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Sample records for immunofluorescence assay testing

  1. Immunofluorescence

    International Nuclear Information System (INIS)

    Bongertz, V.; Castro, B.G.

    1990-01-01

    The advantages of the immunofluorescence assay (IFA) that allowing the specific 'in situ' localization of the antigen-antibody interaction, of paramount importance when complex antigenic preparations, such as for instance tissue sections, are studied. (L.M.J.) [pt

  2. 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.

    Science.gov (United States)

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

    2017-02-01

    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. 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. Sera from 37 BP and 19 pemphigus patients were studied. Associations between tests were assessed using Fisher's exact test. 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. 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.

  3. 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

    Directory of Open Access Journals (Sweden)

    Justyna Gornowicz-Porowska

    2017-02-01

    Full Text Available 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.

  4. Neuromyelitis optica immunoglobulin G in Chinese patients detected by immunofluorescence assay on a monkey brain substrate.

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    Long, Youming; Hu, Xueqiang; Peng, Fuhua; Lu, Zhengqi; Wang, Yuge; Yang, Yu; Qiu, Wei

    2012-01-01

    Serum neuromyelitis optica immunoglobulin G (NMO-IgG) is used as a biomarker to differentiate between neuromyelitis optica (NMO) and multiple sclerosis (MS). However, the original assay is expensive and complex and shows low sensitivity. Here, we investigated the potential of NMO-IgG detection using an indirect immunofluorescence (IIF) assay on monkey brains. NMO-IgG seroprevalence was determined in 168 samples by an IIF assay on a monkey brain substrate. The data were compared with those from a standard mouse brain IIF assay using McNemar and kappa tests. Thirty-one of 50 (62%) NMO patients, 7 of 18 (38.9%) longitudinally extensive transverse myelitis patients, 6 of 57 (10.5%) MS patients, and 5 of 10 (50%) optic neuritis patients were seropositive for NMO-IgG. None of the acute partial transverse myelitis patients (n = 3) or healthy controls (n = 20) was positive. Thus, the sensitivity of the test was 62% for the patients with clinically definite NMO. The specificity was 89.5%, considering the 57 MS patients as the control group. The modified IIF assay on monkey brains and the standard IIF assay based on mouse brains were not significantly different (McNemar test; p = 1.000). The two assays were concordant in 39 seropositive samples and 100 seronegative samples (kappa test; kappa = 0.592, p monkey brain assay was no better than the standard mouse brain IIF assay, we affirmed that NMO-IgG is a sensitive and specific biomarker to differentiate between NMO and MS. Copyright © 2011 S. Karger AG, Basel.

  5. Immunofluorescence Plaque Assay for African Swine Fever Virus

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    Tessler, J.; Hess, W. R.; Pan, I. C.; Trautman, R.

    1974-01-01

    Suitably diluted cell culture adapted African swine fever virus preparations were inoculated on VERO cell monolayers and grown on coverslips. Gum tragacanth was used as an overlay. After three days incubation at 37°C the infected cultures were fixed with acetone and stained with fluorescent antibody conjugate. Fluorescing plaques consisted of 20-30 infected cells. Three statistical criteria for a quantitatively reliable assay were met: the Poisson distribution for plaque counts, linearity of the relationship between the concentration of virus and the plaque count and reproducibility of replicate titrations. The method is suitable for counts up to at least 70 plaques per 5 cm2 coverslip and computed titers are reproducible within 0.16 log units with a total of 300 plaques enumerated. PMID:4279763

  6. Application of a sepharose bead immunofluorescence assay and a solid-phase radioimmunoassay to the bovine leukemia virus system

    International Nuclear Information System (INIS)

    Fiebach, H.; Uckert, W.; Micheel, B.

    1982-01-01

    Several fluorescence assays with bovine leukemia virus (BLV) conjugated to activated Sepharose 4B were used for the detection of BLV and anti-BLV antibodies. These tests were compared with a solid-phase radioimmunoassay and found to be in the same sensitivity range. Sepharose bead immunofluorescence assay and solid-phase radioimmunoassay can be applied to the diagnosis of BLV infection in cattle. (author)

  7. Application of a sepharose bead immunofluorescence assay and a solid-phase radioimmunoassay to the bovine leukemia virus system

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    Fiebach, H.; Uckert, W.; Micheel, B. (Akademie der Wissenschaften der DDR, Berlin. Zentralinstitut fuer Krebsforschung)

    Several fluorescence assays with bovine leukemia virus (BLV) conjugated to activated Sepharose 4B were used for the detection of BLV and anti-BLV antibodies. These tests were compared with a solid-phase radioimmunoassay and found to be in the same sensitivity range. Sepharose bead immunofluorescence assay and solid-phase radioimmunoassay can be applied to the diagnosis of BLV infection in cattle.

  8. Accuracy of rapid influenza diagnostic test and immunofluorescence assay compared to real time RT-PCR in children with influenza A(H1N1pdm09 infection 

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    Aneta Nitsch-Osuch

    2012-10-01

    Full Text Available  Introduction:The influenza burden among children is underestimated. The aim of our study was to estimate the accuracy of the rapid influenza detection test (RIDT BD Directigen™ EZ Flu A B® and direct immunofluorescence assay (DFA used among children with influenza-like illness (ILI consulted in the ambulatory care clinic.Material/Methods:A total of 150 patients were enrolled in the study. Inclusion criteria were: age less than 59 months, presentation of ILI according to the CDC (Centers for Disease Control and Prevention definition (fever >37.8°C, cough and/or sore throat in the absence of another known cause of illness, duration of symptoms shorter than 96 hours. Two nasal swabs and one pharyngeal swab were obtained from patients and tested by RIDT, DFA and real time RT-PCR as the reference method.Results:For influenza A (H1N1pdm09 virus sensitivity of RIDT was 62.2�0(95�0CI 46.5–76.2� specificity 97.1�0(95�0CI 91.8–99.4� PPV 90.3�0(95�0CI 74.3–98� NPV 85.7�0(95�0CI 78.1–91.5� for DFA sensitivity was 60�0(95�0CI 51.9–63.2� specificity 96�0(95�0CI 88.7–98.8� PPV 93.1�0(95�0CI 80.5–98� NPV 72.7�0(95�0CI 67.2–74.9� Analysis of logistic regression revealed that the chance of receiving a true positive result of RIDT was twice as high when the test was conducted during the first 48 hours of symptoms (OR 0.40 vs OR 0.22.Conclusions:The accuracy of RIDT is comparable with DFA and both methods are very specific but moderately sensitive in diagnosis of influenza in young children. Both methods may be recommended for screening for influenza among children.

  9. Systemic lupus erythematosus and the Crithidia luciliae immunofluorescent test

    International Nuclear Information System (INIS)

    Whitehouse, I.J.; Fehr, K.; Wagenhaeuser, F.J.

    1983-01-01

    A comparative study of the Crithidia luciliae immunofluorescence (CL-IF) assay and an adapted Farr radioimmunoassay (RIA), for the measurement of antibodies to native deoxyribonucleic acid, was performed using forty-two sera from patients with systematic lupus erythematosus (SLE) and another forty-two from patients with rheumatoid arthritis. Both assays were specific for SLE. The CL-IF assay was statistically significantly more sensitive than the adapted RIA assay. This significant difference was due to greater sensitivity of the CL-IF assay in the cases of sera from patients with SLE of slight activity. Additional advantages of the CL-IF assay were its use to classify the immunoglobulin types of the antibodies (most commonly IgG or IgM) and to measure complement-fixing antibodies to native deoxyribonucleic acid; it affords a simple method of selecting and following SLE patients at risk of developing severe renal disease. These advantages plus the simplicity and inexpensiveness of the CL-IF assay make it a useful tool, especially for use in small laboratories, for the study of antibodies to native deoxyribonucleic acid in patients with SLE. (orig.) [de

  10. Performance of an ELISA and Indirect Immunofluorescence Assay in Serological Diagnosis of Zoonotic Cutaneous Leishmaniasis in Iran

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    Bahador Sarkari

    2014-01-01

    Full Text Available Serological assays have been extensively evaluated for diagnosis of visceral leishmaniasis (VL and considered as a routine method for diagnosis of VL while these methods are not properly evaluated for diagnosis of cutaneous leishmaniasis (CL. This study aimed to assess the performance of indirect immunofluorescent-antibody test (IFA and enzyme-linked immunosorbent assay (ELISA for serodiagnosis of cutaneous leishmaniasis in Iran. Sixty-one sera samples from parasitologically confirmed CL patients and 50 sera from healthy controls along with 50 sera from non-CL patients were collected. Antigen was prepared from promastigotes and amastigotes of Leishmania major. IFA was used to detect anti-Leishmania IgG while ELISA was used to detect anti-Leishmania IgM, total IgG, or IgG subclasses (IgG1 and 4. ELISA, for detection of total IgG and IgM, showed sensitivity of 83.6% and 84.7% and specificity of 62.7% and 54.6%, respectively. Sensitivity and specificity of ELISA for detecting IgG1 and IgG4 were 64%, 75% and 85%, 49%, respectively. Sensitivity and specificity of IFA were 91.6% and 81%. Conclusion. Findings of this study demonstrated that serological test, especially IFA, can be used for proper diagnosis of CL.

  11. Direct immunofluorescence assay compared to cell culture for the diagnosis of mucocutaneous herpes simplex virus infections in children.

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    Caviness, A Chantal; Oelze, Lindsay L; Saz, Ulas E; Greer, Jewel M; Demmler-Harrison, Gail J

    2010-09-01

    Direct immunofluorescence assay (DFA) is commonly used for the rapid identification of herpes simplex virus (HSV) infection in mucocutaneous lesions, yet little is known about its diagnostic accuracy. To determine the diagnostic yield and accuracy of HSV DFA for the diagnosis of mucocutaneous HSV infection in pediatric patients. Retrospective cross-sectional study of all patients who underwent HSV DFA testing by the Texas Children's Hospital Diagnostic Virology between January 1, 1995 and December 31, 2005. HSV DFA sensitivity, specificity, positive likelihood ratio (LRs), and negative LRs were estimated using viral culture as the reference standard. 659 specimens were submitted for HSV DFA with concurrent viral cultures. Viral cultures were positive for HSV type 1 in 158 (24%) and HSV type 2 in 2 (0.3%). There were 433 different patients with a median age of 8.6 years. Types of lesions were as follows: 50% ulcerative, 26% vesicular, 8% erythema or purpura, 5% pustular, and 11% missing. Of the 659 specimens submitted for HSV DFA, 160 (24%) were inconclusive due to inadequate cells. Of the 499 adequate specimens, overall HSV DFA test accuracy was: sensitivity 61%, specificity 99%, LR positive 40, and LR negative 0.39. A quarter of specimens submitted for HSV DFA testing are not adequate for DFA testing. When HSV DFA can be performed, it is specific, but not sensitive, for the identification of mucocutaneous HSV infection in children. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  12. Evaluation of a Direct Immunofluorescent Assay and/or Conjunctival Cytology for Detection of Canine Distemper Virus Antigen.

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    Athanasiou, Labrini V; Kantere, Maria C; Kyriakis, Constantinos S; Pardali, Dimitra; Adamama Moraitou, Katerina; Polizopoulou, Zoe S

    2018-04-01

    Canine distemper is a common and potentially lethal multisystemic disease caused by the Canine distemper virus (CDV). We evaluated the diagnostic performance of direct immunofluorescent assay (FA) and cytology to detect CDV antigen in conjunctival cells compared with an established polymerase chain reaction (PCR) detection assay used as a gold standard for CDV diagnosis. Samples were collected from 57 young dogs presenting with central nervous system signs compatible with distemper disease. Exfoliative epithelial cells were collected from the right and left conjunctiva of each animal using nylon-bristled cytobrushes for cytology and cotton swabs for FA and PCR. For the direct FA, samples were stained with anti-CDV polyclonal antiserum conjugated to fluorescein isothiocyanate and imaged using a fluorescent microscope. Out of 57 dogs tested, 19 were PCR positive (15 positive in direct FA and 4 positive in cytology, including one that was negative by PCR), whereas 37 dogs were negative in all methods. A good agreement was observed between the FA and PCR, with a κ-value of 0.833 (95% CI: 0.678-0.989). Meanwhile, there was poor agreement between cytology and PCR (κ-value of 0.164; 95% CI: -0.045 to 0.373) and a fair agreement between FA and cytology (κ-value of 0.231; 95% CI: -0.026 to 0.487). Our results indicated a poor performance of cytology for the detection of CDV antigen. In contrast, FA is a 100% specific and an adequately sensitive assay (sensitivity: 78.95%, negative likelihood ratio: 0.21, 95% CI: 0.09-0.50) for antemortem diagnosis of canine distemper.

  13. Determination of cutoff of ELISA and immunofluorescence assay for scrub typhus

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    Nitin Gupta

    2016-01-01

    Full Text Available Background: The most common method employed for diagnosis of scrub typhus is serology. It is widely known that demonstration of ≥4-fold rise in titers of antibody in paired sera is required for diagnosis. However, for guidance of initial treatment, there is a need for rapid diagnosis at the time of admission. Therefore, there is a need for standardized region specific cutoff titers at the time of admission. Materials and Methods: A total of 258 patients of all age groups with clinically suspected scrub typhus over a period of 24 months (October 2013-October 2015 were enrolled. Serum samples of these patients were subjected to immunofluorescent antibody (IFA for immunoglobulin M (IgM (Fuller Labs, USA with dilutions of 1:64, 1:128, 1:256, and 1:512. Serum samples of all 258 patients were subjected to IgM ELISA (Inbios Inc., USA. Any patient with response to antibiotics within 48 h accompanied by either presence of an eschar or positivity by polymerase chain reaction was taken as positive. Receiver operating characteristic (ROC curve was drawn to generate cutoff for these tests. Results: A total of 20 patients were diagnosed as cases of scrub typhus. The ROC curve analysis revealed a cutoff optical density value of 0.87 with sensitivity and specificity of 100% and 94.12%, respectively. ROC curve analysis of IFA revealed sensitivity and specificity of 100% and 93.5%, respectively at 1:64 dilution. Conclusion: Considering cost constraints, centers in and around New Delhi region can use the cutoffs we determined for the diagnosis of scrub typhus.

  14. Indirect immunofluorescence assay for the simultaneous detection of antibodies against clinically important old and new world hantaviruses.

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    Sabine Lederer

    Full Text Available In order to detect serum antibodies against clinically important Old and New World hantaviruses simultaneously, multiparametric indirect immunofluorescence assays (IFAs based on biochip mosaics were developed. Each of the mosaic substrates consisted of cells infected with one of the virus types Hantaan (HTNV, Puumala (PUUV, Seoul (SEOV, Saaremaa (SAAV, Dobrava (DOBV, Sin Nombre (SNV or Andes (ANDV. For assay evaluation, serum IgG and IgM antibodies were analyzed using 184 laboratory-confirmed hantavirus-positive sera collected at six diagnostic centers from patients actively or previously infected with the following hantavirus serotypes: PUUV (Finland, n=97; SEOV (China, n=5; DOBV (Romania, n=7; SNV (Canada, n=23; ANDV (Argentina and Chile, n=52. The control panel comprised 89 sera from healthy blood donors. According to the reference tests, all 184 patient samples were seropositive for hantavirus-specific IgG (n=177; 96% and/or IgM (n=131; 72%, while all control samples were tested negative. In the multiparametric IFA applied in this study, 183 (99% of the patient sera were IgG and 131 (71% IgM positive (accordance with the reference tests: IgG, 96%; IgM, 93%. Overall IFA sensitivity for combined IgG and IgM analysis amounted to 100% for all serotypes, except for SNV (96%. Of the 89 control sera, 2 (2% showed IgG reactivity against the HTNV substrate, but not against any other hantavirus. Due to the high cross-reactivity of hantaviral nucleocapsid proteins, endpoint titrations were conducted, allowing serotype determination in >90% of PUUV- and ANDV-infected patients. Thus, multiparametric IFA enables highly sensitive and specific serological diagnosis of hantavirus infections and can be used to differentiate PUUV and ANDV infection from infections with Murinae-borne hantaviruses (e.g. DOBV and SEOV.

  15. Comparison of enzyme-linked immunosorbent assay, radioimmunoassay, complement fixation, anticomplement immunofluorescence and passive haemaglutination techniques for detecting cytomegalovirus IgG antibody

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    Booth, J C; Hannington, G; Bakir, T M.F.; Stern, H; Kangro, H; Griffiths, P D; Heath, R B [Saint George' s Hospital Medical School, London (UK); Saint Bartholomew' s Hospital, London (UK))

    1982-12-01

    The radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques were found to be comparable in sensitivity and specificity for detecting cytomegalovirus IgG antibody, and 10 to 100 times more sensitive than complement-fixation (CF), anticomplement immunofluorescence (ACIF) and passive haemagglutination (PHA). In screening tests for antibody, the frequency of false-positive and -negative results was 0.6% for RIA and ELISA, 1.5% for CF, 1.6% for ACIF and 3.6% for PHA. PHA was the least satisfactory test, largely because of technical problems.

  16. Direct immunofluorescence and enzyme-linked immunosorbent assays for evaluating chlorinated hydrocarbon degrading bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Brigmon, R.L.; Franck, M.M.; Brey, J.; Fliermans, C.B. [Westinghouse Savannah River, Aiken, SC (United States). Environmental Biotechnology Section; Scott, D.; Lanclos, K. [Medical Coll. of Georgia, Augusta, GA (United States)

    1997-06-01

    Immunological procedures were developed to enumerate chlorinated hydrocarbon degrading bacteria. Polyclonal antibodies (Pabs) were produced by immunizing New Zealand white rabbits against 18 contaminant-degrading bacteria. These included methanotrophic and chlorobenzene (CB) degrading species. An enzyme-linked immunosorbent assay (ELISA) was used to test for specificity and sensitivity of the Pabs. Direct fluorescent antibodies (DFAs) were developed with these Pabs against select methanotrophic bacteria isolated from a trichloroethylene (TCE) contaminated landfill at the Savannah River Site (SRS) and cultures from the American Type Culture Collection (ATCC). Analysis of cross reactivity testing data showed some of the Pabs to be group specific while others were species specific. The threshold of sensitivity for the ELISA is 105 bacteria cells/ml. The DFA can detect as few as one bacterium per ml after concentration. Results from the DFA and ELISA techniques for enumeration of methanotrophic bacteria in groundwater were higher but not significantly different (P < 0.05) compared to indirect microbiological techniques such as MPN. These methods provide useful information on in situ community structure and function for bioremediation applications within 1--4 hours of sampling.

  17. Use of a Granulocyte Immunofluorescence Assay Designed for Humans for Detection of Antineutrophil Cytoplasmic Antibodies in Dogs with Chronic Enteropathies.

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    Florey, J; Viall, A; Streu, S; DiMuro, V; Riddle, A; Kirk, J; Perazzotti, L; Affeldt, K; Wagner, R; Vaden, S; Harris, T; Allenspach, K

    2017-07-01

    Perinuclear antineutrophil cytoplasmic antibodies (pANCA) previously have been shown to be serum markers in dogs with chronic enteropathies, with dogs that have food-responsive disease (FRD) having higher frequencies of seropositivity than dogs with steroid-responsive disease (SRD). The indirect immunofluorescence (IIF) assay used in previous publications is time-consuming to perform, with low interobserver agreement. We hypothesized that a commercially available granulocyte IIF assay designed for humans could be used to detect perinuclear antineutrophil cytoplasmic antibodies in dogs. Forty-four dogs with FRD, 20 dogs with SRD, 20 control dogs, and 38 soft-coated wheaten terrier (SCWT) or SCWT-cross dogs. A granulocyte assay designed for humans was used to detect pANCA, cANCA, and antinuclear antibodies (ANA), as well as antibodies against proteinase-3 protein (PR-3) and myeloperoxidase protein (MPO) in archived serum samples. Sensitivity of the granulocyte assay to predict FRD in dogs was 0.61 (95% confidence interval (CI), 0.45, 0.75), and specificity was 1.00 (95% CI, 0.91, 1.00). A significant association was identified between positive pANCA or cANCA result and diagnosis of FRD (P < 0.0001). Agreement between the two assays to detect ANCA in the same serum samples from SCWT with protein-losing enteropathy/protein-losing nephropathy (PLE/PLN) was substantial (kappa, 0.77; 95% CI, 0.53, 1.00). Eight ANCA-positive cases were positive for MPO or PR-3 antibodies. The granulocyte immunofluorescence assay used in our pilot study was easy and quick to perform. Agreement with the previously published method was good. Copyright © 2017 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

  18. Evaluation of immunofluorescence microscopy and enzyme-linked immunosorbent assay in detection of Cryptosporidium and Giardia infections in asymptomatic dogs

    DEFF Research Database (Denmark)

    Rimhanen-Finne, R.; Enemark, Heidi L.; Kolehmainen, J.

    2007-01-01

    The performance of immunofluorescence microscopy (IF) and enzyme-linked immunosorbent assay (ELISA) in canine feces was evaluated. IF and Cryptosporidium ELISA detected 10(5) oocysts/g, while the detection limit for Giardia ELISA was 10(4) cysts/g. The Cryptosporidium ELISA showed 94% specificity...... zoonotic character of Cryptosporidium and Giardia infections in 150 asymptomatic Finnish dogs from the Helsinki area were studied. The overall proportion of dogs positive for Cryptosporidium was 5% (7/150) and that for Giardia 5% (8/150). In dogs...

  19. Performance analysis of automated evaluation of Crithidia luciliae-based indirect immunofluorescence tests in a routine setting - strengths and weaknesses.

    Science.gov (United States)

    Hormann, Wymke; Hahn, Melanie; Gerlach, Stefan; Hochstrate, Nicola; Affeldt, Kai; Giesen, Joyce; Fechner, Kai; Damoiseaux, Jan G M C

    2017-11-27

    Antibodies directed against dsDNA are a highly specific diagnostic marker for the presence of systemic lupus erythematosus and of particular importance in its diagnosis. To assess anti-dsDNA antibodies, the Crithidia luciliae-based indirect immunofluorescence test (CLIFT) is one of the assays considered to be the best choice. To overcome the drawback of subjective result interpretation that inheres indirect immunofluorescence assays in general, automated systems have been introduced into the market during the last years. Among these systems is the EUROPattern Suite, an advanced automated fluorescence microscope equipped with different software packages, capable of automated pattern interpretation and result suggestion for ANA, ANCA and CLIFT analysis. We analyzed the performance of the EUROPattern Suite with its automated fluorescence interpretation for CLIFT in a routine setting, reflecting the everyday life of a diagnostic laboratory. Three hundred and twelve consecutive samples were collected, sent to the Central Diagnostic Laboratory of the Maastricht University Medical Centre with a request for anti-dsDNA analysis over a period of 7 months. Agreement between EUROPattern assay analysis and the visual read was 93.3%. Sensitivity and specificity were 94.1% and 93.2%, respectively. The EUROPattern Suite performed reliably and greatly supported result interpretation. Automated image acquisition is readily performed and automated image classification gives a reliable recommendation for assay evaluation to the operator. The EUROPattern Suite optimizes workflow and contributes to standardization between different operators or laboratories.

  20. Automated two-site immunofluorescent assay for the measurement of serum chromogranin A.

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    Popovici, Théodora; Moreira, Baptiste; Schlageter, Marie-Hélène; Bories, Phuong-Nhi

    2014-01-01

    Chromogranin A (CgA) is the best-characterized biological marker common to neuroendocrine tumours and is therefore recommended for their diagnosis. The measurement of serum CgA is of great importance for reaching an early diagnosis and thus reducing the delay before treatment is instigated. The Kryptor CgA assay is the first fully automated assay available. The aim of this study was to evaluate its analytical performance. The imprecision and linearity of the Kryptor CgA assay were evaluated. This assay was compared with the Cis Bio CgA RIA assay in 78 serum samples. Its clinical utility was assessed in serum from 229 patients. The study performed on imprecision of Kryptor measurements showed intra- and inter-run CVs ≤ 5%. The study of linearity showed a satisfactory recovery rate for CgA concentrations up to 1200 μg/L. The Kryptor and RIA assays agreed well on the basis of the cut-off values provided by the two manufacturers. The Bland and Altman plot of the values obtained (range: 20-5560 μg/L) provided a mean difference of -10.1 μg/L (SD: 116). The clinical sensitivities of Kryptor CgA for diagnosis of pheochromocytoma and paraganglioma (n 20) and gastroenteropancreatic NETs (n 17) were respectively 100 and 94%. The Kryptor assay for CgA shows reliable analytical and clinical characteristics and allows a fast delivery of results. © 2013.

  1. Indirect micro-immunofluorescence test for detecting type-specific antibodies to herpes simplex virus.

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    Forsey, T; Darougar, S

    1980-02-01

    A rapid indirect micro-immunofluorescence test capable of detecting and differentiating type-specific antibodies to herpes simplex virus is described. The test proved highly sensitive and, in 80 patients with active herpes ocular infection, antibody was detected in 94%. No anti-herpes antibody was detected in a control group of 20 patients with adenovirus infections. Testing of animal sera prepared against herpes simplex virus types 1 and 2 and of human sera from cases of ocular and genital herpes infections showed that the test can differentiate antibodies to the infecting serotypes. Specimens of whole blood, taken by fingerprick, and eye secretions, both collected on cellulose sponges, could be tested by indirect micro-immunofluorescence. Anti-herpes IgG, IgM, and IgA can also be detected.

  2. Isolation, Culture, Functional Assays, and Immunofluorescence of Myofiber-Associated Satellite Cells.

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    Vogler, Thomas O; Gadek, Katherine E; Cadwallader, Adam B; Elston, Tiffany L; Olwin, Bradley B

    2016-01-01

    Adult skeletal muscle stem cells, termed satellite cells, regenerate and repair the functional contractile cells in adult skeletal muscle called myofibers. Satellite cells reside in a niche between the basal lamina and sarcolemma of myofibers. Isolating single myofibers and their associated satellite cells provides a culture system that partially mimics the in vivo environment. We describe methods for isolating and culturing intact individual myofibers and their associated satellite cells from the mouse extensor digitorum longus muscle. Following dissection and isolation of individual myofibers we provide protocols for myofiber transplantation, satellite cell transfection, immune detection of satellite cell antigens, and assays to examine satellite cell self-renewal and proliferation.

  3. A Comparison of Anti-Nuclear Antibody Quantification Using Automated Enzyme Immunoassays and Immunofluorescence Assays

    DEFF Research Database (Denmark)

    Baronaite, Renata; Engelhart, Merete; Mørk Hansen, Troels

    2014-01-01

    using IFA and automated EIA techniques. The IFA results generated by two independent laboratories were compared with the EIA results from antibodies against double-stranded DNA (dsDNA), from ANA screening, and from tests of the seven included subantigens. The final IFA and EIA results for 386 unique......, with Cohen's kappa value of 0.30 (95% confidence interval (CI) = 0.14-0.46), which decreased to 0.23 (95% CI = 0.06-0.40) when the results for dsDNA were omitted. The EIA method was less reliable for assessing nuclear and speckled reactivity patterns, whereas the IFA method presented difficulties detecting...... dsDNA and Ro activity. The automated EIA method was performed in a similar way to the conventional IFA method using HEp-2 cells; thus, automated EIA may be used as a screening test....

  4. Análise TG-ROC de testes de imunofluorescência no diagnóstico de leishmaniose visceral canina Análisis TG-ROC de pruebas de inmunofluorescencia en el diagnóstico de leishmaniasis visceral canina TG-ROC analysis of immunofluorescence assays in canine visceral leishmaniasis diagnosis

    Directory of Open Access Journals (Sweden)

    Rita Maria da Silva

    2009-12-01

    costo-beneficio.OBJECTIVE: To analyze the accuracy of the diagnosis of two protocols of indirect immunofluorescence assays for canine visceral leishmaniasis. METHODS: Dogs from the seroepidemiological survey conducted in an endemic area of the cities of Araçatuba and Andradina, in Northwestern São Paulo state, in 2003, and in a non-endemic area of the metropolitan region of São Paulo, were used to assess two protocols of indirect immunofluorescence assay (IFA for leishmaniasis: one using a Leishmania major heterologous antigen (IFA-BM and another using a Leishmania chagasi homologous antigen (IFA-CH. Two-graph receiver operating characteristic (TG-ROC analysis was used to estimate accuracy. TG-ROC analysis compared 1:20 dilution readings of the homologous antigen (IFA-CH, considered as reference test, with IFA-BM dilutions (heterologous antigen. RESULTS: The 1:20 dilution used in the IFA-CH test showed the best contingency coefficient (0.755 and the highest strength of association between the two variables studied (chi-square=124.3. Thus, it was considered the test reference dilution in comparisons with different IFA-BM test dilutions. The best IFA-BM results were obtained from 1:40 dilutions with the best contingency coefficient (0.680 and highest strength of association (chi-square=80.8. With the change in the cut-off point, recommended for the IFA-BM 1:40 dilution in this analysis, the specificity parameter value rose from 57.5% to 97.7%, even though the 1:80 dilution showed the best sensitivity estimate (80.2%, with the new cut-off point. CONCLUSIONS: TG-ROC analysis can provide important information about diagnostic tests, in addition to offering suggestions on cut-off points that can improve test sensitivity and specificity estimates and assessing these tests in terms of the best cost-benefit ratio.

  5. Surface-biofunctionalized multicore/shell CdTe@SiO2 composite particles for immunofluorescence assay

    Science.gov (United States)

    Jing, Lihong; Li, Yilin; Ding, Ke; Qiao, Ruirui; Rogach, Andrey L.; Gao, Mingyuan

    2011-12-01

    Strongly fluorescent multicore/shell structured CdTe@SiO2 composite particles of ~ 50 nm were synthesized via the reverse microemulsion method by using CdTe quantum dots co-stabilized by thioglycolic acid and thioglycerol. The optical stability of the CdTe@SiO2 composite particles in a wide pH range, under prolonged UV irradiation in pure water, or in different types of physiological buffers was systematically investigated. Towards immunofluorescence assay, both poly(ethylene glycol) (PEG) and carboxyl residues were simultaneously grafted on the surface of the silanol-terminated CdTe@SiO2 composite particles upon further reactions with silane reagents bearing a PEG segment and carboxyl group, respectively, in order to suppress the nonspecific interactions of the silica particles with proteins and meanwhile introduce reactive moieties to the fluorescent particles. Agarose gel electrophoresis, dynamic light scattering and conventional optical spectroscopy were combined to investigate the effectiveness of the surface modifications. Via the surface carboxyl residue, various antibodies were covalently conjugated to the fluorescent particles and the resultant fluorescent probes were used in detecting cancer cells through both direct fluorescent antibody and indirect fluorescent antibody assays, respectively.

  6. Anti-human neutrophil antigen-1a, -1b, and -2 antibodies in neonates and children with immune neutropenias analyzed by extracted granulocyte antigen immunofluorescence assay.

    Science.gov (United States)

    Onodera, Rie; Kurita, Emi; Taniguchi, Kikuyo; Karakawa, Shuhei; Okada, Satoshi; Kihara, Hirotaka; Fujii, Teruhisa; Kobayashi, Masao

    2017-11-01

    Anti-human neutrophil antigen (HNA) antibodies have been implicated in the development of neonatal alloimmune neutropenia (NAN) and autoimmune neutropenia (AIN). There are many conventional assay methods that detect anti-HNA antibodies. However, a method to measure multiple samples and detect several anti-HNA antibodies simultaneously is needed. We developed a new method, the extracted granulocyte antigen immunofluorescence assay (EGIFA), to analyze anti-HNA-1a, -1b, and -2 antibodies in sera. The results obtained by EGIFA were evaluated in comparison with those from several standard assay methods. Anti-HNA antibodies in serum samples from nine familial cases with suspected NAN (n = 19) and children with suspected AIN (n = 88) were also measured by EGIFA. The evaluation of nine serum samples with anti-HNA antibodies suggested that EGIFA demonstrated equivalent specificity and superior sensitivity to monoclonal antibody-specific immobilization of granulocyte antigens and had comparable sensitivity to the granulocyte indirect immunofluorescence test. EGIFA successfully detected anti-HNA-1a or -1b antibodies in seven of nine familial cases with suspected NAN. EGIFA detected anti-HNA antibodies in 40.9% of children with suspected AIN. Among them, isolated anti-HNA-1a or -1b antibody was detected in 4.5 or 12.5% of children, respectively, and anti-HNA-2 antibody was identified in 3.4% of children. The 30.8% (16 of 52) of children negative for anti-HNA antibody by EGIFA were positive for anti-HLA antibody. EGIFA facilitated the measurement of anti-HNA-1a, -1b, and/or -2 antibodies in sera. The prompt measurement of anti-HNA antibodies will improve the diagnosis and clinical management of patients with suspected NAN or AIN. © 2017 AABB.

  7. A Novel High-Content Immunofluorescence Assay as a Tool to Identify at the Single Cell Level γ-Globin Inducing Compounds.

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    Marta Durlak

    Full Text Available The identification of drugs capable of reactivating γ-globin to ameliorate β-thalassemia and Sickle Cell anemia is still a challenge, as available γ-globin inducers still have limited clinical indications. High-throughput screenings (HTS aimed to identify new potentially therapeutic drugs require suitable first-step-screening methods combining the possibility to detect variation in the γ/β globin ratio with the robustness of a cell line. We took advantage of a K562 cell line variant expressing β-globin (β-K562 to set up a new multiplexed high-content immunofluorescence assay for the quantification of γ- and β-globin content at single-cell level. The assay was validated by using the known globin inducers hemin, hydroxyurea and butyric acid and further tested in a pilot screening that confirmed HDACs as targets for γ-globin induction (as proved by siRNA-mediated HDAC3 knockdown and by treatment with HDACs inhibitors entinostat and dacinostat and identified Heme-oxygenases as novel candidate targets for γ-globin induction. Indeed, Heme-oxygenase2 siRNA knockdown as well as its inhibition by Tin protoporphyrin-IX (TinPPIX greatly increased γ-globin expression. This result is particularly interesting as several metalloporphyrins have already been developed for clinical uses and could be tested (alone or in combination with other drugs to improve pharmacological γ-globin reactivation for the treatment of β-hemoglobinopathies.

  8. Aquaporin-4 autoantibodies in neuromyelitis optica spectrum disorders: comparison between tissue-based and cell-based indirect immunofluorescence assays

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    Chan Koon H

    2010-09-01

    Full Text Available Abstract Background Neuromyelitis optica spectrum disorders (NMOSD are severe central nervous system inflammatory demyelinating disorders (CNS IDD characterized by monophasic or relapsing, longitudinally extensive transverse myelitis (LETM and/or optic neuritis (ON. A significant proportion of NMOSD patients are seropositive for aquaporin-4 (AQP4 autoantibodies. We compared the AQP4 autoantibody detection rates of tissue-based indirect immunofluorescence assay (IIFA and cell-based IIFA. Methods Serum of Chinese CNS IDD patients were assayed for AQP4 autoantibodies by tissue-based IIFA using monkey cerebellum and cell-based IIFA using transfected HEK293 cells which express human AQP4 on their cell membranes. Results In total, 128 CNS IDD patients were studied. We found that 78% of NMO patients were seropositive for AQP4 autoantibodies by cell-based IIFA versus 61% by tissue-based IFA (p = 0.250, 75% of patients having relapsing myelitis (RM with LETM were seropositive by cell-based IIFA versus 50% by tissue-based IIFA (p = 0.250, and 33% of relapsing ON patients were seropositive by cell-based IIFA versus 22% by tissue-based IIFA (p = 1.000; however the differences were not statistically significant. All patients seropositive by tissue-based IIFA were also seropositive for AQP4 autoantibodies by cell-based IIFA. Among 29 NMOSD patients seropositive for AQP4 autoantibodies by cell-based IIFA, 20 (69% were seropositive by tissue-based IIFA. The 9 patients seropositive by cell-based IIFA while seronegative by tissue-based IIFA had NMO (3, RM with LETM (3, a single attack of LETM (1, relapsing ON (1 and a single ON attack (1. Among 23 NMO or RM patients seropositive for AQP4 autoantibodies by cell-based IIFA, comparison between those seropositive (n = 17 and seronegative (n = 6 by tissue-based IIFA revealed no differences in clinical and neuroradiological characteristics between the two groups. Conclusion Cell-based IIFA is slightly more sensitive

  9. IgA anti-Actin antibodies in children with celiac disease: comparison of immunofluorescence with Elisa assay in predicting severe intestinal damage

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    Mora Stefano

    2010-03-01

    Full Text Available Abstract Background Previous studies have demonstrated that the presence of serum IgA antibodies against actin filaments (AAA in patients with celiac disease (CD is strongly associated with mucosal damage and severe degrees of villous atrophy. The aims of the present study were (1 to verify the effectiveness of IgA-AAA in newly diagnosed CD patients in a clinical setting (2 to compare the immunofluorescence assay with ELISA assay; (3 to compare the correlation of our IgA anti-tissue transglutaminase antibodies (tTG-Ab class with mucosal intestinal lesions. Methods 90 patients underwent endoscopy and multiple biopsies for suspected CD on the basis of symptoms, in presence of positive tTG-Ab tests. Twenty biopsied and 25 not-biopsied subjects with negative tTG-Ab were tested as control groups. IgA-AAA assays were performed by indirect immunofluorescence using rat epithelial intestinal cells, and by ELISA with a commercial kit. tTG-Ab assay was a radio-binding assay. Intestinal specimens were collected by upper endoscopy and the histological study was done according to the Marsh's classification modified by Oberhuber (M/O. Auto-antibodies assays and histological evaluation have been performed blindly by skilled operators. Results CD diagnosis was confirmed in 82 patients (type I M/O in 2 patients, IIIA in 18 patients, IIIB in 29 patients and IIIC in 33 patients. Two patients with type 1 lesion in presence of positive tTG-Ab and abdominal complaints, started a gluten free diet. The rate of IgA-AAA positivity (sensitivity by IFI and ELISA in histologically proven celiac disease patients, were 5.5% and 25% patients in IIIA, 27.5% and 34.4% patients in IIIB, 78.8% and 75% in IIIC patients, respectively. Patients with normal or nearly normal mucosa, regardless of tTG-Ab status, presented negative IgA-AAA IFI assay. On the other hand, 1 patient with normal mucosa but positive tTG-Ab, also presented positive IgA-AAA ELISA. All healthy non biopsied

  10. An immunofluorescence assay for extracellular matrix components highlights the role of epithelial cells in producing a stable, fibrillar extracellular matrix

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    Omar S. Qureshi

    2017-10-01

    Full Text Available Activated fibroblasts are considered major drivers of fibrotic disease progression through the production of excessive extracellular matrix (ECM in response to signals from damaged epithelial and inflammatory cells. Nevertheless, epithelial cells are capable of expressing components of the ECM, cross-linking enzymes that increase its stability and are sensitive to factors involved in the early stages of fibrosis. We therefore wanted to test the hypothesis that epithelial cells can deposit ECM in response to stimulation in a comparable manner to fibroblasts. We performed immunofluorescence analysis of components of stable, mature extracellular matrix produced by primary human renal proximal tubular epithelial cells and renal fibroblasts in response to cytokine stimulation. Whilst fibroblasts produced a higher basal level of extracellular matrix components, epithelial cells were able to deposit significant levels of fibronectin, collagen I, III and IV in response to cytokine stimulation. In response to hypoxia, epithelial cells showed an increase in collagen IV deposition but not in response to the acute stress stimuli aristolochic acid or hydrogen peroxide. When epithelial cells were in co-culture with fibroblasts we observed significant increases in the level of matrix deposition which could be reduced by transforming growth factor beta (TGF-β blockade. Our results highlight the role of epithelial cells acting as efficient producers of stable extracellular matrix which could contribute to renal tubule thickening in fibrosis.

  11. Automated Processing and Evaluation of Anti-Nuclear Antibody Indirect Immunofluorescence Testing.

    Science.gov (United States)

    Ricchiuti, Vincent; Adams, Joseph; Hardy, Donna J; Katayev, Alexander; Fleming, James K

    2018-01-01

    Indirect immunofluorescence (IIF) is considered by the American College of Rheumatology (ACR) and the international consensus on ANA patterns (ICAP) the gold standard for the screening of anti-nuclear antibodies (ANA). As conventional IIF is labor intensive, time-consuming, subjective, and poorly standardized, there have been ongoing efforts to improve the standardization of reagents and to develop automated platforms for assay incubation, microscopy, and evaluation. In this study, the workflow and performance characteristics of a fully automated ANA IIF system (Sprinter XL, EUROPattern Suite, IFA 40: HEp-20-10 cells) were compared to a manual approach using visual microscopy with a filter device for single-well titration and to technologist reading. The Sprinter/EUROPattern system enabled the processing of large daily workload cohorts in less than 8 h and the reduction of labor hands-on time by more than 4 h. Regarding the discrimination of positive from negative samples, the overall agreement of the EUROPattern software with technologist reading was higher (95.6%) than when compared to the current method (89.4%). Moreover, the software was consistent with technologist reading in 80.6-97.5% of patterns and 71.0-93.8% of titers. In conclusion, the Sprinter/EUROPattern system provides substantial labor savings and good concordance with technologist ANA IIF microscopy, thus increasing standardization, laboratory efficiency, and removing subjectivity.

  12. Automated Processing and Evaluation of Anti-Nuclear Antibody Indirect Immunofluorescence Testing

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    Vincent Ricchiuti

    2018-05-01

    Full Text Available Indirect immunofluorescence (IIF is considered by the American College of Rheumatology (ACR and the international consensus on ANA patterns (ICAP the gold standard for the screening of anti-nuclear antibodies (ANA. As conventional IIF is labor intensive, time-consuming, subjective, and poorly standardized, there have been ongoing efforts to improve the standardization of reagents and to develop automated platforms for assay incubation, microscopy, and evaluation. In this study, the workflow and performance characteristics of a fully automated ANA IIF system (Sprinter XL, EUROPattern Suite, IFA 40: HEp-20-10 cells were compared to a manual approach using visual microscopy with a filter device for single-well titration and to technologist reading. The Sprinter/EUROPattern system enabled the processing of large daily workload cohorts in less than 8 h and the reduction of labor hands-on time by more than 4 h. Regarding the discrimination of positive from negative samples, the overall agreement of the EUROPattern software with technologist reading was higher (95.6% than when compared to the current method (89.4%. Moreover, the software was consistent with technologist reading in 80.6–97.5% of patterns and 71.0–93.8% of titers. In conclusion, the Sprinter/EUROPattern system provides substantial labor savings and good concordance with technologist ANA IIF microscopy, thus increasing standardization, laboratory efficiency, and removing subjectivity.

  13. Computer-assisted enzyme immunoassays and simplified immunofluorescence assays: applications for the diagnostic laboratory and the veterinarian's office.

    Science.gov (United States)

    Jacobson, R H; Downing, D R; Lynch, T J

    1982-11-15

    A computer-assisted enzyme-linked immunosorbent assay (ELISA) system, based on kinetics of the reaction between substrate and enzyme molecules, was developed for testing large numbers of sera in laboratory applications. Systematic and random errors associated with conventional ELISA technique were identified leading to results formulated on a statistically validated, objective, and standardized basis. In a parallel development, an inexpensive system for field and veterinary office applications contained many of the qualities of the computer-assisted ELISA. This system uses a fluorogenic indicator (rather than the enzyme-substrate interaction) in a rapid test (15 to 20 minutes' duration) which promises broad application in serodiagnosis.

  14. [Immunofluorescence assay with Crithidia luciliae for the detection of anti-DNA antibodies. Atypical images and their relationship with Chagas' disease and leishmaniasis].

    Science.gov (United States)

    Griemberg, Gloria; Ferrarotti, Nidia F; Svibel, Graciela; Ravelli, Maria R; Taranto, Nestor J; Malchiodi, Emilio L; Pizzimenti, Maria C

    2006-01-01

    Anti-native DNA antibodies can be detected by indirect immunofluorescence assay with Crithidia luciliae, displaying an annular image due to a kinetoplast containing double stranded DNA. Other structures such as membrane, flagellum and basal corpuscle can be stained as well, showing what is called atypical fluorescent images. As C. luciliae belongs to the Trypanosomatidae family, which include the human pathogens Trypanosoma cruzi and Leishmania spp., it was considered that these atypical images could be caused by cross-reactions. Serological studies for Chagas' disease were performed in 105 serum samples displaying atypical images. Sixty four percent of the samples from non endemic and 78.3% from endemic areas for Chagas' disease showed fluorescence in both, membrane and flagellum (joint image). Fifty samples from normal blood donors and 57 samples from patients with conective tissue diseases were tested with C. luciliae. None of them presented the joint image except for two patients with lupus who were also chagasic. In addition, 54 samples from chagasic patients were studied and all of them presented the joint image. We also studied 46 samples from patients with leishmaniasis from whom 28 were coinfected with T. cruzi. The joint image was observed in 88.0% of the samples with leishmaniasis and in 89.3% of the co-infected samples. The results suggest that C. luciliae could be used as an economical, and of low risk, alternative substrate for the serological diagnosis of Chagas' disease, even though it does not discriminate for Leishmania spp. infection. This study also suggests that whenever atypical images are observed in C. luciliae during the search for anti-DNA antibodies, it would be convenient to submit the patient to clinical and serological tests for the diagnosis of leishmaniosis and Chagas' disease.

  15. Comparison of electron microscopy, enzyme-linked immunosorbent assay, solid-phase radioimmunoassay, and indirect immunofluorescence for detection of human rotavirus antigen in faeces

    Energy Technology Data Exchange (ETDEWEB)

    Birch, C J; Lehmann, N I; Hawker, A J; Marshall, J A; Gust, I D [Fairfield Hospital for Communicable Diseases, Victoria (Australia). Virology Dept.

    1979-07-01

    Four techniques were compared for their practicability, speed, and sensitivity for the detection of human rotavirus. Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) were found to be the most sensitive means of identifying rotavirus and, once processed, up to 40 specimens could be examined daily. Electron microscopy, although less sensitive than these techniques, had the advantage of being able to detect other viral agents present in faecal extracts. Indirect immunofluorescence failed to detect rotavirus as often as the other three methods. In laboratories where routine examination of faecal specimens from patients with gastroenteritis is required, ELISA and RIA are useful alternatives to electron microscopy.

  16. Comparison of electron microscopy, enzyme-linked immunosorbent assay, solid-phase radioimmunoassay, and indirect immunofluorescence for detection of human rotavirus antigen in faeces

    International Nuclear Information System (INIS)

    Birch, C.J.; Lehmann, N.I.; Hawker, A.J.; Marshall, J.A.; Gust, I.D.

    1979-01-01

    Four techniques were compared for their practicability, speed, and sensitivity for the detection of human rotavirus. Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) were found to be the most sensitive means of identifying rotavirus and, once processed, up to 40 specimens could be examined daily. Electron microscopy, although less sensitive than these techniques, had the advantage of being able to detect other viral agents present in faecal extracts. Indirect immunofluorescence failed to detect rotavirus as often as the other three methods. In laboratories where routine examination of faecal specimens from patients with gastroenteritis is required, ELISA and RIA are useful alternatives to electron microscopy. (author)

  17. Application of indirect immunofluorescent test with an improved HEp-2 substrate tranfected with human Ro60/SSA autoantigens

    International Nuclear Information System (INIS)

    Lv Liangjing; Chen Shunle; Gu Yueying; Shen Nan; Bao Chunde; Wang Yuan; Xue Feng; Ye Peng; Yu Chongzhao

    2006-01-01

    To develop an improved substrate for indirect immunofluorescent test (IIF) to detect anti-Ro/SSA autoantibodies, the human 60-kDa Ro/SSA autoantigens (Ro60) cDNAs were obtained from placental cDNA library using PCR and were cloned into the mammalian expression vectorpEGFP-C1. Then the recombinant plasmids were transfected into HEp-2 cells. We con- firmed the overexpression, localization and antigenicity of fusion proteins in transfected cells by means of fluorescence microscopy, immunoblotting and IIF. HEp-2 and HEp-Ro60 was analyzed by IIF using a panel of 10 precipitinpositive anti-Ro human sera simultaneously. Stable expression of Ro60-GFP (green fluorescent protein) fusion proteins maintained ten more generations. And Ro60-GFP kept the antigenicity of Ro and had its own characteristic immunofluorescent pattern in HEp-Ro60 cells. The transfectants dramatically increased the sensitivity of IIF testing (a mean increase of 6.7-fold in endpoint titer, P<0.01). Eight (8/10) positive an- ti-Ro sera showed characteristic immunofluorescent pattern on HEp-Ro60, including two sera which were antinuclear antibodies (ANA) negative on untransfected HEp-2. IIF-ANA in all healthy sera were negative on HEp-Ro60. As a kind of new substrate of IIF, the Ro60 transfectants can be used to detect anti-Ro antibodies. In addition, transfected HEp-2 cells kept the immunofluorescent property of HEp-2 cells in IIF-ANA tests and could be employed as substrate for the routine IIF-ANA detection. The method improved the sensitivity of IIF-ANA. (authors)

  18. Giardia and Cryptosporidium spp. dissemination during wastewater treatment and comparative detection via immunofluorescence assay (IFA), nested polymerase chain reaction (nested PCR) and loop mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Gallas-Lindemann, Carmen; Sotiriadou, Isaia; Plutzer, Judit; Noack, Michael J; Mahmoudi, Mohammad Reza; Karanis, Panagiotis

    2016-06-01

    Environmental water samples from the Lower Rhine area in Germany were investigated via immunofluorescence assays (IFAs), nested polymerase chain reaction (nested PCR) and loop-mediated isothermal amplification (LAMP) to detect the presence of Giardia spp. (n=185) and Cryptosporidium spp. (n=227). The samples were concentrated through filtration or flocculation, and oocysts were purified via centrifugation through a sucrose density gradient. For all samples, IFA was performed first, followed by DNA extraction for the nested PCR and LAMP assays. Giardia cysts were detected in 105 samples (56.8%) by IFA, 62 samples (33.5%) by nested PCR and 79 samples (42.7%) by LAMP. Cryptosporidium spp. were detected in 69 samples (30.4%) by IFA, 95 samples (41.9%) by nested PCR and 99 samples (43.6%) by LAMP. According to these results, the three detection methods are complementary for monitoring Giardia and Cryptosporidium in environmental waters. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Diagnostic utility of a direct immunofluorescence test to detect feline coronavirus antigen in macrophages in effusive feline infectious peritonitis.

    Science.gov (United States)

    Litster, A L; Pogranichniy, R; Lin, T-L

    2013-11-01

    The antemortem diagnosis of feline infectious peritonitis (FIP) remains challenging in clinical practice, since current testing methods have suboptimal diagnostic accuracy. Immunohistochemical testing of biopsy specimens and postmortem examination are the standard diagnostic methods, although direct immunofluorescence (DIF) testing to detect feline coronavirus in macrophages in effusion specimens has been reported to have 100% specificity and has been recommended as an antemortem confirmatory test. The aim of this study was to compare the results of DIF testing in antemortem feline effusions with postmortem results using field samples. Effusion specimens were collected antemortem from 17 cats and tested by DIF, followed by postmortem examination. Histopathological examination of specimens collected at postmortem confirmed FIP in 10/17 cases and ruled out FIP out in 7/17 cases. Antemortem DIF testing was positive in all 10 cases confirmed as FIP at postmortem examination. In the seven cats where FIP was ruled out at postmortem examination, DIF was negative in five cases and positive in the remaining two cases. The calculated sensitivity of DIF testing was 100% and the specificity was 71.4%. Duplicate effusion specimens from eight cats that were initially DIF positive were stored refrigerated (4 °C) or at room temperature (22-25 °C) and subjected to serial DIF testing to determine the duration of positive results. DIF-positive specimens stored at both temperatures retained their positive status for at least 2 days. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Diagnostic value of islet autoantibody assays practised in Russia. 1. Classic immunofluorescence islet cell antibody assay, immunoradiometric glutamic acid decarboxylase antibody assay, and ELISA tyrosine phosphatase antibody and insulin antibody assays

    Directory of Open Access Journals (Sweden)

    Alexei V. Timofeev

    2016-09-01

    Conclusions. We recommend to use ICA, IA-2A and GADA tests surveyed in our study for diagnosis of DM type 1 and differential diagnosis of DM. We don’t recommend IA testing with an Orgentec Anti-Insulin ELISA kit for usage in clinical practice.

  1. A comparative study of an elisa test and an indirect immunofluorescence test for serological diagnosis of Babesia bovis infection

    International Nuclear Information System (INIS)

    Martins, J.R.; Cheong, F.H.; Correa, B.L.; Radley, D.E.; Cereser, V.H.

    1998-01-01

    Detection of antibodies to Babesia bovis in cattle is essential for the understanding of the epidemiology of babesiosis and this study was concerned with comparing the indirect fluorescent antibody with the ELISA. Both assays gave rise to 100% sensitivity whilst the ELISA was shown to be marginally more specific at 98%. The ease of use and low cost of the ELISA would make it the more obvious choice in conducting future serological surveys for this parasite. (author)

  2. Comparison of immunofluorescence and enzyme-linked immunosorbent assay and immunoglobulin G avidity techniques for screening of anti: Toxoplasma antibodies among single serum sample pregnant women in Tabriz, Iran

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    Mehrangiz Rajaii

    2015-01-01

    Full Text Available Background: Congenital toxoplasmosis is that pregnant women acquire the infection during gestation; diagnosis of the acute infection during pregnancy is a complex subject of maternal toxoplasmosis. Thus, the presence of immunoglobulin G (IgG and/or IgM Toxoplasma antibodies in a single serum sample drawn during gestation cannot be used to define whether the infection was recently acquired or chronic. Materials and Methods: At this cross-sectional descriptive study, sera of 391 pregnant women examined and compared. They were in an age range of 21-35 years, referred by gynecologists and infectious disease specialists, during March 2012-April 2013. They have referred, 215 (54.98%, 102 (26%, 74 (18.92% in the first, second and third trimesters of gestation, respectively. For each of them, a questionnaire was completed and serum samples were prepared in an equal condition, examined according to the procedures of indirect immunofluorescence (IIF, enzyme-linked immunosorbent assay (ELISA and IgG Avidity techniques. Results: We have found 111 (28.38% seronegative and 280 (71.61% seropositive cases by IIF and 124 (31.70% seronegative, 267 (68.28% seropositive cases by ELISA. The IgG avidity test confirmed 45 (69.23% and 7 (10.76% doubtful cases of IgM test in IIF and ELISA techniques. Conclusions: This study highlights how to manage pregnant women with toxoplasmosis, especially in a single serum sample condition.

  3. HEMORRHAGIC-FEVER VIRUS-INFECTIONS IN AN ISOLATED RAIN-FOREST AREA OF CENTRAL LIBERIA - LIMITATIONS OF THE INDIRECT IMMUNOFLUORESCENCE SLIDE TEST FOR ANTIBODY SCREENING IN AFRICA

    NARCIS (Netherlands)

    van der Waals, F. W.; Pomeroy, K. L.; Goudsmit, J.; Asher, D. M.; Gajdusek, D. C.

    1986-01-01

    Serum samples from 119 healthy individuals and 106 epilepsy patients inhabiting Grand Bassa County, Liberia, were tested for antibodies to hemorrhagic fever viruses (HFV) by indirect immunofluorescence. E6 Vero cells infected with Lassa fever virus (LAS), Rift Valley Fever virus (RVF), Congo

  4. Longitudinal study of the indirect immunofluorescence and complement fixation tests for diagnosis of chagas' disease in immunosuppressed patients submitted to renal transplantation

    Directory of Open Access Journals (Sweden)

    José Fernando de Castro Figueiredo

    1993-12-01

    Full Text Available Clinical and serological follow-up of 7 patients submitted to renal transplantation and presenting positive serological reactions to Chagas 'disease before immunossupression did not show significant changes in indirect immunofluorescence and complement fixation titres for Chagas ' disease, or signs and symptoms indicating exacerbation of the disease during follow- up. In addition, 18 of 66 recipients of renal transplants considered to be non-chagasic before immunosuppression showed at least one positive result to the indirect immunofluorescence test for Chagas ' disease during the study period. The results suggest that the immunosuppression State induced in chagasic patients submitted to renal transplant did notpromoted exacerbation of the chronic infection in these patients and not interfere with the serological response of chronic chagasics, thus permitting the use of these serologic reactions for diagnostic purposes in these cases. However, the positive results ofthe indirect immunofluorescence test in non- chagasic patients indicate the needforjudicious interpretation ofthe indirect immunofluorescence test for the diagnosis of Chagas' disease in renal transplanted patients.

  5. Enzyme-linked immunosorbent assay (ELISA for measles antibody: a comparison with haemagglutination inhibition, immunofluorescence and plaque neutralization tests Reação imunoenzimática (ELISA para detecção de anticorpos para o vírus do sarampo: comparação com reações de inibição da hema-glutinação, imunofluorescência indireta e neutralização de placas

    Directory of Open Access Journals (Sweden)

    Vanda Akico Ueda Fick de Souza

    1991-02-01

    Full Text Available An enzyme-linked immunosorbent assay (ELISA for measles antibodies was compared with Plaque Neutralization (PRN, Haemagglutination inhibition (HI and Fluorescent antibody (IFA tests in 181 sera from vaccinated children and umbilical cord. Of 179 positive samples by the sensitive PRN, only two, with titers of 8, were negative by ELISA (copositivity of 98.9%. IFA and HI presented, respectively, copo-sitivities of 93.3% and 82.7%. The ELISA presented a high sensitivity as well as a good reproducibility and represents an alternative for the time consuming PRN for detection of low measles antibodies.A reação imunoenzimática (ELISA para determinação de anticorpos para o vírus do sarampo foi comparada com a reação de neutralização de placas (RNP, inibição da hemaglutinação (RIH e imunofluorescência indireta (RIF. Das 179 amostras positivas pela RNP, somente 2, com títulos iguais a 8, se apresentaram negativas por ELISA (copositividade de 98.9%. A RIF e RIH apresentaram, respectivamente, copositividade de 93.3 e 82.7%. ELISA apresentou sensibilidade equivalente à complexa RNP, boa reprodutibilidade e representa uma alternativa para a detecção de baixos títulos de anticorpos contra o sarampo.

  6. Have you stress tested your assay?

    Directory of Open Access Journals (Sweden)

    Zheng Cao

    2016-08-01

    Full Text Available Objectives: When a clinical assay is stressed with extraordinarily high volume of specimens over a short period of time, extra caution may be needed to avoid systematic errors and biases. Here we report our experience with a HgbA1c assay used for high volume wellness screening purpose, to illustrate the importance of stress testing during assay validation. Design and Methods: Over 15,000 whole blood specimens were tested for HgbA1c in a period of 2 months. HgbA1c was tested by an immunoturbidimetric method on a high through-put automation line. The HgbA1c population distribution in our study was compared to that from the NHANES database. Daily distributions of HgbA1c values ≥6%, means and medians were plotted. Correlation studies were performed between the high through-put immunoturbidimetric assay and a medium through-put HPLC method. Results: We observed a shift of HgbA1c distribution to the higher values compared to the NHANES. A bias of 15–20% was noted from further stress testing where large number of samples were batched and tested using the immunoturbidimetric assay. A 5–7% higher bias remained after implementing a cuvette washing program after each HgbA1c sample. We hypothesized this bias was caused by build-up of blood cell fragments in the cuvettes when continuous whole blood samples are run through the system. Our experience suggests stress testing needs to be incorporated early in the test validation process for high volume batched screening applications. This seemingly extra validation step may save significant troubleshooting and retesting efforts down the road. Keywords: Hemoglobin A1c, Immunoturbidimetric assay, HPLC, Quality assurance, Systematic bias, High volume, Automation

  7. A direct view by immunofluorescent comet assay (IFCA) of DNA damage induced by nicking and cutting enzymes, ionizing (137)Cs radiation, UV-A laser microbeam irradiation and the radiomimetic drug bleomycin.

    Science.gov (United States)

    Grigaravicius, Paulius; Rapp, Alexander; Greulich, Karl Otto

    2009-03-01

    In DNA repair research, DNA damage is induced by different agents, depending on the technical facilities of the investigating researchers. A quantitative comparison of different investigations is therefore often difficult. By using a modified variant of the neutral comet assay, where the histone H1 is detected by immunofluorescence [immunofluorescent comet assay (IFCA)], we achieve previously unprecedented resolution in the detection of fragmented chromatin and show that trillions of ultraviolet A photons (of a few eV), billions of bleomycin (BLM) molecules and thousands of gamma quanta (of 662 keV) generate, in first order, similar damage in the chromatin of HeLa cells. A somewhat more detailed inspection shows that the damage caused by 20 Gy ionizing radiation and by a single laser pulse of 10 microJ are comparable, while the damage caused by 12 microg/ml BLM depends highly on the individual cell. Taken together, this work provides a detailed view of DNA fragmentation induced by different treatments and allows comparing them to some extent, especially with respect to the neutral comet assay.

  8. Test procedure for boxed waste assay system

    International Nuclear Information System (INIS)

    Wachter, J.

    1994-01-01

    This document, prepared by Los Alamos National Laboratory's NMT-4 group, details the test methodology and requirements for Acceptance/Qualification testing of a Boxed Waste Assay System (BWAS) designed and constructed by Pajarito Scientific Corporation. Testing of the BWAS at the Plutonium Facility (TA55) at Los Alamos National Laboratory will be performed to ascertain system adherence to procurement specification requirements. The test program shall include demonstration of conveyor handling capabilities, gamma ray energy analysis, and imaging passive/active neutron accuracy and sensitivity. Integral to these functions is the system's embedded operating and data reduction software

  9. Naturally acquired Lawsonia intracellularis infection in pigs studied from weaning to slaughter by indirect immunofluorescence antibody test and polymerase chain reaction on faeces

    DEFF Research Database (Denmark)

    Jensen, Tim Kåre; Vigre, Håkan; Sørensen, Vibeke

    2005-01-01

    by immunofluorescence antibody test compared to 24% by immunohistochemistry on ileal samples. Thus, applied at the time of slaughter the antibody test appeared to be a highly sensitive ante-mortem diagnostic tool for identifying L. intracelluaris exposed pigs with or without current proliferative enteropathy. (c) 2004......The course of naturally acquired Lawsonia intracellularis infection was studied in 41 pigs by testing blood and faeces samples collected four to seven times from before weaning to slaughter 5 months old. At slaughter, a sample of ileum was taken for histopathology. In the first sampling when...... later on shed and/or were seropositive for L. intracellularis. The lowest prevalence of L. intracellularis was observed in 6-13 weeks old pigs and it seemed as though L. intracellularis in early infected pigs only activates a minor antibody response. At slaughter 66% of the pigs were found positive...

  10. Reações de imunofluorescência indireta: algumas modificações de sua técnica Indirect immunofluorescence tests: some modifications of their technique

    Directory of Open Access Journals (Sweden)

    Cláudio Santos Ferreira

    1973-09-01

    Full Text Available Propõem-se modificações de procedimentos rotineiros de reações de imunofluorescência indireta. A eluição das amostras de sangue dessecado contidas em papel-filtro é feita em placas ("godets" com tampas. Alças de níquel-cromo substituem pipetas capilares para a transferência de amostras de eluatos para as lâminas de microscopia. Propõe-se uma técnica rápida e eficiente de lavagem das preparações.Some simplifications intended for routine operations included immunofluorescence tests are proposed. The elution of dried blood samples is performed on test plates (water-color mixing plates, with a lid instead of in test tubes. Nickel-chromium wire loops are substituted for capillary pipettes in transferring eluate samples to microscope slides. A rapid and efficient washing technique is proposed.

  11. Evaluation of a direct immunofluorescent antibody (difma test using Leishmania genus - specific monoclonal antibody in the routine diagnosis of cutaneous leishmaniasis

    Directory of Open Access Journals (Sweden)

    Martha E. Chico

    1995-06-01

    Full Text Available A direct immunofluorescent antibody (DIFMA test using a Leishmania genus- specific monoclonal antibody was evaluated in the routine diagnosis of cutaneous leishmaniasis (CL in Ecuador. This test was compared with the standard diagnostic techniques of scrapings, culture and histology. Diagnostic samples were taken from a total of 90 active dermal ulcers from patients from areas of Ecuador known to be endemic for cutaneous leishmaniasis. DIFMA was positive in all lesions. It was shown to be significantly superior to standard diagnostic methods either alone or in combination. The sensitivity of DIFMA did not diminish with chronicity of lesions. This test proved to be extremely useful in the routine diagnosis of CL because it is highly sensitive, is easy to use and produces rapid results.

  12. A Novel Tool for High-Throughput Screening of Granulocyte-Specific Antibodies Using the Automated Flow Cytometric Granulocyte Immunofluorescence Test (Flow-GIFT

    Directory of Open Access Journals (Sweden)

    Xuan Duc Nguyen

    2011-01-01

    Full Text Available Transfusion-related acute lung injury (TRALI is a severe complication related with blood transfusion. TRALI has usually been associated with antibodies against leukocytes. The flow cytometric granulocyte immunofluorescence test (Flow-GIFT has been introduced for routine use when investigating patients and healthy blood donors. Here we describe a novel tool in the automation of the Flow-GIFT that enables a rapid screening of blood donations. We analyzed 440 sera from healthy female blood donors for the presence of granulocyte antibodies. As positive controls, 12 sera with known antibodies against anti-HNA-1a, -b, -2a; and -3a were additionally investigated. Whole-blood samples from HNA-typed donors were collected and the test cells isolated using cell sedimentation in a Ficoll density gradient. Subsequently, leukocytes were incubated with the respective serum and binding of antibodies was detected using FITC-conjugated antihuman antibody. 7-AAD was used to exclude dead cells. Pipetting steps were automated using the Biomek NXp Multichannel Automation Workstation. All samples were prepared in the 96-deep well plates and analyzed by flow cytometry. The standard granulocyte immunofluorescence test (GIFT and granulocyte agglutination test (GAT were also performed as reference methods. Sixteen sera were positive in the automated Flow-GIFT, while five of these sera were negative in the standard GIFT (anti—HNA 3a, n = 3; anti—HNA-1b, n = 1 and GAT (anti—HNA-2a, n = 1. The automated Flow-GIFT was able to detect all granulocyte antibodies, which could be only detected in GIFT in combination with GAT. In serial dilution tests, the automated Flow-GIFT detected the antibodies at higher dilutions than the reference methods GIFT and GAT. The Flow-GIFT proved to be feasible for automation. This novel high-throughput system allows an effective antigranulocyte antibody detection in a large donor population in order to prevent TRALI due to transfusion of

  13. A novel tool for high-throughput screening of granulocyte-specific antibodies using the automated flow cytometric granulocyte immunofluorescence test (Flow-GIFT).

