portugal. A quarantine health check was performed including a comparative intradermal tuberculosis (IDTB) test. Assessment of the comparative IDTB test at 72 hr revealed a strong positive reaction at the bovine site. Serum was tested with a rapid immunochromatographic assay (TB STAT-PAK) and was positive for tuberculosis antibodies. The tuberculosis tests were repeated 6 wk later with the same positive test outcome. In addition, a broncho-alveolar lavage (BAL) was submitted for mycobacterial culture. The positive IDTB test and TB STAT-PAK results were supported by multiantigen print immunoassay (MAPIA). Based on these results, the animal was suspected to be infected with Mycobacterium tuberculosis complex organisms and was euthanized. No gross or histologic signs of tuberculosis were found at postmortem examination. Mycobacterium interjectum was cultured from the BAL but not from necropsy samples. The antigens used in the TB STAT-PAK and MAPIA tests are reportedly specific for the M. tuberculosis complex, and so it is possible this animal presented with a latent case of tuberculosis or had a previous tuberculosis infection that resolved prior to testing. Cross-reactions with nontuberculous mycobacteria have been described with TB STAT-PAK and MAPIA tests. However, Western blotting analysis using serum from this animal did not recognize M. interjectum proteins of equivalent size to the M. tuberculosis-Mycobacterium bovis proteins recognized in the MAPIA. Thus, antigenic cross-reactivity with M. interjectum can be deemed less likely, but other nontuberculous mycobacterial proteins cannot be ruled out. It is therefore possible that false-positive reactions were obtained. These results highlight the difficulty of diagnosing tuberculosis in the absence of pathology and the presence of

  10. The analytical validation of the Oncotype DX Recurrence Score assay

    PubMed Central

    Baehner, Frederick L

    2016-01-01

    In vitro diagnostic multivariate index assays are highly complex molecular assays that can provide clinically actionable information regarding the underlying tumour biology and facilitate personalised treatment. These assays are only useful in clinical practice if all of the following are established: analytical validation (i.e., how accurately/reliably the assay measures the molecular characteristics), clinical validation (i.e., how consistently/accurately the test detects/predicts the outcomes of interest), and clinical utility (i.e., how likely the test is to significantly improve patient outcomes). In considering the use of these assays, clinicians often focus primarily on the clinical validity/utility; however, the analytical validity of an assay (e.g., its accuracy, reproducibility, and standardisation) should also be evaluated and carefully considered. This review focuses on the rigorous analytical validation and performance of the Oncotype DX® Breast Cancer Assay, which is performed at the Central Clinical Reference Laboratory of Genomic Health, Inc. The assay process includes tumour tissue enrichment (if needed), RNA extraction, gene expression quantitation (using a gene panel consisting of 16 cancer genes plus 5 reference genes and quantitative real-time RT-PCR), and an automated computer algorithm to produce a Recurrence Score® result (scale: 0–100). This review presents evidence showing that the Recurrence Score result reported for each patient falls within a tight clinically relevant confidence interval. Specifically, the review discusses how the development of the assay was designed to optimise assay performance, presents data supporting its analytical validity, and describes the quality control and assurance programmes that ensure optimal test performance over time. PMID:27729940

  11. Diagnostic performance of HPV E6/E7, hTERT, and Ki67 mRNA RT-qPCR assays on formalin-fixed paraffin-embedded cervical tissue specimens from women with cervical cancer.

    PubMed

    Wang, Hye-Young; Kim, Geehyuk; Cho, Hyemi; Kim, Sunghyun; Lee, Dongsup; Park, Sunyoung; Park, Kwang Hwa; Lee, Hyeyoung

    2015-06-01

    Human papillomavirus (HPV) is a major cause of cervical cancer, which is the third most common cancer in women. Human telomerase reverse transcriptase (hTERT) and Ki67 are tumor cell markers indicating cancer cell proliferation in cancer patients, and activation of hTERT and Ki67 leads to progressive cervical carcinogenesis. In the present study, we evaluated the CervicGen HPVE6/E7 mRNA RT-qDx assay, which detects 16 HPV high-risk (HR) genotypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 and 69), and the CervicGen hTERT and Ki67 mRNA RT-qDx assay using 117 formalin-fixed paraffin-embedded (FFPE) cervical cancer tissue samples. The diagnostic validity of the CervicGen HPV RT-qDx assay for detecting histologically proven prevalent squamous cell carcinoma (SCC) was 94% sensitivity, 100% specificity, 77.8% positive predictive value (PPV), and 78.9% negative predictive value (NPV). The most common HPV genotypes detected in FFPE cervical cancer tissue samples were HPV 16 (56%) and HPV 18 (10%). The positivity rate of hTERT and Ki67 mRNA expressions in FFPE cervical cancer tissue samples on RT-qPCR was 65% and 93% respectively. Moreover, the positivity rates were 92% for a combination of HPV E6/E7 and hTERT mRNA expressions, 97% for HPV E6/E7 and Ki67 mRNA expressions, and 99% (99/100) for the combination of HPV E6/E7, hTERT, and Ki67 mRNA expressions. These data showed that SSC FFPE cervical cancer tissue samples correlated more strongly with high Ki67 mRNA expressions than with hTERT mRNA expressions. Notably, hTERT and Ki67 mRNA expression level was increased in high-grade cervical lesions, but was very low in normal samples. Our findings suggest that the combination of HPV E6/E7, hTERT, and Ki67 mRNA expression levels could be used in a complementary manner in diagnosing high-grade cervical lesions. Further studies are required to evaluate these assays as a useful predictive tool for screening low-grade cervical lesions.

  12. Evaluation of the automated ADVIA centaur® XP syphilis assay for serological testing.

    PubMed

    Saw, Sharon; Zhao, Huiqin; Tan, Phyllis; Saw, Betty; Sethi, Sunil

    2017-02-20

    We evaluated the performance of the ADVIA Centaur XP Syphilis assay (Siemens Healthcare Diagnostics, Tarrytown, NY, USA) using samples previously tested on the ARCHITECT i4000SR system (Abbott Diagnostics, Lake Forest, IL, USA) and confirmed by the Treponema pallidum particle agglutination assay (TPPA) (SERODIA-TPPA, Fujirebio Diagnostics Inc., Malvern, PA, USA). Clinical patient information was included to aid resolution of discordant samples where available. Precision, interference, and cross-reactivity were also assessed. Relative to patient clinical status, the sensitivity of both the ADVIA Centaur XP and the ARCHITECT assays was 100% (95% CI, 93.9-100), and the specificity of the ADVIA Centaur XP assay was 95.5% (95% CI, 90.4-98.3), which was slightly higher than that of the ARCHITECT assay at 93.9% (95% CI, 88.4-97.3). Overall agreement relative to patient clinical status was 96.9% (95% CI, 93.3-98.8) for the ADVIA Centaur XP assay and 95.8% (95% CI, 91.9-98.2) for the ARCHITECT assay. Overall agreement between the two automated assays was 96.9% (95% CI, 93.3-98.8). ADVIA Centaur XP assay precision was <5% at all index values tested. No significant interference was observed for lipemia or hemolysis; a small effect was seen with some samples for bilirubin. The assay exhibited no significant cross-reactivity with a number of potential interfering factors. The ADVIA Centaur XP Syphilis assay can be considered a sensitive and accurate assay for identification of treponemal antibodies in screening populations as well as patients presenting with suspicion of syphilitic infection.

  13. Molecular malaria diagnostics: A systematic review and meta-analysis.

    PubMed

    Roth, Johanna M; Korevaar, Daniël A; Leeflang, Mariska M G; Mens, Pètra F

    2016-01-01

    Accurate diagnosis of malaria is essential for identification and subsequent treatment of the disease. Currently, microscopy and rapid diagnostic tests are the most commonly used diagnostics, next to treatment based on clinical signs only. These tests are easy to deploy, but have a relatively high detection limit. With declining prevalence in many areas, there is an increasing need for more sensitive diagnostics. Molecular tools may be a suitable alternative, although costs and technical requirements currently hamper their implementation in resource limited settings. A range of (near) point-of-care diagnostics is therefore under development, including simplifications in sample preparation, amplification and/or read-out of the test. Accuracy data, in combination with technical characteristics, are essential in determining which molecular test, if any, would be the most promising to be deployed. This review presents a comprehensive overview of the currently available molecular malaria diagnostics, ranging from well-known tests to platforms in early stages of evaluation, and systematically evaluates their published accuracy. No important difference in accuracy was found between the most commonly used PCR-based assays (conventional, nested and real-time PCR), with most of them having high sensitivity and specificity, implying that there are no reasons other than practical ones to choose one technique over the other. Loop-mediated isothermal amplification and other (novel) diagnostics appear to be highly accurate as well, with some offering potential to be used in resource-limited settings.

  14. Evaluation of risk and diagnostic value of quantitative assays for anti-Toxoplasma gondii immunoglobulin A (IgA), IgE, and IgM and analytical study of specific IgG in immunodeficient patients.

    PubMed Central

    Pinon, J M; Foudrinier, F; Mougeot, G; Marx, C; Aubert, D; Toupance, O; Niel, G; Danis, M; Camerlynck, P; Remy, G

    1995-01-01

    To determine their prognostic and diagnostic values for toxoplasmosis in immunodepressed subjects, we assayed immunoglobulin A (IgA) and IgE antibodies by means of immunocapture (IC) tests, with revelation done by using a suspension of T. gondii (ICT). We also carried out a simultaneous analytical study of IgG antibodies on cellulose acetate membranes by using the comparative immunological profile method and an enzyme-linked immunofiltration assay (ELIFA). A total of 1,238 samples (serum, cerebrospinal fluid, and aqueous humor from 318 patients) were tested. IgA and IgE antibodies were detected in all heart, kidney, and liver transplant recipients with clinical manifestations of toxoplasmosis; IgA was detected in the aqueous humor of a patient with chorioretinitis. In patients with AIDS-related toxoplasmosis, including the cerebral form, IgA and IgE antibodies or a significant modification of ELIFA IgG values were observed in 38, 19, and 25% of patients, respectively. IgM was detected by ICT only in 12% of patients and aided the diagnosis in 1 of 71 patients. IC tests for specific IgA and IgE alone and combined with ELIFA were positive in 39 and 46% of patients who developed clinical toxoplasmosis, respectively. In a serial study of 16 patients in whom at least one of these three tests was positive, a significant immunological signal sometimes preceded clinical onset by 1, 6, and even 17 months. Similarly, in a group of human immunodeficiency virus-infected patients with evidence of previous exposure to T. gondii but no clinical manifestations, IgA, IgE, and IgA and/or IgE antibodies were detected in only 11, 4, and 12% of patients, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7790453

  15. Lateral flow assays

    PubMed Central

    Koczula, Katarzyna M.

    2016-01-01

    Lateral flow assays (LFAs) are the technology behind low-cost, simple, rapid and portable detection devices popular in biomedicine, agriculture, food and environmental sciences. This review presents an overview of the principle of the method and the critical components of the assay, focusing on lateral flow immunoassays. This type of assay has recently attracted considerable interest because of its potential to provide instantaneous diagnosis directly to patients. The range and interpretation of results and parameters used for evaluation of the assay will also be discussed. The main advantages and disadvantages of LFAs will be summarized and relevant future improvements to testing devices and strategies will be proposed. Finally, the major recent advances and future diagnostic applications in the LFA field will be explored. PMID:27365041

  16. Competitive Protein-binding assay-based Enzyme-immunoassay Method, Compared to High-pressure Liquid Chromatography, Has a Very Lower Diagnostic Value to Detect Vitamin D Deficiency in 9–12 Years Children

    PubMed Central

    Zahedi Rad, Maliheh; Neyestani, Tirang Reza; Nikooyeh, Bahareh; Shariatzadeh, Nastaran; Kalayi, Ali; Khalaji, Niloufar; Gharavi, Azam

    2015-01-01

    Background: The most reliable indicator of Vitamin D status is circulating concentration of 25-hydroxycalciferol (25(OH) D) routinely determined by enzyme-immunoassays (EIA) methods. This study was performed to compare commonly used competitive protein-binding assays (CPBA)-based EIA with the gold standard, high-pressure liquid chromatography (HPLC). Methods: Concentrations of 25(OH) D in sera from 257 randomly selected school children aged 9–11 years were determined by two methods of CPBA and HPLC. Results: Mean 25(OH) D concentration was 22 ± 18.8 and 21.9 ± 15.6 nmol/L by CPBA and HPLC, respectively. However, mean 25(OH) D concentrations of the two methods became different after excluding undetectable samples (25.1 ± 18.9 vs. 29 ± 14.5 nmol/L, respectively; P = 0.04). Based on predefined Vitamin D deficiency as 25(OH) D < 12.5 nmol/L, CPBA sensitivity and specificity were 44.2% and 60.6%, respectively, compared to HPLC. In receiver operating characteristic curve analysis, the best cut-offs for CPBA was 5.8 nmol/L, which gave 82% sensitivity, but specificity was 17%. Conclusions: Though CPBA may be used as a screening tool, more reliable methods are needed for diagnostic purposes. PMID:26330983

  17. Evaluation of the Hologic Panther Transcription-Mediated Amplification Assay for Detection of Mycoplasma genitalium

    PubMed Central

    Costa, A. M.; Su, J.; Lowe, P.; Bradshaw, C. S.; Fairley, C. K.; Garland, S. M.

    2016-01-01

    The detection of Mycoplasma genitalium was evaluated on 1,080 urine samples by the use of a Panther instrument. Overall sensitivity, specificity, positive predictive values, and negative predictive values were 100%, 99.4%, 93.6%, and 100%, respectively. Detection of M. genitalium by the use of the Panther transcription-mediated amplification assay offers a simple, accurate, and sensitive platform for diagnostic laboratories. PMID:27307453

  18. Cell viability assays: introduction.

    PubMed

    Stoddart, Martin J

    2011-01-01

    The measurement of cell viability plays a fundamental role in all forms of cell culture. Sometimes it is the main purpose of the experiment, such as in toxicity assays. Alternatively, cell viability can be used to -correlate cell behaviour to cell number, providing a more accurate picture of, for example, anabolic -activity. There are wide arrays of cell viability methods which range from the most routine trypan blue dye exclusion assay to highly complex analysis of individual cells, such as using RAMAN microscopy. The cost, speed, and complexity of equipment required will all play a role in determining the assay used. This chapter aims to provide an overview of many of the assays available today.

  19. Topoisomerase Assays

    PubMed Central

    Nitiss, John L.; Soans, Eroica; Rogojina, Anna; Seth, Aman; Mishina, Margarita

    2012-01-01

    Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: type I, which makes single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. The protocols described in this unit are of assays used to assess new chemical entities for their ability to inhibit both forms of DNA topoisomerase. Included are an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA and an assay for topoisomerase II based on the decatenation of double-stranded DNA. The preparation of mammalian cell extracts for assaying topoisomerase activity is described, along with a protocol for an ICE assay for examining topoisomerase covalent complexes in vivo and an assay for measuring DNA cleavage in vitro. PMID:22684721

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

    PubMed Central

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

    2016-01-01

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

  1. Highly accurate boronimeter assay of concentrated boric acid solutions

    SciTech Connect

    Ball, R.M. )

    1992-01-01

    The Random-Walk Boronimeter has successfully been used as an on-line indicator of boric acid concentration in an operating commercial pressurized water reactor. The principle has been adapted for measurement of discrete samples to high accuracy and to concentrations up to 6000 ppm natural boron in light water. Boric acid concentration in an aqueous solution is a necessary measurement in many nuclear power plants, particularly those that use boric acid dissolved in the reactor coolant as a reactivity control system. Other nuclear plants use a high-concentration boric acid solution as a backup shutdown system. Such a shutdown system depends on rapid injection of the solution and frequent surveillance of the fluid to ensure the presence of the neutron absorber. The two methods typically used to measure boric acid are the chemical and the physical methods. The chemical method uses titration to determine the ionic concentration of the BO[sub 3] ions and infers the boron concentration. The physical method uses the attenuation of neutrons by the solution and infers the boron concentration from the neutron absorption properties. This paper describes the Random-Walk Boronimeter configured to measure discrete samples to high accuracy and high concentration.

  2. Simple, rapid and accurate molecular diagnosis of acute promyelocytic leukemia by loop mediated amplification technology.

    PubMed

    Spinelli, Orietta; Rambaldi, Alessandro; Rigo, Francesca; Zanghì, Pamela; D'Agostini, Elena; Amicarelli, Giulia; Colotta, Francesco; Divona, Mariadomenica; Ciardi, Claudia; Coco, Francesco Lo; Minnucci, Giulia

    2015-01-01

    The diagnostic work-up of acute promyelocytic leukemia (APL) includes the cytogenetic demonstration of the t(15;17) translocation and/or the PML-RARA chimeric transcript by RQ-PCR or RT-PCR. This latter assays provide suitable results in 3-6 hours. We describe here two new, rapid and specific assays that detect PML-RARA transcripts, based on the RT-QLAMP (Reverse Transcription-Quenching Loop-mediated Isothermal Amplification) technology in which RNA retrotranscription and cDNA amplification are carried out in a single tube with one enzyme at one temperature, in fluorescence and real time format. A single tube triplex assay detects bcr1 and bcr3 PML-RARA transcripts along with GUS housekeeping gene. A single tube duplex assay detects bcr2 and GUSB. In 73 APL cases, these assays detected in 16 minutes bcr1, bcr2 and bcr3 transcripts. All 81 non-APL samples were negative by RT-QLAMP for chimeric transcripts whereas GUSB was detectable. In 11 APL patients in which RT-PCR yielded equivocal breakpoint type results, RT-QLAMP assays unequivocally and accurately defined the breakpoint type (as confirmed by sequencing). Furthermore, RT-QLAMP could amplify two bcr2 transcripts with particularly extended PML exon 6 deletions not amplified by RQ-PCR. RT-QLAMP reproducible sensitivity is 10(-3) for bcr1 and bcr3 and 10(-)2 for bcr2 thus making this assay particularly attractive at diagnosis and leaving RQ-PCR for the molecular monitoring of minimal residual disease during the follow up. In conclusion, PML-RARA RT-QLAMP compared to RT-PCR or RQ-PCR is a valid improvement to perform rapid, simple and accurate molecular diagnosis of APL.

  3. Simple, rapid and accurate molecular diagnosis of acute promyelocytic leukemia by loop mediated amplification technology

    PubMed Central

    Spinelli, Orietta; Rambaldi, Alessandro; Rigo, Francesca; Zanghì, Pamela; D'Agostini, Elena; Amicarelli, Giulia; Colotta, Francesco; Divona, Mariadomenica; Ciardi, Claudia; Coco, Francesco Lo; Minnucci, Giulia

    2015-01-01

    The diagnostic work-up of acute promyelocytic leukemia (APL) includes the cytogenetic demonstration of the t(15;17) translocation and/or the PML-RARA chimeric transcript by RQ-PCR or RT-PCR. This latter assays provide suitable results in 3-6 hours. We describe here two new, rapid and specific assays that detect PML-RARA transcripts, based on the RT-QLAMP (Reverse Transcription-Quenching Loop-mediated Isothermal Amplification) technology in which RNA retrotranscription and cDNA amplification are carried out in a single tube with one enzyme at one temperature, in fluorescence and real time format. A single tube triplex assay detects bcr1 and bcr3 PML-RARA transcripts along with GUS housekeeping gene. A single tube duplex assay detects bcr2 and GUSB. In 73 APL cases, these assays detected in 16 minutes bcr1, bcr2 and bcr3 transcripts. All 81 non-APL samples were negative by RT-QLAMP for chimeric transcripts whereas GUSB was detectable. In 11 APL patients in which RT-PCR yielded equivocal breakpoint type results, RT-QLAMP assays unequivocally and accurately defined the breakpoint type (as confirmed by sequencing). Furthermore, RT-QLAMP could amplify two bcr2 transcripts with particularly extended PML exon 6 deletions not amplified by RQ-PCR. RT-QLAMP reproducible sensitivity is 10−3 for bcr1 and bcr3 and 10−2 for bcr2 thus making this assay particularly attractive at diagnosis and leaving RQ-PCR for the molecular monitoring of minimal residual disease during the follow up. In conclusion, PML-RARA RT-QLAMP compared to RT-PCR or RQ-PCR is a valid improvement to perform rapid, simple and accurate molecular diagnosis of APL. PMID:25815362

  4. The diagnostic performance of an antibody enzyme-linked immunosorbent assay using serum and colostrum to determine the disease status of a Jersey dairy herd infected with Mycobacterium avium subspecies paratuberculosis.

    PubMed

    Jenvey, Caitlin J; Reichel, Michael P; Cockcroft, Peter D

    2016-01-01

    Colostrum may have the ability to improve the diagnostic accuracy of some tests when compared to serum for important livestock diseases because of the high concentrations of immunoglobulins present within this sample type. The ELISA for Johne's disease is one such test, as it suffers from low sensitivity when testing serum samples collected during the subclinical stage of infection. Blood and colostrum samples were collected from 34 Jersey dairy cows and tested for antibodies against Mycobacterium avium subspecies paratuberculosis (MAP) by ELISA. Fecal samples were also collected and tested by a high-throughput Johne's polymerase chain reaction (HT-J PCR) assay and fecal culture (FC), with the latter being used as the reference test. A receiver operating characteristic (ROC) analysis was performed, and the area under the curve (AUC) was calculated. The HT-J PCR and FC results were also compared. Of the 34 cows in this study, 4 had FC results consistent with MAP infection. The HT-J PCR did not identify any FC-positive cows. Using a 1:20 dilution and sample-to-positive (S/P) ratio cutoff threshold of 0.15, the relative sensitivity values of both serum (AUC 0. 56) and colostrum (AUC 0.63) were 0%. With lower sample dilutions, the relative sensitivity values of serum were 0% (1:2, AUC 0.62; 1:5, AUC 0.55); however, the relative sensitivity value of colostrum was 75% (95% confidence interval [CI]: 19-99%) at a dilution of 1:5, S/P ratio cutoff threshold of 0.15, and AUC of 0.73 (95% CI: 0.55-0.87). The testing of colostrum samples for MAP-specific antibodies by ELISA may provide improved identification of animals in the early stages of infection with MAP when compared with serum samples.

  5. Novel multitarget real-time PCR assay for rapid detection of Bordetella species in clinical specimens.

    PubMed

    Tatti, Kathleen M; Sparks, Kansas N; Boney, Kathryn O; Tondella, Maria Lucia

    2011-12-01

    A novel multitarget real-time PCR (RT-PCR) assay for the rapid identification of Bordetella pertussis, B. parapertussis, and B. holmesii was developed using multicopy insertion sequences (ISs) in combination with the pertussis toxin subunit S1 (ptxS1) singleplex assay. The RT-PCR targets for the multiplex assay include IS481, commonly found in B. pertussis and B. holmesii; IS1001 of B. parapertussis; and the IS1001-like sequence of B. holmesii. Overall, 402 Bordetella species and 66 non-Bordetella species isolates were tested in the multitarget assay. Cross-reactivity was found only with 5 B. bronchiseptica isolates, which were positive with IS1001 of B. parapertussis. The lower limit of detection (LLOD) of the multiplex assay was similar to the LLOD of each target in an individual assay format, which was approximately 1 genomic equivalent per reaction for all targets. A total of 197 human clinical specimens obtained during cough-illness outbreak investigations were used to evaluate the multitarget RT-PCR assay. The multiplex assay results from 87 clinical specimens were compared to the individual RT-PCR assay and culture results. The multitarget assay is useful as a diagnostic tool to confirm B. pertussis infections and to rapidly identify other Bordetella species. In conclusion, the use of this multitarget RT-PCR approach increases specificity, while it decreases the amount of time, reagents, and specimen necessary for RT-PCRs used for accurate diagnosis of pertussis-like illness.

  6. Fluorometric assay for aflatoxins

    SciTech Connect

    Chakrabarti, A.G.

    1984-11-01

    The method that is now widely adopted by the government laboratories for the assay of individual aflatoxin components (B/sub 1/, B/sub 2/, G/sub 1/, and G/sub 2/) utilizes a TLC technique. The extraction and clean-up steps of this technique were further researched but the method is still time consuming. It is, therefore, very important to develop a rapid and accurate assay technique for aflatoxins. The current research proposes a technique which utilizes a Turner Fluorometer.

  7. Multiplex Flow Assays

    PubMed Central

    2016-01-01

    Lateral flow or dipstick assays (e.g., home pregnancy tests), where an analyte solution is drawn through a porous membrane and is detected by localization onto a capture probe residing at a specific site on the flow strip, are the most commonly and extensively used type of diagnostic assay. However, after over 30 years of use, these assays are constrained to measuring one or a few analytes at a time. Here, we describe a completely general method, in which any single-plex lateral flow assay is transformed into a multiplex assay capable of measuring an arbitrarily large number of analytes simultaneously. Instead of identifying the analyte by its localization onto a specific geometric location in the flow medium, the analyte-specific capture probe is identified by its association with a specific optically encoded region within the flow medium. The capture probes for nucleic acids, antigens, or antibodies are attached to highly porous agarose beads, which have been encoded using multiple lanthanide emitters to create a unique optical signature for each capture probe. The optically encoded capture probe-derivatized beads are placed in contact with the analyte-containing porous flow medium and the analytes are captured onto the encoded regions as the solution flows through the porous medium. To perform a multiplex diagnostic assay, a solution comprising multiple analytes is passed through the flow medium containing the capture probe-derivatized beads, and the captured analyte is treated with a suitable fluorescent reporter. We demonstrate this multiplex analysis technique by simultaneously measuring DNA samples, antigen–antibody pairs, and mixtures of multiple nucleic acids and antibodies. PMID:27819063

  8. ROM Plus®: accurate point-of-care detection of ruptured fetal membranes

    PubMed Central

    McQuivey, Ross W; Block, Jon E

    2016-01-01

    Accurate and timely diagnosis of rupture of fetal membranes is imperative to inform and guide gestational age-specific interventions to optimize perinatal outcomes and reduce the risk of serious complications, including preterm delivery and infections. The ROM Plus is a rapid, point-of-care, qualitative immunochromatographic diagnostic test that uses a unique monoclonal/polyclonal antibody approach to detect two different proteins found in amniotic fluid at high concentrations: alpha-fetoprotein and insulin-like growth factor binding protein-1. Clinical study results have uniformly demonstrated high diagnostic accuracy and performance characteristics with this point-of-care test that exceeds conventional clinical testing with external laboratory evaluation. The description, indications for use, procedural steps, and laboratory and clinical characterization of this assay are presented in this article. PMID:27274316

  9. ROM Plus(®): accurate point-of-care detection of ruptured fetal membranes.

    PubMed

    McQuivey, Ross W; Block, Jon E

    2016-01-01

    Accurate and timely diagnosis of rupture of fetal membranes is imperative to inform and guide gestational age-specific interventions to optimize perinatal outcomes and reduce the risk of serious complications, including preterm delivery and infections. The ROM Plus is a rapid, point-of-care, qualitative immunochromatographic diagnostic test that uses a unique monoclonal/polyclonal antibody approach to detect two different proteins found in amniotic fluid at high concentrations: alpha-fetoprotein and insulin-like growth factor binding protein-1. Clinical study results have uniformly demonstrated high diagnostic accuracy and performance characteristics with this point-of-care test that exceeds conventional clinical testing with external laboratory evaluation. The description, indications for use, procedural steps, and laboratory and clinical characterization of this assay are presented in this article.

  10. Development of real-time PCR assays for the detection of Moraxella macacae associated with bloody nose syndrome in rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques

    PubMed Central

    Whitehouse, Chris A.; Chase, Kitty; Embers, Monica E.; Kulesh, David A.; Ladner, Jason T.; Palacios, Gustavo F.; Minogue, Timothy D.

    2016-01-01

    Background Moraxella macacae is a recently described bacterial pathogen that causes epistaxis or so-called bloody nose syndrome in captive macaques. The aim of this study was to develop specific molecular diagnostic assays for M. macacae and to determine their performance characteristics. Methods We developed six real-time PCR assays on the Roche LightCycler. The accuracy, precision, selectivity, and limit of detection (LOD) were determined for each assay, in addition to further validation by testing nasal swabs from macaques presenting with epistaxis at the Tulane National Primate Research Center. Results All assays exhibited 100% specificity and were highly sensitive with an LOD of 10 fg for chromosomal assays and 1 fg for the plasmid assay. Testing of nasal swabs from 10 symptomatic macaques confirmed the presence of M. macacae in these animals. Conclusions We developed several accurate, sensitive, and species-specific real-time PCR assays for the detection of M. macacae in captive macaques. PMID:26365904

  11. Enzyme assays.

    PubMed

    Reymond, Jean-Louis; Fluxà, Viviana S; Maillard, Noélie

    2009-01-07

    Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest.

  12. CTL ELISPOT assay.

    PubMed

    Ranieri, Elena; Popescu, Iulia; Gigante, Margherita

    2014-01-01

    Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T cell (CTL) studies have taken advantage with this high-throughput technology by providing insights into quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T cells is of relevance for immune diagnostic. The most utilized Elispot assay is the interferon-gamma (IFN-γ) test, a marker for CD8(+) CTL activation, but Elispot can also be used to distinguish different subsets of activated T cells by using other cytokines such as T-helper (Th) 1-type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21, and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10, and IL-13), and Th17 (IL-17) cells. The reliability of Elispot-generated data, by the evaluation of T cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Miltenyi cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method

  13. Saliva-Based Biosensors: Noninvasive Monitoring Tool for Clinical Diagnostics

    PubMed Central

    Malon, Radha S. P.; Balakrishnan, Malarvili; Córcoles, Emma P.

    2014-01-01

    Saliva is increasingly recognised as an attractive diagnostic fluid. The presence of various disease signalling salivary biomarkers that accurately reflect normal and disease states in humans and the sampling benefits compared to blood sampling are some of the reasons for this recognition. This explains the burgeoning research field in assay developments and technological advancements for the detection of various salivary biomarkers to improve clinical diagnosis, management, and treatment. This paper reviews the significance of salivary biomarkers for clinical diagnosis and therapeutic applications, with focus on the technologies and biosensing platforms that have been reported for screening these biomarkers. PMID:25276835

  14. Integrated diagnostics

    NASA Technical Reports Server (NTRS)

    Hunthausen, Roger J.

    1988-01-01

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

  15. Development of companion diagnostics

    SciTech Connect

    Mankoff, David A.; Edmonds, Christine E.; Farwell, Michael D.; Pryma, Daniel A.

    2015-12-12

    The goal of individualized and targeted treatment and precision medicine requires the assessment of potential therapeutic targets to direct treatment selection. The biomarkers used to direct precision medicine, often termed companion diagnostics, for highly targeted drugs have thus far been almost entirely based on in vitro assay of biopsy material. Molecular imaging companion diagnostics offer a number of features complementary to those from in vitro assay, including the ability to measure the heterogeneity of each patient’s cancer across the entire disease burden and to measure early changes in response to treatment. We discuss the use of molecular imaging methods as companion diagnostics for cancer therapy with the goal of predicting response to targeted therapy and measuring early (pharmacodynamic) response as an indication of whether the treatment has “hit” the target. We also discuss considerations for probe development for molecular imaging companion diagnostics, including both small-molecule probes and larger molecules such as labeled antibodies and related constructs. We then describe two examples where both predictive and pharmacodynamic molecular imaging markers have been tested in humans: endocrine therapy for breast cancer and human epidermal growth factor receptor type 2–targeted therapy. Lastly, the review closes with a summary of the items needed to move molecular imaging companion diagnostics from early studies into multicenter trials and into the clinic.

  16. Development of companion diagnostics

    DOE PAGES

    Mankoff, David A.; Edmonds, Christine E.; Farwell, Michael D.; ...

    2015-12-12

    The goal of individualized and targeted treatment and precision medicine requires the assessment of potential therapeutic targets to direct treatment selection. The biomarkers used to direct precision medicine, often termed companion diagnostics, for highly targeted drugs have thus far been almost entirely based on in vitro assay of biopsy material. Molecular imaging companion diagnostics offer a number of features complementary to those from in vitro assay, including the ability to measure the heterogeneity of each patient’s cancer across the entire disease burden and to measure early changes in response to treatment. We discuss the use of molecular imaging methods asmore » companion diagnostics for cancer therapy with the goal of predicting response to targeted therapy and measuring early (pharmacodynamic) response as an indication of whether the treatment has “hit” the target. We also discuss considerations for probe development for molecular imaging companion diagnostics, including both small-molecule probes and larger molecules such as labeled antibodies and related constructs. We then describe two examples where both predictive and pharmacodynamic molecular imaging markers have been tested in humans: endocrine therapy for breast cancer and human epidermal growth factor receptor type 2–targeted therapy. Lastly, the review closes with a summary of the items needed to move molecular imaging companion diagnostics from early studies into multicenter trials and into the clinic.« less

  17. Development of Companion Diagnostics.

    PubMed

    Mankoff, David A; Edmonds, Christine E; Farwell, Michael D; Pryma, Daniel A

    2016-01-01

    The goal of individualized and targeted treatment and precision medicine requires the assessment of potential therapeutic targets to direct treatment selection. The biomarkers used to direct precision medicine, often termed companion diagnostics, for highly targeted drugs have thus far been almost entirely based on in vitro assay of biopsy material. Molecular imaging companion diagnostics offer a number of features complementary to those from in vitro assay, including the ability to measure the heterogeneity of each patient's cancer across the entire disease burden and to measure early changes in response to treatment. We discuss the use of molecular imaging methods as companion diagnostics for cancer therapy with the goal of predicting response to targeted therapy and measuring early (pharmacodynamic) response as an indication of whether the treatment has "hit" the target. We also discuss considerations for probe development for molecular imaging companion diagnostics, including both small-molecule probes and larger molecules such as labeled antibodies and related constructs. We then describe two examples where both predictive and pharmacodynamic molecular imaging markers have been tested in humans: endocrine therapy for breast cancer and human epidermal growth factor receptor type 2-targeted therapy. The review closes with a summary of the items needed to move molecular imaging companion diagnostics from early studies into multicenter trials and into the clinic.