    Science.gov (United States)

    Nguyen, Xuan Duc; Dengler, Thomas; Schulz-Linkholt, Monika; Klüter, Harald

    2011-02-03

    Transfusion-related acute lung injury (TRALI) is a severe complication related with blood transfusion. TRALI has usually been associated with antibodies against leukocytes. The flow cytometric granulocyte immunofluorescence test (Flow-GIFT) has been introduced for routine use when investigating patients and healthy blood donors. Here we describe a novel tool in the automation of the Flow-GIFT that enables a rapid screening of blood donations. We analyzed 440 sera from healthy female blood donors for the presence of granulocyte antibodies. As positive controls, 12 sera with known antibodies against anti-HNA-1a, -b, -2a; and -3a were additionally investigated. Whole-blood samples from HNA-typed donors were collected and the test cells isolated using cell sedimentation in a Ficoll density gradient. Subsequently, leukocytes were incubated with the respective serum and binding of antibodies was detected using FITC-conjugated antihuman antibody. 7-AAD was used to exclude dead cells. Pipetting steps were automated using the Biomek NXp Multichannel Automation Workstation. All samples were prepared in the 96-deep well plates and analyzed by flow cytometry. The standard granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT) were also performed as reference methods. Sixteen sera were positive in the automated Flow-GIFT, while five of these sera were negative in the standard GIFT (anti-HNA 3a, n = 3; anti-HNA-1b, n = 1) and GAT (anti-HNA-2a, n = 1). The automated Flow-GIFT was able to detect all granulocyte antibodies, which could be only detected in GIFT in combination with GAT. In serial dilution tests, the automated Flow-GIFT detected the antibodies at higher dilutions than the reference methods GIFT and GAT. The Flow-GIFT proved to be feasible for automation. This novel high-throughput system allows an effective antigranulocyte antibody detection in a large donor population in order to prevent TRALI due to transfusion of blood products.

  14. Evaluation of Immunofluorescence Antibody Test Used for the Diagnosis of Canine Leishmaniasis in the Mediterranean Basin: A Systematic Review and Meta-Analysis.

    Directory of Open Access Journals (Sweden)

    Amel Adel

    Full Text Available With an expected sensitivity (Se of 96% and specificity (Sp of 98%, the immunofluorescence antibody test (IFAT is frequently used as a reference test to validate new diagnostic methods and estimate the canine leihmaniasis (CanL true prevalence in the Mediterranean basin. To review the diagnostic accuracy of IFAT to diagnose CanL in this area with reference to its Se and Sp and elucidate the potential causes of their variations, a systematic review was conducted (31 studies for the 26-year period. Three IFAT validation methods stood out: the classical contingency table method, methods based on statistical models and those based on experimental studies. A variation in the IFAT Se and Sp values and cut-off values was observed. For the classical validation method based on a meta-analysis, the Se of IFAT was estimated in this area as 89.86% and 31.25% in symptomatic and asymptomatic dogs, respectively. The Sp of IFAT was estimated in non-endemic and endemic areas as 98.12% and 96.57%, respectively. IFAT can be considered as a good standard test in non-endemic areas for CanL, but its accuracy declines in endemic areas due to the complexity of the disease. Indeed, the accuracy of IFAT is due to the negative results obtained in non-infected dogs from non-endemic areas and to the positive results obtained in sera of symptomatic dogs living in endemic areas. But IFAT results are not unequivocal when it comes to determining CanL infection on asymptomatic dogs living in endemic areas. Statistical methods might be a solution to overcome the lack of gold standard, to better categorize groups of animals investigated, to assess optimal cut-off values and to allow a better estimate of the true prevalence aiming information on preventive/control measures for CanL.

  15. Biological assays for aquatic toxicity testing

    CSIR Research Space (South Africa)

    Slabbert, JL

    1999-10-01

    Full Text Available and management purposes of effluents. If receiving water is used for drinking water purposes, the Ames Salmonella mutagenicity and toad embryo teratogenicity tests should be included in the battery of tests. Some of the rapid microbiotests, the petrozoan oxygen...

  16. Testing fruit quality by photoacoustic spectroscopy assay

    International Nuclear Information System (INIS)

    Popa, C; Dumitras, D C; Patachia, M; Banita, S

    2014-01-01

    This study was conducted with the aim of testing the hypothesis that raspberry and strawberry fruits from nonorganic farming release more ethylene gas compounds compared to organic ones. At the same time, the experiments focused on evaluation of the potential and capabilities of the laser photoacoustic spectroscopy (LPAS) method in the assessment of fruit quality related to the effects of nitrogen. Ethylene gas can be harmful and carcinogenic, because it can accelerate the natural ripening process of physiologically mature fruits and makes the fruits more consistent in size. With the advantages of LPAS, we demonstrate that the concentration of ethylene from nonorganic raspberry and strawberry fruits is greater than from organic ones. (paper)

  17. Discordant results obtained on testing sera from immunocompromised patients for cytomegalovirus IgG by enzyme-linked immunosorbent assay and radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Booth, J C; El Mohandes, L; Tryhorn, Y S [London University (United Kingdom). St. George' s Hospital Medical School, Department of Medical Microbiology; Kangro, H O; Liu, K M [Saint Bartholomew' s Hospital, London (UK)

    1989-10-01

    The investigation of sera from immunocompromised patients for antibody to CMV by ELISA, RIA, immunofluorescence (IF) and complement-fixation (CF) revealed discrepancies that reflected differences in test specificity rather than sensitivity and suggested that for the long-term serological follow-up of such patients it would be advisable not to rely on only a single assay procedure. (author). 12 refs.; 8 figs.; 1 tab.

  18. Recommendations for safety testing with the in vivo comet assay.

    Science.gov (United States)

    Vasquez, Marie Z

    2012-08-30

    While the in vivo comet assay increases its role in regulatory safety testing, deliberations about the interpretation of comet data continue. Concerns can arise regarding comet assay publications with limited data from non-blind testing of positive control compounds and using protocols (e.g. dose concentrations, sample times, and tissues) known to give an expected effect. There may be a tendency towards bias when the validation or interpretation of comet assay data is based on results generated by widely accepted but non-validated assays. The greatest advantages of the comet assay are its sensitivity and its ability to detect genotoxicity in tissues and at sample times that could not previously be evaluated. Guidelines for its use and interpretation in safety testing should take these factors into account. Guidelines should be derived from objective review of data generated by blind testing of unknown compounds dosed at non-toxic concentrations and evaluated in a true safety-testing environment, where the experimental design and conclusions must be defensible. However, positive in vivo comet findings with such compounds are rarely submitted to regulatory agencies and this data is typically unavailable for publication due to its proprietary nature. To enhance the development of guidelines for safety testing with the comet assay, and with the permission of several sponsors, this paper presents and discusses relevant data from multiple GLP comet studies conducted blind, with unknown pharmaceuticals and consumer products. Based on these data and the lessons we have learned through the course of conducting these studies, I suggest significant adjustments to the current conventions, and I provide recommendations for interpreting in vivo comet assay results in situations where risk must be evaluated in the absence of carcinogenicity or clinical data. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Biostatistical analysis of quantitative immunofluorescence microscopy images.

    Science.gov (United States)

    Giles, C; Albrecht, M A; Lam, V; Takechi, R; Mamo, J C

    2016-12-01

    Semiquantitative immunofluorescence microscopy has become a key methodology in biomedical research. Typical statistical workflows are considered in the context of avoiding pseudo-replication and marginalising experimental error. However, immunofluorescence microscopy naturally generates hierarchically structured data that can be leveraged to improve statistical power and enrich biological interpretation. Herein, we describe a robust distribution fitting procedure and compare several statistical tests, outlining their potential advantages/disadvantages in the context of biological interpretation. Further, we describe tractable procedures for power analysis that incorporates the underlying distribution, sample size and number of images captured per sample. The procedures outlined have significant potential for increasing understanding of biological processes and decreasing both ethical and financial burden through experimental optimization. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

  20. Widespread nanoparticle-assay interference: implications for nanotoxicity testing.

    Science.gov (United States)

    Ong, Kimberly J; MacCormack, Tyson J; Clark, Rhett J; Ede, James D; Ortega, Van A; Felix, Lindsey C; Dang, Michael K M; Ma, Guibin; Fenniri, Hicham; Veinot, Jonathan G C; Goss, Greg G

    2014-01-01

    The evaluation of engineered nanomaterial safety has been hindered by conflicting reports demonstrating differential degrees of toxicity with the same nanoparticles. The unique properties of these materials increase the likelihood that they will interfere with analytical techniques, which may contribute to this phenomenon. We tested the potential for: 1) nanoparticle intrinsic fluorescence/absorbance, 2) interactions between nanoparticles and assay components, and 3) the effects of adding both nanoparticles and analytes to an assay, to interfere with the accurate assessment of toxicity. Silicon, cadmium selenide, titanium dioxide, and helical rosette nanotubes each affected at least one of the six assays tested, resulting in either substantial over- or under-estimations of toxicity. Simulation of realistic assay conditions revealed that interference could not be predicted solely by interactions between nanoparticles and assay components. Moreover, the nature and degree of interference cannot be predicted solely based on our current understanding of nanomaterial behaviour. A literature survey indicated that ca. 95% of papers from 2010 using biochemical techniques to assess nanotoxicity did not account for potential interference of nanoparticles, and this number had not substantially improved in 2012. We provide guidance on avoiding and/or controlling for such interference to improve the accuracy of nanotoxicity assessments.

  1. Widespread nanoparticle-assay interference: implications for nanotoxicity testing.

    Directory of Open Access Journals (Sweden)

    Kimberly J Ong

    Full Text Available The evaluation of engineered nanomaterial safety has been hindered by conflicting reports demonstrating differential degrees of toxicity with the same nanoparticles. The unique properties of these materials increase the likelihood that they will interfere with analytical techniques, which may contribute to this phenomenon. We tested the potential for: 1 nanoparticle intrinsic fluorescence/absorbance, 2 interactions between nanoparticles and assay components, and 3 the effects of adding both nanoparticles and analytes to an assay, to interfere with the accurate assessment of toxicity. Silicon, cadmium selenide, titanium dioxide, and helical rosette nanotubes each affected at least one of the six assays tested, resulting in either substantial over- or under-estimations of toxicity. Simulation of realistic assay conditions revealed that interference could not be predicted solely by interactions between nanoparticles and assay components. Moreover, the nature and degree of interference cannot be predicted solely based on our current understanding of nanomaterial behaviour. A literature survey indicated that ca. 95% of papers from 2010 using biochemical techniques to assess nanotoxicity did not account for potential interference of nanoparticles, and this number had not substantially improved in 2012. We provide guidance on avoiding and/or controlling for such interference to improve the accuracy of nanotoxicity assessments.

  2. The comet assay in testing the potential genotoxicity of nanomaterials

    Directory of Open Access Journals (Sweden)

    Amaya Azqueta

    2015-06-01

    Full Text Available In the last two decades the production and use of nanomaterials (NMs has impressively increased. Their small size, given a mass equal to that of the corresponding bulk material, implies an increase in the surface area and consequently in the number of atoms that can be reactive. They possess different physical, chemical and biological properties compared to bulk materials of the same composition, which makes them very interesting and valuable for many different applications in technology, energy, construction, electronics, agriculture, optics, paints, textiles, food, cosmetics, medicine... Toxicological assessment of NMs is crucial; the same properties that make them interesting also make them potentially harmful for health and the environment. However, the term NM covers many different kinds of particle , and so there is no simple, standard approach to assessing their toxicity. NMs can enter the cell, interact with cell components and even penetrate the nucleus and interfere with the genetic material. Among the different branches of toxicology, genotoxicity is a main area of concern since it is closely related with the carcinogenic potential of compounds. The Organisation for Economic Co-operation and Development (OECD has published internationally agreed in vitro and in vivo validated test methods to evaluate different genotoxic endpoints of chemicals, including chromosome and gene mutations, and DNA breaks. However not all the assays are suitable to study the genotoxic potential of NMs as has been shown by the OECD Working Party on Manufactured Nanomaterials (WPMN. Moreover, alterations to DNA bases, which are precursors to mutations and of great importance in elucidating the mechanism of action of NMs, are not covered by the OECD guidelines. The in vivo standard comet assay (which measures DNA breaks and alkali-labile sites was included in the OECD assays battery in September 2014 while the in vitro standard comet assay is currently under

  3. Specific binding-adsorbent assay method and test means

    International Nuclear Information System (INIS)

    1981-01-01

    A description is given of an improved specific binding assay method and test means employing a nonspecific adsorbent for the substance to be determined, particularly hepatitis B surface (HBsub(s)) antigen, in its free state or additionally in the form of its immune complex. The invention is illustrated by 1) the radioimmunoadsorbent assay for HBsub(s) antigen, 2) the radioimmunoadsorbent assay for HBsub(s) antigen in the form of immune complex with antibody, 3) a study of adsorption characteristics of various anion exchange materials for HBsub(s) antigen, 4) the use of hydrophobic adsorbents in a radioimmunoadsorbent assay for HBsub(s) antigen and 5) the radioimmunoadsorbent assay for antibody to HBsub(s) antigen. The advantages of the present method for detecting HBsub(s) antigen compared to previous methods include the manufacturing advantages of eliminating the need for insolubilised anti-HBsub(s) and the advantages of a single incubation step, fewer manipulations, storability of adsorbent materials, increased sensitivity and versatility of detecting HBsub(s) antigen in the form of its immune complex if desired. (U.K.)

  4. Operability test procedure for TRUSAF assayer software upgrade

    International Nuclear Information System (INIS)

    Cejka, C.C.

    1995-01-01

    This OTP is to be used to ensure the operability of the Transuranic Waste Assay System (TRUWAS). The system was upgraded and requires a retest to assure satisfactory operation. The upgrade consists of an AST 486 computer to replace the IBM-PC/XT, and a software upgrade (CNEUT). The software calculations are performed in the same manner as in the previous system (NEUT), however, the new software is written in C Assembly Language. CNEUT is easier to use and far more powerful than the previous program. The TRUWAS is used to verify the TRU content of waste packages sent for storage in the Transuranic Storage and Assay Facility (TRUSAF). The TRUSAF is part of Westinghouse Hanford's certification program for waste to be shipped to the Waste Isolation Pilot Plant (WIPP) in New Mexico. The Transuranic Waste Assayer uses a combination passive-active neutron interrogation system to determine the TRU content of 55-gallon waste drums. The system consists of a shielded assay chamber; Deuterium-Tritium neutron generator; Helium-3 proportional counters; drum handling system; electronics including preamplifier, amplifier, and discriminator for each of the counter packages; and an AST 486 computer/printer system for data acquisition and analysis. The system can detect down to TRU levels of 10 nCi/g in the waste matrix. The equipment to be tested is: Assay Chamber Door Drum Turntable and Automatic Loading Platform Interlocks Assayer Software; and IBM computer/printer software. The objective of the test is to verify that the system is operational with the AST 486 computer, the software used in the new computer system correctly calculates TRU levels, and the new computer system is capable of storing and retrieving data

  5. Estimation of antibodies to human cytomegalovirus by immunofluorescence and radioimmunoassay

    International Nuclear Information System (INIS)

    Jankowski, M.; Gut, W.; Nawrocka, E.

    1980-01-01

    The 125 I labelled IgG fraction against rabbit IgG of goat origin was employed for the detection of CMV IgG and IgM antibodies in the double indirect radioimmunoassay. The results were compared with those obtained in complement fixation, indirect immunofluorescence and anti-complement immunofluorescence tests. The application of labelled anti-fc antisera, instead of antisera against whole IgG in the tests for detection of specific CMV IgG antibody resulted in increased sensitivity of radioimmunoassay and reduction of non-specific cytoplasmatic reactions in preparations stained by indirect immunofluorescence. The absorption of sera with protein A rich staphylococci and aggregates to immunoglobulin eliminated unwanted secondary IgM staining caused by rheumatoid factors both in indirect immunofluorescence and radioimmunoassay tests for CMV antibodies. (author)

  6. The local lymph node assay and skin sensitization testing.

    Science.gov (United States)

    Kimber, Ian; Dearman, Rebecca J

    2010-01-01

    The mouse local lymph node assay (LLNA) is a method for the identification and characterization of skin sensitization hazards. In this context the method can be used both to identify contact allergens, and also determine the relative skin sensitizing potency as a basis for derivation of effective risk assessments.The assay is based on measurement of proliferative responses by draining lymph node cells induced following topical exposure of mice to test chemicals. Such responses are known to be causally and quantitatively associated with the acquisition of skin sensitization and therefore provide a relevant marker for characterization of contact allergic potential.The LLNA has been the subject of exhaustive evaluation and validation exercises and has been assigned Organization for Economic Cooperation and Development (OECD) test guideline 429. Herein we describe the conduct and interpretation of the LLNA.

  7. In Vivo Nanotoxicity Testing using the Zebrafish Embryo Assay.

    Science.gov (United States)

    Rizzo, Larissa Y; Golombek, Susanne K; Mertens, Marianne E; Pan, Yu; Laaf, Dominic; Broda, Janine; Jayapaul, Jabadurai; Möckel, Diana; Subr, Vladimir; Hennink, Wim E; Storm, Gert; Simon, Ulrich; Jahnen-Dechent, Willi; Kiessling, Fabian; Lammers, Twan

    2013-06-10

    Nanoparticles are increasingly used for biomedical purposes. Many different diagnostic and therapeutic applications are envisioned for nanoparticles, but there are often also serious concerns regarding their safety. Given the fact that numerous new nanomaterials are being developed every day, and that not much is known about the long-term toxicological impact of exposure to nanoparticles, there is an urgent need to establish efficient methods for nanotoxicity testing. The zebrafish (Danio rerio) embryo assay has recently emerged as an interesting 'intermediate' method for in vivo nanotoxicity screening, enabling (semi-) high-throughput analyses in a system significantly more complex than cultured cells, but at the same time also less 'invasive' and less expensive than large-scale biocompatibility studies in mice or rats. The zebrafish embryo assay is relatively well-established in the environmental sciences, but it has not yet gained wide notice in the nanomedicine field. Using prototypic polymeric drug carriers, gold-based nanodiagnostics and nanotherapeutics, and iron oxide-based nanodiagnostics, we here show that toxicity testing using zebrafish embryos is easy, efficient and informative, and faithfully reflects, yet significantly extends, cell-based toxicity testing. We therefore expect that the zebrafish embryo assay will become a popular future tool for in vivo nanotoxicity screening.

  8. Comet Assay on Daphnia magna in eco-genotoxicity testing.

    Science.gov (United States)

    Pellegri, Valerio; Gorbi, Gessica; Buschini, Annamaria

    2014-10-01

    Detection of potentially hazardous compounds in water bodies is a priority in environmental risk assessment. For the evaluation and monitoring of water quality, a series of methodologies may be applied. Among them, the worldwide used toxicity tests with organisms of the genus Daphnia is one of the most powerful. In recent years, some attempts were made to utilize Daphnia magna in genotoxicity testing as many of the new environmental contaminants are described as DNA-damaging agents in aquatic organisms. The aim of this research was to develop a highly standardized protocol of the Comet Assay adapted for D. magna, especially regarding the isolation of cells derived from the same tissue (haemolymph) from newborn organisms exposed in vivo. Several methods for haemolymph extraction and different Comet Assay parameters were compared. Electrophoretic conditions were adapted in order to obtain minimum DNA migration in cells derived from untreated organisms and, at the same time, maximum sensitivity in specimens treated with known genotoxicants (CdCl2 and H2O2). Additional tests were performed to investigate if life-history traits of the cladoceran (such as the age of adult organisms that provide newborns, the clutch size of origin, the number of generations reared in standard conditions) and the water composition as well, might influence the response of the assay. This study confirms the potential application of the Comet Assay in D. magna for assessing genotoxic loads in aqueous solution. The newly developed protocol could integrate the acute toxicity bioassay, thus expanding the possibility of using this model species in freshwater monitoring (waters, sediment and soil elutriates) and is in line with the spirit of the EU Water Framework Directive in reducing the number of bioassays that involve medium-sized species. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Comparison of new immunofluorescence method for detection of soy protein in meat products with immunohistochemical, histochemical, and ELISA methods

    Directory of Open Access Journals (Sweden)

    Michaela Petrášová

    2014-01-01

    Full Text Available Soy proteins are commonly used in the food industry thanks to their technological properties. However, soy is, along with cow’s milk, eggs, wheat, peanuts, tree nuts, fish, crustaceans, and molluscs, responsible for around 90% of food allergies, and is also one of the foodstuffs that can cause anaphylaxis. The aim of this work was to compare the immunofluorescence method for the detection of soy protein in meat products purchased from the retail market with other microscopic methods (immunohistochemical and histochemical, with the ELISA reference method and with the confirmatory results. Within the research, 127 meat products purchased in the retail network were examined using the immunofluorescence method used for the detection of soy protein. The method was compared to Enzyme-Linked ImmunoSorbent Assay (ELISA, immunohistochemical, and histochemical methods. According to McNemar’s test, non-compliance between the immunofluorescence method and immunohistochemical method was low. In addition, a significant difference between the fluorescence method and ELISA (P P < 0.01 was found. The immunofluorescence method was also compared with confirmatory results. According to McNemar’s test, non-compliance between the immunofluorescence method and confirmatory results was low. The results showed the possibilities of this new method to detect the content of soy protein in meat products.

  10. Tests for oil/dispersant toxicity: In situ laboratory assays

    International Nuclear Information System (INIS)

    Wright, D.A.; Coelho, G.M.; Aurand, D.V.

    1995-01-01

    As part of its readiness program in oil spill response, the Marine Pollution Control Unit (MPCU), Department of Transport, U.K. conducts annual field trials in the North Sea, approximately 30 nautical miles from the southeast coast of England. The trials take the form of controlled releases of crude oil or Medium Fuel/Gas Oil mix (MFO), with and without the application of Corexit 9527 dispersant. In 1994 and 1995 the authors conducted a series of in situ toxicity bioassays in association with these spills with included 48h LC50 tests for turbot (Scophthalmus maximus) and oyster (Crassostrea gigas) larvae, a 48 h oyster (C. gigas) embryonic development test and two full life-cycle assays using the copepods Acartia tonsa and Tisbe battagliai. Tests were also conducted in the Chesapeake Bay laboratory using estuarine species including the copepod Eurytemora affinis and the inland silverside Menidia beryllina. Here, the authors report on the results of these assays, together with 1996 in situ toxicity data resulting from Norwegian field trials in the northern North Sea

  11. Direct Assay of Filter Media following DEOX Testing

    International Nuclear Information System (INIS)

    R.P. Lind; J.J. Giglio; D.G. Cummings; M.W. Huntley; C.D. Morgan; K.J. Bateman; D.L. Wahlquist; D.A. Sell

    2007-01-01

    The direct assay of filter media by gamma spectrometry following DEOX testing has distinct advantages over analytical chemistry. Prior to using gamma spectrometry for the quantification of cesium (Cs-137), a calibration must be established with known sources since gamma spectrometry yields relative results. Quantitative analytical chemistry, in particular ICP-MS, has been performed on the filter media for comparison to the gamma spectrometry data. The correlation of gamma spectrometry to ICP-MS data is presented to justify the continued use of gamma spectrometry for filter media

  12. Direct assay of filter media following DEOX testing

    International Nuclear Information System (INIS)

    Westphal, B.R.; Lind, R.P.; Giglio, J.J.; Cummings, D.G.; Huntley, M.W.; Morgan, C.D.; Bateman, K.J.; Wahlquist, D.L.; Sell, D.A.

    2007-01-01

    The direct assay of filter media by gamma spectrometry following DEOX testing has distinct advantages over analytical chemistry. Prior to using gamma spectrometry for the quantification of cesium (Cs- 137), a calibration must be established with known sources since gamma spectrometry yields relative results. Quantitative analytical chemistry, in particular ICP-MS, has been performed on the filter media for comparison to the gamma spectrometry data. The correlation of gamma spectrometry to ICP-MS data is presented to justify the continued use of gamma spectrometry for filter media. (authors)

  13. Humoral Immune Response Kinetics in Philander opossum and Didelphis marsupialis Infected and Immunized by Trypanosoma cruzi Employing an Immunofluorescence Antibody Test

    Directory of Open Access Journals (Sweden)

    Ana Paula Legey

    1999-05-01

    Full Text Available Philander opossum and Didelphis marsupialis considered the most ancient mammals and an evolutionary success, maintain parasitism by Trypanosoma cruzi without developing any apparent disease or important tissue lesion. In order to elucidate this well-balanced interaction, we decided to compare the humoral immune response kinetics of the two didelphids naturally and experimentally infected with T. cruzi and immunized by different schedules of parasite antigens, employing an indirect fluorescence antibody test (IFAT. Both didelphids responded with high serological titers to different immunization routes, while the earliest response occurred with the intradermic route. Serological titers of naturally infected P. opossum showed a significant individual variation, while those of D. marsupialis remained stable during the entire follow-up period. The serological titers of the experimentally infected animals varied according to the inoculated strain. Our data suggest that (1 IFAT was sensitive for follow-up of P. opossum in natural and experimental T. cruzi infections; (2 both P. opossum and D. marsupialis are able to mount an efficient humoral immune response as compared to placental mammals; (3 experimentally infected P. opossum and D. marsupialis present distinct patterns of infection, depending on the subpopulation of T. cruzi, (4 the differences observed in the humoral immune responses between P. opossum and D. marsupialis, probably, reflect distinct strategies selected by these animals during their coevolution with T. cruzi.

  14. Highly specific and rapid immuno-fluorescent visualization and detection of E. coli O104:H4 with protein-A coated magnetic beads based LST-MUG assay.

    Science.gov (United States)

    Barizuddin, Syed; Balakrishnan, Baskar; Stringer, R Cody; Dweik, Majed

    2015-08-01

    A method combining immunomagnetic separation and fluorescent sensing was developed to detect Escherichia coli (E. coli) O104:H4. The antibody specific to E. coli O104:H4 was immobilized on protein A-coated magnetic beads. This protein-A-anti E. coli O104:H4 complex was used to bind Fluorescein IsoThioCyanate (FITC) labeled E. coli O104:H4 antigen (whole cell) on it. The goal was to achieve a fluorescently detectable protein-A-anti E. coli O104:H4-E. coli O104:H4 complex on the magnetic beads. Fluorescent microscopy was used to image the magnetic beads. The resulting fluorescence on the beads was due to the FITC labeled antigen binding on the protein-A-anti E. coli O104:H4 immobilized magnetic beads. This visually proves the antigen-antibody binding. The fluorescent imaging results were obtained in 2 h if the minimum available bacteria in the sample were at least 10(5) CFU/ml. If no fluorescence was observed on the magnetic beads during fluorescent imaging, it indicates the bacterial concentration in the sample to be too low for it to have bound to the magnetic beads and hence no detection was possible. To detect bacterial concentration less than 10(5) CFU/ml in the sample, an additional step was required for detection. The magnetic bead complex was added to the LST-MUG (lauryl sulfate tryptose-4-methylumbelliferyl-β-D-glucuronide), a signaling reporter. The E. coli O104:H4 grows in LST-MUG and releases β-glucuronidase enzyme. This enzyme cleaves the MUG substrate that produces 4-methylumbelliferone, a highly fluorescent species. This fluorescence was detected using a spectrofluorometer. The emission peak in the fluorescent spectrum was found to be at 450 nm. The lower and upper detection range for this LST-MUG assay was found to be 2.05×10(5)-4.09×10(8) CFU/ml. The results for the LST-MUG assay for concentrations below 10(5) CFU/ml were ascertained in 8h. The advantages of this technique include the specific detection of bacteria without an enrichment step and

  15. Mutagenicity of Flavonoids Assayed by Bacterial Reverse Mutation (Ames Test

    Directory of Open Access Journals (Sweden)

    Eliana Aparecida Varanda

    2012-05-01

    Full Text Available The mutagenicity of ten flavonoids was assayed by the Ames test, in Salmonella typhimurium strains TA98, TA100 and TA102, with the aim of establishing hydroxylation pattern-mutagenicity relationship profiles. The compounds assessed were: quercetin, kaempferol, luteolin, fisetin, chrysin, galangin, flavone, 3-hydroxyflavone, 5-hydroxyflavone and 7-hydroxyflavone. In the Ames assay, quercetin acted directly and its mutagenicity increased with metabolic activation. In the presence of S9 mix, kaempferol and galangin were mutagenic in the TA98 strain and kaempferol showed signs of mutagenicity in the other strains. The absence of hydroxyl groups, as in flavone, only signs of mutagenicity were shown in strain TA102, after metabolization and, among monohydroxylated flavones (3-hydroxyflavone, 5-hydroxyflavone and 7-hydroxyflavone, the presence of hydroxyl groups only resulted in minor changes. Luteolin and fisetin also showed signs of mutagenicity in strain TA102. Finally, chrysin, which has only two hydroxy groups, at the 5-OH and 7-OH positions, also did not induce mutagenic activity in any of the bacterial strains used, under either activation condition. All the flavonoids were tested at concentrations varying from 2.6 to 30.7 nmol/plate for galangin and 12.1 to 225.0 nmol/plate for other flavonoids. In light of the above, it is necessary to clarify the conditions and the mechanisms that mediate the biological effects of flavonoids before treating them as therapeutical agents, since some compounds can be biotransformed into more genotoxic products; as is the case for galangin, kaempferol and quercetin.

  16. Genotoxicity testing: Comparison of the γH2AX focus assay with the alkaline and neutral comet assays.

    Science.gov (United States)

    Nikolova, Teodora; Marini, Federico; Kaina, Bernd

    2017-10-01

    Genotoxicity testing relies on the quantitative measurement of adverse effects, such as chromosome aberrations, micronuclei, and mutations, resulting from primary DNA damage. Ideally, assays will detect DNA damage and cellular responses with high sensitivity, reliability, and throughput. Several novel genotoxicity assays may fulfill these requirements, including the comet assay and the more recently developed γH2AX assay. Although they are thought to be specific for genotoxicants, a systematic comparison of the assays has not yet been undertaken. In the present study, we compare the γH2AX focus assay with the alkaline and neutral versions of the comet assay, as to their sensitivities and limitations for detection of genetic damage. We investigated the dose-response relationships of γH2AX foci and comet tail intensities at various times following treatment with four prototypical genotoxicants, methyl methanesulfonate (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), mitomycin C, and hydrogen peroxide (H 2 O 2 ) and we tested whether there is a correlation between the endpoints, i.e., alkali-labile sites and DNA strand breaks on the one hand and the cell's response to DNA double-strand breaks and blocked replication forks on the other. Induction of γH2AX foci gave a linear dose response and all agents tested were positive in the assay. The increase in comet tail intensity was also a function of dose; however, mitomycin C was almost completely ineffective in the comet assay, and the doses needed to achieve a significant effect were somewhat higher for some treatments in the comet assay than in the γH2AX foci assay, which was confirmed by threshold analysis. There was high correlation between tail intensity and γH2AX foci for MMS and H 2 O 2 , less for MNNG, and none for mitomycin C. From this we infer that the γH2AX foci assay is more reliable, sensitive, and robust than the comet assay for detecting genotoxicant-induced DNA damage. Copyright © 2017 Elsevier

  17. Immunofluorescence detection of pea protein in meat products.

    Science.gov (United States)

    Petrášová, Michaela; Pospiech, Matej; Tremlová, Bohuslava; Javůrková, Zdeňka

    2016-08-01

    In this study we developed an immunofluorescence method to detect pea protein in meat products. Pea protein has a high nutritional value but in sensitive individuals it may be responsible for causing allergic reactions. We produced model meat products with various additions of pea protein and flour; the detection limit (LOD) of the method for pea flour was 0.5% addition, and for pea protein it was 0.001% addition. The repeatabilities and reproducibilities for samples both positive and negative for pea protein were all 100%. In a blind test with model products and commercial samples, there was no statistically significant difference (p > 0.05) between the declared concentrations of pea protein and flour and the immunofluorescence method results. Sensitivity was 1.06 and specificity was 1.00. These results show that the immunofluorescence method is suitable for the detection of pea protein in meat products.

  18. Comprehensive analysis of sperm DNA fragmentation by five different assays: TUNEL assay, SCSA, SCD test and alkaline and neutral Comet assay.

    Science.gov (United States)

    Ribas-Maynou, J; García-Peiró, A; Fernández-Encinas, A; Abad, C; Amengual, M J; Prada, E; Navarro, J; Benet, J

    2013-09-01

    Sperm DNA fragmentation (SDF) is becoming an important test to assess male infertility. Several different tests are available, but no consensus has yet been reached as to which tests are most predictive of infertility. Few publications have reported a comprehensive analysis comparing these methods within the same population. The objective of this study was to analyze the differences between the five most common methodologies, to study their correlations and to establish their cut-off values, sensitivity and specificity in predicting male infertility. We found differences in SDF between fertile donors and infertile patients in TUNEL, SCSA, SCD and alkaline Comet assays, but none with the neutral Comet assay. The alkaline COMET assay was the best in predicting male infertility followed by TUNEL, SCD and SCSA, whereas the neutral COMET assay had no predictive power. For our patient population, threshold values for infertility were 20.05% for TUNEL assay, 18.90% for SCSA, 22.75% for the SCD test, 45.37% for alkaline Comet and 34.37% for neutral Comet. This work establishes in a comprehensive study that the all techniques except neutral Comet are useful to distinguish fertile and infertile men. © 2013 American Society of Andrology and European Academy of Andrology.

  19. Monkey Feeding Assay for Testing Emetic Activity of Staphylococcal Enterotoxin.

    Science.gov (United States)

    Seo, Keun Seok

    2016-01-01

    Staphylococcal enterotoxins (SEs) are unique bacterial toxins that cause gastrointestinal toxicity as well as superantigenic activity. Since systemic administration of SEs induces superantigenic activity leading to toxic shock syndrome that may mimic enterotoxic activity of SEs such as vomiting and diarrhea, oral administration of SEs in the monkey feeding assay is considered as a standard method to evaluate emetic activity of SEs. This chapter summarizes and discusses practical considerations of the monkey feeding assay used in studies characterizing classical and newly identified SEs.

  20. An Automatic Indirect Immunofluorescence Cell Segmentation System

    Directory of Open Access Journals (Sweden)

    Yung-Kuan Chan

    2014-01-01

    Full Text Available Indirect immunofluorescence (IIF with HEp-2 cells has been used for the detection of antinuclear autoantibodies (ANA in systemic autoimmune diseases. The ANA testing allows us to scan a broad range of autoantibody entities and to describe them by distinct fluorescence patterns. Automatic inspection for fluorescence patterns in an IIF image can assist physicians, without relevant experience, in making correct diagnosis. How to segment the cells from an IIF image is essential in developing an automatic inspection system for ANA testing. This paper focuses on the cell detection and segmentation; an efficient method is proposed for automatically detecting the cells with fluorescence pattern in an IIF image. Cell culture is a process in which cells grow under control. Cell counting technology plays an important role in measuring the cell density in a culture tank. Moreover, assessing medium suitability, determining population doubling times, and monitoring cell growth in cultures all require a means of quantifying cell population. The proposed method also can be used to count the cells from an image taken under a fluorescence microscope.

  1. Avaliação do teste de imunofluorescência indireta para diagnóstico da filariose bancroftiana usando a microfilária de W. bancrofti como antígeno, em Recife-PE, Brasil Evaluation of indirect immunofluorescence test for bancroftian filariasis using Wuchereria bancroft microfilariae as the antigen in Recife, Brazil

    Directory of Open Access Journals (Sweden)

    Gerusa Dreyer

    1991-10-01

    Full Text Available Analisou-se o teste de imunofluorescência indireta com microfilárias de W. bancrofti tratadas pela papaína, como antígeno, amplamente utilizado em Recife para o imunodiagnóstico da filariose linfática. Foram testados soros de 50 pacientes portadores das diversas formas clínicas da doença, incluindo microfilaremia assintomática, eosinofilia pulmonar tropical, elefantíase de membros inferiores, linfagite aguda e quilúria. Para o grupo controle, foram selecionados 50 indivíduos vivendo pelo menos há 5 anos em área endêmica, sem nenhuma evidencia clínica e/ou laboratorial da doença, constituindo os chamados endêmicos normais. A sensibilidade e especificidade do teste, segundo diferentes pontos de corte, mostraram a impossibilidade de diferenciação entre o grupo controle e o grupo sabidamente infectado. Também não foi possível estabelecer correlação entre os títulos encontrados e as diferentes formas clínicas. Foi considerada a existência de reações cruzadas relacionadas a helmintíases intestinais, porém nenhuma relação direta foi encontrada.The authors analysed the indirect immunofluorescence assay, for the diagnosis of bancroftian filariasis using papain treated W. bancrofti microfilariae as antigen, widely used in Recife - Brazil. Sera from 50 patients with several clinical forms of the disease including asymptomatic carriers, tropical pulmonary eosinophilia, elephantiasis, filarial fever and chyluria were analysed. For the control group, 50 individuals were selected, living at least 5 years in endemic area, with neither previous DEC treatment nor clinical-laboratory evidences of the disease, called normals endemic. The sensitivity and specificity were analised taking into account different cut off values. It was not possible to differentiate infected individuals from the control group. It was not even possible to establish any correlation with IMF titers among different clinical presentation of the disease

  2. Test plan for Digface Chemical and Radiation Assay System

    International Nuclear Information System (INIS)

    Akers, D.W.

    1993-07-01

    The Digface Chemical and Radiation Assay System (CRAS) Project will develop a sensor using Prompt Gamma Neutron Activation Analysis (PGNAA) that can detect the present of hazardous chemicals and radioactive materials. The CRAS is being designed for in situ assay of closed drums and contaminated soils for gamma-ray emitting radionuclides and hazardous elements. The CRAS is based upon the use of 252 Cf PGNAA with a germanium gamma-ray spectrometer as the analyzer. Tasks being performed include determining detection limits for a number of hazardous chemicals and assessing matrix and transmission effects through soil. Initial analyses suggest that the technique is applicable to a number of hazardous materials such as trichloroethane and carbon tetrachloride

  3. Ecogenotoxicity testing of aquatic environment by comet assay in plants

    Directory of Open Access Journals (Sweden)

    Anita Mukherjee

    2015-05-01

    Full Text Available One of the goals of environmental monitoring is the detection of potentially hazardous compounds in water. We have set up a standard method to apply the Comet assay in aquatic plants that could be of great interest to evaluate cytotoxicity, genotoxicity and oxidative stress on the same species regarded as most sensitive to environmental pollutants. The aim of the present study was to set up of standardized procedure to evaluate genotoxicity in aquatic plants- Ceratophyllum demersum one that is submerged free floating and the other is Lemna minor - a fresh water floating plant by Comet assay. Electrophoresis and unwinding times were adapted to obtain minimum DNA migration evaluated as tail intensity % or tail moment in the control group and, at the same time maximum sensitivity for DNA damage with known genotoxicants. We further investigated the cytotoxicity and oxidative stress induced in the same species. Based on the repeatability of results obtained we suggest that Ceratophyllum, Lemna can serve as model species and Comet assay could be adopted to monitor the eco-genotoxicity of water pollutants.

  4. Inmunofluorescencia con Crithidia luciliae para la detección de anticuerpos anti-ADN: Imágenes atípicas y su relación con enfermedad de Chagas y leishmaniasis Immunofluorescence assay with Crithidia luciliae for the detection of anti-DNA antibodies: Atypical images and their relationship with Chagas’ disease and leishmaniasis

    Directory of Open Access Journals (Sweden)

    Gloria Griemberg

    2006-02-01

    Full Text Available Los anticuerpos anti-ADN nativo pueden detectarse por inmunofluorescencia indirecta con Crithidia luciliae, observándose tinción fluorescente anular del kinetoplasto que contiene ADN de doble cadena. En algunos casos pueden observarse imágenes fluorescentes en flagelo, membrana y corpúsculo basal, consideradas atípicas. Como C. luciliae pertenece a la familia Trypanosomatidae, que incluye patógenos para el hombre como Trypanosoma cruzi y Leishmaniaspp., se consideró que las imágenes atípicas pudieran deberse a reacciones cruzadas. Se realizaron estudios serológicos para Chagas a 105 muestras provenientes de zona endémica (Corrientes y no endémica (Buenos Aires para T. cruzi que presentaban imágenes atípicas con C. luciliae. La serología para Chagas resultó positiva en el 64.7% de las muestras de Buenos Aires y en el 78.3% de las de Corrientes que presentaban frente a C. luciliae imagen conjunta de membrana y flagelo. No presentaron la imagen conjunta ninguna de las muestras de dadores de sangre normales, ni de pacientes con enfermedades del tejido conectivo, excepto dos con lupus que también eran chagásicos. Todas las muestras de pacientes chagásicos analizadas frente a C. luciliae presentaron la imagen conjunta. Se estudiaron también 46 muestras de pacientes con leishmaniasis, 28 de ellos coinfectados con T. cruzi. La imagen conjunta se observó en el 88.0% de las muestras de leishmaniásicos y en el 78.5% de las de coinfectados. Los resultados sugieren que C. luciliae podría ser un sustrato alternativo, económico y de bajo riesgo para el diagnóstico serológico de enfermedad de Chagas, aunque no discrimina la infección por Leishmania. El hallazgo de la imagen conjunta en la detección de anti-ADN nativo señala la conveniencia de realizar en esos pacientes, estudios clínicos y de laboratorio para enfermedad de Chagas y leishmaniasis.Anti-native DNA antibodies can be detected by indirect immunofluorescence assay with

  5. Thyroglobulin (Tg) Testing Revisited: Tg Assays, TgAb Assays, and Correlation of Results With Clinical Outcomes.

    Science.gov (United States)

    Netzel, Brian C; Grebe, Stefan K G; Carranza Leon, B Gisella; Castro, M Regina; Clark, Penelope M; Hoofnagle, Andrew N; Spencer, Carole A; Turcu, Adina F; Algeciras-Schimnich, Alicia

    2015-08-01

    Measurement of thyroglobulin (Tg) by mass spectrometry (Tg-MS) is emerging as a tool for accurate Tg quantification in patients with anti-Tg autoantibodies (TgAbs). The objective of the study was to perform analytical and clinical evaluations of two Tg-MS assays in comparison with immunometric Tg assays (Tg-IAs) and Tg RIAs (Tg-RIAs) in a cohort of thyroid cancer patients. A total of 589 samples from 495 patients, 243 TgAb-/252 TgAb+, were tested by Beckman, Roche, Siemens-Immulite, and Thermo-Brahms Tg and TgAb assays, two Tg-RIAs, and two Tg-MS assays. The frequency of TgAb+ was 58%, 41%, 27%, and 39% for Roche, Beckman, Siemens-Immulite, and Thermo-Brahms, respectively. In TgAb- samples, clinical sensitivities and specificities of 100% and 74%-100%, respectively, were observed across all assays. In TgAb+ samples, all Tg-IAs demonstrated assay-dependent Tg underestimation, ranging from 41% to 86%. In TgAb+ samples, the use of a common cutoff (0.5 ng/mL) for the Tg-MS, three Tg-IAs, and the USC-RIA improved the sensitivity for the Tg-MSs and Tg-RIAs when compared with the Tg-IAs. In up to 20% of TgAb+ cases, Tg-IAs failed to detect Tg that was detectable by Tg-MS. In Tg-RIAs false-high biases were observed in TgAb+ samples containing low Tg concentrations. Tg-IAs remain the method of choice for Tg quantitation in TgAb- patients. In TgAb+ patients with undetectable Tg by immunometric assay, the Tg-MS will detect Tg in up to 20% additional cases. The Tg-RIA will detect Tg in approximately 35% cases, but a significant proportion of these will be clinical false-positive results. The undetectable Tg-MS seen in approximately 40% of TgAb+ cases in patients with disease need further evaluation.

  6. INEL test plan for evaluating waste assay systems

    International Nuclear Information System (INIS)

    Mandler, J.W.; Becker, G.K.; Harker, Y.D.; Menkhaus, D.E.; Clements, T.L. Jr.

    1996-09-01

    A test bed is being established at the Idaho National Engineering Laboratory (INEL) Radioactive Waste Management Complex (RWMC). These tests are currently focused on mobile or portable radioassay systems. Prior to disposal of TRU waste at the Waste Isolation Pilot Plant (WIPP), radioassay measurements must meet the quality assurance objectives of the TRU Waste Characterization Quality Assurance Program Plan. This test plan provides technology holders with the opportunity to assess radioassay system performance through a three-tiered test program that consists of: (a) evaluations using non-interfering matrices, (b) surrogate drums with contents that resemble the attributes of INEL-specific waste forms, and (c) real waste tests. Qualified sources containing a known mixture and range of radionuclides will be used for the non-interfering and surrogate waste tests. The results of these tests will provide technology holders with information concerning radioassay system performance and provide the INEL with data useful for making decisions concerning alternative or improved radioassay systems that could support disposal of waste at WIPP

  7. INEL test plan for evaluating waste assay systems

    Energy Technology Data Exchange (ETDEWEB)

    Mandler, J.W.; Becker, G.K.; Harker, Y.D.; Menkhaus, D.E.; Clements, T.L. Jr.

    1996-09-01

    A test bed is being established at the Idaho National Engineering Laboratory (INEL) Radioactive Waste Management Complex (RWMC). These tests are currently focused on mobile or portable radioassay systems. Prior to disposal of TRU waste at the Waste Isolation Pilot Plant (WIPP), radioassay measurements must meet the quality assurance objectives of the TRU Waste Characterization Quality Assurance Program Plan. This test plan provides technology holders with the opportunity to assess radioassay system performance through a three-tiered test program that consists of: (a) evaluations using non-interfering matrices, (b) surrogate drums with contents that resemble the attributes of INEL-specific waste forms, and (c) real waste tests. Qualified sources containing a known mixture and range of radionuclides will be used for the non-interfering and surrogate waste tests. The results of these tests will provide technology holders with information concerning radioassay system performance and provide the INEL with data useful for making decisions concerning alternative or improved radioassay systems that could support disposal of waste at WIPP.

  8. (SNP) assay for population stratification test between eastern Asians

    African Journals Online (AJOL)

    Yomi

    2012-01-03

    Jan 3, 2012 ... program STRUCTURE 2.0, which uses a Markov chain Monte. Carlo (MCMC) algorithm to cluster individuals into different cryptic ... HapMap project. .... Evaluation of the 124-plex SNP typing microarray for forensic testing.

  9. A European multicientre study on the comparison of HIV-1 viral loads between VERIS HIV-1 Assay and Roche COBAS® TAQMAN® HIV-1 test, Abbott RealTime HIV-1 Assay, and Siemens VERSANT HIV-1 Assay.

    Science.gov (United States)

    Braun, Patrick; Delgado, Rafael; Drago, Monica; Fanti, Diana; Fleury, Hervé; Hofmann, Jörg; Izopet, Jacques; Kühn, Sebastian; Lombardi, Alessandra; Mancon, Alessandro; Marcos, Mª Angeles; Mileto, Davide; Sauné, Karine; O'Shea, Siobhan; Pérez-Rivilla, Alfredo; Ramble, John; Trimoulet, Pascale; Vila, Jordi; Whittaker, Duncan; Artus, Alain; Rhodes, Daniel

    2017-07-01

    Viral load monitoring is essential for patients under treatment for HIV. Beckman Coulter has developed the VERIS HIV-1 Assay for use on the novel, automated DxN VERIS Molecular Diagnostics System. ¥ OBJECTIVES: Evaluation of the clinical performance of the new quantitative VERIS HIV-1 Assay at multiple EU laboratories. Method comparison with the VERIS HIV-1 Assay was performed with 415 specimens at 5 sites tested with COBAS ® AmpliPrep/COBAS ® TaqMan ® HIV-1 Test, v2.0, 169 specimens at 3 sites tested with RealTime HIV-1 Assay, and 202 specimens from 2 sites tested with VERSANT HIV-1 Assay. Patient monitoring sample results from 4 sites were also compared. Bland-Altman analysis showed the average bias between VERIS HIV-1 Assay and COBAS HIV-1 Test, RealTime HIV-1 Assay, and VERSANT HIV-1 Assay to be 0.28, 0.39, and 0.61 log 10 cp/mL, respectively. Bias at low end levels below 1000cp/mL showed predicted bias to be <0.3 log 10 cp/mL for VERIS HIV-1 Assay versus COBAS HIV-1 Test and RealTime HIV-1 Assay, and <0.5 log 10 cp/mL versus VERSANT HIV-1 Assay. Analysis on 174 specimens tested with the 0.175mL volume VERIS HIV-1 Assay and COBAS HIV-1 Test showed average bias of 0.39 log 10 cp/mL. Patient monitoring results using VERIS HIV-1 Assay demonstrated similar viral load trends over time to all comparators. The VERIS HIV-1 Assay for use on the DxN VERIS System demonstrated comparable clinical performance to COBAS ® HIV-1 Test, RealTime HIV-1 Assay, and VERSANT HIV-1 Assay. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Immunoradiometric assay of lipid A: a test for detecting and quantitating endotoxins of various origins

    Energy Technology Data Exchange (ETDEWEB)

    Nolan, J P; Vladutiu, A O; Moreno, D M; Cohen, S A; Camara, D S [State University of New York, Buffalo (USA). School of Medicine

    1982-11-26

    The ability to measure circulating endotoxin in various disease states has been hampered by the lack of a specific and quantitative assay. The test most commonly used has been the Limulus gelation assay, which measures an enzymatic effect of endotoxin rather than the substance itself. Based on a solid-phase immunoradiometric assay previously developed to detect the specific lipopolysaccharide from Escherichia coli 026, a similar assay has been developed for the lipid A moiety of endotoxins. The assay uses rabbit antibodies to lipid A which do not react with ketodeoxyoctonate, myristic or beta-hydroxymyristic acids, and detects lipid A obtained from endotoxins of various origins after acid hydrolysis of lipopolysaccharide. Experiments in rats given exogenous endotoxin suggest that this assay can be useful for quantitation of bacterial endotoxins in serum and for studying the pathophysiology of experimental endotoxemia.

  11. Desempeño de la prueba de inmunofluorescencia directa en el diagnóstico del virus Influenza A(H1N1 Direct immunofluorescence assay performance in diagnosis of the Influenza A(H1N1 virus

    Directory of Open Access Journals (Sweden)

    Luis Pianciola

    2010-06-01

    Full Text Available El 25 de abril de 2009, a menos de un mes de la detección en México del primer humano con virus Influenza A(H1N1, la enfermedad ya se había propagado a más de 40 países superando los 10 000 casos notificados. Dada su naturaleza impredecible, este tipo de virus requiere métodos diagnósticos apropiados, confiables y seguros, pero que también estén al alcance de los laboratorios clínicos. Mediante el estudio de 291 muestras de pacientes con sospecha de infección por virus Influenza A(H1N1 en Neuquén, Argentina, el presente trabajo compara los dos métodos de diagnóstico utilizados simultáneamente: la prueba de inmunofluorescencia directa (DFA y la de reacción en cadena de la polimerasa en tiempo real (RT-PCR. La DFA dio una sensibilidad de 44,4%, especificidad de 99,6%, valor predictivo positivo de 95,2% y valor predictivo negativo de 90,7%. Los resultados positivos de la metodología pueden considerarse verdaderos positivos. Un resultado negativo no excluye la presencia del virus y la muestra debe examinarse mediante RT-PCR. Del total de 291 muestras, 45 resultaron positivas por RT-PCR y 21 por DFA.By 25 April 2009, less than one month after the first human with Influenza A(H1N1 virus was detected in Mexico, the disease had already spread to more than 40 countries, with over 10 000 cases reported. Due to its unpredictability, this type of virus requires appropriate, reliable, and safe diagnostic methods that are also accessible to clinical laboratories. Through the analysis of 291 samples taken from patients with suspected Influenza A(H1N1 virus infection in Neuquén, Argentina, this study compares the two diagnostic methods used simultaneously: direct immunofluorescence assay (DFA and real-time polymerase chain reaction (RT-PCR. DFA had a sensitivity of 44.4%, a specificity of 99.6%, a positive predictive value of 95.2%, and a negative predictive value of 90.7%. Positive results obtained with this method can be considered true

  12. [Pilot tests using molecular diagnostic assay cervicovaginal infection during pregnancy].

    Science.gov (United States)

    Beltrán-Montoya, J; Escudero-Gontes, S; Martínez-Huerta, N E; Ávila-Vergara, M A; Morales-Hernández, V; Canchola-Sotelo, C; Palacios-González, B; Vadillo-Ortega, F

    2016-08-01

    The prevalence of cervicovaginal infections during pregnancy has been associated with adverse perinatal outcomes however, the actual approach used for diagnosis is not effective. The aim of this study was to compare the diagnosis of vaginal infections in pregnant women using clinical, molecular diagnostic and traditional microbiological culture in a pilot study, to determine the prevalence and association with the development of preterm labor. We performed a nested cross-sectional study composed by 54 women in a cohort of pregnant women in Mexico City. Cervicovaginal infections were evaluated by clinical methods, microbiology culture and a commercially available molecular biology test. Prevalence of cervicovaginal infections during pregnancy was estimated between 28% and 50% according to methodologies. Considering the clinical diagnosis of preterm labor as the gold standard, all diagnostic tests were poor as predictors of preterm labor. Traditional approaches to establish the significance of cervicovaginal infection in pregnancy are exhausted, so be sought new ways to understand this complex relationship. Meanwhile it is recommended to continue to use traditional methods to identify infections during pregnancy in both knowledge of new methods aimed at understanding these relationships are sophisticated.

  13. Evaluation of the HISCL Anti-Treponema pallidum Assay as a Screening Test for Syphilis

    OpenAIRE

    An, Jingna; Chen, Qixia; Liu, Qianqian; Rao, Chenli; Li, Dongdong; Wang, Tingting; Tao, Chuanmin; Wang, Lanlan

    2015-01-01

    The resurgence of syphilis in recent years has become a serious threat to public health worldwide, and the serological detection of specific antibodies against Treponema pallidum remains the most reliable method for laboratory diagnosis of syphilis. This study examined the performance of the recently launched HISCL anti-Treponema pallidum (anti-TP) assay as a screening test for syphilis in a high-volume laboratory. The HISCL anti-TP assay was tested in 300 preselected syphilis-positive sample...

  14. Evaluating the genotoxic effects of workers exposed to lead using micronucleus assay, comet assay and TCR gene mutation test

    International Nuclear Information System (INIS)

    Chen Zhijian; Lou Jianlin; Chen Shijie; Zheng Wei; Wu Wei; Jin Lifen; Deng Hongping; He Jiliang

    2006-01-01

    To evaluate the genotoxic effects of lead (Pb) exposure, 25 workers in a workplace producing storage battery were monitored for three genetic end-points using micronucleus (MN) assay, comet assay and TCR gene mutation test. Twenty-five controls were matched with workers according to age, gender and smoking. The air Pb concentration in the workplace was 1.26 mg/m 3 . All subjects were measured for Pb concentration of blood by atom absorption spectrophotometry. The mean Pb concentration of blood in workers (0.32 mg/l) was significantly higher than that in controls (0.02 mg/l). The results of MN test showed that the mean micronuclei rate (MNR) and mean micronucleated cells rate (MCR) in workers were 9.04 ± 1.51 per mille and 7.76 ± 1.23 per mille , respectively, which were significantly higher than those (2.36 ± 0.42 per mille and 1.92 ± 0.31 per mille ) in controls (P -4 and 1.74 ± 0.17 x 10 -4 , respectively, there was no significant difference between workers and controls (P > 0.05). The results of our study indicated that the genetic damage was detectable in 25 workers occupationally exposed to lead

  15. Evaluation of the Thermo Scientific SureTect Listeria species assay. AOAC Performance Tested Method 071304.

    Science.gov (United States)

    Cloke, Jonathan; Evans, Katharine; Crabtree, David; Hughes, Annette; Simpson, Helen; Holopainen, Jani; Wickstrand, Nina; Kauppinen, Mikko; Leon-Velarde, Carlos; Larson, Nathan; Dave, Keron

    2014-01-01

    The Thermo Scientific SureTect Listeria species Assay is a new real-time PCR assay for the detection of all species of Listeria in food and environmental samples. This validation study was conducted using the AOAC Research Institute (RI) Performance Tested Methods program to validate the SureTect Listeria species Assay in comparison to the reference method detailed in International Organization for Standardization 11290-1:1996 including amendment 1:2004 in a variety of foods plus plastic and stainless steel. The food matrixes validated were smoked salmon, processed cheese, fresh bagged spinach, cantaloupe, cooked prawns, cooked sliced turkey meat, cooked sliced ham, salami, pork frankfurters, and raw ground beef. All matrixes were tested by Thermo Fisher Scientific, Microbiology Division, Basingstoke, UK. In addition, three matrixes (pork frankfurters, fresh bagged spinach, and stainless steel surface samples) were analyzed independently as part of the AOAC-RI-controlled independent laboratory study by the University ofGuelph, Canada. Using probability of detection statistical analysis, a significant difference in favour of the SureTect assay was demonstrated between the SureTect and reference method for high level spiked samples of pork frankfurters, smoked salmon, cooked prawns, stainless steel, and low-spiked samples of salami. For all other matrixes, no significant difference was seen between the two methods during the study. Inclusivity testing was conducted with 68 different isolates of Listeria species, all of which were detected by the SureTect Listeria species Assay. None of the 33 exclusivity isolates were detected by the SureTect Listeria species Assay. Ruggedness testing was conducted to evaluate the performance of the assay with specific method deviations outside of the recommended parameters open to variation, which demonstrated that the assay gave reliable performance. Accelerated stability testing was additionally conducted, validating the assay

  16. A molecular method for typing Herpes simplex virus isolates as an alternative to immunofluorescence methods

    Directory of Open Access Journals (Sweden)

    Abraham A

    2009-01-01

    Full Text Available Background: Typing of Herpes simplex virus (HSV isolates is required to identify the virus isolated in culture. The methods available for this include antigen detection by immunofluorescence (IF assays and polymerase chain reaction (PCR. This study was undertaken to standardize a molecular method for typing of HSV and compare it with a commercial IF reagent for typing. Objectives: To compare a molecular method for typing HSV isolates with a monoclonal antibody (MAb based IF test. Study design : This cross-sectional study utilized four reference strains and 42 HSV isolates obtained from patients between September 1998 and September 2004. These were subjected to testing using an MAb-based IF test and a PCR that detects the polymerase ( pol gene of HSV isolates. Results: The observed agreement of the MAb IF assay with the pol PCR was 95.7%. Fifty four point eight percent (23/42 of isolates tested by IF typing were found to be HSV-1, 40.5% (17/42 were HSV-2, and two (4.8% were untypable using the MAb IF assay. The two untypable isolates were found to be HSV-2 using the pol PCR. In addition, the cost per PCR test for typing is estimated to be around Rs 1,300 (USD 30, whereas the cost per MAb IF test is about Rs 1,500 (USD 35 including all overheads (reagents, instruments, personnel time, and consumables. Conclusion: The pol PCR is a cheaper and more easily reproducible method for typing HSV isolates as compared to the IF test. It could replace the IF-based method for routine typing of HSV isolates as availability of PCR machines (thermal cyclers is now more widespread than fluorescence microscopes in a country like India.

  17. Validity of urinary monoamine assay sales under the "spot baseline urinary neurotransmitter testing marketing model".

    Science.gov (United States)

    Hinz, Marty; Stein, Alvin; Uncini, Thomas

    2011-01-01

    Spot baseline urinary monoamine assays have been used in medicine for over 50 years as a screening test for monoamine-secreting tumors, such as pheochromocytoma and carcinoid syndrome. In these disease states, when the result of a spot baseline monoamine assay is above the specific value set by the laboratory, it is an indication to obtain a 24-hour urine sample to make a definitive diagnosis. There are no defined applications where spot baseline urinary monoamine assays can be used to diagnose disease or other states directly. No peer-reviewed published original research exists which demonstrates that these assays are valid in the treatment of individual patients in the clinical setting. Since 2001, urinary monoamine assay sales have been promoted for numerous applications under the "spot baseline urinary neurotransmitter testing marketing model". There is no published peer-reviewed original research that defines the scientific foundation upon which the claims for these assays are made. On the contrary, several articles have been published that discredit various aspects of the model. To fill the void, this manuscript is a comprehensive review of the scientific foundation and claims put forth by laboratories selling urinary monoamine assays under the spot baseline urinary neurotransmitter testing marketing model.

  18. [Laboratory diagnosis of genital herpes--direct immunofluorescence method].

    Science.gov (United States)

    Majewska, Anna; Romejko-Wolniewicz, Ewa; Zareba-Szczudlik, Julia; Kilijańczyk, Marek; Gajewska, Małgorzata; Młynarczyk, Grazyna

    2013-07-01

    Aim of the study was to determine clinical usefulness of direct immunofluorescence method in the laboratory diagnosis of genital herpes in women. Overall 187 anogenital swabs were collected from 120 women. Using a dacron-tipped applicator 83 swabs were collected from women suspected of genital herpes and 104 from patients with no signs of genital infection. All samples were tested using cell culture (Vero cell line) and then direct immunofluorescence method (DIF) for the identification of antigens of herpes simplex viruses: HSV-1 and HSV-2. Characteristic cytopathic effect (CPE), indicative of alphaherpesvirus infection, was observed in 43.4% of cultures with clinical specimens collected from women with suspected genital herpes and in 29.8% of cultures of clinical specimens taken from patients with no clinical symptoms of genital herpes. Herpes simplex viruses were determined in 73 samples by direct immunofluorescence method after amplification of the virus in cell culture. The DIF test confirmed the diagnosis based on the microscopic CPE observation in 85%. In 15% of samples (taken from pregnant women without clinical signs of infection) we reported positive immunofluorescence in the absence of CPE. The frequency of antigen detection was statistically significantly higher in samples that were positive by culture study (chi-square test with Yates's correction, p genital herpes in swabs taken from the vestibule of the vagina and the vulva. However, there was no statistically significant difference in the frequency of detection of Herpes Simplex Virus antigens in specimens from different parts of the genital tract in both groups of women (chi-square test, p > 0.05). In our study HHV-1 was the main causative agent of genital herpes. The growing worldwide prevalence of genital herpes, challenges with the clinical diagnosis, and availability of effective antiviral therapy are the main reasons for a growing interest in rapid, proper laboratory diagnosis of infected

  19. Epidermis area detection for immunofluorescence microscopy

    Science.gov (United States)

    Dovganich, Andrey; Krylov, Andrey; Nasonov, Andrey; Makhneva, Natalia

    2018-04-01

    We propose a novel image segmentation method for immunofluorescence microscopy images of skin tissue for the diagnosis of various skin diseases. The segmentation is based on machine learning algorithms. The feature vector is filled by three groups of features: statistical features, Laws' texture energy measures and local binary patterns. The images are preprocessed for better learning. Different machine learning algorithms have been used and the best results have been obtained with random forest algorithm. We use the proposed method to detect the epidermis region as a part of pemphigus diagnosis system.

  20. Fluorescence in situ hybridization in combination with the comet assay and micronucleus test in genetic toxicology

    Directory of Open Access Journals (Sweden)

    Hovhannisyan Galina G

    2010-09-01

    Full Text Available Abstract Comet assay and micronucleus (MN test are widely applied in genotoxicity testing and biomonitoring. While comet assay permits to measure direct DNA-strand breaking capacity of a tested agent MN test allows estimating the induced amount of chromosome and/or genome mutations. The potential of these two methods can be enhanced by the combination with fluorescence in situ hybridization (FISH techniques. FISH plus comet assay allows the recognition of targets of DNA damage and repairing directly. FISH combined with MN test is able to characterize the occurrence of different chromosomes in MN and to identify potential chromosomal targets of mutagenic substances. Thus, combination of FISH with the comet assay or MN test proved to be promising techniques for evaluation of the distribution of DNA and chromosome damage in the entire genome of individual cells. FISH technique also permits to study comet and MN formation, necessary for correct application of these methods. This paper reviews the relevant literature on advantages and limitations of Comet-FISH and MN-FISH assays application in genetic toxicology.

  1. Anatomopatologia e imunofluorescência direta e indireta das lesões de pênfigo foliáceo endêmico resistentes à corticoterapia Pathology, direct and indirect immunofluorescent test of lesions of pemphigus foliaceous resistent to corticotherapy

    Directory of Open Access Journals (Sweden)

    Vitor Manoel Silva dos Reis

    1991-04-01

    Full Text Available São estudados 16 doentes de pênfigo foliáceo endêmico (PFE, sob tratamento com corticosteróides, que ainda apresentavam lesões eritemato-pápulo-verrucosas, geralmente hiperpigmentadas, que foram caracterizadas como lesões resistentes à corticoterapia (LRC. O estudo destas lesões foi feito através de anatomopatologia e de imunofluorescência direta (IFD. Anatomopatologicamente essas lesões mostraram tendência à hiperplasia epitelial e clivagem em níveis variáveis na epiderme o que difere dos achados nas lesões recentes do PFE e coincide com os achados nas lesões crônicas do PFE da era pré-corticóide. A IFD da pele lesada foi positiva para IgG em 93,75% dos casos, como ocorre nas fases iniciais do PFE, tendo sido negativa no único caso em que não houve clivagem. Adicionalmente, em oito desses doentes, foram estudados a IFD da pele sã e a imunofluorescência indireta (IFI. A IFD foi positiva em três destes casos e a IFI foi negativa nos oito.A group of 16 patients with endemic pemphigus foliaceus under corticotherapy and still showing erythematous, papulous, verrucous, in general hyperpigmented lesions, which were characterized as cortico therapy resistant lesions, were studied. Such study was made through anatomopathology and direct immunofluorescence (DIF. Anatomopathologically, such lesions showed tendencies to epitelial hyperplasia and cleavage in variable levels at the epidermis what differs from the early lesions of EPF and coincides with the chronic injuries of the EPF of the pre-corticoid era. The DIF of the injured skin was positive for IgG in 93.75% of cases, as it happens in the early stages of EPF, being negative in a simple case in which there was not cleavage. In addition, in eight of those patients, the DIF of the healthy skin and the indirect immunofluorescence (IIF were studied. The DIF was positive in three of these cases and in all eight the IIF was negative.

  2. Endotoxin testing of proteins for parenteral administration using the Mono Mac 6 assay

    DEFF Research Database (Denmark)

    Moesby, Lise; Hansen, E W; Christensen, J D

    2000-01-01

    Pharmaceutical products containing proteins cause problems in testing for endotoxin and pyrogens. Many proteins interfere with the LAL test and the proteins are immunogenic in rabbits. The monocytic cell line Mono Mac 6 is an alternative assay for detection of endotoxin and other pyrogens....

  3. Direct immunofluorescence for the diagnosis of legionellosis

    Directory of Open Access Journals (Sweden)

    David JM Haldane

    1993-01-01

    Full Text Available Culture and direct immunofluorescent microscopy (DFA results for Legionella pneumophila were reviewed over a two-year period. In the first year, a positive result was defined as having at least one morphologically typical fluorescing organism. In the second year, a positive was defined as at least five typical fluorescing organisms. Despite these stricter criteria and other measures to reduce the possibility of reagent contamination, there was no statistically significant difference in the sensitivity or specificity of the DFA in the two years for sputa, deep specimens or overall. Of 37 sputum specimens from infected patients, 16 were positive on DFA. Thirty-two of 38 positive patients were detected by sputum culture. DFA can provide rapid diagnostic information but cannot be used to rule out the diagnosis. Sputum is a useful specimen for the initial laboratory investigation of patients with legionellosis.

  4. Evaluation of the HISCL Anti-Treponema pallidum Assay as a Screening Test for Syphilis.

    Science.gov (United States)

    An, Jingna; Chen, Qixia; Liu, Qianqian; Rao, Chenli; Li, Dongdong; Wang, Tingting; Tao, Chuanmin; Wang, Lanlan

    2015-07-01

    The resurgence of syphilis in recent years has become a serious threat to public health worldwide, and the serological detection of specific antibodies against Treponema pallidum remains the most reliable method for laboratory diagnosis of syphilis. This study examined the performance of the recently launched HISCL anti-Treponema pallidum (anti-TP) assay as a screening test for syphilis in a high-volume laboratory. The HISCL anti-TP assay was tested in 300 preselected syphilis-positive samples, 704 fresh syphilis-negative samples, 48 preselected potentially interfering samples, and 30 "borderline" samples and was compared head to head with the commercially available Lumipulse G TP-N. In this study, the HISCL anti-TP assay was in perfect agreement with the applied testing algorithms with an overall agreement of 100%, comparable to that of Lumipulse G TP-N (99.63%). The sensitivity and specificity of the HISCL anti-TP assay were 100% (95% confidence interval [CI], 98.42% to 100%) and 100% (95% CI, 99.37% to 100%), respectively. Considering the excellent ease of use and automation, high throughput, and its favorable sensitivity and specificity, the HISCL anti-TP assay may represent a new choice for syphilis screening in high-volume laboratories. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  5. Human cryptosporidiosis: detection of specific antibodies in the serum by an indirect immunofluorescence

    Directory of Open Access Journals (Sweden)

    Braz Lúcia M.A.

    1996-01-01

    Full Text Available Cryptosporidium sp., a coccidian parasite usually found in the faeces of cattle, has been recently implicated as an agent of human intestinal disease, mainly in immunocompromised patients. In the study realized, by an indirect immunofluorescence technique, specific immunoglobulins (IgG and IgM have been demonstrated in human serum against Cryptosporidium oocysts. Purified oocysts were used as antigens in the indirect immunofluorecence assay. After analyzing this test in sera from selected groups of patients, the frequency of both specific IgG and IgM of immunocompetent children who were excreting oocysts in their faeces was 62% and in children with negative excretion of oocysts was 20% and 40%, respectively. In adults infected with the human immunodeficiency virus (HIV and who were excreting Cryptosporidium in their stools, the frequency was 57% for IgG but only 2% for IgM. Twenty three percent of immunocompromised adults with not determined excretion of oocysts in their stools had anti-Cryptosporidium IgG in their sera. Children infected with human immunodeficiency virus had no IgM and only 14% had IgG detectable in their sera. The indirect immunoflorescence assay, when used with other parasitological techniques appears to be useful for retrospective population studies and for diagnosis of acute infection. The humoral immune response of HIV positive patients to this protozoan agent needs clarification.

  6. Evaluation of the Thermo Scientific SureTect Salmonella species assay. AOAC Performance Tested Method 051303.

    Science.gov (United States)

    Cloke, Jonathan; Clark, Dorn; Radcliff, Roy; Leon-Velarde, Carlos; Larson, Nathan; Dave, Keron; Evans, Katharine; Crabtree, David; Hughes, Annette; Simpson, Helen; Holopainen, Jani; Wickstrand, Nina; Kauppinen, Mikko

    2014-01-01

    The Thermo Scientific SureTect Salmonella species Assay is a new real-time PCR assay for the detection of Salmonellae in food and environmental samples. This validation study was conducted using the AOAC Research Institute (RI) Performance Tested Methods program to validate the SureTect Salmonella species Assay in comparison to the reference method detailed in International Organization for Standardization 6579:2002 in a variety of food matrixes, namely, raw ground beef, raw chicken breast, raw ground pork, fresh bagged lettuce, pork frankfurters, nonfat dried milk powder, cooked peeled shrimp, pasteurized liquid whole egg, ready-to-eat meal containing beef, and stainless steel surface samples. With the exception of liquid whole egg and fresh bagged lettuce, which were tested in-house, all matrixes were tested by Marshfield Food Safety, Marshfield, WI, on behalf of Thermo Fisher Scientific. In addition, three matrixes (pork frankfurters, lettuce, and stainless steel surface samples) were analyzed independently as part of the AOAC-RI-controlled laboratory study by the University of Guelph, Canada. No significant difference by probability of detection or McNemars Chi-squared statistical analysis was found between the candidate or reference methods for any of the food matrixes or environmental surface samples tested during the validation study. Inclusivity and exclusivity testing was conducted with 117 and 36 isolates, respectively, which demonstrated that the SureTect Salmonella species Assay was able to detect all the major groups of Salmonella enterica subspecies enterica (e.g., Typhimurium) and the less common subspecies of S. enterica (e.g., arizoniae) and the rarely encountered S. bongori. None of the exclusivity isolates analyzed were detected by the SureTect Salmonella species Assay. Ruggedness testing was conducted to evaluate the performance of the assay with specific method deviations outside of the recommended parameters open to variation (enrichment time

  7. Concordant testing results between various Human Papillomavirus assays in primary cervical cancer screening

    DEFF Research Database (Denmark)

    de Thurah, Lena; Bonde, Jesper; Hoa Lam, Janni Uyen

    2018-01-01

    OBJECTIVES: Human Papillomavirus (HPV) assays are increasingly used for primary cervical screening and HPV vaccination effect monitoring. We undertook a systematic literature review to determine the concordance in positive test results (i.e., detection of HPV infections) between Hybrid Capture 2 ...

  8. 40 CFR 798.5395 - In vivo mammalian bone marrow cytogenetics tests: Micronucleus assay.

    Science.gov (United States)

    2010-07-01

    ... Genetic Toxicity § 798.5395 In vivo mammalian bone marrow cytogenetics tests: Micronucleus assay. (a... and documented with data, only this one time point need be sampled. (ii) If a repeated treatment... slides, spread as a smear and stained. (2) Analysis. Slides shall be coded before microscopic analysis...

  9. The validation of waste assay systems during active test at Rokkasho Reprocessing Plant

    International Nuclear Information System (INIS)

    Tamura, Takayuki; Miura, Yasushi; Iwamoto, Tomonori

    2007-01-01

    In order to implement accurate material accountancy at Rokkasho Reprocessing Plant (RRP) as a large scale reprocessing plant, it is necessary to introduce accurate measurement systems not only for mainstream material, but also appropriate measurement systems for solid waste materials. In this sense, the generated wastes by the active test operation have been measured with the Non-Destructive Assay Systems, such as Rokkasho Hulls Measurement System (RHMS) and Waste Crate Assay System (WCAS) for accountancy. This paper describes the experience of the NDA operation and the evaluation results for accountancy. (author)

  10. Anticoccidial efficacy testing: In vitro Eimeria tenella assays as replacement for animal experiments.

    Science.gov (United States)

    Thabet, Ahmed; Zhang, Runhui; Alnassan, Alaa-Aldin; Daugschies, Arwid; Bangoura, Berit

    2017-01-15

    Availability of an accurate in vitro assay is a crucial demand to determine sensitivity of Eimeria spp. field strains toward anticoccidials routinely. In this study we tested in vitro models of Eimeria tenella using various polyether ionophores (monensin, salinomycin, maduramicin, and lasalocid) and toltrazuril. Minimum inhibitory concentrations (MIC 95 , MIC 50/95 ) for the tested anticoccidials were defined based on a susceptible reference (Houghton strain), Ref-1. In vitro sporozoite invasion inhibition assay (SIA) and reproduction inhibition assay (RIA) were applied on sensitive laboratory (Ref-1 and Ref-2) and field (FS-1, FS-2, and FS-3) strains to calculate percent of inhibition under exposure of these strains to the various anticoccidials (%I SIA and%I RIA, respectively). The in vitro data were related to oocyst excretion, lesion scores, performance, and global resistance indices (GI) assessed in experimentally infected chickens. Polyether ionophores applied in the RIA were highly effective at MIC 95 against Ref-1 and Ref-2 (%I RIA ≥95%). In contrast, all tested field strains displayed reduced to low efficacy (%I RIA animal model (p89%) against all strains used in this study. However, adjusted GI (GI adj ) for toltrazuril-treated groups exhibited differences between reference and field strains which might indicate varying sensitivity. RIA is a suitable in vitro tool to detect sensitivity of E. tenella towards polyether ionophores, and may thus help to reduce, replace, or refine use of animal experimentation for in vivo sensitivity assays. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Irradiated cocoa tested in the wing spot assay in Drosophila melanogaster

    International Nuclear Information System (INIS)

    Zimmering, S.; Olvera, O.; Cruces, M.P.; Pimentel, E.; Arceo, C.; Rosa, M.E. de la; Guzman, J.

    1992-01-01

    The result of treatment of Drosophila melanogaster with irradiated cocoa as scored in the somatic wing spot test is described. The test has been used previously in the evaluation of irradiated food and has registrated a significantly greater number of positives among chemicals tested than germ line counterparts. Irradiated cocoa has thus far been reported negative in other mutagenicity assays including those employing salmonella and Drosophila germ cells and mammalian cells. The wing spot test as described in Graf et al. was employed. Females of the genotype mwh were mated with flr 3 /TM3; Ser males. (author). 9 refs.; 1 tab

  12. Tests of equal effect per fraction in microcolony assays of survival after fractionated irradiations

    International Nuclear Information System (INIS)

    Taylor, J.M.G.

    1985-01-01

    H.D Thames, Jr. and H.R. Withers propose a test of an equal effect per fraction in microcolony assays after fractionated radiation, in which the total effect is measured by counting microcolonies derived from surviving cells in a tissue. The factors considered to influence the cytocidal effect per fraction are incomplete repair, repopulation, and synchrony. The statistics used in the method are criticized and conditions are given under which the test should not be used. An alternative method of testing for an equal effect per fraction is proposed. The pros and cons of each test are discussed and compared using some mouse jejunal crypt cell survival data

  13. Repeat testing of low-level HIV-1 RNA: assay performance and implementation in clinical trials.

    Science.gov (United States)

    White, Kirsten; Garner, Will; Wei, Lilian; Eron, Joseph J; Zhong, Lijie; Miller, Michael D; Martin, Hal; Plummer, Andrew; Tran-Muchowski, Cecilia; Lindstrom, Kim; Porter, James; Piontkowsky, David; Light, Angela; Reiske, Heinz; Quirk, Erin

    2018-05-15

    Assess the performance of HIV-1 RNA repeat testing of stored samples in cases of low-level viremia during clinical trials. Prospective and retrospective analysis of randomized clinical trial samples and reference standards. To evaluate assay variability of the Cobas AmpliPrep/Cobas TaqMan HIV-1 Test, v2.0, three separate sources of samples were utilized: the World Health Organization (WHO) HIV reference standard (assayed using 50 independent measurements at six viral loads <200 copies/ml), retrospective analysis of four to six aliquots of plasma samples from four clinical trial participants, and prospective repeat testing of 120 samples from participants in randomized trials with low-level viremia. The TaqMan assay on the WHO HIV-1 RNA standards at viral loads <200 copies/ml performed within the expected variability according to assay specifications. However, standards with low viral loads of 36 and 18 copies/ml reported values of ≥ 50 copies/ml in 66 and 18% of tests, respectively. In participants treated with antiretrovirals who had unexpected viremia of 50-200 copies/ml after achieving <50 copies/ml, retesting of multiple aliquots of stored plasma found <50 copies/ml in nearly all cases upon retesting (14/15; 93%). Repeat testing was prospectively implemented in four clinical trials for all samples with virologic rebound of 50-200 copies/ml (n = 120 samples from 92 participants) from which 42% (50/120) had a retest result of less than 50 copies/ml and 58% (70/120) retested ≥ 50 copies/ml. The TaqMan HIV-1 RNA assay shows variability around 50 copies/ml that affects clinical trial results and may impact clinical practice. In participants with a history of viral load suppression, unexpected low-level viremia may be because of assay variability rather than low drug adherence or true virologic failure. Retesting a stored aliquot of the same sample may differentiate between assay variability and virologic failure as the source of viremia

  14. Use of external metabolizing systems when testing for endocrine disruption in the T-screen assay

    International Nuclear Information System (INIS)

    Taxvig, Camilla; Olesen, Pelle Thonning; Nellemann, Christine

    2011-01-01

    Although, it is well-established that information on the metabolism of a substance is important in the evaluation of its toxic potential, there is limited experience with incorporating metabolic aspects into in vitro tests for endocrine disrupters. The aim of the current study was a) to study different in vitro systems for biotransformation of ten known endocrine disrupting chemicals (EDs): five azole fungicides, three parabens and 2 phthalates, b) to determine possible changes in the ability of the EDs to bind and activate the thyroid receptor (TR) in the in vitro T-screen assay after biotransformation and c) to investigate the endogenous metabolic capacity of the GH3 cells, the cell line used in the T-screen assay, which is a proliferation assay used for the in vitro detection of agonistic and antagonistic properties of compounds at the level of the TR. The two in vitro metabolizing systems tested the human liver S9 mix and the PCB-induced rat microsomes gave an almost complete metabolic transformation of the tested parabens and phthalates. No marked difference the effects in the T-screen assay was observed between the parent compounds and the effects of the tested metabolic extracts. The GH3 cells themselves significantly metabolized the two tested phthalates dimethyl phthalate (DMP) and diethyl phthalate (DEP). Overall the results and qualitative data from the current study show that an in vitro metabolizing system using liver S9 or microsomes could be a convenient method for the incorporation of metabolic and toxicokinetic aspects into in vitro testing for endocrine disrupting effects.

  15. The microculture tetrazolium assay (MTA): another colorimetric method of testing Plasmodium falciparum chemosensitivity.

    Science.gov (United States)

    Delhaes, L; Lazaro, J E; Gay, F; Thellier, M; Danis, M

    1999-01-01

    Malarial lactate dehydrogenase (LDH), which uses 3-acetyl pyridine adenine dinucleotide as coenzyme in a reaction leading to the formation of pyruvate from L-lactate, may be used to study the susceptibility of Plasmodium falciparum to a drug in vitro. Several methods to determine the activity of this enzyme are available. One, the colorimetric method of Makler and colleagues, was modified slightly, by using sodium-2,3-bis-[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5 - carboxanilide (XTT) and following the reaction by measuring the optical density at 450 nm. Using two, culture-adapted strains of P. falciparum, this LDH assay was compared with the unmodified Makler's assay and with the isotopic microtest based on the incorporation of tritium-labelled hypoxanthine. Fresh, clinical P. falciparum isolates were also tested in the presence of several drugs, including chloroquine, mefloquine, quinine, halofantrine, atovaquone and qinghaosu derivatives. The results of the three assays were correlated for all the drugs tested except atovaquone. The two enzymatic assays are non-radioactive, rapid, reliable, inexpensive to perform and semi-automatic. However, they do require an initial parasitaemia of 2% with a haematocrit of 1.8%.

  16. An interferon-gamma release assay test performs well in routine screening for tuberculosis

    DEFF Research Database (Denmark)

    Vestergaard Danielsen, Allan; Fløe, Andreas; Lillebæk, Troels

    2014-01-01

    Introduction: A positive interferon-gamma release assay (IGRA) is regarded as proof of latent Mycobacterium tuberculosis infection. We conducted an evaluation of the IGRA test “T-SPOT.TB” to test its performance during clinical routine use by analysing the positivity rate and odds, effect of season...... and sensitivity. Material and methods: Data from T-SPOT.TB testing together with age and test indications (anti-tumour necrosis factor alpha (TNFα) candidate, contact investigation or suspicion of tuberculosis (TB)) were combined with mycobac­teria culture results. Results: A total of 1,809 patients were tested....... Conclusive results were achieved for 1,780 patients (98.4%). Among these, 4.6% of anti-TNFα candidates, 19.3% of contacts and 24.4% of TB suspects tested positive. Compared with anti-TNFα candidates, the odds for a positive result were significantly higher for contact investigations (odds ratio (OR), mean...

  17. Genotoxicity evaluation of dental restoration nanocomposite using comet assay and chromosome aberration test

    International Nuclear Information System (INIS)

    Musa, Marahaini; Ponnuraj, Kannan Thirumulu; Mohamad, Dasmawati; Rahman, Ismail Ab

    2013-01-01

    Nanocomposite is used as a dental filling to restore the affected tooth, especially in dental caries. The dental nanocomposite (KelFil) for tooth restoration used in this study was produced by the School of Dental Sciences, Universiti Sains Malaysia, Malaysia and is incorporated with monodispersed, spherical nanosilica fillers. The aim of the study was to determine the genotoxic effect of KelFil using in vitro genotoxicity tests. The cytotoxicity and genotoxicity of KelFil was evaluated using MTT assay, comet assay and chromosome aberration tests with or without the addition of a metabolic activation system (S9 mix), using the human lung fibroblast cell line (MRC-5). Concurrent negative and positive controls were included. In the comet assay, no comet formation was found in the KelFil groups. There was a significant difference in tail moment between KelFil groups and positive control (p < 0.05). Similarly, no significant aberrations in chromosomes were noticed in KelFil groups. The mitotic indices of treatment groups and negative control were significantly different from positive controls. Hence, it can be concluded that the locally produced dental restoration nanocomposite (KelFil) is non-genotoxic under the present test conditions. (paper)

  18. Standard test method for nondestructive assay of radioactive material by tomographic gamma scanning

    CERN Document Server

    American Society for Testing and Materials. Philadelphia

    2010-01-01

    1.1 This test method describes the nondestructive assay (NDA) of gamma ray emitting radionuclides inside containers using tomographic gamma scanning (TGS). High resolution gamma ray spectroscopy is used to detect and quantify the radionuclides of interest. The attenuation of an external gamma ray transmission source is used to correct the measurement of the emission gamma rays from radionuclides to arrive at a quantitative determination of the radionuclides present in the item. 1.2 The TGS technique covered by the test method may be used to assay scrap or waste material in cans or drums in the 1 to 500 litre volume range. Other items may be assayed as well. 1.3 The test method will cover two implementations of the TGS procedure: (1) Isotope Specific Calibration that uses standards of known radionuclide masses (or activities) to determine system response in a mass (or activity) versus corrected count rate calibration, that applies to only those specific radionuclides for which it is calibrated, and (2) Respo...

  19. The DNA comet assay and the germination test in detection of food treated by ionizing radiation

    International Nuclear Information System (INIS)

    Huachaca, Nelida Simona Marin

    2002-01-01

    Two methods of irradiated food detection, one biochemical, the comet assay and, other biological, the germination test, were applied in bovine meat and fruit samples. The comet assay detects the damage on DNA caused by ionizing radiation. The germination test evaluates the sensitivity to radiation of seeds as for germination ability, shooting and, rooting. The samples were irradiated in gamma font and electron accelerator. For bovine meat samples, the doses were 0.0; 2.5; 4.5 e 7.0 kGy at chilled condition and, 0.0; 2.5; 4.5; 7.0 e 8.5 kGy at frozen conditions. For fruit samples such as melon, watermelon, apple, orange, papaya and, tomato, the doses were: 0.0; 0.5; 0.75; 1.0; 2.0 e 4.0 kGy. The differences between the gamma rays and the electron beam effects on extent of DNA migration and, on shooting and rooting, showed to be similar. The comet assay, under neutral conditions, permitted to discriminate between irradiated and unirradiated bovine meat samples, until one month of storage. Also, it was possible to distinguish, by the comet assay, the control sample with regard to irradiated fruit, at doses as low as 0,5 kGy. In the germination test, the root length was the best parameter to discriminate irradiated and unirradiated samples of melon, watermelon and tomato, while the germination percent was the best parameter for apple and orange. (author)

  20. Genotoxic and teratogenic potential of marine sediment extracts investigated with comet assay and zebrafish test

    International Nuclear Information System (INIS)

    Kammann, Ulrike; Biselli, Scarlett; Huehnerfuss, Heinrich; Reineke, Ninja; Theobald, Norbert; Vobach, Michael; Wosniok, Werner

    2004-01-01

    Organic extracts of marine sediments from the North Sea and the Baltic Sea were investigated with two toxicity assays. The comet assay based on the fish cell line Epithelioma papulosum cyprini (EPC) was applied to determine the genotoxic potential; zebrafish embryos (Danio rerio) were used to quantify the teratogenic potential of the samples. EC 50 values were calculated from dose-response curves for both test systems. Highest teratogenic and genotoxic effects normalised to total organic carbon (TOC) content were detected in sediment samples of different origins. Polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) are not likely to be the causes of the observed effects, as demonstrated by a two-step fractionation procedure of selected extracts. The toxic potential was more pronounced in fractions having polarity higher than those possessed by PAHs and PCBs. The suitability of the two in vitro test systems for assessing genotoxic and teratogenic effects of marine sediment extracts could be demonstrated. - Capsule: In vitro toxicity assays are used to assess genotoxic and teratogenic effects of environmental extracts

  1. Development of an ATP assay for rapid onboard testing to detect living microorganisms in ballast water

    Science.gov (United States)

    Hyun, Bonggil; Cha, Hyung-Gon; Lee, Nayoung; Yum, Seungshic; Baek, Seung Ho; Shin, Kyoungsoon

    2018-03-01

    Ballast water is a principal pathway for the introduction of pathogens and non-indigenous species to ports worldwide. The International Maritime Organization (IMO) and the United States Coast Guard (USCG) have adopted ballast water management regulations that require, e.g., the installation of shipboard ballast water management systems (BWMS). Rapid and simple analytical methods are needed to monitor whether ballast water disinfection ensures compliance with the discharge standards. In this study laboratory and full scale land-based testing was used to investigate the suitability of an adenosine triphosphate (ATP) assay for quantifying living organisms (≥ 10 and land-based testing the ATP assay also showed a good correlation with the presence of living natural plankton cells in control samples, but the ATP concentration (137 pg mL- 1) was much lower than the ATP guideline. The low ATP concentration in natural plankton cells may reflect a decline in their biological activity because of extended exposure to dark conditions. Although our results need further validation, the ATP assay is a suitable tool for monitoring compliance of ballast water treatment.

  2. Drug-mediated toxicity: illuminating the 'bad' in the test tube by means of cellular assays?

    Science.gov (United States)

    Tralau, Tewes; Luch, Andreas

    2012-07-01

    Health problems are rising worldwide, be it as a consequence of lifestyle and longevity in increasingly affluent societies or due to a sharp rise in bacterial antibiotic resistance. The pharmaceutical industry is caught between high rates of attrition and the rather slow pace of a historically large regulatory system for pharmacological safety. Meanwhile, the past decade has seen a tremendous evolution of the biological toolbox, most notably of cellular assays, stem-cell differentiation and organ-mimicking systems. These systems were readily adapted for lead-compound identification. However, their use as toxicological test systems is lagging behind, not least because of a lack of regulatory acceptance. This review tries to elucidate the scale of the problem and discusses the applicability of the assays currently available, with particular regard to the use of stem cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. A European multicientre study on the comparison of HBV viral loads between VERIS HBV assay and Roche COBAS® TAQMAN® HBV test, Abbott RealTime HBV assay, Siemens VERSANT HBV assay, and Qiagen artus HBV RG kit.

    Science.gov (United States)

    Braun, Patrick; Delgado, Rafael; Drago, Monica; Fanti, Diana; Fleury, Hervé; Izopet, Jacques; Lombardi, Alessandra; Marcos, MaAngeles; Sauné, Karine; O'Shea, Siobhan; Pérez-Rivilla, Alfredo; Ramble, John; Trimoulet, Pascale; Vila, Jordi; Whittaker, Duncan; Artus, Alain; Rhodes, Daniel

    2017-10-01

    Hepatitis B viral load testing is essential to treatment and monitoring decisions in patients with chronic Hepatitis B. Beckman Coulter has developed the VERIS HBV Assay (Veris) for use on the fully automated DxN VERIS Molecular Diagnostics System. 1 OBJECTIVES: To evaluate the clinical performance of the Veris HBV Assay at multiple EU laboratories STUDY DESIGN: Method comparison was performed with a total of 344 plasma specimens from HBV infected patients tested with Veris and COBAS ® TaqMan ® HBV Test (Cobas), 207 specimens tested with Veris and RealTime HBV Assay (RealTime), 86 specimens tested with Veris and VERSANT ® HBV Assay (Versant), and 74 specimens tested with Veris and artus ® HBV RG PCR kit (artus). Bland-Altman analysis showed average bias of -0.46 log 10 IU/mL between Veris and Cobas, -0.46 log 10 IU/mL between Veris and RealTime, -0.36 log 10 IU/mL between Veris and Versant, and -0.12 log 10 IU/mL between Veris and artus. Bias was consistent across the assay range. Patient monitoring results using Veris demonstrated similar viral load trends over time to Cobas, RealTime, and artus. The VERIS HBV Assay demonstrated comparable clinical performance, with varying degrees of negative bias, compared to other currently marketed assays for HBV DNA monitoring. This negative bias should be taken into consideration if switching monitoring methods to Veris. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Local lymph node assay: how testing laboratories apply OECD TG 429 for REACH purposes.

    Science.gov (United States)

    Rovida, Costanza

    2011-01-01

    The Local Lymph Node Assay (LLNA) is the official method for assessing the allergic contact dermatitis potential of chemicals for the purposes of REACH regulation. The LLNA went through a validation process that allowed the delineation of a robust protocol for performing new tests. The OECD accepted this method in 2002 and published OECD TG 429. The European Chemical Agency (ECHA) recently published data that were submitted in the registration dossiers of chemicals. This database was analysed to determine how testing laboratories apply OECD TG 429. This analysis comes after a detailed analysis of four full study reports that were also prepared for REACH purposes. Although the majority of the tests are fully compliant with OECD TG 429, some showed major deviations, and a number of others used more animals than necessary. This suggests that in vivo tests need to be planned more carefully and consciously to obtain meaningful results with the minimum animal number necessary.

  5. Identification of campylobacteria isolated from Danish broilers by phenotypic tests and species-specific PCR assays

    DEFF Research Database (Denmark)

    Wainø, M.; Bang, Dang Duong; Lund, Marianne

    2003-01-01

    campylobacterial cultures, 108 Campylobacter jejuni cultures and 351 campylobacterial cultures other than Camp. jejuni were subjected to various species-specific PCR assays. On the basis of the genotypic tests, it was demonstrated that Camp. jejuni and Camp. coli constituted approx. 99% of all cultures, while...... other species identified were Helicobacter pullorum, Camp. lari and Camp. upsaliensis. However, 29% of the 309 Camp. coli cultures identified by phenotypic tests were hippurate-variable or negative Camp. jejuni cultures, whereas some Camp. lari cultures and unspeciated campylobacter cultures belonged...... and Impact of the Study: Future phenotypic test schemes should be designed to allow a more accurate differentiation of Campylobacter and related species. Preferably, the phenotypic tests should be supplemented with a genotypic strategy to disclose the true campylobacterial species diversity in broilers....

  6. Chemosensitivity testing of primary human renal cell carcinoma by a tetrazolium based microculture assay (MTT).

    Science.gov (United States)

    Mickisch, G; Fajta, S; Keilhauer, G; Schlick, E; Tschada, R; Alken, P

    1990-01-01

    MTT staining procedures have been used in chemosensitivity testing of established cell lines of human and other sources as well as of human leukaemias, but only limited information on its application in primary solid human tumors is presently available. We have evaluated MTT staining in primary human Renal Cell Carcinomas (RCCs), studied various factors interfering with the optimal use, and finally applied it in subsequent chemosensitivity testing. The method depends on the conversion of a water-soluble tetrazolium salt (MTT) to a purple colored formazan precipitate, a reaction effected by enzymes active only in living cells. Single cell suspensions of RCCs were obtained either by enzymatic dispersion or by mechanical dissagregation, filtered through gauze, and purified by Ficoll density centrifugation. Tests were carried out in 96-well microculture plates. 10(4) viable tumor cells per well at 4 h incubation time with 20 micrograms MTT/100 microliters total medium volume yielded best results. Formazan crystals were dissolved with DMSO, and the plates were immediately measured on a microculture plate reader at 540 nm. Under these criteria, linearity of the system could be demonstrated. For chemosensitivity testing, cells were continuously exposed to a number of drugs prior to the MTT staining procedure. Reproducibility of results was assessed and confirmed by culturing RCCs in flasks additionally, resubmitting them after 1, 2, and 4 weeks to the MTT assay. We conclude that the semiautomated MTT assay offers a valid, rapid, reliable and simple method to determine the degree of chemoresistance in primary human RCCs.

  7. Immunofluorescence-based screening identifies germ cell associated microRNA 302 as an antagonist to p63 expression

    DEFF Research Database (Denmark)

    Scheel, Andreas Hans Joachim; Beyer, Ulrike; Agami, Reuven

    2009-01-01

    The tumor suppressor homologue p63 is required for proper skin and limb development, but specific isoforms of it also act as a "guardian of the germline." To gain insight into the regulation of p63 expression, we performed immunofluorescence-based screening assays. Using a large collection of micro...

  8. Tityus serrulatus Scorpion Venom: In Vitro Tests and Their Correlation with In Vivo Lethal Dose Assay

    Directory of Open Access Journals (Sweden)

    Daniela Cajado-Carvalho

    2017-11-01

    Full Text Available Scorpion stings are the main cause of human envenomation in Brazil and, for the treatment of victims, the World Health Organization (WHO recommends the use of antivenoms. The first step to achieve effective antivenom is to use a good quality venom pool and to evaluate it, with LD50 determination as the most accepted procedure. It is, however, time-consuming and requires advanced technical training. Further, there are significant ethical concerns regarding the number of animals required for testing. Hence, we investigated the correspondence between LD50 results, in vitro assays, and a strong correlation with proteolytic activity levels was observed, showing, remarkably, that proteases are potential toxicity markers for Tityus serrulatus venom. The comparison of reversed-phase chromatographic profiles also has a potential application in venoms’ quality control, as there were fewer neurotoxins detected in the venom with high LD50 value. These results were confirmed by mass spectrometry analysis. Therefore, these methods could precede the LD50 assay to evaluate the venom excellence by discriminating—and discarding—poor-quality batches, and, consequently, with a positive impact on the number of animals used. Notably, proposed assays are fast and inexpensive, being technically and economically feasible in Tityus serrulatus venom quality control to produce effective antivenoms.

  9. Screening test for rapid food safety evaluation by menadione-catalysed chemiluminescent assay.

    Science.gov (United States)

    Yamashoji, Shiro; Yoshikawa, Naoko; Kirihara, Masayuki; Tsuneyoshi, Toshihiro

    2013-06-15

    The chemiluminescent assay of menadione-catalysed H2O2 production by living mammalian cells was proposed to be useful for rapid food safety evaluation. The tested foods were extracted with water, ethanol and dimethylsulfoxide, and each extract was incubated with NIH3T3, Neuro-2a and HepG2 cells for 4h. Menadione-catalysed H2O2 production by living mammalian cells exposed to each extract was determined by the chemiluminescent assay requiring only 10 min, and the viability of the cells was estimated as percentage based on H2O2 production by intact cells. In this study the cytotoxicity of food was rated in order of inhibitory effect on H2O2 production by intact cells. The well known natural toxins such as Fusarium mycotoxin, tomato toxin tomatine, potato toxin solanine and marine toxins terodotoxin and brevetoxin could be detected by the above chemiluminescent assay. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. Rapid susceptibility testing of Mycobacterium tuberculosis by bioluminescence assay of mycobacterial ATP

    International Nuclear Information System (INIS)

    Nilsson, L.E.; Hoffner, S.E.; Ansehn, S.

    1988-01-01

    Mycobacterial growth was monitored by bioluminescence assay of mycobacterial ATP. Cultures of Mycobacterium tuberculosis H37Rv and of 25 clinical isolates of the same species were exposed to serial dilutions of ethambutol, isoniazid, rifampin, and streptomycin. A suppression of ATP, indicating growth inhibition, occurred for susceptible but not resistant strains within 5 to 7 days of incubation. Breakpoint concentrations between susceptibility and resistance were determined by comparing these results with those obtained by reference techniques. Full agreement was found in 99% of the assays with the resistance ratio method on Lowenstein-Jensen medium, and 98% of the assays were in full agreement with the radiometric system (BACTEC). A main advantage of the bioluminescence method is its rapidity, with results available as fast as with the radiometric system but at a lower cost and without the need for radioactive culture medium. The method provides kinetic data concerning drug effects within available in vivo drug concentrations and has great potential for both rapid routine susceptibility testing and research applications in studies of drug effects on mycobacteria

  11. Factors influencing the adrenocorticotropin test: role of contemporary cortisol assays, body composition, and oral contraceptive agents

    DEFF Research Database (Denmark)

    Klose, Marianne; Lange, Martin; Rasmussen, Aase Krogh

    2007-01-01

    -hormone levels; corticosteroid-binding globulin levels; and test conditions (fasting/nonfasting, rest/intermittent exercise). METHODS: A 250-microg ACTH test (0800-1000 h) was performed in 100 unmedicated subjects, 13 women taking oral contraception (OC), and six men with nephrotic syndrome. Tests were performed...... fasting supine (n=119), nonfasting supine (n=38), and fasting with intermittent exercise (n=45). Serum cortisol was analyzed by three immunoassays. RESULTS: Even with a negligible between-assay mean bias, individual samples from unmedicated subjects differed by as much as 110 nmol/liter. The normative 2......-binding globulin, fasting/nonfasting, and exercise. Compared with unmedicated subjects, OC women had 2-fold elevated 30-min cortisol (P

  12. Brief Report: Impact of Early Antiretroviral Therapy on the Performance of HIV Rapid Tests and HIV Incidence Assays.

    Science.gov (United States)

    Fogel, Jessica M; Piwowar-Manning, Estelle; Debevec, Barbara; Walsky, Tamara; Schlusser, Katherine; Laeyendecker, Oliver; Wilson, Ethan A; McCauley, Marybeth; Gamble, Theresa; Tegha, Gerald; Soko, Dean; Kumwenda, Johnstone; Hosseinipour, Mina C; Chen, Ying Q; Cohen, Myron S; Eshleman, Susan H

    2017-08-01

    Antiretroviral therapy (ART) can downregulate antibody responses to HIV infection. We evaluated the impact of early vs. delayed ART on the performance of HIV diagnostic and incidence assays. Samples were obtained from 207 participants in the HPTN 052 trial, who were stably suppressed on ART for ≥4 years [Malawi sites; pre-ART CD4 cell count 350-550 cells/mm (early ART arm, N = 180) or ART arm, N = 27)]. Samples were tested with 2 HIV rapid tests and 2 HIV incidence assays; selected samples were also tested with two fourth-generation immunoassays and a Western blot (WB) assay. A pre-ART sample was analyzed if the follow-up sample had a false-negative or weakly-reactive rapid test result, or had an incidence assay result indicative of recent infection (false-recent result). Ten (4.8%) samples had a nonreactive or weakly-reactive rapid test result (7/180 early ART arm, 3/27 delayed ART arm, P = 0.13); one sample had nonreactive fourth-generation assay results and 3 had indeterminate WBs. Forty (18.9%) samples had a false-recent incidence assay result; 16 (7.8%) had false-recent results with both incidence assays. Baseline samples had stronger rapid test and WB bands, higher fourth-generation assay signal-to-cutoff values, and fewer HIV incidence assay results indicative of recent infection. False-negative/weakly-reactive HIV rapid tests and false-recent HIV incidence assay results were observed in virally-suppressed individuals, regardless of pre-ART CD4 cell count. Downregulation of the antibody response to HIV infection in the setting of ART may impact population-level surveys of HIV prevalence and incidence.

  13. Tuberculin skin test and QuantiFERON® assay in young children investigated for tuberculosis in South Africa

    NARCIS (Netherlands)

    Moyo, S.; Isaacs, F.; Gelderbloem, S.; Verver, S.; Hawkridge, A. J.; Hatherill, M.; Tameris, M.; Geldenhuys, H.; Workman, L.; Pai, M.; Hussey, G.; Hanekom, W. A.; Mahomed, H.

    2011-01-01

    Although the literature on interferon-gamma release assays on tuberculosis (TB) in children has increased, data pertaining to young children remain relatively limited. To compare results from the tuberculin skin test (TST) and the QuantiFERON®-TB Gold In-Tube assay (QFT) in children aged <3 years

  14. In-vitro antimycobacterial drug susceptibility testing of non-tubercular mycobacteria by tetrazolium microplate assay.

    Science.gov (United States)

    Sankar, Manimuthu Mani; Gopinath, Krishnamoorthy; Singla, Roopak; Singh, Sarman

    2008-07-11

    Non-tubercular mycobacteria (NTM) has not been given due attention till the recent epidemic of HIV. This has led to increasing interest of health care workers in their biology, epidemiology and drug resistance. However, timely detection and drug susceptibility profiling of NTM isolates are always difficult in resource poor settings like India. Furthermore, no standardized methodology or guidelines are available to reproduce the results with clinical concordance. To find an alternative and rapid method for performing the drug susceptibility assay in a resource limited settings like India, we intended to evaluate the utility of Tetrazolium microplate assay (TEMA) in comparison with proportion method for reporting the drug resistance in clinical isolates of NTM. A total of fifty-five NTM isolates were tested for susceptibility against Streptomycin, Rifampicin, Ethambutol, Ciprofloxacin, Ofloxacin, Azithromycin, and Clarithromycin by TEMA and the results were compared with agar proportion method (APM). Of the 55 isolates, 23 (41.8%) were sensitive to all the drugs and the remaining 32 (58.2%) were resistant to at least one drug. TEMA had very good concordance with APM except with minor discrepancies. Susceptibility results were obtained in the median of 5 to 9 days by TEMA. The NTM isolates were highly sensitive against Ofloxacin (98.18% sensitive) and Ciprofloxacin (90.09% sensitive). M. mucogenicum was sensitive only to Clarithromycin and resistant to all the other drugs tested. The concordance between TEMA and APM ranged between 96.4 - 100%. Tetrazolium Microplate Assay is a rapid and highly reproducible method. However, it must be performed only in tertiary level Mycobacteriology laboratories with proper bio-safety conditions.

  15. Bulk-assay calorimeter: Part 1. System design and operation. Part 2. Calibration and testing

    International Nuclear Information System (INIS)

    Perry, R.B.; Roche, C.T.; Harkness, A.L.; Winslow, G.H.; Youngdahl, G.A.; Lewis, R.N.; Jung, E.A.

    1982-01-01

    The Bulk-Assay Calorimeter is designed to measure the thermal power emitted by plutonium-containing samples. The sample power range of the instrument is 1.4 to 22.4 W. The instrument package consists of the calorimeter measurement chamber, the control circuit power bin, and the data acquisition system. Two sample preheating chambers and five calorimeter canisters for containing the samples are included. A set of 32 test points which monitor voltages at points within the calorimeter and its control circuitry are accessed by the data acquisition system. The use of the test points is described. System start-up and checkout are described. Sample assay and preheater operation procedures are given. The data acquisition system and data analysis software are described. The calorimeter was calibrated at 23 points with heat sources from 1.4 to 22.4 watts. The combined measurement error varied with sample power from 1.4% to 0.1% over the range of calibration measurements. Circuit diagrams for the calorimeter and schematics for the data acquisition system are included

  16. One or two serological assay testing strategy for diagnosis of HBV and HCV infection? The use of predictive modelling.

    Science.gov (United States)

    Parry, John V; Easterbrook, Philippa; Sands, Anita R

    2017-11-01

    Initial serological testing for chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infection is conducted using either rapid diagnostic tests (RDT) or laboratory-based enzyme immunoassays (EIA)s for detection of hepatitis B surface antigen (HBsAg) or antibodies to HCV (anti-HCV), typically on serum or plasma specimens and, for certain RDTs, capillary whole blood. WHO recommends the use of standardized testing strategies - defined as a sequence of one or more assays to maximize testing accuracy while simplifying the testing process and ideally minimizing cost. Our objective was to examine the diagnostic outcomes of a one- versus two-assay serological testing strategy. These data were used to inform recommendations in the 2017 WHO Guidelines on hepatitis B and C testing. Few published studies have compared diagnostic outcomes for one-assay versus two-assay serological testing strategies for HBsAg and anti-HCV. Therefore, the principles of Bayesian statistics were used to conduct a modelling exercise to examine the outcomes of a one-assay versus two-assay testing strategy when applied to a hypothetical population of 10,000 individuals. The resulting model examined the diagnostic outcomes (true and false positive diagnoses; true and false negative diagnoses; positive and negative predictive values as a function of prevalence; and total tests required) for both one-assay and two-assay testing strategies. The performance characteristics assumed for assays used within the testing strategies were informed by WHO prequalification assessment findings and systematic reviews for diagnostic accuracy studies. Each of the presumptive testing strategies (one-assay or two-assay) was modelled at varying prevalences of HBsAg (10%, 2% and 0.4%) and of anti-HCV (40%, 10%, 2% and 0.4%), aimed at representing the range of testing populations typically encountered in WHO Member States. When the two-assay testing strategy was considered, the model assumed the independence of the

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

    Science.gov (United States)

    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.

  18. Comparative assay of fluorescent antibody test results among twelve European National Reference Laboratories using various anti-rabies conjugates

    DEFF Research Database (Denmark)

    Robardet, E.; Andrieu, S.; Rasmussen, Thomas Bruun

    2013-01-01

    Twelve National Reference Laboratories (NRLs) for rabies have undertaken a comparative assay to assess the comparison of fluorescent antibody test (FAT) results using five coded commercial anti-rabies conjugates (Biorad, Bioveta, Fujirebio, Millipore, and SIFIN conjugates). Homogenized positive...

  19. Critical issues with the in vivo comet assay: A report of the comet assay working group in the 6th International Workshop on Genotoxicity Testing (IWGT).

    Science.gov (United States)

    Speit, Günter; Kojima, Hajime; Burlinson, Brian; Collins, Andrew R; Kasper, Peter; Plappert-Helbig, Ulla; Uno, Yoshifumi; Vasquez, Marie; Beevers, Carol; De Boeck, Marlies; Escobar, Patricia A; Kitamoto, Sachiko; Pant, Kamala; Pfuhler, Stefan; Tanaka, Jin; Levy, Dan D

    2015-05-01

    As a part of the 6th IWGT, an expert working group on the comet assay evaluated critical topics related to the use of the in vivo comet assay in regulatory genotoxicity testing. The areas covered were: identification of the domain of applicability and regulatory acceptance, identification of critical parameters of the protocol and attempts to standardize the assay, experience with combination and integration with other in vivo studies, demonstration of laboratory proficiency, sensitivity and power of the protocol used, use of different tissues, freezing of samples, and choice of appropriate measures of cytotoxicity. The standard protocol detects various types of DNA lesions but it does not detect all types of DNA damage. Modifications of the standard protocol may be used to detect additional types of specific DNA damage (e.g., cross-links, bulky adducts, oxidized bases). In addition, the working group identified critical parameters that should be carefully controlled and described in detail in every published study protocol. In vivo comet assay results are more reliable if they were obtained in laboratories that have demonstrated proficiency. This includes demonstration of adequate response to vehicle controls and an adequate response to a positive control for each tissue being examined. There was a general agreement that freezing of samples is an option but more data are needed in order to establish generally accepted protocols. With regard to tissue toxicity, the working group concluded that cytotoxicity could be a confounder of comet results. It is recommended to look at multiple parameters such as histopathological observations, organ-specific clinical chemistry as well as indicators of tissue inflammation to decide whether compound-specific toxicity might influence the result. The expert working group concluded that the alkaline in vivo comet assay is a mature test for the evaluation of genotoxicity and can be recommended to regulatory agencies for use

  20. BMVC test, an improved fluorescence assay for detection of malignant pleural effusions

    International Nuclear Information System (INIS)

    Lin, I-Ting; Tsai, Yu-Lin; Kang, Chi-Chih; Huang, Wei-Chun; Wang, Chiung-Lin; Lin, Mei-Ying; Lou, Pei-Jen; Shih, Jin-Yuan; Wang, Hao-Chien; Wu, Huey-Dong; Tsai, Tzu-Hsiu; Jan, I-Shiow; Chang, Ta-Chau

    2014-01-01

    The diagnosis of malignant pleural effusions is an important issue in the management of malignancy patients. Generally, cytologic examination is a routine diagnostic technique. However, morphological interpretation of cytology is sometimes inconclusive. Here an ancillary method named BMVC test is developed for rapid detection of malignant pleural effusion to improve the diagnostic accuracy at low cost. A simple assay kit is designed to collect living cells from clinical pleural effusion and a fluorescence probe, 3,6-Bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC), is used to illuminate malignant cells. The fluorescence intensity is quantitatively analyzed by ImageJ program. This method yields digital numbers for the test results without any grey zone or ambiguities in the current cytology tests due to intra-observer and inter-observer variability. Comparing with results from double-blind cytologic examination, this simple test gives a good discrimination between malignant and benign specimens with sensitivity of 89.4% (42/47) and specificity of 93.3% (56/60) for diagnosis of malignant pleural effusion. BMVC test provides accurate results in a short time period, and the digital output could assist cytologic examination to become more objective and clear-cut. This is a convenient ancillary tool for detection of malignant pleural effusions

  1. Use of a radiorespirometric assay for testing the antibiotic sensitivity of catheter-associated bacteria

    International Nuclear Information System (INIS)

    Ladd, T.I.; Schmiel, D.; Nickel, J.C.; Costerton, J.W.

    1987-01-01

    A 14 C-radiorespirometric assay was used to show the sensitivity of fixed-film (sessile), catheter-associated and free-living (planktonic) cells of Pseudomonas aeruginosa to varying concentrations (100 micrograms/mL to 1000 micrograms/mL) tobramycin sulfate. This strain of P. aeruginosa has an MIC of 0.6 microgram/ml and an MBC of 50 micrograms/mL when tested by conventional methods. When 14 C-glutamic acid was used as a substrate in this radiorespirometric assay, it could be completed in less than one hour and planktonic samples showed a significant reduction in mineralization activity (evolution of 14 CO 2 ) within eight hours of the antibiotic challenge. These changes in respiratory activity appeared to be dose and time dependent. Within 18 hr. at 1000 micrograms/mL, there was no significant residual respiratory activity in planktonic samples. Some residual respiratory activity was detected, however, in samples exposed to 100 micrograms/mL for 36 hours. The mineralization activity of sessile catheter-associated bacteria was unaffected by four hr. and eight hr. exposures to 1000 micrograms/mL of the antibiotic. A significant reduction in respiratory activity was recorded in catheter samples exposed for 18 hr. or more at each concentration examined. Unlike the planktonic samples, however, the antibiotic challenge failed to eradicate the metabolic activity of the attached bacteria. Antibiotic stressed, catheter-associated bacteria transferred to a post-exposure enrichment broth showed a limited ability to re-establish respiratory activity. This apparent recovery was limited to antibiotic exposures less than 24 hr. and was not observed in planktonic samples. The radioisotopic assay is a non-culture method which can be used to assess the antibiotic sensitivity of both planktonic bacteria and in situ biofilm populations

  2. Integrated sample-to-detection chip for nucleic acid test assays.

    Science.gov (United States)

    Prakash, R; Pabbaraju, K; Wong, S; Tellier, R; Kaler, K V I S

    2016-06-01

    Nucleic acid based diagnostic techniques are routinely used for the detection of infectious agents. Most of these assays rely on nucleic acid extraction platforms for the extraction and purification of nucleic acids and a separate real-time PCR platform for quantitative nucleic acid amplification tests (NATs). Several microfluidic lab on chip (LOC) technologies have been developed, where mechanical and chemical methods are used for the extraction and purification of nucleic acids. Microfluidic technologies have also been effectively utilized for chip based real-time PCR assays. However, there are few examples of microfluidic systems which have successfully integrated these two key processes. In this study, we have implemented an electro-actuation based LOC micro-device that leverages multi-frequency actuation of samples and reagents droplets for chip based nucleic acid extraction and real-time, reverse transcription (RT) PCR (qRT-PCR) amplification from clinical samples. Our prototype micro-device combines chemical lysis with electric field assisted isolation of nucleic acid in a four channel parallel processing scheme. Furthermore, a four channel parallel qRT-PCR amplification and detection assay is integrated to deliver the sample-to-detection NAT chip. The NAT chip combines dielectrophoresis and electrostatic/electrowetting actuation methods with resistive micro-heaters and temperature sensors to perform chip based integrated NATs. The two chip modules have been validated using different panels of clinical samples and their performance compared with standard platforms. This study has established that our integrated NAT chip system has a sensitivity and specificity comparable to that of the standard platforms while providing up to 10 fold reduction in sample/reagent volumes.

  3. Pre-Clinical Testing of Real-Time PCR Assays for Diarrheal Disease Agents of Genera Escherichia and Shigella

    Science.gov (United States)

    2014-05-16

    FOR DIARRHEAL DISEASE AGENTS OF GENERA ESCHERICHIA AND SHIGELLA May 16, 2014 Reporting Period: October 1, 2010 to September 30, 2013...10-2010 - 30-09-2013 PRE-CLINICAL TESTING OF REAL-TIME PCR ASSAYS FOR DIARRHEAL DISEASE AGENTS OF GENERA ESCHERICHIA AND SHIGELLA ...Texas (MOA 2007 - 2013. Agreement No.: DODI 4000.19; AFI 25-201). Pre-clinical test results qualify ETEC and Shigella real-time PCR assays as lead

  4. Testing the genotoxicity of some perfumes with high diethylphthalate (DEP levels using comet assay

    Directory of Open Access Journals (Sweden)

    Iman Al-Saleh

    2015-05-01

    Full Text Available The presence of phthalates in perfumes has gained recently some attention since these chemicals are added sometimes intentionally as a fixative. Our previous study tested 47 branded perfumes sold in Saudi market and found 68% of tested samples had diethylphthalate (DEP, above reported threshold limit of 1 ppm. Of these phthalates, DEP was found to have the highest mean value (1621.63 ppm. These results enticed us to test the potential genotoxicity of 7 brands in which their DEP contents were in the range of 1.06 ppm to 23649.3 ppm in human TK-6 cells using single cell gel electrophoresis assay (comet assay. Cells were exposed to 400 µl perfume for 2 hrs, at room temperature. Tail moment was (TM used as a metric measure for DNA damage and 25 cells per sample were determined by image analysis software. Four perfumes with DEP above 40 ppm produced significant DNA damage in TK-6 cells with TM of 54.27 ± 1.04, n=500 compare to the other 3 perfumes with DEP < 2 ppm (21.94 ± 1.09, n=300, untreated cells (2.99 ± 0.17, n=250 and cells induced with ethanol (33.76 ± 1.4, n=75 or methanol (23.82 ± 1.84, n=100 which are the vehicles used in perfume's preparations. The results suggest that DEP in perfumes might be one of the ingredients that provoked DNA damage; however, further investigation is required confirming our observation.

  5. Herpes Murine Model as a Biological Assay to Test Dialyzable Leukocyte Extracts Activity

    Directory of Open Access Journals (Sweden)

    Nohemí Salinas-Jazmín

    2015-01-01

    Full Text Available Human dialyzable leukocyte extracts (DLEs are heterogeneous mixtures of low-molecular-weight peptides that are released on disruption of peripheral blood leukocytes from healthy donors. DLEs improve clinical responses in infections, allergies, cancer, and immunodeficiencies. Transferon is a human DLE that has been registered as a hemoderivate by Mexican health authorities and commercialized nationally. To develop an animal model that could be used routinely as a quality control assay for Transferon, we standardized and validated a murine model of cutaneous HSV-1 infection. Using this model, we evaluated the activity of 27 Transferon batches. All batches improved the survival of HSV-1-infected mice, wherein average survival rose from 20.9% in control mice to 59.6% in Transferon-treated mice. The activity of Transferon correlated with increased serum levels of IFN-γ and reduced IL-6 and TNF-α concentrations. Our results demonstrate that (i this mouse model of cutaneous herpes can be used to examine the activity of DLEs, such as Transferon; (ii the assay can be used as a routine test for batch release; (iii Transferon is produced with high homogeneity between batches; (iv Transferon does not have direct virucidal, cytoprotective, or antireplicative effects; and (v the protective effect of Transferon in vivo correlates with changes in serum cytokines.

  6. Evaluation of the performance of the reduced local lymph node assay for skin sensitization testing.

    Science.gov (United States)

    Ezendam, Janine; Muller, Andre; Hakkert, Betty C; van Loveren, Henk

    2013-06-01

    The local lymph node assay (LLNA) is the preferred method for classification of sensitizers within REACH. To reduce the number of mice for the identification of sensitizers the reduced LLNA was proposed, which uses only the high dose group of the LLNA. To evaluate the performance of this method for classification, LLNA data from REACH registrations were used and classification based on all dose groups was compared to classification based on the high dose group. We confirmed previous examinations of the reduced LLNA showing that this method is less sensitive compared to the LLNA. The reduced LLNA misclassified 3.3% of the sensitizers identified in the LLNA and misclassification occurred in all potency classes and that there was no clear association with irritant properties. It is therefore not possible to predict beforehand which substances might be misclassified. Another limitation of the reduced LLNA is that skin sensitizing potency cannot be assessed. For these reasons, it is not recommended to use the reduced LLNA as a stand-alone assay for skin sensitization testing within REACH. In the future, the reduced LLNA might be of added value in a weight of evidence approach to confirm negative results obtained with non-animal approaches. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Genotoxicity assessment of cobalt chloride in Eisenia hortensis earthworms coelomocytes by comet assay and micronucleus test.

    Science.gov (United States)

    Ciğerci, İbrahim Hakkı; Ali, Muhammad Muddassir; Kaygısız, Şöhret Yüksek; Liman, Recep

    2016-02-01

    Cobalt and its different compounds are extensively used worldwide and considered as possible environmental pollutant. Earthworms are useful model organism and its different species are used to monitor soil pollution. No study has been found to detect cobalt chloride (CoCl2) genotoxicity in earthworms. So, current study aimed to evaluate CoCl2 induced genotoxicity in Eisenia hortensis earthworms coelomocytes by alkaline comet assay (CA) and micronucleus (MN) test. The earthworms (n = 10 for each group) were exposed to different series of CoCl2 concentrations (100 ppm, 200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm) to find LD50. The LD50 for CoCl2 was found at 226 ppm. Then, doses of LD50/2, LD50 and 2XLD50 for 48 h were used. CA and MN demonstrated the significant increase (P earthworms. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Intra-laboratory validation of a human cell based in vitro angiogenesis assay for testing angiogenesis modulators

    Directory of Open Access Journals (Sweden)

    Jertta-Riina Sarkanen

    2011-01-01

    Full Text Available The developed standardized human cell based in vitro angiogenesis assay was intra-laboratory validated to verify that the method is reliable and relevant for routine testing of modulators of angiogenesis e.g. pharmaceuticals and industrial chemicals. This assay is based on the earlier published method but it was improved and shown to be more sensitive and rapid than the previous assay. The performance of the assay was assessed by using 6 reference chemicals, which are widely used pharmaceuticals that inhibit angiogenesis: acetyl salicylic acid, erlotinib, 2-methoxyestradiol, levamisole, thalidomide, and anti-vascular endothelial growth factor. In the intra-laboratory validation, the sensitivity of the assay (upper and lower limits of detection and linearity of response in tubule formation, batch to batch variation in tubule formation between different Master cell bank batches, and precision as well as the reliability of the assay (reproducibility and repeatability were tested. The pre-set acceptance criteria for the intra-laboratory validation study were met. The relevance of the assay in man was investigated by comparing the effects of reference chemicals and their concentrations to the published human data. The comparison showed a good concordance, which indicates that this human cell based angiogenesis model predicts well the effects in man and has the potential to be used to supplement and/or replace of animal tests.

  9. Electrochemical Genotoxicity Assay Based on a SOS/umu Test Using Hydrodynamic Voltammetry in a Droplet

    Science.gov (United States)

    Kuramitz, Hideki; Sazawa, Kazuto; Nanayama, Yasuaki; Hata, Noriko; Taguchi, Shigeru; Sugawara, Kazuharu; Fukushima, Masami

    2012-01-01

    The SOS/umu genotoxicity assay evaluates the primary DNA damage caused by chemicals from the β-galactosidase activity of S. typhimurium. One of the weaknesses of the common umu test system based on spectrophotometric detection is that it is unable to measure samples containing a high concentration of colored dissolved organic matters, sediment, and suspended solids. However, umu tests with electrochemical detection techniques prove to be a better strategy because it causes less interference, enables the analysis of turbid samples and allows detection even in small volumes without loss of sensitivity. Based on this understanding, we aim to develop a new umu test system with hydrodynamic chronoamperometry using a rotating disk electrode (RDE) in a microliter droplet. PAPG when used as a substrate is not electroactive at the potential at which PAP is oxidized to p-quinone imine (PQI), so the current response of chronoamperometry resulting from the oxidation of PAP to PQI is directly proportional to the enzymatic activity of S. typhimurium. This was achieved by performing genotoxicity tests for 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF-2) and 2-aminoanthracene (2-AA) as model genotoxic compounds. The results obtained in this study indicated that the signal detection in the genotoxicity assay based on hydrodynamic voltammetry was less influenced by the presence of colored components and sediment particles in the samples when compared to the usual colorimetric signal detection. The influence caused by the presence of humic acids (HAs) and artificial sediment on the genotoxic property of selected model compounds such as 4-nitroquinoline-N-oxide (4-NQO), 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), 1,8-dinitropyrene (1,8-DNP) and 1-nitropyrene (1-NP) were also investigated. The results showed that the genotoxicity of 1-NP and MX changed in the presence of 10 mg·L−1 HAs. The genotoxicity of tested chemicals with a high hydrophobicity such as 1,8-DNP

  10. Unsaturated fatty acids show clear elicitation responses in a modified local lymph node assay with an elicitation phase, and test positive in the direct peptide reactivity assay.

    Science.gov (United States)

    Yamashita, Kunihiko; Shinoda, Shinsuke; Hagiwara, Saori; Miyazaki, Hiroshi; Itagaki, Hiroshi

    2015-12-01

    The Organisation for Economic Co-operation and Development (OECD) Test Guidelines (TG) adopted the murine local lymph node assay (LLNA) and guinea pig maximization test (GPMT) as stand-alone skin sensitization test methods. However, unsaturated carbon-carbon double-bond and/or lipid acids afforded false-positive results more frequently in the LLNA compared to those in the GPMT and/or in human subjects. In the current study, oleic, linoleic, linolenic, undecylenic, fumaric, maleic, and succinic acid and squalene were tested in a modified LLNA with an elicitation phase (LLNA:DAE), and in a direct peptide reactivity assay (DPRA) to evaluate their skin-sensitizing potential. Oleic, linoleic, linolenic, undecylenic and maleic acid were positive in the LLNA:DAE, of which three, linoleic, linolenic, and maleic acid were positive in the DPRA. Furthermore, the results of the cross-sensitizing tests using four LLNA:DAE-positive chemicals were negative, indicating a chemical-specific elicitation response. In a previous report, the estimated concentration needed to produce a stimulation index of 3 (EC3) of linolenic acid, squalene, and maleic acid in the LLNA was LLNA. However, the skin-sensitizing potential of all LLNA:DAE-positive chemicals was estimated as weak. These results suggested that oleic, linoleic, linolenic, undecylenic, and maleic acid had skin-sensitizing potential, and that the LLNA overestimated the skin-sensitizing potential compared to that estimated by the LLNA:DAE.

  11. Comet assay in the detection of irradiated garlic; Teste do cometa na deteccao de alho irradiado

    Energy Technology Data Exchange (ETDEWEB)

    Villavicencio, Anna Lucia C.H.; Marin-Huachaca, Nelida Simona; Romanelli, Maria Fernanda [Instituto de Pesquisas Energeticas e Nucleares (IPEN), Sao Paulo, SP (Brazil)]. E-mail: villavic@net.ipen.br; Delincee, Henry [Federal Research Centre for Nutrition - BFE, Karlsruhe (Germany)]. E-mail: henry.delincee@bfe.uni-karlsruhe.de

    2002-07-01

    The increased claim for fresh produce has forced a consensus between nations to pay more attention to the phytosanitary regulations. Inhibition of sprouting of bulbs and tubers by applying ionising radiation is authorised by the National Food Codes in Brazil. The availability of methods for detection of irradiated food will contribute to increase consumers' confidence. A quick and simple screening test to indicate whether a food product has been irradiated or not was utilised in this study. The DNA comet assay was applied to verify whether garlic imported from China had been irradiated or not. This test has already been adopted as a European Standard (EN 13784), for detection of irradiated food. Non-irradiated control samples of garlic and garlic treated with maleic hydrazide were compared with garlic samples irradiated in our department. The unirradiated samples exhibited only limited DNA migration. If samples were irradiated, an increased DNA fragmentation was observed which permitted the discrimination between non-irradiated and irradiated samples. Since the garlic samples from China showed only very limited DNA fragmentation, they were deemed non-irradiated. Thus, this simple screening test was shown to be successful for identification of an irradiation treatment. (author)

  12. Testing for developmental neurotoxicity using a battery of in vitro assays for key cellular events in neurodevelopment.

    Science.gov (United States)

    Harrill, Joshua A; Freudenrich, Theresa; Wallace, Kathleen; Ball, Kenneth; Shafer, Timothy J; Mundy, William R

    2018-04-05

    Medium- to high-throughput in vitro assays that recapitulate the critical processes of nervous system development have been proposed as a means to facilitate rapid testing and identification of chemicals which may affect brain development. In vivo neurodevelopment is a complex progression of distinct cellular processes. Therefore, batteries of in vitro assays that model and quantify effects on a variety of neurodevelopmental processes have the potential to identify chemicals which may affect brain development at different developmental stages. In the present study, the results of concentration-response screening of 67 reference chemicals in a battery of high content imaging and microplate reader-based assays that evaluate neural progenitor cell proliferation, neural proginitor cell apoptosis, neurite initiation/outgrowth, neurite maturation and synaptogenesis are summarized and compared. The assay battery had a high degree of combined sensitivity (87%) for categorizing chemicals known to affect neurodevelopment as active and a moderate degree of combined specificity (71%) for categorizing chemicals not associated with affects on neurodevelopment as inactive. The combined sensitivity of the assay battery was higher compared to any individual assay while the combined specificity of the assay battery was lower compared to any individual assay. When selectivity of effects for a neurodevelopmental endpoint as compared to general cytotoxicity was taken into account, the combined sensitivity of the assay battery decreased (68%) while the combined specificity increased (93%). The identity and potency of chemicals identified as active varied across the assay battery, underscoring the need for use of a combination of diverse in vitro models to comprehensively screen chemicals and identify those which potentially affect neurodevelopment. Overall, these data indicate that a battery of assays which address many different processes in nervous system development may be used to

  13. Evaluation of simple rapid HIV assays and development of national rapid HIV test algorithms in Dar es Salaam, Tanzania.