  18. Accurate Finite Difference Algorithms

    NASA Technical Reports Server (NTRS)

    Goodrich, John W.

    1996-01-01

    Two families of finite difference algorithms for computational aeroacoustics are presented and compared. All of the algorithms are single step explicit methods, they have the same order of accuracy in both space and time, with examples up to eleventh order, and they have multidimensional extensions. One of the algorithm families has spectral like high resolution. Propagation with high order and high resolution algorithms can produce accurate results after O(10(exp 6)) periods of propagation with eight grid points per wavelength.

  19. Accurate monotone cubic interpolation

    NASA Technical Reports Server (NTRS)

    Huynh, Hung T.

    1991-01-01

    Monotone piecewise cubic interpolants are simple and effective. They are generally third-order accurate, except near strict local extrema where accuracy degenerates to second-order due to the monotonicity constraint. Algorithms for piecewise cubic interpolants, which preserve monotonicity as well as uniform third and fourth-order accuracy are presented. The gain of accuracy is obtained by relaxing the monotonicity constraint in a geometric framework in which the median function plays a crucial role.

  20. Microfluidic diagnostics for low-resource settings

    NASA Astrophysics Data System (ADS)

    Hawkins, Kenneth R.; Weigl, Bernhard H.

    2010-02-01

    Diagnostics for low-resource settings need to be foremost inexpensive, but also accurate, reliable, rugged and suited to the contexts of the developing world. Diagnostics for global health, based on minimally-instrumented, microfluidicsbased platforms employing low-cost disposables, has become a very active research area recently-thanks, in part, to new funding from the Bill & Melinda Gates Foundation, the National Institutes of Health, and other sources. This has led to a number of interesting prototype devices that are now in advanced development or clinical validation. These devices include disposables and instruments that perform multiplexed PCR-based assays for enteric, febrile, and vaginal diseases, as well as immunoassays for diseases such as malaria, HIV, and various sexually transmitted diseases. More recently, instrument-free diagnostic disposables based on isothermal nucleic-acid amplification have been developed. Regardless of platform, however, the search for truly low-cost manufacturing methods that would enable affordable systems (at volume, in the appropriate context) remains a significant challenge. Here we give an overview of existing platform development efforts, present some original research in this area at PATH, and reiterate a call to action for more.

  1. Diagnostic challenges for multiplexed protein microarrays.

    PubMed

    Master, Stephen R; Bierl, Charlene; Kricka, Larry J

    2006-11-01

    Multiplexed protein analysis using planar microarrays or microbeads is growing in popularity for simultaneous assays of antibodies, cytokines, allergens, drugs and hormones. However, this new assay format presents several new operational issues for the clinical laboratory, such as the quality control of protein-microarray-based assays, the release of unrequested test data and the use of diagnostic algorithms to transform microarray data into diagnostic results.

  2. Diagnostic and vaccine chapter.

    PubMed

    Wolfram, J H; Kokanov, S K; Verkhovsky, O A

    2010-10-01

    The first report in this chapter describes the development of a killed composite vaccine. This killed vaccine is non-infectious to humans, other animals, and the environment. The vaccine has low reactivity, is non-abortive, and does not induce pathomorphological alterations to the organs of vaccinated animals. The second report of this chapter describes the diagnostic value of a competitive enzyme-linked immunosorbent assay for detecting Brucella-specific antibodies and its ability to discriminate vaccinated cattle from infected cattle. The results indicated that the competitive enzyme-linked immunosorbent assay is more sensitive than traditional tests for detecting antibodies to Brucella abortus in naturally and experimentally infected cattle.

  3. A multi-analyte assay for the non-invasive detection of bladder cancer.

    PubMed

    Goodison, Steve; Chang, Myron; Dai, Yunfeng; Urquidi, Virginia; Rosser, Charles J

    2012-01-01

    Accurate urinary assays for bladder cancer (BCa) detection would benefit both patients and healthcare systems. Through genomic and proteomic profiling of urine components, we have previously identified a panel of biomarkers that can outperform current urine-based biomarkers for the non-invasive detection of BCa. Herein, we report the diagnostic utility of various multivariate combinations of these biomarkers. We performed a case-controlled validation study in which voided urines from 127 patients (64 tumor bearing subjects) were analyzed. The urinary concentrations of 14 biomarkers (IL-8, MMP-9, MMP-10, SDC1, CCL18, PAI-1, CD44, VEGF, ANG, CA9, A1AT, OPN, PTX3, and APOE) were assessed by enzyme-linked immunosorbent assay (ELISA). Diagnostic performance of each biomarker and multivariate models were compared using receiver operating characteristic curves and the chi-square test. An 8-biomarker model achieved the most accurate BCa diagnosis (sensitivity 92%, specificity 97%), but a combination of 3 of the 8 biomarkers (IL-8, VEGF, and APOE) was also highly accurate (sensitivity 90%, specificity 97%). For comparison, the commercial BTA-Trak ELISA test achieved a sensitivity of 79% and a specificity of 83%, and voided urine cytology detected only 33% of BCa cases in the same cohort. These data show that a multivariate urine-based assay can markedly improve the accuracy of non-invasive BCa detection. Further validation studies are under way to investigate the clinical utility of this panel of biomarkers for BCa diagnosis and disease monitoring.

  4. Pre-Clinical Evaluation of a Real-Time PCR Assay on a Portable Instrument as a Possible Field Diagnostic Tool: Experiences from the Testing of Clinical Samples for African and Classical Swine Fever Viruses.

    PubMed

    Liu, L; Luo, Y; Accensi, F; Ganges, L; Rodríguez, F; Shan, H; Ståhl, K; Qiu, H-J; Belák, S

    2016-06-16

    African swine fever (ASF) and classical swine fever (CSF) are two highly infectious transboundary animal diseases (TADs) that are serious threats to the pig industry worldwide, including in China, the world's largest pork producer. In this study, a duplex real-time PCR assay was developed for the rapid detection and differentiation of African swine fever virus (ASFV) and classical swine fever virus (CSFV). The assay was performed on a portable, battery-powered PCR thermocycler with a low sample throughput (termed as 'T-COR4 assay'). The feasibility and reliability of the T-COR4 assay as a possible field method was investigated by testing clinical samples collected in China. When evaluated with reference materials or samples from experimental infections, the assay performed in a reliable manner, producing results comparable to those obtained from stationary PCR platforms. Of 59 clinical samples, 41 had results identical to a two-step CSFV real-time PCR assay. No ASFV was detected in these samples. The T-COR4 assay was technically easy to perform and produced results within 3 h, including sample preparation. In combination with a simple sample preparation method, the T-COR4 assay provides a new tool for the field diagnosis and differentiation of ASF and CSF, which could be of particular value in remote areas.

  5. Diagnostic hematology of reptiles.

    PubMed

    Stacy, Nicole I; Alleman, A Rick; Sayler, Katherine A

    2011-03-01

    The hematologic evaluation of reptiles is an indispensable diagnostic tool in exotic veterinary practice. The diversity of reptile species, their characteristic physiologic features, and effects of intrinsic and extrinsic factors present unique challenges for accurate interpretation of the hemogram. Combining the clinical presentation with hematologic findings provides valuable information in the diagnosis and monitoring of disease and helps guide the clinician toward therapy and further diagnostic testing. This article outlines the normal and pathologic morphology of blood cells of reptile species. The specific comparative aspects of reptiles are emphasized, and structural and functional abnormalities in the reptilian hemogram are described.

  6. Diagnostic and prognostic epigenetic biomarkers in cancer.

    PubMed

    Costa-Pinheiro, Pedro; Montezuma, Diana; Henrique, Rui; Jerónimo, Carmen

    2015-01-01

    Growing cancer incidence and mortality worldwide demands development of accurate biomarkers to perfect detection, diagnosis, prognostication and monitoring. Urologic (prostate, bladder, kidney), lung, breast and colorectal cancers are the most common and despite major advances in their characterization, this has seldom translated into biomarkers amenable for clinical practice. Epigenetic alterations are innovative cancer biomarkers owing to stability, frequency, reversibility and accessibility in body fluids, entailing great potential of assay development to assist in patient management. Several studies identified putative epigenetic cancer biomarkers, some of which have been commercialized. However, large multicenter validation studies are required to foster translation to the clinics. Herein we review the most promising epigenetic detection, diagnostic, prognostic and predictive biomarkers for the most common cancers.

  7. LAMP Detection Assays for Boxwood Blight Pathogens: A Comparative Genomics Approach

    PubMed Central

    Malapi-Wight, Martha; Demers, Jill E.; Veltri, Daniel; Marra, Robert E.; Crouch, Jo Anne

    2016-01-01

    Rapid and accurate molecular diagnostic tools are critical to efforts to minimize the impact and spread of emergent pathogens. The identification of diagnostic markers for novel pathogens presents several challenges, especially in the absence of information about population diversity and where genetic resources are limited. The objective of this study was to use comparative genomics datasets to find unique target regions suitable for the diagnosis of two fungal species causing a newly emergent blight disease of boxwood. Candidate marker regions for loop-mediated isothermal amplification (LAMP) assays were identified from draft genomes of Calonectria henricotiae and C. pseudonaviculata, as well as three related species not associated with this disease. To increase the probability of identifying unique targets, we used three approaches to mine genome datasets, based on (i) unique regions, (ii) polymorphisms, and (iii) presence/absence of regions across datasets. From a pool of candidate markers, we demonstrate LAMP assay specificity by testing related fungal species, common boxwood pathogens, and environmental samples containing 445 diverse fungal taxa. This comparative-genomics-based approach to the development of LAMP diagnostic assays is the first of its kind for fungi and could be easily applied to diagnostic marker development for other newly emergent plant pathogens. PMID:27199028

  8. LAMP detection assays for boxwood blight pathogens: A comparative genomics approach

    SciTech Connect

    Malapi-Wight, Martha; Demers, Jill E.; Veltri, Daniel; Marra, Robert E.; Crouch, Jo Anne

    2016-05-20

    Rapid and accurate molecular diagnostic tools are critical to efforts to minimize the impact and spread of emergent pathogens. The identification of diagnostic markers for novel pathogens presents several challenges, especially in the absence of information about population diversity and where genetic resources are limited. The objective of this study was to use comparative genomics datasets to find unique target regions suitable for the diagnosis of two fungal species causing a newly emergent blight disease of boxwood. Candidate marker regions for loop-mediated isothermal amplification (LAMP) assays were identified from draft genomes of Calonectria henricotiae and C. pseudonaviculata, as well as three related species not associated with this disease. To increase the probability of identifying unique targets, we used three approaches to mine genome datasets, based on (i) unique regions, (ii) polymorphisms, and (iii) presence/absence of regions across datasets. From a pool of candidate markers, we demonstrate LAMP assay specificity by testing related fungal species, common boxwood pathogens, and environmental samples containing 445 diverse fungal taxa. In conclusion, this comparative-genomics-based approach to the development of LAMP diagnostic assays is the first of its kind for fungi and could be easily applied to diagnostic marker development for other newly emergent plant pathogens.

  9. LAMP detection assays for boxwood blight pathogens: A comparative genomics approach

    DOE PAGES

    Malapi-Wight, Martha; Demers, Jill E.; Veltri, Daniel; ...

    2016-05-20

    Rapid and accurate molecular diagnostic tools are critical to efforts to minimize the impact and spread of emergent pathogens. The identification of diagnostic markers for novel pathogens presents several challenges, especially in the absence of information about population diversity and where genetic resources are limited. The objective of this study was to use comparative genomics datasets to find unique target regions suitable for the diagnosis of two fungal species causing a newly emergent blight disease of boxwood. Candidate marker regions for loop-mediated isothermal amplification (LAMP) assays were identified from draft genomes of Calonectria henricotiae and C. pseudonaviculata, as well asmore » three related species not associated with this disease. To increase the probability of identifying unique targets, we used three approaches to mine genome datasets, based on (i) unique regions, (ii) polymorphisms, and (iii) presence/absence of regions across datasets. From a pool of candidate markers, we demonstrate LAMP assay specificity by testing related fungal species, common boxwood pathogens, and environmental samples containing 445 diverse fungal taxa. In conclusion, this comparative-genomics-based approach to the development of LAMP diagnostic assays is the first of its kind for fungi and could be easily applied to diagnostic marker development for other newly emergent plant pathogens.« less

  10. Companion diagnostics and molecular imaging-enhanced approaches for oncology clinical trials

    PubMed Central

    Van Heertum, Ronald L; Scarimbolo, Robert; Ford, Robert; Berdougo, Eli; O’Neal, Michael

    2015-01-01

    In the era of personalized medicine, diagnostic approaches are helping pharmaceutical and biotechnology sponsors streamline the clinical trial process. Molecular assays and diagnostic imaging are routinely being used to stratify patients for treatment, monitor disease, and provide reliable early clinical phase assessments. The importance of diagnostic approaches in drug development is highlighted by the rapidly expanding global cancer diagnostics market and the emergent attention of regulatory agencies worldwide, who are beginning to offer more structured platforms and guidance for this area. In this paper, we highlight the key benefits of using companion diagnostics and diagnostic imaging with a focus on oncology clinical trials. Nuclear imaging using widely available radiopharmaceuticals in conjunction with molecular imaging of oncology targets has opened the door to more accurate disease assessment and the modernization of standard criteria for the evaluation, staging, and treatment responses of cancer patients. Furthermore, the introduction and validation of quantitative molecular imaging continues to drive and optimize the field of oncology diagnostics. Given their pivotal role in disease assessment and treatment, the validation and commercialization of diagnostic tools will continue to advance oncology clinical trials, support new oncology drugs, and promote better patient outcomes. PMID:26392755

  11. Companion diagnostics and molecular imaging-enhanced approaches for oncology clinical trials.

    PubMed

    Van Heertum, Ronald L; Scarimbolo, Robert; Ford, Robert; Berdougo, Eli; O'Neal, Michael

    2015-01-01

    In the era of personalized medicine, diagnostic approaches are helping pharmaceutical and biotechnology sponsors streamline the clinical trial process. Molecular assays and diagnostic imaging are routinely being used to stratify patients for treatment, monitor disease, and provide reliable early clinical phase assessments. The importance of diagnostic approaches in drug development is highlighted by the rapidly expanding global cancer diagnostics market and the emergent attention of regulatory agencies worldwide, who are beginning to offer more structured platforms and guidance for this area. In this paper, we highlight the key benefits of using companion diagnostics and diagnostic imaging with a focus on oncology clinical trials. Nuclear imaging using widely available radiopharmaceuticals in conjunction with molecular imaging of oncology targets has opened the door to more accurate disease assessment and the modernization of standard criteria for the evaluation, staging, and treatment responses of cancer patients. Furthermore, the introduction and validation of quantitative molecular imaging continues to drive and optimize the field of oncology diagnostics. Given their pivotal role in disease assessment and treatment, the validation and commercialization of diagnostic tools will continue to advance oncology clinical trials, support new oncology drugs, and promote better patient outcomes.

  12. Diagnostic application of H3N8-specific equine influenza real-time reverse transcription polymerase chain reaction assays for the detection of Canine influenza virus in clinical specimens.

    PubMed

    Lu, Zhengchun; Dubovi, Edward J; Zylich, Nancy C; Crawford, P Cynda; Sells, Stephen; Go, Yun Young; Loynachan, Alan T; Timoney, Peter J; Chambers, Thomas M; Balasuriya, Udeni B R

    2010-11-01

    The objective of the current study was to determine the capability of 3 recently described one-step TaqMan real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assays targeting the nucleoprotein (NP), matrix (M), and hemagglutinin (HA) genes of H3N8 Equine influenza virus (EIV NP, EIV M, and EIV HA3 assays, respectively) to detect Canine influenza virus (CIV). The assays were initially evaluated with nucleic acid extracted from tissue culture fluid (TCF) containing the A/canine/FL/43/04 strain of Influenza A virus associated with the 2004 canine influenza outbreak in Florida. The EIV NP, EIV M, and EIV HA3 assays could detect CIV nucleic acid at threshold cycle (Ct) values of 16.31, 23.71, and 15.28, respectively. Three assays using TCF or allantoic fluid (AF) samples containing CIV (n  =  13) and archived canine nasal swab samples (n  =  20) originally submitted for laboratory diagnosis of CIV were further evaluated. All TCF and AF samples, together with 10 nasal swab samples that previously tested positive for virus by attempted isolation in embryonated hens' eggs or Madin-Darby canine kidney cells, were positive in all 3 real-time RT-PCR assays. None of the 3 assays detected the H1N1 Swine influenza virus strain in current circulation. These findings demonstrate that previously described real-time RT-PCR assays targeting NP, M, and H3 HA gene segments of H3N8 EIV are also valuable for the diagnosis of CIV infection in dogs. The assays could expedite the detection and identification of CIV.

  13. Molecular and biological diagnostic tests for monitoring benzimidazole resistance in human soil-transmitted helminths.

    PubMed

    Diawara, Aïssatou; Schwenkenbecher, Jan M; Kaplan, Ray M; Prichard, Roger K

    2013-06-01

    In endemic countries with soil-transmitted helminths mass drug administration with albendazole or mebendazole are being implemented as a control strategy. However, it is well known in veterinary helminths that the use of the same benzimidazole drugs can place selection on the β-tubulin gene, leading to resistance. Given the concern that resistance could arise in human soil-transmitted helminths, there is an urgent need to develop accurate diagnostic tools for monitoring resistance. In this study, we developed molecular assays to detect putative resistance genetic changes in Ascaris lumbricoides, Trichuris trichiura, and hookworms, and we optimized an egg hatch assay for the canine hookworm Ancylostoma caninum and applied it to Necator americanus. Both assays were tested on field samples. The molecular assays demonstrated their reproducibility and capacity to detect the presence of worms carrying putative resistance-associated genetic changes. However, further investigations are needed to validate our molecular and biological tests on additional field isolates.

  14. Hexosaminidase assays.

    PubMed

    Wendeler, Michaela; Sandhoff, Konrad

    2009-11-01

    beta-Hexosaminidases (EC 3.2.1.52) are lysosomal enzymes that remove terminal beta-glycosidically bound N-acetylglucosamine and N-acetylgalactosamine residues from a number of glycoconjugates. Reliable assay systems are particularly important for the diagnosis of a family of lysosomal storage disorders, the GM2 gangliosidoses that result from inherited beta-hexosaminidase deficiency. More recently, aberrant hexosaminidase levels have also been found to be associated with a variety of inflammatory diseases. Apart from patient testing and carrier screening, practical in vitro assays are indispensable for the characterization of knock-out mice with potentially altered hexosaminidase activities, for detailed structure-function studies aimed at elucidating the enzymatic mechanism, and to characterize newly described enzyme variants from other organisms. The purpose of this article is to discuss convenient hexosaminidase assay procedures for these and other applications, using fluorogenic or chromogenic artificial substrates as well as the physiological glycolipid substrate GM2. Attempts are also made to provide an overview of less commonly used alternative techniques and to introduce recent developments enabling high-throughput screening for enzyme inhibitors.

  15. Development of Rapid Isothermal Amplification Assays for Detection of Phytophthora spp. in Plant Tissue.

    PubMed

    Miles, Timothy D; Martin, Frank N; Coffey, Michael D

    2015-02-01

    Several isothermal amplification techniques recently have been developed that are tolerant of inhibitors present in many plant extracts, which can reduce the need for obtaining purified DNA for running diagnostic assays. One such commercially available technique that has similarities with real-time polymerase chain reaction (PCR) for designing primers and a labeled probe is recombinase polymerase amplification (RPA). This technology was used to develop two simple and rapid approaches for detection of Phytophthora spp.: one genus-specific assay multiplexed with a plant internal control and the other species-specific assays for Phytophthora ramorum and P. kernoviae. All assays were tested for sensitivity (ranging from 3 ng to 1 fg of DNA) and specificity using DNA extracted from more than 136 Phytophthora taxa, 21 Pythium spp., 1 Phytopythium sp., and a wide range of plant species. The lower limit of linear detection using purified DNA was 200 to 300 fg of DNA in all pathogen RPA assays. Six different extraction buffers were tested for use during plant tissue maceration and the assays were validated in the field by collecting 222 symptomatic plant samples from over 50 different hosts. Only 56 samples were culture positive for Phytophthora spp. whereas 91 were positive using the Phytophthora genus-specific RPA test and a TaqMan real-time PCR assay. A technique for the generation of sequencing templates from positive RPA amplifications to confirm species identification was also developed. These RPA assays have added benefits over traditional technologies because they are rapid (results can be obtained in as little as 15 min), do not require DNA extraction or extensive training to complete, use less expensive portable equipment than PCR-based assays, and are significantly more specific than current immunologically based methods. This should provide a rapid, field-deployable capability for pathogen detection that will facilitate point-of-sample collection processing

  16. Real-Time PCR Assay for the Identification of the Brown Marmorated Stink Bug (Halyomorpha halys)

    PubMed Central

    Dhami, Manpreet K.; Dsouza, Melissa; Waite, David W.; Anderson, Diane; Li, Dongmei

    2016-01-01

    The brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae), is a gregarious crop pest that has rapidly spread across the world in the last two decades. It is an excellent hitchhiker species, especially as an over-wintering adult. During this period it is often associated with non-biological commodities such as shipping containers and machinery that travel long distances. Inadequate identification keys and similarity to common species has assisted its spread across Europe, while accurate identification from immature stages or eggs is not possible. We developed a real-time TaqMan PCR assay for the accurate and sensitive detection of the brown marmorated stink bug from all life stages. The assay performance against required diagnostic criterion and within a quarantine framework are described. PMID:26955631

  17. Multilaboratory Comparison of Quantitative PCR Assays for Detection and Quantification of Fusarium virguliforme from Soybean Roots and Soil.

    PubMed

    Kandel, Yuba R; Haudenshield, James S; Srour, Ali Y; Islam, Kazi Tariqul; Fakhoury, Ahmad M; Santos, Patricia; Wang, Jie; Chilvers, Martin I; Hartman, Glen L; Malvick, Dean K; Floyd, Crystal M; Mueller, Daren S; Leandro, Leonor F S

    2015-12-01

    The ability to accurately detect and quantify Fusarium virguliforme, the cause of sudden death syndrome (SDS) in soybean, in samples such as plant root tissue and soil is extremely valuable for accurate disease diagnoses and to address research questions. Numerous quantitative real-time polymerase chain reaction (qPCR) assays have been developed for this pathogen but their sensitivity and specificity for F. virguliforme have not been compared. In this study, six qPCR assays were compared in five independent laboratories using the same set of DNA samples from fungi, plants, and soil. Multicopy gene-based assays targeting the ribosomal DNA intergenic spacer (IGS) or the mitochondrial small subunit (mtSSU) showed relatively high sensitivity (limit of detection [LOD] = 0.05 to 5 pg) compared with a single-copy gene (FvTox1)-based assay (LOD = 5 to 50 pg). Specificity varied greatly among assays, with the FvTox1 assay ranking the highest (100%) and two IGS assays being slightly less specific (95 to 96%). Another IGS assay targeting four SDS-causing fusaria showed lower specificity (70%), while the two mtSSU assays were lowest (41 and 47%). An IGS-based assay showed consistently highest sensitivity (LOD = 0.05 pg) and specificity and inclusivity above 94% and, thus, is suggested as the most useful qPCR assay for F. virguliforme diagnosis and quantification. However, specificity was also above 94% in two other assays and their selection for diagnostics and research will depend on objectives, samples, and materials used. These results will facilitate both fundamental and disease management research pertinent to SDS.

  18. Assays for determination of protein concentration.

    PubMed

    Olson, Bradley J S C; Markwell, John

    2007-05-01

    Biochemical analysis of proteins relies on accurate quantitation of protein concentration. This unit describes how to perform commonly used protein assays, e.g., Lowry, Bradford, BCA, and UV spectroscopic protein assays. The primary focus of the unit is assay selection, emphasizing sample and buffer compatibility. Protein assay standard curves and data processing fundamentals are discussed in detail. This unit also details high-throughput adaptations of the commonly used protein assays, and also contains a protocol for BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels, which is reliable, inexpensive, and quick.

  19. Assays for determination of protein concentration.

    PubMed

    Olson, Bradley J S C; Markwell, John

    2007-09-01

    Biochemical analysis of proteins relies on accurate quantitation of protein concentration. This appendix describes how to perform commonly used protein assays, e.g., Lowry, Bradford, BCA, and UV spectroscopic protein assays. The primary focus of the appendix is assay selection, emphasizing sample and buffer compatibility. Protein assay standard curves and data processing fundamentals are discussed in detail. This appendix also details high-throughput adaptations of the commonly used protein assays, and also contains a protocol for BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels, which is reliable, inexpensive, and quick.

  20. Accurate quantum chemical calculations

    NASA Technical Reports Server (NTRS)

    Bauschlicher, Charles W., Jr.; Langhoff, Stephen R.; Taylor, Peter R.

    1989-01-01

    An important goal of quantum chemical calculations is to provide an understanding of chemical bonding and molecular electronic structure. A second goal, the prediction of energy differences to chemical accuracy, has been much harder to attain. First, the computational resources required to achieve such accuracy are very large, and second, it is not straightforward to demonstrate that an apparently accurate result, in terms of agreement with experiment, does not result from a cancellation of errors. Recent advances in electronic structure methodology, coupled with the power of vector supercomputers, have made it possible to solve a number of electronic structure problems exactly using the full configuration interaction (FCI) method within a subspace of the complete Hilbert space. These exact results can be used to benchmark approximate techniques that are applicable to a wider range of chemical and physical problems. The methodology of many-electron quantum chemistry is reviewed. Methods are considered in detail for performing FCI calculations. The application of FCI methods to several three-electron problems in molecular physics are discussed. A number of benchmark applications of FCI wave functions are described. Atomic basis sets and the development of improved methods for handling very large basis sets are discussed: these are then applied to a number of chemical and spectroscopic problems; to transition metals; and to problems involving potential energy surfaces. Although the experiences described give considerable grounds for optimism about the general ability to perform accurate calculations, there are several problems that have proved less tractable, at least with current computer resources, and these and possible solutions are discussed.

  1. Endodontic diagnostic terminology update.

    PubMed

    McClannahan, Scott B; Baisden, Michael K; Bowles, Walter R

    2011-01-01

    Determination of the etiology of the patient's chief complaint and a correct diagnosis are paramount prior to a recommendation of endodontic therapy. Reproduction of the patient's chief complaint is critical. If the chief complaint cannot be reproduced, consider consultation with or referral to an endodontist or orofacial pain specialist. The diagnostic terminology presented in this update provides for a more accurate description and communication of the health or pathological conditions of both pulpal and apical tissues. This information is summarized in Table I.

  2. Beamlet laser diagnostics

    SciTech Connect

    Burkhart, S.C.; Behrendt, W.C.; Smith, I.

    1996-06-01

    Beamlet is instrumented extensively to monitor the performance of the overall laser system and many of its subsystems. Beam diagnostics, installed in key locations, are used to fully characterize the beam during its propagation through the multipass cavity and the laser`s output section. This article describes the diagnostics stations located on Beamlet and discusses the design, calibration, and performance of the Beamlet calorimeters. The authors used Nova`s diagnostics packages to develop the Beamlet design to determine beam energy, spatial profile, temporal profile, and other beam parameters. Technologic improvements within the last several years in controls, charge-coupled device (CCD) cameras, and fast oscilloscopes have allowed the authors to obtain more accurate measurements on the Beamlet laser system. They briefly cover some of these techniques, including a description of their LabVIEW based data acquisition system.

  3. A review of platelet secretion assays for the diagnosis of inherited platelet secretion disorders.

    PubMed

    Mumford, Andrew D; Frelinger, Andrew L; Gachet, Christian; Gresele, Paolo; Noris, Patrizia; Harrison, Paul; Mezzano, Diego

    2015-07-01

    Measurement of platelet granule release to detect inherited platelet secretion disorders (IPSDs) is essential for the evaluation of patients with abnormal bleeding and is necessary to distinguish which granule sub-types are affected and whether there is abnormal granule bio-synthesis or secretion. The radioactive serotonin incorporation and release assay, described before 1970, is still considered the "gold standard" test to assess platelet δ-granule release, although is unsuitable for clinical diagnostic laboratories. Luciferin-based assays, such as lumiaggregometry, are the most widely performed alternatives, although these methods do not distinguish defects in δ-granule biosynthesis from defects in secretion. Platelet α-granule release is commonly evaluated using flow cytometry by measuring surface exposure of P-selectin after platelet activation. However, this assay has poor sensitivity for some α-granule disorders. Only few studies have been published with more recently developed assays and no critical reviews on these methods are available. In this review, we describe the rationale for developing robust and accurate laboratory tests of platelet granule release and describe the characteristics of the currently available tests. We identify an unmet need for further systematic evaluation of new assays and for standardisation of methodologies for clinical diagnostic laboratories.

  4. A home-brew real-time PCR assay for reliable detection and quantification of mature miR-122.

    PubMed

    Naderi, Mahmood; Abdul Tehrani, Hossein; Soleimani, Masoud; Shabani, Iman; Hashemi, Seyed Mahmoud

    2015-09-01

    miR-122 is a liver-specific miRNA that has significant gene expression alterations in response to specific pathophysiological circumstances of liver such as drug-induced liver injury, hepatocellular carcinoma, and hepatitis B and C virus infections. Therefore, accurate and precise quantification of miR-122 is very important for clinical diagnostics. However, because of the lack of in vitro diagnostics assays for miR-122 detection and quantification of the existence of an open-source assay could inevitably provide external evaluation by other researchers and the chance of promoting the assay when required. The aim of this study was to develop a Taqman real-time polymerase chain reaction assay, which is capable of robust and reliable quantification of miR-122 in different sample types. We used stem loop methodology to design a specific Taqman real-time polymerase chain reaction assay for miR-122. This technique enabled us to reliably and reproducibly quantify short-length oligonucleotides such as miR-122. The specificity, sensitivity, interassay and intra-assay, and the dynamic range of the assay were experimentally determined by their respective methodology. The assay had a linear dynamic range of 3E to 4.8E miR-122 copies/reaction and the limit of detection was determined to be between 960 and 192 copies/reaction with 95% confidence interval. The assay gave a coefficient of variation for the Ct values of <1.4% and 0.78% for intra-assay and interassay, respectively. Taking into account that miR-122 is expressed in >50,000 copies per hepatocyte, this assay is able to suffice the need for reliable detection and quantification of this miRNA. Therefore, this study can be considered as a start point for standardizing miR-122 quantification.