    Science.gov (United States)

    Lyamuya, Eligius F; Aboud, Said; Urassa, Willy K; Sufi, Jaffer; Mbwana, Judica; Ndugulile, Faustin; Massambu, Charles

    2009-02-18

    Suitable algorithms based on a combination of two or more simple rapid HIV assays have been shown to have a diagnostic accuracy comparable to double enzyme-linked immunosorbent assay (ELISA) or double ELISA with Western Blot strategies. The aims of this study were to evaluate the performance of five simple rapid HIV assays using whole blood samples from HIV-infected patients, pregnant women, voluntary counseling and testing attendees and blood donors, and to formulate an alternative confirmatory strategy based on rapid HIV testing algorithms suitable for use in Tanzania. Five rapid HIV assays: Determine HIV-1/2 (Inverness Medical), SD Bioline HIV 1/2 3.0 (Standard Diagnostics Inc.), First Response HIV Card 1-2.0 (PMC Medical India Pvt Ltd), HIV1/2 Stat-Pak Dipstick (Chembio Diagnostic System, Inc) and Uni-Gold HIV-1/2 (Trinity Biotech) were evaluated between June and September 2006 using 1433 whole blood samples from hospital patients, pregnant women, voluntary counseling and testing attendees and blood donors. All samples that were reactive on all or any of the five rapid assays and 10% of non-reactive samples were tested on a confirmatory Inno-Lia HIV I/II immunoblot assay (Immunogenetics). Three hundred and ninety samples were confirmed HIV-1 antibody positive, while 1043 were HIV negative. The sensitivity at initial testing of Determine, SD Bioline and Uni-Gold was 100% (95% CI; 99.1-100) while First Response and Stat-Pak had sensitivity of 99.5% (95% CI; 98.2-99.9) and 97.7% (95% CI; 95.7-98.9), respectively, which increased to 100% (95% CI; 99.1-100) on repeat testing. The initial specificity of the Uni-Gold assay was 100% (95% CI; 99.6-100) while specificities were 99.6% (95% CI; 99-99.9), 99.4% (95% CI; 98.8-99.7), 99.6% (95% CI; 99-99.9) and 99.8% (95% CI; 99.3-99.9) for Determine, SD Bioline, First Response and Stat-Pak assays, respectively. There was no any sample which was concordantly false positive in Uni-Gold, Determine and SD Bioline assays. An

  14. The Vitotox and ToxTracker assays: A two-test combination for quick and reliable assessment of genotoxic hazards.

    Science.gov (United States)

    Ates, Gamze; Favyts, Dorien; Hendriks, Giel; Derr, Remco; Mertens, Birgit; Verschaeve, Luc; Rogiers, Vera; Y Doktorova, Tatyana

    2016-11-01

    To ensure safety for humans, it is essential to characterize the genotoxic potential of new chemical entities, such as pharmaceutical and cosmetic substances. In a first tier, a battery of in vitro tests is recommended by international regulatory agencies. However, these tests suffer from inadequate specificity: compounds may be wrongly categorized as genotoxic, resulting in unnecessary, time-consuming, and expensive in vivo follow-up testing. In the last decade, novel assays (notably, reporter-based assays) have been developed in an attempt to overcome these drawbacks. Here, we have investigated the performance of two in vitro reporter-based assays, Vitotox and ToxTracker. A set of reference compounds was selected to span a variety of mechanisms of genotoxic action and applicability domains (e.g., pharmaceutical and cosmetic ingredients). Combining the performance of the two assays, we achieved 93% sensitivity and 79% specificity for prediction of gentoxicity for this set of compounds. Both assays permit quick high-throughput analysis of drug candidates, while requiring only small quantities of the test substances. Our study shows that these two assays, when combined, can be a reliable method for assessment of genotoxicity hazard. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Testing for myositis specific autoantibodies: Comparison between line blot and immunoprecipitation assays in 57 myositis sera.

    Science.gov (United States)

    Cavazzana, Ilaria; Fredi, Micaela; Ceribelli, Angela; Mordenti, Cristina; Ferrari, Fabio; Carabellese, Nice; Tincani, Angela; Satoh, Minoru; Franceschini, Franco

    2016-06-01

    To analyze the performance of a line blot assay for the identification of autoantibodies in sera of patients affected by myositis, compared with immunoprecipitation (IP) as gold standard. 66 sera of patients with myositis (23 polymyositis, 8 anti-synthetase syndromes, 29 dermatomyositis and 6 overlap syndromes) were tested by commercial LB (Euroimmun, Lubeck, Germany); 57 sera were analyzed also by IP of K562 cell extract radiolabeled with (35)S-methionine. Inter-rater agreement was calculated with Cohen's k coefficient. Myositis-specific antibodies (MSA) were detected in 36/57 sera (63%) by IP and in 39/66 sera (59%) by LB. The most frequent MSA found by LB were anti-Jo1 and anti-Mi2 found in 15% (10/66) of sera, followed by anti-NXP2 and anti-SRP detected in 106% (7/66) of sera. Anti-TIF1gamma and anti-MDA5 were found in 6 (9%) and 5 sera (7.6%), respectively. A good agreement between methods was found only for anti-TIF1γ, anti-MDA5 and anti-NXP-2 antibodies, while a moderate agreement was estimated for anti-Mi2 and anti-EJ. By contrast, a high discordance rate for the detection of anti-Jo1 antibodies was evident (k: 0.3). Multiple positivity for MSA were found in 11/66 (17%) by LB and 0/57 by IP (p: 0001). Comparing the clinical features of these 11 sera, we found total discrepancies between assays in 3 sera (27.3%), a relative discrepancy due to the occurrence of one discordant autoantibody (not confirmed by IP) in 5 cases (45.5%) and a total discrepancy between LB and IP results, but with a relative concordance with clinical features were found in other 3 sera (27.3%). The semiquantitative results do not support the interpretation of the data. The use of LB assay allowed the detection of new MSA, such as anti-MDA5, anti-MJ and anti-TIF1gamma antibodies, previously not found with routine methods. However, the high prevalence of multiple positivities and the high discondant rate of anti-Jo1 antibodies could create some misinterpretation of the results from the

  16. Human Sperm Bioassay for Reprotoxicity Testing in Embryo Culture Media: Some Practical Considerations in Reducing the Assay Time

    Directory of Open Access Journals (Sweden)

    Amjad Hossain

    2010-01-01

    Full Text Available Human sperm assay (HSA is a preferred in house quality control and proficiency test (PT practiced in fertility laboratories. HSA is performed over varying durations, apparently without following set criteria. To better understand the assay time required for reprotoxicity testing in embryo culture media, we compared American-Association-of-Bioanalysts-(AAB- administered HSA data to our own assay performed using PT samples obtained from AAB. Participating laboratories were required to culture sperm for 48 hours to determine media acceptability. Conclusions drawn from 48- and 24-hour observations were the same, suggesting that HSA could identify reprotoxic media in less time than required by AAB. Our assay revealed that changes in motility grade in adulterated media are significantly different from those in control media. Furthermore, grade changes can be identified earlier than differences in motility loss between samples. Analyzing motility and motility quality together provides a method for establishing an optimal time for HSA.

  17. Comparison of DNA comet assay and germination test (half-embryo-test) in gamma-irradiated cherry seeds

    International Nuclear Information System (INIS)

    Todoroki, Setsuko; Hayashi, Toru

    2002-01-01

    Cherry fruits were irradiated with gamma-rays at doses up to 200Gy (effective dose for disinfestation of codling moth), and DNA strand break in seed embryos was investigated by using alkaline comet assay. Immediately after irradiation (≥100Gy), DNA from embryos produced comets with a long and wide tail due to fragmentation. In control cells, DNA relaxed and produced comet with very short tail (with few strand break). After 72h storage, DNA from fruits irradiated at 200 Gy showed comets with little tail and tail moment of comets was same as un-irradiated control. These results indicate that the strand breaks of DNA caused by irradiation in fresh seed embryo are repaired during storage. On the contrary, the ability of germination lost by irradiation did not restored, a dose of 100Gy and more retarded shoot elongation. In cherries irradiated at 100Gy, the shooting percentage was less than 50% at 4th day after incubation. Germination test (Half embryo test) can be discriminate between irradiated and un-irradiated cherries. (author)

  18. Comparison of tetrazolium colorimetric and [3H]-uridine assays for in vitro chemosensitivity testing.

    Science.gov (United States)

    Ford, C H; Richardson, V J; Tsaltas, G

    1989-01-01

    We have routinely used a [3H]-uridine microplate assay for assessing chemosensitivity. A colorimetric assay with the advantages of safety, cost and simplicity has previously been described and relies on the ability of living cells to reduce a soluble tetrazolium salt, 3-4,5-dimethylthiazol-2,5-diphenyl-tetrazolium bromide (MMT), into an insoluble formazan precipitate. We compared the chemosensitivity of 14 human tumour cell lines of colonic, lung and cervical carcinoma origin to doxorubicin, vindesine or vindesine immunoconjugates in both the [3H]-uridine assay and a modified MTT assay to evaluate whether we could change to the non-radiolabelled method. Correlation between the concentration of drug causing 50% inhibition of cell growth (IC50) for these agents between the two assays was very poor. However, taking account of recent reports in the literature, we modified the MTT assay by removing serum-containing medium and using dimethyl sulphoxide to solubilise the formazan precipitate. This considerably improved the correlation between the assays for doxorubicin (r = 0.871; P = 0.001) and vindesine (r = 0.981; P less than 0.001). Our data indicates that the MTT assay can be used to replace the [3H]-uridine assay for chemosensitivity screening, but further modifications are necessary to improve the sensitivity and decrease the problem of cell loss after washing, which was noted with some adherent cell lines.

  19. Testing candidate genes for attention-deficit/hyperactivity disorder in fruit flies using a high throughput assay for complex behavior

    DEFF Research Database (Denmark)

    Rohde, Palle Duun; Madsen, Lisbeth Strøm; Arvidson, Sandra Marie Neumann

    2016-01-01

    Fruit flies are important model organisms for functional testing of candidate genes in multiple disciplines, including the study of human diseases. Here we use a high-throughput locomotor activity assay to test the response on activity behavior of gene disruption in Drosophila melanogaster. The aim...

  20. Continuing assessment of the 5 day sodium carbonate-ammonium nitrate extraction assay as an indicator test for silicon fertilizers

    Science.gov (United States)

    The five day sodium carbonate-ammonium nitrate extraction assay has been proposed by the AAFPCO as a standard test to identify fertilizers that provide plant-available Si. A single-lab validation test was previously performed; however, the analysis lacked any correlation to a grow-out study. To do...

  1. Individual response to ionising radiation: What predictive assay(s) to choose?; Reponse individuelle aux radiations ionisantes: quel(s) test(s) predictif(s) choisir?

    Energy Technology Data Exchange (ETDEWEB)

    Granzotto, A.; Viau, M.; Devic, C.; Maalouf, M.; Thomas, Ch.; Vogin, G.; Foray, N. [Inserm, U836, groupe de radiobiologie, institut des neurosciences, chemin Fortune-Ferrini, 38042 Grenoble (France); Granzotto, A.; Vogin, G.; Balosso, J. [Centre de hadrontherapie Etoile, 69008 Lyon (France); Joubert, A. [Societe Magelis, 84160 Cadenet (France); Maalouf, M. [Centre national d' etudes spatiales, 75001 Paris (France); Vogin, G.; Colin, C. [EA 3738, faculte de medecine, Lyon-Sud, 69921 Oullins (France); Malek, K.; Balosso, J. [Service de radiotherapie, CHU A.-Michallon, 38042 Grenoble (France); Colin, C. [Service de radiologie, CHU Lyon-Sud, 69490 Pierre-Benite (France)

    2011-02-15

    Individual response to ionizing radiation is an important information required to apply an efficient radiotherapy treatment against tumour and to avoid any adverse effects in normal tissues. In 1981, Fertil and Malaise have demonstrated that the post-irradiation local tumor control determined in vivo is correlated with clonogenic cell survival assessed in vitro. Furthermore, these authors have reminded the relevance of the concept of intrinsic radiosensitivity that is specific to each individual organ (Fertil and Malaise, 1981) [1]. To date, since clonogenicity assays are too time-consuming and do not provide any other molecular information, a plethora of research groups have attempted to determine the molecular bases of intrinsic radiosensitivity in order to propose reliable and faster predictive assays. To this aim, several approaches have been developed. Notably, the recent revolution in genomic and proteomics technologies is providing a considerable number of data but their link with radiosensitivity still remains to be elucidated. On another hand, the systematic screening of some candidate genes potentially involved in the radiation response is highlighting the complexity of the molecular and cellular mechanisms of DNA damage sensing and signalling and shows that an abnormal radiation response is not necessarily due to the impairment of one single protein. Finally, more modest approaches consisting in focusing some specific functions of DNA repair seem to provide more reliable clues to predict over-acute reactions caused by radiotherapy. In this review, we endeavored to analyse the contributions of these major approaches to predict human radiosensitivity. (authors)

  2. Role of direct immunofluorescence in the diagnosis of glomerulonephritis

    Directory of Open Access Journals (Sweden)

    Archana C Buch

    2015-01-01

    Full Text Available Background: Immunofluorescence microscopy is a vital tool for the diagnosis of glomerular diseases. This study was carried out to study patterns of glomerulonephritis (GN and to record the sensitivity of direct immunofluorescence (DIF in renal lesions. The DIF findings were correlated with clinical and histopathology findings and discrepancies were analyzed. Materials and Methods: The cross-sectional analytical study was conducted during the period July 2011 to July 2013 at a tertiary care Hospital, Department of Pathology. A total of 75 renal biopsies were received for routine and immunofluorescence studies in which histopathology and clinical data were reviewed and analyzed. Results: The sensitivity of DIF was 87.9% and specificity was 70.5%. The maximum number of cases were seen in the age group 41-50 years. The pattern of GN by DIF was minimal change disease (MCD in 24%, IgA nephropathy in 13%, focal segmental glomerulosclerosis in 9% and membranoproliferative glomerulonephritis in 8% of the cases. Twelve histopathologically proven cases of GN were negative on DIF. One case of MCD on histopathology was diagnosed as IgM nephropathy based on DIF. Conclusion: Direct immunofluorescence forms an important diagnostic tool in reaching the exact diagnosis in various types of GN presenting with overlapping clinical and histomorphological features.

  3. Rapid antigen detection test for respiratory syncytial virus diagnosis as a diagnostic tool.

    Science.gov (United States)

    Mesquita, Flávio da Silva; Oliveira, Danielle Bruna Leal de; Crema, Daniela; Pinez, Célia Miranda Nunes; Colmanetti, Thaís Cristina; Thomazelli, Luciano Matsumia; Gilio, Alfredo Elias; Vieira, Sandra Elisabeth; Martinez, Marina Baquerizo; Botosso, Viviane Fongaro; Durigon, Edison Luiz

    The aim of this study was to evaluate the QuickVue ® RSV Test Kit (QUIDEL Corp, CA, USA) as a screening tool for respiratory syncytial virus in children with acute respiratory disease in comparison with the indirect immunofluorescence assay as gold standard. In Brazil, rapid antigen detection tests for respiratory syncytial virus are not routinely utilized as a diagnostic tool, except for the diagnosis of dengue and influenza. The authors retrospectively analyzed 486 nasopharyngeal aspirate samples from children under age 5 with acute respiratory infection, between December 2013 and August 2014, the samples were analyzed by indirect immunofluorescence assay and QuickVue ® RSV Test kit. Samples with discordant results were analyzed by real time PCR and nucleotide sequencing. From 313 positive samples by immunofluorescence assays, 282 (90%) were also positive by the rapid antigen detection test, two were positive only by rapid antigen detection test, 33 were positive only by immunofluorescence assays, and 171 were positive by both methods. The 35 samples with discordant results were analyzed by real time PCR; the two samples positive only by rapid antigen detection test and the five positive only by immunofluorescence assays were also positive by real time PCR. There was no relation between the negativity by QuickVue ® RSV Test and viral load or specific strain. The QuickVue ® RSV Test showed sensitivity of 90%, specificity of 98.8%, predictive positive value of 99.3%, and negative predictive value of 94.6%, with accuracy of 93.2% and agreement κ index of 0.85 in comparison to immunofluorescence assay. This study demonstrated that the QuickVue ® RSV Test Kit can be effective in early detection of Respiratory syncytial virus in nasopharyngeal aspirate and is reliable for use as a diagnostic tool in pediatrics. Copyright © 2016 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

  4. Rapid antigen detection test for respiratory syncytial virus diagnosis as a diagnostic tool,

    Directory of Open Access Journals (Sweden)

    Flávio da Silva Mesquita

    Full Text Available Abstract Objective: The aim of this study was to evaluate the QuickVue® RSV Test Kit (QUIDEL Corp, CA, USA as a screening tool for respiratory syncytial virus in children with acute respiratory disease in comparison with the indirect immunofluorescence assay as gold standard. In Brazil, rapid antigen detection tests for respiratory syncytial virus are not routinely utilized as a diagnostic tool, except for the diagnosis of dengue and influenza. Methods: The authors retrospectively analyzed 486 nasopharyngeal aspirate samples from children under age 5 with acute respiratory infection, between December 2013 and August 2014, the samples were analyzed by indirect immunofluorescence assay and QuickVue® RSV Test kit. Samples with discordant results were analyzed by real time PCR and nucleotide sequencing. Results: From 313 positive samples by immunofluorescence assays, 282 (90% were also positive by the rapid antigen detection test, two were positive only by rapid antigen detection test, 33 were positive only by immunofluorescence assays, and 171 were positive by both methods. The 35 samples with discordant results were analyzed by real time PCR; the two samples positive only by rapid antigen detection test and the five positive only by immunofluorescence assays were also positive by real time PCR. There was no relation between the negativity by QuickVue® RSV Test and viral load or specific strain. The QuickVue® RSV Test showed sensitivity of 90%, specificity of 98.8%, predictive positive value of 99.3%, and negative predictive value of 94.6%, with accuracy of 93.2% and agreement κ index of 0.85 in comparison to immunofluorescence assay. Conclusions: This study demonstrated that the QuickVue® RSV Test Kit can be effective in early detection of Respiratory syncytial virus in nasopharyngeal aspirate and is reliable for use as a diagnostic tool in pediatrics.

  5. [IFN-gamma enzyme-linked immunospot assay versus PPD tuberculin skin test in the diagnosis of tuberculous epididymitis].

    Science.gov (United States)

    Huang, Hao; Yang, Xi-Fei; Deng, Qun-Yi; Li, Bing; Liu, Guo-Hui; Zhang, Jie-Yun; Yang, Da-Fei

    2012-06-01

    To explore the potential application of IFN-gamma enzyme-linked immunospot (ELISPOT) assay in the diagnosis of tuberculous epididymitis (TE) by comparing ELISPOT assay with the traditional purified protein derivative (PPD) tuberculin skin test. We examined 13 TE patients using an in-house ELISPOT kit, another 11 TE patients by PPD skin testing, and 57 healthy male volunteers by parallel test with both the methods. Twelve (92.3%) of the 13 TE cases were positive on ELISPOT assay, and 10 (90.9%) of the 11 TE cases positive on PPD skin test, with no statistically significant differences between the two groups (P > 0.05). Among the 57 healthy male volunteers, 8 (14.0%) were positive on ELISPOT, and 28 (49.1%) positive on PPD test, the latter significantly higher than the former (P PPD test in the examination of tuberculous epididymitis. As for specificity, ELISPOT assay seems better than PPD test in differentiating tuberculous epididymitis patients from healthy males.

  6. Diagnostic performance of a commercial immunoblot assay for myositis antibody testing.

    Science.gov (United States)

    Bundell, Chris; Rojana-Udomsart, Arada; Mastaglia, Frank; Hollingsworth, Peter; McLean-Tooke, Andrew

    2016-06-01

    The objective of this study was to establish a population based reference range for a commercial immunoblot assay detecting myositis specific autoantibodies (MSAs) and myositis associated autoantibodies (MAAs), and to assess the diagnostic performance of this reference range against the manufacturer's recommended ranges in a myositis patient cohort. A total of 124 patients from a myositis cohort and 197 healthy controls were serologically assessed using a commercial immunoblot containing eleven autoantigens (Jo-1, EJ, OJ, PL7, PL12, Mi-2, SRP, Ku, PMScl75, PMScl100 and Ro52) according to the manufacturer's instructions. Use of the manufacturer's reference ranges resulted in detection of MSAs in 19.4% of myositis patients and 9.1% of controls; MAAs were detected in 41.1% of myositis patients and 14.2% of controls. Reference values derived from the healthy control population resulted in significant differences in cut-off values for some autoantibodies, particularly Ro52 and PMScl75. Use of local reference ranges reduced detection of MSAs to 16.9% of myositis patients and 3% of healthy controls, with MAAs 23.4% of patients and 2% of healthy controls. Application of population based reference ranges resulted in significant differences in detection of MSAs and MAAs compared to the manufacturer's recommended ranges. Cut-off levels should be assessed to ensure suitability for the population tested. Copyright © 2016. Published by Elsevier B.V.

  7. Analysis of five streptokinase formulations using the euglobulin lysis test and the plasminogen activation assay

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    Couto L.T.

    2004-01-01

    Full Text Available Streptokinase, a 47-kDa protein isolated and secreted by most group A, C and G ß-hemolytic streptococci, interacts with and activates human protein plasminogen to form an active complex capable of converting other plasminogen molecules to plasmin. Our objective was to compare five streptokinase formulations commercially available in Brazil in terms of their activity in the in vitro tests of euglobulin clot formation and of the hydrolysis of the plasmin-specific substrate S-2251(TM. Euglobulin lysis time was determined using a 96-well microtiter plate. Initially, human thrombin (10 IU/ml and streptokinase were placed in individual wells, clot formation was initiated by the addition of plasma euglobulin, and turbidity was measured at 340 nm every 30 s. In the second assay, plasminogen activation was measured using the plasmin-specific substrate S-2251(TM. Streptase(TM was used as the reference formulation because it presented the strongest fibrinolytic activity in the euglobulin lysis test. The Unitinase(TM and Solustrep(TM formulations were the weakest, showing about 50% activity compared to the reference formulation. All streptokinases tested activated plasminogen but significant differences were observed. In terms of total S-2251(TM activity per vial, Streptase(TM (75.7 ± 5.0 units and Streptonase(TM (94.7 ± 4.6 units had the highest activity, while Unitinase(TM (31.0 ± 2.4 units and Strek(TM (32.9 ± 3.3 units had the weakest activity. Solustrep(TM (53.3 ± 2.7 units presented intermediate activity. The variations among the different formulations for both euglobulin lysis test and chromogenic substrate hydrolysis correlated with the SDS-PAGE densitometric results for the amount of 47-kDa protein. These data show that the commercially available clinical streptokinase formulations vary significantly in their in vitro activity. Whether these differences have clinical implications needs to be investigated.

  8. DETECTION OF ANTIBODIES TO CANDIDA ALBICANS GERM TUBE BY IMMUNOFLUORESCENCE IN IMMUNOSUPPRESSED MICE WITH EXPERIMENTAL SYSTEMIC CANDIDIASIS

    Directory of Open Access Journals (Sweden)

    F. Zaini

    2007-07-01

    Full Text Available "nThe increasing incidence of systemic candidiasis, which parallels the use of invasive and immunosuppressive medical procedures, necessitates development of rapid and cost effective tests for diagnosis of systemic candidiasis. Therefore in this study 85 mice were first immunosuppressed by cyclophosphamide and then infected by Candida albicans NCPF 3153. Other 85 mice were employed as control. The case and control mice were bled and then autopsied. Hearts and kidneys were checked by direct, histopathological and cultural examination for systemic candidiasis. The 85 sera from histological proven cases and 85 control mice were adsorbed with heat killed blastospores of same strain of C. albicans. Anti-Candida albicans germ tube antibodies were detected by indirect immunofluorescence assay for diagnosis of invasive candidiasis in case and control mice. In addition, sera from 35 mice with proven cryptococcosis were also tested. While 84 mice with proven systemic candidiasis (100% had anti-germ tube antibodies, these antibodies were absent in all controls and mice with cryptococcosis. The specificity was 100%, indicating a high degree of discrimination was possible between systemic candidiasis and cryptococcosis in the mice studied. It must be concluded that anti-germ tube responses did not appear to be significantly reduced in immunocompromised mice.

  9. Utilization of DNA comet assay and half embryo test to identify irradiated lentil

    International Nuclear Information System (INIS)

    Romanelli, Maria Fernanda; Villavicencio, Anna Lucia C.H.

    2001-01-01

    Full text: Legumes make an important contribution to human nutrition on a worldwide basis. Insect infestation cause extensive damage to stored grains. Over the last few decades some countries adopted food irradiation as a safe food process. Radiation's processing on foods improves hygienic quality and extends their shelf life. The use of radiation treatment to reduce the microbial population and thereby extend the shelf life in legumes has been reported in many papers. Irradiation has been shown to be an effective pest control method for these commodities and a good alternative to prohibited methyl bromide. Radiation disinfestation can facilitate trade in foods that often harbor insect pests of quarantine importance. Although the wholesomeness of irradiated food is no longer a question there is a need for irradiation control in the international trade of foods, in order to enhance the consumer confidence in the regulation. As a screening methods to identify irradiated lentils, processed by e-beam as a food treatment to disinfestation, the DNA Comet Assay and Half Embryo tests were performed. The methodologies used in this work are based upon biological changes that occur in Brazilian lentils. The samples were irradiated in an electron beam accelerator facility of Radiation Dynamics Inc., USA (E=1,5 MeV, l=25 mA). The irradiation doses were 0,7; 1,4 and 3,0 kGy at dry conditions. The thickness of samples was less than 0,5 cm. A sensitive technique to detect DNA fragmentation is the microgel electrophoresis of single cells or nuclei, also called 'comet assay'. Since the large molecule of DNA is an easy target for ionizing radiation, changes in DNA offer potential as a detection method. It is restricted to foods that have not been subjected to heat or other treatments, which also cause DNA fragmentation. Lentil samples were crushed with a mortar and pestle and was transferred to 3ml ice-cold PBS. This suspension was stirred for 5 minutes and filtered. 100μl cell

  10. Comparison of 2 molecular assays and a serologic test in diagnosing Mycoplasma pneumoniae infection in paediatrics patients.

    Science.gov (United States)

    Blanco, Silvia; Fuenzalida, Loreto; Bas, Albert; Prat, Cristina; Ramírez, Aida; Matas, Lurdes; Rodrigo, Carlos; Ausina, Vicente

    2011-12-01

    Two commercial polymerase chain reaction (PCR) assays (a real-time PCR [Cepheid] and an oligochromatographic test [Speed-oligo]) and 1 serology test (Serodia-Myco II) for detecting Mycoplasma pneumoniae in nasopharyngeal aspirates and serum samples were studied. Among the 145 samples, 32 serum pairs were serologically positive for M. pneumoniae. Of these, in 30 nasopharyngeal aspirates, M. pneumoniae was detected using the real-time PCR assay and 25 using Speed-oligo, corresponding to a sensitivity of 93.7% and 78.1%, respectively. Among the 94 samples with negative serology, we only obtained 1 positive result by real-time PCR assay. In the group of samples from healthy children, no positive results were obtained. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. A comparative study of Mono Mac 6 cells, isolated mononuclear cells and Limulus amoebocyte lysate assay in pyrogen testing

    DEFF Research Database (Denmark)

    Moesby, Lise; Jensen, S; Hansen, E W

    1999-01-01

    ) and Staphylococcus aureus was comparable to that of MNC. Aspergillus niger and Candida albicans induced IL-6 in isolated MNC, but not in MM6. The detection limit for Salmonella typhimurium in the MM6 assay was comparable to that of the LAL assay. As expected, S. aureus and C. albicans did not show any LAL activity......Pyrogen induced secretion of interleukin 6 (IL-6) in Mono Mac 6 (MM6) cells was measured. The ability of the MM6 cell culture to detect pyrogens was compared to the Limulus amoebocyte lysate (LAL) test and isolated mononuclear cells (MNC). The detection limit of MM6 for lipopolysaccharide (LPS....... A. niger and Influenza virus showed some activity in the LAL test, but could not be detected by MM6 cells. In conclusion, the MM6 assay is a good supplement to the current pyrogen assays for detection of LPS, S. aureus and S. typhimurium, but the MM6 assay could not detect A. niger, C. albicans...

  12. Assessing genotoxicity of diuron on Drosophila melanogaster by the wing-spot test and the wing imaginal disk comet assay.

    Science.gov (United States)

    Peraza-Vega, Ricardo I; Castañeda-Sortibrán, América N; Valverde, Mahara; Rojas, Emilio; Rodríguez-Arnaiz, Rosario

    2017-05-01

    The aim of this study was to evaluate the genotoxicity of the herbicide diuron in the wing-spot test and a novel wing imaginal disk comet assay in Drosophila melanogaster. The wing-spot test was performed with standard (ST) and high-bioactivation (HB) crosses after providing chronic 48 h treatment to third instar larvae. A positive dose-response effect was observed in both crosses, but statistically reduced spot frequencies were registered for the HB cross compared with the ST. This latter finding suggests that metabolism differences play an important role in the genotoxic effect of diuron. To verify diuron's ability to produce DNA damage, a wing imaginal disk comet assay was performed after providing 24 h diuron treatment to ST and HB third instar larvae. DNA damage induced by the herbicide had a significantly positive dose-response effect even at very low concentrations in both strains. However, as noted for the wing-spot test, a significant difference between strains was not observed that could be related to the duration of exposure between both assays. A positive correlation between the comet assay and the wing-spot test was found with regard to diuron genotoxicity.

  13. Evaluation of simple rapid HIV assays and development of national rapid HIV test algorithms in Dar es Salaam, Tanzania

    Directory of Open Access Journals (Sweden)

    Mbwana Judica

    2009-02-01

    Full Text Available Abstract Background Suitable algorithms based on a combination of two or more simple rapid HIV assays have been shown to have a diagnostic accuracy comparable to double enzyme-linked immunosorbent assay (ELISA or double ELISA with Western Blot strategies. The aims of this study were to evaluate the performance of five simple rapid HIV assays using whole blood samples from HIV-infected patients, pregnant women, voluntary counseling and testing attendees and blood donors, and to formulate an alternative confirmatory strategy based on rapid HIV testing algorithms suitable for use in Tanzania. Methods Five rapid HIV assays: Determine™ HIV-1/2 (Inverness Medical, SD Bioline HIV 1/2 3.0 (Standard Diagnostics Inc., First Response HIV Card 1–2.0 (PMC Medical India Pvt Ltd, HIV1/2 Stat-Pak Dipstick (Chembio Diagnostic System, Inc and Uni-Gold™ HIV-1/2 (Trinity Biotech were evaluated between June and September 2006 using 1433 whole blood samples from hospital patients, pregnant women, voluntary counseling and testing attendees and blood donors. All samples that were reactive on all or any of the five rapid assays and 10% of non-reactive samples were tested on a confirmatory Inno-Lia HIV I/II immunoblot assay (Immunogenetics. Results Three hundred and ninety samples were confirmed HIV-1 antibody positive, while 1043 were HIV negative. The sensitivity at initial testing of Determine, SD Bioline and Uni-Gold™ was 100% (95% CI; 99.1–100 while First Response and Stat-Pak had sensitivity of 99.5% (95% CI; 98.2–99.9 and 97.7% (95% CI; 95.7–98.9, respectively, which increased to 100% (95% CI; 99.1–100 on repeat testing. The initial specificity of the Uni-Gold™ assay was 100% (95% CI; 99.6–100 while specificities were 99.6% (95% CI; 99–99.9, 99.4% (95% CI; 98.8–99.7, 99.6% (95% CI; 99–99.9 and 99.8% (95% CI; 99.3–99.9 for Determine, SD Bioline, First Response and Stat-Pak assays, respectively. There was no any sample which was

  14. Testing human sperm chemotaxis: how to detect biased motion in population assays.

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    Leah Armon

    Full Text Available Biased motion of motile cells in a concentration gradient of a chemoattractant is frequently studied on the population level. This approach has been particularly employed in human sperm chemotactic assays, where the fraction of responsive cells is low and detection of biased motion depends on subtle differences. In these assays, statistical measures such as population odds ratios of swimming directions can be employed to infer chemotactic performance. Here, we report on an improved method to assess statistical significance of experimentally determined odds ratios and discuss the strong impact of data correlations that arise from the directional persistence of sperm swimming.

  15. Immunofluorescence in multiple tissues utilizing serum from a patient affected by systemic lupus erythematosus

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    Piotr Brzezinski

    2012-01-01

    Full Text Available Introduction: Lupus erythematosus is a chronic, inflammatory autoimmune disease that can affect multiple organs. Lupus can affect many parts of the body, especially in systemic lupus erythematosus (SLE; affected tissues may include the joints, skin, kidneys, heart, lungs, blood vessels, and brain. Case report: A 46-year-old female presented with pruritus, photosensitivity and edema of the cheeks of about 2 years duration, and was evaluated by a dermatologist. On examination, multiple telangiectasias were present on the cheeks, with erythema, edema and a malar rash observed. A review of systems documented breathing difficulty and pleuitic pain, joint pain and joint edema, photosensitivity, cardiac dysrhythmia, and periodic pain in the back close to the kidneys. Methods: Skin biopsies for hematoxylin and eosin testing, as well for direct and indirect immunofluorescence were performed, in addition to multiple diagnostic blood tests, chest radiography and directed immunologic testing. Results: The blood testing showed elevated C-reactive protein. Direct and indirect immunofluorescence testing utilizing monkey esophagus, mouse and pig heart and kidney, normal human eyelid skin and veal brain demonstrated strong reactivity to several components of smooth muscle, nerves, blood vessels, skin basement membrane zone and sweat gland ducts and skin meibomian glands. Anti-endomysium antibodies were detected as well as others, especially using FITC conjugated Complement/C1q, FITC conjugated anti-human immunoglobulin IgG and FITC conjugated anti-human fibrinogen. Conclusions: We conclude that both direct and indirect immunofluorescence using several substrates can unveil previously undocumented autoantibodies in multiple organs in lupus erythematosus, and that these findings could be utilized to complement existing diagnostic testing for this disorder.

  16. Alkaline Comet Assay and Micronucleus Test Parameters in Children Exposed to Diagnostic X-Ray Examination

    International Nuclear Information System (INIS)

    Gajski, G.; Geric, M.; Garaj-Vrhovac, V.; Milkovic, Dj.; Beck, N.; Ranogajec-Komor, M.; Miljanic, S.; Knezevic, Z.

    2011-01-01

    Chest radiograms represent the basic radiological examination of thorax and are the most frequently performed radiological diagnostic procedure in the child population. Understanding the risks of low doses of radiation is an important aspect in the risk benefit analysis in paediatric populations. To provide the best care for the young patients the effects of radiation should be minimized thus chest X-rays must be performed by highest standards to ensure that the young patient has the lowest risk possible. Since children are the most sensitive to radiation, there is a need for follow up of the young populations that receive these X-ray diagnostic examinations. Follow up would be especially advisable for children that are at higher risk of radiation induced damage, for example children with a predisposition to DNA damage, or for children that are constantly exposed to numerous radiological examinations due to their illness. In that manner, present study was undertaken to evaluate application of different dosimetry systems in conjunction with alkaline comet assay and micronucleus test for the assessment of different types of DNA and chromosomal alterations in child population exposed to acute diagnostic X-rays examination. For that purpose doses were measured using thermoluminescence (TL) and radiophotoluminescent (RPL) dosimetry systems. The study demonstrated that immediately after exposure to diagnostic X-irradiation, mean percentage of DNA in tail of the comets, which is indirect measures of DNA damage, was significantly changed. The same was noticed for mean total number of micronuclei as well. It was shown that children with pulmonary diseases subjected to diagnostic procedure develop a significant increase in mean total number of each measured parameter which are the biomarkers of genetic damage for carcinogenesis, than prior to diagnostic procedure and that interindividual differences exist for each monitored child. Our results show that genetic damage arises

  17. A preclinical physiological assay to test modulation of knee joint pain in the spinal cord: effects of oxycodone and naproxen.

    Science.gov (United States)

    Miranda, Jason A; Stanley, Phil; Gore, Katrina; Turner, Jamie; Dias, Rebecca; Rees, Huw

    2014-01-01

    Sensory processing in the spinal cord during disease states can reveal mechanisms for novel treatments, yet very little is known about pain processing at this level in the most commonly used animal models of articular pain. Here we report a test of the prediction that two clinically effective compounds, naproxen (an NSAID) and oxycodone (an opiate), are efficacious in reducing the response of spinal dorsal horn neurons to noxious knee joint rotation in the monosodium iodoacetate (MIA) sensitized rat. The overall objective for these experiments was to develop a high quality in vivo electrophysiology assay to confidently test novel compounds for efficacy against pain. Given the recent calls for improved preclinical experimental quality we also developed and implemented an Assay Capability Tool to determine the quality of our assay and ensure the quality of our results. Spinal dorsal horn neurons receiving input from the hind limb knee joint were recorded in anesthetized rats 14 days after they were sensitized with 1 mg of MIA. Intravenous administered oxycodone and naproxen were each tested separately for their effects on phasic, tonic, ongoing and afterdischarge action potential counts in response to innocuous and noxious knee joint rotation. Oxycodone reduced tonic spike counts more than the other measures, doing so by up to 85%. Tonic counts were therefore designated the primary endpoint when testing naproxen which reduced counts by up to 81%. Both reductions occurred at doses consistent with clinically effective doses for osteoarthritis. These results demonstrate that clinically effective doses of standard treatments for osteoarthritis reduce pain processing measured at the level of the spinal cord for two different mechanisms. The Assay Capability Tool helped to guide experimental design leading to a high quality and robust preclinical assay to use in discovering novel treatments for pain.

  18. Pregnancy does not affect HIV incidence test results obtained using the BED capture enzyme immunoassay or an antibody avidity assay.

    Directory of Open Access Journals (Sweden)

    Oliver Laeyendecker

    2010-10-01

    Full Text Available Accurate incidence estimates are needed for surveillance of the HIV epidemic. HIV surveillance occurs at maternal-child health clinics, but it is not known if pregnancy affects HIV incidence testing.We used the BED capture immunoassay (BED and an antibody avidity assay to test longitudinal samples from 51 HIV-infected Ugandan women infected with subtype A, C, D and intersubtype recombinant HIV who were enrolled in the HIVNET 012 trial (37 baseline samples collected near the time of delivery and 135 follow-up samples collected 3, 4 or 5 years later. Nineteen of 51 women were also pregnant at the time of one or more of the follow-up visits. The BED assay was performed according to the manufacturer's instructions. The avidity assay was performed using a Genetic Systems HIV-1/HIV-2 + O EIA using 0.1M diethylamine as the chaotropic agent.During the HIVNET 012 follow-up study, there was no difference in normalized optical density values (OD-n obtained with the BED assay or in the avidity test results (% when women were pregnant (n = 20 results compared to those obtained when women were not pregnant (n = 115; for BED: p = 0.9, generalized estimating equations model; for avidity: p = 0.7, Wilcoxon rank sum. In addition, BED and avidity results were almost exactly the same in longitudinal samples from the 18 women who were pregnant at only one study visit during the follow-up study (p = 0.6, paired t-test.These results from 51 Ugandan women suggest that any changes in the antibody response to HIV infection that occur during pregnancy are not sufficient to alter results obtained with the BED and avidity assays. Confirmation with larger studies and with other HIV subtypes is needed.

  19. Detection of Bim and Puma in mouse hair follicles using immunofluorescence and TUNEL assay double staining

    Czech Academy of Sciences Publication Activity Database

    Veselá, Barbora; Matalová, Eva

    2015-01-01

    Roč. 90, č. 8 (2015), s. 587-593 ISSN 1052-0295 Grant - others:GA ČR(CZ) GAP502/12/1285 Program:GA Institutional support: RVO:67985904 Keywords : apoptosis * Bim * development Subject RIV: ED - Physiology Impact factor: 1.078, year: 2015

  20. Environmental Technology Verification Report for Abraxis Ecologenia® 17β-Estradiol (E2) Microplate Enzyme-Linked Immunosorbent Assay (ELISA) Test Kits

    Science.gov (United States)

    This verification test was conducted according to procedures specifiedin the Test/QA Planfor Verification of Enzyme-Linked Immunosorbent Assay (ELISA) Test Kis for the Quantitative Determination of Endocrine Disrupting Compounds (EDCs) in Aqueous Phase Samples. Deviations to the...

  1. Analytical characteristics and comparative evaluation of Aptima HCV quant Dx assay with the Abbott RealTime HCV assay and Roche COBAS AmpliPrep/COBAS TaqMan HCV quantitative test v2.0.

    Science.gov (United States)

    Worlock, A; Blair, D; Hunsicker, M; Le-Nguyen, T; Motta, C; Nguyen, C; Papachristou, E; Pham, J; Williams, A; Vi, M; Vinluan, B; Hatzakis, A

    2017-04-04

    The Aptima HCV Quant Dx assay (Aptima assay) is a fully automated quantitative assay on the Panther® system. This assay is intended for confirmation of diagnosis and monitoring of HCV RNA in plasma and serum specimens. The purpose of the testing described in this paper was to evaluate the performance of the Aptima assay. The analytical sensitivity, analytical specificity, precision, and linearity of the Aptima assay were assessed. The performance of the Aptima assay was compared to two commercially available HCV assays; the Abbott RealTime HCV assay (Abbott assay, Abbott Labs Illinois, USA) and the Roche COBAS Ampliprep/COBAS Taqman HCV Quantitative Test v2.0 (Roche Assay, Roche Molecular Systems, Pleasanton CA, USA). The 95% Lower Limit of Detection (LoD) of the assay was determined from dilutions of the 2nd HCV WHO International Standard (NIBSC 96/798 genotype 1) and HCV positive clinical specimens in HCV negative human plasma and serum. Probit analysis was performed to generate the 95% predicted detection limits. The Lower Limit of Quantitation (LLoQ) was established for each genotype by diluting clinical specimens and the 2nd HCV WHO International Standard (NIBSC 96/798 genotype 1) in HCV negative human plasma and serum. Specificity was determined using 200 fresh and 536 frozen HCV RNA negative clinical specimens including 370 plasma specimens and 366 serum specimens. Linearity for genotypes 1 to 6 was established by diluting armored RNA or HCV positive clinical specimens in HCV negative serum or plasma from 8.08 log IU/mL to below 1 log IU/mL. Precision was tested using a 10 member panel made by diluting HCV positive clinical specimens or spiking armored RNA into HCV negative plasma and serum. A method comparison was conducted against the Abbott assay using 1058 clinical specimens and against the Roche assay using 608 clinical specimens from HCV infected patients. In addition, agreement between the Roche assay and the Aptima assay using specimens with low

  2. Cytotoxicity Test Based on Human Cells Labeled with Fluorescent Proteins: Fluorimetry, Photography, and Scanning for High-Throughput Assay.

    Science.gov (United States)

    Kalinina, Marina A; Skvortsov, Dmitry A; Rubtsova, Maria P; Komarova, Ekaterina S; Dontsova, Olga A

    2018-06-01

    High- and medium-throughput assays are now routine methods for drug screening and toxicology investigations on mammalian cells. However, a simple and cost-effective analysis of cytotoxicity that can be carried out with commonly used laboratory equipment is still required. The developed cytotoxicity assays are based on human cell lines stably expressing eGFP, tdTomato, mCherry, or Katushka2S fluorescent proteins. Red fluorescent proteins exhibit a higher signal-to-noise ratio, due to less interference by medium autofluorescence, in comparison to green fluorescent protein. Measurements have been performed on a fluorescence scanner, a plate fluorimeter, and a camera photodocumentation system. For a 96-well plate assay, the sensitivity per well and the measurement duration were 250 cells and 15 min for the scanner, 500 cells and 2 min for the plate fluorimeter, and 1000 cells and less than 1 min for the camera detection. These sensitivities are similar to commonly used MTT (tetrazolium dye) assays. The used scanner and the camera had not been previously applied for cytotoxicity evaluation. An image processing scheme for the high-resolution scanner is proposed that significantly diminishes the number of control wells, even for a library containing fluorescent substances. The suggested cytotoxicity assay has been verified by measurements of the cytotoxicity of several well-known cytotoxic drugs and further applied to test a set of novel bacteriotoxic compounds in a medium-throughput format. The fluorescent signal of living cells is detected without disturbing them and adding any reagents, thus allowing to investigate time-dependent cytotoxicity effects on the same sample of cells. A fast, simple and cost-effective assay is suggested for cytotoxicity evaluation based on mammalian cells expressing fluorescent proteins and commonly used laboratory equipment.

  3. Quantitative Immunofluorescence Analysis of Nucleolus-Associated Chromatin.

    Science.gov (United States)

    Dillinger, Stefan; Németh, Attila

    2016-01-01

    The nuclear distribution of eu- and heterochromatin is nonrandom, heterogeneous, and dynamic, which is mirrored by specific spatiotemporal arrangements of histone posttranslational modifications (PTMs). Here we describe a semiautomated method for the analysis of histone PTM localization patterns within the mammalian nucleus using confocal laser scanning microscope images of fixed, immunofluorescence stained cells as data source. The ImageJ-based process includes the segmentation of the nucleus, furthermore measurements of total fluorescence intensities, the heterogeneity of the staining, and the frequency of the brightest pixels in the region of interest (ROI). In the presented image analysis pipeline, the perinucleolar chromatin is selected as primary ROI, and the nuclear periphery as secondary ROI.

  4. Clinical evaluation of a helicase-dependant amplification (HDA)-based commercial assay for the simultaneous detection of HSV-1 and HSV-2.

    Science.gov (United States)

    Teo, Jeanette W P; Chiang, Donald; Jureen, Roland; Lin, Raymond T P

    2015-11-01

    In this study, we evaluate the performance of a commercial assay, the AmpliVue HSV 1+2 Assay (Quidel), which employs HDA for the detection of both HSV-1 and HSV-2. The assay was tested on 307 clinical specimens (genital, oral, and dermal). When compared to shell vial virus culture and immunofluorescence typing of HSV, the positive percent agreement and negative percent agreement values were 98.2% and 90.9%, respectively. Excellent assay performance was demonstrated. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Testing the utility of fluorescent proteins in Mimulus lewisii by an Agrobacterium-mediated transient assay.

    Science.gov (United States)

    Ding, Baoqing; Yuan, Yao-Wu

    2016-04-01

    The Agrobacterium -mediated transient expression assay by leaf infiltration in Mimulus lewisii is robust. Fluorescent proteins EGFP, EYFP and DsRed give bright fluorescence signals in the infiltrated tissue. Mimulus lewisii is an emerging developmental genetic model system. Recently developed genomic and genetic resources and a stable transformation protocol have greatly facilitated the identification and functional characterization of genes controlling the development of ecologically important floral traits using this species. To further expedite gene and protein function analyses in M. lewisii, we adopted and simplified the Agrobacterium-mediated transient gene expression method routinely used in tobacco plants. With the validated transient assay, we examined the performance of fluorescent proteins EGFP, EYFP and DsRed in M. lewisii. All three proteins gave bright fluorescence signals when transiently expressed in agroinfiltrated leaves. Furthermore, we demonstrated the utility of fluorescent proteins in M. lewisii by showing the nuclear localization of Reduced Carotenoid Pigmentation 1 (RCP1), a recently discovered R2R3-MYB transcription factor that regulates carotenoid pigmentation during flower development. Both the transient assay and the fluorescent proteins are valuable additions to the M. lewisii toolbox, making this emerging genetic and developmental model system even more powerful.

  6. Application of firefly luciferase assay for adenosine triphosphate (ATP) to antimicrobial drug sensitivity testing

    Science.gov (United States)

    Picciolo, G. L.; Tuttle, S. A.; Schrock, C. G.; Deming, J. W.; Barza, M. J.; Wienstein, L.; Chappelle, E. W.

    1977-01-01

    The development of a rapid method for determining microbial susceptibilities to antibiotics using the firefly luciferase assay for adenosine triphosphate (ATP) is documented. The reduction of bacterial ATP by an antimicrobial agent was determined to be a valid measure of drug effect in most cases. The effect of 12 antibiotics on 8 different bacterial species gave a 94 percent correlation with the standard Kirby-Buer-Agar disc diffusion method. A 93 percent correlation was obtained when the ATP assay method was applied directly to 50 urine specimens from patients with urinary tract infections. Urine samples were centrifuged first to that bacterial pellets could be suspended in broth. No primary isolation or subculturing was required. Mixed cultures in which one species was predominant gave accurate results for the most abundant organism. Since the method is based on an increase in bacterial ATP with time, the presence of leukocytes did not interfere with the interpretation of results. Both the incubation procedure and the ATP assays are compatible with automation.

  7. Comparative evaluation of the INNO-LIA syphilis score and the MarDx Treponema pallidum immunoglobulin G Marblot test assays for the serological diagnosis of syphilis.

    Science.gov (United States)

    Lam, T K; Lau, H Y; Lee, Y P; Fung, S M; Leung, W L; Kam, K M

    2010-02-01

    We evaluated the performance of two immunoblot assays: the INNO-LIA Syphilis Score (LIA) and the MarDx T. pallidum IgG Marblot Test (TWB), as compared with that of the Murex ICE Syphilis enzyme immunoassay (EIA), the Serodia Treponema pallidum particle agglutination (TPPA) assay and the fluorescent treponemal antibody-absorption (FTA-abs) assay, for the serological diagnosis of syphilis using serum samples of 135 attendees of the social hygiene clinics of the Department of Health in Hong Kong newly diagnosed with syphilis and provided with clinical stages (39 in primary, 20 in secondary, 18 in early latent and 58 in latent of unknown duration) and of 43 normal healthy subjects between October and December 2004. The differences in the overall sensitivities of the LIA assay and the EIA/TPPA/FTA-abs assays were not statistically significant (P > 0.05) whereas the overall sensitivity of the TWB assay was significantly lower (P FTA-abs assays. The LIA assay had an overall sensitivity of 94.1% (95% CI 88.7-97.0%) whereas the TWB assay 65.2% (95% CI 56.8-72.7%). Both the LIA and the TWB assays have a specificity of 100%. When consensus results were derived from the most predominant results of the EIA, the TPPA and the FTA-abs assays, the LIA assay had a positive agreement with the consensus results of 98.5% (95% CI 94.5-99.6%) whereas the TWB assay 68.2% (95% CI 59.8-75.6%). Therefore, the LIA assay performed significantly better (P < 0.05) than the TWB assay. The LIA assay can be considered to be a valid alternative confirmatory test for the serological diagnosis of syphilis.

  8. Diagnostic significance of colloid body deposition in direct immunofluorescence

    Directory of Open Access Journals (Sweden)

    Chularojanamontri Leena

    2010-01-01

    Full Text Available Background: Colloid bodies (CB in direct immunofluorescence (DIF studies are usually found in interface dermatitis. Furthermore, CB can be found in various skin diseases and even in normal skin. Aim: To evaluate the diagnostic value of CB deposits in DIF studies. Methods: From 1996-2007, data from 502 patients where DIF studies showed immunoreactants at CB were enrolled. The definite diagnoses of these patients were based on clinical, histopathological and immunofluorescent findings. The results of DIF studies were analyzed. Results: Immunoreactants at CB were detected in 44.4%, 43.8%, 4.2%, 3.8%, and 2.2% of interface dermatitis, vasculitis, autoimmune vesiculobullous disease, panniculitis, and scleroderma/morphea, respectively. The most common immunoreactant deposit of all diseases was Immunoglobulin M (IgM. Brighter intensity and higher quantity of CB was detected frequently in the group with interface dermatitis. Conclusions: Immunoreactant deposits at CB alone can be found in various diseases but a strong intensity and high quantity favor the diagnosis of interface dermatitis. CB plus dermoepidermal junction (DEJ deposits are more common in interface dermatitis than any other disease. Between lichen planus (LP and discoid lupus erythematosus (DLE, CB alone is more common in LP; whereas, CB plus DEJ and superficial blood vessel (SBV is more common in DLE. The most common pattern in both diseases is CB plus DEJ. The quantity and intensity of CB in LP is higher than in DLE.

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

    Science.gov (United States)

    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

  10. Evaluation of a new serological test for syphilis based on chemiluminescence assay in a tertiary care hospital

    Directory of Open Access Journals (Sweden)

    Aseem K Tiwari

    2015-01-01

    Full Text Available Context: Syphilis is a transfusion transmissible infections and it is mandatory to do serological test for syphilis (STS on all donor blood samples. STS is usually based on detection of antibodies against the cardiolipin-lecithin antigen or against the Treponema-specific antigen. STS with good sensitivity and specificity helps enhance blood safety and consolidation of STS along with other transfusion transmittable infections such as human immunodeficiency virus, hepatitis-C virus, and hepatitis-B virus helps in reducing the errors and enhances efficiency. Aims: This study was designed to evaluate the performance of newly introduced VITROS ® syphilis Treponema pallidum agglutination (TPA assay based on enhanced chemiluminescence principle for its analytical performance for use as a STS on donor blood samples at a tertiary care health center in National Capital Region, India. Materials and Methods: A total of 108 random blood units collected from the donors (both voluntary and replacement donors and 28 known syphilis sero-reactive samples stored at −20°C, were used to evaluate the performance of VITROS ® syphilis TPA assay based on enhanced chemiluminescence assay on VITROS ® ECiQ immunodiagnostics system along with its analytical performance in terms of its sensitivity, precision, cross-reactivity and interference studies. Results: VITROS ® syphilis TPA showed 100% sensitivity and specificity with precision (20 days study of <10% co-efficient of variation. There was no cross-reactivity with other viral and auto-immune antibodies. No interference was observed from endogenous interfering substances like free hemoglobin or fats. Conclusions: Performance of the VITROS ® syphilis TPA assay meets the requirements for its use as STS in blood bank, thus allowing consolidation with other transfusion transmittable infections screening assay on chemiluminescence platform, which is highly valuable for optimizing workflow and efficiency.

  11. Evaluation of a new serological test for syphilis based on chemiluminescence assay in a tertiary care hospital.

    Science.gov (United States)

    Tiwari, Aseem K; Pandey, Prashant K; Dara, Ravi C; Rawat, Ganesh S; Raina, Vimarsh; Bhargava, Richa

    2015-01-01

    Syphilis is a transfusion transmissible infections and it is mandatory to do serological test for syphilis (STS) on all donor blood samples. STS is usually based on detection of antibodies against the cardiolipin-lecithin antigen or against the Treponema-specific antigen. STS with good sensitivity and specificity helps enhance blood safety and consolidation of STS along with other transfusion transmittable infections such as human immunodeficiency virus, hepatitis-C virus, and hepatitis-B virus helps in reducing the errors and enhances efficiency. This study was designed to evaluate the performance of newly introduced VITROS(®) syphilis Treponema pallidum agglutination (TPA) assay based on enhanced chemiluminescence principle for its analytical performance for use as a STS on donor blood samples at a tertiary care health center in National Capital Region, India. A total of 108 random blood units collected from the donors (both voluntary and replacement donors) and 28 known syphilis sero-reactive samples stored at -20°C, were used to evaluate the performance of VITROS(®) syphilis TPA assay based on enhanced chemiluminescence assay on VITROS(®) ECiQ immunodiagnostics system along with its analytical performance in terms of its sensitivity, precision, cross-reactivity and interference studies. VITROS(®) syphilis TPA showed 100% sensitivity and specificity with precision (20 days study) of endogenous interfering substances like free hemoglobin or fats. Performance of the VITROS(®) syphilis TPA assay meets the requirements for its use as STS in blood bank, thus allowing consolidation with other transfusion transmittable infections screening assay on chemiluminescence platform, which is highly valuable for optimizing workflow and efficiency.

  12. Radiometric cytolysis inhibition assay, a new rapid test for neutralizing antibodies to intact and trypsin-cleaved poliovirus

    International Nuclear Information System (INIS)

    Hovi, T.; Roivainen, M.

    1989-01-01

    We have developed a new rapid test, the radiometric cytolysis inhibition assay (RACINA), for the determination of neutralizing poliovirus antibodies. HeLa cells prelabeled with 51 Cr, [ 3 H]leucine, or, preferentially, with [ 3 H]uridine are used as sensitive quantitative indicators of residual infectious virus. Both suspensions and monolayer cultures of the indicator cells can be used. Neutralization of a fraction of a high-titer virus preparation can be scored after the first replication cycle at 8 to 10 h. By lowering the incubation temperature to 30 degree C, the completion of the cytolysis due to the first replication cycle of poliovirus was delayed beyond 21 h. This makes it possible to use the RACINA, unlike the standard microneutralization assay, for measuring antibodies to trypsin-cleaved polioviruses. The RACINA was found to be as sensitive as and more reproducible than the standard microneutralization assay in the measurement of neutralizing poliovirus antibodies. The RACINA is a rapid and reliable test for neutralizing antibodies and in principle it may be applicable for quantitation of neutralizing antibodies to other cytolytic agents as well

  13. Biomonitoring of agricultural workers exposed to pesticide mixtures in Guerrero state, Mexico, with comet assay and micronucleus test.

    Science.gov (United States)

    Carbajal-López, Yolanda; Gómez-Arroyo, Sandra; Villalobos-Pietrini, Rafael; Calderón-Segura, María Elena; Martínez-Arroyo, Amparo

    2016-02-01

    The aim of this study was to evaluate the genotoxic effect of pesticides in exfoliated buccal cells of workers occupationally exposed in Guerrero, Mexico, using the comet assay and the micronucleus test. The study compared 111 agricultural workers in three rural communities (Arcelia 62, Ajuchitlan 13, and Tlapehuala 36), with 60 non-exposed individuals. All the participants were males. The presence of DNA damage was investigated in the exfoliated buccal cells of study participants with the comet assay and the micronucleus (MN) test; comet tail length was evaluated in 100 nuclei and 3000 epithelial cells of each individual, respectively; other nuclear anomalies such as nuclear buds, karyolysis, karyorrhexis, and binucleate cells were also evaluated. Study results revealed that the tail migration of DNA and the frequency of MN increased significantly in the exposed group, which also showed nuclear anomalies associated with cytotoxic or genotoxic effect. No positive correlation was noted between exposure time and tail length and micronuclei frequencies. No significant effect on genetic damage was observed as a result of age, smoking, and alcohol consumption. The MN and comet assay in exfoliated buccal cells are useful and minimally invasive methods for monitoring genetic damage in individuals exposed to pesticides. This study provided valuable data for establishing the possible risk to human health associated with pesticide exposure.

  14. Monitoring and assay procedures for krypton-85 used in circulatory tests

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Jr., J. M.; Williams, K. D.

    1963-11-15

    During the past five years, krypton-85 has been used extensively at the National Institutes of health and by the heart Catheterization laboratory in the identification of circulatory shunts. Several methods of assaying the incoming shipments of radioactive gas are given. These consist of ionization chamber measurements, gamma ray spectrometry, and gross photon counting of the cylinder surface with an end-window Geiger-Mueller tube. A device (consisting of a log-count-rate meter, recorder, and G.M. tube) is used to continuously monitor the laboratory. The method of calibrating this device is presented. A means for removing contaminated air from the vicinity of the operating table was devised. The results of air clearance studies of the room are included. (auth)

  15. Screening health care workers with interferon-γ release assay versus tuberculin skin test: impact on costs and adherence to testing (the SWITCH study).

    Science.gov (United States)

    Wrighton-Smith, Peter; Sneed, Laurie; Humphrey, Frances; Tao, Xuguang; Bernacki, Edward

    2012-07-01

    To determine the price point at which an interferon-γ release assay (IGRA) is less costly than a tuberculin skin test (TST) for health care employee tuberculosis screening. A multidecision tree-based cost model incorporating inputs gathered from time-motion studies and parallel testing by IGRA and TST was conducted in a subset of our employees. Administering a TST testing program costs $73.20 per person screened, $90.80 per new hire, and $63.42 per annual screen. Use of an IGRA for employee health testing is cost saving at an IGRA test cost of $54.83 or less per test and resulted in higher completion rates because of the elimination of the need for a second visit to interpret the TST. Using an IGRA for employee health screening can be an institutional cost saving and results in higher compliance rates.

  16. The identification of carbapenemase types in Enterobacteriaceae by using molecular assay and phenotyping confirmation tests.

    Science.gov (United States)

    Genc, Ozlem; Aksu, Evrim; Gulcan, Aynur

    2016-06-01

    In this study, we aimed to identify the molecular carbapenemase types of the Enterobacteriaceae isolates and to evaluate the performance of manually prepared and commercially available combination disc methods and the modified Hodge test. One hundred and forty carbapenemase producing isolates and 45 isolates as control group were included in our study. The Xpert CARBA-R test was used as the molecular method. Antibiotic susceptibility tests were performed using combined discs, manually prepared with APBA (3-aminophenyl boronic acid), DPA (dipicolinic acid), EDTA (Ethylene diamine tetra acetic acid), cloxacillin supplements and Mastdiscs Combi-D70C that includes four antibiotic discs with specific inhibitors and temocillin discs. The modified Hodge test was performed on all isolates. OXA-48 gene was identified in 129 isolates , the NDM gene was identified in 10 isolates and VIM in one isolate. Thirty inaccurate results (30/185, 16%) were detected by using the manually prepared confirmation test. The sensitivity and specificity of this test were identified respectively 85% and 73%. Also, the sensitivity and specificity of the Mastdiscs Combi-D70C were identified as 100%. Negative results were detected in 3 NDM isolates with the use of a modified Hodge test. Sensitivity and specificity were calculated for the modified Hodge test respectively 97% and 100%. Finally, molecular methods provide results rapidly but they are not always easily accessible. The modified Hodge test can be used only for screening as a first step test and is not one of the tests that can identify the type of the carbapenemase. When carbapenem-resistant Enterobacteriaceae are detected, a commercial kit like Mastdiscs Combi-D70 may be preferred instead of the manually prepared phenotypic verification tests. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Comparison of PCR, culture, and serological tests for diagnosis of Mycoplasma pneumoniae respiratory tract infection in children

    NARCIS (Netherlands)

    Dorigo-Zetsma, J. W.; Zaat, S. A.; Wertheim-van Dillen, P. M.; Spanjaard, L.; Rijntjes, J.; van Waveren, G.; Jensen, J. S.; Angulo, A. F.; Dankert, J.

    1999-01-01

    For diagnosis of Mycoplasma pneumoniae infection we compared two rapid tests, PCR and the immunoglobulin M immunofluorescence assay (IgM IFA), with culture and the complement fixation test (CFT), in a prospective study among 92 children with respiratory tract infection and 74 controls. Based on

  18. Quality assurance for HIV point-of-care testing and treatment monitoring assays

    Science.gov (United States)

    Sandstrom, Paul; Denny, Thomas N.; Hurlston, Mackenzie; Ball, Terry B.; Peeling, Rosanna W.; Boeras, Debrah I.

    2016-01-01

    In 2015, UNAIDS launched the 90-90-90 targets aimed at increasing the number of people infected with HIV to become aware of their status, access antiretroviral therapies and ultimately be virally suppressed. To achieve these goals, countries may need to scale up point-of-care (POC) testing in addition to strengthening central laboratory services. While decentralising testing increases patient access to diagnostics, it presents many challenges with regard to training and assuring the quality of tests and testing. To ensure synergies, the London School of Hygiene & Tropical Medicine held a series of consultations with countries with an interest in quality assurance and their implementing partners, and agreed on an external quality assessment (EQA) programme to ensure reliable results so that the results lead to the best possible care for HIV patients. As a result of the consultations, EQA International was established, bringing together EQA providers and implementers to develop a strategic plan for countries to establish national POC EQA programmes and to estimate the cost of setting up and maintaining the programme. With the dramatic increase in the number of proficiency testing panels required for thousands of POC testing sites across Africa, it is important to facilitate technology transfer from global EQA providers to a network of regional EQA centres in Africa for regional proficiency testing panel production. EQA International will continue to identify robust and cost-effective EQA technologies for quality POC testing, integrating novel technologies to support sustainable country-owned EQA programmes in Africa. PMID:28879133

  19. IFN-γ release assay conversions and reversions. Challenges with serial testing in U.S. health care workers.

    Science.gov (United States)

    Joshi, Manish; Monson, Thomas P; Joshi, Anita; Woods, Gail L

    2014-03-01

    IFN-γ release assays (IGRAs) including the QuantiFERON-TB gold in-tube test (QFT-GIT) are increasingly used in place of the tuberculin skin test (TST) in surveillance programs for Mycobacterium tuberculosis infection in the United States. However, data on conversions, reversions, and predictive value of QFT in such programs for health care workers (HCWs) are limited. The purpose of this study is to assess long-term reproducibility and conversion and reversion rates of QFT-GIT among HCWs who underwent serial testing at a tertiary care center in the United States. Retrospective chart review of HCWs at the Central Arkansas Veterans Healthcare System (CAVHS) who underwent serial testing with QFT-GIT as a part of their employee screening between November 1, 2008 and January 31, 2011. A total of 2,303 HCWs had at least 2 QFT-GITs 1 year apart. The initial QFT-GIT was positive for 69 and 2 were indeterminate. Of these 69 HCWs, 31 (45%) reverted on repeat testing, and 25 of 31 (80.6%) HCWs who reverted had a negative look-back TST. Of the 2,232 HCWs with an initial negative QFT-GIT, 71 (3.2%) converted on repeat testing. A third QFT-GIT assay was performed in 41 of the 71 converters and 90% (37 of 41) reverted back to negative. Only two HCWs had TST and QFT-GIT conversion. Poor IGRA reproducibility and a low predictive value of QFT-GIT conversions indicate that QFT-GIT with current interpretation criteria should not be used for serial screening of U.S. HCWs. Negative TSTs have higher reproducibility than QFT-GIT for serial testing of HCWs in low tuberculosis incidence settings.

  20. An enzyme-linked immunosorbent assay and a gold-nanoparticle based immuno chromatographic test for amatoxins using recombinant antibody

    International Nuclear Information System (INIS)

    He, Kuo; Zhao, Ruiping; Wang, Lixia; Feng, Tingting; Wei, Dong; Zhang, Xiuyuan

    2016-01-01

    The authors describe two kinds of rapid assays for the determination of amatoxins in mushrooms. The first is an enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase. The second is a rapid immuno chromatographic assay that uses colloidal gold as a red label (CG-ICA). Both are based on the use of a well-characterized recombinant single chain variable fragment antibody (named scFv-A4). The half-maximum inhibition concentrations (IC50) of α-amanitin, β-amanitin and γ-amanitin are 78, 85 and 90 ng⋅mL"-"1, and the limits of detection (LODs; for IC15) are 1.9, 2.1 and 2.8 ng⋅mL"-"1. The method was applied to the determination of amanitins in mushrooms, and the LODs for α-amanitin, β-amanitin and γ-amanitin in mushroom samples were found to be 4.9, 6.4 and 8.3 ng⋅mL"-"1. The visual minimum detection limits of the optimized CGIA are 4 and 6 ng⋅mL"-"1 for mushroom samples. The test can be performed within 10 min. The results of the analysis of spiked samples showed that the CG-IA can rapidly and semi-quantitatively quantify amatoxins in mushroom samples on site and at low costs. (author)

  1. Continuing Assessment of the 5-Day Sodium Carbonate-Ammonium Nitrate Extraction Assay as an Indicator Test for Silicon Fertilizers.