  5. BIOACCESSIBILITY TESTS ACCURATELY ESTIMATE ...

    EPA Pesticide Factsheets

    Hazards of soil-borne Pb to wild birds may be more accurately quantified if the bioavailability of that Pb is known. To better understand the bioavailability of Pb to birds, we measured blood Pb concentrations in Japanese quail (Coturnix japonica) fed diets containing Pb-contaminated soils. Relative bioavailabilities were expressed by comparison with blood Pb concentrations in quail fed a Pb acetate reference diet. Diets containing soil from five Pb-contaminated Superfund sites had relative bioavailabilities from 33%-63%, with a mean of about 50%. Treatment of two of the soils with P significantly reduced the bioavailability of Pb. The bioaccessibility of the Pb in the test soils was then measured in six in vitro tests and regressed on bioavailability. They were: the “Relative Bioavailability Leaching Procedure” (RBALP) at pH 1.5, the same test conducted at pH 2.5, the “Ohio State University In vitro Gastrointestinal” method (OSU IVG), the “Urban Soil Bioaccessible Lead Test”, the modified “Physiologically Based Extraction Test” and the “Waterfowl Physiologically Based Extraction Test.” All regressions had positive slopes. Based on criteria of slope and coefficient of determination, the RBALP pH 2.5 and OSU IVG tests performed very well. Speciation by X-ray absorption spectroscopy demonstrated that, on average, most of the Pb in the sampled soils was sorbed to minerals (30%), bound to organic matter 24%, or present as Pb sulfate 18%. Ad

  6. The use of immunoglobulin light chain assays in the diagnosis of paraprotein-related kidney disease

    PubMed Central

    Yadav, Punit; Leung, Nelson; Sanders, Paul W.; Cockwell, Paul

    2016-01-01

    Kidney involvement is common in paraprotein-related diseases. A diversity of clinical presentations and histopathological features can occur secondary to tissue injury caused by precipitation or deposition of a clonal immunoglobulin, usually an immunoglobulin light chain. The paraprotein is either produced by multiple myeloma or by a clone of B-cell lineage that does not fulfill diagnostic criteria for multiple myeloma. The recent introduction of serum immunoglobulin free light chain assays, which accurately quantify both light chain isotypes to produce a ratio that indicates the presence or absence of a light chain paraprotein, is a major clinical development. However, as the interpretation of the assay can be challenging, the aim of this review is to clarify the role of serum and urinary light chain assays in the screening and diagnosis of paraprotein-related kidney disease. PMID:25296094

  7. Development of forensic assay signatures for ebolaviruses.

    PubMed

    Song, Jian; Doggett, Norman; Wren, Melinda; Burr, Tom; Fenimore, P W; Hatcher, Eneida L; Bruno, William J; Li, Po-E; Stubben, Chris; Wolinsky, Murray

    2015-03-01

    Ebolaviruses are a diverse group of RNA viruses comprising five different species, four of which cause fatal hemorrhagic fever in humans. Because of their high infectivity and lethality, ebolaviruses are considered major biothreat agents. Although detection assays exist, no forensic assays are currently available. Here, we report the development of forensic assays that differentiate ebolaviruses. We performed phylogenetic analyses and identified canonical SNPs for all species, major clades and isolates. TaqMan-MGB allelic discrimination assays based on these SNPs were designed, screened against synthetic RNA templates, and validated against ebolavirus genomic RNAs. A total of 45 assays were validated to provide 100% coverage of the species and variants with additional resolution at the isolate level. These assays enabled accurate forensic analysis on 4 "unknown" ebolaviruses. Unknowns were correctly classified to species and variant. A goal of providing resolution below the isolate level was not successful. These high-resolution forensic assays allow rapid and accurate genotyping of ebolaviruses for forensic investigations.

  8. Oestradiol assays: fitness for purpose?

    PubMed

    Middle, Jonathan G; Kane, John W

    2009-11-01

    In this review we discuss the analytical inadequacies of oestradiol assays in relation to the clinical requirements for performing them, and make recommendations for their improvement. The measurement of oestradiol can be requested in a number of clinical scenarios (precocious puberty, infertility, assisted conception, hormone replacement therapy). The very wide dynamic range of oestradiol concentrations is a huge challenge for routine assays, which they are unlikely to meet on theoretical as well as practical grounds. The EQA performance of oestradiol assays in terms of trueness, comparability, recovery and analytical sensitivity leaves much to be desired and indicates that calibration is compromised by poor analytical specificity. To make oestradiol assays fit for purpose requires concerted action by all stakeholders to define analytical quality specifications for the various clinical scenarios involved, and then to encourage concerted action by the diagnostic industry to use the steroid reference measurement system to improve specificity, trueness and traceability.

  9. Neisseria Species Identification Assay for the Confirmation of Neisseria gonorrhoeae-Positive Results of the COBAS Amplicor PCR▿

    PubMed Central

    Mangold, Kathy A.; Regner, MaryAnn; Tajuddin, Mohammed; Tajuddin, Aamair M.; Jennings, Lawrence; Du, Hongyan; Kaul, Karen L.

    2007-01-01

    Screening assays for Neisseria gonorrhoeae exhibit low positive predictive values, particularly in low-prevalence populations. A new real-time PCR assay that detects and identifies individual Neisseria spp. using melt curve analysis was compared to two previously published supplementary assays. NsppID, a 16S rRNA real-time PCR/melt curve assay developed to distinguish N. gonorrhoeae from other Neisseria spp., was compared to real-time PCR assays targeting genes reportedly specific for N. gonorrhoeae, the cppB gene and the porA pseudogene. A total of 408 clinical specimens (324 female endocervical swabs and 84 male urine or urogenital swab specimens) were screened using the COBAS Amplicor assay for Chlamydia trachomatis and N. gonorrhoeae (CT/NG) (Roche Diagnostics, Indianapolis, IN) followed by confirmatory testing via real-time PCR. The NsppID assay detected Neisseria spp. in 150/181 COBAS-positive specimens (82%), including six dual infections, and identified N. gonorrhoeae in 102 (56%) specimens. Sixty-nine of 181 (38%) specimens were positive for N. gonorrhoeae by porA pseudogene, and 115/181 (64%) were positive for cppB. However, cppB was also positive in 15% of COBAS-negative specimens, more than either NsppID (4%) or porA pseudogene (2%) assays. The porA pseudogene assay had the highest specificity for both genders but the lowest sensitivity, especially in female specimens. NsppID had a slightly lower specificity but greater sensitivity and overall accuracy. The least desirable confirmatory assay was cppB, due to poor specificity. The NsppID assay is an accurate confirmatory assay for N. gonorrhoeae detection. In addition, the NsppID assay can identify the non-N. gonorrhoeae species responsible for the majority of false-positive results from the COBAS Amplicor CT/NG assay. PMID:17360838

  10. Establishment and Comparison of Two Different Diagnostic Platforms for Detection of DENV1 NS1 Protein

    PubMed Central

    Tang, Yin-Liang; Chiu, Chien-Yu; Lin, Chun-Yu; Huang, Chung-Hao; Chen, Yen-Hsu; Destura, Raul V.; Chao, Day-Yu; Wu, Han-Chung

    2015-01-01

    Dengue virus (DENV) infection is currently at pandemic levels, with populations in tropical and subtropical regions at greatest risk of infection. Early diagnosis and management remain the cornerstone for good clinical outcomes, thus efficient and accurate diagnostic technology in the early stage of the disease is urgently needed. Serotype-specific monoclonal antibodies (mAbs) against the DENV1 nonstructural protein 1 (NS1), DA12-4, DA13-2, and DA15-3, which were recently generated using the hybridoma technique, are suitable for use in diagnostic platforms. Immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA) and Western blot analysis further confirmed the serotype specificity of these three monoclonal antibodies. The ELISA-based diagnostic platform was established using the combination of two highly sensitive mAbs (DA15-3 and DB20-6). The same combination was also used for the flow cytometry-based diagnostic platform. We report here the detection limits of flow cytometry-based and ELISA-based diagnostic platforms using these mAbs to be 0.1 and 1 ng/mL, respectively. The collected clinical patient serum samples were also assayed by these two serotyping diagnostic platforms. The sensitivity and specificity for detecting NS1 protein of DENV1 are 90% and 96%, respectively. The accuracy of our platform for testing clinical samples is more advanced than that of the two commercial NS1 diagnostic platforms. In conclusion, our platforms are suitable for the early detection of NS1 protein in DENV1 infected patients. PMID:26610481

  11. Accurate spectral color measurements

    NASA Astrophysics Data System (ADS)

    Hiltunen, Jouni; Jaeaeskelaeinen, Timo; Parkkinen, Jussi P. S.

    1999-08-01

    Surface color measurement is of importance in a very wide range of industrial applications including paint, paper, printing, photography, textiles, plastics and so on. For a demanding color measurements spectral approach is often needed. One can measure a color spectrum with a spectrophotometer using calibrated standard samples as a reference. Because it is impossible to define absolute color values of a sample, we always work with approximations. The human eye can perceive color difference as small as 0.5 CIELAB units and thus distinguish millions of colors. This 0.5 unit difference should be a goal for the precise color measurements. This limit is not a problem if we only want to measure the color difference of two samples, but if we want to know in a same time exact color coordinate values accuracy problems arise. The values of two instruments can be astonishingly different. The accuracy of the instrument used in color measurement may depend on various errors such as photometric non-linearity, wavelength error, integrating sphere dark level error, integrating sphere error in both specular included and specular excluded modes. Thus the correction formulas should be used to get more accurate results. Another question is how many channels i.e. wavelengths we are using to measure a spectrum. It is obvious that the sampling interval should be short to get more precise results. Furthermore, the result we get is always compromise of measuring time, conditions and cost. Sometimes we have to use portable syste or the shape and the size of samples makes it impossible to use sensitive equipment. In this study a small set of calibrated color tiles measured with the Perkin Elmer Lamda 18 and the Minolta CM-2002 spectrophotometers are compared. In the paper we explain the typical error sources of spectral color measurements, and show which are the accuracy demands a good colorimeter should have.

  12. Clonality Testing in Veterinary Medicine: A Review With Diagnostic Guidelines.

    PubMed

    Keller, S M; Vernau, W; Moore, P F

    2016-07-01

    The accurate distinction of reactive and neoplastic lymphoid proliferations can present challenges. Given the different prognoses and treatment strategies, a correct diagnosis is crucial. Molecular clonality assays assess rearranged lymphocyte antigen receptor gene diversity and can help differentiate reactive from neoplastic lymphoid proliferations. Molecular clonality assays are commonly used to assess atypical, mixed, or mature lymphoid proliferations; small tissue fragments that lack architecture; and fluid samples. In addition, clonality testing can be utilized to track neoplastic clones over time or across anatomic sites. Molecular clonality assays are not stand-alone tests but useful adjuncts that follow clinical, morphologic, and immunophenotypic assessment. Even though clonality testing provides valuable information in a variety of situations, the complexities and pitfalls of this method, as well as its dependency on the experience of the interpreter, are often understated. In addition, a lack of standardized terminology, laboratory practices, and interpretational guidelines hinders the reproducibility of clonality testing across laboratories in veterinary medicine. The objectives of this review are twofold. First, the review is intended to familiarize the diagnostic pathologist or interested clinician with the concepts, potential pitfalls, and limitations of clonality testing. Second, the review strives to provide a basis for future harmonization of clonality testing in veterinary medicine by providing diagnostic guidelines.

  13. [Novel methods for dementia diagnostics].

    PubMed

    Wiltfang, J

    2015-04-01

    Novel diagnostic methods, such as cerebrospinal fluid-based neurochemical dementia diagnostics (CSF-NDD) and [18F] amyloid positron emission tomography (PET) are meanwhile recommended for specific indications by international guidelines for the improved early and differential diagnostics of multigenic (sporadic) Alzheimer's dementia (AD). In the case of CSF-NDD the German neuropsychiatric guidelines have already been validated on the S3 level of evidence (http://www.DGPPN.de) and the additional consideration of [18F] amyloid-PET in the current update of the guidelines is to be expected. By means of CSF-NDD and/or [18F] amyloid-PET a predictive diagnosis of incipient (preclinical) AD is also possible for patients at high risk for AD who are in prodromal stages, such as mild cognitive impairment (MCI). As accompanying (secondary) preventive therapy of AD cannot be offered a predictive molecular dementia diagnostics is not recommended by the German neuropsychiatric dementia guidelines (http://www.DGPPN.de). However, novel diagnostic approaches, which offer molecular positive diagnostics of AD have already gained high relevance in therapy research as they allow promising preventive treatment avenues to be validated directly in the clinical trial. Moreover, future blood-based dementia diagnostics by means of multiplex assays is becoming increasingly more feasible; however, so far corresponding proteomic or epigenetic assays could not be consistently validated in independent studies.

  14. Assay strategies and methods for phospholipases

    SciTech Connect

    Reynolds, L.J.; Washburn, W.N.; Deems, R.A.; Dennis, E.A.

    1991-01-01

    Of the general considerations discussed, the two issues which are most important in choosing an assay are (1) what sensitivity is required to assay a particular enzyme and (2) whether the assay must be continuous. One can narrow the options further by considering substrate availability, enzyme specificity, assay convenience, or the presence of incompatible side reactions. In addition, the specific preference of a particular phospholipase for polar head group, micellar versus vesicular substrates, and anionic versus nonionic detergents may further restrict the options. Of the many assays described in this chapter, several have limited applicability or serious drawbacks and are not commonly employed. The most commonly used phospholipase assays are the radioactive TLC assay and the pH-stat assay. The TLC assay is probably the most accurate, sensitive assay available. These aspects often outweigh the disadvantages of being discontinuous, tedious, and expensive. The radioactive E. coli assay has become popular recently as an alternative to the TLC assay for the purification of the mammalian nonpancreatic phospholipases. The assay is less time consuming and less expensive than the TLC assay, but it is not appropriate when careful kinetics are required. Where less sensitivity is needed, or when a continuous assay is necessary, the pH-stat assay is often employed. With purified enzymes, when free thiol groups are not present, a spectrophotometric thiol assay can be used. This assay is {approximately} as sensitive as the pH-stat assay but is more convenient and more reproducible, although the substrate is not available commercially. Despite the many assay choices available, the search continues for a convenient, generally applicable assay that is both sensitive and continuous.

  15. [Thalassaemia diagnostics].

    PubMed

    Kusters, Elske; Kerkhoffs, Jean-Louis H; van Rossum, André P

    2014-01-01

    The thalassaemias are characterised by quantitative aberrations in the production of the globin chains that make up haemoglobin, and are a subgroup of the haemoglobinopathies. In this LabQuiz we show how thalassaemia carrier status can be indicated in the results of regular laboratory tests, and discuss the laboratory diagnostics that can confirm or rule out thalassaemia. In these two cases we will present a man of Moroccan descent, and two brothers of Filipino descent, all with anaemia and microcytosis. We show it is possible to differentiate between iron-deficiency anaemia and thalassaemia carrier status on the basis of a complete blood count and measurement of ferritin levels, and which laboratory diagnostics can be subsequently performed in order to confirm a suspicion of thalassaemia. The background section discusses the properties and pitfalls of routine laboratory diagnostics for the thalassaemias, and thalassaemia diagnostics in the Dutch newborn screening programme.

  16. Comparison of two immunochromatographic assays and the indirect immunofluorescence antibody test for diagnosis of Trypanosoma cruzi infection in dogs in south central Louisiana.

    PubMed

    Nieto, Prixia D; Boughton, Roger; Dorn, Patricia L; Steurer, Frank; Raychaudhuri, Syamal; Esfandiari, Javan; Gonçalves, Edson; Diaz, James; Malone, John B

    2009-11-12

    Two rapid tests evaluated in dogs considered to be of high risk of infection with the Chagas parasite Trypanosoma cruzi using two immunochromatographic assays: Trypanosoma Detect for canine, InBios, Seattle, WA and CHAGAS STAT-PAK assay, Chembio Diagnostic Systems, Medford, NY, in south central Louisiana. For this purpose a serological survey was carried out in a total of 122 dogs and a serum bank was created. These 122 animals were first tested by IFAT that was used as the standard test. From the serum bank 50 samples were tested using the two rapid Chagas assays and results compared to the standard test IFAT. The serological survey using IFAT showed a prevalence of T. cruzi infection in 22.1% of the tested dogs. In the immunochromatographic assays, 13 and 11 animals were positive on rapid assay: Trypanosoma Detect for canine, InBios and CHAGAS STAT-PAK, Chembio Diagnostic Systems, respectively compared to 11 positive by IFAT. These two immunochromatographic tests have shown high susceptibility and specificity compared to our standard method IFAT. The rapid, easy and accurate screening assays used in conjunction with confirmatory tests, would be an excellent tool for veterinarians to diagnose T. cruzi infection. Early detection of T. cruzi infection may prevent complications through an effective treatment. Greater awareness by veterinarians of the risk, clinical findings, history along with diagnostic methods will contribute greatly to an understanding of the true prevalence of Chagas disease in dogs in Louisiana.

  17. Thioaptamer Diagnostic System (TDS)

    NASA Technical Reports Server (NTRS)

    Yang, Xianbin

    2015-01-01

    AM Biotechnologies, LLC, in partnership with Sandia National Laboratories, has developed a diagnostic device that quickly detects sampled biomarkers. The TDS quickly quantifies clinically relevant biomarkers using only microliters of a single sample. The system combines ambient-stable, long shelf-life affinity assays with handheld, microfluidic gel electrophoresis affinity assay quantification technology. The TDS is easy to use, operates in microgravity, and permits simultaneous quantification of 32 biomarkers. In Phase I of the project, the partners demonstrated that a thioaptamer assay used in the microfluidic instrument could quantify a specific biomarker in serum in the low nanomolar range. The team also identified novel affinity agents to bone-specific alkaline phosphatase (BAP) and demonstrated their ability to detect BAP with the microfluidic instrument. In Phase II, AM Biotech expanded the number of ambient affinity agents and demonstrated a TDS prototype. In the long term, the clinical version of the TDS will provide a robust, flight-tested diagnostic capability for space exploration missions.

  18. Rapid and Accurate Detection of Mycobacterium tuberculosis in Sputum Samples by Cepheid Xpert MTB/RIF Assay—A Clinical Validation Study

    PubMed Central

    Rachow, Andrea; Zumla, Alimuddin; Heinrich, Norbert; Rojas-Ponce, Gabriel; Mtafya, Bariki; Reither, Klaus; Ntinginya, Elias N.; O'Grady, Justin; Huggett, Jim; Dheda, Keertan; Boehme, Catharina; Perkins, Mark; Saathoff, Elmar; Hoelscher, Michael

    2011-01-01

    Background A crucial impediment to global tuberculosis control is the lack of an accurate, rapid diagnostic test for detection of patients with active TB. A new, rapid diagnostic method, (Cepheid) Xpert MTB/RIF Assay, is an automated sample preparation and real-time PCR instrument, which was shown to have good potential as an alternative to current reference standard sputum microscopy and culture. Methods We performed a clinical validation study on diagnostic accuracy of the Xpert MTB/RIF Assay in a TB and HIV endemic setting. Sputum samples from 292 consecutively enrolled adults from Mbeya, Tanzania, with suspected TB were subject to analysis by the Xpert MTB/RIF Assay. The diagnostic performance of Xpert MTB/RIF Assay was compared to standard sputum smear microscopy and culture. Confirmed Mycobacterium tuberculosis in a positive culture was used as a reference standard for TB diagnosis. Results Xpert MTB/RIF Assay achieved 88.4% (95%CI = 78.4% to 94.9%) sensitivity among patients with a positive culture and 99% (95%CI = 94.7% to 100.0%) specificity in patients who had no TB. HIV status did not affect test performance in 172 HIV-infected patients (58.9% of all participants). Seven additional cases (9.1% of 77) were detected by Xpert MTB/RIF Assay among the group of patients with clinical TB who were culture negative. Within 45 sputum samples which grew non-tuberculous mycobacteria the assay's specificity was 97.8% (95%CI = 88.2% to 99.9%). Conclusions The Xpert MTB/RIF Assay is a highly sensitive, specific and rapid method for diagnosing TB which has potential to complement the current reference standard of TB diagnostics and increase its overall sensitivity. Its usefulness in detecting sputum smear and culture negative patients needs further study. Further evaluation in high burden TB and HIV areas under programmatic health care settings to ascertain applicability, cost-effectiveness, robustness and local acceptance are required. PMID:21738575

  19. Invasive mycoses: diagnostic challenges.

    PubMed

    Ostrosky-Zeichner, Luis

    2012-01-01

    Despite the availability of newer antifungal drugs, outcomes for patients with invasive fungal infections (IFIs) continue to be poor, in large part due to delayed diagnosis and initiation of appropriate antifungal therapy. Standard histopathologic diagnostic techniques are often untenable in at-risk patients, and culture-based diagnostics typically are too insensitive or nonspecific, or provide results after too long a delay for optimal IFI management. Newer surrogate markers of IFIs with improved sensitivity and specificity are needed to enable earlier diagnosis and, ideally, to provide prognostic information and/or permit therapeutic monitoring. Surrogate assays should also be accessible and easy to implement in the hospital. Several nonculture-based assays of newer surrogates are making their way into the medical setting or are currently under investigation. These new or up-and-coming surrogates include antigens/antibodies (mannan and antimannan antibodies) or fungal metabolites (d-arabinitol) for detection of invasive candidiasis, the Aspergillus cell wall component galactomannan used to detect invasive aspergillosis, or the fungal cell wall component and panfungal marker β-glucan. In addition, progress continues with use of polymerase chain reaction- or other nucleic acid- or molecular-based assays for diagnosis of either specific or generic IFIs, although the various methods must be better standardized before any of these approaches can be more fully implemented into the medical setting. Investigators are also beginning to explore the possibility of combining newer surrogate markers with each other or with more standard diagnostic approaches to improve sensitivity, specificity, and capacity for earlier diagnosis, at a time when fungal burden is still relatively low and more responsive to antifungal therapy.

  20. Implementation of Rapid Molecular Infectious Disease Diagnostics: the Role of Diagnostic and Antimicrobial Stewardship.

    PubMed

    Messacar, Kevin; Parker, Sarah K; Todd, James K; Dominguez, Samuel R

    2017-03-01

    New rapid molecular diagnostic technologies for infectious diseases enable expedited accurate microbiological diagnoses. However, diagnostic stewardship and antimicrobial stewardship are necessary to ensure that these technologies conserve, rather than consume, additional health care resources and optimally affect patient care. Diagnostic stewardship is needed to implement appropriate tests for the clinical setting and to direct testing toward appropriate patients. Antimicrobial stewardship is needed to ensure prompt appropriate clinical action to translate faster diagnostic test results in the laboratory into improved outcomes at the bedside. This minireview outlines the roles of diagnostic stewardship and antimicrobial stewardship in the implementation of rapid molecular infectious disease diagnostics.

  1. A molecular-beacon-based asymmetric PCR assay for easy visualization of amplicons in the diagnosis of trichomoniasis.

    PubMed

    Sonkar, Subash C; Sachdev, Divya; Mishra, Prashant K; Kumar, Anita; Mittal, Pratima; Saluja, Daman

    2016-12-15

    The currently available nucleic acid amplification tests (NAATs) for trichomoniasis are accurate, quick and confirmative with superior sensitivity than traditional culture-based microbiology assays. However, these assays are associated with problems of carry over contamination, false positive results, requirement of technical expertise for performance and detection of end product. Hence, a diagnostic assay with easy visualization of the amplified product will be profitable. An in-house, rapid, sensitive, specific molecular-beacon-based PCR assay, using primers against pfoB gene of Trichomonas vaginalis, was developed and evaluated using dry ectocervical swabs (n=392) from symptomatic females with vaginal discharge. Total DNA was isolated and used as template for the PCR assays. The performance and reproducibility of PCR assay was evaluated by composite reference standard (CRS). For easy visualization of the amplified product, molecular-beacon was designed and amplicons were visualized directly using fluorescent handheld dark reader or by Micro-Plate Reader. Molecular-beacons are single-stranded hairpin shaped nucleic acid probes composed of a stem, with fluorophore/quencher pair and a loop region complementary to the desired DNA. The beacon-based PCR assay designed in the present study is highly specific as confirmed by competition experiments and extremely sensitive with detection limit of 20fg of genomic DNA (3-4 pathogens). The minimum infrastructure requirement and ease to perform the assay makes this method highly useful for resource poor countries for better disease management.

  2. Whole blood assay for trypsin activity using polyanionic focusing gel electrophoresis.

    PubMed

    Lefkowitz, Roy B; Schmid-Schönbein, Geert W; Heller, Michael J

    2010-07-01

    The measurement of trypsin activity directly in blood is important for the development of novel diagnostics and for biomedical research. Presently, most degradative enzyme assays require sample preparation, making them time consuming, costly, and less accurate. We recently demonstrated a simple and rapid electrophoretic assay for the measurement of trypsin activity directly in whole blood. This assay utilizes a charge-changing fluorescent peptide substrate that produces a positively charged fluorescent product fragment upon cleavage by the target enzyme. This fragment is then rapidly separated from whole blood by electrophoresis and quantified with a fluorescent detector. In this study, we demonstrate that polyanionic poly-L-glutamic acid-doped polyacrylamide gels can focus the fluorescent cleavage product and markedly improve the LODs of the assay. A LOD of 2 pg in 6 microL (0.3 ng/mL) in whole human blood was achieved after a 1-h reaction of enzyme and substrate followed by 10 min of electrophoresis. This is 50- to 200-fold better than the estimated reference levels for trypsin (15-60 ng/mL) in blood. This straightforward technique now allows for the rapid measurement of clinically relevant levels of trypsin activity in microliter volumes of whole blood, providing a useful tool for the development of novel point-of-care diagnostics.

  3. A novel, single nucleotide polymorphism-based assay to detect 22q11 deletions.

    PubMed

    Funke, Birgit H; Brown, Alison C; Ramoni, Marco F; Regan, Maura E; Baglieri, Chris; Finn, Christine T; Babcock, Melanie; Shprintzen, Robert J; Morrow, Bernice E; Kucherlapati, Raju

    2007-01-01

    Velocardiofacial syndrome, DiGeorge syndrome, and conotruncal anomaly face syndrome, now collectively referred to as 22q11deletion syndrome (22q11DS) are caused by microdeletions on chromosome 22q11. The great majority ( approximately 90%) of these deletions are 3 Mb in size. The remaining deleted patients have nested break-points resulting in overlapping regions of hemizygosity. Diagnostic testing for the disorder is traditionally done by fluorescent in situ hybridization (FISH) using probes located in the proximal half of the region common to all deletions. We developed a novel, high-resolution single-nucleotide polymorphism (SNP) genotyping assay to detect 22q11 deletions. We validated this assay using DNA from 110 nondeleted controls and 77 patients with 22q11DS that had previously been tested by FISH. The assay was 100% sensitive (all deletions were correctly identified). Our assay was also able to detect a case of segmental uniparental disomy at 22q11 that was not detected by the FISH assay. We used Bayesian networks to identify a set of 17 SNPs that are sufficient to ascertain unambiguously the deletion status of 22q11DS patients. Our SNP based assay is a highly accurate, sensitive, and specific method for the diagnosis of 22q11 deletion syndrome.

  4. Barcoded microchips for biomolecular assays.

    PubMed

    Zhang, Yi; Sun, Jiashu; Zou, Yu; Chen, Wenwen; Zhang, Wei; Xi, Jianzhong Jeff; Jiang, Xingyu

    2015-01-20

    Multiplexed assay of analytes is of great importance for clinical diagnostics and other analytical applications. Barcode-based bioassays with the ability to encode and decode may realize this goal in a straightforward and consistent manner. We present here a microfluidic barcoded chip containing several sets of microchannels with different widths, imitating the commonly used barcode. A single barcoded microchip can carry out tens of individual protein/nucleic acid assays (encode) and immediately yield all assay results by a portable barcode reader or a smartphone (decode). The applicability of a barcoded microchip is demonstrated by human immunodeficiency virus (HIV) immunoassays for simultaneous detection of three targets (anti-gp41 antibody, anti-gp120 antibody, and anti-gp36 antibody) from six human serum samples. We can also determine seven pathogen-specific oligonucleotides by a single chip containing both positive and negative controls.

  5. 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

  6. DIAGNOSTICS OF BNL ERL

    SciTech Connect

    POZDEYEV,E.; BEN-ZVI, I.; CAMERON, P.; GASSNER, D.; KAYRAN, D.; ET AL.

    2007-06-25

    The ERL Prototype project is currently under development at the Brookhaven National Laboratory. The ERL is expected to demonstrate energy recovery of high-intensity beams with a current of up to a few hundred milliamps, while preserving the emittance of bunches with a charge of a few nanocoulombs produced by a high-current SRF gun. To successfully accomplish this task the machine will include beam diagnostics that will be used for accurate characterization of the three dimensional beam phase space at the injection and recirculation energies, transverse and longitudinal beam matching, orbit alignment, beam current measurement, and machine protection. This paper outlines requirements on the ERL diagnostics and describes its setup and modes of operation.

  7. 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

  8. Unmet Diagnostic Needs in Infectious Disease

    PubMed Central

    Blaschke, Anne J.; Hersh, Adam L.; Beekmann, Susan E.; Ince, Dilek; Polgreen, Philip M.; Hanson, Kimberly E.

    2014-01-01

    Accurate diagnosis is critical to providing appropriate care in infectious diseases. New technologies for infectious disease diagnostics are emerging, but gaps remain in test development and availability. The Emerging Infections Network surveyed Infectious Diseases physicians to assess unmet diagnostic needs. Responses reflected the urgent need to identify drug-resistant infections and highlighted the potential for early diagnosis to improve antibiotic stewardship. Information gained from this survey can help inform recommendations for new diagnostic test development in the future. PMID:25456043

  9. Total cholesterol performance of Abell–Levy–Brodie–Kendall reference measurement procedure: Certification of Japanese in-vitro diagnostic assay manufacturers through CDC’s Cholesterol Reference Method Laboratory Network☆

    PubMed Central

    Nakamura, Masakazu; Iso, Hiroyasu; Kitamura, Akihiko; Imano, Hironori; Kiyama, Masahiko; Yokoyama, Shinji; Kayamori, Yuzo; Koyama, Isao; Nishimura, Kunihiro; Nakai, Michikazu; Dasti, Mahnaz; Vesper, Hubert W.; Teramoto, Tamio; Miyamoto, Yoshihiro

    2015-01-01

    Background Accurate measurement of total cholesterol (TC) is important for cardiovascular disease risk management. The US Centers for Disease Control and Prevention (CDC) and Cholesterol Reference Method Laboratory Network (CRMLN) perform Abell–Levy–Brodie–Kendall (AK) reference measurement procedure (RMP) for TC as a secondary reference method, and implement Certification Protocol for Manufacturers. Japanese CRMLN laboratory at Osaka performed the AK RMP for 22 years, and conducted TC certification for reagent/calibrator/instrument systems of six Japanese manufacturers every 2 years for 16 years. Osaka TC performance was examined and compared to CDC’s reference values. Methods AK RMP involved sample hydrolysis, cholesterol extraction, and determination of cholesterol levels by spectrophotometry. The Certification Protocol for Manufacturers includes comparison with AK RMP using at least 40 fresh specimens. Demonstration of average bias ≤3% and total coefficient of variation ≤3% qualified an analytical system for certification. Results In the AK RMP used in the Osaka CRMLN laboratory, the regression equation for measuring TC was y (Osaka) = 1.000x (CDC) + 0.032 (n = 619, R2 = 1.000). Six Japanese manufacturers had allowable performance for certification. Conclusions The AK RMP for TC measurement was accurate, precise, and stable for 22 years. Six Japanese manufacturers were certified for 16 years. PMID:25818239

  10. Contribution to diagnostics/prognostics of tuberculosis in children. I. New methods of assaying zinc and simultaneously copper and zinc in diluted sera by flame atomic-absorption spectrometry.

    PubMed

    Luterotti, Svjetlana; Kordić, Tončica Vukman; Dodig, Slavica

    2015-09-01

    In an attempt to provide a reliable status of metal ions in children, new methods of analysis of children's sera are proposed. New flame atomic-absorption spectrometric (FAAS) methods are simple, cost- and time-effective and, above all, labor-, reagent- and sample-saving. Two methods were suggested: method A for simultaneous determination of Cu and Zn from 5-fold diluted sera, and method B, for assaying zinc alone in 10-fold diluted samples. Both methods are based on a single-step sample pretreatment (deproteinization with 3 mol dm-3 HCl). Method A uses a single-step calibration with a mixed standard. The main advantage of method B is an additional reduction in sample consumption. Both methods were fully validated against reference methods. Accuracy, sensitivity and precision have proven them to be comparable to the reference methods in terms of analytical performance, and applicable to analyses of children's sera.