    Science.gov (United States)

    Zellner, Wendy; Friedrich, Russell L; Kim, Sujin; Sturtz, Douglas; Frantz, Jonathan; Altland, James; Krause, Charles

    2015-01-01

    The 5-day sodium carbonate-ammonium nitrate extraction assay (5-day method) has been recognized by the American Association of Plant Food Control Officials as a validated test method to identify fertilizers or beneficial substances that provide plant-available silicon (Si). The test method used the molybdenum blue colorimetric assay to quantify percentage Si; however, laboratories may use inductively coupled plasma optical emission spectroscopy (ICP-OES) for elemental analysis. To examine the use of either colorimetric or ICP-OES methods for Si determination, the 5-day method was performed on the following Si-containing compounds; wollastonite, sand, biochar, and a basic oven furnace (BOF) slag. Grow-out studies using Zinnia elegans were also performed using varying rates of the wollastonite, biochar, and BOF slag. Our results show using the 5-day method, wollastonite had the highest extracted amounts of silicic acid (H4SiO4) at 4% followed by biochar (2%), BOF slag (1%), and sand (0%). Extraction values calculated using either the molybdenum blue colorimetric assay or ICP-OES for detection of the H4SiO4 had a significant correlation, supporting the application of either detection method for this type of analysis. However, when extracted values were compared to amounts of Si taken up by the plants, the 5-day method overestimated both wollastonite and biochar. While this method is a valid indicator test for determining a soluble Si source, other plant species and methods should be perused to potentially provide more quantitative analyses for plant-available Si content of all materials.

  2. A comprehensive statistical classifier of foci in the cell transformation assay for carcinogenicity testing.

    Science.gov (United States)

    Callegaro, Giulia; Malkoc, Kasja; Corvi, Raffaella; Urani, Chiara; Stefanini, Federico M

    2017-12-01

    The identification of the carcinogenic risk of chemicals is currently mainly based on animal studies. The in vitro Cell Transformation Assays (CTAs) are a promising alternative to be considered in an integrated approach. CTAs measure the induction of foci of transformed cells. CTAs model key stages of the in vivo neoplastic process and are able to detect both genotoxic and some non-genotoxic compounds, being the only in vitro method able to deal with the latter. Despite their favorable features, CTAs can be further improved, especially reducing the possible subjectivity arising from the last phase of the protocol, namely visual scoring of foci using coded morphological features. By taking advantage of digital image analysis, the aim of our work is to translate morphological features into statistical descriptors of foci images, and to use them to mimic the classification performances of the visual scorer to discriminate between transformed and non-transformed foci. Here we present a classifier based on five descriptors trained on a dataset of 1364 foci, obtained with different compounds and concentrations. Our classifier showed accuracy, sensitivity and specificity equal to 0.77 and an area under the curve (AUC) of 0.84. The presented classifier outperforms a previously published model. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Development in Assay Methods for in Vitro Antimalarial Drug Efficacy Testing: A Systematic Review

    Directory of Open Access Journals (Sweden)

    Shweta Sinha

    2017-10-01

    Full Text Available The emergence and spread of drug resistance are the major challenges in malaria eradication mission. Besides various strategies laid down by World Health Organization, such as vector management, source reduction, early case detection, prompt treatment, and development of new diagnostics and vaccines, nevertheless the need for new and efficacious drugs against malaria has become a critical priority on the global malaria research agenda. At several screening stages, millions of compounds are screened (1,000–2,000,000 compounds per screening campaign, before pre-clinical trials to select optimum lead. Carrying out in vitro screening of antimalarials is very difficult as different assay methods are subject to numerous sources of variability across different laboratories around the globe. Despite this, in vitro screening is an essential part of antimalarial drug development as it enables to resource various confounding factors such as host immune response and drug–drug interaction. Therefore, in this article, we try to illustrate the basic necessity behind in vitro study and how new methods are developed and subsequently adopted for high-throughput antimalarial drug screening and its application in achieving the next level of in vitro screening based on the current approaches (such as stem cells.

  4. Private sector tuberculosis prevention in the US: Characteristics associated with interferon-gamma release assay or tuberculin skin testing.

    Science.gov (United States)

    Stockbridge, Erica L; Miller, Thaddeus L; Carlson, Erin K; Ho, Christine

    2018-01-01

    To determine whether latent tuberculosis infection risk factors are associated with an increased likelihood of latent tuberculosis infection testing in the US private healthcare sector. A national sample of medical and pharmacy claims representing services rendered January 2011 through December 2013 for 3,997,986 commercially insured individuals in the US who were 0 to 64 years of age. We used multivariable logistic regression models to determine whether TB/LTBI risk factors were associated with an increased likelihood of Interferon-Gamma Release Assay (IGRA) or Tuberculin Skin Test (TST) testing in the private sector. 4.31% (4.27-4.34%) received at least one TST/IGRA test between 2011 and 2013 while 1.69% (1.67-1.72%) received a TST/IGRA test in 2013. Clinical risk factors associated with a significantly increased likelihood of testing included HIV, immunosuppressive therapy, exposure to tuberculosis, a history of tuberculosis, diabetes, tobacco use, end stage renal disease, and alcohol use disorder. Other significant variables included gender, age, asthma, the state tuberculosis rate, population density, and percent of foreign-born persons in a county. Private sector TST/IGRA testing is not uncommon and testing varies with clinical risk indicators. Thus, the private sector can be a powerful resource in the fight against tuberculosis. Analyses of administrative data can inform how best to leverage private sector healthcare toward tuberculosis prevention activities.

  5. A comparative clinical study of efficacy of microimmuno assay with WIDAL-test in enteric fever in children

    Directory of Open Access Journals (Sweden)

    Gandarajapura Nagaraj Madhu

    2014-01-01

    Full Text Available The diagnosis of typhoid fever in young children is also a dilemma because of its manifestations and typical presentation may not be seen in all cases. Antibodies to Salmonella typhi antigen are developed in the human body, which can be detected as a diagnostic test for the enteric fever. Objective: This study was undertaken to compare the efficacy of WIDAL-test with micro-immunoassay (dot enzyme immunosorbent assay. Method: 40 cases of clinically suspected enteric fever cases were included in this study. Result: In the present study, nearly 92% were positive for micro immunoassay (dot-enzyme immunosorbent assay by Enterocheck-WB kit, 80% were positive for WIDAL and only 15% were culture positive. Immunoassay positive, but WIDAL negative cases were 20%, whereas WIDAL positive and immunoassay negative cases were only 7.5%. The positive predictivity of micro-immunoassay in diagnosing enteric fever is better than WIDAL both in 1st and 2nd week of illness. Micro-immunoassay done in the study was rapid in diagnosing the case. Conclusion: It is concluded from the present study that the micro-immunoassay (Enterocheck-WB is better than WIDAL-test in the diagnosis of enteric fever in children.

  6. Development of sample assay system equipped with 3He Alternative Neutron Detectors (ASAS). (2) Results of ASAS measurement test

    International Nuclear Information System (INIS)

    Tanigawa, Masafumi; Mukai, Yasunobu; Kurita, Tsutomu; Makino, Risa; Nakamura, Hironobu; Tobita, Hiroshi; Ohzu, Akira; Kureta, Masatoshi; Seya, Michio

    2015-01-01

    Against the background of the serious shortage of 3 He gas, design and development of a new detector equipped ZnS/ 10 B 2 O 3 ceramic scintillation neutron detectors in JAEA, with the support of the government (the Ministry of Education, Culture, Sports, Science and Technology). The design of the alternative 3 He detector is referred from INVS (INVentory Sample assay system (HLNCC (High Level Neutron Coincidence Counter) type)) which is being used for the verification of MOX powder etc. and is named it as ASAS (Alternative Sample Assay System). In order to prove the Pu quantitative performance as an alternative technology, several measurement tests and comparison test with INVS were conducted using ASAS. In these tests, evaluation of fundamental performance (counting efficiency and die-away time) and uncertainty evaluations were implemented. As a result, although fundamental performance of ASAS was not achieved to the one of INVS, we could confirm that ASAS has almost the same Pu quantitative performance including measurement uncertainty as that of INVS. (author)

  7. Genetic mouse embryo assay: improving performance and quality testing for assisted reproductive technology (ART) with a functional bioassay.

    Science.gov (United States)

    Gilbert, Rebecca S; Nunez, Brandy; Sakurai, Kumi; Fielder, Thomas; Ni, Hsiao-Tzu

    2016-03-24

    Growing concerns about safety of ART on human gametes, embryos, clinical outcomes and long-term health of offspring require improved methods of risk assessment to provide functionally relevant assays for quality control testing and pre-clinical studies prior to clinical implementation. The one-cell mouse embryo assay (MEA) is the most widely used for development and quality testing of human ART products; however, concerns exist due to the insensitivity/variability of this bioassay which lacks standardization and involves subjective analysis by morphology alone rather than functional analysis of the developing embryos. We hypothesized that improvements to MEA by the use of functional molecular biomarkers could enhance sensitivity and improve detection of suboptimal materials/conditions. Fresh one-cell transgenic mouse embryos with green fluorescent protein (GFP) expression driven by Pou6f1 or Cdx2 control elements were harvested and cultured to blastocysts in varied test and control conditions to compare assessment by standard morphology alone versus the added dynamic expression of GFP for screening and selection of critical raw materials and detection of suboptimal culture conditions. Transgenic mouse embryos expressing functionally relevant biomarkers of normal early embryo development can be used to monitor the developmental impact of culture conditions. This novel approach provides a superior MEA that is more meaningful and sensitive for detection of embryotoxicity than morphological assessment alone.

  8. Cytotoxicity test of 40, 50 and 60% citric acid as dentin conditioner by using MTT assay on culture cell line

    Directory of Open Access Journals (Sweden)

    Christian Khoswanto

    2008-09-01

    Full Text Available Background: Open dentin is always covered by smear layer, therefore before restoration is performed, cavity or tooth which has been prepared should be clean from dirt. The researchers suggested that clean dentin surface would reach effective adhesion between resin and tooth structure, therefore dentin conditioner like citric acid was used to reach the condition. Even though citric acid is not strong acid but it can be very erosive to oral mucous. Several requirements should be fulfilled for dental product such as non toxic, non irritant, biocompatible and should not have negative effect against local, systemic or biological environment. Cytotoxicity test was apart of biomaterial evaluation and needed for standard screening. Purpose: This study was to know the cytotoxicity of 40, 50, 60% citric acid as dentin conditioner using MTT assay. Method: This study is an experimental research using the Post-Test Only Control Group Design. Six samples of each 40, 50 and 60% citric acid for citotoxicity test using MTT assay. The density of optic formazan indicated the number of living cells. All data were statistically analyzed by one way ANOVA. Result: The percentage of living cells in 40, 50 and 60% citric acid were 95.14%, 93.42% and 93.14%. Conclusion: Citric acid is non toxic and safe to be used as dentine conditioner.

  9. Comparison of indirect immunofluorescence test for measles antibodies with haemagglutination inhibition and plaque neutralization tests Comparação da reação de imunofluorescência indireta para o vírus do sarampo com as reações de inibição da hemaglutinação e neutralização por redução de placas

    Directory of Open Access Journals (Sweden)

    Vanda Akico Ueda Fick de Souza

    1990-10-01

    Full Text Available Indirect Immunofluorescence (IFA, Plaque Reduction Neutralization (PRN and Haemagglutination Inhibition (HI tests for measles antibodies were carried out in 197 sera obtained from umbilical cord and vaccinated children. The IFA was also applied to blood samples collected with filter paper. IFA results demonstrated that the test is relatively simple to perform, with good reproducibility for different antigen lots. Good correlation was obtained between IFA, PRN and HI antibody titers. Better correlation was demonstrated with IFA and PRN than with HI and PRN tests. Sensitivity of IFA in detecting antibody was less effective than PRN, however more effective than HI using rhesus monkey red blood cells. PRN antibody titers over 100 were detected by IFA but not by HI (9.7% with negative results. IFA may be of considerable practical use and able to substitute HI in Seroepidemiological surveys and to evaluate vaccine efficacy. It also can be simplified by employing filter paper collected samples.As reações de imunofluorescência indireta (RIF, neutralização por redução de placas (RNP e inibição da hemaglutinação (RIH para detecção de anticorpos para o vírus do sarampo foram aplicadas a 197 soros provenientes de cordão umbilical e de crianças vacinadas contra o sarampo. Avaliou-se ainda a aplicação da RIF em amostras colhidas em papel de filtro. A RIF apresentou-se como uma prova de execução relativamente simples, de boa reprodutibilidade com diferentes partidas de antígeno. Observou-se boa correlação entre os títulos de anticorpos obtidos por RIF, RNP e RIH. Com a RNP, a RIF apresentou maior correlação que a RIH. A sensibilidade da RIF na detecção de anticorpos contra o sarampo foi menor que RNP, porém superior à da RIH com hemácias de macaco rhesus. Anticorpos com títulos superiores a 100 pela RNP foram sistematicamente detectados pela RIF, mas não por RIH (9,7% de resultados negativos. A RIF pode ser de grande utilidade

  10. Evaluation of the Luciferase Assay-Based In Vitro Elicitation Test for Serum IgE

    Directory of Open Access Journals (Sweden)

    Ryosuke Nakamura

    2012-01-01

    Results: The area under the ROC curves was highest in the EXiLE test (0.977, followed by CAP-FEIA (0.926 and degranulation (0.810. At an optimal cutoff range (1.648-1.876 calculated from the ROC curve of the EXiLE test, sensitivity and specificity were 0.944 and 0.917, respectively. A 95% positive predictive value was given at a cutoff level of 2.054 (fold increase in luciferase expression by logistic regression analysis. Conclusions: In contrast to in vivo tests, the EXiLE test appears to be a useful tool in diagnosing patients suspected of having IgE-dependent EW allergy without the risk of severe systemic reactions.

  11. Antigen spot test (AST): a highly sensitive assay for the detection of antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Herbrink, P; van Bussel, F J; Warnaar, S O [Rijksuniversiteit Leiden (Netherlands)

    1982-02-12

    A method is described for detection of antibodies by means of nitrocellulose or diazobenzyloxymethyl (DBM) paper on which various antigens have been spotted. The sensitivity of this antigen spot test (AST) is comparable with that of RIA and ELISA. The method requires only nanogram amounts of antigen. Since a variety of antigens can be spotted on a single piece of nitrocellulose or DBM paper, this antigen spot test is especially useful for specificity controls on antibodies.

  12. Quality assurance for HIV point-of-care testing and treatment monitoring assays

    Directory of Open Access Journals (Sweden)

    Adrienne F.A. Meyers

    2016-10-01

    Full Text Available In 2015, UNAIDS launched the 90-90-90 targets aimed at increasing the number of peopleinfected with HIV to become aware of their status, access antiretroviral therapies and ultimatelybe virally suppressed. To achieve these goals, countries may need to scale up point-of-care (POC testing in addition to strengthening central laboratory services. While decentralisingtesting increases patient access to diagnostics, it presents many challenges with regard totraining and assuring the quality of tests and testing. To ensure synergies, the London Schoolof Hygiene & Tropical Medicine held a series of consultations with countries with an interestin quality assurance and their implementing partners, and agreed on an external qualityassessment (EQA programme to ensure reliable results so that the results lead to the bestpossible care for HIV patients. As a result of the consultations, EQA International wasestablished, bringing together EQA providers and implementers to develop a strategic planfor countries to establish national POC EQA programmes and to estimate the cost of setting upand maintaining the programme. With the dramatic increase in the number of proficiencytesting panels required for thousands of POC testing sites across Africa, it is important tofacilitate technology transfer from global EQA providers to a network of regional EQA centresin Africa for regional proficiency testing panel production. EQA International will continue toidentify robust and cost-effective EQA technologies for quality POC testing, integrating noveltechnologies to support sustainable country-owned EQA programmes in Africa.

  13. Padronização de ensaio imunoenzimático para pesquisa de anticorpos das classes IgM e IgG anti-Toxoplasma gondii e comparação com a técnica de imunofluorescência indireta Standardization of enzyme-linked immunosorbent assay ELISA to detect anti-Toxoplasma gondii IgM and IgG antibodies, and comparison with the indirect immunofluorescence technique

    Directory of Open Access Journals (Sweden)

    Cláudia Maria Antunes Uchôa

    1999-12-01

    Full Text Available A sorologia tem sido o método de escolha para o diagnóstico da toxoplasmose. Devido a isto, padronizamos um ensaio imunoenzimático (ELISA e comparamos seus resultados com a técnica de imunofluorescência indireta (IFI. A técnica padronizada apresentou na pesquisa de IgG sensibilidade (S de 96,7% e especificidade (E de 75%, com valor de predição de positividade (VPP de 83,3% e de negatividade (VPN de 94,7%, com uma concordância ajustada (K de 73,5%. A IFI apresentou S de 83,8%, E de 79,1% com VPP de 83,8 % e VPN de 79,1% com K de 63%. A concordância bruta entre os dois testes (ELISA/IFI foi de 88,3% para pesquisa de IgG e de 81,5% para pesquisa de IgM, sendo o K de 70,8% para IgG e de 1,3% para IgM, sendo o índice de correlação (r de 0,556 para IgG e de -0,023 para IgM. Podemos concluir que a ELISA-IgG padronizada é indicada nos processos de triagem sorológica, sendo a ELISA-IgM desaconselhada uma vez que apresentou baixos índices de concordância ajustada com a técnica de referência, sugerindo pouca confiabilidade dos resultados.Serology has been the most popular method to diagnose toxoplasmosis. Accordingly, this study standardizes an enzyme-linked immunosorbent assay (ELISA and compares its results with the IFI technique. In the IgG detection test, the standardized technique presented a sensibility (S of 96.77%, a specificity (SP of 75%, with a positive predictive value (PPV of 83.33%, a negative predictive value (NPV of 94.74%, and an adjusted concordance (K of 73.50%. The IFI exhibited 83.87% for S, 79.16% for SP, 83.81% for PPV, 79.16% for NPV, and 63% for K. The rough concordance between these two tests (ELISA/IFI was 88.35% for the IgG detection test and 81.55% for the IgM detection test. K was 70.82% and 1.31% for IgG and IgM, respectively, the correlation index (r being 0.556 for IgG and -0.023 for IgM. We can conclude that standardized ELISA-IgG is indicated in serologic selection processes, whereas the ELISA-IgM is

  14. Testing a dual-fluorescence assay to monitor the viability of filamentous cyanobacteria.

    Science.gov (United States)

    Johnson, Tylor J; Hildreth, Michael B; Gu, Liping; Zhou, Ruanbao; Gibbons, William R

    2015-06-01

    Filamentous cyanobacteria are currently being engineered to produce long-chain organic compounds, including 3rd generation biofuels. Because of their filamentous morphology, standard methods to quantify viability (e.g., plate counts) are not possible. This study investigated a dual-fluorescence assay based upon the LIVE/DEAD® BacLight™ Bacterial Viability Kit to quantify the percent viability of filamentous cyanobacteria using a microplate reader in a high throughput 96-well plate format. The manufacturer's protocol calls for an optical density normalization step to equalize the numbers of viable and non-viable cells used to generate calibration curves. Unfortunately, the isopropanol treatment used to generate non-viable cells released a blue pigment that altered absorbance readings of the non-viable cell solution, resulting in an inaccurate calibration curve. Thus we omitted this optical density normalization step, and carefully divided cell cultures into two equal fractions before the isopropanol treatment. While the resulting calibration curves had relatively high correlation coefficients, their use in various experiments resulted in viability estimates ranging from below 0% to far above 100%. We traced this to the apparent inaccuracy of the propidium iodide (PI) dye that was to stain only non-viable cells. Through further analysis via microplate reader, as well as confocal and wide-field epi-fluorescence microscopy, we observed non-specific binding of PI in viable filamentous cyanobacteria. While PI will not work for filamentous cyanobacteria, it is possible that other fluorochrome dyes could be used to selectively stain non-viable cells. This will be essential in future studies for screening mutants and optimizing photobioreactor system performance for filamentous cyanobacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. RNA biology in a test tube--an overview of in vitro systems/assays.

    Science.gov (United States)

    Roca, Xavier; Karginov, Fedor V

    2012-01-01

    In vitro systems have provided a wealth of information in the field of RNA biology, as they constitute a superior and sometimes the unique approach to address many important questions. Such cell-free methods can be sorted by the degree of complexity of the preparation of enzymatic and/or regulatory activity. Progress in the study of pre-mRNA processing has largely relied on traditional in vitro methods, as these reactions have been recapitulated in cell-free systems. The pre-mRNA capping, editing, and cleavage/polyadenylation reactions have even been reconstituted using purified components, and the enzymes responsible for catalysis have been characterized by such techniques. In vitro splicing using nuclear or cytoplasmic extracts has yielded clues on spliceosome assembly, kinetics, and mechanisms of splicing and has been essential to elucidate the function of splicing factors. Coupled systems have been important to functionally connect distinct processes, like transcription and splicing. Extract preparation has also been adapted to cells from a variety of tissues and species, revealing general versus species-specific mechanisms. Cell-free assays have also been applied to newly discovered pathways such as those involving small RNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs), and Piwi-interacting RNAs (piRNAs). The first two pathways have been well characterized largely by in vitro methods, which need to be developed for piRNAs. Finally, new techniques, such as single-molecule studies, are continuously being established, providing new and important insights into the field. Thus, in vitro approaches have been, are, and will continue being at the forefront of RNA research. Copyright © 2012 John Wiley & Sons, Ltd.

  16. Standard test method for plutonium assay by plutonium (III) diode array spectrophotometry

    CERN Document Server

    American Society for Testing and Materials. Philadelphia

    2002-01-01

    1.1 This test method describes the determination of total plutonium as plutonium(III) in nitrate and chloride solutions. The technique is applicable to solutions of plutonium dioxide powders and pellets (Test Methods C 697), nuclear grade mixed oxides (Test Methods C 698), plutonium metal (Test Methods C 758), and plutonium nitrate solutions (Test Methods C 759). Solid samples are dissolved using the appropriate dissolution techniques described in Practice C 1168. The use of this technique for other plutonium-bearing materials has been reported (1-5), but final determination of applicability must be made by the user. The applicable concentration range for plutonium sample solutions is 10–200 g Pu/L. 1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard. 1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropria...

  17. The diagnostic sensitivity of dengue rapid test assays is significantly enhanced by using a combined antigen and antibody testing approach.

    Directory of Open Access Journals (Sweden)

    Scott R Fry

    2011-06-01

    Full Text Available BACKGROUND: Serological tests for IgM and IgG are routinely used in clinical laboratories for the rapid diagnosis of dengue and can differentiate between primary and secondary infections. Dengue virus non-structural protein 1 (NS1 has been identified as an early marker for acute dengue, and is typically present between days 1-9 post-onset of illness but following seroconversion it can be difficult to detect in serum. AIMS: To evaluate the performance of a newly developed Panbio® Dengue Early Rapid test for NS1 and determine if it can improve diagnostic sensitivity when used in combination with a commercial IgM/IgG rapid test. METHODOLOGY: The clinical performance of the Dengue Early Rapid was evaluated in a retrospective study in Vietnam with 198 acute laboratory-confirmed positive and 100 negative samples. The performance of the Dengue Early Rapid in combination with the IgM/IgG Rapid test was also evaluated in Malaysia with 263 laboratory-confirmed positive and 30 negative samples. KEY RESULTS: In Vietnam the sensitivity and specificity of the test was 69.2% (95% CI: 62.8% to 75.6% and 96% (95% CI: 92.2% to 99.8 respectively. In Malaysia the performance was similar with 68.9% sensitivity (95% CI: 61.8% to 76.1% and 96.7% specificity (95% CI: 82.8% to 99.9% compared to RT-PCR. Importantly, when the Dengue Early Rapid test was used in combination with the IgM/IgG test the sensitivity increased to 93.0%. When the two tests were compared at each day post-onset of illness there was clear differentiation between the antigen and antibody markers. CONCLUSIONS: This study highlights that using dengue NS1 antigen detection in combination with anti-glycoprotein E IgM and IgG serology can significantly increase the sensitivity of acute dengue diagnosis and extends the possible window of detection to include very early acute samples and enhances the clinical utility of rapid immunochromatographic testing for dengue.

  18. The performance of the SEPT9 gene methylation assay and a comparison with other CRC screening tests: A meta-analysis.

    Science.gov (United States)

    Song, Lele; Jia, Jia; Peng, Xiumei; Xiao, Wenhua; Li, Yuemin

    2017-06-08

    The SEPT9 gene methylation assay is the first FDA-approved blood assay for colorectal cancer (CRC) screening. Fecal immunochemical test (FIT), FIT-DNA test and CEA assay are also in vitro diagnostic (IVD) tests used in CRC screening. This meta-analysis aims to review the SEPT9 assay performance and compare it with other IVD CRC screening tests. By searching the Ovid MEDLINE, EMBASE, CBMdisc and CJFD database, 25 out of 180 studies were identified to report the SEPT9 assay performance. 2613 CRC cases and 6030 controls were included, and sensitivity and specificity were used to evaluate its performance at various algorithms. 1/3 algorithm exhibited the best sensitivity while 2/3 and 1/1 algorithm exhibited the best balance between sensitivity and specificity. The performance of the blood SEPT9 assay is superior to that of the serum protein markers and the FIT test in symptomatic population, while appeared to be less potent than FIT and FIT-DNA tests in asymptomatic population. In conclusion, 1/3 algorithm is recommended for CRC screening, and 2/3 or 1/1 algorithms are suitable for early detection for diagnostic purpose. The SEPT9 assay exhibited better performance in symptomatic population than in asymptomatic population.

  19. Performance of the Abbott RealTime MTB RIF/INH resistance assay when used to test Mycobacterium tuberculosis specimens from Bangladesh

    Directory of Open Access Journals (Sweden)

    Kostera J

    2018-05-01

    Full Text Available Joshua Kostera, Gregor Leckie, Klara Abravaya, Hong Wang Abbott Molecular, Abbott Laboratories, Des Plaines, IL, USA Introduction: The Abbott RealTime MTB RIF/INH Resistance Assay (RT MTB RIF/INH is an assay for the detection of rifampicin (RIF- and/or isoniazid (INH-resistant Mycobacterium tuberculosis (MTB. The assay can be used to test sputum, bronchial alveolar lavage, and N-Acetyl-L-Cysteine (NALC/NaOH pellets prepared from these samples. The assay can be used in direct testing mode, or in reflex mode following a MTB positive result produced by its companion assay, Abbott RT MTB. Methods: In this study, the direct testing mode was used to test paired sputum and NALC/NaOH pellets prepared from sputum collected from Bangladesh TB patients. One hundred and thirty two paired samples were tested. Results: The RT MTB RIF/INH inhibition rate was 0%. One hundred and twenty-two paired samples had results above the assay limit of detection and were analyzed by comparing with results from phenotypic drug sensitivity testing, GeneXpert MTB/RIF (Xpert, and MTBDR plus (Hain. RT MTB RIF/INH results were in good agreement with those of GeneXpert and Hain. Conclusion: The ability of this assay to detect RIF and INH resistance may contribute to the global control of multidrug resistant tuberculosis. Keywords: tuberculosis, rifampicin, isoniazid, resistance

  20. Immunofluorescence detection of milk protein in meat products

    Directory of Open Access Journals (Sweden)

    Michaela Petrášová

    2015-05-01

    Full Text Available Nowadays there are various vegetable protein additives intended for the manufacture of meat products in the food industry. These ingredients include both, plant-origin as well as animal-origin proteins. The most common vegetable additives include various types of flour, starch, fiber and plant protein. Among animal proteins, the most commonly used are plasma, collagen or milk protein. Milk protein is added to meat products due to its functional properties, such as emulsifying fats, improving the holding capacity of meat, improving juiciness, gel-forming capacity and affecting the taste of the product. Usage of these proteins, however, is currently limited by the effective legislation, not only in order to prevent consumer deception, but also because of their potential impact on consumers' health of. Thus, this issue has received considerable attention not only in the Czech Republic, but also globally. The main risk is the impossibility of selecting a suitable foodstuff for individuals with potential allergic reactions. The only option for allergic consumers to protect themselves is to strictly exclude the given allergen from their diet. Although the number of studies dealing with the reduction or loss of allergenicity is increasing, yet these practices are not common. Most of the population suffering from food allergies is thus still dependent on strict exclusion of foodstuffs causing adverse allergic reactions from their diet. Detection of allergens in foodstuffs is unfortunately quite difficult due to the fact that they occur in trace amounts and are often masked by different parts of the foodstuff. This research dealt with the detection of milk protein in meat products purchased in the market network of the Czech Republic, whereas declaration given by the manufacturer on the packaging for the small meat products purchased from the market was used to verify the detection of milk protein by the immunofluorescence method. 20 products were

  1. Immunofluorescent determination of wheat protein in meat products

    Directory of Open Access Journals (Sweden)

    Michaela Petrášová

    2014-02-01

    foodstuffs is relatively difficult because of the fact that they occur in trace amounts and are often masked by various parts of the product. This paper deals with detection of wheat protein in meat products bought in the retail network of the Czech Republic. Ten cooked meat products, especially types of sausages and soft salami which stated wheat protein in their composition, were examined. The samples were processed using the method of immunofluorescence and stained with Texas Red fluorochrome. The presence of wheat protein was demonstrated in all the examined meat products. From the results it follows that the method of immunofluorescence is suitable for detection of wheat protein in meat products. Normal 0 21 false false false CS JA X-NONE

  2. EUROPattern Suite technology for computer-aided immunofluorescence microscopy in autoantibody diagnostics.

    Science.gov (United States)

    Krause, C; Ens, K; Fechner, K; Voigt, J; Fraune, J; Rohwäder, E; Hahn, M; Danckwardt, M; Feirer, C; Barth, E; Martinetz, T; Stöcker, W

    2015-04-01

    Antinuclear autoantibodies (ANA) are highly informative biomarkers in autoimmune diagnostics. The increasing demand for effective test systems, however, has led to the development of a confusingly large variety of different platforms. One of them, the indirect immunofluorescence (IIF), is regarded as the common gold standard for ANA screening, as described in a position statement by the American College of Rheumatology in 2009. Technological solutions have been developed aimed at standardization and automation of IIF to overcome methodological limitations and subjective bias in IIF interpretation. In this review, we present the EUROPattern Suite, a system for computer-aided immunofluorescence microscopy (CAIFM) including automated acquisition of digital images and evaluation of IIF results. The system was originally designed for ANA diagnostics on human epithelial cells, but its applications have been extended with the latest system update version 1.5 to the analysis of antineutrophil cytoplasmic antibodies (ANCA) and anti-dsDNA antibodies. © The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  3. Comparison of two interferon-gamma assays and tuberculin skin test for tracing tuberculosis contacts

    NARCIS (Netherlands)

    Arend, Sandra M.; Thijsen, Steven F. T.; Leyten, Eliane M. S.; Bouwman, John J. M.; Franken, Willeke P. J.; Koster, Ben F. P. J.; Cobelens, Frank G. J.; van Houte, Arend-Jan; Bossink, Ailko W. J.

    2007-01-01

    The tuberculin skin test (TST) has low specificity. QuantiFERON-TB Gold (QFT-G) and T-SPOT.TB are based on interferon (IFN)-gamma responses to Mycobacterium tuberculosis-specific antigens. A novel in-tube format of QFT-G (QFT-GIT) offers logistical advantages. To compare TST, QFT-GIT, and T-SPOT.TB

  4. VIRAL TESTING USING BIOLOGICAL AND SEROLOGICAL ASSAY FOR MOST IMPORTANT VIRUSES TO PLUM

    Directory of Open Access Journals (Sweden)

    Catita Plopa

    2014-12-01

    Full Text Available Establishing an accurate diagnosis in terms of viral for propagation of fruit tree is very important, it represents the most effective method of protection against viruses. Based on these considerations the primary objective of this study is to detect viruses with the highest incidence in plum by biological and ELISA serological methods, to a number of 85 samples taken from 17 varieties. Serologic testing on DAS-ELISA diagnosed 3 positive samples to Plum pox virus (PPV, 2 positives sample to Prunus necrotic ring spot virus (PNRSV and one positive sample to Prune dwarf virus (PDV. There were not positive samples to Apple chlorotic leaf spot virus (ACLSV. The tests conducted on woody indicator plants by grafting on protect conditions and after 3-24 months assured of diagnosis for PPV, PDV, PNRSV and ACLSV viruses. The biological indicators: ‘GF 305’, ‘Tuleu dulce’ and ‘Vânăt de Italia’, have shown symptoms for PNRSV for two samples.On biological indicator ‘Vânăt de Italia’ and ‘Tuleu dulce’ not appeared symptoms for ‘Centenar’variety tested for PPV, although the symptoms were obvious on ‘GF 305’ indicator, but viral infection was confirmed by ELISA test. Symptoms that indicate the presence of PDV occurred by ‘Vânăt de Italia’ biological indicator.

  5. Use of external metabolizing systems when testing for endocrine disruption in the T-screen assay

    DEFF Research Database (Denmark)

    Taxvig, Camilla; Olesen, Pelle Thonning; Nellemann, Christine Lydia

    2011-01-01

    Although, it is well-established that information on the metabolism of a substance is important in the evaluation of its toxic potential, there is limited experience with incorporating metabolic aspects into in vitro tests for endocrine disrupters. The aim of the current study was a) to study...

  6. Assaying the Mutagenic Potential of ELF Radiation through Reverse Mutagenesis via the Ames Test

    National Research Council Canada - National Science Library

    Moga, Paul

    1996-01-01

    ...) on certain strains of Salmonella typhimurium. The strain of S-typhimurium used in the Ames test has a mutation on one gene of the histidine operon which prevents it from growing and replicating without the presence of histidine in the media...

  7. Metrological tests of a 200 L calibration source for HPGE detector systems for assay of radioactive waste drums

    International Nuclear Information System (INIS)

    Boshkova, T.; Mitev, K.

    2016-01-01

    In this work we present test procedures, approval criteria and results from two metrological inspections of a certified large volume "1"5"2Eu source (drum about 200 L) intended for calibration of HPGe gamma assay systems used for activity measurement of radioactive waste drums. The aim of the inspections was to prove the stability of the calibration source during its working life. The large volume source was designed and produced in 2007. It consists of 448 identical sealed radioactive sources (modules) apportioned in 32 transparent plastic tubes which were placed in a wooden matrix which filled the drum. During the inspections the modules were subjected to tests for verification of their certified characteristics. The results show a perfect compliance with the NIST basic guidelines for the properties of a radioactive certified reference material (CRM) and demonstrate the stability of the large volume CRM-drum after 7 years of operation. - Highlights: • Large (200 L) volume drum source designed, produced and certified as CRM in 2007. • Source contains 448 identical sealed radioactive "1"5"2Eu sources (modules). • Two metrological inspections in 2011 and 2014. • No statistically significant changes of the certified characteristics over time. • Stable calibration source for HPGe-gamma radioactive waste assay systems.

  8. Early postnatal diagnosis of hereditary spherocytosis by combining light microscopy, acidified glycerol lysis test and eosin-5'-maleimide binding assay.

    Science.gov (United States)

    Andres, Oliver; Eber, Stefan; Speer, Christian P

    2015-12-01

    Exact diagnosis of hereditary spherocytosis (HS) is widely considered unreliable around birth. However, early postnatal diagnosis at the beginning of congenital hemolysis may be essential for managing neonatal anemia and hemolytic icterus, identifying those at high risk for severe hyperbilirubinemia, irreversible kernicterus, or sudden need for red cell transfusion. We analyzed 37 blood samples from neonates or infants up to six weeks of life that had been collected in-house or shipped to our laboratory due to suspected red cell membrane disorder. By combining assessment of red cell morphology, acidified glycerol lysis test (AGLT), and eosin-5'-maleimide (EMA) binding assay, we were able to clearly exclude HS in 22 and confirm HS in 10 patients, of which one had undergone red cell transfusion prior to blood sampling. Assessment of red cell morphology and normal test results allowed diagnosis of infantile pyknocytosis or Heinz body anemia in three neonates. Re-evaluation of five patients with inconsistent results of AGLT and EMA binding led to confirmation of HS in two cases. Automated analysis of hematologic parameters revealed elevated proportion of hyperdense cells to be a highly significant indicator for HS in neonatal infants. We showed that assessment of red cell morphology in combination with AGLT and EMA binding assay is a reliable basis for confirming or rejecting suspected diagnosis of HS even in neonates. Our data underline the necessity for blood sampling and laboratory exploration in suspected red cell membrane or enzyme defects at the earliest occasion.

  9. Assaying embryotoxicity in the test tube: current limitations of the embryonic stem cell test (EST) challenging its applicability domain.

    Science.gov (United States)

    Riebeling, Christian; Hayess, Katrin; Peters, Annelieke K; Steemans, Margino; Spielmann, Horst; Luch, Andreas; Seiler, Andrea E M

    2012-05-01

    Testing for embryotoxicity in vitro is an attractive alternative to animal experimentation. The embryonic stem cell test (EST) is such a method, and it has been formally validated by the European Centre for the Validation of Alternative Methods. A number of recent studies have underscored the potential of this method. However, the EST performed well below the 78% accuracy expected from the validation study using a new set of chemicals and pharmaceutical compounds, and also of toxicity criteria, tested to enlarge the database of the validated EST as part of the Work Package III of the ReProTect Project funded within the 6th Framework Programme of the European Union. To assess the performance and applicability domain of the EST we present a detailed review of the substances and their effects in the EST being nitrofen, ochratoxin A, D-penicillamine, methylazoxymethanol, lovastatin, papaverine, warfarin, β-aminopropionitrile, dinoseb, furosemide, doxylamine, pravastatin, and metoclopramide. By delineation of the molecular mechanisms of the substances we identify six categories of reasons for misclassifications. Some of these limitations might also affect other in vitro methods assessing embryotoxicity. Substances that fall into these categories need to be included in future validation sets and in validation guidelines for embryotoxicity testing. Most importantly, we suggest conceivable improvements and additions to the EST which will resolve most of the limitations.

  10. Comparison of agar gel immunodiffusion test, enzyme-linked immunosorbent assay and PCR in diagnostics of enzootic bovine leukosis

    Directory of Open Access Journals (Sweden)

    Malovrh Tadej

    2005-01-01

    Full Text Available Bovine leukaemia virus (BLV is a retrovirus that induces a chronic infection in cattle. Once infected, cattle remain virus carriers for life and start to show an antibody response within a few weeks after infection. Eradication and control of the disease are based on early diagnostics and segregation of the carriers. The choice of a diagnostic method depends on the eradication programme, money resources and characteristics of the herd to be analysed. The agar gel immunodiffusion (AGID test has been the serological test of choice for routine diagnosis of serum samples. Nevertheless, in more recent years, the enzyme-linked immunosorbent assay (ELISA has replaced the AGID for large scale testing. For this purpose, commercially available BLV-ELISA kits were compared to the AGID and to the polymerase chain reaction (PCR method performed with two sets of primers, amplifying env region. The ELISA kit based on the p24 core protein was found to be less specific and served as a screening test. The ELISA kit based on the envelope glycoprotein (gpSI served as a verification test and gave a good correlation with the AGID test and PCR method. However, ELISA showed a higher sensitivity than AGID. The p24 based ELiSA was useful for screening a large number of samples, whereas gp51 based ELISA, AGID and PCR were more important for detecting the antibody response against the individual BLV-proteins and therefore for verification of the infection with BLV.

  11. Evaluation of the BYG Carba Test, a New Electrochemical Assay for Rapid Laboratory Detection of Carbapenemase-Producing Enterobacteriaceae

    Science.gov (United States)

    Yunus, Sami; Massart, Marion; Huang, Te-Din; Glupczynski, Youri

    2015-01-01

    Accurate detection of carbapenemase-producing Enterobacteriaceae (CPE) constitutes a major laboratory diagnostic challenge. We evaluated an electrochemical technique (the BYG Carba test) which allows detection of CPE in less than 35 min. The BYG Carba test was first validated in triplicate against 57 collection isolates with previously characterized β-lactam resistance mechanisms (OXA-48, n = 12; KPC, n = 8; NDM, n = 8; VIM, n = 8; IMP, n = 3; GIM, n = 1; GES-6, n = 1; no carbapenemase, n = 16) and against a panel of 10 isolates obtained from the United Kingdom National External Quality Assessment Service (NEQAS). The test was then evaluated prospectively against 324 isolates referred to the national reference center for suspicion of CPE. The BYG Carba test results were compared with those obtained with the Carba NP test using multiplex PCR sequencing as the gold standard. Of the 57 collection and the 10 NEQAS isolates, all but one GES-6-producing isolate were correctly identified by the Carba BYG test. Among the 324 consecutive Enterobacteriaceae isolates tested prospectively, 146 were confirmed as noncarbapenemase producers by PCR while 178 harbored a carbapenemase gene (OXA-48, n = 117; KPC, n = 25; NDM, n = 23; and VIM, n = 13). Prospectively, in comparison with PCR results, the BYG Carba test displayed 95% sensitivity and 100% specificity versus 89% and 100%, respectively, for the Carba NP test. The BYG Carba test is a novel, rapid, and efficient assay based on an electro-active polymer biosensing technology discriminating between CPE and non-CPE. The precise electrochemical signal (electrochemical impedance variations) allows the establishment of real-time objective measurement and interpretation criteria which should facilitate the accreditation process of this technology. PMID:26637378

  12. Metrological tests of a 200 L calibration source for HPGE detector systems for assay of radioactive waste drums.

    Science.gov (United States)

    Boshkova, T; Mitev, K

    2016-03-01

    In this work we present test procedures, approval criteria and results from two metrological inspections of a certified large volume (152)Eu source (drum about 200L) intended for calibration of HPGe gamma assay systems used for activity measurement of radioactive waste drums. The aim of the inspections was to prove the stability of the calibration source during its working life. The large volume source was designed and produced in 2007. It consists of 448 identical sealed radioactive sources (modules) apportioned in 32 transparent plastic tubes which were placed in a wooden matrix which filled the drum. During the inspections the modules were subjected to tests for verification of their certified characteristics. The results show a perfect compliance with the NIST basic guidelines for the properties of a radioactive certified reference material (CRM) and demonstrate the stability of the large volume CRM-drum after 7 years of operation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Avoidance bio-assays may help to test the ecological significance of soil pollution

    International Nuclear Information System (INIS)

    Martinez Aldaya, Maite; Lors, Christine; Salmon, Sandrine; Ponge, Jean-Francois

    2006-01-01

    We measured the short-term (100 min) avoidance of a soil heavily polluted by hydrocarbons by the soil springtail Folsomia candida, at six rates of dilution in a control, unpolluted soil. We compared the results with those of long-term (40-day) population tests. Five strains were compared, of varying geographical and ecological origin. When pure, the polluted soil was lethal in the long-term and avoided in the short-term by all strains. Avoidance tests, but not population tests, were able to discriminate between strains. Avoidance thresholds differed among strains. Two ecological consequences of the results were discussed: (i) toxic compounds may kill soil animals or deprive them from food, resulting in death of populations, (ii) pollution spots can be locally deprived of fauna because of escape movements of soil animals. Advantages and limitations of the method have been listed, together with proposals for their wider use in soil ecology and ecotoxicology. - Polluted soils are avoided by soil animals, a phenomenon which can be used as a cheap, sensitive tool for the early detection of environmental risk

  14. IFDOTMETER: A New Software Application for Automated Immunofluorescence Analysis.

    Science.gov (United States)

    Rodríguez-Arribas, Mario; Pizarro-Estrella, Elisa; Gómez-Sánchez, Rubén; Yakhine-Diop, S M S; Gragera-Hidalgo, Antonio; Cristo, Alejandro; Bravo-San Pedro, Jose M; González-Polo, Rosa A; Fuentes, José M

    2016-04-01

    Most laboratories interested in autophagy use different imaging software for managing and analyzing heterogeneous parameters in immunofluorescence experiments (e.g., LC3-puncta quantification and determination of the number and size of lysosomes). One solution would be software that works on a user's laptop or workstation that can access all image settings and provide quick and easy-to-use analysis of data. Thus, we have designed and implemented an application called IFDOTMETER, which can run on all major operating systems because it has been programmed using JAVA (Sun Microsystems). Briefly, IFDOTMETER software has been created to quantify a variety of biological hallmarks, including mitochondrial morphology and nuclear condensation. The program interface is intuitive and user-friendly, making it useful for users not familiar with computer handling. By setting previously defined parameters, the software can automatically analyze a large number of images without the supervision of the researcher. Once analysis is complete, the results are stored in a spreadsheet. Using software for high-throughput cell image analysis offers researchers the possibility of performing comprehensive and precise analysis of a high number of images in an automated manner, making this routine task easier. © 2015 Society for Laboratory Automation and Screening.

  15. In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

    KAUST Repository

    Castagnetti, Francesco

    2017-06-13

    Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and agerelated muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

  16. Direct immunofluorescence of normal skin in rheumatoid arthritis.

    Science.gov (United States)

    Fitzgerald, O M; Barnes, L; Woods, R; McHugh, L; Barry, C; O'Loughlin, S

    1985-11-01

    The clinical significance of previously described immunoglobulin and complement deposition in the superficial dermal vessel walls of patients with rheumatoid arthritis is unknown. In the present study, skin biopsies were obtained from the normal forearm and buttock of 48 unselected patients with rheumatoid arthritis and were examined by direct immunofluorescence (IF) for the presence of immunoglobulin (IgG,A,M) and complement (C3) in the vessel walls. Deposits of C3, IgM or IgG were detected in 10 patients. Five patients had deposits at the forearm sample alone, four patients had deposits at both biopsy sites, while one patient was positive at the buttock alone. Clinical features were similar in patients with and without vessel IF. However, patients with IF were significantly more seropositive with lower levels of complement and raised levels of serum IgA and IgM. There was also an increased level of circulating IgG immune complexes in these patients. Further analysis following exclusion of seronegative patients revealed similar results. This study suggests that the presence of vessel IF identifies a subgroup of patients who have evidence of more severe immunological disturbance.

  17. [Standardized indirect immunofluorescence. Differentiation of mitochondrial, microsomal and ribosomal antibodies].

    Science.gov (United States)

    Storch, W

    1977-02-15

    By an extensive standardisation of the indirect immunofluorescence for the demonstration espeially of mitochondrial antibodies we succeeded in recognizing atypical fluorescence patterns and in describing their exact localisation. On the basis of absorption studies with mitochondrias, microsomas and ribosomas by comparative observation of sections of liver, stomach and kidneys of rats the preferred sort of reaction and the intensity of fluorescence of antibodies against mitochondria, microsomas and ribosomas were empirically established. Antimitochondrial antibodies react above all with the parietal cells of the stomach and the distal epithelia of the tubulus of the kidney. Antibodies against microsomas of liver and kidney are characterized by a brilliant diffuse cytoplasmatic fluorescence of the hepatocytes and by a comparatively weaker fluorescence of exclusively proximal tubuli of the kidneys of rats. Antibodies against ribosomas lead to a fluorescence especially of the main cells of the stomach. The differentiation of several cytoplasmatic antibodies is among others of interest for the diagnosis of certain autoimmune diseases. Although there are numerous still unclear findings and "overlap" phenomena the existence of high titre antibodies against mitochondrias speaks for a primarily biliary cirrhosis or a pseudo-LE-syndrome, the existence of antibodies against microsomas of kidney and liver of rats for a special form of a chronically active hepatitis and the existence of the very rare antibodies against ribosomas for an active lupus erythematodes disseminatus.

  18. Determining the size and concentration dependence of gold nanoparticles in vitro cytotoxicity (IC50) test using WST-1 assay

    International Nuclear Information System (INIS)

    Rosli, Nur Shafawati binti; Rahman, Azhar Abdul; Aziz, Azlan Abdul; Shamsuddin, Shaharum

    2015-01-01

    Gold nanoparticles (AuNPs) received a great deal of attention for biomedical applications, especially in diagnostic imaging and therapeutics. Even though AuNPs have potential benefits in biomedical applications, the impact of AuNPs on human and environmental health still remains unclear. The use of AuNPs which is a high-atomic-number materials, provide advantages in terms of radiation dose enhancement. However, before this can become a clinical reality, cytotoxicity of the AuNPs has to be carefully evaluated. Cytotoxicity test is a rapid, standardized test that is very sensitive to determine whether the nanoparticles produced are harmful or benign on cellular components. In this work the size and concentration dependence of AuNPs cytotoxicity in breast cancer cell lines (MCF-7) are tested by using WST-1 assay. The sizes of AuNPs tested were 13 nm, 50 nm, and 70 nm. The cells were seeded in the 96-well plate and were treated with different concentrations of AuNPs by serial dilution for each size of AuNPs. The high concentration of AuNPs exhibit lower cell viability compared to low concentration of AuNPs. We quantified the toxicity of AuNPs in MCF-7 cell lines by determining the IC 50 values in WST-1 assays. The IC 50 values (inhibitory concentrations that effected 50% growth inhibition) of 50 nm AuNPs is lower than 13 nm and 70 nm AuNPs. Mean that, 50nm AuNPs are more toxic to the MCF-7 cells compared to smaller and larger sizes AuNPs. The presented results clearly indicate that the cytotoxicity of AuNPs depend not only on the concentration, but also the size of the nanoparticles

  19. Comparative determination of the rheumatic factor by means of agglutination, immunofluorescence and radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Jaeger, L.; Storz, H.; Hein, G.; Schlenvoigt, G. (Friedrich-Schiller-Universitaet, Jena (German Democratic Republic). Bereich Medizin)

    1982-01-01

    The rheumatic factor (RF) was determined by means of agglutination, immunofluorescence (IF) test and radioimmunoassay (RIPEGA) in random groups of 56 patients with rheumatoid arthritis (RA), 13 patients with seronegative RA and 39 patients with psoriasis arthropathica. All three methods are of equal value with regard to the number of positive results. Further classification of seronegative patients, i.e. patients with a negative agglutination reaction and the clinical symptoms of RA is possible with the IF method and, above all, by means of RIPEGA. But because of the comprehensive test devices the two methods are only an alternative. Titer differences are attributed to the different indication principles and the immunological heterogeneity of RF. An improvement of the diagnosis of activity was not possible.

  20. Comparative determination of the rheumatic factor by means of agglutination, immunofluorescence and radioimmunoassay

    International Nuclear Information System (INIS)

    Jaeger, L.; Storz, H.; Hein, G.; Schlenvoigt, G.

    1982-01-01

    The rheumatic factor (RF) was determined by means of agglutination, immunofluorescence (IF) test and radioimmunoassay (RIPEGA) in random groups of 56 patients with rheumatoid arthritis (RA), 13 patients with seronegative RA and 39 patients with psoriasis arthropathica. All three methods are of equal value with regard to the number of positive results. Further classification of seronegative patients, i.e. patients with a negative agglutination reaction and the clinical symptoms of RA is possible with the IF method and, above all, by means of RIPEGA. But because of the comprehensive test devices the two methods are only an alternative. Titer differences are attributed to the different indication principles and the immunological heterogeneity of RF. An improvement of the diagnosis of activity was not possible. (author)

  1. Design and Validation Testing of TruckScan to Assay Large Sacks of Fukushima Radioactive Debris on a Truck

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Atsuo [Canberra-Japan (Japan); Bronson, Frazier [Canberra Industries Inc. (United States)

    2015-07-01

    As a result of the March 2011 earthquake and resulting tsunami in Japan, there was a serious accident at the Fukushima Dai-ichi Nuclear Power Plant. This accident has contaminated soil and vegetation in a wide area around the plant. Decontamination projects over the last 4 years have resulted in large numbers of 1 cubic meter canvas bags of debris, commonly called Super Sacks [SS]. These are currently stored nearby where they were generated, but starting in 2015, they will be moved to various Interim Storage Facilities [ISF]. Trucks will typically carry 8-20 of these SSs. When the trucks arrive at the ISF they need to be rapidly sorted into groups according to radioactivity level, for efficient subsequent processing. Canberra Industries, Inc. [CI] has designed a new truck monitoring system 'TruckScan' for use at these ISFs. The TruckScan system must measure the entire truck loaded with multiple closely packed SSs, and generate a nuclide specific assay report showing the radioactivity in each individual SS. The Canberra-Japan office, along with Obayashi Corporation have performed validation testing to demonstrate to the regulatory authorities that the proposed technique was sufficiently accurate. These validation tests were conducted at a temporary storage area in Fukushima prefecture. Decontaminated waste of various representative types and of various levels of radioactivity was gathered and mixed to create homogeneous volumes. These volumes were sampled multiple times and assayed with laboratory HPGe detectors to determine the reference concentration of each pile. Multiple SSs were loaded from each pile. Some of the SSs were filled 50% full, others 75% full, and others 100% full, to represent the typical loading configuration of the existing SSs in the field. The content of the SSs are either sand, soil, or vegetation with densities ranging from 0.3 g/cc - 1.6 g/cc. These SSs with known concentrations of Cs-134 and Cs-137 were then loaded onto trucks in

  2. Design and Validation Testing of TruckScan to Assay Large Sacks of Fukushima Radioactive Debris on a Truck

    International Nuclear Information System (INIS)

    Suzuki, Atsuo; Bronson, Frazier

    2015-01-01

    As a result of the March 2011 earthquake and resulting tsunami in Japan, there was a serious accident at the Fukushima Dai-ichi Nuclear Power Plant. This accident has contaminated soil and vegetation in a wide area around the plant. Decontamination projects over the last 4 years have resulted in large numbers of 1 cubic meter canvas bags of debris, commonly called Super Sacks [SS]. These are currently stored nearby where they were generated, but starting in 2015, they will be moved to various Interim Storage Facilities [ISF]. Trucks will typically carry 8-20 of these SSs. When the trucks arrive at the ISF they need to be rapidly sorted into groups according to radioactivity level, for efficient subsequent processing. Canberra Industries, Inc. [CI] has designed a new truck monitoring system 'TruckScan' for use at these ISFs. The TruckScan system must measure the entire truck loaded with multiple closely packed SSs, and generate a nuclide specific assay report showing the radioactivity in each individual SS. The Canberra-Japan office, along with Obayashi Corporation have performed validation testing to demonstrate to the regulatory authorities that the proposed technique was sufficiently accurate. These validation tests were conducted at a temporary storage area in Fukushima prefecture. Decontaminated waste of various representative types and of various levels of radioactivity was gathered and mixed to create homogeneous volumes. These volumes were sampled multiple times and assayed with laboratory HPGe detectors to determine the reference concentration of each pile. Multiple SSs were loaded from each pile. Some of the SSs were filled 50% full, others 75% full, and others 100% full, to represent the typical loading configuration of the existing SSs in the field. The content of the SSs are either sand, soil, or vegetation with densities ranging from 0.3 g/cc - 1.6 g/cc. These SSs with known concentrations of Cs-134 and Cs-137 were then loaded onto trucks in

  3. Inkjet printing lanthanide doped nanorods test paper for visual assays of nitroaromatic explosives

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Liang [Department of Materials Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230026 (China); Institute of Intelligent Machines, Chinese Academy of Sciences, Hefei, Anhui 230031 (China); Mei, Qingsong [Institute of Intelligent Machines, Chinese Academy of Sciences, Hefei, Anhui 230031 (China); Yang, Lei [Department of Chemical Physics, University of Science and Technology of China, Hefei, Anhui 230026 (China); Zhang, Cheng; Liu, Renyong [Institute of Intelligent Machines, Chinese Academy of Sciences, Hefei, Anhui 230031 (China); Han, Mingyong, E-mail: my-han@imre.a-star.edu.sg [Institute of Intelligent Machines, Chinese Academy of Sciences, Hefei, Anhui 230031 (China); ASTAR, Inst Mat Res and Engn, Singapore 117602 (Singapore); Zhang, Ruilong [Department of Materials Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230026 (China); Institute of Intelligent Machines, Chinese Academy of Sciences, Hefei, Anhui 230031 (China); Zhang, Zhongping, E-mail: zpzhang@iim.ac.cn [Institute of Intelligent Machines, Chinese Academy of Sciences, Hefei, Anhui 230031 (China)

    2013-11-13

    Graphical abstract: -- Highlights: •A test paper was used for visualization of explosive 2,4,6-trinitrophenol (TNP) by the naked eye. •TNP can strongly quench the phosphorescence of NaGdF{sub 4}:Ce/Tb nanorods. •Polyethylenimine (PEI) molecules facilitate the formation of uniform NaGdF{sub 4} nanorods. •PEI molecules provide specific recognized sites for TNP by the acid–base pairing interaction. -- Abstract: The facile and sensitive strategies for detections of nitroaromatic explosives are highly desirable in many challenging environments, especially for homeland security against terrorism. Here, we inkjet printed polyethylenimine (PEI)-coated Ce, Tb co-doped NaGdF{sub 4} nanorods (NaGdF{sub 4}:Ce/Tb NRs) onto common filter paper to construct test paper for visual and instant detections of a typical explosive 2,4,6-trinitrophenol (TNP). Polyethylenimine molecules not only facilitate the formation of uniform NaGdF{sub 4} nanorods but also provide specific recognized sites for TNP by the acid–base pairing interaction. The resultant TNP bound at the surface of PEI-coated NaGdF{sub 4}:Ce/Tb NRs can strongly quench the phosphorescence with a remarkably high quenching constant by the charge transfer mechanism from NaGdF{sub 4}:Ce/Tb NRs to TNP. By printing of the probe on a piece of filter paper, trace amounts of TNP can be visually detected by the appearance of a dark color against a bright green background under a UV lamp. This test paper can detect TNP as low as 0.45 ng mm{sup −2} by the naked eye, which provides a potential application in the rapid, on-line detections of explosives.

  4. Inkjet printing lanthanide doped nanorods test paper for visual assays of nitroaromatic explosives

    International Nuclear Information System (INIS)

    Hong, Liang; Mei, Qingsong; Yang, Lei; Zhang, Cheng; Liu, Renyong; Han, Mingyong; Zhang, Ruilong; Zhang, Zhongping

    2013-01-01

    Graphical abstract: -- Highlights: •A test paper was used for visualization of explosive 2,4,6-trinitrophenol (TNP) by the naked eye. •TNP can strongly quench the phosphorescence of NaGdF 4 :Ce/Tb nanorods. •Polyethylenimine (PEI) molecules facilitate the formation of uniform NaGdF 4 nanorods. •PEI molecules provide specific recognized sites for TNP by the acid–base pairing interaction. -- Abstract: The facile and sensitive strategies for detections of nitroaromatic explosives are highly desirable in many challenging environments, especially for homeland security against terrorism. Here, we inkjet printed polyethylenimine (PEI)-coated Ce, Tb co-doped NaGdF 4 nanorods (NaGdF 4 :Ce/Tb NRs) onto common filter paper to construct test paper for visual and instant detections of a typical explosive 2,4,6-trinitrophenol (TNP). Polyethylenimine molecules not only facilitate the formation of uniform NaGdF 4 nanorods but also provide specific recognized sites for TNP by the acid–base pairing interaction. The resultant TNP bound at the surface of PEI-coated NaGdF 4 :Ce/Tb NRs can strongly quench the phosphorescence with a remarkably high quenching constant by the charge transfer mechanism from NaGdF 4 :Ce/Tb NRs to TNP. By printing of the probe on a piece of filter paper, trace amounts of TNP can be visually detected by the appearance of a dark color against a bright green background under a UV lamp. This test paper can detect TNP as low as 0.45 ng mm −2 by the naked eye, which provides a potential application in the rapid, on-line detections of explosives

  5. Dosimetry of irradiation models. The 96-well clonogenic assay for testing radiosensitivity of cell lines

    International Nuclear Information System (INIS)

    Kulmala, J.; Rantanen, V.; Turku Univ.; Pekkola-Heino, K.; Turku Univ.; Tuominen, J.; Grenman, R.; Turku Univ.

    1995-01-01

    Radiation experiments with cells in single cell suspension in test tubes and on 96-well plates were carried out and compared. The cells originated from cell lines established from carcinomas of the floor of the mouth and from endometrical carcinoma. Two irradiation models were constructed. Both models allowed the absorbed doses to the cells to be administered with a high accuracy in both experimental settings (better than 5.0%). These irradiation models were compared on cancer cell lines with dissimilar inherent radiation sensitivity and histologic type (UM-SCC-1 resistant, UM-SCC-14A sensitive, and UT-EC-2B highly sensitive); various radiation doses were used. The fractions of surviving cells as a function of radiation dose were compared: there was no significant difference between cells irradiated in test tubes and cells irradiated in 96-well plates. Thus, if the absorbed doses in cells suspended in a tube and in a plate were the same, the survival was similar regardless of the type of irradiation model. (orig.)

  6. Usefulness of Two Aspergillus PCR Assays and Aspergillus Galactomannan and β-d-Glucan Testing of Bronchoalveolar Lavage Fluid for Diagnosis of Chronic Pulmonary Aspergillosis.

    Science.gov (United States)

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

    2017-06-01

    We evaluated the usefulness of an Aspergillus galactomannan (GM) test, a β-d-glucan (βDG) test, and two different Aspergillus PCR assays of bronchoalveolar lavage fluid (BALF) samples for the diagnosis of chronic pulmonary aspergillosis (CPA). BALF samples from 30 patients with and 120 patients without CPA were collected. We calculated the sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio for each test individually and in combination with other tests. The optical density index values, as determined by receiver operating characteristic analysis, for the diagnosis of CPA were 0.5 and 100 for GM and βDG testing of BALF, respectively. The sensitivity and specificity of the GM test, βDG test, and PCR assays 1 and 2 were 77.8% and 90.0%, 77.8% and 72.5%, 86.7% and 84.2%, and 66.7% and 94.2%, respectively. A comparison of the PCR assays showed that PCR assay 1 had a better sensitivity, a better negative predictive value, and a better negative likelihood ratio and PCR assay 2 had a better specificity, a better positive predictive value, and a better positive likelihood ratio. The combination of the GM and βDG tests had the highest diagnostic odds ratio. The combination of the GM and βDG tests on BALF was more useful than any single test for diagnosing CPA. Copyright © 2017 American Society for Microbiology.

  7. Mutagenicity assayed by dominant lethality testing in mice fed a combined gamma-irradiated diet

    International Nuclear Information System (INIS)

    Rupova, I.; Katsarova, Ts.; Bajrakova, A.; Baev, I.; Tencheva, S.

    1980-01-01

    Mice fed a combined gamma-irradiated diet were examined for a mutagenic effect using the dominant lethality test. Their feed contained the following irradiated ingredients: 20% maize, 10% dried plums, and 5% walnut kernels. Taking into account cycle duration in spermatogenesis and oogenesis, males were fed this special diet throughout 56 days, and females throughout 21 days. The experiments involved three animal groups: (1) fed the special diet containing irradiated ingredients; (2) fed the special diet but with the ingredients nonirradiated; and (3) fed standard vivarium diet. Matings to provide the first generation were between one parent fed the special diet and a partner fed standard diet. With an adequate number of implants examined on day 16 of gestation, embryonic death rate was not found to be increased; hence, induction of dominant lethality from consumption of irradiated diet failed to be demonstrated

  8. The local lymph node assay compared with the human maximization test as an indicator of allergic potency in humans using patch test clinic populations.

    Science.gov (United States)

    Zaghi, Danny; Maibach, Howard I

    2009-01-01

    The human maximization test (HMT) is a method to evaluate potency in humans, while the local lymph node assay (LLNA) is a test method that allows for the measuring of the allergic potency of a substance in a rodent. It has been proposed that an EC3 value (the value obtained by the LLNA test, ie, the concentration of an allergen leading to a 3-fold increase of baseline proliferation rate) would be a reliable indicator for a compound's allergic potency in humans. This paper compares the correlation between the EC3 value of a compound and its allergic occurrence in the general population with the correlation between the HMT of the compound and its allergic occurrence in the general population, to determine the relationship to potency. The correlation values when outliers were removed from the sample were -0.56 and -0.71 for LLNA and HMT, respectively, suggesting that there is a possible 20% error margin in LLNA's ability to predict potency. The data also suggest that other factors (such as exposure) could play up to a 30% role in the determination of allergic occurrence in the general population. The potency assays might be made more clinically relevant for predicting allergic frequencies by including a frequency factor and other factors in its dermatotoxicological interpretation.

  9. A comparison of titers of anti-Brucella antibodies of naturally infected and healthy vaccinated cattle by standard tube agglutination test, microtiter plate agglutination test, indirect hemagglutination assay, and indirect enzyme-linked immunosorbent assay

    Directory of Open Access Journals (Sweden)

    Anju Mohan

    2016-07-01

    Full Text Available Aim: We determined the antibody response in cattle naturally infected with brucellosis and normal healthy adult cattle vaccinated during calf hood with strain 19. Materials and Methods: The antibody titers were measured by standard tube agglutination test (STAT, microtiter plate agglutination test (MAT, indirect hemagglutination assay (IHA, and indirect enzyme-linked immunosorbent assay (iELISA as per standard protocols. Results: The mean STAT titers were 1.963±0.345 in infected cattle and 1.200±0.155 in healthy vaccinated cattle. The difference was extremely significant (p<0.0001. The mean MAT titers were 2.244±0.727 in infected cattle and 1.200±0.155 in healthy vaccinated cattle. The difference was very significant (p<0.005. The mean IHA titers in infected cattle were 2.284±0.574, and those in healthy vaccinated cattle were 1.200±0.155. The difference was extremely significant (p=0.0002. However, the difference in mean iELISA titers of infected cattle (1.3678±0.014 and healthy vaccinated cattle (1.367±0.014 was non-significant. The infected animals showed very high titers of agglutinating antibodies compared to the vaccinated animals. However, it cannot be ascertained whether these antibodies are due to vaccine or response to infection. Since the infected animals had been vaccinated earlier, the current infection may suggest that vaccination was unable to induce protective levels of antibody. The heightened antibody response after infection may also indicate a secondary immune response to the antigens common to the vaccine strain and wild Brucella organisms. Conclusion: The brucellosis infected animals showed very high titers of agglutinating antibodies compared to the vaccinated animals.