  11. Diagnostic Performance of the New Version (v2.0) of GenoType MTBDRsl Assay for Detection of Resistance to Fluoroquinolones and Second-Line Injectable Drugs: a Multicenter Study

    PubMed Central

    Tagliani, Elisa; Cabibbe, Andrea M.; Miotto, Paolo; Borroni, Emanuele; Toro, Juan Carlos; Mansjö, Mikael; Hoffner, Sven; Hillemann, Doris; Zalutskaya, Aksana; Skrahina, Alena

    2015-01-01

    Resistance to fluoroquinolones (FLQ) and second-line injectable drugs (SLID) is steadily increasing, especially in eastern European countries, posing a serious threat to effective tuberculosis (TB) infection control and adequate patient management. The availability of rapid molecular tests for the detection of extensively drug-resistant TB (XDR-TB) is critical in areas with high rates of multidrug-resistant TB (MDR-TB) and XDR-TB and limited conventional drug susceptibility testing (DST) capacity. We conducted a multicenter study to evaluate the performance of the new version (v2.0) of the Genotype MTBDRsl assay compared to phenotypic DST and sequencing on a panel of 228 Mycobacterium tuberculosis isolates and 231 smear-positive clinical specimens. The inclusion of probes for the detection of mutations in the eis promoter region in the MTBDRsl v2.0 test resulted in a higher sensitivity for detection of kanamycin resistance for both direct and indirect testing (96% and 95.4%, respectively) than that seen with the original version of the assay, whereas the test sensitivities for detection of FLQ resistance remained unchanged (93% and 83.6% for direct and indirect testing, respectively). Moreover, MTBDRsl v2.0 showed better performance characteristics than v1.0 for the detection of XDR-TB, with high specificity and sensitivities of 81.8% and 80.4% for direct and indirect testing, respectively. MTBDRsl v2.0 thus represents a reliable test for the rapid detection of resistance to second-line drugs and a useful screening tool to guide the initiation of appropriate MDR-TB treatment. PMID:26179309

  12. Laboratory Diagnostics of Botulism

    PubMed Central

    Lindström, Miia; Korkeala, Hannu

    2006-01-01

    Botulism is a potentially lethal paralytic disease caused by botulinum neurotoxin. Human pathogenic neurotoxins of types A, B, E, and F are produced by a diverse group of anaerobic spore-forming bacteria, including Clostridium botulinum groups I and II, Clostridium butyricum, and Clostridium baratii. The routine laboratory diagnostics of botulism is based on the detection of botulinum neurotoxin in the patient. Detection of toxin-producing clostridia in the patient and/or the vehicle confirms the diagnosis. The neurotoxin detection is based on the mouse lethality assay. Sensitive and rapid in vitro assays have been developed, but they have not yet been appropriately validated on clinical and food matrices. Culture methods for C. botulinum are poorly developed, and efficient isolation and identification tools are lacking. Molecular techniques targeted to the neurotoxin genes are ideal for the detection and identification of C. botulinum, but they do not detect biologically active neurotoxin and should not be used alone. Apart from rapid diagnosis, the laboratory diagnostics of botulism should aim at increasing our understanding of the epidemiology and prevention of the disease. Therefore, the toxin-producing organisms should be routinely isolated from the patient and the vehicle. The physiological group and genetic traits of the isolates should be determined. PMID:16614251

  13. Loop-Mediated Isothermal Amplification Assay for Identification of Five Human Plasmodium Species in Malaysia.

    PubMed

    Lau, Yee-Ling; Lai, Meng-Yee; Fong, Mun-Yik; Jelip, Jenarun; Mahmud, Rohela

    2016-02-01

    The lack of rapid, affordable, and accurate diagnostic tests represents the primary hurdle affecting malaria surveillance in resource- and expertise-limited areas. Loop-mediated isothermal amplification (LAMP) is a sensitive, rapid, and cheap diagnostic method. Five species-specific LAMP assays were developed based on 18S rRNA gene. Sensitivity and specificity of LAMP results were calculated as compared with microscopic examination and nested polymerase chain reaction. LAMP reactions were highly sensitive with the detection limit of one copy for Plasmodium vivax, Plasmodium falciparum, and Plasmodium malariae and 10 copies for Plasmodium knowlesi and Plasmodium ovale. LAMP positively detected all human malaria species in all positive samples (N = 134; sensitivity = 100%) within 35 minutes. All negative samples were not amplified by LAMP (N = 67; specificity = 100%). LAMP successfully detected two samples with very low parasitemia. LAMP may offer a rapid, simple, and reliable test for the diagnosis of malaria in areas where malaria is prevalent.

  14. Fiber-optic immuno-biosensor for rapid and accurate detection of nerve growth factor in human blood.

    PubMed

    Tang, Liang; Cha, Yong-Mei; Li, Hongmei; Chen, Peng-Sheng; Lin, Shien-Fong

    2006-01-01

    An accurate and rapid assay of cardiac nerve growth factor (NGF) levels in blood can provide physicians with critical information regarding myocardial injury and neural remodeling in cardiac tissues to identify patients at risk of impending heart attack, thereby enabling them to receive appropriate lifesaving treatment more quickly. Currently used assay methods, such as enzyme-linked immunosorbent assay (ELISA), are usually time-consuming (hours to days), expensive and technically complicated. In this paper, we described the development and clinical study of a rapid and sensitive method for detection and quantification of NGF in human blood plasma. This method utilizes a fiber-optic, immuno-biosensing system which performs a fluorophore-mediated sandwich immunoassay on the surface of an optical fiber. Physiological concentrations of NGF could be quantified in both buffer and human blood plasma samples within 5 minutes. The NGF concentrations determined by the fiberoptic sensor were comparable to those by the gold standard, ELISA. Preliminary study of NGF assay in cardiac patient plasma samples showed a great potential of the fiber-optic sensor as a rapid diagnostic and prognostic tool in clinical applications.

  15. FEL-accelerator related diagnostics

    SciTech Connect

    Kevin Jordan; David Douglas; Stephen V. Benson; Pavel Evtuschenko

    2007-08-02

    Free Electron Lasers (FEL) present a unique set of beam parameters to the diagnostics suite. The FEL requires characterization of the full six dimensional phase space of the electron beam at the wiggler and accurate alignment of the electron beam to the optical mode of the laser. In addition to the FEL requirements on the diagnostics suite, the Jefferson Lab FEL is operated as an Energy Recovered Linac (ERL) which imposes additional requirements on the diagnostics. The ERL aspect of the Jefferson Lab FEL requires that diagnostics operate over a unique dynamic range and operate with simultaneous transport of the accelerated and energy recovered beams. This talk will present how these challenges are addressed at the Jefferson Lab FEL.

  16. Rapid diagnostic tests for malaria.

    PubMed

    Visser, Theodoor; Daily, Jennifer; Hotte, Nora; Dolkart, Caitlin; Cunningham, Jane; Yadav, Prashant

    2015-12-01

    Maintaining quality, competitiveness and innovation in global health technology is a constant challenge for manufacturers, while affordability, access and equity are challenges for governments and international agencies. In this paper we discuss these issues with reference to rapid diagnostic tests for malaria. Strategies to control and eliminate malaria depend on early and accurate diagnosis. Rapid diagnostic tests for malaria require little training and equipment and can be performed by non-specialists in remote settings. Use of these tests has expanded significantly over the last few years, following recommendations to test all suspected malaria cases before treatment and the implementation of an evaluation programme to assess the performance of the malaria rapid diagnostic tests. Despite these gains, challenges exist that, if not addressed, could jeopardize the progress made to date. We discuss recent developments in rapid diagnostic tests for malaria, highlight some of the challenges and provide suggestions to address them.

  17. Optical assay for biotechnology and clinical diagnosis.

    PubMed

    Moczko, Ewa; Cauchi, Michael; Turner, Claire; Meglinski, Igor; Piletsky, Sergey

    2011-08-01

    In this paper, we present an optical diagnostic assay consisting of a mixture of environmental-sensitive fluorescent dyes combined with multivariate data analysis for quantitative and qualitative examination of biological and clinical samples. The performance of the assay is based on the analysis of spectrum of the selected fluorescent dyes with the operational principle similar to electronic nose and electronic tongue systems. This approach has been successfully applied for monitoring of growing cell cultures and identification of gastrointestinal diseases in humans.

  18. Molecular diagnostic methods for invasive fungal disease: the horizon draws nearer?

    PubMed

    Halliday, C L; Kidd, S E; Sorrell, T C; Chen, S C-A

    2015-04-01

    Rapid, accurate diagnostic laboratory tests are needed to improve clinical outcomes of invasive fungal disease (IFD). Traditional direct microscopy, culture and histological techniques constitute the 'gold standard' against which newer tests are judged. Molecular diagnostic methods, whether broad-range or fungal-specific, have great potential to enhance sensitivity and speed of IFD diagnosis, but have varying specificities. The use of PCR-based assays, DNA sequencing, and other molecular methods including those incorporating proteomic approaches such as matrix-assisted laser desorption ionisation-time of flight mass spectroscopy (MALDI-TOF MS) have shown promising results. These are used mainly to complement conventional methods since they require standardisation before widespread implementation can be recommended. None are incorporated into diagnostic criteria for defining IFD. Commercial assays may assist standardisation. This review provides an update of molecular-based diagnostic approaches applicable to biological specimens and fungal cultures in microbiology laboratories. We focus on the most common pathogens, Candida and Aspergillus, and the mucormycetes. The position of molecular-based approaches in the detection of azole and echinocandin antifungal resistance is also discussed.

  19. Beyond skin testing: state of the art and new horizons in food allergy diagnostic testing.

    PubMed

    Caubet, Jean-Christoph; Sampson, Hugh A

    2012-02-01

    Food allergy affects approximately 1% to 10.8% of the general population, and its prevalence seems to be increasing. An accurate diagnosis is particularly important because a misdiagnosis could lead to life-threatening reactions or to unnecessary restrictive diets. However, allergy tests currently used in clinical practice have limited accuracy, and an oral food challenge, considered as the gold standard, is often required to confirm or exclude a food allergy. This article reviews several promising novel approaches for the diagnosis of food allergy, such as new molecular diagnostic technologies and functional assays, along with their potential clinical applications.

  20. Microarray Technologies in Fungal Diagnostics.

    PubMed

    Rupp, Steffen

    2017-01-01

    Microarray technologies have been a major research tool in the last decades. In addition they have been introduced into several fields of diagnostics including diagnostics of infectious diseases. Microarrays are highly parallelized assay systems that initially were developed for multiparametric nucleic acid detection. From there on they rapidly developed towards a tool for the detection of all kind of biological compounds (DNA, RNA, proteins, cells, nucleic acids, carbohydrates, etc.) or their modifications (methylation, phosphorylation, etc.). The combination of closed-tube systems and lab on chip devices with microarrays further enabled a higher automation degree with a reduced contamination risk. Microarray-based diagnostic applications currently complement and may in the future replace classical methods in clinical microbiology like blood cultures, resistance determination, microscopic and metabolic analyses as well as biochemical or immunohistochemical assays. In addition, novel diagnostic markers appear, like noncoding RNAs and miRNAs providing additional room for novel nucleic acid based biomarkers. Here I focus an microarray technologies in diagnostics and as research tools, based on nucleic acid-based arrays.

  1. Evaluation of Direct Colorimetric MTT Assay for Rapid Detection of Rifampicin and Isoniazid Resistance in Mycobacterium tuberculosis

    PubMed Central

    Woldemeskel, Dawit; Gessesse, Amare

    2016-01-01

    With the spread of multidrug-resistant tuberculosis (MDR-TB) strains there is an increasing need for new accurate and cost-effective methods for a rapid diagnostic and drug susceptibility testing (DST), particularly in low-income countries where tuberculosis is hyperendemic. A colorimetric assay using 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT) has been suggested as a promising method for DST, especially to rifampicin. In this study, we standardized and evaluated the MTT assay for a rapid direct detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis strains from sputum specimens using Lowenstein-Jensen (LJ) culture medium as a gold standard. The MTT assay sensitivity, specificity, positive and negative predictive values for rifampicin were 100%, 86%, 100%, 99%, respectively. For isoniazid, the MTT assay had a 100% sensitivity, specificity, positive and negative predictive values. Interestingly, the MTT assay gave interpretable results within two weeks for 94% of the samples compared to 7–14 weeks for LJ media. Overall, an excellent agreement was observed between MTT assay and LJ proportion method (Kappa, 0.91 for rifampicin and 1.00 for isoniazid). In conclusion, the direct colorimetric MTT assay simultaneously detects susceptible and resistant strains of M. tuberculosis within three weeks. It significantly shortens the time required to obtain a DST result and could be a reliable alternative method for rapid detection of drug-resistant TB strains in high-TB-burden resource-limited settings. PMID:28030634

  2. Integrated process for the purification and immobilization of the envelope protein domain III of dengue virus type 2 expressed in Rachiplusia nu larvae and its potential application in a diagnostic assay.

    PubMed

    Smith, María Emilia; Targovnik, Alexandra Marisa; Cerezo, Julieta; Morales, María Alejandra; Miranda, María Victoria; Talou, Julián Rodríguez

    2016-11-23

    Dengue incidence has grown dramatically in the last years, with about 40% of the world population at risk of infection. Recently, a vaccine developed by Sanofi Pasteur has been registered, but only in a few countries. Moreover, specific antiviral drugs are not available. Thus, an efficient and accurate diagnosis is important for disease management. To develop a low-cost immunoassay for dengue diagnosis, in the present study we expressed the envelope protein domain III of dengue virus type 2 in Rachiplusia nu larvae by infection with a recombinant baculovirus. The antigen was expressed as a fusion to hydrophobin I (DomIIIHFBI) to easily purify it by an aqueous two-phase system (ATPS) and to efficiently immobilize it in immunoassay plates. A high level of recombinant DomIIIHFBI was obtained in R. nu, where yields reached 4.5 mg per g of larva. Also, we were able to purify DomIIIHFBI by an ATPS with 2% of Triton X-114, reaching a yield of 73% and purity higher than 80% in a single purification step. The recombinant DomIIIHFBI was efficiently immobilized in hydrophobic surface plates. The immunoassay we developed with the immobilized antigen was able to detect IgG specific for dengue virus type 2 in serum samples and not for other serotypes.

  3. A guide for clinicians in the evaluation of emerging molecular diagnostics for newly diagnosed prostate cancer.

    PubMed

    Canfield, Steven E; Kibel, Adam S; Kemeter, Michael J; Febbo, Phillip G; Lawrence, H Jeffrey; Moul, Judd W

    2014-01-01

    Prostate-specific antigen (PSA) screening is associated with a decline in prostate cancer-related mortality. However, screening has also led to overdiagnosis and overtreatment of clinically insignificant tumors. Recently, certain national guidelines (eg, US Preventive Services Task Force) have recommended against PSA screening, which may lead to a reverse-stage migration. Although many prostate tumors are indolent at presentation, others are aggressive and are appropriate targets for treatment interventions. Utilization of molecular markers may improve our ability to measure tumor biology and allow better discrimination of indolent and aggressive tumors at diagnosis. Many emerging commercial molecular diagnostic assays have been designed to provide more accurate risk stratification for newly diagnosed prostate cancer. Unfamiliarity with molecular diagnostics may make it challenging for some clinicians to navigate and interpret the medical literature to ascertain whether particular assays are appropriately developed and validated for clinical use. Herein, the authors provide a framework for practitioners to use when assessing new tissue-based molecular assays. This review outlines aspects of assay development, clinical and analytic validation and clinical utility studies, and regulatory issues, which collectively determine whether tests (1) are actionable for specific clinical indications, (2) measurably influence treatment decisions, and (3) are sufficiently validated to warrant incorporation into clinical practice.

  4. Development of an albumin copper binding (ACuB) assay to detect ischemia modified albumin.

    PubMed

    Eom, Ji-Eun; Lee, Eunyoung; Jeon, Kyung-Hwa; Sim, Jeongeun; Suh, Minah; Jhon, Gil-Ja; Kwon, Youngjoo

    2014-01-01

    Myocardial ischemia (MI) induces many changes in the body, including pH decrease and electrolyte imbalance. No obvious symptoms of MI appear until irreversible cellular injuries occur. Since early treatment is critical for recovery from ischemia, the development of reliable diagnostic tool is demanded to detect the early ischemic status. Ischemia modified albumin (IMA), formed by cleavage of the last two amino acids of the human serum albumin (HSA) N-terminus, has been considered so far as the most trustworthy and accurate marker for the investigation of ischemia. IMA levels are elevated in plasma within a few minutes of ischemic onset, and may last for up to 6 h. In the present study, we developed a novel assay for the examination of IMA levels to ameliorate the known albumin cobalt binding (ACB) test established previously. We observed a stronger copper ion bound to the HSA N-terminal peptide than cobalt ion by HPLC and ESI-TOF mass spectrometric analyses. The copper ion was employed with lucifer yellow (LY), a copper-specific reagent to develop a new albumin copper binding (ACuB) assay. The parameters capable of affecting the assay results were optimized, and the finally-optimized ACuB assay was validated. The result of the IMA level measurement in normal versus stroke rat serum suggests that the ACuB assay is likely to be a reliable and sensitive method for the detection of ischemic states.

  5. Fluorescence-based assays for in vitro analysis of cell adhesion and migration.

    PubMed

    Spessotto, Paola; Lacrima, Katia; Nicolosi, Pier Andrea; Pivetta, Eliana; Scapolan, Martina; Perris, Roberto

    2009-01-01

    Cell adhesion and cell migration are two primary cellular phenomena for which in vitro approaches may be exploited to effectively dissect the individual events and underlying molecular mechanisms. The use of assays dedicated to the analysis of cell adhesion and migration in vitro also afford an efficient way of conducting larger basic and applied research screenings on the factors affecting these processes and are potentially exploitable in the context of routine diagnostic, prognostic, and predictive tests in the biological and medical fields. Therefore, there is a longstanding continuum in the interest in devising more rationale such assays and major contributions in this direction have been provided by the advent of procedures based on fluorescence cell tagging, the design of instruments capable of detecting fluorescent signals with high sensitivity, and informatic tools allowing sophisticated elaboration of data generated through these instruments. In this report, we describe three representative fluorescence-based model assays for the qualitative and quantitative assessment of cell adhesion and cell locomotion in static and dynamic conditions. The assays are easily performed, accurate and reproducible, and can be automated for high-to-medium throughput screenings of cell behavior in vitro. Performance of the assays involves the use of certain dedicated disposable accessories, which are commercially available, and a few instruments that, due to their versatility, can be regarded as constituents of a more generic laboratory setup.

  6. Accurate Evaluation of Quantum Integrals

    NASA Technical Reports Server (NTRS)

    Galant, D. C.; Goorvitch, D.; Witteborn, Fred C. (Technical Monitor)

    1995-01-01

    Combining an appropriate finite difference method with Richardson's extrapolation results in a simple, highly accurate numerical method for solving a Schrodinger's equation. Important results are that error estimates are provided, and that one can extrapolate expectation values rather than the wavefunctions to obtain highly accurate expectation values. We discuss the eigenvalues, the error growth in repeated Richardson's extrapolation, and show that the expectation values calculated on a crude mesh can be extrapolated to obtain expectation values of high accuracy.

  7. Dried plasma spots in the diagnosis of tuberculosis: IP-10 release assay on filter paper

    PubMed Central

    Aabye, Martine G.; Latorre, Irene; Diaz, Jessica; Maldonado, Jose; Mialdea, Irene; Eugen-Olsen, Jesper; Ravn, Pernille; Dominguez, Jose; Ruhwald, Morten

    2013-01-01

    Interferon (IFN)-γ release assays (IGRAs) are probably the most accurate tests for the detection of latent Mycobacterium tuberculosis infection, but IGRAs are labour intensive and the transport of samples over longer distances is difficult. IFN-γ-induced protein (IP)-10 is expressed at 100-fold higher levels than IFN-γ, and IP-10 release assays have comparable performance to IGRAs. The aim of this study was to explore the diagnostic potential of a novel IP-10 release assay based on dried plasma spots (DPS). The presence of IP-10 and IFN-γ was determined in plasma and in DPS by ELISA. Diagnostic algorithms for plasma and DPS tests for IP-10 were developed on a training cohort comprising 60 tuberculosis (TB) patients and 59 healthy controls. Diagnostic accuracy was assessed in a validation cohort comprising 78 TB patients and 98 healthy controls. Plasma was measured in Spain and DPS samples were sent to Denmark using the conventional postal service for analysis. IP-10 was readily detectable in both plasma and DPS, and correlation was excellent (r2 = 0.95). QuantiFERON-TB Gold In-Tube (QFT-TB) and IP-10 in DPS and plasma rendered comparable sensitivity (78%, 82% and 84%, respectively), specificity (100%, 97% and 97%, respectively) and indeterminate rates (p>0.55). The DPS-based IP-10 test has comparable diagnostic accuracy to the QFT-TB and samples can be sent via conventional mail over long distances for analysis without affecting the results. PMID:23349445

  8. Calibration issues for neutron diagnostics

    SciTech Connect

    Sadler, G.J.; Adams, J.M.; Barnes, C.W.

    1997-12-01

    The performance of diagnostic systems are limited by their weakest constituents, including their calibration issues. Neutron diagnostics are notorious for problems encountered while determining their absolute calibrations, due mainly to the nature of the neutron transport problem. In order to facilitate the determination of an accurate and precise calibration, the diagnostic design should be such as to minimize the scattered neutron flux. ITER will use a comprehensive set of neutron diagnostics--comprising radial and vertical neutron cameras, neutron spectrometers, a neutron activation system and internal and external fission chambers--to provide accurate measurements of fusion power and power densities as a function of time. The calibration of such an important diagnostic system merits careful consideration. Some thoughts have already been given to this subject during the conceptual design phase in relation to the time-integrated neutron activation and time-dependent neutron yield monitors. However, no overall calibration strategy has been worked out so far. This paper represents a first attempt to address this vital issue. Experience gained from present large tokamaks (JET, TFTR and JT60U) and proposals for ITER are reviewed. The need to use a 14-MeV neutron generator as opposed to radioactive sources for in-situ calibration of D-T diagnostics will be stressed. It is clear that the overall absolute determination of fusion power will have to rely on a combination of nuclear measuring techniques, for which the provision of accurate and independent calibrations will constitute an ongoing process as ITER moves from one phase of operation to the next.

  9. Validity of a PCR assay in CSF for the diagnosis of neurocysticercosis

    PubMed Central

    Campoverde, Alfredo; Romo, Matthew L.; García, Lorena; Piedra, Luis M.; Pacurucu, Mónica; López, Nelson; Aguilar, Jenner; López, Sebastian; Vintimilla, Luis C.; Toral, Ana M.; Peña-Tapia, Pablo

    2017-01-01

    Objective: To prospectively evaluate the validity of a PCR assay in CSF for the diagnosis of neurocysticercosis (NC). Methods: We conducted a multicenter, prospective case-control study, recruiting participants from 5 hospitals in Cuenca, Ecuador, from January 2015 to February 2016. Cases fulfilled validated diagnostic criteria for NC. For each case, a neurosurgical patient who did not fulfill the diagnostic criteria for NC was selected as a control. CT and MRI, as well as a CSF sample, were collected from both cases and controls. The diagnostic criteria to identify cases were used as a reference standard. Results: Overall, 36 case and 36 control participants were enrolled. PCR had a sensitivity of 72.2% (95% confidence interval [CI] 54.8%–85.8%) and a specificity of 100.0% (95% CI 90.3%–100.0%). For parenchymal NC, PCR had a sensitivity of 42.9% (95% CI 17.7%–71.1%), and for extraparenchymal NC, PCR had a sensitivity of 90.9% (95% CI 70.8%–98.9%). Conclusions: This study demonstrated the usefulness of this PCR assay in CSF for the diagnosis of NC. PCR may be particularly helpful for diagnosing extraparenchymal NC when neuroimaging techniques have failed. Classification of evidence: This study provides Class III evidence that CSF PCR can accurately identify patients with extraparenchymal NC. PMID:28105460

  10. Ultrasensitive microanalytical diagnostic methods for rickettsial pathogens

    SciTech Connect

    Hatch, A. V.

    2012-03-01

    A strategic CRADA was established between Sandia National Laboratories (SNL) and the University of Texas Medical Branch (UTMB) at Galveston to address pressing needs for US protection against biological weapons of mass destruction (WMD) and emerging infectious diseases. The combination of unique expertise and facilities at UTMB and SNL enabled interdisciplinary research efforts in the development of rapid and accurate diagnostic methods for early detection of trace priority pathogen levels. Outstanding postdoctoral students were also trained at both institutions to help enable the next generation of scientists to tackle the challenging interdisciplinary problems in the area of biodefense and emerging infectious diseases. Novel approaches to diagnostics were developed and the both the speed of assays as well as the detection sensitivity were improved by over an order of magnitude compared to traditional methods. This is a significant step toward more timely and specific detection of dangerous infections. We developed in situ polymerized porous polymer monoliths that can be used as (1) size exclusion elements for capture and processing of rickettsial infected cells from a sample, (2) photopatternable framework for grafting high densities of functionalized antibodies/fluorescent particles using novel monolith chemistry. Grafting affinity reagents specific to rickettsial particles enables rapid, ultra-sensitive assays by overcoming transport limitations of traditional planar assay approaches. We have selectively trapped particles and bacteria at the cell trap and have also detected picomolar mouse IL-6 captured with only 20 minutes total incubation times using the densely patterned monolith framework. As predicted, the monolith exhibits >10x improvements in both capture speed and capture density compared to traditional planar approaches. The most significant advancements as part of this CRADA is the optimization of techniques allowing the detection of <10 rickettsial

  11. Accurate detection and quantitation of heteroplasmic mitochondrial point mutations by pyrosequencing.

    PubMed

    White, Helen E; Durston, Victoria J; Seller, Anneke; Fratter, Carl; Harvey, John F; Cross, Nicholas C P

    2005-01-01

    Disease-causing mutations in mitochondrial DNA (mtDNA) are typically heteroplasmic and therefore interpretation of genetic tests for mitochondrial disorders can be problematic. Detection of low level heteroplasmy is technically demanding and it is often difficult to discriminate between the absence of a mutation or the failure of a technique to detect the mutation in a particular tissue. The reliable measurement of heteroplasmy in different tissues may help identify individuals who are at risk of developing specific complications and allow improved prognostic advice for patients and family members. We have evaluated Pyrosequencing technology for the detection and estimation of heteroplasmy for six mitochondrial point mutations associated with the following diseases: Leber's hereditary optical neuropathy (LHON), G3460A, G11778A, and T14484C; mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS), A3243G; myoclonus epilepsy with ragged red fibers (MERRF), A8344G, and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP)/Leighs: T8993G/C. Results obtained from the Pyrosequencing assays for 50 patients with presumptive mitochondrial disease were compared to those obtained using the commonly used diagnostic technique of polymerase chain reaction (PCR) and restriction enzyme digestion. The Pyrosequencing assays provided accurate genotyping and quantitative determination of mutational load with a sensitivity and specificity of 100%. The MELAS A3243G mutation was detected reliably at a level of 1% heteroplasmy. We conclude that Pyrosequencing is a rapid and robust method for detecting heteroplasmic mitochondrial point mutations.

  12. Microdroplet chain array for cell migration assays.

    PubMed

    Ma, Yan; Pan, Jian-Zhang; Zhao, Shi-Ping; Lou, Qi; Zhu, Ying; Fang, Qun

    2016-11-29

    Establishing cell migration assays in multiple different microenvironments is important in the study of tissue repair and regeneration, cancer progression, atherosclerosis, and arthritis. In this work, we developed a miniaturized and massive parallel microfluidic platform for multiple cell migration assays combining the traditional membrane-based cell migration technique and the droplet-based microfluidic technique. Nanoliter-scale droplets are flexibly assembled as building blocks based on a porous membrane to form microdroplet chains with diverse configurations for different assay modes. Multiple operations including in-droplet 2D/3D cell culture, cell co-culture and cell migration induced by a chemoattractant concentration gradient in droplet chains could be flexibly performed with reagent consumption in the nanoliter range for each assay and an assay scale-up to 81 assays in parallel in one microchip. We have applied the present platform to multiple modes of cell migration assays including the accurate cell migration assay, competitive cell migration assay, biomimetic chemotaxis assay, and multifactor cell migration assay based on the organ-on-a-chip concept, for demonstrating its versatility, applicability, and potential in cell migration-related research.

  13. 21 CFR 864.7250 - Erythropoietin assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... erythropoietin (an enzyme that regulates the production of red blood cells) in serum or urine. This assay provides diagnostic information for the evaluation of erythrocytosis (increased total red cell mass) and anemia. (b) Classification. Class II. The special control for this device is FDA's “Document for...

  14. Salivary diagnostics

    PubMed Central

    Lee, J.M.; Garon, E.; Wong, D.T.

    2010-01-01

    The ability to monitor health status, disease onset and progression, and treatment outcome through non-invasive means is a most desirable goal in the health care promotion and delivery. There are three prerequisites to materialize this goal: specific biomarkers associated with a health or disease state; a non-invasive approach to detect and monitor the biomarkers; and the technologies to discriminate the biomarkers. A national initiative catalyzed by the National Institute of Dental & Craniofacial Research (NIDCR) has created a roadmap to achieve these goals through the use of oral fluids as the diagnostic medium to scrutinize the health and/or disease status of individuals. Progress has shown this is an ideal opportunity to bridge state of the art saliva-based biosensors, optimized to disease discriminatory salivary biomarkers, for diagnostic applications. Oral fluid being the ‘mirror of body’ is a perfect medium to be explored for health and disease surveillance. The translational applications and opportunities are enormous. PMID:19627522

  15. Molecular diagnostics and parasitic disease.

    PubMed

    Vasoo, Shawn; Pritt, Bobbi S

    2013-09-01

    Molecular parasitology represents an emerging field in microbiology diagnostics. Although most assays use nonstandardized, laboratory-developed methods, a few commercial systems have recently become available and are slowly being introduced into larger laboratories. In addition, a few methodologies show promise for use in field settings in which parasitic infections are endemic. This article reviews the available techniques and their applications to major parasitic diseases such as malaria, leishmaniasis, and trichomoniasis.

  16. High Frequency QRS ECG Accurately Detects Cardiomyopathy

    NASA Technical Reports Server (NTRS)

    Schlegel, Todd T.; Arenare, Brian; Poulin, Gregory; Moser, Daniel R.; Delgado, Reynolds

    2005-01-01

    High frequency (HF, 150-250 Hz) analysis over the entire QRS interval of the ECG is more sensitive than conventional ECG for detecting myocardial ischemia. However, the accuracy of HF QRS ECG for detecting cardiomyopathy is unknown. We obtained simultaneous resting conventional and HF QRS 12-lead ECGs in 66 patients with cardiomyopathy (EF = 23.2 plus or minus 6.l%, mean plus or minus SD) and in 66 age- and gender-matched healthy controls using PC-based ECG software recently developed at NASA. The single most accurate ECG parameter for detecting cardiomyopathy was an HF QRS morphological score that takes into consideration the total number and severity of reduced amplitude zones (RAZs) present plus the clustering of RAZs together in contiguous leads. This RAZ score had an area under the receiver operator curve (ROC) of 0.91, and was 88% sensitive, 82% specific and 85% accurate for identifying cardiomyopathy at optimum score cut-off of 140 points. Although conventional ECG parameters such as the QRS and QTc intervals were also significantly longer in patients than controls (P less than 0.001, BBBs excluded), these conventional parameters were less accurate (area under the ROC = 0.77 and 0.77, respectively) than HF QRS morphological parameters for identifying underlying cardiomyopathy. The total amplitude of the HF QRS complexes, as measured by summed root mean square voltages (RMSVs), also differed between patients and controls (33.8 plus or minus 11.5 vs. 41.5 plus or minus 13.6 mV, respectively, P less than 0.003), but this parameter was even less accurate in distinguishing the two groups (area under ROC = 0.67) than the HF QRS morphologic and conventional ECG parameters. Diagnostic accuracy was optimal (86%) when the RAZ score from the HF QRS ECG and the QTc interval from the conventional ECG were used simultaneously with cut-offs of greater than or equal to 40 points and greater than or equal to 445 ms, respectively. In conclusion 12-lead HF QRS ECG employing

  17. Point-of-Care Diagnostics in Low Resource Settings: Present Status and Future Role of Microfluidics

    PubMed Central

    Sharma, Shikha; Zapatero-Rodríguez, Julia; Estrela, Pedro; O’Kennedy, Richard

    2015-01-01

    The inability to diagnose numerous diseases rapidly is a significant cause of the disparity of deaths resulting from both communicable and non-communicable diseases in the developing world in comparison to the developed world. Existing diagnostic instrumentation usually requires sophisticated infrastructure, stable electrical power, expensive reagents, long assay times, and highly trained personnel which is not often available in limited resource settings. This review will critically survey and analyse the current lateral flow-based point-of-care (POC) technologies, which have made a major impact on diagnostic testing in developing countries over the last 50 years. The future of POC technologies including the applications of microfluidics, which allows miniaturisation and integration of complex functions that facilitate their usage in limited resource settings, is discussed The advantages offered by such systems, including low cost, ruggedness and the capacity to generate accurate and reliable results rapidly, are well suited to the clinical and social settings of the developing world. PMID:26287254

  18. Point-of-Care Diagnostics in Low Resource Settings: Present Status and Future Role of Microfluidics.

    PubMed

    Sharma, Shikha; Zapatero-Rodríguez, Julia; Estrela, Pedro; O'Kennedy, Richard

    2015-08-13

    The inability to diagnose numerous diseases rapidly is a significant cause of the disparity of deaths resulting from both communicable and non-communicable diseases in the developing world in comparison to the developed world. Existing diagnostic instrumentation usually requires sophisticated infrastructure, stable electrical power, expensive reagents, long assay times, and highly trained personnel which is not often available in limited resource settings. This review will critically survey and analyse the current lateral flow-based point-of-care (POC) technologies, which have made a major impact on diagnostic testing in developing countries over the last 50 years. The future of POC technologies including the applications of microfluidics, which allows miniaturisation and integration of complex functions that facilitate their usage in limited resource settings, is discussed The advantages offered by such systems, including low cost, ruggedness and the capacity to generate accurate and reliable results rapidly, are well suited to the clinical and social settings of the developing world.

  19. Evaluation of Raman spectroscopy in comparison to commonly performed dengue diagnostic tests

    NASA Astrophysics Data System (ADS)

    Khan, Saranjam; Ullah, Rahat; Khurram, Muhammad; Ali, Hina; Mahmood, Arshad; Khan, Ajmal; Ahmed, Mushtaq

    2016-09-01

    This study demonstrates the evaluation of Raman spectroscopy as a rapid diagnostic test in comparison to commonly performed tests for an accurate detection of dengue fever in human blood sera. Blood samples of 104 suspected dengue patients collected from Holy Family Hospital, Rawalpindi, Pakistan, have been used in this study. Out of 104 samples, 52 (50%) were positive based on immunoglobulin G (IgG), whereas 54 (52%) were positive based on immunoglobulin M (IgM) antibody tests. For the determination of the diagnostic capabilities of Raman spectroscopy, accuracy, sensitivity, specificity and false positive rate have been calculated in comparison to normally performed IgM and IgG captured enzyme-linked immunosorbent assay tests. Accuracy, precision, specificity, and sensitivity for Raman spectroscopy in comparison to IgM were found to be 66%, 70%, 72%, and 61%, whereas based on IgG they were 47%, 46%, 52%, and 43%, respectively.

  20. Principles and applications of polymerase chain reaction in medical diagnostic fields: a review

    PubMed Central

    Valones, Marcela Agne Alves; Guimarães, Rafael Lima; Brandão, Lucas André Cavalcanti; de Souza, Paulo Roberto Eleutério; de Albuquerque Tavares Carvalho, Alessandra; Crovela, Sergio

    2009-01-01

    Recent developments in molecular methods have revolutionized the detection and characterization of microorganisms in a broad range of medical diagnostic fields, including virology, mycology, parasitology, microbiology and dentistry. Among these methods, Polymerase Chain Reaction (PCR) has generated great benefits and allowed scientific advancements. PCR is an excellent technique for the rapid detection of pathogens, including those difficult to culture. Along with conventional PCR techniques, Real-Time PCR has emerged as a technological innovation and is playing an ever-increasing role in clinical diagnostics and research laboratories. Due to its capacity to generate both qualitative and quantitative results, Real-Time PCR is considered a fast and accurate platform. The aim of the present literature review is to explore the clinical usefulness and potential of both conventional PCR and Real-Time PCR assays in diverse medical fields, addressing its main uses and advances. PMID:24031310

  1. Rotorcraft Diagnostics

    NASA Technical Reports Server (NTRS)

    Haste, Deepak; Azam, Mohammad; Ghoshal, Sudipto; Monte, James

    2012-01-01

    Health management (HM) in any engineering systems requires adequate understanding about the system s functioning; a sufficient amount of monitored data; the capability to extract, analyze, and collate information; and the capability to combine understanding and information for HM-related estimation and decision-making. Rotorcraft systems are, in general, highly complex. Obtaining adequate understanding about functioning of such systems is quite difficult, because of the proprietary (restricted access) nature of their designs and dynamic models. Development of an EIM (exact inverse map) solution for rotorcraft requires a process that can overcome the abovementioned difficulties and maximally utilize monitored information for HM facilitation via employing advanced analytic techniques. The goal was to develop a versatile HM solution for rotorcraft for facilitation of the Condition Based Maintenance Plus (CBM+) capabilities. The effort was geared towards developing analytic and reasoning techniques, and proving the ability to embed the required capabilities on a rotorcraft platform, paving the way for implementing the solution on an aircraft-level system for consolidation and reporting. The solution for rotorcraft can he used offboard or embedded directly onto a rotorcraft system. The envisioned solution utilizes available monitored and archived data for real-time fault detection and identification, failure precursor identification, and offline fault detection and diagnostics, health condition forecasting, optimal guided troubleshooting, and maintenance decision support. A variant of the onboard version is a self-contained hardware and software (HW+SW) package that can be embedded on rotorcraft systems. The HM solution comprises components that gather/ingest data and information, perform information/feature extraction, analyze information in conjunction with the dependency/diagnostic model of the target system, facilitate optimal guided troubleshooting, and offer

  2. Detection of shigella in lettuce by the use of a rapid molecular assay with increased sensitivity

    PubMed Central

    Jiménez, Kenia Barrantes; McCoy², Clyde B.; Achí, Rosario

    2010-01-01

    A Multiplex Polymerase Chain Reaction (PCR) assay to be used as an alternative to the conventional culture method in detecting Shigella and enteroinvasive Escherichia coli (EIEC) virulence genes ipaH and ial in lettuce was developed. Efficacy and rapidity of the molecular method were determined as compared to the conventional culture. Lettuce samples were inoculated with different Shigella flexneri concentrations (from 10 CFU/ml to 107 CFU/ml). DNA was extracted directly from lettuce after inoculation (direct-PCR) and after an enrichment step (enrichment PCR). Multiplex PCR detection limit was 104CFU/ml, diagnostic sensitivity and specificity were 100% accurate. An internal amplification control (IAC) of 100 bp was used in order to avoid false negative results. This method produced results in 1 to 2 days while the conventional culture method required 5 to 6 days. Also, the culture method detection limit was 106 CFU/ml, diagnostic sensitivity was 53% and diagnostic specificity was 100%. In this study a Multiplex PCR method for detection of virulence genes in Shigella and EIEC was shown to be effective in terms of diagnostic sensitivity, detection limit and amount of time as compared to Shigella conventional culture. PMID:24031579

  3. Development, optimization, and validation of a Classical swine fever virus real-time reverse transcription polymerase chain reaction assay.

    PubMed

    Eberling, August J; Bieker-Stefanelli, Jill; Reising, Monica M; Siev, David; Martin, Barbara M; McIntosh, Michael T; Beckham, Tammy R

    2011-09-01

    Classical swine fever (CSF) is an economically devastating disease of pigs. Instrumental to the control of CSF is a well-characterized assay that can deliver a rapid, accurate diagnosis prior to the onset of clinical signs. A real-time fluorogenic-probe hydrolysis (TaqMan) reverse transcription polymerase chain reaction (RT-PCR) for CSF was developed by the United States Department of Agriculture (USDA) at the Plum Island Animal Disease Center (CSF PIADC assay) and evaluated for analytical and diagnostic sensitivity and specificity. A well-characterized panel including Classical swine fever virus (CSFV), Bovine viral diarrhea virus (BVDV), and Border disease virus (BDV) isolates was utilized in initial feasibility and optimization studies. The assay was initially designed and validated for use on the ABI 7900HT using the Qiagen QuantiTect® Probe RT-PCR chemistry. However, demonstrating equivalency with multiple one-step RT-PCR chemistries and PCR platforms increased the versatility of the assay. Limit of detection experiments indicated that the Qiagen QuantiTect® Multiplex (NoROX) and the Invitrogen SuperScript® III RT-PCR kits were consistently the most sensitive one-step chemistries for use with the CSF PIADC primer/probe set. Analytical sensitivity of the CSF PIADC assay ranged from <1-2.95 log(10) TCID(50)/ml on both the ABI 7900HT and ABI 7500 platforms. The CSF PIADC assay had 100% diagnostic sensitivity and specificity when tested on a panel of 152 clinical samples from the Dominican Republic and Colombia. The ability to perform this newly developed assay in 96-well formats provides an increased level of versatility for use in CSF surveillance programs.

  4. Molecular classification of cancer with the 92-gene assay in cytology and limited tissue samples

    PubMed Central

    Brachtel, Elena F.; Operaña, Theresa N.; Sullivan, Peggy S.; Kerr, Sarah E.; Cherkis, Karen A.; Schroeder, Brock E.; Dry, Sarah M.; Schnabel, Catherine A.

    2016-01-01

    Background Detailed molecular evaluation of cytology and limited tissue samples is increasingly becoming the standard for cancer care. Reproducible and accurate diagnostic approaches with reduced demands on cellularity are an ongoing unmet need. This study evaluated the performance of a 92-gene assay for molecular diagnosis of tumor type/subtype in cytology and limited tissue samples. Methods Clinical validation of accuracy for the 92-gene assay in limited tissue samples such as cytology cell blocks, core biopsies and small excisions was conducted in a blinded multi-institutional study (N = 109, 48% metastatic, 53% grade II and III). Analytical success rate and diagnostic utility were evaluated in a consecutive series of 644 cytology cases submitted for clinical testing. Results The 92-gene assay demonstrated 91% sensitivity (95% CI [0.84, 0.95]) for tumor classification, with high accuracy maintained irrespective of specimen type (100%, 92%, and 86% in FNA/cytology cell blocks, core biopsies, and small excisions, respectively; p = 0.26). The assay performed equally well for metastatic versus primary tumors (90% vs 93%, p = 0.73), and across histologic grades (100%, 90%, 89%, in grades I, II, and III, respectively; p = 0.75). In the clinical case series, a molecular diagnosis was reported in 87% of the 644 samples, identifying 23 different tumor types and allowing for additional mutational analysis in selected cases. Conclusions These findings demonstrate high accuracy and analytical success rate of the 92-gene assay, supporting its utility in the molecular diagnosis of cancer for specimens with limited tissue. PMID:27034010

  5. Setting a standard: the limulus amebocyte lysate assay and the assessment of microbial contamination on spacecraft surfaces.

    PubMed

    Morris, Heather C; Monaco, Lisa A; Steele, Andrew; Wainwright, Norm

    2010-10-01

    Historically, colony-forming units as determined by plate cultures have been the standard unit for microbiological analysis of environmental samples, medical diagnostics, and products for human use. However, the time and materials required make plate cultures expensive and potentially hazardous in the closed environments of future NASA missions aboard the International Space Station and missions to other Solar System targets. The Limulus Amebocyte Lysate (LAL) assay is an established method for ensuring the sterility and cleanliness of samples in the meat-packing and pharmaceutical industries. Each of these industries has verified numerical requirements for the correct interpretation of results from this assay. The LAL assay is a rapid, point-of-use, verified assay that has already been approved by NASA Planetary Protection as an alternate, molecular method for the examination of outbound spacecraft. We hypothesize that standards for molecular techniques, similar to those used by the pharmaceutical and meat-packing industries, need to be set by space agencies to ensure accurate data interpretation and subsequent decision making. In support of this idea, we present research that has been conducted to relate the LAL assay to plate cultures, and we recommend values obtained from these investigations that could assist in interpretation and analysis of data obtained from the LAL assay.

  6. Oral vs. salivary diagnostics

    NASA Astrophysics Data System (ADS)

    Marques, Joana; Corby, Patricia M.; Barber, Cheryl A.; Abrams, William R.; Malamud, Daniel

    2015-05-01

    The field of "salivary diagnostics" includes studies utilizing samples obtained from a variety of sources within the oral cavity. These samples include; whole unstimulated saliva, stimulated whole saliva, duct saliva collected directly from the parotid, submandibular/sublingual glands or minor salivary glands, swabs of the buccal mucosa, tongue or tonsils, and gingival crevicular fluid. Many publications state "we collected saliva from subjects" without fully describing the process or source of the oral fluid. Factors that need to be documented in any study include the time of day of the collection, the method used to stimulate and collect the fluid, and how much fluid is being collected and for how long. The handling of the oral fluid during and post-collection is also critical and may include addition of protease or nuclease inhibitors, centrifugation, and cold or frozen storage prior to assay. In an effort to create a standard protocol for determining a biomarker's origin we carried out a pilot study collecting oral fluid from 5 different sites in the mouth and monitoring the concentrations of pro- and anti-inflammatory cytokines detected using MesoScaleDiscovery (MSD) electrochemiluminesence assays. Our data suggested that 3 of the cytokines are primarily derived from the submandibular gland, while 7 of the cytokines come from a source other than the major salivary glands such as the minor salivary glands or cells in the oral mucosae. Here we review the literature on monitoring biomarkers in oral samples and stress the need for determining the blood/saliva ratio when a quantitative determination is needed and suggest that the term oral diagnostic be used if the source of an analyte in the oral cavity is unknown.

  7. In-line sensor for accurate rf power measurements

    NASA Astrophysics Data System (ADS)

    Gahan, D.; Hopkins, M. B.

    2005-10-01

    An in-line sensor has been constructed with 50Ω characteristic impedance to accurately measure rf power dissipated in a matched or unmatched load with a view to being implemented as a rf discharge diagnostic. The physical construction and calibration technique are presented. The design is a wide band, hybrid directional coupler/current-voltage sensor suitable for fundamental and harmonic power measurements. A comparison with a standard wattmeter using dummy load impedances shows that this in-line sensor is significantly more accurate in mismatched conditions.

  8. In-line sensor for accurate rf power measurements

    SciTech Connect

    Gahan, D.; Hopkins, M.B.

    2005-10-15

    An in-line sensor has been constructed with 50 {omega} characteristic impedance to accurately measure rf power dissipated in a matched or unmatched load with a view to being implemented as a rf discharge diagnostic. The physical construction and calibration technique are presented. The design is a wide band, hybrid directional coupler/current-voltage sensor suitable for fundamental and harmonic power measurements. A comparison with a standard wattmeter using dummy load impedances shows that this in-line sensor is significantly more accurate in mismatched conditions.

  9. New Insights into Butyrylcholinesterase Activity Assay: Serum Dilution Factor as a Crucial Parameter

    PubMed Central

    Jońca, Joanna; Żuk, Monika; Wasąg, Bartosz; Janaszak-Jasiecka, Anna; Lewandowski, Krzysztof; Wielgomas, Bartosz; Waleron, Krzysztof; Jasiecki, Jacek

    2015-01-01

    Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman’s method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay. PMID:26444431

  10. 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.

  11. Assuring reliable performance of antibiotic assay media.

    PubMed

    Freeman, K A; Johnson, D P; Garth, M A

    1977-11-01

    The Microbiological Assay Branch of the National Center for Antibiotics Analysis assays over 100,000 samples of antibiotic products annually, using more than 1000 Ib dehydrated media. The media must be consistently dependable to produce accurate, reliable test results. To assure that the supply of media will meet the established requirements, each lot before purchase is subjected to a series of trials designed to examine growth support, sensitivity, and behavioral and physical factors. Actual antibiotic assays are conducted with the test medium, and performance is rated against a control medium. Controls on the system reduce the variables to allow appraisal of the medium itself.

  12. Microbiologic assay of space hardware.

    NASA Technical Reports Server (NTRS)

    Favero, M. S.

    1971-01-01

    Review of the procedures used in the microbiological examination of space hardware. The general procedure for enumerating aerobic and anaerobic microorganisms and spores is outlined. Culture media and temperature-time cycles used for incubation are reviewed, along with assay systems designed for the enumeration of aerobic and anaerobic spores. The special problems which are discussed are involved in the precise and accurate enumeration of microorganisms on surfaces and in the neutralization of viable organisms buried inside solid materials that could be released to a planet's surface if the solid should be fractured. Special attention is given to sampling procedures including also the indirect techniques of surface assays of space hardware such as those using detachable or fallout strips. Some data on comparative levels of microbial contamination on lunar and planetary spacecraft are presented.

  13. Development of an Assay for the Detection of PrPres in Blood and Urine Based on PMCA Assay and ELISA Methods

    DTIC Science & Technology

    2007-09-01

    diagnostic assay. The assay in the pilot form is developed with plasma from hamsters infected with the 263K strain of scrapie . The same assay can be...adapted to human PrP test. In this funding period we completed the optimization of the conditions for proteinase K (PK) digestion of PrPres in scrapie ...alone10. We have been developing a prototype assay for TSE infection using hamsters infected with the 263K stain of scrapie . In our current assay

  14. Malignant Catarrhal Fever: Understanding Molecular Diagnostics in Context of Epidemiology

    PubMed Central

    Li, Hong; Cunha, Cristina W.; Taus, Naomi S.

    2011-01-01

    Malignant catarrhal fever (MCF) is a frequently fatal disease, primarily of ruminants, caused by a group of gammaherpesviruses. Due to complexities of pathogenesis and epidemiology in various species, which are either clinically-susceptible or reservoir hosts, veterinary clinicians face significant challenges in laboratory diagnostics. The recent development of specific assays for viral DNA and antibodies has expanded and improved the inventory of laboratory tests and opened new opportunities for use of MCF diagnostics. Issues related to understanding and implementing appropriate assays for specific diagnostic needs must be addressed in order to take advantage of molecular diagnostics in the laboratory. PMID:22072925

  15. Instrumentation and diagnostics

    SciTech Connect

    Nakaishi, C.V.; Bedick, R.C.

    1990-12-01

    This Technology Status Report describes research and accomplishments for the Instrumentation and Diagnostics (I D) Projects within the Advanced Research and Technology Development (AR TD) Program of the United States Department of Energy (DOE) Office of Fossil Energy (FE). Process understanding and control can be improved through the development of advanced instrumentation and diagnostics. The thrust of the I D Projects is to further develop existing measurement and control techniques for application to advanced coal-based technologies. Project highlights are: an inductively coupled plasma (ICP) instrument has been developed to analyze trace elements in gasification and combustion process streams. An in situ two-color Mie scattering technique with LSS can simultaneously measure the size, velocity, and elemental composition of coal particles during combustion. A high-temperature, fluorescence thermometry technique has accurately measured gas temperatures during field testing in combustion and gasification environments. Expert systems have been developed to improve the control of advanced coal-based processes. Capacitance flowmeters were developed to determine the mass flowrate, solid volume fraction, and particle velocities of coal slurries. 32 refs., 9 figs.

  16. The development, evaluation and performance of molecular diagnostics for detection of Mycobacterium tuberculosis.

    PubMed

    Bates, Matthew; Zumla, Alimuddin

    2016-01-01

    The unique pathogenesis of tuberculosis (TB) poses several barriers to the development of accurate diagnostics: a) the establishment of life-long latency by Mycobacterium tuberculosis (M.tb) after primary infection confounds the development of classical antibody or antigen based assays; b) our poor understanding of the molecular pathways that influence progression from latent to active disease; c) the intracellular nature of M.tb infection in tissues means that M.tb and/or its components, are not readily detectable in peripheral specimens; and d) the variable presence of M.tb bacilli in specimens from patients with extrapulmonary TB or children. The literature on the current portfolio of molecular diagnostics tests for TB is reviewed here and the developmental pipeline is summarized. Also reviewed are data from recently published operational research on the GeneXpert MTB/RIF assay and discussed are the lessons that can be taken forward for the design of studies to evaluate the impact of TB diagnostics.

  17. Important hemoprotozoan diseases of livestock: Challenges in current diagnostics and therapeutics: An update

    PubMed Central

    Maharana, Biswa Ranjan; Tewari, Anup Kumar; Saravanan, Buddhi Chandrasekaran; Sudhakar, Naduvanahalli Rajanna

    2016-01-01

    Hemoprotozoan parasites pose a serious threat to the livestock population in terms of mortality, reduced milk yield and lowered draft power. Diagnosis of these diseases often poses a challenging task. Needless to say that impact of disease in health and productivity is huge though a fair economic assessment on the quantum of economic loss associated is yet to be worked out from India. The diagnosis of hemoprotozoan infections largely depends on various laboratory-based diagnostic methods as the clinical manifestations are often inconspicuous and non-specific. Traditional diagnostic methods rely on microscopical demonstration of infective stages in blood or tissue fluids. However, it is laborious, lesser sensitive, and cannot differentiate between morphologically similar organisms. Recent development in the technologies has opened new avenues for improvement in the accurate diagnosis of parasitic infections. Serological tests are simple, fast but lack specificity. With advent of molecular techniques, as DNA hybridization assays, polymerase chain reaction and its modifications ensure the detection of infection in the latent phase of the disease. Nucleic acid-based assays are highly sensitive, free from immunocompetence and can differentiate between morphologically similar parasites. With the advent of newer diagnostics complemented with traditional ones will be of huge help for targeted selective treatment with better chemotherapeutic agents. PMID:27284225

  18. Prospective comparison of the diagnostic potential of real-time PCR, double-sandwich enzyme-linked immunosorbent assay for galactomannan, and a (1-->3)-beta-D-glucan test in weekly screening for invasive aspergillosis in patients with hematological disorders.

    PubMed

    Kawazu, Masahito; Kanda, Yoshinobu; Nannya, Yasuhito; Aoki, Katsunori; Kurokawa, Mineo; Chiba, Shigeru; Motokura, Toru; Hirai, Hisamaru; Ogawa, Seishi

    2004-06-01

    The establishment of an optimal noninvasive method for diagnosing invasive aspergillosis (IA) is needed to improve the management of this life-threatening infection in patients with hematological disorders, and a number of noninvasive tests for IA that target different fungal components, including galactomannan, (1-->3)-beta-d-glucan (BDG), and Aspergillus DNA, have been developed. In this study, we prospectively evaluated the diagnostic potential of three noninvasive tests for IA that were used in a weekly screening strategy: the double-sandwich enzyme-linked immunosorbent assay (ELISA) for galactomannan (Platelia Aspergillus), a real-time PCR assay for Aspergillus DNA (GeniQ-Asper), and an assay for BDG (beta-glucan Wako). We analyzed 149 consecutive treatment episodes in 96 patients with hematological disorders who were at high risk for IA and diagnosed 9 proven IA cases, 2 probable IA cases, and 13 possible invasive fugal infections. In a receiver-operating characteristic (ROC) analysis, the area under the ROC curve was greatest for ELISA, using two consecutive positive results (0.97; P = 0.036 for ELISA versus PCR, P = 0.055 for ELISA versus BDG). Based on the ROC curve, the cutoff for the ELISA could be reduced to an optical density index (O.D.I.) of 0.6. With the use of this cutoff for ELISA and cutoffs for PCR and BDG that give a comparable level of specificity, the sensitivity/specificity/positive predictive value/negative predictive value of the ELISA and the PCR and BDG tests were 1.00/0.93/0.55/1.00, 0.55/0.93/0.40/0.96, and 0.55/0.93/0.40/0.96, respectively. In conclusion, among these weekly screening tests for IA, the double-sandwich ELISA test was the most sensitive at predicting the diagnosis of IA in high-risk patients with hematological disorders, using a reduced cutoff of 0.6 O.D.I.

  19. Prospective Comparison of the Diagnostic Potential of Real-Time PCR, Double-Sandwich Enzyme-Linked Immunosorbent Assay for Galactomannan, and a (1→3)-β-d-Glucan Test in Weekly Screening for Invasive Aspergillosis in Patients with Hematological Disorders

    PubMed Central

    Kawazu, Masahito; Kanda, Yoshinobu; Nannya, Yasuhito; Aoki, Katsunori; Kurokawa, Mineo; Chiba, Shigeru; Motokura, Toru; Hirai, Hisamaru; Ogawa, Seishi

    2004-01-01

    The establishment of an optimal noninvasive method for diagnosing invasive aspergillosis (IA) is needed to improve the management of this life-threatening infection in patients with hematological disorders, and a number of noninvasive tests for IA that target different fungal components, including galactomannan, (1→3)-β-d-glucan (BDG), and Aspergillus DNA, have been developed. In this study, we prospectively evaluated the diagnostic potential of three noninvasive tests for IA that were used in a weekly screening strategy: the double-sandwich enzyme-linked immunosorbent assay (ELISA) for galactomannan (Platelia Aspergillus), a real-time PCR assay for Aspergillus DNA (GeniQ-Asper), and an assay for BDG (β-glucan Wako). We analyzed 149 consecutive treatment episodes in 96 patients with hematological disorders who were at high risk for IA and diagnosed 9 proven IA cases, 2 probable IA cases, and 13 possible invasive fugal infections. In a receiver-operating characteristic (ROC) analysis, the area under the ROC curve was greatest for ELISA, using two consecutive positive results (0.97; P = 0.036 for ELISA versus PCR, P = 0.055 for ELISA versus BDG). Based on the ROC curve, the cutoff for the ELISA could be reduced to an optical density index (O.D.I.) of 0.6. With the use of this cutoff for ELISA and cutoffs for PCR and BDG that give a comparable level of specificity, the sensitivity/specificity/positive predictive value/negative predictive value of the ELISA and the PCR and BDG tests were 1.00/0.93/0.55/1.00, 0.55/0.93/0.40/0.96, and 0.55/0.93/0.40/0.96, respectively. In conclusion, among these weekly screening tests for IA, the double-sandwich ELISA test was the most sensitive at predicting the diagnosis of IA in high-risk patients with hematological disorders, using a reduced cutoff of 0.6 O.D.I. PMID:15184460

  20. Diagnostics in Japan's microgravity experiments

    NASA Technical Reports Server (NTRS)

    Kadota, Toshikazu

    1995-01-01

    The achievement of the combustion research under microgravity depends substantially on the availability of diagnostic systems. The non-intrusive diagnostic systems are potentially applicable for providing the accurate, realistic and detailed information on momentum, mass and energy transport, complex gas phase chemistry, and phase change in the combustion field under microgravity. The non-intrusive nature of optical instruments is essential to the measurement of combustion process under microgravity which is very nervous to any perturbation. However, the implementation of the non-intrusive combustion diagnostic systems under microgravity is accompanied by several constraints. Usually, a very limited space is only available for constructing a highly sophisticated system which is so sensitive that it is easily affected by the magnitude of the gravitational force, vibration and heterogeneous field of temperature and density of the environments. The system should be properly adjusted prior to the experiment. Generally, it is quite difficult to tune the instruments during measurements. The programmed sequence of operation should also be provided. Extensive effort has been toward the development of non-intrusive diagnostic systems available for the combustion experiments under microgravity. This paper aims to describe the current art and the future strategy on the non-intrusive diagnostic systems potentially applicable to the combustion experiments under microgravity in Japan.

  1. Applied genomics: data mining reveals species-specific malaria diagnostic targets more sensitive than 18S rRNA.

    PubMed

    Demas, Allison; Oberstaller, Jenna; DeBarry, Jeremy; Lucchi, Naomi W; Srinivasamoorthy, Ganesh; Sumari, Deborah; Kabanywanyi, Abdunoor M; Villegas, Leopoldo; Escalante, Ananias A; Kachur, S Patrick; Barnwell, John W; Peterson, David S; Udhayakumar, Venkatachalam; Kissinger, Jessica C

    2011-07-01

    Accurate and rapid diagnosis of malaria infections is crucial for implementing species-appropriate treatment and saving lives. Molecular diagnostic tools are the most accurate and sensitive method of detecting Plasmodium, differentiating between Plasmodium species, and detecting subclinical infections. Despite available whole-genome sequence data for Plasmodium falciparum and P. vivax, the majority of PCR-based methods still rely on the 18S rRNA gene targets. Historically, this gene has served as the best target for diagnostic assays. However, it is limited in its ability to detect mixed infections in multiplex assay platforms without the use of nested PCR. New diagnostic targets are needed. Ideal targets will be species specific, highly sensitive, and amenable to both single-step and multiplex PCRs. We have mined the genomes of P. falciparum and P. vivax to identify species-specific, repetitive sequences that serve as new PCR targets for the detection of malaria. We show that these targets (Pvr47 and Pfr364) exist in 14 to 41 copies and are more sensitive than 18S rRNA when utilized in a single-step PCR. Parasites are routinely detected at levels of 1 to 10 parasites/μl. The reaction can be multiplexed to detect both species in a single reaction. We have examined 7 P. falciparum strains and 91 P. falciparum clinical isolates from Tanzania and 10 P. vivax strains and 96 P. vivax clinical isolates from Venezuela, and we have verified a sensitivity and specificity of ∼100% for both targets compared with a nested 18S rRNA approach. We show that bioinformatics approaches can be successfully applied to identify novel diagnostic targets and improve molecular methods for pathogen detection. These novel targets provide a powerful alternative molecular diagnostic method for the detection of P. falciparum and P. vivax in conventional or multiplex PCR platforms.

  2. Synergism between particle-based multiplexing and microfluidics technologies may bring diagnostics closer to the patient.

    PubMed

    Derveaux, S; Stubbe, B G; Braeckmans, K; Roelant, C; Sato, K; Demeester, J; De Smedt, S C

    2008-08-01

    In the field of medical diagnostics there is a growing need for inexpensive, accurate, and quick high-throughput assays. On the one hand, recent progress in microfluidics technologies is expected to strongly support the development of miniaturized analytical devices, which will speed up (bio)analytical assays. On the other hand, a higher throughput can be obtained by the simultaneous screening of one sample for multiple targets (multiplexing) by means of encoded particle-based assays. Multiplexing at the macro level is now common in research labs and is expected to become part of clinical diagnostics. This review aims to debate on the "added value" we can expect from (bio)analysis with particles in microfluidic devices. Technologies to (a) decode, (b) analyze, and (c) manipulate the particles are described. Special emphasis is placed on the challenges of integrating currently existing detection platforms for encoded microparticles into microdevices and on promising microtechnologies that could be used to down-scale the detection units in order to obtain compact miniaturized particle-based multiplexing platforms.

  3. Diagnostic modalities.

    PubMed

    Elstob, Alison; Gonsalves, Michael; Patel, Uday

    2016-12-01

    The incidental detection of small renal masses on imaging undertaken to evaluate unrelated symptoms or conditions is an increasingly common occurrence. Accurate imaging characterisation is fundamental to determining optimum patient management. The goals of imaging small renal masses include determining whether a lesion is solid or cystic, if there are signs of biological aggressiveness and whether the lesion is likely benign or malignant. The current imaging practices and the evidence supporting the use of different imaging modalities for the characterisation of small renal masses are discussed. CT remains the primary imaging modality and is able to classify most masses into surgical or non-surgical lesions. MRI and contrast enhanced ultrasound are most often employed to problem solve in lesions deemed indeterminate on contrast enhanced CT or for patients in which CECT is contraindicated. Percutaneous biopsy should be considered in lesions that remain indeterminate after initial imaging investigations. Given the central role of imaging in the management of small renal masses, all multidisciplinary team members involved in renal cancer care should have an understanding of the performance of the different imaging modalities.

  4. Role of molecular diagnostics in the management of infectious disease emergencies.

    PubMed

    Krishna, Neel K; Cunnion, Kenji M

    2012-11-01

    In the setting of infectious disease emergencies, rapid and accurate identification of the causative agent is critical to optimizing antimicrobial therapy in a timely manner. It is clearly evident that the age of molecular diagnostics is now upon us, with real-time PCR becoming the standard of diagnosis for many infectious disease emergencies in either monoplex or multiplex format. Other molecular techniques such as whole or partial genome sequencing, microarrays, broad-range PCR, restriction fragment length polymorphisms, and molecular typing are also being used. However, for most small clinical laboratories, implementation of these advanced molecular techniques is not feasible owing to the high cost of instrumentation and reagents. If these tests are not available in-house, samples can be sent to national reference laboratories (eg, Mayo Medical Laboratories and Quest Diagnostics) for real-time PCR assays that can be completed in 1 day. It is anticipated that over time commercial real-time PCR tests and instrumentation will become more standardized and affordable, allowing individual laboratories to conduct tests locally, thus further reducing turnaround time. Although real-time PCR has been proved to expand our diagnostic capability, it must be stressed that such molecular methodology constitutes only an additional tool in the diagnosis of infectious diseases in emergency situations. Phenotypic methodologies (staining, cultures, biochemical tests, and serology) still play a critical role in identifying, confirming, and providing antibiotic susceptibility testing for many microbial pathogens. As multiplex assays become increasingly available, there will be even greater temptation for taking a “shotgun” approach to diagnostic testing. These new technologies will not substitute for a proper history and physical examination leading to a thoughtful differential diagnosis. None the less, these new molecular tests increase the capability of the diagnostician to

  5. RAS - Screens & Assays

    Cancer.gov

    A primary goal of the RAS Initiative is to develop assays for RAS activity, localization, and signaling and adapt those assays so they can be used for finding new drug candidates. Explore the work leading to highly validated screening protocols.

  6. Next-generation confirmatory disease diagnostics

    NASA Astrophysics Data System (ADS)

    Lin, Robert; Gerver, Rachel; Karns, Kelly; Apori, Akwasi A.; Denisin, Aleksandra K.; Herr, Amy E.

    2014-06-01

    Microfluidic tools are advancing capabilities in screening diagnostics for use in near-patient settings. Here, we review three case studies to illustrate the flexibility and analytical power offered by microanalytical tools. We first overview a near-patient tool for detection of protein markers found in cerebrospinal fluid (CSF), as a means to identify the presence of cerebrospinal fluid in nasal mucous - an indication that CSF is leaking into the nasal cavity. Microfluidic design allowed integration of several up-stream preparatory steps and rapid, specific completion of the human CSF protein assay. Second, we overview a tear fluid based assay for lactoferrin, a protein produced in the lacrimal gland, then secreted into tear fluid. Tear Lf is a putative biomarker for primary SS. A critical contribution of this and related work being measurement of Lf, even in light of well-known and significant matrix interactions and losses during the tear fluid collection and preparation. Lastly, we review a microfluidic barcode platform that enables rapid measurement of multiple infectious disease biomarkers in human sera. The assay presents a new approach to multiplexed biomarker detection, yet in a simple straight microchannel - thus providing a streamlined, simplified microanalytical platform, as is relevant to robust operation in diagnostic settings. We view microfluidic design and analytical chemistry as the basis for emerging, sophisticated assays that will advance not just screening diagnostic technology, but confirmatory assays, sample preparation and handling, and thus introduction and utilization of new biomarkers and assay formats.