  10. Usefulness of a single-assay chemiluminescence test (Tularaemia VIRCLIA IgG + IgM monotest) for the diagnosis of human tularemia. Comparison of five serological tests.

    Science.gov (United States)

    Cubero, África; Durántez, Carlos; Almaraz, Ana; Fernández-Lago, Luis; Gutiérrez, María P; Castro, María J; Bratos, Miguel A; Simarro, María; March, Gabriel A; Orduña, Antonio

    2018-04-01

    The aim of this work was to ascertain the usefulness of a new commercially-available single-assay chemiluminescence test (CHT) for the diagnosis of human tularemia (Tularaemia VIRCLIA IgG + IgM monotest, Vircell, Santa Fe, Granada, Spain). A total of 773 sera from 773 patients including 364 initial sera from patients with diagnosed tularemia, patients with suspected tularemia not confirmed (100), healthy people (152), patients with serology positive to Brucella (97), patients diagnosed with other infectious diseases (30), and patients diagnosed with autoimmune diseases (30) were included. All sera were tested by CHT, "in-house" microagglutination test (MAT), immunochromatographic test (ICT) (Virapid Tularaemia, Vircell, Santa Fe Granada, Spain), and "in-house" ELISA IgG, and ELISA IgM. Of the total initial sera, 334 (sensitivity 91.8%) were positive in the CHT, 332 (sensitivity 91.2%) in the MAT, 330 (sensitivity 90.7%) in the ICT, and 328 (sensitivity 90.1%) in the ELISA IgG and ELISA IgM tests. The specificity of the CHT was 96.7%; of the MAT, 100%; of the ICT, 98.7%; and of the ELISA IgG and ELISA IgM, 97.4%. In the group of patients with serology positive to Brucella, at least 12.4% of sera were positive in tularemia tests (12.4% in ELISA IgM, 13.4% in MAT, 14.4% in ICT, and 15.5% in CHT and ELISA IgG). In conclusion, CHT presents a sensitivity and specificity in early diagnosis of human tularemia, similar to MAT, ICT, and ELISA IgG and ELISA IgM. Its single assay design allows lower costs, especially in areas of low endemicity or inter-epidemic periods.

  11. Community Laboratory Testing for Cryptosporidium: Multicenter Study Retesting Public Health Surveillance Stool Samples Positive for Cryptosporidium by Rapid Cartridge Assay with Direct Fluorescent Antibody Testing.

    Directory of Open Access Journals (Sweden)

    Dawn M Roellig

    Full Text Available Cryptosporidium is a common cause of sporadic diarrheal disease and outbreaks in the United States. Increasingly, immunochromatography-based rapid cartridge assays (RCAs are providing community laboratories with a quick cryptosporidiosis diagnostic method. In the current study, the Centers for Disease Control and Prevention (CDC, the Association of Public Health Laboratories (APHL, and four state health departments evaluated RCA-positive samples obtained during routine Cryptosporidium testing. All samples underwent "head to head" re-testing using both RCA and direct fluorescence assay (DFA. Community level results from three sites indicated that 54.4% (166/305 of Meridian ImmunoCard STAT! positives and 87.0% (67/77 of Remel Xpect positives were confirmed by DFA. When samples were retested by RCA at state laboratories and compared with DFA, 83.3% (155/186 of Meridian ImmunoCard STAT! positives and 95.2% (60/63 of Remel Xpect positives were confirmed. The percentage of confirmed community results varied by site: Minnesota, 39.0%; New York, 63.9%; and Wisconsin, 72.1%. The percentage of confirmed community results decreased with patient age; 12.5% of community positive tests could be confirmed by DFA for patients 60 years of age or older. The percentage of confirmed results did not differ significantly by sex, storage temperature, time between sample collection and testing, or season. Findings from this study demonstrate a lower confirmation rate of community RCA positives when compared to RCA positives identified at state laboratories. Elucidating the causes of decreased test performance in order to improve overall community laboratory performance of these tests is critical for understanding the epidemiology of cryptosporidiosis in the United States (US.

  12. Cytotoxic drug sensitivity testing of tumor cells from patients with ovarian carcinoma using the fluorometric microculture cytotoxicity assay (FMCA).

    Science.gov (United States)

    Csoka, K; Larsson, R; Tholander, B; Gerdin, E; de la Torre, M; Nygren, P

    1994-08-01

    The automated fluorometric microculture cytotoxicity assay (FMCA) is based on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate (FDA) to fluorescein by viable cells after a 72-hr culture period in microtiter plates. The FMCA was adopted for chemosensitivity testing of tumor cells from patients with ovarian carcinoma. Thirty-seven samples of solid tumors and malignant effusions were obtained from 35 patients at diagnosis or relapse. Tumor cells from solid samples and effusions were prepared by enzymatic digestion and centrifugation, respectively, followed by Percoll or Ficoll purification. The fluorescence was proportional to the number of cells/well and considerably higher in tumor cells than in contaminating normal cells. The effect of up to 19 cytotoxic drugs was successfully assessed in 70% of the samples and there was a good correlation between drug sensitivity data reported by the FMCA and the DiSC assay performed in parallel. The overall drug sensitivity pattern in vitro corresponded well to the clinical experience. The effect of cisplatin varied considerably between patients and resistance was found also in cases not previously exposed to cytotoxic drugs. The FMCA is a rapid and simple method that seems to report clinically relevant cytotoxic drug sensitivity data in ovarian carcinomas. In the future, this method may contribute to optimizing chemotherapy by assisting in individualized drug selection and new drug development.

  13. A Dual Electrochemical Sensor Based on a Test-strip Assay for the Quantitative Determination of Albumin and Creatinine.

    Science.gov (United States)

    Yasukawa, Tomoyuki; Kiba, Yuya; Mizutani, Fumio

    2015-01-01

    A dual-electrochemical sensor based on a test-strip assay with immunochemistry and enzyme reactions has been developed for the determination of albumin and creatinine. Each nitrocellulose membrane with an immobilization area of an anti-albumin antibody or three enzymes was prepared in the device with three working electrodes for measuring albumin, creatinine, and ascorbic acid, as well as an Ag/AgCl electrode used as a counter/pseudo-reference electrode. The reactions of three enzymes were initiated by flowing a solution containing creatinine to detect an oxidation current of hydrogen peroxide. A sandwich-type immunocomplex was formed by albumin and antibody labeled with glucose oxidase (GOx). Captured GOx catalyzed the reduction of Fe(CN)6(3-) to Fe(CN)6(4-), which was oxidized electrochemically to determine the captured albumin. The responses for creatinine and albumin increased with the concentrations in millimolar order and over the range 18.75 - 150 μg mL(-1), respectively. The present sensor would be a distinct demonstration for producing quantitative dual-assays for various biomolecules used for clinical diagnoses.

  14. Comparative genotoxicity testing of Rhine river sediment extracts using the comet assay with permanent fish cell lines (RTG-2 and RTL-W1) and the Ames test

    Energy Technology Data Exchange (ETDEWEB)

    Kosmehl, T.; Braunbeck, T.; Hollert, H. [Dept. of Zoology, Aquatic Ecology and Toxicology Section, Univ. of Heidelberg (Germany); Krebs, F.; Manz, W. [German Federal Inst. of Hydrology, Koblenz (Germany); Erdinger, L. [Dept. for Hygiene and Medical Microbiology, Inst. for Hygiene, Univ. of Heidelberg (Germany)

    2004-07-01

    Whilst at least in Germany assessment strategies on the basis of chemical analysis and acute toxicity data dominated the last decades, the development of more specific biological endpoints and biomarkers in ecotoxicology is required in order to arrive at a good ecological potential and good chemical status of surface waters in the European river basins until the year 2015, as required by the European Water Framework Directive. Since sediments have for long been known to function both as a sink and as a source of pollutants in aquatic systems, and since part of the particle-associated substances have frequently been demonstrated to cause mutagenic and carcinogenic effects in aquatic organisms, particularly in fish, there is, among other requirements, an urgent need to develop, standardize and implement integrated vertebrate-based test systems addressing genotoxicity into recent sediment investigation strategies. Thus, the present study was designed to compare the suitability of two commonly used test systems, the comet assay and the Ames test, for the evaluation of the ecotoxicological burden of surface and core sediment samples from the river Rhine. Methods (or main features). In order to determine the importance of inherent enzymatic activities, two permanent fish cell lines with different biotransformation capacities, RTL-W1 and RTG-2, were compared with respect to their capability of detecting genotoxic effects in 18 surface and core sediment samples from 9 locations along the river Rhine in the comet assay with and without exogenous bioactivation. For further comparison, as a prokaryotic mutagenicity assay, the Salmonella plate incorporation assay (Ames test) with the test strains TA98 and TA100 with and without exogenous metabolic activation was used. Results and discussion. Whereas all sediment extracts induced genotoxic effects in the comet assay with RTL-W1 cells, only 12 out of 18 sediment extracts revealed significant genotoxicity in the tests with the

  15. Identification of irradiated wheat by germination test, DNA comet assay and electron spin resonance

    Science.gov (United States)

    Barros, Adilson C.; Freund, Maria Teresa L.; Villavicencio, Ana Lúcia C. H.; Delincée, Henry; Arthur, Valter

    2002-03-01

    In several countries, there has been an increase in the use of radiation for food processing thus improving the quality and sanitary conditions, inhibiting pathogenic microorganisms, delaying the natural aging process and so extending product lifetime. The need to develop analytical methods to detect these irradiated products is also increasing. The goal of this research was to identify wheat irradiated using different radiation doses. Seeds were irradiated with a gamma 60Co source (Gammacell 220 GC) in the Centro de Energia Nuclear na Agricultura and the Instituto de Pesquisas Energéticas e Nucleares. Dose rate used were 1.6 and 5.8kGy/h. Applied doses were 0.0, 0.10, 0.25, 0.50, 0.75, 1.0, and 2.0kGy. After irradiation, seeds were analysed over a 6 month period. Three different detection methods were employed to determine how irradiation had modified the samples. Screening methods consisted of a germination test measuring the inhibition of shooting and rooting and analysis of DNA fragmentation. The method of electron spin resonance spectroscopy allowed a better dosimetric evaluation. These techniques make the identification of irradiated wheat with different doses possible.

  16. Test and standardization of a radiometric method for free testosterone assay in biological materials

    International Nuclear Information System (INIS)

    Mathor, M.B.

    1983-01-01

    The principles of equilibrium dialysis technique using semi-permeable cellulose membranes were analysed and a modification was presented for the calculation of the unbound or free fraction of plasma steroids. The results were compared with those from the classical way of calculation used in the equilibrium dialysis technique in which it is necessary to include the change in weight of the dialysis bags. Technical aspects of the method were studied such as weight variation of the dialysis bags during dialysis, comparison of the results obtained using multiple plasma dilutions or one dilution only and comparing serum and plasma. The results obtained with the multiple dilutions technique were similar to those with non-diluted plasma while significantly different from those using one dilution only. The operational characteristics of the method were evaluated showing its specificity at least by the dilution test, besides being precise considering intra and interassay reproductibility. The mean + - s.d. of the percent free testosterone levels were 2.57 + - 0.45% and 1.67 + - 0.30% for males and females at the mid-follicular phase respectively. (Author) [pt

  17. Porous silicon mass spectrometry as an alternative confirmatory assay for compliance testing of methadone.

    Science.gov (United States)

    Guinan, Taryn M; Neldner, Declan; Stockham, Peter; Kobus, Hilton; Della Vedova, Christopher B; Voelcker, Nicolas H

    2017-05-01

    Porous silicon based surface-assisted laser desorption ionization mass spectrometry (pSi SALDI-MS) is an analytical technique well suited for high throughput analysis of low molecular weight compounds from biological samples. A potential application of this technology is the compliance monitoring of opioid addiction programmes, where methadone is used as a pharmacological treatment for drugs such as heroin. Here, we present the detection and quantification of methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) from water and clinical samples (saliva, urine, and plasma) from opioid dependent participants using pSi SALDI-MS. A one-step solvent phase extraction using chloroform was developed for the detection of methadone from clinical samples for analysis by pSi SALDI-MS. Liquid chromatography-mass spectrometry (LC-MS) was used as a comparative technique for the quantification of methadone from clinical saliva and plasma samples. In all cases, we obtained a good correlation of pSi SALDI-MS and LC-MS results, suggesting that pSi SALDI-MS may be an alternative procedure for high-throughput screening and quantification for application in opioid compliance testing. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  18. A spheroid-based 3-D culture model for pancreatic cancer drug testing, using the acid phosphatase assay

    International Nuclear Information System (INIS)

    Wen, Z.; Liao, Q.; Hu, Y.; You, L.; Zhou, L.; Zhao, Y.

    2013-01-01

    Current therapy for pancreatic cancer is multimodal, involving surgery and chemotherapy. However, development of pancreatic cancer therapies requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Compared to two-dimensional culture of cell monolayer, three-dimensional (3-D) models more closely mimic native tissues, since the tumor microenvironment established in 3-D models often plays a significant role in cancer progression and cellular responses to the drugs. Accumulating evidence has highlighted the benefits of 3-D in vitro models of various cancers. In the present study, we have developed a spheroid-based, 3-D culture of pancreatic cancer cell lines MIAPaCa-2 and PANC-1 for pancreatic drug testing, using the acid phosphatase assay. Drug efficacy testing showed that spheroids had much higher drug resistance than monolayers. This model, which is characteristically reproducible and easy and offers rapid handling, is the preferred choice for filling the gap between monolayer cell cultures and in vivo models in the process of drug development and testing for pancreatic cancer

  19. A spheroid-based 3-D culture model for pancreatic cancer drug testing, using the acid phosphatase assay

    Directory of Open Access Journals (Sweden)

    Z. Wen

    2013-08-01

    Full Text Available Current therapy for pancreatic cancer is multimodal, involving surgery and chemotherapy. However, development of pancreatic cancer therapies requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Compared to two-dimensional culture of cell monolayer, three-dimensional (3-D models more closely mimic native tissues, since the tumor microenvironment established in 3-D models often plays a significant role in cancer progression and cellular responses to the drugs. Accumulating evidence has highlighted the benefits of 3-D in vitro models of various cancers. In the present study, we have developed a spheroid-based, 3-D culture of pancreatic cancer cell lines MIAPaCa-2 and PANC-1 for pancreatic drug testing, using the acid phosphatase assay. Drug efficacy testing showed that spheroids had much higher drug resistance than monolayers. This model, which is characteristically reproducible and easy and offers rapid handling, is the preferred choice for filling the gap between monolayer cell cultures and in vivo models in the process of drug development and testing for pancreatic cancer.

  20. Hormone assay

    International Nuclear Information System (INIS)

    Eisentraut, A.M.

    1977-01-01

    An improved radioimmunoassay is described for measuring total triiodothyronine or total thyroxine levels in a sample of serum containing free endogenous thyroid hormone and endogenous thyroid hormone bound to thyroid hormone binding protein. The thyroid hormone is released from the protein by adding hydrochloric acid to the serum. The pH of the separated thyroid hormone and thyroid hormone binding protein is raised in the absence of a blocking agent without interference from the endogenous protein. 125 I-labelled thyroid hormone and thyroid hormone antibodies are added to the mixture, allowing the labelled and unlabelled thyroid hormone and the thyroid hormone antibody to bind competitively. This results in free thyroid hormone being separated from antibody bound thyroid hormone and thus the unknown quantity of thyroid hormone may be determined. A thyroid hormone test assay kit is described for this radioimmunoassay. It provides a 'single tube' assay which does not require blocking agents for endogenous protein interference nor an external solid phase sorption step for the separation of bound and free hormone after the competitive binding step; it also requires a minimum number of manipulative steps. Examples of the assay are given to illustrate the reproducibility, linearity and specificity of the assay. (UK)

  1. Test of equal effect per fraction and estimation of initial clonogen number in microcolony assays of survival after fractionated irradiation

    International Nuclear Information System (INIS)

    Thames, H.D.; Withers, H.R.

    1980-01-01

    In the use of multifraction microcolony assays to infer the low-dose response of in situ renewal systems such as intestinal crypts, the assumption of equal effect per dose fraction is required. Moreover, the construction of a cell-survival curve requires knowledge of the initial count of cells capable of repopulating each renewal structure. We describe a method of designing fractionation protocols which provides a regression estimate of the initial number of clonogens per renewal structure and a test of the hypothesis of equal effect per fraction. The essential factor in the experimental design is the use of common dose fractions (use of the same dose per fraction in series with different numbers of fractions). Applications of the method to data for which the assumption of equal effect per fraction holds (four-hour fractionation interval murine testis study) and does not hold (one-hour fractionation interval murine jejunal crypt study) are presented. (author)

  2. Simultaneous detection of dual biomarkers from humans exposed to organophosphorus pesticides by combination of immunochromatographic test strip and ellman assay.

    Science.gov (United States)

    Yang, Mingming; Zhao, Yuting; Wang, Limin; Paulsen, Michael; Simpson, Christopher D; Liu, Fengquan; Du, Dan; Lin, Yuehe

    2018-05-01

    A novel sandwich immunoassay based immunochromatographic test strip (ICTS) has been developed for simultaneously measuring both butyrylcholinesterase (BChE) activity and the total amount of BChE (including inhibited and active enzyme) from 70 μLpost-exposure human plasma sample. The principle of this method is based on the BChE monoclonal antibody (MAb) capable of acting as both capture antibody and detection antibody. The BChE MAb which was immobilized on the test line was able to recognize both organophosphorus BChE adducts (OP-BChE) and BChE and provided equal binding affinity, permitting detection of the total enzyme amount in post-exposure human plasma samples. The formed immunocomplexes on the test line can further be excised from the test-strip for subsequent off-line measurement of BChE activity using the Ellman assay. Therefore, dual biomarkers of BChE activity and phosphorylation (OP-BChE) will be obtained simultaneously. The whole sandwich-immunoassay was performed on one ICTS, greatly reducing analytical time. The ICTS sensor showed excellent linear responses for assaying total amount of BChE and active BChE ranging from 0.22 to 3.58nM and 0.22-7.17nM, respectively. Both the signal detection limits are 0.10nM. We validated the practical application of the proposed method to measure 124 human plasma samples from orchard workers and cotton farmers with long-term exposure to organophosphorus pesticides (OPs). The results were in highly agreement with LC/MS/MS which verified our method is extremely accurate. Combining the portability and rapidity of test strip and the compatibility of BChE MAb as both capture antibody and detection antibody, the developed method provides a baseline-free, low-cost and rapid tool for in-field monitoring of OP exposures. Copyright © 2018 Elsevier B.V. All rights reserved.

  3. Staining pattern classification of antinuclear autoantibodies based on block segmentation in indirect immunofluorescence images.

    Directory of Open Access Journals (Sweden)

    Jiaqian Li

    Full Text Available Indirect immunofluorescence based on HEp-2 cell substrate is the most commonly used staining method for antinuclear autoantibodies associated with different types of autoimmune pathologies. The aim of this paper is to design an automatic system to identify the staining patterns based on block segmentation compared to the cell segmentation most used in previous research. Various feature descriptors and classifiers are tested and compared in the classification of the staining pattern of blocks and it is found that the technique of the combination of the local binary pattern and the k-nearest neighbor algorithm achieve the best performance. Relying on the results of block pattern classification, experiments on the whole images show that classifier fusion rules are able to identify the staining patterns of the whole well (specimen image with a total accuracy of about 94.62%.

  4. Development and implementation of tPA clot lysis activity assay using ACL TOP™ hemeostasis testing system in QC laboratories

    Directory of Open Access Journals (Sweden)

    Lichun Huang

    2017-12-01

    Full Text Available This report describes the design, development, validation and long-term performance of tPA clot lysis activity assay using Advanced Chemistry Line Total Operational Performance (ACL TOP™ Homeostasis Testing System. The results of the study demonstrated robust and stable performance of the analytical method. The accuracy of the assay, expressed by percent recovery is 98–99%. The intermediate precision and repeatability precision, expressed as Relative Standard Deviation (RSD, was 3% and less than 2% respectively. The validated range is from 70% to 130% of the target potency of 5.8 × 105 IU/mg. The linearity of this range, expressed in correlation coefficient, is 0.997. After the assay is transferred to a QC laboratory, the assay retained high accuracy and precision with a success rate of >99%. Keywords: Potency assay, Clot lysis, Comparability, Automation

  5. Interlaboratory testing of porcine sera for antibodies to porcine circovirus type 2

    DEFF Research Database (Denmark)

    McNair, I.; Marshall, M.; McNeilly, F.

    2004-01-01

    A panel of 20 porcine sera was distributed to 5 laboratories across Europe and Canada. Each center was requested to test the sera for the presence of porcine circovirus type 2 antibodies using the routine assays, indirect immunofluorescence assay (IFA) and indirect immunoperoxidase monolayer assa...... than did IFA, and paraformaldehyde gave higher titers than did acetone or ethyl alcohol. This report highlights the need for standardized procedures and biologicals for this virus....

  6. Antibodies to UV irradiated DNA: the monitoring of DNA damage by ELISA and indirect immunofluorescence

    Energy Technology Data Exchange (ETDEWEB)

    Wani, A A; Gibson-D' Ambrosio, R E; D' Ambrosio, S M [Ohio State Univ., Columbus (USA). Dept. of Radiology

    1984-10-01

    The enzyme-linked immunosorbant assay (ELISA) was modified to (1) characterize antibodies raised in rabbits against UV-irradiated single-stranded DNA (UVssDNA) complexed with methylated BSA and (2) directly detect pyrimidine dimers in irradiated DNA. The antisera specifically bound to UVssDNA, UVpoly(dT) and to a limited extent to UVdsDNA and UVpoly(dC). Fifty per cent of the maximum antibody binding was observed at a 1-5000 dilution against UVssDNA. Binding to ssDNA and poly(dT) was observed only at much higher concentrations of antibody, whereas no binding to double stranded DNA (dsDNA) was observed. The extent of binding of the antibody was dependent on the UV dose to DNA and the concentration of antigen immobilized on the plate. The ability of various irradiated molecules, DNA, homopolymers and linkers to act as inhibitors of antibody binding establishes that the antigenic determinants are mainly thymine homodimers with lower affinity for cytosine dimers. Potential usefulness of the antibodies to directly quantitate pyrimidine dimers in cells exposed to UV radiation was determined by indirect immunofluorescence. Flow cytometric analysis of immunostained human lymphocytes irradiated with 254 nm radiation indicated that greater than 50% of the population had significantly higher fluorescent intensity than unirradiated cells.

  7. Reproducibility of microbial mutagenicity assays. I. Tests with Salmonella typhimurium and Escherichia coli using a standardized protocol

    International Nuclear Information System (INIS)

    Dunkel, V.C.; Zeiger, E.; Brusick, D.; McCoy, E.; McGregor, D.; Mortelmans, K.; Rosenkranz, H.S.; Simmon, V.F.

    1984-01-01

    The Salmonella/microsome test developed by Ames and his coworkers has been widely used in the evaluation of chemicals for genotoxic potential. Although the value of this assay is well recognized, there have been no comprehensive studies on the interlaboratory reproducibility of the method using a standardized protocol. A program was therefore initiated to compare the results obtained in four laboratories from testing a series of coded mutagens and nonmutagens using a standardized protocol. Additional objectives of this study were to compare male Fisher 344 rat, B6C3F1 mouse, and Syrian hamster liver S-9 preparations for the activation of chemicals; to compare Aroclor 1254-induced liver S-9 from all three species with the corresponding non-induced liver S-9's; and to compare the response of Escherichia coli WP-2 uvrA with the Salmonella typhimurium tester strains recommended by Ames. Since a primary use of in vitro microbial mutagenesis tests is the identification of potential carcinogens by their mutagenicity, the authors decided to compare the animal species and strains used by the National Cancer Institute/National Toxicology Program (NCI/NTP) for animal carcinogenicity studies

  8. In vitro testing of chemotherapeutic drug combinations in acute myelocytic leukaemia using the fluorometric microculture cytotoxicity assay (FMCA).

    Science.gov (United States)

    Larsson, R; Fridborg, H; Kristensen, J; Sundström, C; Nygren, P

    1993-05-01

    The fluorometric microculture cytotoxicity assay (FMCA) was employed for analysing the effect of different chemotherapeutic drug combinations and their single constituents in 44 cases of acute myelocytic leukaemia (AML). A large heterogeneity with respect to cell kill was observed for all combinations tested, the interactions ranging from antagonistic to synergistic in terms of the multiplicative concept for drug interactions. However, an 'additive' model provided a significantly better fit of the data compared to the effect of the most active single agent of the combination (Dmax) for several common antileukaemic drug combinations. When the two interaction models were related to treatment outcome 38% of the non-responders showed preference for the additive model whereas the corresponding figure for responders was 80%. Overall, in 248 of 290 (85%) tests performed with drug combinations, there was an agreement between the effect of the combination and that of the most active single component. Direct comparison of Dmax and the combination for correlation with clinical outcome demonstrated only minor differences in the ability to predict drug resistance. The results show that FMCA appear to report drug interactions in samples from patients with AML in accordance with clinical experience. Furthermore, testing single agents as a substitute for drug combinations may be adequate for detection of clinical drug resistance to combination therapy in AML.

  9. Clinical evaluation of thyrotropin-releasing hormone (TRH) test with a sensitive immunoradiometric thyrotropin (TSH) assay kit

    International Nuclear Information System (INIS)

    Nakamura, Saeko; Demura, Reiko; Yamanaka, Yukako; Ishiwatari, Naoko; Jibiki, Kazuko; Odagiri, Emi; Demura, Hiroshi

    1987-01-01

    Thyrotropin-releasing hormone (TRH) test was performed using a commercially available immunoradiometric thyrotropin (TSH) assay kit (RIA-gnost hTSH) in patients with endocrine diseases. The basal serum concentration of TSH ranged from 0.2 to 2.9 μU/ml in healthy subjects. The values for endocrine diseases, except for Graves' disease, were almost within the normal range. A significant increase in TSH values caused by TRH test was observed in females compared with males (4.4 - 24.7 μU/ml vs 4.1 - 12.3 μU/ml). In cases of Graves' disease, there was a good correlation between the basal TSH value and the response of TSH to TRH. However, in the other endocrine diseases, including acromegaly, prolactinoma, anorexia nervosa, Cushing syndrome, and hypopituitarism, the response of TSH to TRH did not necessarily correlated with the basal TSH value. TRH test would be of value in elucidating pathophysiologic features, as well as in accurately diagnosing secretion reserve of TSH. (Namekawa, K.)

  10. Clinical evaluation of thyrotropin-releasing hormone (TRH) test with a sensitive immunoradiometric thyrotropin (TSH) assay kit

    Energy Technology Data Exchange (ETDEWEB)

    Nakamura, Saeko; Demura, Reiko; Yamanaka, Yukako; Ishiwatari, Naoko; Jibiki, Kazuko; Odagiri, Emi; Demura, Hiroshi

    1987-10-01

    Thyrotropin-releasing hormone (TRH) test was performed using a commercially available immunoradiometric thyrotropin (TSH) assay kit (RIA-gnost hTSH) in patients with endocrine diseases. The basal serum concentration of TSH ranged from 0.2 to 2.9 ..mu..U/ml in healthy subjects. The values for endocrine diseases, except for Graves' disease, were almost within the normal range. A significant increase in TSH values caused by TRH test was observed in females compared with males (4.4 - 24.7 ..mu..U/ml vs 4.1 - 12.3 ..mu..U/ml). In cases of Graves' disease, there was a good correlation between the basal TSH value and the response of TSH to TRH. However, in the other endocrine diseases, including acromegaly, prolactinoma, anorexia nervosa, Cushing syndrome, and hypopituitarism, the response of TSH to TRH did not necessarily correlated with the basal TSH value. TRH test would be of value in elucidating pathophysiologic features, as well as in accurately diagnosing secretion reserve of TSH. (Namekawa, K.).

  11. Mutagenicity testing in the Salmonella typhimurium assay of phenolic compounds and phenolic fractions obtained from smokehouse smoke condensates.

    Science.gov (United States)

    Pool, B L; Lin, P Z

    1982-08-01

    Smokehouse smoke, which is used for flavouring meat products, was investigated for its mutagenic activity in the Salmonella typhimurium assay. We were chiefly concerned with the fractions free of polycyclic aromatic hydrocarbons but containing phenol compounds, which are responsible for the preservative and aromatizing properties of the smoke. The most abundantly occurring phenol compounds (phenol, cresols, 2,4-dimethylphenol, brenzcatechine, syringol, eugenol, vanilline and guaiacol) gave negative results when they were tested for mutagenicity at five concentrations up to 5000 micrograms/plate, with and without S-9 mix, using five strains of S. typhimurium. Even when phenol was further investigated in a variety of test conditions, no induction of his+ revertants was observed. When smokehouse smoke was condensed and fractionated the majority of the various phenolic fractions also gave negative results when tested at five concentrations using five strains of S. typhimurium. However there was a slight increase in the number of revertants in a few cases. The presence in the phenolic fractions of very small amounts of mutagenic impurities, the nature of which needs further investigation, cannot be excluded. These results support the further development of non-hazardous smoke-aroma preparations, based on the phenolic components of smokehouse smoke.

  12. Mutagenicity testing in the Salmonella typhimurium assay of phenolic compounds and phenolic fractions obtained from smokehouse smoke condensates

    Energy Technology Data Exchange (ETDEWEB)

    Pool, B.L.; Lin, P.Z.

    1982-08-01

    Smokehouse smoke, which is used for flavouring meat products, was investigated for its mutagenic activity in the Salmonella typhimurium assay. We were chiefly concerned with the fractions free of polycyclic aromatic hydrocarbons but containing phenol compounds, which are responsible for the preservative and aromatizing properties of the smoke. The most abundantly occurring phenol compounds (phenol, cresols, 2,4-dimethylphenol, brenzcatechine, syringol, eugenol, vanilline and guaiacol) gave negative results when they were tested for mutagenicity at five concentrations up to 5000 micrograms/plate, with and without S-9 mix, using five strains of S. typhimurium. Even when phenol was further investigated in a variety of test conditions, no induction of his+ revertants was observed. When smokehouse smoke was condensed and fractionated the majority of the various phenolic fractions also gave negative results when tested at five concentrations using five strains of S. typhimurium. However there was a slight increase in the number of revertants in a few cases. The presence in the phenolic fractions of very small amounts of mutagenic impurities, the nature of which needs further investigation, cannot be excluded. These results support the further development of non-hazardous smoke-aroma preparations, based on the phenolic components of smokehouse smoke.

  13. Simple PCR assays improve the sensitivity of HIV-1 subtype B drug resistance testing and allow linking of resistance mutations.

    Directory of Open Access Journals (Sweden)

    Jeffrey A Johnson

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

  14. Comparison of three stool antigen assays with the 13C- urea breath test for the primary diagnosis of Helicobacter pylori infection and monitoring treatment outcome.

    LENUS (Irish Health Repository)

    Hooton, Carmel

    2012-02-03

    BACKGROUND: The urea breath test (UBT) is the gold-standard non-invasive test for the detection of Helicobacter pylori infection, however, the lack of availability of the UBT due to the high cost of the test, and in particular the need for expensive analytical instrumentation, limits the usefulness of this method. Stool antigen assays may offer an alternative non-invasive method for the diagnosis of infection. OBJECTIVE: To compare the accuracy of three stool antigen assays (HpSA, IDEIA HpStAR, and ImmunoCard STAT) against the UBT for the primary diagnosis of H. pylori infection and for monitoring treatment outcome. METHODS: A total of 102 patients attending two gastroenterology day-case clinics for the investigation of dyspepsia were included. Each patient provided breath and stool samples for analysis. Patients who tested positive for H. pylori by the validated UBT were prescribed triple therapy and invited to return for repeat breath and stool sample analysis 6 weeks post-treatment. RESULTS: Of the 102 patients tested, 48 were diagnosed with H. pylori infection by the UBT. The HpSA assay interpreted 38 of these as positive (79% sensitive). Of the 54 UBT-negative patients the HpSA assay interpreted all 54 as negative (100% specific). The IDEIA HpStAR assay correctly identified 44 patients as positive (92% sensitive) and 50 as negative (92.5% specific). The ImmunoCard STAT assay interpreted 38 patients as positive (79% sensitive) and 52 as negative (96.3% specific). CONCLUSION: The findings indicate that the IDEIA HpStAR stool antigen kit is the most accurate assay of the three assays evaluated, and possibly represents a viable alternative to the UBT for the primary diagnosis of H. pylori infection and for monitoring treatment outcome.

  15. HIV Incidence Estimates Using the Limiting Antigen Avidity EIA Assay at Testing Sites in Kiev City, Ukraine: 2013-2014

    Science.gov (United States)

    Kruglov, Yuri; Yurchenko, Alexander

    2016-01-01

    Objective To estimate HIV incidence and highlight the characteristics of persons at greatest risk of HIV in the Ukraine capital, Kiev. Method Residual samples from newly-diagnosed persons attending the Kiev City AIDS Centre were tested for evidence of recent HIV infection using an avidity assay. Questions on possible risk factors for HIV acquisition and testing history were introduced. All persons (≥16yrs) presenting for an HIV test April’13–March’14 were included. Rates per 100,000 population were calculated using region-specific denominators. Results During the study period 6370 individuals tested for HIV. Of the 467 individuals newly-diagnosed with HIV, 21 had insufficient samples for LAg testing. Of the remaining 446, 39 (8.7%) were classified as recent with an avidity index <1.5ODn, 10 were reclassified as long-standing as their viral load was <1000 copies/mL, resulting in 29 (6.5%) recent HIV infections. The only independent predictor for a recent infection was probable route of exposure, with MSM more likely to present with a recent infection compared with heterosexual contact [Odds Ratio 8.86; 95%CI 2.65–29.60]. We estimated HIV incidence at 21.5 per 100,000 population, corresponding to 466 new infections. Using population estimates for MSM and PWID, incidence was estimated to be between 2289.6 and 6868.7/100,000 MSM, and 350.4 for PWID. Conclusion A high proportion of persons newly-infected remain undiagnosed, with MSM disproportionally affected with one in four newly-HIV-diagnosed and one in three recently-HIV-infected. Our findings should be used for targeted public health interventions and health promotion. PMID:27276170

  16. HIV Incidence Estimates Using the Limiting Antigen Avidity EIA Assay at Testing Sites in Kiev City, Ukraine: 2013-2014.

    Directory of Open Access Journals (Sweden)

    Ruth Simmons

    Full Text Available To estimate HIV incidence and highlight the characteristics of persons at greatest risk of HIV in the Ukraine capital, Kiev.Residual samples from newly-diagnosed persons attending the Kiev City AIDS Centre were tested for evidence of recent HIV infection using an avidity assay. Questions on possible risk factors for HIV acquisition and testing history were introduced. All persons (≥16yrs presenting for an HIV test April'13-March'14 were included. Rates per 100,000 population were calculated using region-specific denominators.During the study period 6370 individuals tested for HIV. Of the 467 individuals newly-diagnosed with HIV, 21 had insufficient samples for LAg testing. Of the remaining 446, 39 (8.7% were classified as recent with an avidity index <1.5ODn, 10 were reclassified as long-standing as their viral load was <1000 copies/mL, resulting in 29 (6.5% recent HIV infections. The only independent predictor for a recent infection was probable route of exposure, with MSM more likely to present with a recent infection compared with heterosexual contact [Odds Ratio 8.86; 95%CI 2.65-29.60]. We estimated HIV incidence at 21.5 per 100,000 population, corresponding to 466 new infections. Using population estimates for MSM and PWID, incidence was estimated to be between 2289.6 and 6868.7/100,000 MSM, and 350.4 for PWID.A high proportion of persons newly-infected remain undiagnosed, with MSM disproportionally affected with one in four newly-HIV-diagnosed and one in three recently-HIV-infected. Our findings should be used for targeted public health interventions and health promotion.

  17. HIV Incidence Estimates Using the Limiting Antigen Avidity EIA Assay at Testing Sites in Kiev City, Ukraine: 2013-2014.

    Science.gov (United States)

    Simmons, Ruth; Malyuta, Ruslan; Chentsova, Nelli; Karnets, Iryna; Murphy, Gary; Medoeva, Antonia; Kruglov, Yuri; Yurchenko, Alexander; Copas, Andrew; Porter, Kholoud

    2016-01-01

    To estimate HIV incidence and highlight the characteristics of persons at greatest risk of HIV in the Ukraine capital, Kiev. Residual samples from newly-diagnosed persons attending the Kiev City AIDS Centre were tested for evidence of recent HIV infection using an avidity assay. Questions on possible risk factors for HIV acquisition and testing history were introduced. All persons (≥16yrs) presenting for an HIV test April'13-March'14 were included. Rates per 100,000 population were calculated using region-specific denominators. During the study period 6370 individuals tested for HIV. Of the 467 individuals newly-diagnosed with HIV, 21 had insufficient samples for LAg testing. Of the remaining 446, 39 (8.7%) were classified as recent with an avidity index <1.5ODn, 10 were reclassified as long-standing as their viral load was <1000 copies/mL, resulting in 29 (6.5%) recent HIV infections. The only independent predictor for a recent infection was probable route of exposure, with MSM more likely to present with a recent infection compared with heterosexual contact [Odds Ratio 8.86; 95%CI 2.65-29.60]. We estimated HIV incidence at 21.5 per 100,000 population, corresponding to 466 new infections. Using population estimates for MSM and PWID, incidence was estimated to be between 2289.6 and 6868.7/100,000 MSM, and 350.4 for PWID. A high proportion of persons newly-infected remain undiagnosed, with MSM disproportionally affected with one in four newly-HIV-diagnosed and one in three recently-HIV-infected. Our findings should be used for targeted public health interventions and health promotion.

  18. Performance of two Aspergillus IgG EIA assays compared with the precipitin test in chronic and allergic aspergillosis.

    Science.gov (United States)

    Baxter, C G; Denning, D W; Jones, A M; Todd, A; Moore, C B; Richardson, M D

    2013-04-01

    Detection of Aspergillus IgG antibodies is important in the diagnosis of chronic pulmonary aspergillosis and allergic bronchopulmonary aspergillosis. Immunoprecipitation techniques to detect these antibodies appear to lack sensitivity and accurate quantitation compared with enzyme immunoassays (EIA). This study assessed the performance of two commercial EIAs compared with counterimmunoelectrophoresis (CIE). This was a prospective cohort study of 175 adult patients with chronic or allergic pulmonary aspergillosis. Aspergillus IgG antibodies were detected using CIE, Phadia ImmunoCap Aspergillus IgG and Bio-Rad Platelia Aspergillus IgG. Inter-assay reproducibility was determined for each method and 25 patients had two serum samples analysed within a 6-month interval. When compared with CIE, both ImmunoCap and Platelia Aspergillus IgG had good sensitivity (97 and 93%, respectively) for detection of Aspergillus IgG antibodies. The level of agreement between the two EIAs for positive results was good, but the concentration of antibodies was not correlated between the tests or with CIE titre. ImmunoCap IgG inter-assay coefficient of variation was 5%, whereas Platelia IgG was 33%. Median ImmunoCap IgG values for CPA and allergic aspergillosis were 95 and 32 mg/L, respectively, whereas Platelia IgG values were >80 and 6 AU/mL. The direction of CIE titre change over 6 months was mirrored by ImmunoCap IgG levels in 92% of patients, and by Platelia IgG in 72% of patients. Both ImmunoCap and Platelia Aspergillus IgG EIAs are sensitive measures of Aspergillus IgG antibodies compared with CIE. However, ImmunoCap appears to have better reproducibility and may be more suitable for monitoring patient disease. © 2012 The Authors Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.

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

    Science.gov (United States)

    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. Copyright 2009 Wiley-Liss, Inc.

  20. Direct nitrate reductase assay versus microscopic observation drug susceptibility test for rapid detection of MDR-TB in Uganda.

    Directory of Open Access Journals (Sweden)

    Freddie Bwanga

    Full Text Available The most common method for detection of drug resistant (DR TB in resource-limited settings (RLSs is indirect susceptibility testing on Lowenstein-Jensen medium (LJ which is very time consuming with results available only after 2-3 months. Effective therapy of DR TB is therefore markedly delayed and patients can transmit resistant strains. Rapid and accurate tests suitable for RLSs in the diagnosis of DR TB are thus highly needed. In this study we compared two direct techniques--Nitrate Reductase Assay (NRA and Microscopic Observation Drug Susceptibility (MODS for rapid detection of MDR-TB in a high burden RLS. The sensitivity, specificity, and proportion of interpretable results were studied. Smear positive sputum was collected from 245 consecutive re-treatment TB patients attending a TB clinic in Kampala, Uganda. Samples were processed at the national reference laboratory and tested for susceptibility to rifampicin and isoniazid with direct NRA, direct MODS and the indirect LJ proportion method as reference. A total of 229 specimens were confirmed as M. tuberculosis, of these interpretable results were obtained in 217 (95% with either the NRA or MODS. Sensitivity, specificity and kappa agreement for MDR-TB diagnosis was 97%, 98% and 0.93 with the NRA; and 87%, 95% and 0.78 with the MODS, respectively. The median time to results was 10, 7 and 64 days with NRA, MODS and the reference technique, respectively. The cost of laboratory supplies per sample was low, around 5 USD, for the rapid tests. The direct NRA and MODS offered rapid detection of resistance almost eight weeks earlier than with the reference method. In the study settings, the direct NRA was highly sensitive and specific. We consider it to have a strong potential for timely detection of MDR-TB in RLS.

  1. Factor VII assay performance: an analysis of the North American Specialized Coagulation Laboratory Association proficiency testing results.

    Science.gov (United States)

    Zantek, N D; Hsu, P; Refaai, M A; Ledford-Kraemer, M; Meijer, P; Van Cott, E M

    2013-06-01

    The performance of factor VII (FVII) assays currently used by clinical laboratories was examined in North American Specialized Coagulation Laboratory Association (NASCOLA) proficiency tests. Data from 12 surveys conducted between 2008 and 2010, involving 20 unique specimens plus four repeat-tested specimens, were analyzed. The number of laboratories per survey was 49-54 with a total of 1224 responses. Numerous reagent/instrument combinations were used. For FVII > 80 or 50 U/dL, among commonly used methods, one thromboplastin and one calibrator produced results 5-6 U/dL higher and another thromboplastin and calibrator produced results 5-6 U/dL lower than all other methods, and human thromboplastin differed from rabbit by +7.6 U/dL. Preliminary evidence suggests these differences could be due to the calibrator. For FVII <50 U/dL, differences among the commonly used reagents and calibrators were generally not significant. © 2013 Blackwell Publishing Ltd.

  2. A dual-docking microfluidic cell migration assay (D2-Chip) for testing neutrophil chemotaxis and the memory effect.

    Science.gov (United States)

    Yang, Ke; Wu, Jiandong; Xu, Guoqing; Xie, Dongxue; Peretz-Soroka, Hagit; Santos, Susy; Alexander, Murray; Zhu, Ling; Zhang, Michael; Liu, Yong; Lin, Francis

    2017-04-18

    Chemotaxis is a classic mechanism for guiding cell migration and an important topic in both fundamental cell biology and health sciences. Neutrophils are a widely used model to study eukaryotic cell migration and neutrophil chemotaxis itself can lead to protective or harmful immune actions to the body. While much has been learnt from past research about how neutrophils effectively navigate through a chemoattractant gradient, many interesting questions remain unclear. For example, while it is tempting to model neutrophil chemotaxis using the well-established biased random walk theory, the experimental proof was challenged by the cell's highly persistent migrating nature. A special experimental design is required to test the key predictions from the random walk model. Another question that has interested the cell migration community for decades concerns the existence of chemotactic memory and its underlying mechanism. Although chemotactic memory has been suggested in various studies, a clear quantitative experimental demonstration will improve our understanding of the migratory memory effect. Motivated by these questions, we developed a microfluidic cell migration assay (so-called dual-docking chip or D 2 -Chip) that can test both the biased random walk model and the memory effect for neutrophil chemotaxis on a single chip enabled by multi-region gradient generation and dual-region cell alignment. Our results provide experimental support for the biased random walk model and chemotactic memory for neutrophil chemotaxis. Quantitative data analyses provide new insights into neutrophil chemotaxis and memory by making connections to entropic disorder, cell morphology and oscillating migratory response.

  3. A new rapid colourimetric method for testing Mycobacterium tuberculosis susceptibility to isoniazid and rifampicin: a crystal violet decolourisation assay

    Directory of Open Access Journals (Sweden)

    Ahmet Yilmaz Coban

    2014-04-01

    Full Text Available The aim of this study was to investigate the performance of a new and accurate method for the detection of isoniazid (INH and rifampicin (RIF resistance among Mycobacterium tuberculosis isolates using a crystal violet decolourisation assay (CVDA. Fifty-five M. tuberculosis isolates obtained from culture stocks stored at -80ºC were tested. After bacterial inoculation, the samples were incubated at 37ºC for seven days and 100 µL of CV (25 mg/L stock solution was then added to the control and sample tubes. The tubes were incubated for an additional 24-48 h. CV (blue/purple was decolourised in the presence of bacterial growth; thus, if CV lost its colour in a sample containing a drug, the tested isolate was reported as resistant. The sensitivity, specificity, positive predictive value, negative predictive value and agreement for INH were 92.5%, 96.4%, 96.1%, 93.1% and 94.5%, respectively, and 88.8%, 100%, 100%, 94.8% and 96.3%, respectively, for RIF. The results were obtained within eight-nine days. This study shows that CVDA is an effective method to detect M. tuberculosis resistance to INH and RIF in developing countries. This method is rapid, simple and inexpensive. Nonetheless, further studies are necessary before routine laboratory implementation.

  4. Variability in the recognition of distinctive immunofluorescence patterns in different brands of HEp-2 cell slides

    Directory of Open Access Journals (Sweden)

    Alessandra Dellavance

    2013-06-01

    Full Text Available INTRODUCTION: Indirect immunofluorescence on HEp-2 cells is considered the gold standard for the detection of autoantibodies against cellular antigens. However, the culture conditions, cell fixation and permeabilization processes interfere directly in the preservation and spatial distribution of antigens. Therefore, one can assume that certain peculiarities in the processing of cellular substrate may affect the recognition of indirect immunofluorescence patterns associated with several autoantibodies. OBJECTIVE: To evaluate a panel of serum samples representing nuclear, nucleolar, cytoplasmic, mitotic apparatus, and chromosome plate patterns on HEp-2 cell substrates from different suppliers. MATERIALS AND METHODS: Seven blinded observers, independent from the three selected reference centers, evaluated 17 samples yielding different nuclear, nucleolar, cytoplasmic and mitotic apparatus patterns on HEp-2 cell slides from eight different brands. The slides were coded to maintain confidentiality of both brands and participating centers. RESULTS: The 17 HEp-2 cell patterns were identified on most substrates. Nonetheless, some slides showed deficit in the expression of several patterns: nuclear coarse speckled/U1-ribonucleoprotein associated with antibodies against RNP (U1RNP, centromeric protein F (CENP-F, proliferating cell nuclear antigen (PCNA, cytoplasmic fine speckled associated with anti-Jo-1 antibodies (histidyl synthetase, nuclear mitotic apparatus protein 1 (NuMA-1 and nuclear mitotic apparatus protein 2 (NuMA-2. CONCLUSION: Despite the overall good quality of the assessed HEp-2 substrates, there was considerable inconsistency in results among different commercial substrates. The variations may be due to the evaluated batches, hence generalizations cannot be made as to the respective brands. It is recommended that each new batch or new brand be tested with a panel of reference sera representing the various patterns.

  5. Comparison of Brucella immunoglobulin M and G flow assays with serum agglutination and 2-mercaptoethanol tests in the diagnosis of brucellosis

    NARCIS (Netherlands)

    Zeytinoğlu, Ayşin; Turhan, Ajda; Altuğlu, Imre; Bilgiç, Altinay; Abdoel, Theresia H.; Smits, Henk L.

    2006-01-01

    The diagnostic value of Brucella IgM/IgG flow assays was evaluated in comparison with serum agglutination and 2-mercaptoethanol tests by testing a selection of serum samples submitted to the laboratory because of clinical suspicion of brucellosis. All 39 admission and 11 follow-up samples that

  6. Risk of iatrogenic human immunodeficiency virus infection through transfusion of blood tested by inappropriately stored or expired rapid antibody assays in a Zambian hospital

    NARCIS (Netherlands)

    Consten, E. C.; van der Meer, J. T.; de Wolf, F.; Heij, H. A.; Henny, P. C.; van Lanschot, J. J.

    1997-01-01

    BACKGROUND: The purpose of this study was to estimate the risk of human immunodeficiency virus (HIV) infection via the transfusion of blood tested by inappropriately stored or expired rapid antibody assays in Zambia. STUDY DESIGN AND METHODS: Surgical patients (n = 370) were tested with antibody

  7. Dual Testing Algorithm of BED-CEIA and AxSYM Avidity Index Assays Performs Best in Identifying Recent HIV Infection in a Sample of Rwandan Sex Workers

    NARCIS (Netherlands)

    Braunstein, Sarah L.; Nash, Denis; Kim, Andrea A.; Ford, Ken; Mwambarangwe, Lambert; Ingabire, Chantal M.; Vyankandondera, Joseph; van de Wijgert, Janneke H. H. M.

    2011-01-01

    To assess the performance of BED-CEIA (BED) and AxSYM Avidity Index (Ax-AI) assays in estimating HIV incidence among female sex workers (FSW) in Kigali, Rwanda. Eight hundred FSW of unknown HIV status were HIV tested; HIV-positive women had BED and Ax-AI testing at baseline and ≥12 months later to

  8. Test of mutagenicity of an irradiated standard diet for laboratory animals in the host-mediated assay with salmonella typhimurium TA 1530

    International Nuclear Information System (INIS)

    Muenzner, R.; Renner, H.W.

    1976-01-01

    Feed irradiated at a dose of 3 Mrad was tested for mutagenic activity in the host-mediated assay with the mouse as host and Salmonella typhimurium TA 1530 as indicator organism. In the in vivo and in the in vitro comparative test the irradiated feed showed no mutagenic effect. (orig.) [de

  9. Detection of Lawsonia intracellularis in formalin-fixed porcine intestinal tissue samples: comparison of immunofluorescence and in-situ hybridization, and evaluation of the effects of controlled autolysis.

    Science.gov (United States)

    Jensen, T K; Boesen, H T; Vigre, H; Boye, M

    2010-01-01

    Two methods, an immunofluorescence assay (IFA; with a Lawsonia intracellularis-specific monoclonal antibody) and fluorescent in-situ hybridization (FISH; with a specific oligonucleotide probe targeting 16S ribosomal RNA of the bacterium), were compared for their ability to detect L. intracellularis (the cause of porcine proliferative enteritis [PE]) in formalin-fixed samples of intestinal tissue. Of 69 intestinal samples with gross lesions of PE, 63 were positive by both FISH and IFA, but six were positive only by IFA. This indicated that the sensitivity of FISH was 91% that of IFA. However, both methods had a specificity of 100%. Fifty normal porcine intestines were negative by both tests. IFA was much less susceptible than FISH to the effects of autolysis. Thus, three of nine samples from pigs with PE were FISH-negative after being kept at 20 degrees C for 4 days, and seven were FISH negative after 2 weeks; after 4 weeks at this temperature, however, six of the nine samples were still IFA positive. After being kept at 4 degrees C for 12 weeks, the majority of samples (> or = 66%) were positive by both methods.

  10. Integration of an optical CMOS sensor with a microfluidic channel allows a sensitive readout for biological assays in point-of-care tests.

    Science.gov (United States)

    Van Dorst, Bieke; Brivio, Monica; Van Der Sar, Elfried; Blom, Marko; Reuvekamp, Simon; Tanzi, Simone; Groenhuis, Roelf; Adojutelegan, Adewole; Lous, Erik-Jan; Frederix, Filip; Stuyver, Lieven J

    2016-04-15

    In this manuscript, a microfluidic detection module, which allows a sensitive readout of biological assays in point-of-care (POC) tests, is presented. The proposed detection module consists of a microfluidic flow cell with an integrated Complementary Metal-Oxide-Semiconductor (CMOS)-based single photon counting optical sensor. Due to the integrated sensor-based readout, the detection module could be implemented as the core technology in stand-alone POC tests, for use in mobile or rural settings. The performance of the detection module was demonstrated in three assays: a peptide, a protein and an antibody detection assay. The antibody detection assay with readout in the detection module proved to be 7-fold more sensitive that the traditional colorimetric plate-based ELISA. The protein and peptide assay showed a lower limit of detection (LLOD) of 200 fM and 460 fM respectively. Results demonstrate that the sensitivity of the immunoassays is comparable with lab-based immunoassays and at least equal or better than current mainstream POC devices. This sensitive readout holds the potential to develop POC tests, which are able to detect low concentrations of biomarkers. This will broaden the diagnostic capabilities at the clinician's office and at patient's home, where currently only the less sensitive lateral flow and dipstick POC tests are implemented. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Using standard serology blood tests to diagnose latent syphilis

    Directory of Open Access Journals (Sweden)

    G. L. Katunin

    2016-01-01

    Full Text Available Goal. To conduct a comparative assessment of the results of regulated serological tests obtained as a result of blood tests in patients suffering from latent syphilis. Materials and methods. The authors examined 187 patient medical records with newly diagnosed latent syphilis in FGBU GNTsDK (State Research Center for Dermatology, Venereology and Cosmetology, Health Ministry of the Russian Federation, in 2006-2015. The results of patient blood tests were analyzed with the use of non-treponemal (microprecipitation test/RPR and treponemal (passive hemagglutination test, immune-enzyme assay (IgA, IgM, IgG, IFabs, immunofluorescence test and Treponema pallidum immobilization test serology tests. Results. According to the results of blood tests of latent syphilis patients, the largest number of positive results was obtained as a result of treponemal serology tests such as immune-enzyme assay (100%, passive hemagglutination test (100% and IFabs (100%. The greatest number of negative results was observed in non-treponemal (microprecipitation test/RPR serology tests: in 136 (72.7% patients; evidently positive results (4+ test results were obtained in 8 (4.3% patients only. According to the results of a comparative analysis of blood tests in patients suffering from latent syphilis obtained with the use of treponemal serology tests, the greatest number of evidently positive results (4+ was noted for the passive hemagglutination test (67.9%. Negative treponemal test results were obtained with the use of the immunofluorescence test and Treponema pallidum immobilization test (21.9% and 11.8% of cases, respectively. Moreover, weakly positive results prevailed for the immunofluorescence test: in 65 (34.7% patients. Conclusion. These data confirm that the following treponemal tests belong to the most reliable ones for revealing patients suffering from latent syphilis: immune-enzyme assay, passive hemagglutination test and IFabs.

  12. Evaluation of the tuberculin skin test and the interferon-γ release assay for TB screening in French healthcare workers

    Directory of Open Access Journals (Sweden)

    Raffi Francois

    2009-11-01

    Full Text Available Abstract Introduction Using French cut-offs for the Tuberculin Skin Test (TST, results of the TST were compared with the results of an Interferon-γ Release Assay (IGRA in Healthcare Workers (HCW after contact to AFB-positive TB patients. Methods Between May 2006 and May 2007, a total of 148 HCWs of the University Hospital in Nantes, France were tested simultaneously with IGRA und TST. A TST was considered to indicate recent latent TB infection (LTBI if an increase of >10 mm or if TST ≥ 15 mm for those with no previous TST result was observed. For those with a positive TST, chest X-ray was performed and preventive chemotherapy was offered. Results All HCWs were BCG-vaccinated. The IGRA was positive in 18.9% and TST ≥ 10 mm was observed in 65.5%. A recent LTBI was believed to be highly probable in 30.4% following TST. Agreement between IGRA and TST was low (kappa 0.041. In 10 (16.7% out of 60 HCWs who needed chest X-ray following TST the IGRA was positive. In 9 (20% out of 45 HCWs to whom preventive chemotherapy was offered following TST the IGRA was positive. Of those considered TST-negative following the French guidelines, 20.5% were IGRA-positive. In a two-step strategy - positive TST verified by IGRA - 18 out of 28 (64.3% IGRA-positive HCWs would not have been detected using French guidelines for TST interpretation. Conclusion The introduction of IGRA in contact tracings of BCG-vaccinated HCWs reduces X-rays and preventive chemotherapies. Increasing the cut-off for a positive TST does not seem to be helpful to overcome the effect of BCG vaccination on TST.

  13. A sensitive, reproducible and objective immunofluorescence analysis method of dystrophin in individual fibers in samples from patients with duchenne muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Chantal Beekman

    Full Text Available Duchenne muscular dystrophy (DMD is characterized by the absence or reduced levels of dystrophin expression on the inner surface of the sarcolemmal membrane of muscle fibers. Clinical development of therapeutic approaches aiming to increase dystrophin levels requires sensitive and reproducible measurement of differences in dystrophin expression in muscle biopsies of treated patients with DMD. This, however, poses a technical challenge due to intra- and inter-donor variance in the occurrence of revertant fibers and low trace dystrophin expression throughout the biopsies. We have developed an immunofluorescence and semi-automated image analysis method that measures the sarcolemmal dystrophin intensity per individual fiber for the entire fiber population in a muscle biopsy. Cross-sections of muscle co-stained for dystrophin and spectrin have been imaged by confocal microscopy, and image analysis was performed using Definiens software. Dystrophin intensity has been measured in the sarcolemmal mask of spectrin for each individual muscle fiber and multiple membrane intensity parameters (mean, maximum, quantiles per fiber were calculated. A histogram can depict the distribution of dystrophin intensities for the fiber population in the biopsy. This method was tested by measuring dystrophin in DMD, Becker muscular dystrophy, and healthy muscle samples. Analysis of duplicate or quadruplicate sections of DMD biopsies on the same or multiple days, by different operators, or using different antibodies, was shown to be objective and reproducible (inter-assay precision, CV 2-17% and intra-assay precision, CV 2-10%. Moreover, the method was sufficiently sensitive to detect consistently small differences in dystrophin between two biopsies from a patient with DMD before and after treatment with an investigational compound.

  14. Detection of antinuclear antibodies by solid-phase immunoassays and immunofluorescence analysis

    DEFF Research Database (Denmark)

    Fenger, Mogens; Wiik, Allan; Høier-Madsen, Mimi

    2004-01-01

    -established rheumatic disorders, the newly developed EIA in which HEp-2 extracts were included had sensitivities and specificities comparable to or in some instances better than the IFA. The assays without HEp-2 extracts included had significantly lower sensitivities and specificities. In the outpatient population, up...... to 51% of patients had positive ANA tests that did not correspond to classic ANA-associated disease. However, in the assays in which the HEp-2 extracts were not included, the false-positive rate was ... and was mostly extracts included had a low sensitivity but a high specificity, particularly in nonselected populations. The choice of test is highly dependent on the clinical setting in which...

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

    Science.gov (United States)

    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

  16. A comparative evaluation of in vitro skin sensitisation tests: the human cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA).

    Science.gov (United States)

    Ashikaga, Takao; Sakaguchi, Hitoshi; Sono, Sakiko; Kosaka, Nanae; Ishikawa, Makie; Nukada, Yuko; Miyazawa, Masaaki; Ito, Yuichi; Nishiyama, Naohiro; Itagaki, Hiroshi

    2010-08-01

    We previously developed the human cell-line activation test (h-CLAT) in vitro skin sensitisation test, based on our reported finding that a 24-hour exposure of THP-1 cells (a human monocytic leukaemia cell line) to sensitisers is sufficient to induce the augmented expression of CD86 and CD54. The aim of this study is to confirm the predictive value of h-CLAT for skin sensitisation activity by employing a larger number of test chemicals. One hundred chemicals were selected, according to their categorisation in the local lymph node assay (LLNA), as being: extreme, strong, moderate and weak sensitisers, and non-sensitisers. The correlation of the h-CLAT results with the LLNA results was 84%. There were some false negatives (e.g. benzoyl peroxide, hexyl cinnamic aldehyde) and some false positives (e.g. 1-bromobutane, diethylphthalate). Eight out of the 9 false negatives (89%) were water-insoluble chemicals. The h-CLAT could positively predict not only extreme and strong sensitisers, but also moderate and weak sensitisers, though the detection rates of weak sensitisers and non-sensitisers were comparatively low. Some sensitisers enhanced both CD86 and CD54 levels, and some enhanced the level of only one of them. The use of the combination of CD86 and CD54 induction as a positive indicator, improved the accuracy of the test. In conclusion, the h-CLAT is expected to be a useful cell-based in vitro method for predicting skin sensitisation potential. 2010 FRAME.

  17. In vitro genotoxicity of neutral red after photo-activation and metabolic activation in the Ames test, the micronucleus test and the comet assay.

    Science.gov (United States)

    Guérard, Melanie; Zeller, Andreas; Singer, Thomas; Gocke, Elmar

    2012-07-04

    Neutral red (Nr) is relatively non-toxic and is widely used as indicator dye in many biological test systems. It absorbs visible light and is known to act as a photosensitizer, involving the generation of reactive oxygen species (type-I reaction) and singlet oxygen (type-II reaction). The mutagenicity of Nr was determined in the Ames test (with Salmonella typhimurium strains TA1535, TA97, TA98, TA98NR, TA100, and TA102) with and without metabolic activation, and with and without photo-activation on agar plates. Similarly to the situation following metabolic activation, photo-mutagenicity of Nr was seen with all Salmonella strains tested, albeit with different effects between these strains. To our knowledge, Nr is the only photo-mutagen showing such a broad action. Since the effects are also observed in strains not known to be responsive to ROS, this indicates that ROS production is not the sole mode of action that leads to photo-genotoxicity. The reactive species produced by irradiation are short-lived as pre-irradiation of an Nr solution did not produce mutagenic effects when added to the bacteria. In addition, mutagenicity in TA98 following irradiation was stronger than in the nitroreductase-deficient strain TA98NR, indicating that nitro derivatives that are transformed by bacterial nitroreductase to hydroxylamines appear to play a role in the photo-mutagenicity of Nr. Photo-genotoxicity of Nr was further investigated in the comet assay and micronucleus test in L5178Y cells. Concentration-dependent increases in primary DNA damage and in the frequency of micronuclei were observed after irradiation. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. Immunofluorescence pattern of antinuclear antibody and its association with autoantibody profile in systemic lupus erythematosus

    Directory of Open Access Journals (Sweden)

    Sadia Sharmin

    2016-08-01

    Full Text Available Background: Antinuclear antibody (ANA is useful in the diagnosis of systemic lupus erythematosus (SLE. Association of specific autoantibodies with the immunofluorescence pattern of ANA in SLE as noted in Western literature has been taken as reference in all over the world. However, in Bangladesh such research work or data correlating the autoantibodies and their ANA patterns is inadequate. Objective: To identify an association between immunofluorescence patterns of antinuclear antibody on HEp-2 cell and more specific antinuclear reactivities (e.g. anti-dsDNA and anti-extractable nuclear antigen in the serum samples of SLE patients.Methods: Serum samples of 37 SLE patients who were diagnosed by ARA (American Rheumatism Association classification criteria and laboratory tests, attending at lupus clinic of Bangabandhu Sheikh Mujib Medical University (BSMMU during the study period of six months were subjected for ANA testing by Indirect Imrnunofluorescence (IIF on HEp-2 cell, anti-dsDNA by ELISA and anti- extractable nuclear antigen (anti-ENA by Dot Immunoblot. Dot blot strips were tested for anti-Sm, anti-RNP, anti-SSA/Ro, and anti-SSB/La. Results: Out of 37 SLE patients 32 (86.5% cases were ANA positive by IIF on HEp-2 cell. ANA positive sera exhibited three fluorescence patterns such as speckled (43.7%, peripheral (34.3% and homogenous pattern (21.8%. Peripheral pattern (100% was strongly associated with anti-dsDNA (p<0.05 and homogenous pattern (85.7% was also predominantly associated with anti-dsDNA (p<0.05. Speckled pattern (85.6% was significantly associated with anti-ENA (p<0.05. Anti-dsDNA was positive in 75% of SLE cases and majority (45.8% of which showed peripheral pattern whereas anti-ENA was positive in 48.6% cases and majority (70.5% of which showed speckled pattern. The most commonly identified antinuclear autoreactivity was directed towards anti-RNP (22.2% then anti-Sm (16.6%, anti-SSA (16.6% and anti-SSB (11.1 %. Multiple anti

  19. SRTC Spreadsheet to Determine Relative Percent Difference (RPD) for Duplicate Waste Assay Results and to Perform the RPD Acceptance Test

    International Nuclear Information System (INIS)

    Casella, V.R.

    2002-01-01

    This report documents the calculations and logic used for the Microsoft(R) Excel spreadsheet that is used at the 773-A Solid Waste Assay Facility for evaluating duplicate analyses, and validates that the spreadsheet is performing these functions correctly

  20. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas

    2013-01-21

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling the macroscopic observation. Such tests are most often used to explore antibody-antigen reactions. Agglutination has been used for protein assays using a biotin/streptavidin system as well as a hybridization based assay. The agglutination systems are prone to selftermination of the linking analyte, prone to active site saturation and loss of agglomeration at high analyte concentrations. We investigated the molecular target/ligand interaction, explaining the common agglutination problems related to analyte self-termination, linkage of the analyte to the same bead instead of different microbeads. We classified the agglutination process into three kinds of assays: a two- component assay, a three-component assay and a stepped three- component assay. Although we compared these three kinds of assays for recognizing DNA and protein molecules, the assay can be used for virtually any molecule, including ions and metabolites. In total, the optimized assay permits detecting analytes with high sensitivity in a short time, 5 min, at room temperature. Such a system is appropriate for POC testing.