  7. Assays of Serum Testosterone.

    PubMed

    Herati, Amin S; Cengiz, Cenk; Lamb, Dolores J

    2016-05-01

    The diagnosis of male hypogonadism depends on an assessment of the clinical signs and symptoms of hypogonadism and serum testosterone level. Current clinical laboratory testosterone assay platforms include immunoassays and mass spectrometry. Despite significant advances to improve the accuracy and precision of the currently available assays, limited comparability exists between assays at the lower and upper extremes of the testosterone range. Because of this lack of comparability, there is no current gold standard assay for the assessment of total testosterone levels.

  8. Evaluation of a new rapid molecular diagnostic system for Plasmodium falciparum combined with DNA filter paper, loop-mediated isothermal amplification, and melting curve analysis.

    PubMed

    Yamamura, Mariko; Makimura, Koichi; Ota, Yasuo

    2009-01-01

    Falciparum malaria is a fatal infection without immediate diagnosability or treatment. There are shortages of clinicians and examiners skilled in the treatment of malaria in non-endemic countries, including Japan. This study was performed to evaluate a novel rapid molecular diagnostic system consisting of loop-mediated isothermal amplification (LAMP) combined with DNA filter paper (FTA card) and melting curve analysis. Combining LAMP with melting curve analysis enabled diagnosis of Plasmodium falciparum more accurately with relative ease. FTA cards could be used to clarify problems regarding storage, infectivity, and transportation. The LAMP assay was carried out at a constant temperature of 63 degrees C for 90 min. The diagnostic system (malaria-LAMP) accurately diagnosed malaria (47 samples from Thailand and 50 from Zimbabwe) with 97.8% sensitivity and 85.7% specificity as compared with microscopic methods, indicating the usefulness of this combined system.

  9. Accelerating Biomedical Research in Designing Diagnostic Assays, Drugs, and Vaccines

    DTIC Science & Technology

    2010-10-01

    structures by ab initio folding us- ing the Rosetta code, a computationally intensive method. We structurally compare the approach’s derived models to a...attempts are unsuccessful, then the computationally intensive ab initio Rosetta program is used. The ab initio models are annotated by structurally...Biololgy, vol. 3, no. 64, 2009; doi:10.1186/1752-0509-3-64. 12. Y. Chushak and M.O. Stone , “In Silico Selection of RNA Aptamers,” Nucleic Acids Research

  10. Oligonucleotide Fingerprint Identification for Microarray-Based Pathogen Diagnostic Assays

    DTIC Science & Technology

    2006-10-01

    Yersinia species), or a set of organisms that may or may not have any phylogenetic relationship (e.g. to detect a viral or a bacterial family) and (2) the... phylogenetic tree or published data. The target and neighbor will contain common DNA sequences, which, obviously, cannot be used as DNA fingerprints. DNA... Yersinia pestis and 250 fingerprints for Francisella tularensis are presented. Availability: The implemented algorithm is available upon request

  11. Concordance in diagnostic testing for respiratory pathogens of Bighorn Sheep

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Reliable diagnostic tests are essential for disease investigation and management. This is particularly true for diseases of free-ranging wildlife where sampling is logistically difficult precluding retesting. Clinical assays for wildlife diseases frequently vary among laboratories because of lack ...

  12. Companion diagnostics: the key to personalized medicine. Foreword.

    PubMed

    Jørgensen, Jan Trøst

    2015-02-01

    This special focus issue of Expert Review of Molecular Diagnostics on in vitro companion diagnostics aims to provide the reader with up-to-date knowledge on this fast-evolving area of medical research. Companion diagnostics takes up a central role in the development of targeted drugs and to a large extent, the success of this type of therapy depends on their performance. Companion diagnostic assays have a single patient as a point of reference and they will be decisive for the move toward a more precise and individualized pharmacotherapy. The 'first generation' of companion diagnostic assays relies on single biomarker detection but with our increasing understanding of disease pathophysiology a new generation of assays is under development, which will be based on patient profiling and multiplex platforms.

  13. Chronic Meningitis: Simplifying a Diagnostic Challenge.

    PubMed

    Baldwin, Kelly; Whiting, Chris

    2016-03-01

    Chronic meningitis can be a diagnostic dilemma for even the most experienced clinician. Many times, the differential diagnosis is broad and encompasses autoimmune, neoplastic, and infectious etiologies. This review will focus on a general approach to chronic meningitis to simplify the diagnostic challenges many clinicians face. The article will also review the most common etiologies of chronic meningitis in some detail including clinical presentation, diagnostic testing, treatment, and outcomes. By using a case-based approach, we will focus on the key elements of clinical presentation and laboratory analysis that will yield the most rapid and accurate diagnosis in these complicated cases.

  14. Updates on chikungunya epidemiology, clinical disease, and diagnostics.

    PubMed

    Sam, I-Ching; Kümmerer, Beate M; Chan, Yoke-Fun; Roques, Pierre; Drosten, Christian; AbuBakar, Sazaly

    2015-04-01

    Chikungunya virus (CHIKV) is an Aedes-borne alphavirus, historically found in Africa and Asia, where it caused sporadic outbreaks. In 2004, CHIKV reemerged in East Africa and spread globally to cause epidemics, including, for the first time, autochthonous transmission in Europe, the Middle East, and Oceania. The epidemic strains were of the East/Central/South African genotype. Strains of the Asian genotype of CHIKV continued to cause outbreaks in Asia and spread to Oceania and, in 2013, to the Americas. Acute disease, mainly comprising fever, rash, and arthralgia, was previously regarded as self-limiting; however, there is growing evidence of severe but rare manifestations, such as neurological disease. Furthermore, CHIKV appears to cause a significant burden of long-term morbidity due to persistent arthralgia. Diagnostic assays have advanced greatly in recent years, although there remains a need for simple, accurate, and affordable tests for the developing countries where CHIKV is most prevalent. This review focuses on recent important work on the epidemiology, clinical disease and diagnostics of CHIKV.

  15. Toxoplasma gondii: history and diagnostic test development.

    PubMed

    Wyrosdick, Heidi M; Schaefer, John J

    2015-12-01

    Toxoplasma gondii is a protozoa that causes toxoplasmosis in people and other animals. It is considered one of the most common parasitic infections in the world due to its impressive range of hosts, widespread environmental contamination and the diverse means by which animals can be infected. Despite its ubiquity and numerous ongoing research efforts into both its basic biology and clinical management, many aspects of diagnosis and management of this disease are poorly understood. The range of diagnostic options that is available for veterinary diagnostic investigators are notably more limited than those available to medical diagnosticians, making accurate interpretation of each test result critical. The current review joins other reviews on the parasite with a particular emphasis on the history and continued development of diagnostic tests that are useful for veterinary diagnostic investigations. An understanding of the strengths and shortcomings of current diagnostic techniques will assist veterinary and public health officials in formulating effective treatment and control strategies in diverse animal populations.

  16. New Technologies for Diagnosing Pediatric Tumors Expert Opinion on Medical Diagnostics

    PubMed Central

    Wei, Jun S.; Badgett, Thomas C.; Khan, Javed

    2008-01-01

    Background The completion of Human Genome Project (HGP) has paved the way for novel, more detailed and accurate molecular diagnostic classification of cancer. With the information from the HGP, cancers can be categorized not only on the morphology or limited immunohistological markers, but according to their “molecular fingerprints” such as gene expression profiles. Technologies detecting these signatures have been developed to simultaneously measure multiple genes or proteins in one assay with high sensitivity and specificity. Objective To evaluate potential innovative novel methods of diagnosis and prognosis in pediatric cancers. Methods We selected a variety of promising new diagnostic technologies utilizing molecular signatures which harness the results from HGP including DNA microarray, bead-based detection system, multiplexed RT-PCR, MesoScale Discovery (MSD), and isotope-coded affinity tag (ICAT), as well as their applications in biomarker discovery for pediatric tumors. Label-free detection technologies and the obstacles for taking these new diagnostic technologies from the bench to the bedside are also discussed. Conclusion The use of molecular signatures is gaining acceptance in clinical practice. However, technical challenges need to be addressed before incorporating these new technologies into current diagnostic and prognostic schema. PMID:19554203

  17. Identification and Differentiation of Verticillium Species and V. longisporum Lineages by Simplex and Multiplex PCR Assays

    PubMed Central

    Inderbitzin, Patrik; Davis, R. Michael; Bostock, Richard M.; Subbarao, Krishna V.

    2013-01-01

    Accurate species identification is essential for effective plant disease management, but is challenging in fungi including Verticillium sensu stricto (Ascomycota, Sordariomycetes, Plectosphaerellaceae), a small genus of ten species that includes important plant pathogens. Here we present fifteen PCR assays for the identification of all recognized Verticillium species and the three lineages of the diploid hybrid V. longisporum. The assays were based on DNA sequence data from the ribosomal internal transcribed spacer region, and coding and non-coding regions of actin, elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase and tryptophan synthase genes. The eleven single target (simplex) PCR assays resulted in amplicons of diagnostic size for V. alfalfae, V. albo-atrum, V. dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii, V. nonalfalfae, V. nubilum, V. tricorpus, V. zaregamsianum, and Species A1 and Species D1, the two undescribed ancestors of V. longisporum. The four multiple target (multiplex) PCR assays simultaneously differentiated the species or lineages within the following four groups: Verticillium albo-atrum, V. alfalfae and V. nonalfalfae; Verticillium dahliae and V. longisporum lineages A1/D1, A1/D2 and A1/D3; Verticillium dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii and V. tricorpus; Verticillium isaacii, V. klebahnii and V. tricorpus. Since V. dahliae is a parent of two of the three lineages of the diploid hybrid V. longisporum, no simplex PCR assay is able to differentiate V. dahliae from all V. longisporum lineages. PCR assays were tested with fungal DNA extracts from pure cultures, and were not evaluated for detection and quantification of Verticillium species from plant or soil samples. The DNA sequence alignments are provided and can be used for the design of additional primers. PMID:23823707

  18. Assessing Biases in the Evaluation of Classification Assays for HIV Infection Recency.

    PubMed

    Patterson-Lomba, Oscar; Wu, Julia W; Pagano, Marcello

    2015-01-01

    Identifying recent HIV infection cases has important public health and clinical implications. It is essential for estimating incidence rates to monitor epidemic trends and evaluate the effectiveness of interventions. Detecting recent cases is also important for HIV prevention given the crucial role that recently infected individuals play in disease transmission, and because early treatment onset can improve the clinical outlook of patients while reducing transmission risk. Critical to this enterprise is the development and proper assessment of accurate classification assays that, based on cross-sectional samples of viral sequences, help determine infection recency status. In this work we assess some of the biases present in the evaluation of HIV recency classification algorithms that rely on measures of within-host viral diversity. Particularly, we examine how the time since infection (TSI) distribution of the infected subjects from which viral samples are drawn affect performance metrics (e.g., area under the ROC curve, sensitivity, specificity, accuracy and precision), potentially leading to misguided conclusions about the efficacy of classification assays. By comparing the performance of a given HIV recency assay using six different TSI distributions (four simulated TSI distributions representing different epidemic scenarios, and two empirical TSI distributions), we show that conclusions about the overall efficacy of the assay depend critically on properties of the TSI distribution. Moreover, we demonstrate that an assay with high overall classification accuracy, mainly due to properly sorting members of the well-represented groups in the validation dataset, can still perform notoriously poorly when sorting members of the less represented groups. This is an inherent issue of classification and diagnostics procedures that is often underappreciated. Thus, this work underscores the importance of acknowledging and properly addressing evaluation biases when proposing

  19. Mouse Assay for Determination of Arsenic Bioavailability in Contaminated Soils

    EPA Science Inventory

    Background: Accurate assessment of human exposure estimates from arsenic-contaminated soils depends upon estimating arsenic (As) soil bioavailability. Development of bioavailability assays provides data needed for human health risk assessments and supports development and valida...

  20. Reporter phage and breath tests: emerging phenotypic assays for diagnosing active tuberculosis, antibiotic resistance, and treatment efficacy.

    PubMed

    Jain, Paras; Thaler, David S; Maiga, Mamoudou; Timmins, Graham S; Bishai, William R; Hatfull, Graham F; Larsen, Michelle H; Jacobs, William R

    2011-11-15

    The rapid and accurate diagnosis of active tuberculosis (TB) and its drug susceptibility remain a challenge. Phenotypic assays allow determination of antibiotic susceptibilities even if sequence data are not available or informative. We review 2 emerging diagnostic approaches, reporter phage and breath tests, both of which assay mycobacterial metabolism. The reporter phage signal, Green fluorescent protein (GFP) or β-galactosidase, indicates transcription and translation inside the recipient bacilli and its attenuation by antibiotics. Different breath tests assay, (1) exhaled antigen 85, (2) mycobacterial urease activity, and (3) detection by trained rats of disease-specific odor in sputum, have also been developed. When compared with culture, reporter phage assays shorten the time for initial diagnosis of drug susceptibility by several days. Both reporter phage and breath tests have promise as early markers to determine the efficacy of treatment. While sputum often remains smear and Mycobacterium tuberculosis DNA positive early in the course of efficacious antituberculous treatment, we predict that both breath and phage tests will rapidly become negative. If this hypothesis proves correct, phage assays and breath tests could become important surrogate markers in early bactericidal activity (EBA) studies of new antibiotics.

  1. A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses

    PubMed Central

    Wadhwa, Ashutosh; Wilkins, Kimberly; Gao, Jinxin; Condori Condori, Rene Edgar; Gigante, Crystal M.; Zhao, Hui; Ma, Xiaoyue; Ellison, James A.; Greenberg, Lauren; Velasco-Villa, Andres; Orciari, Lillian

    2017-01-01

    Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high

  2. Detection of ALK rearrangements in lung cancer patients using a homebrew PCR assay.

    PubMed

    Yu, Hui; Chang, JianHua; Liu, Fang; Wang, Qifeng; Lu, YongMing; Zhang, ZhuanXu; Shen, Jiabing; Zhai, Qing; Meng, Xia; Wang, Jialei; Ye, Xun

    2017-01-31

    Lung cancer patients with anaplastic lymphoma kinase (ALK) rearrangements are candidates for targeted therapeutics. However, patients must be tested with a companion diagnostic assay to realize their ALK rearrangement status. We analyzed the publicly available E-GEOD-31210 microarray dataset and identified a non-coding RNA, sweyjawbu, which is strongly associated with ALK rearrangements. We validated these results using quantitative real-time PCR in an independent cohort consisting of 4 cell lines and 83 clinical samples. We could differentiate between ALK rearrangement-positive and -negative lung cancer samples by comparing sweyjawbu expression. Additionally, ALK rearrangement status was determined by comparing the expression of the 5' and 3' regions of the ALK transcript or by detecting known ALK hybrid subtypes. Thus, using our homebrew PCR assay, we were able to accurately detect ALK rearrangements, which could be used for diagnostic screening of lung cancer patients. The prototype could potentially be transferred to an automatic multiplex PCR platform (FilmArray) to differentiate between ALK rearrangement-positive and -negative patients in point-of-care settings.

  3. A GeXP-Based Assay for Simultaneous Detection of Multiple Viruses in Hospitalized Children with Community Acquired Pneumonia

    PubMed Central

    Wang, Le; Zhao, Mengchuan; Shi, Zhongren; Feng, Zhishan; Guo, Weiwei; Yang, Shuo; Liu, Lanping; Li, Guixia

    2016-01-01

    The GeXP-based assay has recently been developed for simultaneous detection of multiple pathogens. So far, the application of the GeXP assay to test larger clinical samples has hardly been reported. Community-acquired pneumonia (CAP) is the leading cause of death in children worldwide and a substantial proportion of childhood CAP is caused by viruses. Rapid and accurate diagnosis of virus infection is important for the clinical management of CAP. In this study, we explored the GeXP assay for simultaneous detection of 20 types/subtypes of viruses in hospitalized children with CAP. A total of 1699 nasopharyngeal swabs were prospectively collected and viral nucleic acid was extracted and assayed. Using viral genomic DNA or RNA as template, we showed that at the concentration of 104 copies of DNA or RNA of each virus/μl, all 20 target viruses were simultaneously identified by the GeXP assay. Fifteen control microorganisms, in contrast, failed to be amplified by the assay. About 65% of cases tested in this study had viral infection, with patients aged <3 years having a 70% positive rate, significantly higher than that in patients aged > 3 years (40%). The most frequently detected virus was RSV followed by PIV3, HRV, ADV and HBoV. Seasonal distribution analysis revealed that RSV was the most predominant in autumn and winter, while in spring and summer PIV3 and RSV were the most frequently identified with similar positive percentages. One hundred twenty randomly-chosen samples tested by the GeXP assay were re-evaluated by mono-RT-PCR, the results showed 97.5% diagnosis agreement between these 2 methods. Our findings suggest that the GeXP assay could be a valuable diagnostic tool for virus infection in pediatric patients with CAP. PMID:27627439

  4. On numerically accurate finite element

    NASA Technical Reports Server (NTRS)

    Nagtegaal, J. C.; Parks, D. M.; Rice, J. R.

    1974-01-01

    A general criterion for testing a mesh with topologically similar repeat units is given, and the analysis shows that only a few conventional element types and arrangements are, or can be made suitable for computations in the fully plastic range. Further, a new variational principle, which can easily and simply be incorporated into an existing finite element program, is presented. This allows accurate computations to be made even for element designs that would not normally be suitable. Numerical results are given for three plane strain problems, namely pure bending of a beam, a thick-walled tube under pressure, and a deep double edge cracked tensile specimen. The effects of various element designs and of the new variational procedure are illustrated. Elastic-plastic computation at finite strain are discussed.

  5. Colorimetric protein assay techniques.

    PubMed

    Sapan, C V; Lundblad, R L; Price, N C

    1999-04-01

    There has been an increase in the number of colorimetric assay techniques for the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coefficient of variation/laboratory-to-laboratory variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro-Kjeldahl technique, quantitative amino acid analysis or the biuret assay. In a complex mixture it might be inappropriate to focus on a general method of protein determination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the 'gold standard' for a given protein, this method needs to be used routinely for calibration.

  6. An overview of the diagnostic tools.

    PubMed

    Coste, J

    2013-09-01

    Prions are unconventional infectious agents that cause fatal neurological illnesses such as Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy, and scrapie. Variant CJD can occur via blood transfusions. However, as no screening assay is available, uncertainties remain over the prevalence of vCJD in asymptomatic blood donors. Development of a diagnostic assay is therefore a primary objective. Little is known about the nature, distribution and level of infectivity in human blood and we have to rely on assumptions made from animal models. Ideally, two types of assays are required: a rapid high-throughput assay to routinely screen all blood donations and a confirmatory assay to ensure that all positive results from initial screening are true positives. Key event in prion disease is thought to be the conversion of normal cellular prion protein PrPc to a misfolded aggregated form termed PrP(TSE). This specific characteristic has been exploited to develop some tests.

  7. Assay validation of the cardiac isoform of troponin I in double crested cormorant (Phalacrocorax auritus) plasma for diagnosis of cardiac damage.

    PubMed

    Daigneault, Melissa; Harr, Kendal E; Dean, Karen M; Bursian, Steven J

    2017-03-14

    Cardiac abnormalities, initially found in Deepwater Horizon weathered MC252 crude oil exposed Double Crested Cormorants (DCCOs) upon gross necropsy, were further investigated using echocardiography. Clinical and statistically significant changes including decreased ventricular myocardial contractility and arrhythmia were elucidated by echocardiography and interpreted by boarded cardiologists as potentially life threatening. The objective of this investigation was to initiate development of an antemortem, sensitive blood screening test for cardiac damage due to oil exposure of avian species. An assay for the cardiac isoform of troponin I (cTnI) which is known to be highly cross-reactive across mammalian species was chosen and analytically validated in DCCO. This is the first time this test has been analytically validated in avian species. All plasma samples from birds assessed as healthy had trace concentrations (<0.016ng/ml). The assays was precise and accurate revealing a coefficient of variation <3% and an R(2)>0.99. Diagnostic investigation revealed that the test appears to have diagnostic potential for the diagnosis of cardiomyocyte damage. Diagnostic sensitivity and specificity were 91% and 73% in this laboratory population. Due to an equivocal sample population in which health could not be proven, further investigation is needed to diagnostically validate troponin I in the assessment of oil exposure in DCCO.

  8. Tuberculosis diagnostics: innovating to make an impact.

    PubMed

    Ghanashyam, Bharathi

    2011-04-01

    The 'International Symposium on TB Diagnostics: Innovating to Make an Impact' was organized by the International Centre for Genetic Engineering & Biotechnology, New Delhi, India, on December 16-17, 2010, with sponsorship support from the Bill & Melinda Gates Foundation, Foundation for Innovative New Diagnostics and AERAS Global TB Vaccine Foundation. This highly successful symposium attracted more than 300 participants from India and several other countries and covered several aspects of TB diagnostics, including recent scientific advances in TB diagnostics, progress made in expanding the TB diagnostics pipeline including a portfolio of WHO-endorsed, validated new tools and improved technologies, the successful development of newer molecular assays that have the potential to be used at the point of treatment and the growing contributions of emerging economies such as India. In addition to highlighting the positive aspects of TB diagnostics, the symposium speakers also highlighted the need to focus on worrisome aspects of TB diagnosis, including widespread abuse of inappropriate tests that can prevent the use of good diagnostics, lack of quality assurance in laboratories, lack of adequate regulation of diagnostics and how these can pose a major challenge for roll-out and implementation of new tools. The symposium ended with a very stimulating discussion on how India can become a global leader in TB innovations.

  9. Immunosensors in Clinical Laboratory Diagnostics.

    PubMed

    Justino, Celine I L; Duarte, Armando C; Rocha-Santos, Teresa A P

    2016-01-01

    The application of simple, cost-effective, rapid, and accurate diagnostic technologies for detection and identification of cardiac and cancer biomarkers has been a central point in the clinical area. Biosensors have been recognized as efficient alternatives for the diagnostics of various diseases due to their specificity and potential for application on real samples. The role of nanotechnology in the construction of immunological biosensors, that is, immunosensors, has contributed to the improvement of sensitivity, since they are based in the affinity between antibody and antigen. Other analytes than biomarkers such as hormones, pathogenic bacteria, and virus have also been detected by immunosensors for clinical point-of-care applications. In this chapter, we first introduced the various types of immunosensors and discussed their applications in clinical diagnostics over the recent 6 years, mainly as point-of-care technologies for the determination of cardiac and cancer biomarkers, hormones, pathogenic bacteria, and virus. The future perspectives of these devices in the field of clinical diagnostics are also evaluated.

  10. Absolute nuclear material assay

    DOEpatents

    Prasad, Manoj K [Pleasanton, CA; Snyderman, Neal J [Berkeley, CA; Rowland, Mark S [Alamo, CA

    2012-05-15

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  11. Absolute nuclear material assay

    DOEpatents

    Prasad, Manoj K.; Snyderman, Neal J.; Rowland, Mark S.

    2010-07-13

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  12. Automated diagnostic kiosk for diagnosing diseases

    DOEpatents

    Regan, John Frederick; Birch, James Michael

    2014-02-11

    An automated and autonomous diagnostic apparatus that is capable of dispensing collection vials and collections kits to users interesting in collecting a biological sample and submitting their collected sample contained within a collection vial into the apparatus for automated diagnostic services. The user communicates with the apparatus through a touch-screen monitor. A user is able to enter personnel information into the apparatus including medical history, insurance information, co-payment, and answer a series of questions regarding their illness, which is used to determine the assay most likely to yield a positive result. Remotely-located physicians can communicate with users of the apparatus using video tele-medicine and request specific assays to be performed. The apparatus archives submitted samples for additional testing. Users may receive their assay results electronically. Users may allow the uploading of their diagnoses into a central databank for disease surveillance purposes.

  13. Huntington Disease: Molecular Diagnostics Approach.

    PubMed

    Bastepe, Murat; Xin, Winnie

    2015-10-06

    Huntington disease (HD) is caused by expansion of a CAG trinucleotide repeat in the first exon of the Huntingtin (HTT) gene. Molecular testing of Huntington disease for diagnostic confirmation and disease prediction requires detection of the CAG repeat expansion. There are three main types of HD genetic testing: (1) diagnostic testing to confirm or rule out disease, (2) presymptomatic testing to determine whether an at-risk individual inherited the expanded allele, and (3) prenatal testing to determine whether the fetus has inherited the expanded allele. This unit includes protocols that describe the complementary use of polymerase chain reactions (PCR) and Southern blot hybridization to accurately measure the CAG trinucleotide repeat size and interpret the test results. In addition, an indirect linkage analysis that does not reveal the unwanted parental HD status in a prenatal testing will also be discussed.

  14. Accurate ab Initio Spin Densities.

    PubMed

    Boguslawski, Katharina; Marti, Konrad H; Legeza, Ors; Reiher, Markus

    2012-06-12

    We present an approach for the calculation of spin density distributions for molecules that require very large active spaces for a qualitatively correct description of their electronic structure. Our approach is based on the density-matrix renormalization group (DMRG) algorithm to calculate the spin density matrix elements as a basic quantity for the spatially resolved spin density distribution. The spin density matrix elements are directly determined from the second-quantized elementary operators optimized by the DMRG algorithm. As an analytic convergence criterion for the spin density distribution, we employ our recently developed sampling-reconstruction scheme [J. Chem. Phys.2011, 134, 224101] to build an accurate complete-active-space configuration-interaction (CASCI) wave function from the optimized matrix product states. The spin density matrix elements can then also be determined as an expectation value employing the reconstructed wave function expansion. Furthermore, the explicit reconstruction of a CASCI-type wave function provides insight into chemically interesting features of the molecule under study such as the distribution of α and β electrons in terms of Slater determinants, CI coefficients, and natural orbitals. The methodology is applied to an iron nitrosyl complex which we have identified as a challenging system for standard approaches [J. Chem. Theory Comput.2011, 7, 2740].

  15. Broad base biological assay using liquid based detection assays

    SciTech Connect

    Milanovich, F; Albala, J; Colston, B; Langlois, R; Venkateswaren, K

    2000-10-31

    organization, and DNA replication and repair. Understanding the complexities of these interactions is a fundamental step towards comprehending key aspects of disease biochemistry. This past year, using the LA technology, we were able to confirm the dynamics of a well characterized three protein, bacterial DNA repair mechanism--UvrABC. Next fiscal year we will begin studying the less characterized mammalian homologous recombinational DNA repair pathway examining the protein/protein and protein/DNA interactions of RAD51B/C. In the second thrust, we are looking at a model human disease state to assess the application of the LA in highly parallel and rapid medical diagnostics. In collaboration with researchers at UCSF and the California Department of Public Health we are developing a multiplex assay for the determination of Herpes-8 exposure (a cancer inducing virus) in aids patients. We have successfully demonstrated a 8-plex assay and will extend to 20-plex in the near future. In a parallel effort we will develop an 18-plex assay for detecting antibodies to all vaccine-preventable childhood viral infections. Finally we are developing a concept that would utilize the bead assay in the simplest possible form. After microbead capture of the biomarker sample and a fluorescent reporter in solution, the beads are trapped on an ordered dipstick array. The color of each bead is used to identify the biomarker, while the fluorescent reporter measures its concentration. This concept, MIDS, would enable widespread use of the technology by reducing the capital investment required while greatly simplifying its operation and maintenance.

  16. 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.

  17. Mass Spectrometry Based Ultrasensitive DNA Methylation Profiling Using Target Fragmentation Assay.

    PubMed

    Lin, Xiang-Cheng; Zhang, Ting; Liu, Lan; Tang, Hao; Yu, Ru-Qin; Jiang, Jian-Hui

    2016-01-19

    Efficient tools for profiling DNA methylation in specific genes are essential for epigenetics and clinical diagnostics. Current DNA methylation profiling techniques have been limited by inconvenient implementation, requirements of specific reagents, and inferior accuracy in quantifying methylation degree. We develop a novel mass spectrometry method, target fragmentation assay (TFA), which enable to profile methylation in specific sequences. This method combines selective capture of DNA target from restricted cleavage of genomic DNA using magnetic separation with MS detection of the nonenzymatic hydrolysates of target DNA. This method is shown to be highly sensitive with a detection limit as low as 0.056 amol, allowing direct profiling of methylation using genome DNA without preamplification. Moreover, this method offers a unique advantage in accurately determining DNA methylation level. The clinical applicability was demonstrated by DNA methylation analysis using prostate tissue samples, implying the potential of this method as a useful tool for DNA methylation profiling in early detection of related diseases.

  18. Development and Validation of a Real-Time PCR Assay for Rapid Detection of Two-Spotted Spider Mite, Tetranychus urticae (Acari: Tetranychidae).

    PubMed

    Li, Dongmei; Fan, Qing-Hai; Waite, David W; Gunawardana, Disna; George, Sherly; Kumarasinghe, Lalith

    2015-01-01

    Spider mites of the genus Tetranychus are difficult to identify due to their limited diagnostic characters. Many of them are morphologically similar and males are needed for species-level identification. Tetranychus urticae is a common interception and non-regulated pest at New Zealand's borders, however, most of the intercepted specimens are females and the identification was left at Tetranychus sp. Consequently, the shipments need to be fumigated. DNA sequencing and PCR-restriction fragment length polymorphism (PCR-RFLP) protocols could be used to facilitate the accurate identification. However, in the context of border security practiced in New Zealand, insect identifications are required to be provided within four hours of receiving the samples; thus, those molecular methods are not sufficient to meet this requirement. Therefore, a real-time PCR TaqMan assay was developed for identification of T. urticae by amplification of a 142 bp Internal Transcribed Spacer (ITS) 1 sequence. The developed assay is rapid, detects all life stages of T. urticae within three hours, and does not react with closely related species. Plasmid DNA containing ITS1 sequence of T. uritcae was serially diluted and used as standards in the real-time PCR assay. The quantification cycle (Cq) value of the assay depicted a strong linear relationship with T. urticae DNA content, with a regression coefficient of 0.99 and efficiency of 98%. The detection limit was estimated to be ten copies of the T. urticae target region. The assay was validated against a range of T. urticae specimens from various countries and hosts in a blind panel test. Therefore the application of the assay at New Zealand will reduce the unnecessary fumigation and be beneficial to both the importers and exporters. It is expected that the implementation of this real-time PCR assay would have wide applications in diagnostic and research agencies worldwide.

  19. Development and Validation of a Real-Time PCR Assay for Rapid Detection of Two-Spotted Spider Mite, Tetranychus urticae (Acari: Tetranychidae)

    PubMed Central

    Li, Dongmei; Fan, Qing-Hai; Waite, David W.; Gunawardana, Disna; George, Sherly; Kumarasinghe, Lalith

    2015-01-01

    Spider mites of the genus Tetranychus are difficult to identify due to their limited diagnostic characters. Many of them are morphologically similar and males are needed for species-level identification. Tetranychus urticae is a common interception and non-regulated pest at New Zealand’s borders, however, most of the intercepted specimens are females and the identification was left at Tetranychus sp. Consequently, the shipments need to be fumigated. DNA sequencing and PCR-restriction fragment length polymorphism (PCR-RFLP) protocols could be used to facilitate the accurate identification. However, in the context of border security practiced in New Zealand, insect identifications are required to be provided within four hours of receiving the samples; thus, those molecular methods are not sufficient to meet this requirement. Therefore, a real-time PCR TaqMan assay was developed for identification of T. urticae by amplification of a 142 bp Internal Transcribed Spacer (ITS) 1 sequence. The developed assay is rapid, detects all life stages of T. urticae within three hours, and does not react with closely related species. Plasmid DNA containing ITS1 sequence of T. uritcae was serially diluted and used as standards in the real-time PCR assay. The quantification cycle (Cq) value of the assay depicted a strong linear relationship with T. urticae DNA content, with a regression coefficient of 0.99 and efficiency of 98%. The detection limit was estimated to be ten copies of the T. urticae target region. The assay was validated against a range of T. urticae specimens from various countries and hosts in a blind panel test. Therefore the application of the assay at New Zealand will reduce the unnecessary fumigation and be beneficial to both the importers and exporters. It is expected that the implementation of this real-time PCR assay would have wide applications in diagnostic and research agencies worldwide. PMID:26147599

  20. An isothermal, label-free, and rapid one-step RNA amplification/detection assay for diagnosis of respiratory viral infections.

    PubMed

    Koo, Bonhan; Jin, Choong Eun; Lee, Tae Yoon; Lee, Jeong Hoon; Park, Mi Kyoung; Sung, Heungsup; Park, Se Yoon; Lee, Hyun Jung; Kim, Sun Mi; Kim, Ji Yeun; Kim, Sung-Han; Shin, Yong

    2017-04-15

    Recently, RNA viral infections caused by respiratory viruses, such as influenza, parainfluenza, respiratory syncytial virus, coronavirus, and Middle East respiratory syndrome-coronavirus (MERS-CoV), and Zika virus, are a major public health threats in the world. Although myriads of diagnostic methods based on RNA amplification have been developed in the last decades, they continue to lack speed, sensitivity, and specificity for clinical use. A rapid and accurate diagnostic method is needed for appropriate control, including isolation and treatment of the patients. Here, we report an isothermal, label-free, one-step RNA amplification and detection system, termed as iROAD, for the diagnosis of respiratory diseases. It couples a one-step isothermal RNA amplification method and a bio-optical sensor for simultaneous viral RNA amplification/detection in a label-free and real-time manner. The iROAD assay offers a one-step viral RNA amplification/detection example to rapid analysis (<20min). The detection limit of iROAD assay was found to be 10-times more sensitive than that of real-time reverse transcription-PCR method. We confirmed the clinical utility of the iROAD assay by detecting viral RNAs obtained from 63 human respiratory samples. We envision that the iROAD assay will be useful and potentially adaptable for better diagnosis of emerging infectious diseases including respiratory diseases.

  1. Accurate Quantification of Cardiovascular Biomarkers in Serum Using Protein Standard Absolute Quantification (PSAQ™) and Selected Reaction Monitoring*

    PubMed Central

    Huillet, Céline; Adrait, Annie; Lebert, Dorothée; Picard, Guillaume; Trauchessec, Mathieu; Louwagie, Mathilde; Dupuis, Alain; Hittinger, Luc; Ghaleh, Bijan; Le Corvoisier, Philippe; Jaquinod, Michel; Garin, Jérôme; Bruley, Christophe; Brun, Virginie

    2012-01-01

    Development of new biomarkers needs to be significantly accelerated to improve diagnostic, prognostic, and toxicity monitoring as well as therapeutic follow-up. Biomarker evaluation is the main bottleneck in this development process. Selected Reaction Monitoring (SRM) combined with stable isotope dilution has emerged as a promising option to speed this step, particularly because of its multiplexing capacities. However, analytical variabilities because of upstream sample handling or incomplete trypsin digestion still need to be resolved. In 2007, we developed the PSAQ™ method (Protein Standard Absolute Quantification), which uses full-length isotope-labeled protein standards to quantify target proteins. In the present study we used clinically validated cardiovascular biomarkers (LDH-B, CKMB, myoglobin, and troponin I) to demonstrate that the combination of PSAQ and SRM (PSAQ-SRM) allows highly accurate biomarker quantification in serum samples. A multiplex PSAQ-SRM assay was used to quantify these biomarkers in clinical samples from myocardial infarction patients. Good correlation between PSAQ-SRM and ELISA assay results was found and demonstrated the consistency between these analytical approaches. Thus, PSAQ-SRM has the capacity to improve both accuracy and reproducibility in protein analysis. This will be a major contribution to efficient biomarker development strategies. PMID:22080464

  2. Development and Characterization of Probe-Based Real Time Quantitative RT-PCR Assays for Detection and Serotyping of Foot-And-Mouth Disease Viruses Circulating in West Eurasia.

    PubMed

    Jamal, Syed M; Belsham, Graham J

    2015-01-01

    Rapid and accurate diagnosis of foot-and-mouth disease (FMD) and virus serotyping are of paramount importance for control of this disease in endemic areas where vaccination is practiced. Ideally this virus characterization should be achieved without the need for virus amplification in cell culture. Due to the heterogeneity of FMD viruses (FMDVs) in different parts of the world, region specific diagnostic tests are required. In this study, hydrolysable probe-based real time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays were developed for specific detection and serotyping of the FMDVs currently circulating in West Eurasia. These assays were evaluated, in parallel with pan-FMDV diagnostic assays and earlier serotype-specific assays, using field samples originating from Pakistan and Afghanistan containing FMD viruses belonging to different sublineages of O-PanAsia, A-Iran05 and Asia-1 (Group-II and Group-VII (Sindh-08)). In addition, field samples from Iran and Bulgaria, containing FMDVs belonging to the O-PanAsiaANT-10 sublineage were also tested. Each of the three primer/probe sets was designed to be specific for just one of the serotypes O, A and Asia-1 of FMDV and detected the RNA from the target viruses with cycle threshold (CT) values comparable with those obtained with the serotype-independent pan-FMDV diagnostic assays. No cross-reactivity was observed in these assays between the heterotypic viruses circulating in the region. The assays reported here have higher diagnostic sensitivity (100% each for serotypes O and Asia-1, and 92% [95% CI = 81.4-100%] for serotype A positive samples) and specificity (100% each for serotypes O, A and Asia-1 positive samples) for the viruses currently circulating in West Eurasia compared to the serotyping assays reported earlier. Comparisons of the sequences of the primers and probes used in these assays and the corresponding regions of the circulating viruses provided explanations for the poor

  3. Diagnostic imaging for aortic dissection.

    PubMed

    Kapustin, Andrew J; Litt, Harold I

    2005-01-01

    Diagnostic imaging for aortic dissection has dramatically changed in recent years. Previously, imaging consisted of conventional X-ray radiography, followed by invasive catheter angiography. Now imaging of dissection is performed primarily with multidetector CT, and to a lesser extent, with ultrasound and MRI. Catheter angiography is used primarily as a means of treating complications. Which modality to choose depends on patient factors, physician preference, and differences in availability of state-of-the-art equipment. All three modalities are highly accurate in experienced hands and have revolutionized the detection and evaluation of this condition.

  4. Development and evaluation of a simple assay for Marburg virus detection using a reverse transcription-loop-mediated isothermal amplification method.

    PubMed

    Kurosaki, Yohei; Grolla, Allen; Fukuma, Aiko; Feldmann, Heinz; Yasuda, Jiro

    2010-07-01

    Marburg virus (MARV) causes a severe hemorrhagic fever in humans with a high mortality rate. The rapid and accurate identification of the virus is required to appropriately provide infection control and outbreak management. Here, we developed and evaluated a one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the rapid and simple detection of MARV. By combining two sets of primers specific for the Musoke and Ravn genetic lineages, a multiple RT-LAMP assay detected MARV strains of both lineages, and no cross-reactivity with other hemorrhagic fever viruses (Ebola virus and Lassa virus) was observed. The assay could detect 10(2) copies of the viral RNA per tube within 40 min by real-time monitoring of the turbidities of the reaction mixtures. The assay was further evaluated using viral RNA extracted from clinical specimens collected in the 2005 Marburg hemorrhagic fever outbreak in Angola and yielded positive results for samples containing MARV at greater than 10(4) 50% tissue culture infective doses/ml, exhibiting 78% (14 of 18 samples positive) consistency with the results of a reverse transcription-PCR assay carried out in the field laboratory. The results obtained by both agarose gel electrophoresis and naked-eye judgment indicated that the RT-LAMP assay developed in this study is an effective tool for the molecular detection of MARV. Furthermore, it seems suitable for use for field diagnostics or in laboratories in areas where MARV is endemic.

  5. Evaluation of Commercially Available Serologic Diagnostic Tests for Chikungunya Virus

    PubMed Central

    Flusin, Olivier; Panella, Amanda; Tenebray, Bernard; Lanciotti, Robert; Leparc-Goffart, Isabelle

    2014-01-01

    Chikungunya virus (CHIKV) is present or emerging in dengue virus–endemic areas. Infections caused by these viruses share some common signs/symptoms, but prognosis, patient care, and persistent symptoms differ. Thus, accurate diagnostic methods are essential for differentiating the infections. We evaluated 4 CHIKV serologic diagnostic tests, 2 of which showed poor sensitivity and specificity. PMID:25418184

  6. A comparison of the Genie and western blot assays in confirmatory testing for HIV-1 antibody.

    PubMed

    Chan, E L; Sidaway, F; Horsman, G B

    1996-03-01

    The Genie HIV-1/2 kit (Sanofi Diagnostics Pasteur, Montreal, Quebec), a synthetic-peptide solid-phase enzyme immunoassay, was evaluated as a confirmatory assay for HIV-1 antibodies in comparison with Western blot (BioRad, Hercules, CA, USA) on 50 stored HIV-1 antibody-positive sera and the 137 sera yielding repeated positive results in the conventional EIA screen out of 13405 fresh patient sera from Saskatchewan in 1993. The stored HIV-1-positive sera were uniformly positive in the Genie test. Of the 137 EIA screen-positive sera, 33 were uniformly positive and 64 were uniformly negative in Genie and Western blot; 36 were Genie-negative and indeterminate by Western blot; and four were Genie indeterminate, of which one was negative and three were indeterminate by Western blot. All HIV-1 Western blot-indeterminate and Genie-interdeterminate sera were negative in radio-immunoprecipitation assay (RIPA) and Western blot for HIV-1 and HIV-2 antibodies performed by a reference laboratory. Genie gave an accurate definitive result for 97% of EIA positive sera compared with 71% for Western blot. There was excellent correlation between Genie, Western blot and RIPA results. However, the Genie assay was faster, less costly and yielded fewer indeterminate results than Western blot in confirmatory testing for HIV-1 antibodies.

  7. Diagnostic and Prognostic Biomarkers in Melanoma

    PubMed Central

    Leininger, Jennifer; Hamby, Carl; Safai, Bijan

    2014-01-01

    Melanoma is a lethal melanocytic neoplasm. Unfortunately, the histological diagnosis can be difficult at times. Distinguishing ambiguous melanocytic neoplasms that are benign nevi from those that represent true melanoma is important both for treatment and prognosis. Diagnostic biomarkers currently used to assist in the diagnosis of melanoma are usually specific only for melanocytic neoplasms and not necessarily for their ability to metastasize. Traditional prognostic biomarkers include depth of invasion and mitotic count. Newer diagnostic and prognostic biomarkers utilize immunohistochemical staining as well as ribonucleic acid, micro-ribonucleic acid, and deoxyribonucleic acid assays and fluorescence in situ hybridization. Improved diagnostic and prognostic biomarkers are of increasing importance in the treatment of melanoma with the development of newer and more targeted therapies. Herein, the authors review many of the common as well as newer diagnostic and prognostic biomarkers used in melanoma. PMID:25013535

  8. Magnetic bead-quantum dot assay for detection of a biomarker for traumatic brain injury

    NASA Astrophysics Data System (ADS)

    Kim, Chloe; Searson, Peter C.

    2015-10-01

    Current diagnostic methods for traumatic brain injury (TBI), which accounts for 15% of all emergency room visits, are limited to neuroimaging modalities. The challenges of accurate diagnosis and monitoring of TBI have created the need for a simple and sensitive blood test to detect brain-specific biomarkers. Here we report on an assay for detection of S100B, a putative biomarker for TBI, using antibody-conjugated magnetic beads for capture of the protein, and antibody-conjugated quantum dots for optical detection. From Western Blot, we show efficient antigen capture and concentration by the magnetic beads. Using magnetic bead capture and quantum dot detection in serum samples, we show a wide detection range and detection limit below the clinical cut-off level.Current diagnostic methods for traumatic brain injury (TBI), which accounts for 15% of all emergency room visits, are limited to neuroimaging modalities. The challenges of accurate diagnosis and monitoring of TBI have created the need for a simple and sensitive blood test to detect brain-specific biomarkers. Here we report on an assay for detection of S100B, a putative biomarker for TBI, using antibody-conjugated magnetic beads for capture of the protein, and antibody-conjugated quantum dots for optical detection. From Western Blot, we show efficient antigen capture and concentration by the magnetic beads. Using magnetic bead capture and quantum dot detection in serum samples, we show a wide detection range and detection limit below the clinical cut-off level. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr05608j

  9. Bicinchoninic acid (BCA) assay in low volume.

    PubMed

    Bainor, Anthony; Chang, Lyra; McQuade, Thomas J; Webb, Brian; Gestwicki, Jason E

    2011-03-15

    The BCA assay is a colorimetric method for estimating protein concentration. In 96-well plates, the relationship between protein content and absorbance is nearly linear over a wide range; however, performance is reduced in lower volume. To overcome this limitation, we performed the BCA assays in opaque, white 384-well plates. These plates emit fluorescence between 450-600 nm when excited at 430 nm; thus, their fluorescence is quenched by the BCA chromophore (λ(max) 562 nm). This arrangement allowed accurate determination of protein content using only 2 μL of sample. Moreover, soluble flourescein could replace the white plates, creating a homogenous format.

  10. Intelligent diagnostics systems

    NASA Technical Reports Server (NTRS)

    Mcquiston, Barbara M.; Dehoff, Ronald L.

    1992-01-01

    Intelligent systems have been applied to today's problems and could also be applied to space operations integrity. One of these systems is the XMAN tool designed for 'troubleshooting' jet engines. XMAN is the eXpert MAiNtenance tool developed to be an expert information analysis tool which stores trending and diagnostic data on Air Force engines. XMAN operates with a 'network topology' which follows a flow chart containing engine management information reports required by the governments technical order procedures. With XMAN technology, the user is able to identify engine problems by presenting the assertions of the fault isolation logic and attempting to satisfy individual assertions by referring to the databases created by an engine monitoring system. The troubleshooting process requires interaction between the technician and the computer to acquire new evidence form auxiliary maintenance tests corroboration of analytical results to accurately diagnose equipment malfunctions. This same technology will be required for systems which are functioning in space either with an onboard crew, or with an unmanned system. The technology and lessons learned developing this technology while suggesting definite applications for its use with developing space systems are addressed.

  11. Gamma Interferon Release Assays for Detection of Mycobacterium tuberculosis Infection

    PubMed Central

    Denkinger, Claudia M.; Kik, Sandra V.; Rangaka, Molebogeng X.; Zwerling, Alice; Oxlade, Olivia; Metcalfe, John Z.; Cattamanchi, Adithya; Dowdy, David W.; Dheda, Keertan; Banaei, Niaz

    2014-01-01

    SUMMARY Identification and treatment of latent tuberculosis infection (LTBI) can substantially reduce the risk of developing active disease. However, there is no diagnostic gold standard for LTBI. Two tests are available for identification of LTBI: the tuberculin skin test (TST) and the gamma interferon (IFN-γ) release assay (IGRA). Evidence suggests that both TST and IGRA are acceptable but imperfect tests. They represent indirect markers of Mycobacterium tuberculosis exposure and indicate a cellular immune response to M. tuberculosis. Neither test can accurately differentiate between LTBI and active TB, distinguish reactivation from reinfection, or resolve the various stages within the spectrum of M. tuberculosis infection. Both TST and IGRA have reduced sensitivity in immunocompromised patients and have low predictive value for progression to active TB. To maximize the positive predictive value of existing tests, LTBI screening should be reserved for those who are at sufficiently high risk of progressing to disease. Such high-risk individuals may be identifiable by using multivariable risk prediction models that incorporate test results with risk factors and using serial testing to resolve underlying phenotypes. In the longer term, basic research is necessary to identify highly predictive biomarkers. PMID:24396134

  12. R&D ERL: Diagnostics

    SciTech Connect

    Gassner, D.

    2010-01-01

    The Energy Recovery Linac (ERL) prototype project is currently under development at the Brookhaven National Laboratory. The ERL is expected to demonstrate energy recovery of high intensity beams with a current of up to a few hundred milliamps, while preserving the emittance of bunches with a charge of a few nanocoulombs produced by a high current SRF gun. To successfully accomplish this task the machine will include beam diagnostics that will be used for accurate characterization of the three dimensional beam phase space at the injection and recirculation energies, transverse and longitudinal beam matching, orbit alignment, beam current measurement, and machine protection. This report outlines requirements on the ERL diagnostics and describes its setup and modes of operation. The BNL Prototype ERL is an R&D effort aimed at reducing risks and costs associated with the proposed RHIC II electron cooler and eRHIC collider. The ERL will serve as a test bed for developing and testing instrumentation and studying physics and technological issues relevant to very high current ERL's. The prototype ERL, mated to a high current SRF gun, is expected to demonstrate production and energy recovery of high intensity, low emittance beams with a current of up to a few hundred milliamps. To successfully accomplish this task the ERL will include beam diagnostics required to characterize and tune beam parameters, as well as for machine protection. A preliminary diagnostics plan was presented in earlier publications. In this report, we describe the diagnostics presently planned to provide the data needed to meet these goals.

  13. Diagnostic Algorithm Benchmarking

    NASA Technical Reports Server (NTRS)

    Poll, Scott

    2011-01-01

    A poster for the NASA Aviation Safety Program Annual Technical Meeting. It describes empirical benchmarking on diagnostic algorithms using data from the ADAPT Electrical Power System testbed and a diagnostic software framework.

  14. Prenatal Genetic Diagnostic Tests

    MedlinePlus

    ... Education & Events Advocacy For Patients About ACOG Prenatal Genetic Diagnostic Tests Home For Patients Search FAQs Prenatal ... Pamphlets - Spanish FAQ164, September 2016 PDF Format Prenatal Genetic Diagnostic Tests Pregnancy What is prenatal genetic testing? ...

  15. Sensitivities of antigen detection and PCR assays greatly increased compared to that of the standard culture method for screening for group B streptococcus carriage in pregnant women.

    PubMed

    Rallu, Fabien; Barriga, Peter; Scrivo, Carole; Martel-Laferrière, Valérie; Laferrière, Céline

    2006-03-01

    Group B streptococcus (GBS) is a major cause of serious infections in neonates. The 2002 revised guidelines of the Centers for Disease Control and Prevention (CDC) for the prevention of perinatal GBS disease recommend that all pregnant women be screened for GBS carriage at between 35 and 37 weeks of gestation and that intrapartum antibiotic prophylaxis be given to carriers. We studied the performances of four different GBS detection assays in the context of antenatal screening. Between May and August 2004, the 605 vaginorectal swab specimens received at our bacteriology laboratory for GBS antenatal detection were tested by the four assays. The standard culture method was done according to the CDC recommendations. The three experimental assays performed with the growth from the selective enrichment (LIM) broth (Todd-Hewitt broth with 15 mug/ml nalidixic acid and 10 mug/ml colistin) after overnight incubation were a GBS antigen detection assay (PathoDx) and two PCR assays (for cfb and scpB). The most accurate assay was the scpB PCR (sensitivity, 99.6%; specificity, 100%), followed by the cfb PCR (sensitivity, 75.3%; specificity, 100%), GBS antigen detection (sensitivity, 57.3%; specificity, 99.5%), and standard culture (sensitivity, 42.3%; specificity, 100%). The GBS antigen detection assay was found to be more sensitive than the standard culture method, and moreover, the assay has a low cost and is easy to perform in all obstetrical centers which have access to the most basic of diagnostic microbiology services. We believe that antigen detection on incubated LIM broth should replace the standard culture method for screening for GBS carriage at 35 to 37 weeks of gestation. The impact of the greater sensitivities of PCR assays on the diminution of neonatal GBS infections remains to be demonstrated.

  16. Diagnostics for ITER

    SciTech Connect

    Donne, A. J. H.; Hellermann, M. G. von; Barnsley, R.

    2008-10-22

    After an introduction into the specific challenges in the field of diagnostics for ITER (specifically high level of nuclear radiation, long pulses, high fluxes of particles to plasma facing components, need for reliability and robustness), an overview will be given of the spectroscopic diagnostics foreseen for ITER. The paper will describe both active neutral-beam based diagnostics as well as passive spectroscopic diagnostics operating in the visible, ultra-violet and x-ray spectral regions.

  17. Malaria Diagnostics in Clinical Trials

    PubMed Central

    Murphy, Sean C.; Shott, Joseph P.; Parikh, Sunil; Etter, Paige; Prescott, William R.; Stewart, V. Ann

    2013-01-01

    Malaria diagnostics are widely used in epidemiologic studies to investigate natural history of disease and in drug and vaccine clinical trials to exclude participants or evaluate efficacy. The Malaria Laboratory Network (MLN), managed by the Office of HIV/AIDS Network Coordination, is an international working group with mutual interests in malaria disease and diagnosis and in human immunodeficiency virus/acquired immunodeficiency syndrome clinical trials. The MLN considered and studied the wide array of available malaria diagnostic tests for their suitability for screening trial participants and/or obtaining study endpoints for malaria clinical trials, including studies of HIV/malaria co-infection and other malaria natural history studies. The MLN provides recommendations on microscopy, rapid diagnostic tests, serologic tests, and molecular assays to guide selection of the most appropriate test(s) for specific research objectives. In addition, this report provides recommendations regarding quality management to ensure reproducibility across sites in clinical trials. Performance evaluation, quality control, and external quality assessment are critical processes that must be implemented in all clinical trials using malaria tests. PMID:24062484

  18. DNA Microarray-Based Diagnostics.

    PubMed

    Marzancola, Mahsa Gharibi; Sedighi, Abootaleb; Li, Paul C H

    2016-01-01

    The DNA microarray technology is currently a useful biomedical tool which has been developed for a variety of diagnostic applications. However, the development pathway has not been smooth and the technology has faced some challenges. The reliability of the microarray data and also the clinical utility of the results in the early days were criticized. These criticisms added to the severe competition from other techniques, such as next-generation sequencing (NGS), impacting the growth of microarray-based tests in the molecular diagnostic market.Thanks to the advances in the underlying technologies as well as the tremendous effort offered by the research community and commercial vendors, these challenges have mostly been addressed. Nowadays, the microarray platform has achieved sufficient standardization and method validation as well as efficient probe printing, liquid handling and signal visualization. Integration of various steps of the microarray assay into a harmonized and miniaturized handheld lab-on-a-chip (LOC) device has been a goal for the microarray community. In this respect, notable progress has been achieved in coupling the DNA microarray with the liquid manipulation microsystem as well as the supporting subsystem that will generate the stand-alone LOC device.In this chapter, we discuss the major challenges that microarray technology has faced in its almost two decades of development and also describe the solutions to overcome the challenges. In addition, we review the advancements of the technology, especially the progress toward developing the LOC devices for DNA diagnostic applications.

  19. Immuno-Chromatographic Wicking Assay for the Rapid Detection of Chikungunya Viral Antigens in Mosquitoes (Diptera: Culicidae).

    PubMed

    Hinson, Juanita M; Davé, Sonia; McMenamy, Scott S; Davé, Kirti; Turell, Michael J

    2015-07-01

    The outbreak of disease caused by chikungunya virus (CHIKV) in 2006 and the recent spread of this virus to the Americas in 2013 indicate the potential for this virus to spread and cause significant disease. However, there are currently no accurate and reliable field-usable, diagnostic methods to provide critical, real-time information for early detection of CHIKV within the vector populations in order to implement appropriate vector control and personal protective measures. In this article, we report the ability of an immuno-chromatographic assay developed by VecTOR Test Systems Inc. to detect CHIKV in a pool of female Aedes mosquitoes containing a single CHIKV-infected mosquito. The CHIKV dipstick assay was simple to use, did not require a cold chain, and provided clear results within 1 h. It was highly specific and did not cross-react with samples spiked with a variety of other alpha, bunya, and flaviviruses. The CHIKV assay can provide real-time critical information on the presence of CHIKV in mosquitoes to public health personnel. Results from this assay will allow a rapid threat assessment and the focusing of vector control measures in high-risk areas.

  20. A paper-based lateral flow assay for morphine.

    PubMed

    Teerinen, Tuija; Lappalainen, Timo; Erho, Tomi

    2014-09-01

    Morphine was used as a model analyte to examine the possibility of using cellulose, physically modified by papermaking and converting techniques, as a capillary matrix in a lateral flow type of diagnostic assay. This research was directed toward low-cost, disposable, and portable paper-based diagnostics, with the aim of addressing the analytical performance of paper as a substrate in the analysis for drugs of abuse. Antibody Fab fragments were used as sensing molecules, and gold nanoparticle detection was employed. Inkjet printing was used to pattern sensing biomolecules as detection zones on paper. To validate the usefulness of paper as a diagnostic platform, the principle of a direct sandwich assay, based on immunocomplex formation between morphine and the anti-morphine Fab fragment and detection of the formed immunocomplex by another Fab fragment, was implemented. Results were compared with that achieved by using nitrocellulose as a reference material. Possible interfering from the sample matrix on assay quality was investigated with spiked oral fluid samples. Under optimized conditions, a visually assessed limit of detection for the sandwich assay was 1 ng/mL, indicating that the paper-based test devices developed in this work can perform screening for drugs of abuse and can fulfill the requirement for a sensitive assay in diagnostically relevant ranges.

  1. 38 CFR 4.46 - Accurate measurement.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2013-07-01 2013-07-01 false Accurate measurement. 4... RATING DISABILITIES Disability Ratings The Musculoskeletal System § 4.46 Accurate measurement. Accurate measurement of the length of stumps, excursion of joints, dimensions and location of scars with respect...

  2. 38 CFR 4.46 - Accurate measurement.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2012-07-01 2012-07-01 false Accurate measurement. 4... RATING DISABILITIES Disability Ratings The Musculoskeletal System § 4.46 Accurate measurement. Accurate measurement of the length of stumps, excursion of joints, dimensions and location of scars with respect...

  3. 38 CFR 4.46 - Accurate measurement.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2010-07-01 2010-07-01 false Accurate measurement. 4... RATING DISABILITIES Disability Ratings The Musculoskeletal System § 4.46 Accurate measurement. Accurate measurement of the length of stumps, excursion of joints, dimensions and location of scars with respect...

  4. 38 CFR 4.46 - Accurate measurement.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2014-07-01 2014-07-01 false Accurate measurement. 4... RATING DISABILITIES Disability Ratings The Musculoskeletal System § 4.46 Accurate measurement. Accurate measurement of the length of stumps, excursion of joints, dimensions and location of scars with respect...

  5. 38 CFR 4.46 - Accurate measurement.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2011-07-01 2011-07-01 false Accurate measurement. 4... RATING DISABILITIES Disability Ratings The Musculoskeletal System § 4.46 Accurate measurement. Accurate measurement of the length of stumps, excursion of joints, dimensions and location of scars with respect...

  6. CPTAC Assay Portal: a repository of targeted proteomic assays

    SciTech Connect

    Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John; Kennedy, Jacob; Mani, DR; Zimmerman, Lisa J.; Meyer, Matthew R.; Mesri, Mehdi; Rodriguez, Henry; Abbateillo, Susan E.; Boja, Emily; Carr, Steven A.; Chan, Daniel W.; Chen, Xian; Chen, Jing; Davies, Sherri; Ellis, Matthew; Fenyo, David; Hiltket, Tara; Ketchum, Karen; Kinsinger, Christopher; Kuhn, Eric; Liebler, Daniel; Lin, De; Liu, Tao; Loss, Michael; MacCoss, Michael; Qian, Weijun; Rivers, Robert; Rodland, Karin D.; Ruggles, Kelly; Scott, Mitchell; Smith, Richard D.; Thomas, Stefani N.; Townsend, Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Paulovich, Amanda G.

    2014-06-27

    To address these issues, the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as a public repository of well-characterized quantitative, MS-based, targeted proteomic assays. The purpose of the CPTAC Assay Portal is to facilitate widespread adoption of targeted MS assays by disseminating SOPs, reagents, and assay characterization data for highly characterized assays. A primary aim of the NCI-supported portal is to bring together clinicians or biologists and analytical chemists to answer hypothesis-driven questions using targeted, MS-based assays. Assay content is easily accessed through queries and filters, enabling investigators to find assays to proteins relevant to their areas of interest. Detailed characterization data are available for each assay, enabling researchers to evaluate assay performance prior to launching the assay in their own laboratory.

  7. Tube-Forming Assays.

    PubMed

    Brown, Ryan M; Meah, Christopher J; Heath, Victoria L; Styles, Iain B; Bicknell, Roy

    2016-01-01

    Angiogenesis involves the generation of new blood vessels from the existing vasculature and is dependent on many growth factors and signaling events. In vivo angiogenesis is dynamic and complex, meaning assays are commonly utilized to explore specific targets for research into this area. Tube-forming assays offer an excellent overview of the molecular processes in angiogenesis. The Matrigel tube forming assay is a simple-to-implement but powerful tool for identifying biomolecules involved in angiogenesis. A detailed experimental protocol on the implementation of the assay is described in conjunction with an in-depth review of methods that can be applied to the analysis of the tube formation. In addition, an ImageJ plug-in is presented which allows automatic quantification of tube images reducing analysis times while removing user bias and subjectivity.

  8. ISHM-oriented adaptive fault diagnostics for avionics based on a distributed intelligent agent system

    NASA Astrophysics Data System (ADS)

    Xu, Jiuping; Zhong, Zhengqiang; Xu, Lei

    2015-10-01

    In this paper, an integrated system health management-oriented adaptive fault diagnostics and model for avionics is proposed. With avionics becoming increasingly complicated, precise and comprehensive avionics fault diagnostics has become an extremely complicated task. For the proposed fault diagnostic system, specific approaches, such as the artificial immune system, the intelligent agents system and the Dempster-Shafer evidence theory, are used to conduct deep fault avionics diagnostics. Through this proposed fault diagnostic system, efficient and accurate diagnostics can be achieved. A numerical example is conducted to apply the proposed hybrid diagnostics to a set of radar transmitters on an avionics system and to illustrate that the proposed system and model have the ability to achieve efficient and accurate fault diagnostics. By analyzing the diagnostic system's feasibility and pragmatics, the advantages of this system are demonstrated.

  9. The evolving potential of companion diagnostics.

    PubMed

    Khoury, Joseph D

    2016-01-01

    The scope of companion diagnostics in cancer has undergone significant shifts in the past few years, with increased development of targeted therapies and novel testing platforms. This has provided new opportunities to effect unprecedented paradigm shifts in the application of personalized medicine principles for patients with cancer. These shifts involve assay platforms, analytes, regulations, and therapeutic approaches. As opportunities involving each of these facets of companion diagnostics expand, close collaborations between key stakeholders should be enhanced to ensure optimal performance characteristics and patient outcomes.

  10. Doped colorimetric assay liposomes

    DOEpatents

    Charych, Deborah; Stevens, Raymond C.

    2001-01-01

    The present invention provides compositions comprising colorimetric assay liposomes. The present invention also provides methods for producing colorimetric liposomes and calorimetric liposome assay systems. In preferred embodiments, these calorimetric liposome systems provide high levels of sensitivity through the use of dopant molecules. As these dopants allow the controlled destabilization of the liposome structure, upon exposure of the doped liposomes to analyte(s) of interest, the indicator color change is facilitated and more easily recognized.

  11. Multiple log potash assay

    NASA Astrophysics Data System (ADS)

    Hill, D. G.

    1993-10-01

    A five-mineral multiple-log potash assay technique has been successfully applied to evaluate potash-rich intervals in evaporite sequences. The technique is able to distinguish economic potash minerals from non-economic potash minerals and from other non-potash radioactive minerals. It can be applied on location, using a programmable calculator or microcomputer, providing near real-time logs of potash mineral concentrations. Log assay values show good agreement with core wet chemistry analyses.

  12. Performance Characteristics of Current-Generation Immulite 2000 TORCH Assays

    PubMed Central

    Centonze, A. R.; Tonolli, E.

    2013-01-01

    The performances of seven Immulite 2000 (Siemens Healthcare Diagnostics) TORCH (Toxoplasma gondii, other microorganisms, rubella virus, cytomegalovirus, and herpes simplex virus) assays were evaluated in comparison with the performances of the ETI-MAX 3000 (DiaSorin) TORCH assays. The two systems demonstrated good agreement, and given their sensitivity, specificity, and positive predictive value, they can be used with confidence for TORCH prenatal screening. PMID:23175287

  13. Target Diagnostics Supports NIF's Path to Ignition

    SciTech Connect

    Shelton, R

    2011-12-07

    The physics requirements derived from the National Ignition Facility (NIF) experimental campaigns are leading to a wide variety of target diagnostics. Software development for the control and analysis of these diagnostics is included in the NIF Integrated Computer Control System, Diagnostic Control System and Data Visualization. These projects implement the configuration, controls, data analysis and visual representation of most of these diagnostics. To date, over 40 target diagnostics have been developed to support NIF experiments. In 2011 diagnostics were developed or enhanced to measure Ignition performance in a high neutron yield environment. Performance is optimized around four key variables: Adiabat (a) which is the strength and timing of four shocks delivered to the target, Velocity (V) of the imploding target, Mix (M) is the uniformity of the burn, and the Shape (S) of the imploding Deuterium Tritium (DT) hot spot. The diagnostics used to measure each of these parameters is shown in figure 1. Adiabat is measured using the Velocity Interferometer System for Any Reflector (VISAR) diagnostic consisting of three streak cameras. To provide for more accurate adiabat measurements the VISAR streak cameras were enhanced in FY11 with a ten comb fiducial signal controller to allow for post shot correction of the streak camera sweep non-linearity. Mix is measured by the Neutron Time of Flight (NTOF) and Radiochemical Analysis of Gaseous Samples (RAGS) diagnostics. To accommodate high neutron yield shots, NTOF diagnostic controls are being modified to use Mach Zehnder interferometer signals to allow the digitizers to be moved from near the target chamber to the neutron shielded diagnostic mezzanine. In December 2011 the first phase of RAGS diagnostic commissioning will be completed. This diagnostic will analyze the tracers that are added to NIF target capsules that undergo nuclear reactions during the shot. These gases are collected and purified for nuclear counting by

  14. Rapid Accurate Identification of Bacterial and Viral Pathogens

    SciTech Connect

    Dunn, John

    2007-03-09

    The goals of this program were to develop two assays for rapid, accurate identification of pathogenic organisms at the strain level. The first assay "Quantitative Genome Profiling or QGP" is a real time PCR assay with a restriction enzyme-based component. Its underlying concept is that certain enzymes should cleave genomic DNA at many sites and that in some cases these cuts will interrupt the connection on the genomic DNA between flanking PCR primer pairs thereby eliminating selected PCR amplifications. When this occurs the appearance of the real-time PCR threshold (Ct) signal during DNA amplification is totally eliminated or, if cutting is incomplete, greatly delayed compared to an uncut control. This temporal difference in appearance of the Ct signal relative to undigested control DNA provides a rapid, high-throughput approach for DNA-based identification of different but closely related pathogens depending upon the nucleotide sequence of the target region. The second assay we developed uses the nucleotide sequence of pairs of shmi identifier tags (-21 bp) to identify DNA molecules. Subtle differences in linked tag pair combinations can also be used to distinguish between closely related isolates..