  1. Correlation of antinuclear antibody immunofluorescence patterns with immune profile using line immunoassay in the Indian scenario

    Directory of Open Access Journals (Sweden)

    Sebastian Wendy

    2010-07-01

    Full Text Available Background: Immunity status, individual response to disease and types of antibodies produced are well known to vary from person to person, place to place and probably from population to population. A broad spectrum of specific auto antibodies that have so far been associated with specific rheumatic diseases, as noted in Western literature, has been well taken as a reference standard all over the world. There is neither research work nor any data correlating the auto antibodies and their antinuclear antibody (ANA patterns with the immunoprofile in the Indian population to date. Aims: To understand a definite association between ANA patterns and specific antibodies in the serum in the Indian study population and to document similarities / differences with the West. Settings and Design: This prospective and retrospective double blind study was undertaken on the South Indian population referred for ANA testing by Indirect Immunofluorescence method and by immunoline methods. Materials and Methods: Serum samples of patients from a random South Indian population who sought medical help for rheumatic disease were subjected for ANA testing by indirect immunofluorescence (IIF method and line immunoassay during the study period of 27 months. Serum samples were processed in dilution of 1:100 using HEp - 2010 / liver biochip (Monkey (EUROIMMUN AG. The serum samples which were further processed for line immunoassay were treated in 1:100 dilution on nylon strips coated with recombinant and purified antigens as discrete lines with plastic backing (EUROIMMUN AG coated with antigens nRNP / Sm, Sm, SSA, Ro-52, SSB, Scl-70, PM-Scl, PCNA, Jo-1, CENP-B, dsDNA, nucleosomes, histones, ribosomal protein-P, anti-mitochondrial antibodies (AMA-M2 along with a control band. The analysis was done by comparing the intensity of the reaction with positive control line by image analysis. Results: The antinuclear antibody indirect immunofluorescence (ANA - IIF patterns obtained

  2. Specificity of the Tuberculin Skin Test and the T-SPOT."TB" Assay among Students in a Low-Tuberculosis Incidence Setting

    Science.gov (United States)

    Talbot, Elizabeth A.; Harland, Dawn; Wieland-Alter, Wendy; Burrer, Sherry; Adams, Lisa V.

    2012-01-01

    Objective: Interferon-[gamma] release assays (IGRAs) are an important tool for detecting latent "Mycobacterium tuberculosis" infection (LTBI). Insufficient data exist about IGRA specificity in college health centers, most of which screen students for LTBI using the tuberculin skin test (TST). Participants: Students at a low-TB incidence college…

  3. Modification of the BAX System PCR assay for detecting Salmonella in beef, produce, and soy protein isolate. Performance Tested Method 100201.

    Science.gov (United States)

    Peng, Linda X; Wallace, Morgan; Andaloro, Bridget; Fallon, Dawn; Fleck, Lois; Delduco, Dan; Tice, George

    2011-01-01

    The BAX System PCR assay for Salmonella detection in foods was previously validated as AOAC Research Institute (RI) Performance Tested Method (PTM) 100201. New studies were conducted on beef and produce using the same media and protocol currently approved for the BAX System PCR assay for E. coli O157:H7 multiplex (MP). Additionally, soy protein isolate was tested for matrix extension using the U.S. Food and Drug Administration-Bacteriological Analytical Manual (FDA-BAM) enrichment protocols. The studies compared the BAX System method to the U.S. Department of Agriculture culture method for detecting Salmonella in beef and the FDA-BAM culture method for detecting Salmonella in produce and soy protein isolate. Method comparison studies on low-level inoculates showed that the BAX System assay for Salmonella performed as well as or better than the reference method for detecting Salmonella in beef and produce in 8-24 h enrichment when the BAX System E. coli O157:H7 MP media was used, and soy protein isolate in 20 h enrichment with lactose broth followed by 3 h regrowth in brain heart infusion broth. An inclusivity panel of 104 Salmonella strains with diverse serotypes was tested by the BAX System using the proprietary BAX System media and returned all positive results. Ruggedness factors involved in the enrichment phase were also evaluated by testing outside the specified parameters, and none of the factors examined affected the performance of the assay.

  4. Comparison of an enzyme linked immunosorbent assay (ELISA) and a radioallergosorbent test (RAST) for detection of IgE antibodies to Brugia malayi

    NARCIS (Netherlands)

    Wahyuni, Sitti; van Ree, Ronald; Mangali, Andarias; Supali, Taniawati; Yazdanbakhsh, Maria; Sartono, Erliyani

    2003-01-01

    The enzyme linked immunosorbent assay (ELISA) for specific IgE antibodies to Brugia malayi was compared with the radioallergosorbent test (RAST) for use in immunoepidemiological studies of lymphatic filariasis. Sera used were from individuals (aged 5-82 years) living in an area endemic for lymphatic

  5. Radioreceptor opioid assay

    International Nuclear Information System (INIS)

    Miller, R.J.; Chang, K.-J.

    1981-01-01

    A radioreceptor assay is described for assaying opioid drugs in biological fluids. The method enables the assay of total opioid activity, being specific for opioids as a class but lacking specificity within the class. A radio-iodinated opioid and the liquid test sample are incubated with an opiate receptor material. The percentage inhibition of the binding of the radio-iodinated compound to the opiate receptor is calculated and the opioid activity of the test liquid determined from a standard curve. Examples of preparing radio-iodinated opioids and assaying opioid activity are given. A test kit for the assay is described. Compared to other methods, this assay is cheap, easy and rapid. (U.K.)

  6. Application of the micronucleus test and comet assay in Trachemys callirostris erythrocytes as a model for in situ genotoxic monitoring.

    Science.gov (United States)

    Zapata, Lina M; Bock, Brian C; Orozco, Luz Yaneth; Palacio, Jaime A

    2016-05-01

    Trachemys callirostris is a turtle species endemic to northern South America. In northern Colombia it occurs in the middle and lower Magdalena River drainage and its principal tributaries (lower Cauca and San Jorge rivers) and in other minor drainages such as the lower Sinú River. In recent years, industrial, agricultural, and mining activities have altered natural habitats in Colombia where this species occurs, and many of the pollutants released there are known to induce genetic alterations in wildlife species. The micronucleus test and comet assay are two of the most widely used methods to characterize DNA damage induced by physical and chemical agents in wildlife species, but have not been employed previously for genotoxic evaluations in T. callirostris. The goal of this study was to optimize these genotoxic biomarkers for T. callirostris erythrocytes in order to establish levels of DNA damage in this species and thereby evaluate its potential as a sentinel species for monitoring genotoxic effects in freshwater environments in northern Colombia. Both genotoxic techniques were applied on peripheral blood erythrocytes from 20 captive-reared T. callirostris individuals as a negative control, as well as from samples obtained from 49 individuals collected in Magangué (Magdalena River drainage) and 24 individuals collected in Lorica (Sinú River drainage) in northern Colombia. Negative control individuals exhibited a baseline frequency of micronuclei of 0.78±0.58 and baseline values for comet tail length and tail moment of 3.34±0.24µm and 10.70±5.5, respectively. In contrast, samples from both field sites exhibited significantly greater evidence of genotoxic effects for both tests. The mean MN frequencies in the samples from Magangué and Lorica were 8.04±7.08 and 12.19±12.94, respectively. The mean tail length for samples from Magangué and Lorica were 5.78±3.18 and 15.46±7.39, respectively. Finally, the mean tail moment for samples from Magangué and

  7. The DNA comet assay and the germination test in detection of food treated by ionizing radiation; Teste do cometa e teste de germinacao na deteccao do tratamento de alimentos com a radiacao ionizante

    Energy Technology Data Exchange (ETDEWEB)

    Huachaca, Nelida Simona Marin

    2002-07-01

    Two methods of irradiated food detection, one biochemical, the comet assay and, other biological, the germination test, were applied in bovine meat and fruit samples. The comet assay detects the damage on DNA caused by ionizing radiation. The germination test evaluates the sensitivity to radiation of seeds as for germination ability, shooting and, rooting. The samples were irradiated in gamma font and electron accelerator. For bovine meat samples, the doses were 0.0; 2.5; 4.5 e 7.0 kGy at chilled condition and, 0.0; 2.5; 4.5; 7.0 e 8.5 kGy at frozen conditions. For fruit samples such as melon, watermelon, apple, orange, papaya and, tomato, the doses were: 0.0; 0.5; 0.75; 1.0; 2.0 e 4.0 kGy. The differences between the gamma rays and the electron beam effects on extent of DNA migration and, on shooting and rooting, showed to be similar. The comet assay, under neutral conditions, permitted to discriminate between irradiated and unirradiated bovine meat samples, until one month of storage. Also, it was possible to distinguish, by the comet assay, the control sample with regard to irradiated fruit, at doses as low as 0,5 kGy. In the germination test, the root length was the best parameter to discriminate irradiated and unirradiated samples of melon, watermelon and tomato, while the germination percent was the best parameter for apple and orange. (author)

  8. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas; Castro, David; Foulds, Ian G.; Parameswaran, Ash M.; Sumanpreet, K. Chhina

    2013-01-01

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling

  9. Determination of low tetanus or diphtheria antitoxin titers in sera by a toxin neutralization assay and a modified toxin-binding inhibition test

    Directory of Open Access Journals (Sweden)

    M.H. Sonobe

    2007-01-01

    Full Text Available A method for the screening of tetanus and diphtheria antibodies in serum using anatoxin (inactivated toxin instead of toxin was developed as an alternative to the in vivo toxin neutralization assay based on the toxin-binding inhibition test (TOBI test. In this study, the serum titers (values between 1.0 and 19.5 IU measured by a modified TOBI test (Modi-TOBI test and toxin neutralization assays were correlated (P < 0.0001. Titers of tetanus or diphtheria antibodies were evaluated in serum samples from guinea pigs immunized with tetanus toxoid, diphtheria-tetanus or triple vaccine. For the Modi-TOBI test, after blocking the microtiter plates, standard tetanus or diphtheria antitoxin and different concentrations of guinea pig sera were incubated with the respective anatoxin. Twelve hours later, these samples were transferred to a plate previously coated with tetanus or diphtheria antitoxin to bind the remaining anatoxin. The anatoxin was then detected using a peroxidase-labeled tetanus or diphtheria antitoxin. Serum titers were calculated using a linear regression plot of the results for the corresponding standard antitoxin. For the toxin neutralization assay, L+/10/50 doses of either toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus detection, or in guinea pigs for anti-diphtheria detection. Both assays were suitable for determining wide ranges of antitoxin levels. The linear regression plots showed high correlation coefficients for tetanus (r² = 0.95, P < 0.0001 and for diphtheria (r² = 0.93, P < 0.0001 between the in vitro and the in vivo assays. The standardized method is appropriate for evaluating titers of neutralizing antibodies, thus permitting the in vitro control of serum antitoxin levels.

  10. A quantum dot-immunofluorescent labeling method to investigate the interactions between a crinivirus and its whitefly vector

    Directory of Open Access Journals (Sweden)

    James C. K. Ng

    2013-04-01

    Full Text Available Successful vector-mediated plant virus transmission entails an intricate but poorly understood interplay of interactions among virus, vector, and plant. The complexity of interactions requires continually improving/evaluating tools and methods for investigating the determinants that are central to mediating virus transmission. A recent study using an organic fluorophore (Alexa Fluor-based immunofluorescent localization assay demonstrated that specific retention of Lettuce infectious yellows virus (LIYV virions in the anterior foregut or cibarium of its whitefly vector is required for virus transmission. Continuous exposure of organic fluorophore to high excitation light intensity can result in diminished or loss of signals, potentially confounding the identification of important interactions associated with virus transmission. This limitation can be circumvented by incorporation of photostable fluorescent nanocrystals, such as quantum dots (QDs, into the assay. We have developed and evaluated a QD-immunofluorescent labeling method for the in vitro and in situ localization of LIYV virions based on the recognition specificity of streptavidin-conjugated QD605 (S-QD605 for biotin-conjugated anti-LIYV IgG (B-αIgG. IgG biotinylation was verified in a blot overlay assay by probing SDS-PAGE separated B-αIgG with S-QD605. Immunoblot analyses of LIYV using B-αIgG and S-QD605 resulted in a virus detection limit comparable to that of DAS-ELISA. In membrane feeding experiments, QD signals were observed in the anterior foregut or cibarium of virion-fed whitefly vectors but absent in those of virion-fed whitefly non-vectors. Specific virion retention in whitefly vectors corresponded with successful virus transmission. A fluorescence photobleaching assay of viruliferous whiteflies fed B-αIgG and S-QD605 vs. those fed anti-LIYV IgG and Alexa Fluor 488-conjugated IgG revealed that QD signal was stable and deteriorated ∼7 to 8 fold slower than that of Alexa

  11. Tuberculin skin testing and treatment modulates interferon-gamma release assay results for latent tuberculosis in migrants.

    Directory of Open Access Journals (Sweden)

    Matthew K O'Shea

    Full Text Available Identifying latent tuberculosis infection (LTBI in people migrating from TB endemic regions to low incidence countries is an important control measure. However, no prospective longitudinal comparisons between diagnostic tests used in such migrant populations are available.To compare commercial interferon (IFN-gamma release assays (IGRAs and the tuberculin skin test (TST for diagnosing LTBI in a migrant population, and the influence of antecedent TST and LTBI treatment on IGRA performance.This cohort study, performed from February to September 2012, assessed longitudinal IGRA and TST responses in Nepalese military recruits recently arrived in the UK. Concomitant T-SPOT.TB, QFT-GIT and TST were performed on day 0, with IGRAs repeated 7 and 200 days later, following treatment for LTBI if necessary.166 Nepalese recruits were prospectively assessed. At entry, 21 individuals were positive by T-SPOT.TB and 8 individuals by QFT-GIT. There was substantial agreement between TST and T-SPOT.TB positives at baseline (71.4% agreement; κ = 0.62; 95% CI:0.44-0.79, but only moderate concordance between positive IGRAs (38.1% agreement; κ = 0.46; 95% CI:0.25-0.67. When reassessed 7 days following TST, numbers of IGRA-positive individuals changed from 8 to 23 for QFT-GIT (p = 0.0074 and from 21 to 23 for T-SPOT.TB (p = 0.87. This resulted in an increase in IGRA concordance to substantial (64.3% agreement; κ = 0.73; 95% CI:0.58-0.88. Thus, in total on day 0 and day 7 after testing, 29 out of 166 participants (17.5% provided a positive IGRA and of these 13 were TST negative. Two hundred days after the study commenced and three months after treatment for LTBI was completed by those who were given chemoprophylaxis, 23 and 21 participants were positive by T-SPOT.TB or QFT-GIT respectively. When individual responses were examined longitudinally within this population 35% of the day 7 QFT-GIT-positive, and 19% T-SPOT.TB-positive individuals, were

  12. Detection of viral infection by immunofluorescence in formalin-fixed tissues, pretreated with trypsin

    Directory of Open Access Journals (Sweden)

    O. M. Barth

    1988-06-01

    Full Text Available The presence of viral antigen in sections from formalin-fixed and paraffin-embedded human tissues was demonstrated by trypsin digestion followed by direct or indirect immunofluorescence. The specimens may be used for retrospective diagnosis. The immunofluorescence technique has to be adapted to the suspected virus infection on the basis of previous histopathology study. Variations of trypsin concentration time and temperature of incubation, expose different viral antigens and have to be previously tested for each unknown system. For measles virus detection in lung a stronger digestion has to be applied as compared to adenovirus or respiratory disease viruses in the same tisue. Flavivirus in liver tissue needs a weaker digestion. The reproducibility of the method makes it useful as a routine technique in diagnosis of virus infection.A presença de antígeno viral em cortes de tecidos humanos fixados em formol e emblocados em parafina foi demonstrada pela digestão com tripsina foi demonstrada pela ingestão com tripsina seguida de imunofluorescência direta ou indireta. Os espécimens podem ser utilizados para diagnoses retrospectivas. A técnica da imunofluorescência deve ser adaptada à infecção viral suspeita segundo diagnosie histopatológica prévia. Os parâmetros para a digestão do tecido pela tripsina, relacionados à concentração, duração de atuação e temperatura, expõem diferentes antígenos virais e devem ser previamente testados para cada sistema a ser estabelecido. Uma digestão mais intensa deve ser aplicada para a detecção do vírus do sarampo em tecido pulmonar do que para adenovírus ou vírus respiratório sincicial no mesmo tecido. Por outro lado, o vírus da febre amarela em tecido de fígado necessita de uma digestão mais fraca.

  13. Improved testing of recent HIV-1 infections with the BioRad avidity assay compared to the limiting antigen avidity assay and BED Capture enzyme immunoassay: evaluation using reference sample panels from the German Seroconverter Cohort.

    Directory of Open Access Journals (Sweden)

    Andrea Hauser

    Full Text Available BACKGROUND: The variety and limitations of current laboratory methods for estimating HIV-incidence has driven attempts to improve and standardize the performance of serological 'Tests for Recent HIV-Infections' (TRI. Primary and follow-up HIV-1 positive plasma samples from individuals with well-defined dates of infection collected as part of the German Seroconverter Cohort provided specimens highly suitable for use in comparing the performance of three TRIs: the AWARE™ BED™ EIA HIV-1 Incidence test (BED-CEIA, Genetic systems HIV-1/HIV-2 Plus O EIA antibody avidity-based assay (BioRad Avidity and Sedia™ HIV-1 LAg Avidity EIA (LAg Avidity. METHODS: The evaluation panel included 180 specimens: 44 from antiretroviral (ARV-naïve individuals with recently acquired HIV-infection (≤ 130 days; 25 B and 19 non-B subtypes and 136 from long-term (>12 months infected individuals [101 ARV-naïve subtype B, 16 non-B subtypes, 14 ARV-treated individuals, 5 slow progressors (SLP]. RESULTS: For long-term infected, ARV-naïve individuals the false recent rates (FRR of both the BioRad and LAg Avidity assays were 2% (2/101 for subtype B and 6% (1/16 for subtype 'non-B', while the FRR of the BED-CEIA was 7% (7/101 for subtype B and 25% (4/16 for subtype 'non-B' (all p>0.05. Misclassification of ARV-treated individuals and SLP was rare by LAg (1/14, 0/5 and BioRad Avidity assays (2/14, 1/5 but more frequent by BED-CEIA (5/14, 3/5. Among recently-infected individuals (subtype B, 60% (15/25 were correctly classified by BED-CEIA, 88% (22/25 by BioRad Avidity and significantly fewer by LAg (48%, 12/25 compared to BioRad Avidity (p = 0.005 with a higher true-recency rate among non-B infections for all assays. CONCLUSIONS: This study using well-characterized specimens demonstrated lower FRRs for both avidity methods than with the BED-CEIA. For recently infected individuals the BioRad Avidity assay was shown to give the most accurate results.

  14. Development of an in vitro Assay, based on the BioFilm Ring Test®, for Rapid Profiling of Biofilm-Growing Bacteria

    Directory of Open Access Journals (Sweden)

    Enea Gino Di Domenico

    2016-09-01

    Full Text Available Microbial biofilm represents a major virulence factor associated with chronic and recurrent infections. Pathogenic bacteria embedded in biofilms are highly resistant to environmental and chemical agents, including antibiotics and therefore difficult to eradicate. Thus, reliable tests to assess biofilm formation by bacterial strains as well as the impact of chemicals or antibiotics on biofilm formation represent desirable tools for a most effective therapeutic management and microbiological risk control. Current methods to evaluate biofilm formation are usually time-consuming, costly, and hardly applicable in the clinical setting.The aim of the present study was to develop and assess a simple and reliable in vitro procedure for the characterization of biofilm-producing bacterial strains for future clinical applications based on the BioFilm Ring Test® (BRT technology. The procedure developed for clinical testing (cBRT can provide an accurate and timely (5 hours measurement of biofilm formation for the most common pathogenic bacteria seen in clinical practice. The results gathered by the cBRT assay were in agreement with the traditional crystal violet (CV staining test, according to the kappa coefficient test (kappa = 0.623. However, the cBRT assay showed higher levels of specificity (92.2% and accuracy (88.1% as compared to CV. The results indicate that this procedure offers an easy, rapid and robust assay to test microbial biofilm and a promising tool for clinical microbiology.

  15. A feasibility study of adenosine triphosphate-based chemotherapy response assay (ATP-CRA) as a chemosensitivity test for lung cancer.

    Science.gov (United States)

    Kang, Shin Myung; Park, Moo Suk; Chang, Joon; Kim, Se Kyu; Kim, Haeryoung; Shin, Dong-Hwan; Chung, Kyung Young; Kim, Dae Joon; Sohn, Joo Hyuk; Choi, Sung Ho; Kim, Jeongmi; Yoon, Eun Jin; Kim, Joo-Hang

    2005-08-01

    A chemosensitivity test can reflect the differences in responses of individual cancer patients to chemotherapeutic agents. The adenosine triphosphate-based chemotherapy response assay (ATP-CRA) is an accurate method, which does not require a large amount of tissue specimen. So far, no studies have evaluated the utility of the ATP-CRA in Korea. Therefore, we investigated the clinical usefulness of the ATP-CRA in 53 patients with lung cancer. Tumor tissues were obtained from bronchoscopic biopsies or surgical resections. The validity of ATP-CRA was assessed focusing on the success rate, experimental error level (intraassay mean coefficient of variation [CV]) and reproducibility. The overall success rate of ATP-CRA was 90.6% (48/53). Normal cells were effectively eliminated from the tumor tissues with the use of ficoll gradient centrifugation and immunomagnetic separation, which was confirmed using loss of heterozygosity analysis of the 3p deletion. The mean CV of ATP assays was 10.5+/-4.6%. The reproducibility of ATP assays was 94+/-3.8%. The results of the ATP assays were reported to physicians within 7 days of specimen collection. More than 6 anticancer drugs were tested on the tumor specimens obtained from bronchoscopic biopsies. The ATP-CRA is a stable, accurate and potentially practical chemosensitivity test in patients with lung cancer.

  16. Comparative analysis of the intracerebral mouse protection test and serological method for potency assays of pertussis component in DTP vaccine

    Directory of Open Access Journals (Sweden)

    Denise Cristina Souza Matos

    2012-06-01

    Full Text Available The aim of this study was to compare the PSPT standardized in-house as an alternative to MPT for potency assays of pertussis component. Statistical analyses have showed similar pertussis potency values when PSPT was compared to MPT. Significant correlation between the potency results obtained by in vivo and in vitro assays was also been observed. Results by PSPT have demonstrated reproducibility and accuracy for potency pertussis control and this approach has been considered promising for use at least during the steps of production.

  17. Lack of genotoxicity of potassium iodate in the alkaline comet assay and in the cytokinesis-block micronucleus test. Comparison to potassium bromate.

    Science.gov (United States)

    Poul, J M; Huet, S; Godard, T; Sanders, P

    2004-02-01

    Iodine could be added to the diet of human population in the form of iodide or iodate but iodate had not been adequately tested for genotoxicity and carcinogenicity. In the present study, genotoxic effects of potassium iodate were evaluated in vitro using the alkaline comet assay and the cytokinesis-block micronucleus assay on CHO cells and compared to halogenate salt analogues potassium bromate and chlorate and also to their respective reduced forms (potassium iodide, bromide and chloride). The results showed that the comet assay failed to detect the presence of DNA damage after a treatment of cells by potassium iodate for concentrations up to 10 mM. This absence of primary DNA damage was confirmed in the cytokinesis-block micronucleus assay. In the same way, results showed that potassium chlorate as well as potassium iodide, bromide and chloride did not induced DNA damage in the alkaline comet assay for doses up to 10 mM. By contrast, potassium bromate exposure led to an increase in both DNA damage and frequency of micronucleated cells. The repair of bromate-induced DNA damage was incomplete 24 h after the end of treatment. These results seem to indicate that potassium bromate would induce DNA damage by several mechanisms besides oxidative stress.

  18. HIV incidence in rural South Africa: comparison of estimates from longitudinal surveillance and cross-sectional cBED assay testing.

    Directory of Open Access Journals (Sweden)

    Till Bärnighausen

    Full Text Available The BED IgG-Capture Enzyme Immunoassay (cBED assay, a test of recent HIV infection, has been used to estimate HIV incidence in cross-sectional HIV surveys. However, there has been concern that the assay overestimates HIV incidence to an unknown extent because it falsely classifies some individuals with non-recent HIV infections as recently infected. We used data from a longitudinal HIV surveillance in rural South Africa to measure the fraction of people with non-recent HIV infection who are falsely classified as recently HIV-infected by the cBED assay (the long-term false-positive ratio (FPR and compared cBED assay-based HIV incidence estimates to longitudinally measured HIV incidence.We measured the long-term FPR in individuals with two positive HIV tests (in the HIV surveillance, 2003-2006 more than 306 days apart (sample size n = 1,065. We implemented four different formulae to calculate HIV incidence using cBED assay testing (n = 11,755 and obtained confidence intervals (CIs by directly calculating the central 95(th percentile of incidence values. We observed 4,869 individuals over 7,685 person-years for longitudinal HIV incidence estimation. The long-term FPR was 0.0169 (95% CI 0.0100-0.0266. Using this FPR, the cross-sectional cBED-based HIV incidence estimates (per 100 people per year varied between 3.03 (95% CI 2.44-3.63 and 3.19 (95% CI 2.57-3.82, depending on the incidence formula. Using a long-term FPR of 0.0560 based on previous studies, HIV incidence estimates varied between 0.65 (95% CI 0.00-1.32 and 0.71 (95% CI 0.00-1.43. The longitudinally measured HIV incidence was 3.09 per 100 people per year (95% CI 2.69-3.52, after adjustment to the sex-age distribution of the sample used in cBED assay-based estimation.In a rural community in South Africa with high HIV prevalence, the long-term FPR of the cBED assay is substantially lower than previous estimates. The cBED assay performs well in HIV incidence estimation if the locally

  19. Detection of serum antitrichomonal antibodies in urogenital trichomoniasis by immunofluorescence.

    Directory of Open Access Journals (Sweden)

    Bhatt R

    1992-04-01

    Full Text Available Trichomonas vaginalis is a frequently encountered genital pathogen in both males and females. In females, vaginitis due to this parasite is one of the most common manifestation. The indirect fluorescent technique (IFA test was carried out to detect antitrichomonal antibodies in 370 female patients using whole cell antigen. Seventy one (19.18% gave positive reaction for either of the class IgG, IgM and IgA antibodies. The level of the IgG class antibodies was found to be higher i.e. 58 (81.69% than IgM 11 (15.27% antibodies, which may be suggestive of past infection or a prolonged manifestation by the organisms.

  20. Effective testing for pulmonary tuberculosis using Xpert MTB/RIF assay for stool specimens in immunocompetent Pakistani children

    Directory of Open Access Journals (Sweden)

    Zahra Hasan

    2016-01-01

    Conclusion: Use of Xpert MTB/RIF assay for stool-based diagnosis of pulmonary TB in immunocompetent children is useful in a resource poor setting. This is a valuable and noninvasive diagnostic alternative for the diagnosis of childhood TB and can be adapted by pediatric arms of national TB programs.

  1. Thyroglobulin (Tg) recovery testing with quantitative Tg antibody measurement for determining interference in serum Tg assays in differentiated thyroid carcinoma

    NARCIS (Netherlands)

    Persoon, ACM; Links, TP; Wilde, J; Sluiter, WJ; Wolffenbuttel, BHR; van den Ouweland, JMW

    Background: Thyroglobulin (Tg) measurements are complicated by interference from Tg autoantibodies (TgAbs) or heterophilic antibodies (HAMAs). We used a new automated immunochemiluminometric assay (ICMA) with Tg recovery (TgR) on the Nichols Advantage (R) platform to reassess the clinical utility of

  2. Unique Nanoparticle Properties Confound Fluorescent Based Assays Widely Employed in Their In Vitro Toxicity Testing and Ranking

    Science.gov (United States)

    Nanomaterials are a diverse collection of novel materials that exhibit at least one dimension less than 100 nm and display unique chemical and physical properties due to their nanoscale size. An emphasis has been put on developing high throughput screening (HTS) assays to charac...

  3. Comparison of mRNA splicing assay protocols across multiple laboratories: recommendations for best practice in standardized clinical testing.

    Science.gov (United States)

    Whiley, Phillip J; de la Hoya, Miguel; Thomassen, Mads; Becker, Alexandra; Brandão, Rita; Pedersen, Inge Sokilde; Montagna, Marco; Menéndez, Mireia; Quiles, Francisco; Gutiérrez-Enríquez, Sara; De Leeneer, Kim; Tenés, Anna; Montalban, Gemma; Tserpelis, Demis; Yoshimatsu, Toshio; Tirapo, Carole; Raponi, Michela; Caldes, Trinidad; Blanco, Ana; Santamariña, Marta; Guidugli, Lucia; de Garibay, Gorka Ruiz; Wong, Ming; Tancredi, Mariella; Fachal, Laura; Ding, Yuan Chun; Kruse, Torben; Lattimore, Vanessa; Kwong, Ava; Chan, Tsun Leung; Colombo, Mara; De Vecchi, Giovanni; Caligo, Maria; Baralle, Diana; Lázaro, Conxi; Couch, Fergus; Radice, Paolo; Southey, Melissa C; Neuhausen, Susan; Houdayer, Claude; Fackenthal, Jim; Hansen, Thomas Van Overeem; Vega, Ana; Diez, Orland; Blok, Rien; Claes, Kathleen; Wappenschmidt, Barbara; Walker, Logan; Spurdle, Amanda B; Brown, Melissa A

    2014-02-01

    Accurate evaluation of unclassified sequence variants in cancer predisposition genes is essential for clinical management and depends on a multifactorial analysis of clinical, genetic, pathologic, and bioinformatic variables and assays of transcript length and abundance. The integrity of assay data in turn relies on appropriate assay design, interpretation, and reporting. We conducted a multicenter investigation to compare mRNA splicing assay protocols used by members of the ENIGMA (Evidence-Based Network for the Interpretation of Germline Mutant Alleles) consortium. We compared similarities and differences in results derived from analysis of a panel of breast cancer 1, early onset (BRCA1) and breast cancer 2, early onset (BRCA2) gene variants known to alter splicing (BRCA1: c.135-1G>T, c.591C>T, c.594-2A>C, c.671-2A>G, and c.5467+5G>C and BRCA2: c.426-12_8delGTTTT, c.7988A>T, c.8632+1G>A, and c.9501+3A>T). Differences in protocols were then assessed to determine which elements were critical in reliable assay design. PCR primer design strategies, PCR conditions, and product detection methods, combined with a prior knowledge of expected alternative transcripts, were the key factors for accurate splicing assay results. For example, because of the position of primers and PCR extension times, several isoforms associated with BRCA1, c.594-2A>C and c.671-2A>G, were not detected by many sites. Variation was most evident for the detection of low-abundance transcripts (e.g., BRCA2 c.8632+1G>A Δ19,20 and BRCA1 c.135-1G>T Δ5q and Δ3). Detection of low-abundance transcripts was sometimes addressed by using more analytically sensitive detection methods (e.g., BRCA2 c.426-12_8delGTTTT ins18bp). We provide recommendations for best practice and raise key issues to consider when designing mRNA assays for evaluation of unclassified sequence variants.

  4. Trospium chloride has no effect on memory testing and is assay undetectable in the central nervous system of older patients with overactive bladder.

    Science.gov (United States)

    Staskin, D; Kay, G; Tannenbaum, C; Goldman, H B; Bhashi, K; Ling, J; Oefelein, M G

    2010-08-01

    Muscarinic receptors in the brain play an important role in cognitive function, especially memory, and there is growing awareness that specific antimuscarinic drugs for overactive bladder (OAB) may have adverse central nervous system (CNS) effects. Selection of an antimuscarinic OAB drug with reduced potential for CNS effects could be especially beneficial in the elderly people, in whom even the modest cognitive impairment may negatively affect independence. The purpose of the study is to determine if trospium chloride is assay detectable in the CNS of older adults with OAB and to assess whether deterioration of memory occurs in these individuals. Twelve cognitively intact older adults (>or=65-75 years old) with OAB were given extended-release trospium chloride 60 mg once daily over a 10-day period to achieve plasma steady-state levels. Standardised memory testing (Hopkins Verbal Learning Test-Revised and Brief Visuospatial Memory Test-Revised) was performed predose and postdose. Cerebrospinal spinal fluid (CSF) and plasma samples were drawn on day 10 and assayed for trospium chloride. Predose (day 0) and postdose (day 10) results on the memory tests were compared using a reliable change index to assess a meaningful change in learning or memory. Trospium chloride levels in all the CSF samples (n = 72) of all participants were assay undetectable (memory testing revealed no significant net drug effect on learning or recall. This is the first study to investigate for the presence of an OAB antimuscarinic in the human brain, performed by assaying for concentrations of trospium chloride and correlating with simultaneous clinical cognitive safety measures. The results of both pharmacological and neuropsychological testing support the hypothesis of a lack of detectable CNS penetration for the quaternary amine trospium chloride.

  5. Immunofluorescent detection in the ovary of host antibodies against a secretory ferritin injected into female Haemaphysalis longicornis ticks.

    Science.gov (United States)

    Galay, Remil Linggatong; Matsuo, Tomohide; Hernandez, Emmanuel Pacia; Talactac, Melbourne Rio; Kusakisako, Kodai; Umemiya-Shirafuji, Rika; Mochizuki, Masami; Fujisaki, Kozo; Tanaka, Tetsuya

    2018-04-01

    Due to the continuous threat of ticks and tick-borne diseases to human and animal health worldwide, and the drawbacks of chemical acaricide application, many researchers are exploring vaccination as an alternative tick control method. Earlier studies have shown that host antibodies can circulate in the ticks, but it has not been confirmed whether these antibodies can be passed on to the eggs. We previously reported that ticks infesting rabbits immunized with a recombinant secretory ferritin of Haemaphysalis longicornis (HlFER2) had reduced egg production and hatching. Here we attempted to detect the presence of antibodies against HlFER2 in the ovary and eggs of female ticks through immunofluorescent visualization. Purified anti-HlFER2 antibodies or rabbit IgG for control was directly injected to engorged female H. longicornis. Ovaries and eggs after oviposition were collected and prepared for an indirect immunofluorescent antibody test. Positive fluorescence was detected in ovaries one day post-injection of anti-HlFER2 antibodies. Through silencing of Hlfer2 gene, we also determined whether the injected antibodies can specifically bind to native HlFER2. Immunofluorescence was observed in the oocytes of dsLuciferase control ticks injected with anti-HlFER2 antibodies, but not in the oocytes of Hlfer2-silenced ticks also injected with anti-HlFER2 antibodies. Our current findings suggest that host antibodies can be passed on to the oocytes, which is significant in formulating a vaccine that can disrupt tick reproduction. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. The BD Onclarity HPV assay on SurePath collected samples meets the International Guidelines for Human Papillomavirus Test Requirements for Cervical Screening

    DEFF Research Database (Denmark)

    Ejegod, Ditte; Bottari, Fabio; Pedersen, Helle

    2016-01-01

    This study describes a validation of the BD Onclarity HPV (Onclarity) assay using the international guidelines for HPV test requirements for cervical cancer screening of women 30 years and above using Danish SurePath screening samples. The clinical specificity (0.90, 95% CI: 0.88-0.91) and sensit......This study describes a validation of the BD Onclarity HPV (Onclarity) assay using the international guidelines for HPV test requirements for cervical cancer screening of women 30 years and above using Danish SurePath screening samples. The clinical specificity (0.90, 95% CI: 0.......88-0.91) and sensitivity (0.97, 95% CI: 0.87-1.0) of the Onclarity assay were shown to be non-inferior to the reference assay (specificity 0.90, 95% CI: 0.88-0.92, sensitivity 0.98, 95% CI: 0.91-1.0). The intra-laboratory reproducibility of Onclarity was 97% with a lower confidence bound of 96% (kappa value: 0...

  7. H5N1-SeroDetect EIA and rapid test: a novel differential diagnostic assay for serodiagnosis of H5N1 infections and surveillance.

    Science.gov (United States)

    Khurana, Surender; Sasono, Pretty; Fox, Annette; Nguyen, Van Kinh; Le, Quynh Mai; Pham, Quang Thai; Nguyen, Tran Hien; Nguyen, Thanh Liem; Horby, Peter; Golding, Hana

    2011-12-01

    Continuing evolution of highly pathogenic (HP) H5N1 influenza viruses in wild birds with transmission to domestic poultry and humans poses a pandemic threat. There is an urgent need for a simple and rapid serological diagnostic assay which can differentiate between antibodies to seasonal and H5N1 strains and that could provide surveillance tools not dependent on virus isolation and nucleic acid technologies. Here we describe the establishment of H5N1 SeroDetect enzyme-linked immunosorbent assay (ELISA) and rapid test assays based on three peptides in HA2 (488-516), PB1-F2 (2-75), and M2e (2-24) that are highly conserved within H5N1 strains. These peptides were identified by antibody repertoire analyses of H5N1 influenza survivors in Vietnam using whole-genome-fragment phage display libraries (GFPDLs). To date, both platforms have demonstrated high levels of sensitivity and specificity in detecting H5N1 infections (clade 1 and clade 2.3.4) in Vietnamese patients as early as 7 days and up to several years postinfection. H5N1 virus-uninfected individuals in Vietnam and the United States, including subjects vaccinated with seasonal influenza vaccines or with confirmed seasonal virus infections, did not react in the H5N1-SeroDetect assays. Moreover, sera from individuals vaccinated with H5N1 subunit vaccine with moderate anti-H5N1 neutralizing antibody titers did not react positively in the H5N1-SeroDetect ELISA or rapid test assays. The simple H5N1-SeroDetect ELISA and rapid tests could provide an important tool for large-scale surveillance for potential exposure to HP H5N1 strains in both humans and birds.

  8. Double-label immunofluorescence method for simultaneous detection of adenovirus and herpes simplex virus from the eye.

    Science.gov (United States)

    Walpita, P; Darougar, S

    1989-07-01

    The development and application of a double-label immunofluorescence method which has the potential to screen for single or dual infections from any site, in single shell vial cultures, is described. In this study, a total of 1,141 ocular specimens were inoculated in shell vials, centrifuged at 15,000 X g for 1 h, incubated at 37 degrees C for 48 h, and fixed in methanol at room temperature for 15 min. The virus inclusions were detected by staining with a double-label indirect immunofluorescence procedure using mixtures of appropriate first antibodies, followed by fluorescein- and rhodamine-conjugated second antibodies. Each specimen was also inoculated in parallel by the conventional virus isolation method. The sensitivity and specificity of the double-label shell vial procedure were comparable to those with the conventional method, and the former test took only 48 h to complete. The test offers a rapid and simple single-vial procedure which allows for individual or simultaneous detection of multiple pathogens. It results in savings in time and cost over the conventional virus isolation method and other shell vial procedures.

  9. Evaluation of the diagnostic value of serologic microagglutination testing and a polymerase chain reaction assay for diagnosis of acute leptospirosis in dogs in a referral center.

    Science.gov (United States)

    Fraune, Claudia Kümmerle; Schweighauser, Ariane; Francey, Thierry

    2013-05-15

    To determine the diagnostic value of a serologic microagglutination test (MAT) and a PCR assay on urine and blood for the diagnosis of leptospirosis in dogs with acute kidney injury (AKI). Cross-sectional study. Animals-76 dogs with AKI in a referral hospital (2008 to 2009). Dogs' leptospirosis status was defined with a paired serologic MAT against a panel of 11 Leptospira serovars as leptospirosis-associated (n = 30) or nonleptospirosis-associated AKI (12). In 34 dogs, convalescent serologic testing was not possible, and leptospirosis status was classified as undetermined. The diagnostic value of the MAT single acute or convalescent blood sample was determined in dogs in which leptospirosis status could be classified. The diagnostic value of a commercially available genus-specific PCR assay was evaluated by use of 36 blood samples and 20 urine samples. Serologic acute testing of an acute blood sample had a specificity of 100% (95% CI, 76% to 100%), a sensitivity of 50% (33% to 67%), and an accuracy of 64% (49% to 77%). Serologic testing of a convalescent blood sample had a specificity of 92% (65% to 99%), a sensitivity of 100% (87% to 100%), and an accuracy of 98% (88% to 100%). Results of the Leptospira PCR assay were negative for all samples from dogs for which leptospirosis status could be classified. Serologic MAT results were highly accurate for diagnosis of leptospirosis in dogs, despite a low sensitivity for early diagnosis. In this referral setting of dogs pretreated with antimicrobials, testing of blood and urine samples with a commercially available genus-specific PCR assay did not improve early diagnosis.

  10. Validation of clinical testing for warfarin sensitivity: comparison of CYP2C9-VKORC1 genotyping assays and warfarin-dosing algorithms.

    Science.gov (United States)

    Langley, Michael R; Booker, Jessica K; Evans, James P; McLeod, Howard L; Weck, Karen E

    2009-05-01

    Responses to warfarin (Coumadin) anticoagulation therapy are affected by genetic variability in both the CYP2C9 and VKORC1 genes. Validation of pharmacogenetic testing for warfarin responses includes demonstration of analytical validity of testing platforms and of the clinical validity of testing. We compared four platforms for determining the relevant single nucleotide polymorphisms (SNPs) in both CYP2C9 and VKORC1 that are associated with warfarin sensitivity (Third Wave Invader Plus, ParagonDx/Cepheid Smart Cycler, Idaho Technology LightCycler, and AutoGenomics Infiniti). Each method was examined for accuracy, cost, and turnaround time. All genotyping methods demonstrated greater than 95% accuracy for identifying the relevant SNPs (CYP2C9 *2 and *3; VKORC1 -1639 or 1173). The ParagonDx and Idaho Technology assays had the shortest turnaround and hands-on times. The Third Wave assay was readily scalable to higher test volumes but had the longest hands-on time. The AutoGenomics assay interrogated the largest number of SNPs but had the longest turnaround time. Four published warfarin-dosing algorithms (Washington University, UCSF, Louisville, and Newcastle) were compared for accuracy for predicting warfarin dose in a retrospective analysis of a local patient population on long-term, stable warfarin therapy. The predicted doses from both the Washington University and UCSF algorithms demonstrated the best correlation with actual warfarin doses.

  11. Clinical Comparison of the Treponema pallidum CAPTIA Syphilis-G Enzyme Immunoassay with the Fluorescent Treponemal Antibody Absorption Immunoglobulin G Assay for Syphilis Testing

    OpenAIRE

    Halling, V. W.; Jones, M. F.; Bestrom, J. E.; Wold, A. D.; Rosenblatt, J. E.; Smith, T. F.; Cockerill, F. R.

    1999-01-01

    Recently, a treponema-specific immunoglobulin G (IgG) enzyme immunoassay (EIA), the CAPTIA Syphilis-G (Trinity Biotech, Jamestown, N.Y.), has become available as a diagnostic test for syphilis. A total of 89 stored sera previously tested by the fluorescent treponemal antibody absorption (FTA-ABS) IgG assay were evaluated by the CAPTIA EIA. The FTA-ABS IgG procedure was performed by technologists unblinded to results of rapid plasmid reagin (RPR) testing of the same specimens. Borderline CAPTI...

  12. Mutagenic effects of tributyltin and inorganic lead (Pb II on the fish H. malabaricus as evaluated using the comet assay and the piscine micronucleus and chromosome aberration tests

    Directory of Open Access Journals (Sweden)

    Ferraro Marcos Vinícius M.

    2004-01-01

    Full Text Available Genotoxicity studies on toxic metals and their organic compounds are very important, especially so in the investigation of the effects of these compounds on the aquatic environments where they tend to accumulate. The use of endemic aquatic organisms as biological sentinels has proved useful to environmental monitoring. We assessed the mutagenic potential of tributyltin (TBT and inorganic lead (PbII using samples of the fish Hoplias malabaricus (commonly called traíra using the comet assay and the piscine micronucleus and chromosome aberration tests. Eighteen H. malabaricus were acclimatized in three individual aquariums, each containing six fish, six fish being exposed to 0.3 mg/g of body weight (bw of TBT, six to 21 mg/g bw of PbII and six being used as controls. Exposure to TBT and PbII was achieved by feeding the fish every five days with Astyanax (a small fish that is part of the normal diet of H. malabaricus which had been injected with solutions of TBT, PbII or with water (the control group. After two months the H. malabaricus were sacrificed and their peripheral blood collected and subjected to the comet and micronucleus assays, the chromosome aberration assay being conducted using kidney-tissue. Although the comet assay showed now mutagenic effects at the lead concentrations used but encountered results with TBT, the micronucleus and chromosome aberrations assays both indicated that TBT and PbII are potentially mutagenic (p < 0.01, the micronucleus assay showing morphological alterations of the nucleus.

  13. Evaluation of GenoType® MTBDRplus assay for rapid detection of drug susceptibility testing of multi-drug resistance tuberculosis in Northern India

    Directory of Open Access Journals (Sweden)

    Anand Kumar Maurya

    2013-01-01

    Full Text Available Background: The problem of multi-drug resistance tuberculosis (MDR-TB is growing in several hotspots throughout the world. Rapid and accurate diagnosis of MDR-TB is crucial to facilitate early treatment and to reduce its spread in the community. The aim of the present study was to evaluate the new, novel GenoType® MTBDRplus assay for rapid detection of drug susceptibility testing (DST of MDR-TB cases in Northern India. Materials and Methods: A total of 550 specimens were collected from highly suspected drug resistant from pulmonary and extra-pulmonary TB cases. All the specimens were processed by Ziehl- Neelsen staining, culture, differentiation by the GenoType® CM assay, first line DST using BacT/ALERT 3D system and GenoType® MTBDRplus assay. The concordance of the GenoType® MTBDRplus assay was calculated in comparison with conventional DST results. Results: Overall the sensitivity for detection of rifampicin, isoniazid and MDR-TB resistance by GenoType® MTBDRplus assay was 98.0%, 98.4% and 98.2% respectively. Out of 55 MDR-TB strains, 45 (81.8%, 52 (94.5% and 17 (30.9% strains showed mutation in rpoB, katG and inhA genes respectively (P < 0.05. The most prominent mutations in rpoB, katG and inhA genes were; 37 (67.3% in S531L, 52 (94.5% in S315T1 and 11 (20% in C15T regions respectively (P < 0.05. Conclusions: Our study demonstrated a high concordance between the GenoType® MTBDRplus assay resistance patterns and those were observed by conventional DST with good sensitivity, specificity with short turnaround times and to control new cases of MDR-TB in countries with a high prevalence of MDR-TB.

  14. Electrically detected displacement assay (EDDA): a practical approach to nucleic acid testing in clinical or medical diagnosis.

    Science.gov (United States)

    Liepold, P; Kratzmüller, T; Persike, N; Bandilla, M; Hinz, M; Wieder, H; Hillebrandt, H; Ferrer, E; Hartwich, G

    2008-07-01

    This paper introduces the electrically detected displacement assay (EDDA), a electrical biosensor detection principle for applications in medical and clinical diagnosis, and compares the method to currently available microarray technologies in this field. The sensor can be integrated into automated systems of routine diagnosis, but may also be used as a sensor that is directly applied to the polymerase chain reaction (PCR) reaction vessel to detect unlabeled target amplicons within a few minutes. Major aspects of sensor assembly like immobilization procedure, accessibility of the capture probes, and prevention from nonspecific target adsorption, that are a prerequisite for a robust and reliable performance of the sensor, are demonstrated. Additionally, exemplary results from a human papillomavirus assay are presented.

  15. The first FDA marketing authorizations of next-generation sequencing technology and tests: challenges, solutions and impact for future assays.

    Science.gov (United States)

    Bijwaard, Karen; Dickey, Jennifer S; Kelm, Kellie; Težak, Živana

    2015-01-01

    The rapid emergence and clinical translation of novel high-throughput sequencing technologies created a need to clarify the regulatory pathway for the evaluation and authorization of these unique technologies. Recently, the US FDA authorized for marketing four next generation sequencing (NGS)-based diagnostic devices which consisted of two heritable disease-specific assays, library preparation reagents and a NGS platform that are intended for human germline targeted sequencing from whole blood. These first authorizations can serve as a case study in how different types of NGS-based technology are reviewed by the FDA. In this manuscript we describe challenges associated with the evaluation of these novel technologies and provide an overview of what was reviewed. Besides making validated NGS-based devices available for in vitro diagnostic use, these first authorizations create a regulatory path for similar future instruments and assays.

  16. A novel assay for detecting antibodies to cytochrome P4502D6, the molecular target of liver kidney microsomal antibody type 1.

    Science.gov (United States)

    Kerkar, N; Ma, Y; Hussain, M; Muratori, L; Targett, C; Williams, R; Bianchi, F B; Mieli-Vergani, G; Vergani, D

    1999-03-04

    Liver Kidney Microsomal type 1 (LKM1) antibody, the diagnostic marker of autoimmune hepatitis type 2, is also found in a proportion of patients with hepatitis C virus infection (HCV). It is detected conventionally by the subjective immunofluorescence technique. Our aim was to establish a simple and objective enzyme-linked immunosorbent assay (ELISA) that measures antibodies to cytochrome P4502D6 (CYP2D6), the target of LKM1. An indirect ELISA using eukaryotically expressed CYP2D6 was designed. Absorbance values obtained against a reference microsomal preparation were subtracted from those obtained against a microsomal preparation over-expressing CYP2D6, thus removing the non-CYP2D6-specific reaction. Sera from 51 LKM1 positive patients (21 autoimmune hepatitis and 30 with HCV infection), 111 LKM1 negative patients with chronic liver disease (including 20 with HCV infection) and 43 healthy controls were tested. Of 51 patients positive by immunofluorescence, 48 were also positive by ELISA while all the 154 LKM1 negative subjects were also negative by ELISA. There was a high degree of association between IFL and ELISA as demonstrated by a kappa reliability value of 0.96. The absorbance values by ELISA correlated with immunofluorescence LKM1 titres both in autoimmune hepatitis (r = 0.74, p < 0.001) and HCV infection (r = 0.67, p < 0.001). The simple, objective ELISA described has the potential to replace the standard immunofluorescence technique.

  17. Evaluation of enzyme-linked immunosorbent and alternative assays for detection of HIV antibodies using panels of Brasilian sera Avaliação de testes sorológicos para a detecção de anticorpos anti-HIV em painéis de soros de brasileiros

    Directory of Open Access Journals (Sweden)

    Jairo Ivo-Dos-Santos

    1990-04-01

    Full Text Available Sera from 472 Brazilian subjects, confirmed to be either positive or negative for HIV antibodies and comprising the total clinical spectrum of HIV infection, were utilized in the evaluation of six commercially available enzyme-linked immunosorbent assays (ELISA, as well as of four alternative assays, namely indirect immunofluorescence (IIF, passive hemagglutination (PHA, dot blot and Karpas AIDS cell test. The sensitivities ranged from 100% (Abbott and Roche ELISA to 84.2% (PHA and the specificities ranged from 99.3% (IIF to 80.2% (PHA. The sensitivity and specificity of the PHA and the sensitivity of the Karpas cell test were significantly lower than those of the other tests. Although the IFF and dot blot had good sensitivities and specificities, the six ELISA were more attractive than those tests when other parameters such as ease of reading and duration of assay were considered.Os soros de 472 brasileiros, confirmados como sendo positivos ou negativos em relação à presença de anticorpos anti-HIV e compreendendo todo o espectro clínico da infecção, foram utilizados na avaliação de seis ensaios imunoenzimáticos comerciais (ELISA, bem como de quatro testes alternativos tais como imunofluorescência indireta (IFI, hemaglutinação passiva (HP, dot blot e Karpas AIDS cell test. As sensibilidades variaram de 100% (ELISA Abbott e Roche a 84,2% (HP e as especificidades variaram de 99,3% (IFI a 80,2% (HP. A sensibilidade e especificidade da HP e a sensibilidade do Karpas AIDS cell test foram significativamente menores que os outros ensaios. Embora a IFI e o dot blot tivessem apresentado uma boa sensibilidade e especificidade, os ensaios imunoenzimáticos (ELISA foram mais adequados para serem utilizados em triagem quando outros parâmetros tais como facilidade de leitura e interpretação dos resultados e duração dos ensaios foram considerados.

  18. Use of commercially available rabbit monoclonal antibodies for immunofluorescence double staining

    DEFF Research Database (Denmark)

    Bzorek, M.; Stamp, I.M.; Frederiksen, L.

    2008-01-01

    Immunohistochemistry, that is, the use of polyclonal and monoclonal antibodies to detect cell and tissue antigens at a microscopical level is a powerful tool for both research and diagnostic purposes. Especially in the field of hematologic disease, there is often a need to detect several antigens...... synchronously, and we report here a fast and easy technique for demonstrating more than 1 antigen in 1 slide using immunofluorescence. We have used commercially available rabbit monoclonal antibodies (Cyclin D1, CD3, CD5, CD23, etc.) paired with mouse monoclonal antibodies (CD7, CD20, CD79a, Pax-5, etc.......) for double immunofluorescence labeling on paraffin-embedded tissue sections. Commercially available rabbit monoclonal antibodies in combination with mouse monoclonal antibodies proved useful in double immunofluorescence labeling on paraffin-embedded tissue, and all combinations used yielded excellent results...

  19. Improved detection of Pneumocystis carinii by an immunofluorescence technique using monoclonal antibodies

    DEFF Research Database (Denmark)

    Orholm, M; Holten-Andersen, W; Lundgren, Jens Dilling

    1990-01-01

    To assess whether a recently developed indirect immunofluorescent stain using monoclonal antibodies was more sensitive in detecting Pneumocystis carinii than the combination of Giemsa and methenamine silver nitrate stains which has routinely been used in the laboratory, 88 lavage fluid specimens...... silver nitrate and toluidine blue O. Immunofluorescence using the monoclonal antibodies from the NIH was significantly more sensitive than any other single staining method and than the combination of Giemsa and methenamine silver nitrate staining. The study also showed that the cytospin centrifuge...

  20. Optimization of Single- and Dual-Color Immunofluorescence Protocols for Formalin-Fixed, Paraffin-Embedded Archival Tissues.

    Science.gov (United States)

    Kajimura, Junko; Ito, Reiko; Manley, Nancy R; Hale, Laura P

    2016-02-01

    Performance of immunofluorescence staining on archival formalin-fixed paraffin-embedded human tissues is generally not considered to be feasible, primarily due to problems with tissue quality and autofluorescence. We report the development and application of procedures that allowed for the study of a unique archive of thymus tissues derived from autopsies of individuals exposed to atomic bomb radiation in Hiroshima, Japan in 1945. Multiple independent treatments were used to minimize autofluorescence and maximize fluorescent antibody signals. Treatments with NH3/EtOH and Sudan Black B were particularly useful in decreasing autofluorescent moieties present in the tissue. Deconvolution microscopy was used to further enhance the signal-to-noise ratios. Together, these techniques provide high-quality single- and dual-color fluorescent images with low background and high contrast from paraffin blocks of thymus tissue that were prepared up to 60 years ago. The resulting high-quality images allow the application of a variety of image analyses to thymus tissues that previously were not accessible. Whereas the procedures presented remain to be tested for other tissue types and archival conditions, the approach described may facilitate greater utilization of older paraffin block archives for modern immunofluorescence studies. © 2016 The Histochemical Society.

  1. Investigation of OMNIgene·SPUTUM performance in delayed tuberculosis testing by smear, culture, and Xpert MTB/RIF assays in Uganda

    Directory of Open Access Journals (Sweden)

    C.D. Kelly-Cirino

    2017-06-01

    Full Text Available OMNIgene·SPUTUM (OM-S is a sample transport reagent designed to work with all tuberculosis diagnostics while eliminating the need for cold chain. OM-S-treated sputum samples were assayed in several tests after multiday holds. Raw sputa from 100 patients underwent direct smear microscopy, were manually split and assigned to the OM-S group [OM-S added at collection (no other processing required and tested after 0- to 5-day holds at room temperature] or standard-of-care (SOC group (NaOH/N-acetyl l-cysteine decontamination, all tested on day of collection. Concentrated smear microscopy, Lowenstein Jensen (LJ culture, and mycobacteria growth indicator tube (MGIT culture were performed. For patients with negative direct smear, a second sample was split, with SOC (raw sputum and OM-S portions (sediment tested in the Xpert MTB/RIF (Xpert assay. OM-S group and SOC group results were strongly concordant on all four tests [range, 89% (MGIT–97% (Xpert]. OM-S MGIT, LJ, and Xpert tests were in statistical agreement with SOC MGIT as reference. OM-S specimens had lower culture contamination rates (3% vs. 10% LJ; 2% vs. 5% MGIT but required, on average, 5.6 additional days to become MGIT-positive. The findings suggest that samples held/transported in OM-S are compatible with smear microscopy, LJ or MGIT culture, and Xpert, and perform comparably to fresh sputum samples. Larger feasibility studies are warranted.

  2. Endometrial carcinoma in vitro chemosensitivity testing of single and combination chemotherapy regimens using the novel microculture kinetic apoptosis assay: implications for endometrial cancer treatment.

    Science.gov (United States)

    Ballard, Karen S; Homesley, Howard D; Hodson, Charles; Presant, Cary A; Rutledge, James; Hallquist, Allan; Perree, Mathieu

    2010-03-01

    The in vitro microculture kinetic (MiCK) apoptosis assay has been used to predict single or combination chemotherapy response in leukemia patients. This feasibility study addressed MiCK in endometrial cancer specimens. Endometrial cancer specimens from total abdominal hysterectomies were processed at a central laboratory. Single cell suspensions of viable endometrial cancer cells were plated in individual wells. Single and combination regimens were tested: combinations of doxorubicin, cisplatin, and paclitaxel and carboplatin and paclitaxel (Gynecologic Oncology Group [GOG] 209 endometrial cancer phase III trial arms) as well as single agent testing with paclitaxel, carboplatin, doxorubicin, cisplatin, ifosfamide, and vincristine (active agents in GOG trials). Apoptosis was measured continuously over 48 hours. Fifteen of nineteen patients had successful assays. The highest mean chemo sensitivity was noted in the combination of cisplatin, doxorubicin, and paclitaxel with lower mean chemosensitivity for carboplatin and paclitaxel. Combination chemotherapy had higher chemosensitivity than single drug chemotherapy. However, in 25% of patients a single drug had higher chemosensitivity than combination chemotherapy. As single agents, ifosfamide, cisplatin, and paclitaxel had the highest kinetic unit values. Using a panel of agents simulating clinical dose regimens, the MiCK assay was feasible in evaluating in vitro chemosensitivity of endometrial cancer. MiCK assay results correlated with GOG clinical trial results. However, 25% of patients might be best treated with single agent chemotherapy selected by MiCK. Ifosfamide, cisplatin, and paclitaxel appear to have high activity as single agents. MiCK may be useful in future new drug testing and individualizing endometrial cancer patient's chemotherapy management.

  3. Code verification and Y2K testing, calibration, testing, and installation of the radionuclide assay system-photon (RAS-P) at multiple sites for the Savannah River Site

    International Nuclear Information System (INIS)

    Hodge, C.A.

    2000-01-01

    The Radionuclide Assay System - Photon (RAS-P) is a near-field, transmission-corrected assay system developed for measurement of the actinide content of relatively homogeneous waste generated by facility operations. It is intended for use by facility operations personnel, and has features to enhance its usefulness and efficiency. These include multinuclide assay capability, automatic (off-shift) collection of background and straight-through transmission source data, enforcement of measurement control requirements, Go-NoGo or Assay modes, password protection, and reporting of total fissile gram equivalent values. System hardware consists of a shielded high-resolution germanium detector, a turntable, a shielded transmission source and shutter assembly, and a desktop computer and laser printer mounted on a compact frame. RAS-P was designed to assay the contents of cylindrical containers up to 30 inches diameter by 32 inches high, boxes up to 30 inches diagonal by 32 inches high, and HE PA filters up to 2 x 2 x 1 feet. Prior to installation at the Savannah River Site (SRS), code validation, system performance, and assurance against Y2K effects all were confirmed. Code validation was accomplished using spreadsheet calculations that were independent of the original code to calculate intermediate and final result produced by RAS-P. System testing was performed by repeated operation of the instrument under all required circumstances. Y2K testing was performed simultaneously with code validation following a protocol prescribed by the SRS Y2K subcommittee that required assays with dates varying throughout the expected useful life of the RAS-P, particularly those bracketing Y2K boundaries. Performance history has been compiled demonstrating reliability (system availability), diversability (the ability to alter assay parameters and obtain results quickly), and measurement control characteristics

  4. Oestrogenic activity of a textile industrial wastewater treatment plant effluent evaluated by the E-screen test and MELN gene-reporter luciferase assay

    Energy Technology Data Exchange (ETDEWEB)

    Schiliro, Tiziana, E-mail: tiziana.schiliro@unito.it [Department of Public Health and Microbiology, University of Torino, Via Santena 5bis, 10126 Torino (Italy); Porfido, Arianna [Department of Public Health and Microbiology, University of Torino, Via Santena 5bis, 10126 Torino (Italy); Spina, Federica; Varese, Giovanna Cristina [Department of Life Sciences and Systems Biology, University of Torino, Viale Mattioli 25, 10125 Torino (Italy); Gilli, Giorgio [Department of Public Health and Microbiology, University of Torino, Via Santena 5bis, 10126 Torino (Italy)

    2012-08-15

    This study quantified the biological oestrogenic activity in the effluent of a textile industrial wastewater treatment plant (IWWTP) in northwestern Italy. Samples of the IWWTP effluent were collected monthly, both before and after tertiary treatment (ozonation). After solid phase extraction, all samples were subjected to two in vitro tests of total estrogenic activity, the human breast cancer cell line (MCF-7 BUS) proliferation assay, or E-screen test, and the luciferase-transfected human breast cancer cell line (MELN) gene-reporter assay, to measure the 17{beta}-oestradiol equivalent quantity (EEQ). In the E-screen test, the mean EEQ values were 2.35 {+-} 1.68 ng/L pre-ozonation and 0.72 {+-} 0.58 ng/L post-ozonation; in the MELN gene-reporter luciferase assay, the mean EEQ values were 4.18 {+-} 3.54 ng/L pre-ozonation and 2.53 {+-} 2.48 ng/L post-ozonation. These results suggest that the post-ozonation IWWTP effluent had a lower oestrogenic activity (simple paired t-tests, p < 0.05). The average reduction of estrogenic activity of IWWTP effluent after ozonation was 67 {+-} 26% and 52 {+-} 27% as measured by E-screen test and MELN gene-reporter luciferase assay, respectively. There was a positive and significant correlation between the two tests (Rho S = 0.650, p = 0.022). This study indicates that the environmental risk is low because oestrogenic substances are deposited into the river via IWWTP at concentrations lower than those at which chronic exposure has been reported to affect the endocrine system of living organisms. -- Highlights: Black-Right-Pointing-Pointer The two in vitro tests are suited for oestrogenic activity assessment in textile WWTP. Black-Right-Pointing-Pointer There is a significant correlation between the results of the two in vitro tests. Black-Right-Pointing-Pointer The oestrogenic activity of the effluent is reduced by ozonation. Black-Right-Pointing-Pointer The input of estrogenic substances into the river via textile WWTP is low.

  5. Standard test method for non-destructive assay of nuclear material in waste by passive and active neutron counting using a differential Die-away system

    CERN Document Server

    American Society for Testing and Materials. Philadelphia

    2009-01-01

    1.1 This test method covers a system that performs nondestructive assay (NDA) of uranium or plutonium, or both, using the active, differential die-away technique (DDT), and passive neutron coincidence counting. Results from the active and passive measurements are combined to determine the total amount of fissile and spontaneously-fissioning material in drums of scrap or waste. Corrections are made to the measurements for the effects of neutron moderation and absorption, assuming that the effects are averaged over the volume of the drum and that no significant lumps of nuclear material are present. These systems are most widely used to assay low-level and transuranic waste, but may also be used for the measurement of scrap materials. The examples given within this test method are specific to the second-generation Los Alamos National Laboratory (LANL) passive-active neutron assay system. 1.1.1 In the active mode, the system measures fissile isotopes such as 235U and 239Pu. The neutrons from a pulsed, 14-MeV ne...

  6. Feasibility of the fluorometric microculture cytotoxicity assay (FMCA) for cytotoxic drug sensitivity testing of tumor cells from patients with acute lymphoblastic leukemia.

    Science.gov (United States)

    Nygren, P; Kristensen, J; Jonsson, B; Sundström, C; Lönnerholm, G; Kreuger, A; Larsson, R

    1992-11-01

    The automated fluorometric microculture cytotoxicity assay (FMCA) was used for chemotherapeutic drug sensitivity testing of fresh and cryopreserved tumor cells from patients with acute lymphoblastic leukemia (ALL) at diagnosis and relapse. The technique success rate was 87% for fresh and 81% for cryopreserved samples. Up to 16 different cytotoxic drugs were routinely tested, but neither asparaginase nor methotrexate produced dose-response related cell kill. FMCA data showed good correlation to the well established Disc assay and the drug sensitivity reported by the FMCA was in good agreement with known clinical activity. Samples from children and initial ALL tended to be more drug sensitive than those from adults and ALL at relapse, respectively. For 36 samples clinical outcome was correlated to the quartile position in comparison to all other samples for the most in vitro active drug actually given to the patient. For patients with samples in the first, second, third, and fourth quartiles, the probabilities of complete remission were 89, 57, 38, and 0%, respectively. Using the median value as cut-off line, the sensitivity and specificity of the assay were 87 and 62%, respectively. It is concluded that the FMCA with a minimum of effort and with high success rate report clinically relevant drug sensitivity profiles for ALL.