  15. Diagnostics for Fast Ignition Science

    SciTech Connect

    MacPhee, A; Akli, K; Beg, F; Chen, C; Chen, H; Clarke, R; Hey, D; Freeman, R; Kemp, A; Key, M; King, J; LePape, S; Link, A; Ma, T; Nakamura, N; Offermann, D; Ovchinnikov, V; Patel, P; Phillips, T; Stephens, R; Town, R; Wei, M; VanWoerkom, L; Mackinnon, A

    2008-05-06

    The concept for Electron Fast Ignition Inertial Confinement Fusion demands sufficient laser energy be transferred from the ignitor pulse to the assembled fuel core via {approx}MeV electrons. We have assembled a suite of diagnostics to characterize such transfer. Recent experiments have simultaneously fielded absolutely calibrated extreme ultraviolet multilayer imagers at 68 and 256eV; spherically bent crystal imagers at 4 and 8keV; multi-keV crystal spectrometers; MeV x-ray bremmstrahlung and electron and proton spectrometers (along the same line of sight); nuclear activation samples and a picosecond optical probe based interferometer. These diagnostics allow careful measurement of energy transport and deposition during and following laser-plasma interactions at extremely high intensities in both planar and conical targets. Augmented with accurate on-shot laser focal spot and pre-pulse characterization, these measurements are yielding new insight into energy coupling and are providing critical data for validating numerical PIC and hybrid PIC simulation codes in an area that is crucial for many applications, particularly fast ignition. Novel aspects of these diagnostics and how they are combined to extract quantitative data on ultra high intensity laser plasma interactions are discussed, together with implications for full-scale fast ignition experiments.

  16. High Energy Laser Diagnostic Sensors

    NASA Astrophysics Data System (ADS)

    Luke, James R.; Goddard, Douglas N.; Lewis, Jay; Thomas, David

    2010-10-01

    Recent advancements in high energy laser (HEL) sources have outpaced diagnostic tools capable of accurately quantifying system performance. Diagnostic tools are needed that allow system developers to measure the parameters that define HEL effectiveness. The two critical parameters for quantifying HEL effectiveness are the irradiance on target and resultant rise in target temperature. Off-board sensing has its limitations, including unpredictable changes in the reflectivity of the target, smoke and outgassing, and atmospheric distortion. On-board sensors overcome the limitations of off-board techniques but must survive high irradiance levels and extreme temperatures. We have developed sensors for on-target diagnostics of high energy laser beams and for the measurement of the thermal response of the target. The conformal sensors consist of an array of quantum dot photodetectors and resistive temperature detectors. The sensor arrays are lithographically fabricated on flexible substrates and can be attached to a variety of laser targets. We have developed a nanoparticle adhesive process that provides good thermal contact with the target and that ensures the sensor remains attached to the target for as long as the target survives. We have calibrated the temperature and irradiance sensors and demonstrated them in a HEL environment.

  17. Specific assays of hemostasis proteins: fibrinogen.

    PubMed

    Palareti, G; Maccaferri, M

    1990-01-01

    Fibrinogen levels are considered a useful indicator in several pathological conditions and recent epidemiological studies have indicated a relationship between fibrinogen levels and increased risk of cardiovascular disease. An accurate measurement of this protein is therefore recommended and the Italian Committee for Standardization of Methods in Hematology and Laboratory has carried out a collaborative study to determine accuracy, precision and comparability of results obtained by six different methods, i.e., 1. Blombäck and Blombäck method, 2. clotting assay according to von Clauss, 3. radial immunodiffusion according to Mancini et al., 4. total amount of clottable fibrinogen by means of turbidimetric assay according to Ellis and Stransky, and 5. with ChromotimeSystem, 6. prothrombin time (PT)-derived fibrinogen assay on ACL coagulometer. The most accurate resulted the von Clauss method, but only if calibrated with an internal standard; in fact, when the manufacturer's tables are used, the method proved to be highly inaccurate. The best precision, both intra- and between-laboratory, was obtained by the PT-derived test on ACL. On the basis of this still incomplete evaluation of the CISMEL study data, we can conclude that: i. some methods used in clinical laboratories give accurate results only after adequate calibration; ii. a reference standard pool may be a valid tool for calibration and for a better between-laboratory comparability; iii. a predilution of the samples with high fibrinogen levels seems indicated; iv. automation markedly increases the precision of methods.

  18. SNAP Assay Technology.

    PubMed

    O'Connor, Thomas P

    2015-12-01

    The most widely used immunoassay configuration is the enzyme-linked immunosorbent assay (ELISA) because the procedure produces highly sensitive and specific results and generally is easy to use. By definition, ELISAs are immunoassays used to detect a substance (typically an antigen or antibody) in which an enzyme is attached (conjugated) to one of the reactants and an enzymatic reaction is used to amplify the signal if the substance is present. Optimized ELISAs include several steps that are performed in sequence using a defined protocol that typically includes application of sample and an enzyme-conjugated antibody or antigen to an immobilized reagent, followed by wash and enzyme reaction steps. The SNAP assay is an in-clinic device that performs each of the ELISA steps in a timed sequential fashion with little consumer interface. The components and mechanical mechanism of the assay device are described. Detailed descriptions of features of the assay, which minimize nonspecific binding and enhance the ability to read results from weak-positive samples, are given. Basic principles used in assays with fundamentally different reaction mechanisms, namely, antigen-detection, antibody-detection, and competitive assays are given. Applications of ELISA technology, which led to the development of several multianalyte SNAP tests capable of testing for up to 6 analytes using a single-sample and a single-SNAP device are described.

  19. Serum selenium assay following serum ferritin assay

    SciTech Connect

    Stevens, R.G.; Morris, J.S.; Hann, H.L.; Pulsipher, B.; Stahlhut, M.W.

    1986-08-01

    Stored serum samples can be an important research resource into the etiology of cancer. These sera cannot be replaced and should therefore be used to best advantage. In previous epidemiologic studies, only single serum constituents have been assayed in individual serum samples. For example, serum ferritin has been examined in samples stored for as long as 10 years at -20C for a possible relation with general mortality (1) and cancer death (2). Ferritin is the tissue iron-storage protein and is therefore subject to denaturation. Serum selenium has also been examined in relation to cancer risk in a prospective manner by using stored frozen serum samples (3, 4). The interactions of a variety of serum factors in relation to cancer risk would be a desirable research goal, except that the amounts of serum typically available in frozen serum banks are less than 1 ml. It was the purpose of this investigation to determine if a radioimmunoassay for ferritin affected a subsequent neutron activation assay for selenium on the same 0.1 ml serum sample.

  20. An assay for measurement of protein adsorption to glass vials.

    PubMed

    Varmette, Elizabeth; Strony, Brianne; Haines, Daniel; Redkar, Rajendra

    2010-01-01

    Protein adsorption to primary packaging is one of the problems faced by biopharmaceutical drug companies. An assay was developed to quantify loss of proteins to glass vial surfaces. The assay involves the labeling of protein with a fluorescent dye, incubation of the labeled protein with the vial surface, elution of the adsorbed protein using a stripping buffer, and determination of fluorescence of the adsorbed protein using a fluorometer. The assay is simple to set up, accurate, sensitive, and flexible. The assay can be modified for indirect measurement of protein adsorption and offers an attractive alternative for researchers to quantify protein adsorption to glass vials and syringes.

  1. Nuclear Resonance Fluorescence for Materials Assay

    SciTech Connect

    Quiter, Brian J.; Ludewigt, Bernhard; Mozin, Vladimir; Prussin, Stanley

    2009-06-29

    This paper discusses the use of nuclear resonance fluorescence (NRF) techniques for the isotopic and quantitative assaying of radioactive material. Potential applications include age-dating of an unknown radioactive source, pre- and post-detonation nuclear forensics, and safeguards for nuclear fuel cycles Examples of age-dating a strong radioactive source and assaying a spent fuel pin are discussed. The modeling work has ben performed with the Monte Carlo radiation transport computer code MCNPX, and the capability to simulate NRF has bee added to the code. Discussed are the limitations in MCNPX?s photon transport physics for accurately describing photon scattering processes that are important contributions to the background and impact the applicability of the NRF assay technique.

  2. Nuclear Resonance Fluorescence for Materials Assay

    SciTech Connect

    Quiter, Brian; Ludewigt, Bernhard; Mozin, Vladimir; Prussin, Stanley

    2009-06-05

    This paper discusses the use of nuclear resonance fluorescence (NRF) techniques for the isotopic and quantitative assaying of radioactive material. Potential applications include age-dating of an unknown radioactive source, pre- and post-detonation nuclear forensics, and safeguards for nuclear fuel cycles Examples of age-dating a strong radioactive source and assaying a spent fuel pin are discussed. The modeling work has ben performed with the Monte Carlo radiation transport computer code MCNPX, and the capability to simulate NRF has bee added to the code. Discussed are the limitations in MCNPX's photon transport physics for accurately describing photon scattering processes that are important contributions to the background and impact the applicability of the NRF assay technique.

  3. Highly Accurate Antibody Assays for Early and Rapid Detection of Tuberculosis in African and Asian Elephants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tuberculosis (TB) in elephants is a re-emerging zoonotic disease caused primarily by Mycobacterium tuberculosis. Current methods for screening and diagnosis rely on trunk wash culture, which has serious limitations due to low test sensitivity, slow turn-around time, and variable sample quality. Inn...

  4. Tumorsphere assay provides more accurate prediction of in vivo responses to chemotherapeutics

    PubMed Central

    Kim, Soyoung; Alexander, Caroline M.

    2014-01-01

    Although the sphere culture system has been widely used in stem cell biology, its application for drug screening is limited due to lack of standardized, rapid analytical tools. To optimize sphere cultures for in vitro screening of drugs, we evaluated the properties of primary tumor cells growing as tumorspheres and compared their chemosensitivity to those of cells growing in monolayer. Most cells in tumorsphere cultures were quiescent whereas cells in monolayer culture had a high mitotic index. Moreover, doxorubicin showed better cytotoxicity than paclitaxel in the sphere cultures, but their efficacy was reversed in the monolayer cultures. Importantly, the response of cytotoxic outcomes for suspension cultures matched the in vivo response better than monolayer cultures, providing support for the use of short term suspension cultures of primary cells as a model for drug testing. PMID:24158677

  5. Cell Culture Assay for Human Noroviruses [response

    SciTech Connect

    Straub, Tim M.; Honer Zu Bentrup, Kerstin; Orosz Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza A.; Nickerson, Cheryl A.

    2007-07-01

    We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

  6. Predictive assay for cancer targets

    NASA Astrophysics Data System (ADS)

    Suess, Amanda; Nguyen, Christine; Sorensen, Karen; Montgomery, Jennifer; Souza, Brian; Kulp, Kris; Dugan, Larry; Christian, Allen

    2005-11-01

    Early detection of cancer is a key element in successful treatment of the disease. Understanding the particular type of cancer involved, its origins and probable course, is also important. PhIP (2-amino-1- methyl-6 phenylimidazo [4,5-b]pyridine), a heterocyclic amine produced during the cooking of meat at elevated temperatures, has been shown to induce mammary cancer in female, Sprague-Dawley rats. Tumors induced by PhIP have been shown to contain discreet cytogenetic signature patterns of gains and losses using comparative genomic hybridization (CGH). To determine if a protein signature exists for these tumors, we are analyzing expression levels of the protein products of the above-mentioned tumors in combination with a new bulk protein subtractive assay. This assay produces a panel of antibodies against proteins that are either on or off in the tumor. Hybridization of the antibody panel onto a 2-D gel of tumor or control protein will allow for identification of a distinct protein signature in the tumor. Analysis of several gene databases has identified a number of rat homologs of human cancer genes located in these regions of gain and loss. These genes include the oncogenes c-MYK, ERBB2/NEU, THRA and tumor suppressor genes EGR1 and HDAC3. The listed genes have been shown to be estrogen-responsive, suggesting a possible link between delivery of bio-activated PhIP to the cell nucleus via estrogen receptors and gene-specific PhIP-induced DNA damage, leading to cell transformation. All three tumors showed similar silver staining patterns compared to each other, while they all were different than the control tissue. Subsequent screening of these genes against those from tumors know to be caused by other agents may produce a protein signature unique to PhIP, which can be used as a diagnostic to augment optical and radiation-based detection schemes.

  7. Predictive Assay For Cancer Targets

    SciTech Connect

    Suess, A; Nguyen, C; Sorensen, K; Montgomery, J; Souza, B; Kulp, K; Dugan, L; Christian, A

    2005-09-19

    Early detection of cancer is a key element in successful treatment of the disease. Understanding the particular type of cancer involved, its origins and probable course, is also important. PhIP (2-amino-1-methyl-6 phenylimidazo [4,5-b]pyridine), a heterocyclic amine produced during the cooking of meat at elevated temperatures, has been shown to induce mammary cancer in female, Sprague-Dawley rats. Tumors induced by PhIP have been shown to contain discreet cytogenetic signature patterns of gains and losses using comparative genomic hybridization (CGH). To determine if a protein signature exists for these tumors, we are analyzing expression levels of the protein products of the above-mentioned tumors in combination with a new bulk protein subtractive assay. This assay produces a panel of antibodies against proteins that are either on or off in the tumor. Hybridization of the antibody panel onto a 2-D gel of tumor or control protein will allow for identification of a distinct protein signature in the tumor. Analysis of several gene databases has identified a number of rat homologs of human cancer genes located in these regions of gain and loss. These genes include the oncogenes c-MYK, ERBB2/NEU, THRA and tumor suppressor genes EGR1 and HDAC3. The listed genes have been shown to be estrogen-responsive, suggesting a possible link between delivery of bio-activated PhIP to the cell nucleus via estrogen receptors and gene-specific PhIP-induced DNA damage, leading to cell transformation. All three tumors showed similar silver staining patterns compared to each other, while they all were different than the control tissue. Subsequent screening of these genes against those from tumors know to be caused by other agents may produce a protein signature unique to PhIP, which can be used as a diagnostic to augment optical and radiation-based detection schemes.

  8. [Diagnostic criteria for neuromyelitisoptica spectrum disorders].

    PubMed

    Belova, A N; Boiko, A N; Belova, E M

    2016-01-01

    The review is devoted to revised international diagnostic criteria for neuromyelitisoptica spectrum disorders (NMOSD).Current diagnostic criteria allow NMOSD diagnosis not only for serum aquaporin-4 immunoglobulin G antibodies (AQP4-IgG)-seropositive patients but for AQP4-IgG-seronegative patients as well. New criteria are expected to make NMOSD diagnosis earlier and more accurate as well as to facilitate the differentiation with multiple sclerosis. Furthermore, unify international criteria should help to perform comparable epidemiologic studies and clinical trials of new drugs for NMOSD.

  9. Commissioning activities of the initial magnetic diagnostics for KSTAR tokamak

    NASA Astrophysics Data System (ADS)

    Lee, Sang Gon; Gyo Bak, Jun; Mie Ka, Eun

    2007-11-01

    The initial magnetic diagnostics for the KSTAR superconducting tokamak including three Rogowski coils, five flux/voltage loops, and sixty-four magnetic field probes have been successfully installed. The Rogowski coils, flux/voltage loops, and magnetic field probes measure the total plasma current, poloidal flux and loop voltage, and local poloidal magnetic field for the plasma position control and equilibrium studies, respectively. Accurate position measurements after installation for all of these initial magnetic diagnostics and in situ calibration for the Rogowski coils were finished. Data acquisition systems for these initial magnetic diagnostics are currently under preparation. Detail commissioning activities before the first plasma from these initial magnetic diagnostics will be presented.

  10. Review of Two Decades of Cholera Diagnostics – How Far Have We Really Come?

    PubMed Central

    Dick, Michal H.; Guillerm, Martine; Moussy, Francis; Chaignat, Claire-Lise

    2012-01-01

    Background Cholera, an ancient scourge, continues to inflict high rates of mortality today. The rising incidence of epidemics in areas of poor sanitation and crowding highlight the need for better epidemic prevention and early response. Such interventions require the availability of rapid and accurate diagnostic techniques to trigger timely response and mitigate the scale of the outbreak. The current gold standard of bacterial culture is inadequate for rapid diagnosis, highlighting the overarching neglect of field diagnostic needs. This paper was written to support the World Health Organisation's Global Task Force on Cholera Control mandated Cholera and diarrhoeal disease laboratory Network (CholdiNet) in devising a protocol for the validation of Rapid Diagnostic Tests (RDTs) for Vibrio cholerae. The status of diagnostic tools for Vibrio cholerae is assessed, describing products that have been commercialised over the last two decades and discussing their peer-reviewed evaluation. Method Review of post-1990 peer-reviewed and grey literature on rapid diagnostic tests for Vibrio cholerae. Results Since 1990, twenty four diagnostic tests have been developed for the detection of Vibrio cholerae in human faecal samples. Fourteen of these have also been described in the literature, with rapid chromatographic-immuno assays (CIA) featuring strongly. Polymerase chain reaction (PCR) assays maintain the ability to detect the lowest amount of bacteria; however CIAs achieve both low detection thresholds and high sensitivity and specificity, making them possible candidates for use in field conditions. Field and laboratory studies were performed in a wide range of settings demonstrating variability in performance, however only a few of these studies were sufficiently stringent, highlighting five RDTs that showed promise in field conditions; COAT, IP cholera dipstick, SMART, IP dipstick and Medicos. In light of non-independent reporting, the authors would like to see these five

  11. Companion diagnostics in oncology - current status and future aspects.

    PubMed

    Jørgensen, Jan Trøst

    2013-01-01

    A large number of targeted anticancer drugs are currently under development and most of them will have a companion diagnostic linked to their use. If a diagnostic assay is developed in conjunction with a targeted anticancer drug, such an assay will later end up determining the conditions for the use of the drug after its approval. The assay then becomes a kind of 'gatekeeper' in relation to which patients should be treated with the drug in question. This 'gatekeeper' role implies that companion diagnostic assays must live up to the same regulatory standards and requirements known from drug development. The assays must have proven to be analytically robust and reliable and to have demonstrated clinical utility before they are routinely used in the clinic. In the drug-diagnostic codevelopment model, several 'traditional' study designs have been used to demonstrate clinical utility. However, if we are to benefit from the increasing knowledge provided by molecular oncology, new ways should be developed to demonstrate the clinical utility of drug-diagnostic combinations. The development of such an approach will require a rethinking at different levels and is likely to include a number of ethical, regulatory and practical challenges.

  12. Comparative endpoint sensitivity of in vitro estrogen agonist assays.

    PubMed

    Dreier, David A; Connors, Kristin A; Brooks, Bryan W

    2015-07-01

    Environmental and human health implications of endocrine disrupting chemicals (EDCs), particularly xenoestrogens, have received extensive study. In vitro assays are increasingly employed as diagnostic tools to comparatively evaluate chemicals, whole effluent toxicity and surface water quality, and to identify causative EDCs during toxicity identification evaluations. Recently, the U.S. Environmental Protection Agency (USEPA) initiated ToxCast under the Tox21 program to generate novel bioactivity data through high throughput screening. This information is useful for prioritizing chemicals requiring additional hazard information, including endocrine active chemicals. Though multiple in vitro and in vivo techniques have been developed to assess estrogen agonist activity, the relative endpoint sensitivity of these approaches and agreement of their conclusions remain unclear during environmental diagnostic applications. Probabilistic hazard assessment (PHA) approaches, including chemical toxicity distributions (CTD), are useful for understanding the relative sensitivity of endpoints associated with in vitro and in vivo toxicity assays by predicting the likelihood of chemicals eliciting undesirable outcomes at or above environmentally relevant concentrations. In the present study, PHAs were employed to examine the comparative endpoint sensitivity of 16 in vitro assays for estrogen agonist activity using a diverse group of compounds from the USEPA ToxCast dataset. Reporter gene assays were generally observed to possess greater endpoint sensitivity than other assay types, and the Tox21 ERa LUC BG1 Agonist assay was identified as the most sensitive in vitro endpoint for detecting an estrogenic response. When the sensitivity of this most sensitive ToxCast in vitro endpoint was compared to the human MCF-7 cell proliferation assay, a common in vitro model for biomedical and environmental monitoring applications, the ERa LUC BG1 assay was several orders of magnitude less

  13. Homogeneous, bioluminescent proteasome assays.

    PubMed

    O'Brien, Martha A; Moravec, Richard A; Riss, Terry L; Bulleit, Robert F

    2015-01-01

    Protein degradation is mediated predominantly through the ubiquitin-proteasome pathway. The importance of the proteasome in regulating degradation of proteins involved in cell-cycle control, apoptosis, and angiogenesis led to the recognition of the proteasome as a therapeutic target for cancer. The proteasome is also essential for degrading misfolded and aberrant proteins, and impaired proteasome function has been implicated in neurodegerative and cardiovascular diseases. Robust, sensitive assays are essential for monitoring proteasome activity and for developing inhibitors of the proteasome. Peptide-conjugated fluorophores are widely used as substrates for monitoring proteasome activity, but fluorogenic substrates can exhibit significant background and can be problematic for screening because of cellular autofluorescence or interference from fluorescent library compounds. Furthermore, fluorescent proteasome assays require column-purified 20S or 26S proteasome (typically obtained from erythrocytes), or proteasome extracts from whole cells, as their samples. To provide assays more amenable to high-throughput screening, we developed a homogeneous, bioluminescent method that combines peptide-conjugated aminoluciferin substrates and a stabilized luciferase. Using substrates for the chymotrypsin-like, trypsin-like, and caspase-like proteasome activities in combination with a selective membrane permeabilization step, we developed single-step, cell-based assays to measure each of the proteasome catalytic activities. The homogeneous method eliminates the need to prepare individual cell extracts as samples and has adequate sensitivity for 96- and 384-well plates. The simple "add and read" format enables sensitive and rapid proteasome assays ideal for inhibitor screening.

  14. SIGMA RECEPTOR BINDING ASSAYS

    PubMed Central

    CHU, UYEN B.; RUOHO, ARNOLD E.

    2016-01-01

    Sigma receptors belong to a class of small molecule-regulated, primarily endoplasmic reticulum (ER) membrane-associated receptors, of which there are two subtypes: the Sigma-1 receptor (S1R) and the Sigma-2 receptor (S2R). Both S1R and S2R bind to a number of drugs including antipsychotic, haloperidol, and the opioid analgesic, (+)-pentazocine. Sigma receptors are implicated in multiple disease pathologies associated with the nervous system including diseases affecting motor control such as Amyotrophic Lateral Sclerosis (ALS) and Alzeimher's disease. This unit describes methods for the pharmacological characterization of S1R and S2R using radioligand-binding assays. In the first section, radioligand saturation binding assay to determine receptor densities and competitive inhibition assays to characterize affinities of novel compounds are presented for S1R using the selective S1R ligand, [3H]-(+)-pentazocine. The second section describes radioligand saturation binding assay and competitive inhibition assays for the S2R using a non-selective S1R and S2R ligand, [3H]-1,3-di(2-tolyl)guanidine ([3H]-DTG). PMID:26646191

  15. Production of monoclonal antibodies for detection of Citrus leprosis virus C in enzyme-linked immuno-assays and immunocapture reverse transcription-polymerase chain reaction.

    PubMed

    Choudhary, Nandlal; Roy, Avijit; Govindarajulu, A; Nakhla, M K; Levy, L; Brlansky, R H

    2014-09-01

    Citrus leprosis virus C (CiLV-C) causes damage in citrus production in the South and Central America. Since closely related types of citrus viruses have recently been described monoclonal antibodies (MAbs) are needed for accurate and sensitive diagnosis of CiLV-C. In this study, MAbs to the expressed coat protein of CiLV-C were produced for serological detection of CiLV-C in crude extracts of infected tissues in double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA), dot blot immunosorbent assays (DBIA) and immuonocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) procedures. Monoclonal antibodies were developed in mice to the purified expressed coat protein of CiLV-C. The published standard protocols of DAS-ELISA, DBIA and IC-RT-PCR were followed for the detection of coat protein p29 of CiLV-C in the crude extracts of CiLV-C infected tissues. Two monoclonal antibodies, designated G10 and C11, were identified from four potential candidates for the specific and sensitive detection of coat protein p29 of CiLV-C in the crude citrus extracts of CiLV-C infected tissues in DAS-ELISA, whereas G10 was also selected based on performance for use in the DBIA and IC-RT-PCR diagnostic assays. Sensitivity analysis comparing the three methods for detection of coat protein p29 of CiLV-C determined that IC-RT-PCR was more sensitive than DAS-ELISA and DBIA. The creation of MAbs to CiLV-C allows for the sensitive and accurate detection of the virus from CiLV-C infected citrus leaf tissues. Successful detection of the virus in three diagnostic assays formats provides flexibility to diagnosticians who can use either ELISA or DBIA for screening large numbers of samples, and IC-RT-PCR for rapid, sensitive confirmation testing.

  16. Rapid and Quantitative Detection of Leifsonia xyli subsp. xyli in Sugarcane Stalk Juice Using a Real-Time Fluorescent (TaqMan) PCR Assay

    PubMed Central

    Fu, Hua-Ying; Sun, Sheng-Ren; Wang, Jin-Da; Ahmad, Kashif; Wang, Heng-Bo; Chen, Ru-Kai

    2016-01-01

    Ratoon stunting disease (RSD) of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agent Leifsonia xyli subsp. xyli (Lxx). A TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR) assay was established in this study for the quantification of Lxx detection in sugarcane stalk juice. A pair of PCR primers (Pat1-QF/Pat1-QR) and a fluorogenic probe (Pat1-QP) targeting the Part1 gene of Lxx were used for the qPCR assay. The assay had a detection limit of 100 copies of plasmid DNA and 100 fg of Lxx genomic DNA, which was 100-fold more sensitive than the conventional PCR. Fifty (28.7%) of 174 stalk juice samples from two field trials were tested to be positive by qPCR assay, whereas, by conventional PCR, only 12.1% (21/174) were tested to be positive with a published primer pair CxxITSf#5/CxxITSr#5 and 15.5% (27/174) were tested to be positive with a newly designed primer pair Pat1-F2/Pat1-R2. The new qPCR assay can be used as an alternative to current diagnostic methods for Lxx, especially when dealing with certificating a large number of healthy cane seedlings and determining disease incidence accurately in commercial fields. PMID:27725937

  17. Use of cross-reactive serological assays for detecting novel pathogens in wildlife: assessing an appropriate cutoff for henipavirus assays in African bats.

    PubMed

    Peel, Alison J; McKinley, Trevelyan J; Baker, Kate S; Barr, Jennifer A; Crameri, Gary; Hayman, David T S; Feng, Yan-Ru; Broder, Christopher C; Wang, Lin-Fa; Cunningham, Andrew A; Wood, James L N

    2013-11-01

    Reservoir hosts of novel pathogens are often identified or suspected as such on the basis of serological assay results, prior to the isolation of the pathogen itself. Serological assays might therefore be used outside of their original, validated scope in order to infer seroprevalences in reservoir host populations, until such time that specific diagnostic assays can be developed. This is particularly the case in wildlife disease research. The absence of positive and negative control samples and gold standard diagnostic assays presents challenges in determining an appropriate threshold, or 'cutoff', for the assay that enables differentiation between seronegative and seropositive individuals. Here, multiple methods were explored to determine an appropriate cutoff for a multiplexed microsphere assay that is used to detect henipavirus antibody binding in fruit bat plasma. These methods included calculating multiples of 'negative' control assay values, receiver operating characteristic curve analyses, and Bayesian mixture models to assess the distribution of assay outputs for classifying seropositive and seronegative individuals within different age classes. As for any diagnostic assay, the most appropriate cutoff determination method and value selected must be made according to the aims of the study. This study is presented as an example for others where reference samples, and assays that have been characterised previously, are absent.

  18. Rover waste assay system

    SciTech Connect

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J.

    1997-11-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched {sup 235}U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for {sup 137}Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs.

  19. Recombinase Polymerase Amplification Assay for Rapid Detection of Francisella tularensis

    PubMed Central

    Euler, Milena; Wang, Yongjie; Otto, Peter; Tomaso, Herbert; Escudero, Raquel; Anda, Pedro; Hufert, Frank T.

    2012-01-01

    Several real-time PCR approaches to develop field detection for Francisella tularensis, the infectious agent causing tularemia, have been explored. We report the development of a novel qualitative real-time isothermal recombinase polymerase amplification (RPA) assay for use on a small ESEQuant Tube Scanner device. The analytical sensitivity and specificity were tested using a plasmid standard and DNA extracts from infected rabbit tissues. The assay showed a performance comparable to real-time PCR but reduced the assay time to 10 min. The rapid RPA method has great application potential for field use or point-of-care diagnostics. PMID:22518861

  20. Recombinase polymerase amplification assay for rapid detection of Francisella tularensis.

    PubMed

    Euler, Milena; Wang, Yongjie; Otto, Peter; Tomaso, Herbert; Escudero, Raquel; Anda, Pedro; Hufert, Frank T; Weidmann, Manfred

    2012-07-01

    Several real-time PCR approaches to develop field detection for Francisella tularensis, the infectious agent causing tularemia, have been explored. We report the development of a novel qualitative real-time isothermal recombinase polymerase amplification (RPA) assay for use on a small ESEQuant Tube Scanner device. The analytical sensitivity and specificity were tested using a plasmid standard and DNA extracts from infected rabbit tissues. The assay showed a performance comparable to real-time PCR but reduced the assay time to 10 min. The rapid RPA method has great application potential for field use or point-of-care diagnostics.

  1. Advances in paper-based point-of-care diagnostics.

    PubMed

    Hu, Jie; Wang, ShuQi; Wang, Lin; Li, Fei; Pingguan-Murphy, Belinda; Lu, Tian Jian; Xu, Feng

    2014-04-15

    Advanced diagnostic technologies, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), have been widely used in well-equipped laboratories. However, they are not affordable or accessible in resource-limited settings due to the lack of basic infrastructure and/or trained operators. Paper-based diagnostic technologies are affordable, user-friendly, rapid, robust, and scalable for manufacturing, thus holding great potential to deliver point-of-care (POC) diagnostics to resource-limited settings. In this review, we present the working principles and reaction mechanism of paper-based diagnostics, including dipstick assays, lateral flow assays (LFAs), and microfluidic paper-based analytical devices (μPADs), as well as the selection of substrates and fabrication methods. Further, we report the advances in improving detection sensitivity, quantification readout, procedure simplification and multi-functionalization of paper-based diagnostics, and discuss the disadvantages of paper-based diagnostics. We envision that miniaturized and integrated paper-based diagnostic devices with the sample-in-answer-out capability will meet the diverse requirements for diagnosis and treatment monitoring at the POC.

  2. Clonogenic Assay: Adherent Cells

    PubMed Central

    Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T.; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C.

    2011-01-01

    The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant

  3. Performance of Simplexa dengue molecular assay compared to conventional and SYBR green RT-PCR for detection of dengue infection in Indonesia.

    PubMed

    Sasmono, R Tedjo; Aryati, Aryati; Wardhani, Puspa; Yohan, Benediktus; Trimarsanto, Hidayat; Fahri, Sukmal; Setianingsih, Tri Y; Meutiawati, Febrina

    2014-01-01

    Diagnostic tests based on detection of dengue virus (DENV) genome are available with varying sensitivities and specificities. The Simplexa Dengue assay (Focus Diagnostics) is a newly developed real-time RT-PCR method designed to detect and serotype DENV simultaneously. To assess the performance of the Simplexa Dengue assay, we performed comparison with conventional RT-PCR and SYBR Green real-time RT-PCR on patients sera isolated from eight cities across Indonesia, a dengue endemic country. A total of 184 sera that were confirmed using NS1 and/or IgM and IgG ELISA were examined. Using conventional and SYBR Green real-time RT-PCR, we detected DENV in 53 (28.8%) and 81 (44.0%) out of 184 sera, respectively. When the Simplexa Dengue assay was employed, the detection rate was in