  7. BLIND TRIALS EVALUATING IN VITRO INFECTIVITY OF CRYPTOSPORIDIUM PARVUM OOCYSTS USING CELL CULTURE IMMUNOFLUORESCENCE

    Science.gov (United States)

    An optimized cell culture-immunofluorescence (IFA) procedure, using the HCT-8 cell line, was evaluated in 'blind' trials to determine the sensitivity and reproducibility for measuring infectivity of flow cytometry prepared inocula of C. parvum oocysts. In separate trials, suspens...

  8. Clinical comparison of the Treponema pallidum CAPTIA syphilis-G enzyme immunoassay with the fluorescent treponemal antibody absorption immunoglobulin G assay for syphilis testing.

    Science.gov (United States)

    Halling, V W; Jones, M F; Bestrom, J E; Wold, A D; Rosenblatt, J E; Smith, T F; Cockerill, F R

    1999-10-01

    Recently, a treponema-specific immunoglobulin G (IgG) enzyme immunoassay (EIA), the CAPTIA Syphilis-G (Trinity Biotech, Jamestown, N.Y.), has become available as a diagnostic test for syphilis. A total of 89 stored sera previously tested by the fluorescent treponemal antibody absorption (FTA-ABS) IgG assay were evaluated by the CAPTIA EIA. The FTA-ABS IgG procedure was performed by technologists unblinded to results of rapid plasmid reagin (RPR) testing of the same specimens. Borderline CAPTIA-positive samples (antibody indices of >/=0.650 and 0.900, the sample was considered positive. Thirteen of 89 (15%) samples had discrepant results. Compared to the FTA-ABS assay, the CAPTIA EIA had a sensitivity and specificity and positive and negative predictive values of 70.7, 97.9, 96.7, and 79.7%, respectively. In another analysis, discrepancies between results were resolved by repeated FTA-ABS testing (technologists were blinded to previous RPR results) and patient chart reviews. Seven CAPTIA-negative samples which were previously interpreted (unblinded) as minimally reactive by the FTA method were subsequently interpreted (blinded) as nonreactive. One other discrepant sample (CAPTIA negative and FTA-ABS positive [at an intensity of 3+], unblinded) was FTA negative with repeated testing (blinded). For the five remaining discrepant samples, chart reviews indicated that one patient (CAPTIA negative and FTA-ABS positive [minimally reactive], blinded) had possible syphilis. These five samples were also evaluated and found to be negative by another treponema-specific test, the Treponema pallidum microhemagglutination assay. Therefore, after repeated testing and chart reviews, 2 of the 89 (2%) samples had discrepant results; the adjusted sensitivity, specificity, and positive and negative predictive values were 96.7, 98.3, 96.7, and 98.3%, respectively. This study demonstrates that the CAPTIA IgG EIA is a reliable method for syphilis testing and that personnel performing tests

  9. Evaluation of an egg yolk enzyme-linked immunosorbent assay antibody test and its use to assess the prevalence of Mycoplasma gallisepticum infection in laying hens in Italy

    Directory of Open Access Journals (Sweden)

    Marco Tamba

    2010-01-01

    Full Text Available The prevalence of Mycoplasma gallisepticum infection in commercial layers was established by the presence of antibodies in eggs. Saline-extracted yolks were used with a commercial enzyme-linked immunosorbent assay kit. For the prevalence study, yolks from 30 eggs were obtained from each of 66 flocks coming from 36 layer farms. The prevalence of egg antibodies to Mycoplasma gallisepticum was 33.3% in single-age farms and 77.8% in multi-age farms. In 27 flocks, antibody titers were compared with results obtained from blood samples taken in the same flock and in the same period and analyzed with the same kit. This study has confirmed that egg yolk enzyme-linked immunosorbent assay antibody test is a suitable and practical approach for assessing the flock prevalence of Mycoplasma gallisepticum infection in layer hens.

  10. A Spectrophotometric Assay for Robust Viability Testing of Seed Batches Using 2,3,5-Triphenyl Tetrazolium Chloride: Using Hordeum vulgare L. as a Model

    Directory of Open Access Journals (Sweden)

    Laura Lopez Del Egido

    2017-05-01

    Full Text Available A comparative analysis was carried out of published methods to assess seed viability using 2,3,5-triphenyltetrazolium chloride (TTC based assays of seed batches. The tests were carried out on seeds of barley (Hordeum vulgare cv. Optic as a model. We established that 10% [w/v] trichloroacetic acid (TCA/methanol is superior to the acetone and methanol-only based methods: allowing the highest recovery of formazan and the lowest background optical density (OD readings, across seed lots comprising different ratios of viable and dead seeds. The method allowed a linear-model to accurately capture the statistically significant relationship between the quantity of formazan that could be extracted using the method we developed and the seed temperature-response, and seed viability as a function of artificially aged seed lots. Other quality control steps are defined to help ensure the assay is robust and these are reported in a Standard Operating Procedure.

  11. Modified batch anaerobic digestion assay for testing efficiencies of trace metal additives to enhance methane production of energy crops.

    Science.gov (United States)

    Brulé, Mathieu; Bolduan, Rainer; Seidelt, Stephan; Schlagermann, Pascal; Bott, Armin

    2013-01-01

    Batch biochemical methane potential (BMP) assays to evaluate the methane yield of biogas substrates such as energy crops are usually carried out with undiluted inoculum. A BMP assay was performed on two energy crops (green cuttings and grass silage). Anaerobic digestion was performed both with and without supplementation of three commercial additives containing trace metals in liquid, solid or adsorbed form (on clay particles). In order to reveal positive effects of trace metal supplementation on the methane yield, besides undiluted inoculum, 3-fold and 10-fold dilutions of the inoculum were applied for substrate digestion. Diluted inoculum variants were supplemented with both mineral nutrients and pH-buffering substances to prevent a collapse of the digestion process. As expected, commercial additives had no effect on the digestion process performed with undiluted inoculum, while significant increases of methane production through trace element supplementation could be observed on the diluted variants. The effect of inoculum dilution may be twofold: (1) decrease in trace metal supplementation from the inoculum and (2) reduction in the initial number of bacterial cells. Bacteria require higher growth rates for substrate degradation and hence have higher trace element consumption. According to common knowledge of the biogas process, periods with volatile fatty acids accumulation and decreased pH may have occurred in the course ofanaerobic digestion. These effects may have led to inhibition, not only ofmethanogenes and acetogenes involved in the final phases of methane production, but also offibre-degrading bacterial strains involved in polymer hydrolysis. Further research is required to confirm this hypothesis.

  12. The New Xpert MTB/RIF Ultra: Improving Detection of Mycobacterium tuberculosis and Resistance to Rifampin in an Assay Suitable for Point-of-Care Testing.

    Science.gov (United States)

    Chakravorty, Soumitesh; Simmons, Ann Marie; Rowneki, Mazhgan; Parmar, Heta; Cao, Yuan; Ryan, Jamie; Banada, Padmapriya P; Deshpande, Srinidhi; Shenai, Shubhada; Gall, Alexander; Glass, Jennifer; Krieswirth, Barry; Schumacher, Samuel G; Nabeta, Pamela; Tukvadze, Nestani; Rodrigues, Camilla; Skrahina, Alena; Tagliani, Elisa; Cirillo, Daniela M; Davidow, Amy; Denkinger, Claudia M; Persing, David; Kwiatkowski, Robert; Jones, Martin; Alland, David

    2017-08-29

    The Xpert MTB/RIF assay (Xpert) is a rapid test for tuberculosis (TB) and rifampin resistance (RIF-R) suitable for point-of-care testing. However, it has decreased sensitivity in smear-negative sputum, and false identification of RIF-R occasionally occurs. We developed the Xpert MTB/RIF Ultra assay (Ultra) to improve performance. Ultra and Xpert limits of detection (LOD), dynamic ranges, and RIF-R rpoB mutation detection were tested on Mycobacterium tuberculosis DNA or sputum samples spiked with known numbers of M. tuberculosis H37Rv or Mycobacterium bovis BCG CFU. Frozen and prospectively collected clinical samples from patients suspected of having TB, with and without culture-confirmed TB, were also tested. For M. tuberculosis H37Rv, the LOD was 15.6 CFU/ml of sputum for Ultra versus 112.6 CFU/ml of sputum for Xpert, and for M. bovis BCG, it was 143.4 CFU/ml of sputum for Ultra versus 344 CFU/ml of sputum for Xpert. Ultra resulted in no false-positive RIF-R specimens, while Xpert resulted in two false-positive RIF-R specimens. All RIF-R-associated M. tuberculosis rpoB mutations tested were identified by Ultra. Testing on clinical sputum samples, Ultra versus Xpert, resulted in an overall sensitivity of 87.5% (95% confidence interval [CI], 82.1, 91.7) versus 81.0% (95% CI, 74.9, 86.2) and a sensitivity on sputum smear-negative samples of 78.9% (95% CI, 70.0, 86.1) versus 66.1% (95% CI, 56.4, 74.9). Both tests had a specificity of 98.7% (95% CI, 93.0, 100), and both had comparable accuracies for detection of RIF-R in these samples. Ultra should significantly improve TB detection, especially in patients with paucibacillary disease, and may provide more-reliable RIF-R detection. IMPORTANCE The Xpert MTB/RIF assay (Xpert), the first point-of-care assay for tuberculosis (TB), was endorsed by the World Health Organization in December 2010. Since then, 23 million Xpert tests have been procured in 130 countries. Although Xpert showed high overall sensitivity and

  13. Tuberculosis screening of new hospital employees: compliance, clearance to work time, and cost using tuberculin skin test and interferon-gamma release assays.

    Science.gov (United States)

    Foster-Chang, Sarah A; Manning, Mary L; Chandler, Laura

    2014-11-01

    Selection of the most suitable test(s) for detection of Mycobacterium tuberculosis (TB) infection should be based on purpose, setting, effectiveness, and cost. Two tests are available to screen for latent TB: the tuberculin skin test (TST) and the more recent interferon-gamma release assays (IGRAs). Based on the administrative, logistic, and technical ease of use, an IGRA trial was initiated by the occupational health department at an urban Veteran's Administration health care facility for TB screening of new employees. As a result, new employees completing the pre-placement process within the organization's designated 14 days increased from 77% to 97%, new employee clearance to work time decreased from 13.18 to 5.91 days, and new employee TB screening costs were reduced by 40%. The IGRA is an acceptable alternative to the TST and has significant potential to improve the process of pre-placement TB screening. Copyright 2014, SLACK Incorporated.

  14. Standard test method for nondestructive assay of nuclear material in scrap and waste by passive-Active neutron counting using 252Cf shuffler

    CERN Document Server

    American Society for Testing and Materials. Philadelphia

    2008-01-01

    1.1 This test method covers the nondestructive assay of scrap and waste items for U, Pu, or both, using a 252Cf shuffler. Shuffler measurements have been applied to a variety of matrix materials in containers of up to several 100 L. Corrections are made for the effects of matrix material. Applications of this test method include measurements for safeguards, accountability, TRU, and U waste segregation, disposal, and process control purposes (1, 2, 3). 1.1.1 This test method uses passive neutron coincidence counting (4) to measure the 240Pu-effective mass. It has been used to assay items with total Pu contents between 0.03 g and 1000 g. It could be used to measure other spontaneously fissioning isotopes such as Cm and Cf. It specifically describes the approach used with shift register electronics; however, it can be adapted to other electronics. 1.1.2 This test method uses neutron irradiation with a moveable Cf source and counting of the delayed neutrons from the induced fissions to measure the 235U equiva...

  15. Rapid Identification of Dengue Virus Serotypes Using Monoclonal Antibodies in an Indirect Immunofluorescence Test.

    Science.gov (United States)

    1982-06-18

    encephalitis(TBH-28), West Nile(E-101), Yellow fever(French neurotropic and 17D strains), and Zika . Two Sandfly Fever viruses (213452 and Candiru) were...were provided as first passage isolates ( Aedes pseudoscutellaris cells, AP-61) or human serum from recent dengue virus patients. African isolates... viruses of the Phlebovirus genus (Table 1). Several monoclonal antibody preparations reacted solely with dengue virus serotypes. Two preparations (13E7 and

  16. Interlaboratory validation of 1% pluronic l92 surfactant as a suitable, aqueous vehicle for testing pesticide formulations using the murine local lymph node assay.

    Science.gov (United States)

    Boverhof, Darrell R; Wiescinski, Connie M; Botham, Phil; Lees, David; Debruyne, Eric; Repetto-Larsay, Marina; Ladics, Gregory; Hoban, Denise; Gamer, Armin; Remmele, Marina; Wang-Fan, Weizheng; Ullmann, Ludwig G; Mehta, Jyotigna; Billington, Richard; Woolhiser, Michael R

    2008-09-01

    The mouse local lymph node assay (LLNA) has become the preferred test for evaluating the dermal sensitization potential of chemicals and requirements are now emerging for its use in the evaluation of their formulated products, especially in the European Union. However, despite its widespread use and extensive validation, the use of this assay for directly testing mixtures and formulated products has been questioned, which could lead to repeat testing using multiple animal models. As pesticide formulations are typically a specific complex blend of chemicals for use as aqueous-based dilutions, traditional vehicles prescribed for the LLNA may change the properties of these formulations leading to inaccurate test results and hazard identification. The objective of this study was to evaluate the effectiveness of an aqueous solution of Pluronic L92 block copolymer surfactant (L92) as a vehicle in the mouse LLNA across five laboratories. Three chemicals with known sensitization potential and four pesticide formulations for which the sensitization potential in guinea pigs and/or humans had previously been assessed were used. Identical LLNA protocols and test materials were used in the evaluation. Assessment of the positive control chemicals, hexylcinnamaldehyde, formaldehyde, and potassium dichromate revealed positive results when using 1% aqueous L92 as the vehicle. Furthermore, results for these chemicals were reproducible among the five laboratories and demonstrated consistent relative potency determinations. The four pesticide formulations diluted in 1% aqueous L92 also demonstrated reproducible results in the LLNA among the five laboratories. Results for these test materials were also consistent with those generated previously using guinea pigs or from human experience. These data support testing aqueous compatible chemicals or pesticide formulations using the mouse LLNA, and provide additional support for the use of 1% aqueous L92 as a suitable, aqueous-based vehicle.

  17. External quality assurance of fibrinogen assays using normal plasma: results of the 2008 College of American Pathologists proficiency testing program in coagulation.

    Science.gov (United States)

    Cunningham, Mark T; Olson, John D; Chandler, Wayne L; Van Cott, Elizabeth M; Eby, Charles S; Teruya, Jun; Hollensead, Sandra C; Adcock, Dorothy M; Allison, Paul M; Kottke-Marchant, Kandice K; Smith, Marc D

    2012-07-01

    Proper diagnosis and therapy of fibrinogen deficiency requires high-quality fibrinogen assays. To assess the interlaboratory bias, precision, and grading of fibrinogen assays used by laboratories participating in the United States College of American Pathologists proficiency testing program in coagulation. Two identical vials of normal plasma were sent to more than 3500 laboratories. Participants measured fibrinogen levels using local methods. Fifty different fibrinogen methods were evaluated. All-method bias was 8.3% (range of method-specific biases, 0.0%-27.0%) and all-method coefficient of variation was 7.7% (range of method-specific coefficients of variation, 0.7%-25.8%). After controlling for reagent/instrument type, mean fibrinogen levels were 11.6% higher for prothrombin time-based reagents compared to Clauss (P graded as pass or fail using a target range of ±20% from the method mean (total pass rate, 98.8%). Total fail rate was 3.0-fold lower for mechanical instruments compared to photo-optical (0.5% versus 1.5%, P  =  .001). Nonetheless many photo-optical methods had very high precision and very low fail rates. Fibrinogen assays showed highly variable methodology and performance characteristics. Bias, precision, and grading were affected by the type of reagent or instrument used.

  18. The Culture Repopulation Ability (CRA) Assay and Incubation in Low Oxygen to Test Antileukemic Drugs on Imatinib-Resistant CML Stem-Like Cells.

    Science.gov (United States)

    Cheloni, Giulia; Tanturli, Michele

    2016-01-01

    Chronic myeloid leukemia (CML) is a stem cell-driven disorder caused by the BCR/Abl oncoprotein, a constitutively active tyrosine kinase (TK). Chronic-phase CML patients are treated with impressive efficacy with TK inhibitors (TKi) such as imatinib mesylate (IM). However, rather than definitively curing CML, TKi induces a state of minimal residual disease, due to the persistence of leukemia stem cells (LSC) which are insensitive to this class of drugs. LSC persistence may be due to different reasons, including the suppression of BCR/Abl oncoprotein. It has been shown that this suppression follows incubation in low oxygen under appropriate culture conditions and incubation times.Here we describe the culture repopulation ability (CRA) assay, a non-clonogenic assay capable - together with incubation in low oxygen - to reveal in vitro stem cells endowed with marrow repopulation ability (MRA) in vivo. The CRA assay can be used, before moving to animal tests, as a simple and reliable method for the prescreening of drugs potentially active on CML and other leukemias with respect to their activity on the more immature leukemia cell subsets.

  19. A comparison of indirect immunofluorescence and electron microscopy for the diagnosis of some haemorrhagic viruses in cell cultures.

    Science.gov (United States)

    El Mekki, A A; van der Groen, G

    1981-09-01

    Yellow fever, dengue (types 1, 2 and 4), Chikungunya, Rift Valley fever, Ebola, Marburg, and Lassa viruses were inoculated into susceptible cell cultures and daily investigated by indirect immunofluorescence (IFA) and electron microscopy (EM) with a view to achieve an early detection-identification of these agents. Compared to the other cell lines tested (Vero, BHK-21 and Aedes albopictus), CV-1 cells were found to be more sensitive. Viral antigens were detected by IFA from a few hours post inoculation (CHIK and RVF) to a maximum of 3 days (YF and EBO). For most of the viruses studied, the cytopathic effect (CPE) commenced 2-3 days after the detection of viral antigens. Virus particles were detected by EM only in the case of EBO, MBG and LAS, before any CPE was observed in cell cultures.

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

    Science.gov (United States)

    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 (

  1. Cytokine Release Assays as Tests for Exposure to Leishmania, and for Confirming Cure from Leishmaniasis, in Solid Organ Transplant Recipients.

    Directory of Open Access Journals (Sweden)

    Eugenia Carrillo

    Full Text Available Spain has one of the world's largest pools of organ donors and is a global leader in terms of the number of transplants it performs. The current outbreak of leishmaniasis in Fuenlabrada (in the southwest of the region of Madrid, Spain has involved 600 clinical cases since late 2009 (prevalence 0.2%. It may therefore be wise to monitor the town's transplanted population for Leishmania infantum; its members are immunosuppressed and at greater risk of infection and relapse following treatment. The present work examines the use of cytokine release assays to determine the prevalence of Leishmania infection in this population, and to confirm recovery following treatment for visceral leishmaniasis (VL. The humoral and cellular immune responses to L. infantum were characterized in 63 solid organ transplant (SOT recipients from Fuenlabrada, 57 of whom reported no previous episode of VL (NVL subjects, and six of whom had been cured of VL (CVL subjects. Seventeen subjects (12 NVL and 5 CVL showed a patent lymphoproliferative response to soluble Leishmania antigen (SLA. Stimulation of peripheral blood mononuclear cell cultures and of whole blood with SLA led to the production of different combinations of cytokines that might serve to confirm Leishmania infection or recovery from VL and help prevent cured patients from relapsing into this serious condition.

  2. Assay of anti-HBs antibodies using a recombinant antigen and latex particle counting: comparison with five commercial tests.

    Science.gov (United States)

    Galanti, L M; Cornu, C; Masson, P L; Robert, A R; Becheanu, D; Lamy, M E; Cambiaso, C L

    1991-05-01

    An assay of anti-HBs antibodies based on agglutination of latex particles coated with recombinant HBs-antigen was compared with Abbott radioimmunoassay (Abbott-RIA), which uses a human plasma-derived antigen. The population examined consisted of 76 Abbott-RIA anti-HBs-negative prevaccinated subjects and 1044 serum samples anti-HBs found positive by Abbott-RIA, including 283 samples of subjects vaccinated either with a human plasma-derived vaccine (group A; n = 180) or with a recombinant vaccine (group B; n = 103). Correlation coefficients between the two techniques were respectively r = 0.89 for the whole population (n = 1044), r = 0.98 in group A and r = 0.74 in group B. Anti-HBs titres were higher with latex than with RIA in group B as shown by the regression slopes: latex = 508 + 1.11 RIA in group A and latex = -1138 + 3.97 RIA in group B, suggesting that some vaccinated subjects from group B produced antibodies against epitopes proper to the recombinant antigen. In the prevaccinated population and in group A, the latex results were compared with those of radioimmunoassays (Abbott, Sorin) and enzyme immunoassays (Behring, Roche, Pasteur). Only the Roche-EIA detected anti-HBs in the prevaccinated subjects. The correlation between the various immunoassays was r greater than 0.96 only for values higher than 100 IU/l.

  3. Direct Agglutination Test and Enzyme Linked Immunosorbent Assay with Urine Samples for the Diagnosis of Visceral Leishma-niasis

    Directory of Open Access Journals (Sweden)

    Sarkari B

    2007-07-01

    Full Text Available Background: Visceral leishmaniasis (VL or Kala azar is an infectious disease caused by various species of Leishmania parasites. The aim of this study was to detect and compare the presence of anti-Leishmania antibodies in the urine of vis-ceral leishmaniasis patients using ELISA and DAT methods."nMethods: A total of 30 urine samples were collected from VL patients referred to Shiraz (southeast of Iran hospitals. Moreover 31 urine samples were collected from healthy individuals and patients with other diseases such as malaria, brucellosis, hydatidosis and cutaneous leishmaniasis. Collected samples were examined to detect anti-Leishmania antibod-ies in urine, using ELISA and DAT."nResults: Anti-Leishmania antibody was detected in urine of 18 out of 30 (60% VL patients by DAT while ELISA detected anti-Leishmania antibodies in urine of 28 out of 30 (93.3% of VL cases. Sensitivity and specificity of urine-based DAT was 60% and 83.9%, respectively while sensitivity and specificity of urine-based ELISA were 93.3% and 93.5%, corre-spondingly. "nConclusion: Urine-based DAT and ELISA have a reasonable specificity and sensitivity in diagnosis of VL. Accordingly, urine-based ELISA might be a suitable alternative for serum based assays for diagnosis of VL.

  4. The E-screen test and the MELN gene-reporter assay used for determination of estrogenic activity in fruits and vegetables in relation to pesticide residues.

    Science.gov (United States)

    Schilirò, Tiziana; Porfido, Arianna; Longo, Annalisa; Coluccia, Sara; Gilli, Giorgio

    2013-12-01

    Endocrine-disrupting chemicals (EDCs) may lead to adverse systemic effects by interfering with normal hormone homeostasis, and diet is considered to be among the main routes of EDC exposure. The present study investigated the total estrogenic activity of fruits and vegetables by calculating the 17-β-estradiol equivalent quantity (EEQ) using two in vitro tests: the human breast cancer cell line (MCF-7 BUS) proliferation assay (E-screen test) and the luciferase-transfected human breast cancer cell line (MELN) gene-reporter assay. Of the 24 analyzed fruits and vegetables, 14 contained from 1 to 4 pesticide residues in concentrations ranging from 0.02 to 1.19 ppm, whereas the other 10 did not contain any pesticide residues. The EEQ values for all positive samples ranged from 0.010 to 0.616 μg/100g for the above in vitro tests. Our study demonstrates that estrogenic activity was present in fruits and vegetables and that the concentration of allowable pesticide residues and EEQ values were positively correlated; however, no correlation was found by comparing the estrogenic activity and the intrinsic content of phytoestrogens obtained from the available literature. A theoretical adult dietary intake of 0.7-0.9 ng EEQ/L/day from fruits and vegetables was calculated. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Semi-automated relative quantification of cell culture contamination with mycoplasma by Photoshop-based image analysis on immunofluorescence preparations.

    Science.gov (United States)

    Kumar, Ashok; Yerneni, Lakshmana K

    2009-01-01

    Mycoplasma contamination in cell culture is a serious setback for the cell-culturist. The experiments undertaken using contaminated cell cultures are known to yield unreliable or false results due to various morphological, biochemical and genetic effects. Earlier surveys revealed incidences of mycoplasma contamination in cell cultures to range from 15 to 80%. Out of a vast array of methods for detecting mycoplasma in cell culture, the cytological methods directly demonstrate the contaminating organism present in association with the cultured cells. In this investigation, we report the adoption of a cytological immunofluorescence assay (IFA), in an attempt to obtain a semi-automated relative quantification of contamination by employing the user-friendly Photoshop-based image analysis. The study performed on 77 cell cultures randomly collected from various laboratories revealed mycoplasma contamination in 18 cell cultures simultaneously by IFA and Hoechst DNA fluorochrome staining methods. It was observed that the Photoshop-based image analysis on IFA stained slides was very valuable as a sensitive tool in providing quantitative assessment on the extent of contamination both per se and in comparison to cellularity of cell cultures. The technique could be useful in estimating the efficacy of anti-mycoplasma agents during decontaminating measures.

  6. Diagnostic accuracy of a loop-mediated isothermal PCR assay for detection of Orientia tsutsugamushi during acute Scrub Typhus infection.

    Science.gov (United States)

    Paris, Daniel H; Blacksell, Stuart D; Nawtaisong, Pruksa; Jenjaroen, Kemajittra; Teeraratkul, Achara; Chierakul, Wirongrong; Wuthiekanun, Vanaporn; Kantipong, Pacharee; Day, Nicholas P J

    2011-09-01

    There is an urgent need to develop rapid and accurate point-of-care (POC) technologies for acute scrub typhus diagnosis in low-resource, primary health care settings to guide clinical therapy. In this study we present the clinical evaluation of loop-mediated isothermal PCR assay (LAMP) in the context of a prospective fever study, including 161 patients from scrub typhus-endemic Chiang Rai, northern Thailand. A robust reference comparator set comprising following 'scrub typhus infection criteria' (STIC) was used: a) positive cell culture isolate and/or b) an admission IgM titer ≥1∶12,800 using the 'gold standard' indirect immunofluorescence assay (IFA) and/or c) a 4-fold rising IFA IgM titer and/or d) a positive result in at least two out of three PCR assays. Compared to the STIC criteria, all PCR assays (including LAMP) demonstrated high specificity ranging from 96-99%, with sensitivities varying from 40% to 56%, similar to the antibody based rapid test, which had a sensitivity of 47% and a specificity of 95%. The diagnostic accuracy of the LAMP assay was similar to realtime and nested conventional PCR assays, but superior to the antibody-based rapid test in the early disease course. The combination of DNA- and antibody-based detection methods increased sensitivity with minimal reduction of specificity, and expanded the timeframe of adequate diagnostic coverage throughout the acute phase of scrub typhus.

  7. Assessment of cosmetic ingredients in the in vitro reconstructed human epidermis test method EpiSkin™ using HPLC/UPLC-spectrophotometry in the MTT-reduction assay.

    Science.gov (United States)

    Alépée, N; Hibatallah, J; Klaric, M; Mewes, K R; Pfannenbecker, U; McNamee, P

    2016-06-01

    Cosmetics Europe recently established HPLC/UPLC-spectrophotometry as a suitable alternative endpoint detection system for measurement of formazan in the MTT-reduction assay of reconstructed human tissue test methods irrespective of the test system involved. This addressed a known limitation for such test methods that use optical density for measurement of formazan and may be incompatible for evaluation of strong MTT reducer and/or coloured chemicals. To build on the original project, Cosmetics Europe has undertaken a second study that focuses on evaluation of chemicals with functionalities relevant to cosmetic products. Such chemicals were primarily identified from the Scientific Committee on Consumer Safety (SCCS) 2010 memorandum (addendum) on the in vitro test EpiSkin™ for skin irritation testing. Fifty test items were evaluated in which both standard photometry and HPLC/UPLC-spectrophotometry were used for endpoint detection. The results obtained in this study: 1) provide further support for Within Laboratory Reproducibility of HPLC-UPLC-spectrophotometry for measurement of formazan; 2) demonstrate, through use a case study with Basazol C Blue pr. 8056, that HPLC/UPLC-spectrophotometry enables determination of an in vitro classification even when this is not possible using standard photometry and 3) addresses the question raised by SCCS in their 2010 memorandum (addendum) to consider an endpoint detection system not involving optical density quantification in in vitro reconstructed human epidermis skin irritation test methods. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Evaluation and validation of a single-dilution potency assay based upon serology of vaccines containing diphtheria toxoid: analysis for consistency in production and testing at the laboratory of the Control of Biological Products of the RIVM

    NARCIS (Netherlands)

    Akkermans AM; Hendriksen CFM; Marsman FR; de Jong WH; van de Donk HJM

    1993-01-01

    A single-dilution assay can be a valid procedure to demonstrate that a product exceeds the minimal requirement given for potency provided that consistency in production and testing has been proven. Information is presented justifying the use of a single dilution assay based upon quantitative

  9. Local lymph node assay (LLNA): comparison of different protocols by testing skin-sensitizing epoxy resin system components.

    Science.gov (United States)

    Gamer, Armin O; Nies, Eberhard; Vohr, Hans-Werner

    2008-12-01

    Thirteen epoxy resin system components were tested in the LLNA with regard to their sensitizing potency. Lymph node stimulation was quantified not only by measuring the incorporation of [3H]-thymidine into the ear lymph nodes but also the counts of cells recovered from these organs. Equivalent figures were obtained with both endpoints used for the evaluation of lymph node cell proliferation if the reference stimulation indices were adjusted. When dissolved in acetone, all test substances showed skin-sensitizing potential, mainly on the boundary between "strong" and "moderate" according to common potency evaluation schemes. Replacing acetone with acetone/olive oil (4:1) as a vehicle for four selected test items, resulted in considerably lower estimated concentrations for sensitization induction. The challenges in comparing the results obtained by different LLNA variations are discussed.

  10. Determination of total galactose from dried blood spots—extensive assay evaluation of a CE-marked test-kit

    OpenAIRE

    Fingerhut, R; Torresani, T

    2013-01-01

    Most newborn screening laboratories use CE-marked or FDA-approved test-kits, like in routine clinical chemistry. National regulations require only minimal evaluation from the customer, if the test-kits are used as specified by the manufacturer. The microtiter-based kit-concept is often based on the perception, that the laboratory always processes whole microtiter plates. However, in the daily routine, this is rather a rare exception, which leads to much higher costs per newborn, compared to t...

  11. Comparison of dose-responses of contact allergens using the guinea pig maximization test and the local lymph node assay

    NARCIS (Netherlands)

    Och, F.M.M. van; Vandebriel, R.J.; Prinsen, M.K.; Jong, W.H. de; Slob, W.; Loveren, H. van

    2001-01-01

    The guinea pig maximization test (GPMT) has been used as a method for the prediction of skin sensitizing potential for over 30 years. Besides hazard identification, risk assessment of sensitizing chemicals requires the assessment of potency. For the determination of potency based on lowest effective

  12. Preliminary assays for lemongrass essential oil ecotoxicological test in D. similis and C. silvestrii submitted to gamma radiation

    International Nuclear Information System (INIS)

    Gimiliani, Giovana T.; Rogero, Sizue O.; Rogero, Jose R.; Cruz, Aurea S.

    2011-01-01

    Pharmaceutical products are of great interest in ecotoxicological studies due to being found some of these products in the superficial waters and sediments, water and sewage treatment effluents. It was verified an increase of insect repellent chemical products in the aquatic environment because of the increase of diseases transmitted by mosquitoes like dengue. As these compounds show toxicity, the use of essential oils natural products with repellent properties is increasing and the literature about the impact in the aquatic environment is scarce. The hydric frame would suffer natural radiation and radiations from energy generation nuclear plants impacts fall out of tests and nuclear accidents. There is no universal definition of environmental protection and there are few studies on radiation effects in the aquatic environment. In this study was determined the lemon grass essential oil toxicity level as well as the lethal dose of ionizing radiation, LD 50 , in aquatic organisms. Cytotoxicity test was performed by in vitro neutral red uptake method in NCTC clone L929 cell line. In the LD 50 test aquatic organisms were submitted to gamma radiation. The essential oil of lemongrass Cymbopogon flexuosus showed cytotoxicity index IC 50 about 50μg.mL -1 . The LD 50 for Daphnia similis was 242 Gy and Ceriodaphnia silvestrii about 525 Gy. Studies will be continued with acute and chronic ecotoxicological tests of lemongrass essential oil in natural organisms and in organisms submitted to gamma radiation, utilizing the results obtained in this work. (author)

  13. Comparison of Culture, Cytotoxin Assay and Two Eia Tests with Clinical Diagnosis of Clostridium difficile-Associated Diarrhea

    Directory of Open Access Journals (Sweden)

    Marilyn Binning

    1994-01-01

    Full Text Available Objective: The most common etiology of infectious diarrhea in hospitalized patients is Clostridium difficile. No single laboratory test yields a definitive diagnosis. Four methods were evaluated for their sensitivity and specificity in patients who had clinically defined C difficile-associated diarrhea.

  14. Preliminary assays for lemongrass essential oil ecotoxicological test in D. similis and C. silvestrii submitted to gamma radiation

    Energy Technology Data Exchange (ETDEWEB)

    Gimiliani, Giovana T.; Rogero, Sizue O.; Rogero, Jose R., E-mail: gtgimiliani@ipen.br [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Cruz, Aurea S. [Instituto Adolfo Lutz, Sao Paulo, SP (Brazil). Culturas Celulares

    2011-07-01

    Pharmaceutical products are of great interest in ecotoxicological studies due to being found some of these products in the superficial waters and sediments, water and sewage treatment effluents. It was verified an increase of insect repellent chemical products in the aquatic environment because of the increase of diseases transmitted by mosquitoes like dengue. As these compounds show toxicity, the use of essential oils natural products with repellent properties is increasing and the literature about the impact in the aquatic environment is scarce. The hydric frame would suffer natural radiation and radiations from energy generation nuclear plants impacts fall out of tests and nuclear accidents. There is no universal definition of environmental protection and there are few studies on radiation effects in the aquatic environment. In this study was determined the lemon grass essential oil toxicity level as well as the lethal dose of ionizing radiation, LD{sub 50}, in aquatic organisms. Cytotoxicity test was performed by in vitro neutral red uptake method in NCTC clone L929 cell line. In the LD{sub 50} test aquatic organisms were submitted to gamma radiation. The essential oil of lemongrass Cymbopogon flexuosus showed cytotoxicity index IC{sub 50} about 50{mu}g.mL{sup -1}. The LD{sub 50} for Daphnia similis was 242 Gy and Ceriodaphnia silvestrii about 525 Gy. Studies will be continued with acute and chronic ecotoxicological tests of lemongrass essential oil in natural organisms and in organisms submitted to gamma radiation, utilizing the results obtained in this work. (author)

  15. Sensitivity of direct immunofluorescence in oral diseases. Study of 125 cases.

    Science.gov (United States)

    Sano, Susana Mariela; Quarracino, María Cecilia; Aguas, Silvia Cristina; González, Ernestina Jesús; Harada, Laura; Krupitzki, Hugo; Mordoh, Ana

    2008-05-01

    Direct immunofluorescence (DIF) is widely used for the diagnosis of bullous diseases and other autoimmune pathologies such as oral lichen planus. There is no evidence in the literature on how the following variants influence the detection rate of DIF: intraoral site chosen for the biopsy, perilesional locus or distant site from the clinical lesion, number of biopsies and instrument used. to determine if the following variants influenced the sensitivity (detection rate): intraoral site chosen for the biopsy, perilesional or distant site from the clinical lesion, number of biopsies and instrument used (punch or scalpel). A retrospective study was done at the Cátedra de Patología y Clínica Bucodental II at the Facultad de Odontología, Universidad de Buenos Aires; 136 clinical medical histories were revised for the period March 2000 - March 2005 corresponding to patients with clinical diagnosis of OLP and bullous diseases (vulgar pemphigus, bullous pemphigoid and cicatricial pemphigoid). DIF detection rate was 65.8% in patients with OLP, 66.7% in cicatricial pemphigoid patients, in bullous pemphigoid 55.6%, in pemphigus vulgaris 100%, and in those cases in which certain diagnosis could not be obtained, the DIF positivity rate was 45.5% (Pearson chi(2) (4)= 21.5398 Pr= 0.000). There was no statistically significant difference between the different sites of biopsy (Fisher exact test: 0.825). DIF detection rate in perilesional biopsies was 66.1% and in those distant from the site of clinical lesion was 64.7% (Pearson chi(2) v1)= 0.0073 Pr= 0.932. When the number of biopsies were incremented, DIF detection rate also incremented (Pearson chi(2) = 8.7247 Pr= 0.003). The biopsies taken with punch had a higher detection rate than those taken with scalpel (39.1% versus 71.7%) (Pearson chi(2) = 49.0522 Pr= 0.000). While not statistically significant, the tendency outlined in this study indicates there are intraoral regions in which the detection rate of the DIF technique is

  16. Indirect immunofluorescence staining of Chlamydia trachomatis inclusions in microculture plates with monoclonal antibodies.

    Science.gov (United States)

    Zapata, M; Chernesky, M; Mahony, J

    1984-06-01

    Indirect immunofluorescence (IF) staining, using a monoclonal antibody, detected two- to fourfold more inclusions than did iodine staining. Of 274 clinical specimens, 53 (19.3%) were positive by IF on passage 1 as compared with 33 (12%) by iodine staining (P less than 0.005). IF-stained inclusions in McCoy cells in the bottom of microculture wells were readily viewed with a long-focal-length objective at a magnification of 250 X.

  17. Indirect immunofluorescence staining of Chlamydia trachomatis inclusions in microculture plates with monoclonal antibodies.

    OpenAIRE

    Zapata, M; Chernesky, M; Mahony, J

    1984-01-01

    Indirect immunofluorescence (IF) staining, using a monoclonal antibody, detected two- to fourfold more inclusions than did iodine staining. Of 274 clinical specimens, 53 (19.3%) were positive by IF on passage 1 as compared with 33 (12%) by iodine staining (P less than 0.005). IF-stained inclusions in McCoy cells in the bottom of microculture wells were readily viewed with a long-focal-length objective at a magnification of 250 X.

  18. Immunofluorescent staining of nuclear antigen in lymphoid cells transformed by Herpesvirus papio (HVP).

    Science.gov (United States)

    Schmitz, H

    1981-01-01

    An improved fixation method for antigen detection in lymphoblastoid cells is described. Herpesvirus papio nuclear antigen (HUPNA) could be stained in several transformed lymphoid cell lines by anti-complement immunofluorescence (ACIF). Antibody to HUPNA was detected in many human sera containing antibodies to Epstein-Barr virus capsid and nuclear antigen (EBNA). Rheumatoid arthritis sera showed a high incidence of both anti-EBNA and anti-HUPNA antibodies.

  19. Assay system

    International Nuclear Information System (INIS)

    Patzke, J.B.; Rosenberg, B.J.

    1984-01-01

    The accuracy of assays for monitoring concentrations of basic drugs in biological fluids containing a 1 -acid glycoproteins, such as blood (serum or plasma), is improved by the addition of certain organic phosphate compounds to minimize the ''protein effect.'' Kits containing the elements of the invention are also disclosed

  20. Synthesis of carboxyl-capped and bright YVO4:Eu,Bi nanoparticles and their applications in immunochromatographic test strip assay

    International Nuclear Information System (INIS)

    Luo, Min; Sun, Tian-Ying; Wang, Jia-Hong; Yang, Peng; Gan, Liang; Liang, Li-Lei; Yu, Xue-Feng; Gong, Xing-Hou

    2013-01-01

    Graphical abstract: - Highlights: • The morphology and properties of YVO 4 :Eu,Bi nanoparticles were investigated. • YVO 4 :Eu,Bi were coupled with IgG for bioprobes due to their good properties. • YVO 4 :Eu,Bi were applied to immunochromatographic test strip assay. - Abstract: Carboxyl-capped YVO 4 :Eu,Bi nanoparticles with average diameter of ∼10 nm were synthesized via a copolymer of phosphono and carboxylic acid mediated hydrothermal method. Under a 350 nm ultraviolet light excitation, the YVO 4 :Eu,Bi NPs exhibit sharp and bright red emission peaked at 615 nm and with highest quantum yield of ∼43%. Furthermore, the nanoparticles show good water/buffer stability and feasible bioconjugation benefiting from the carboxylic groups on their surface. Based on these kind optical and surface properties of the YVO 4 :Eu,Bi nanoparticles, an immunochromatographic test strip assay for quantitative determination of human IgG was achieved. This protocol can be extended to other rare-earth nanoparticles with the purpose of developing bioprobes for desired applications

  1. Fibrinogen Demonstration in Oral Lichen Planus: An Immunofluorescence Study on Archival Tissues.

    Science.gov (United States)

    Shirol, Pallavi D; Naik, Veena; Kale, Alka

    2015-10-01

    Lichen planus is a premalignant condition with minimal diagnostic aids. This study is an attempt to use paraffin embedded sections of lichen planus with immunofluorescein stain and to evaluate the immunofluorescent sections to establish pattern of fibrinogen deposition. Thirty-five paraffin embedded sections of old and new cases of oral lichen planus (study group) and five normal oral mucosa (control group) were chosen. Two sections of each (H & E) case were taken, one was stained with hematoxylin and eosin and another with fluorescein isothiocynate conjugate (FITC) polyclonal rabbit antibody against fibrinogen. Fluorescent findings were examined with a fluorescent microscope. A high statistical significant correlation was found in respect to fluorescence positivity, intensity of fluorescence and distribution of fluorescence each with p < 0.0001 and fluorescence at blood vessel walls (p = 0.0003). This study suggested that paraffin embedded sections can be successfully used in direct immunofluorescence staining in routine set up where only formalin fixed tissues are received. Paraffin embedded sections can be successfully used in direct immunofluorescence staining when only formalin fixed tissues are received.

  2. Standard test method for nondestructive assay of special nuclear material holdup using Gamma-Ray spectroscopic methods

    CERN Document Server

    American Society for Testing and Materials. Philadelphia

    2007-01-01

    1.1 This test method describes gamma-ray methods used to nondestructively measure the quantity of 235U, or 239Pu remaining as holdup in nuclear facilities. Holdup occurs in all facilities where nuclear material is processed, in process equipment, in exhaust ventilation systems and in building walls and floors. 1.2 This test method includes information useful for management, planning, selection of equipment, consideration of interferences, measurement program definition, and the utilization of resources (1, 2, 3, 4). 1.3 The measurement of nuclear material hold up in process equipment requires a scientific knowledge of radiation sources and detectors, transmission of radiation, calibration, facility operations and error analysis. It is subject to the constraints of the facility, management, budget, and schedule; plus health and safety requirements; as well as the laws of physics. The measurement process includes defining measurement uncertainties and is sensitive to the form and distribution of the material...

  3. Determining the size and concentration dependence of gold nanoparticles in vitro cytotoxicity (IC{sub 50}) test using WST-1 assay

    Energy Technology Data Exchange (ETDEWEB)

    Rosli, Nur Shafawati binti; Rahman, Azhar Abdul [School of Physics, Universiti Sains Malaysia, 11800, Pulau Pinang (Malaysia); Aziz, Azlan Abdul [School of Physics, Universiti Sains Malaysia, 11800, Pulau Pinang (Malaysia); Nano-Biotechnology Research and Innovation (NanoBRI), Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800, Pulau Pinang (Malaysia); Shamsuddin, Shaharum [Nano-Biotechnology Research and Innovation (NanoBRI), Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800, Pulau Pinang (Malaysia); School of Health Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia)

    2015-04-24

    Gold nanoparticles (AuNPs) received a great deal of attention for biomedical applications, especially in diagnostic imaging and therapeutics. Even though AuNPs have potential benefits in biomedical applications, the impact of AuNPs on human and environmental health still remains unclear. The use of AuNPs which is a high-atomic-number materials, provide advantages in terms of radiation dose enhancement. However, before this can become a clinical reality, cytotoxicity of the AuNPs has to be carefully evaluated. Cytotoxicity test is a rapid, standardized test that is very sensitive to determine whether the nanoparticles produced are harmful or benign on cellular components. In this work the size and concentration dependence of AuNPs cytotoxicity in breast cancer cell lines (MCF-7) are tested by using WST-1 assay. The sizes of AuNPs tested were 13 nm, 50 nm, and 70 nm. The cells were seeded in the 96-well plate and were treated with different concentrations of AuNPs by serial dilution for each size of AuNPs. The high concentration of AuNPs exhibit lower cell viability compared to low concentration of AuNPs. We quantified the toxicity of AuNPs in MCF-7 cell lines by determining the IC{sub 50} values in WST-1 assays. The IC{sub 50} values (inhibitory concentrations that effected 50% growth inhibition) of 50 nm AuNPs is lower than 13 nm and 70 nm AuNPs. Mean that, 50nm AuNPs are more toxic to the MCF-7 cells compared to smaller and larger sizes AuNPs. The presented results clearly indicate that the cytotoxicity of AuNPs depend not only on the concentration, but also the size of the nanoparticles.

  4. Development of a microarray-based assay for efficient testing of new HSP70/DnaK inhibitors.

    Science.gov (United States)

    Mohammadi-Ostad-Kalayeh, Sona; Hrupins, Vjaceslavs; Helmsen, Sabine; Ahlbrecht, Christin; Stahl, Frank; Scheper, Thomas; Preller, Matthias; Surup, Frank; Stadler, Marc; Kirschning, Andreas; Zeilinger, Carsten

    2017-12-15

    A facile method for testing ATP binding in a highly miniaturized microarray environment using human HSP70 and DnaK from Mycobacterium tuberculosis as biological targets is reported. Supported by molecular modelling studies we demonstrate that the position of the fluorescence label on ATP has a strong influence on the binding to human HSP70. Importantly, the label has to be positioned on the adenine ring and not to the terminal phosphate group. Unlabelled ATP displaced bound Cy5-ATP from HSP70 in the micromolar range. The affinity of a well-known HSP70 inhibitor VER155008 for the ATP binding site in HSP70 was determined, with a EC 50 in the micromolar range, whereas reblastin, a HSP90-inhibitor, did not compete for ATP in the presence of HSP70. The applicability of the method was demonstrated by screening a small compound library of natural products. This unraveled that terphenyls rickenyl A and D, recently isolated from cultures of the fungus Hypoxylon rickii, are inhibitors of HSP70. They compete with ATP for the chaperone in the range of 29 µM (Rickenyl D) and 49 µM (Rickenyl A). Furthermore, the microarray-based test system enabled protein-protein interaction analysis using full-length HSP70 and HSP90 proteins. The labelled full-length human HSP90 binds with a half-maximal affinity of 5.5 µg/ml (∼40 µM) to HSP70. The data also demonstrate that the microarray test has potency for many applications from inhibitor screening to target-oriented interaction studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Comparison of a flow assay for brucellosis antibodies with the reference cELISA test in West African Bos indicus.

    Directory of Open Access Journals (Sweden)

    Barend M deC Bronsvoort

    Full Text Available Brucellosis is considered by the Food and Agricultural Organisation and the World Health Organisation as one of the most widespread zoonoses in the world. It is a major veterinary public health challenge as animals are almost exclusively the source of infection for people. It is often undiagnosed in both human patients and the animal sources and it is widely acknowledged that the epidemiology of brucellosis in humans and animals is poorly understood, particularly in sub-Saharan Africa. It is therefore important to develop better diagnostic tools in order to improve our understanding of the epidemiology and also for use in the field for disease control and eradication. As with any new diagnostic test, it is essential that it is validated in as many populations as possible in order to characterise its performance and improve the interpretation of its results. This paper describes a comparison between a new lateral flow assasy (LFA for bovine brucellosis and the widely used cELISA in a no gold standard analysis to estimate test performance in this West African cattle population. A Bayesian formulation of the Hui-Walter latent class model incorporated previous studies' data on sensitivity and specificity of the cELISA. The results indicate that the new LFA is very sensitive (approximately 87% and highly specific (approximately 97%. The analysis also suggests that the current cut-off of the cELSIA may not be optimal for this cattle population but alternative cut-offs did not significantly change the estimates of the LFA. This study demonstrates the potential usefulness of this simple to use test in field based surveillance and control which could be easily adopted for use in developing countries with only basic laboratory facilities.

  6. Comparison of a Flow Assay for Brucellosis Antibodies with the Reference cELISA Test in West African Bos indicus

    Science.gov (United States)

    Bronsvoort, Barend M. deC.; Koterwas, Bronwyn; Land, Fiona; Handel, Ian G.; Tucker, James; Morgan, Kenton L.; Tanya, Vincent N.; Abdoel, Theresia H.; Smits, Henk L.

    2009-01-01

    Brucellosis is considered by the Food and Agricultural Organisation and the World Health Organisation as one of the most widespread zoonoses in the world. It is a major veterinary public health challenge as animals are almost exclusively the source of infection for people. It is often undiagnosed in both human patients and the animal sources and it is widely acknowledged that the epidemiology of brucellosis in humans and animals is poorly understood, particularly in sub-Saharan Africa. It is therefore important to develop better diagnostic tools in order to improve our understanding of the epidemiology and also for use in the field for disease control and eradication. As with any new diagnostic test, it is essential that it is validated in as many populations as possible in order to characterise its performance and improve the interpretation of its results. This paper describes a comparison between a new lateral flow assasy (LFA) for bovine brucellosis and the widely used cELISA in a no gold standard analysis to estimate test performance in this West African cattle population. A Bayesian formulation of the Hui-Walter latent class model incorporated previous studies' data on sensitivity and specificity of the cELISA. The results indicate that the new LFA is very sensitive (∼87%) and highly specific (∼97%). The analysis also suggests that the current cut-off of the cELSIA may not be optimal for this cattle population but alternative cut-offs did not significantly change the estimates of the LFA. This study demonstrates the potential usefulness of this simple to use test in field based surveillance and control which could be easily adopted for use in developing countries with only basic laboratory facilities. PMID:19381332

  7. Impact of grey zone sample testing by enzyme-linked immunosorbent assay in enhancing blood safety: Experience at a tertiary care hospital in North India.

    Science.gov (United States)

    Solanki, Archana; Singh, Abhay; Chaudhary, Rajendra

    2016-01-01

    Enzyme-linked immunosorbent assay (ELISA) used for screening blood donors for transfusion transmitted infections (TTIs) can sometimes fail to detect blood donors who are recently infected or possessing the low strength of pathogen. Estimation of a grey zone in ELISA testing and repeat testing of grey zone samples can further help in reducing the risks of TTI in countries where nucleic acid amplification testing for TTIs is not feasible. Grey zone samples with optical density (OD) lying between cut-off OD and 10% below the cut-off OD (cut-off OD × 0.9) were identified during routine ELISA testing. On performing repeat ELISA testing on grey zone samples in duplicate, the samples showing both OD value below grey zone were marked nonreactive, and samples showing one or both OD value in the grey zone were marked indeterminate. The samples on repeat testing showing one or both OD above cut-off value were marked positive. About 119 samples (77 for hepatitis B virus [HBV], 23 for human immunodeficiency virus [HIV], and 19 for hepatitis C virus [HCV]) were found to be in grey zone. On repeat testing of these samples in duplicate, 70 (58.8%) samples (45 for HBV, 12 for HIV, and 13 for HCV) were found to be reactive. Six (5%) samples (four for HBV, one for HIV, and one for HCV) were found to be indeterminate. Seventy donors initially screened negative, were found out to be potentially infectious on repeat grey zone testing. Thus, estimation of grey zone samples with repeat testing can further enhance the safety of blood transfusion.

  8. Use of HPLC/UPLC-spectrophotometry for detection of formazan in in vitro Reconstructed human Tissue (RhT)-based test methods employing the MTT-reduction assay to expand their applicability to strongly coloured test chemicals.

    Science.gov (United States)

    Alépée, N; Barroso, J; De Smedt, A; De Wever, B; Hibatallah, J; Klaric, M; Mewes, K R; Millet, M; Pfannenbecker, U; Tailhardat, M; Templier, M; McNamee, P

    2015-06-01

    A number of in vitro test methods using Reconstructed human Tissues (RhT) are regulatory accepted for evaluation of skin corrosion/irritation. In such methods, test chemical corrosion/irritation potential is determined by measuring tissue viability using the photometric MTT-reduction assay. A known limitation of this assay is possible interference of strongly coloured test chemicals with measurement of formazan by absorbance (OD). To address this, Cosmetics Europe evaluated use of HPLC/UPLC-spectrophotometry as an alternative formazan measurement system. Using the approach recommended by the FDA guidance for validation of bio-analytical methods, three independent laboratories established and qualified their HPLC/UPLC-spectrophotometry systems to reproducibly measure formazan from tissue extracts. Up to 26 chemicals were then tested in RhT test systems for eye/skin irritation and skin corrosion. Results support that: (1) HPLC/UPLC-spectrophotometry formazan measurement is highly reproducible; (2) formazan measurement by HPLC/UPLC-spectrophotometry and OD gave almost identical tissue viabilities for test chemicals not exhibiting colour interference nor direct MTT reduction; (3) independent of the test system used, HPLC/UPLC-spectrophotometry can measure formazan for strongly coloured test chemicals when this is not possible by absorbance only. It is therefore recommended that HPLC/UPLC-spectrophotometry to measure formazan be included in the procedures of in vitro RhT-based test methods, irrespective of the test system used and the toxicity endpoint evaluated to extend the applicability of these test methods to strongly coloured chemicals. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Options to Expand HIV Viral Load Testing in South Africa: Evaluation of the GeneXpert® HIV-1 Viral Load Assay.

    Directory of Open Access Journals (Sweden)

    Natasha Gous

    Full Text Available Expansion of HIV viral load (VL testing services are required to meet increased targets for monitoring patients on antiretroviral treatment. South Africa currently tests >4million VLs per annum in 16 highly centralised, automated high-throughput laboratories. The Xpert HIV-1 VL assay (Cepheid was evaluated against in-country predicates, the Roche Cobas Taqmanv2 and Abbott HIV-1RT, to investigate options for expanding VL testing using GeneXpert's random access, polyvalent capabilities and already established footprint in South Africa with the Xpert MTB/RIF assay (207 sites. Additionally, the performance of Xpert HIV-1VL on alternative, off-label specimen types, Dried Blood Spots (DBS and whole blood, was investigated.Precision, accuracy (agreement and clinical misclassification (1000cp/ml of Xpert HIV-1VL plasma was compared to Taqmanv2 (n = 155 and Abbott HIV-1 RT (n = 145. Misclassification of Xpert HIV-1VL was further tested on DBS (n = 145 and whole blood (n = 147.Xpert HIV-1VL demonstrated 100% concordance with predicate platforms on a standardised frozen, plasma panel (n = 42 and low overall percentage similarity CV of 1.5% and 0.9% compared to Taqmanv2 and Abbott HIV-1 RT, respectively. On paired plasma clinical specimens, Xpert HIV-1VL had low bias (SD 0.32-0.37logcp/ml and 3% misclassification at the 1000cp/ml threshold compared to Taqmanv2 (fresh and Abbott HIV-1 RT (frozen, respectively. Xpert HIV-1VL on whole blood and DBS increased misclassification (upward by up to 14% with increased invalid rate. All specimen testing was easy to perform and compatible with concurrent Xpert MTB/RIF Tuberculosis testing on the same instrument.The Xpert HIV-1VL on plasma can be used interchangeably with existing predicate platforms in South Africa. Whole blood and DBS testing requires further investigation, but polyvalency of the GeneXpert offers a solution to extending VL testing services.

  10. In vitro mutagenicity assay (Ames test and phytochemical characterization of seeds oil of Helianthus annuus Linné (sunflower

    Directory of Open Access Journals (Sweden)

    Nelma de Mello Silva Oliveira

    Full Text Available The objective of this research was to investigate the genotoxic potential of the oil of H. annuus L. (sunflower seeds via the Ames test as well as its oxidative properties and lipid composition. The pre-incubation method, system metabolic activation (S9 fraction and five S. typhimurium strains (TA97, TA98, TA100, TA1535 and TA102 were employed for the Ames test. The oxidative stability and fatty acid composition were analyzed by standard methods and gas chromatography. A revertant analysis showed no significant differences between the treatment doses (10–200 μl/plate and the negative controls, regardless of S9+ and S9−, and included all of the S. typhimurium strains. Chromatographic analysis showed high levels of polyunsaturated fatty acids, followed by monounsaturated, saturated and total trans-isomers. Among the polyunsaturated, monounsaturated and saturated fatty acids, linoleic, oleic and palmitic acids predominated. The results suggest that the sunflower oil is not genotoxic as indicated by frameshift mutations and base pair substitutions regardless of the treatment dose, but shows dose-dependent toxicity. The oxidative properties of the sunflower oil were consistent with the requirements of national and international standards. However, its composition could also indicate phytotherapeutic properties. Keywords: Helianthus annuus L., Sunflower oil, Genetic toxicity, Gas chromatography

  11. Removal of Lipid from Serum Increases Coherence between Brucellosis Rapid Agglutination Test and Enzyme-linked Immunosorbent Assay in Bears in Alaska, USA.

    Science.gov (United States)

    Godfroid, Jacques; Beckmen, Kimberlee; Helena Nymo, Ingebjørg

    2016-10-01

    In cases of chronic Brucella spp. infection, results of the rose bengal plate test (RBPT) and indirect enzyme-linked immunosorbent assay (ELISA) should be coherent, as reported in controlled conditions in the literature. We compared RBPT and ELISA results in 58 Alaska grizzly bears ( Ursus arctos horribilis), eight Kodiak brown bears ( Ursus arctos middendorffi), and six Alaska Peninsula brown bears ( Ursus arctos gyas). Of the 72 bears tested, 42 (58%) were ELISA positive and 53 (73%) were RBPT positive. However, the coherence between the tests was only fair (K=0.37, SE=0.11), suggesting that either the serologic results were not compatible with Brucella spp. infection or that there was a technical problem with the tests. To address a potential technical problem, we performed a 30-min chloroform/centrifugation cleanup. Following cleanup, the ELISA identified 43 positives (59%) and the RBPT identified 47 (65%), and the coherence between the tests was much improved (K=0.80, SE=0.07). We recommend cleaning wildlife sera with a high lipid content before performing RBPT and performing RBPT and ELISA in parallel to assess coherence. Our results suggest that Alaskan brown bears have been exposed to Brucella spp.

  12. Evaluation of an Immunochromatographic Strip (Xenostrip –Tv Test for Diagnosis of Vaginal Trichomoniasis Compared with Wet Mount and PCR Assay

    Directory of Open Access Journals (Sweden)

    MH Feiz-Haddad

    2008-09-01

    Full Text Available "nBackground: Trichomoniasis, caused by Trichomonas vaginalis, is one of the most common sexually transmitted infections in the world. Diagnosis of T. vaginalis is performed by different methods, including wet mount, culture, serological methods and PCR, which required laboratory equipments and expert laboratory personnel. The aim of this study was evaluation of immunochromatographic strip test (Xenostrip-Tv for diagnosis of vaginal trichomoniasis compared with wet mount and PCR assay."nMethods: In this prospective study vaginal swabs were obtained from 100 women with genital complaints demanding a speculum examination, referred to Imam Khomeini and Amir Kabir hospitals in Ahwaz, Khuzestan Province. Samples were first examined by wet mount and Xenostrip-Tv. PCR assay was performed in the next step using TVK3 and TVK7 primers initially. The positive samples were then confirmed by the second PCR assay using TVA5-1 and TVA6 primers."nResults: PCR with TVA5-1 and TVA6 primers was determined as gold standard. The wet mount as well as Xenostrip-Tv sensitivity and specificity were 73.3% and 100%, respectively in comparison with gold standard. The sensitivity and specificity of PCR with primers TVK3 and TVK7 were also determined as 100% and 96.6%, respectively. The infection rates were 14% for wet mount and Xenostrip-Tv, 21% for PCR with primers TVK3 plus TVK7 and 19% with the gold standard PCR using TVA5-1 and TVA6 primers."nConclusion: Xenostrip- Tv could be used for diagnosis of vaginal trichomoniasis in regions with no laboratory diagnostic facilities.

  13. In vitro drug sensitivity testing of tumor cells from patients with non-Hodgkin's lymphoma using the fluorometric microculture cytotoxicity assay.

    Science.gov (United States)

    Nygren, P; Hagberg, H; Glimelius, B; Sundström, C; Kristensen, J; Christiansen, I; Larsson, R

    1994-01-01

    Tumor cell drug sensitivity is an important determinant of chemotherapy response. Its measurement in vitro would aid in therapy individualization and new drug development. The fluorometric microculture cytotoxicity assay (FMCA), based on production by viable cells of fluorescent fluorescein after 3 days of culture, was used for cytotoxic drug sensitivity testing of 73 samples of tumor cells from patients with non-Hodgkin's lymphoma (NHL). The technical success rate was 92%, and FMCA data showed good correlation to the Disc assay. NHL samples were considerably more drug sensitive than were samples from in vivo resistant tumors. There was no obvious difference in drug sensitivity for high- vs. low-grade or untreated vs. previously treated low-grade NHL. For 26 patients, clinical outcome was correlated to in vitro response giving a sensitivity and specificity of 93 and 48%, respectively. Cross-resistance between standard drugs was frequent in vitro. Resistance modulators potentiated the effect of vincristine and doxorubicin in 10-29% of the samples, most frequently from previously treated patients. The FMCA seems to report clinically relevant drug sensitivity data for NHL, and thus it could serve as a tool for optimization of chemotherapy in the future.

  14. Effect of Pregnancy on Interferon Gamma Release Assay and Tuberculin Skin Test Detection of Latent TB Infection Among HIV-Infected Women in a High Burden Setting.

    Science.gov (United States)

    LaCourse, Sylvia M; Cranmer, Lisa M; Matemo, Daniel; Kinuthia, John; Richardson, Barbra A; Horne, David J; John-Stewart, Grace

    2017-05-01

    Peripartum immunologic changes may affect latent tuberculosis infection (LTBI) diagnostic performance among HIV-infected women. HIV-infected women were serially tested with tuberculin skin test (TST) and interferon gamma release assay [QuantiFERON TB Gold In-tube (QFT)] in pregnancy and 6 weeks postpartum in Kenya. Prevalence, sensitivity and agreement, and correlates of QFT/TST positivity were assessed. Quantitative QFT mitogen and Mycobacterium tuberculosis antigen (Mtb-Ag) responses were compared by peripartum stage. Incidence of test conversion at 6 weeks postpartum was evaluated in baseline TST-/QFT- women. Among 100 HIV-infected women, median age was 26 years, median CD4 was 555 cells per cubic millimeter, and 88% were on antiretrovirals. More women were QFT+ than TST+ in both pregnancy (35.4% vs. 13.5%, P = 0.001) and postpartum (29.6% vs. 14.8%, P pregnancy vs. postpartum, and specifically among persistently QFT+ women (Mtb-Ag: 3.46 vs. 4.48 IU/mL, P = 0.007). QFT indeterminate rate was higher in pregnancy (16%) compared with postpartum (0%) because of lower mitogen response. QFT identified >2-fold more women with LTBI compared with TST in pregnancy and postpartum. Lower QFT Mtb-Ag and mitogen responses in pregnancy compared with postpartum suggest that pregnancy-associated immunologic changes may influence LTBI test performance.

  15. Screening of latent tuberculosis infection among health care workers working in Hajj pilgrimage area in Saudi Arabia, using interferon gamma release assay and tuberculin skin test.

    Science.gov (United States)

    Bukhary, Zakeya A; Amer, Soliman M; Emara, Magdy M; Abdalla, Mohammad E; Ali, Sahar A

    2018-01-01

    Interferon gamma release assays (IGRA) is highly specific for Mycobacterium tuberculosis and is the preferred test in BCG-vaccinated individuals. The few studies that have screened health care workers (HCWs) in Saudi Arabia for latent tuberculosis infection (LTBI) using IGRA have varied in agreement with the traditional tuberculin skin test (TST). Assess the prevalence of LTBI among HCWs working in the Hajj pilgrimage using IGRA and TST and measuring their agreement. Cross-sectional prospective. Multiple non-tertiary care hospitals. HCWs who worked during the Hajj pilgrimage in Saudi Arabia in December 2015. Data was collected by standarized questionnaire. Samples were drawn and analyzed by standard methods. The prevalence of LTBI among HCW and the agreement by kappa statistic between QFT-GIT and TST. 520 subjects. Nurses accounted for 30.7% of the sample and physicians, 19.2%. The majority were BCG vaccinated (98.5%). There were a total of 56 positive by QFT-GIT and the LTBI rate was 10.8%. In 50 QFT positive/476 TST negative the LTBI rate was 10.5% in discordant tests, and in 6 QFT positive/44 TST positive it was 13.6% in concordant tests. The overall agreement between both tests was poor-83% and kappa was 0.02. LTBI prevalence was associated with longer employment (13.1 [9.2] years). The QFT-GIT positive test was significantly higher in physicians (P=.02) and in HCWs working in chest hospitals 16/76 (21.05%) (P=.001). Agreement between the tests was poor. QFT-GIT detected LTBI when TST was negative in HCWs who had a history of close contact with TB patients. A second step TST was not feasible within 2-3 weeks. None.

  16. Comparison of a newly developed automated and quantitative hepatitis C virus (HCV) core antigen test with the HCV RNA assay for clinical usefulness in confirming anti-HCV results.

    Science.gov (United States)

    Kesli, Recep; Polat, Hakki; Terzi, Yuksel; Kurtoglu, Muhammet Guzel; Uyar, Yavuz

    2011-12-01

    Hepatitis C virus (HCV) is a global health care problem. Diagnosis of HCV infection is mainly based on the detection of anti-HCV antibodies as a screening test with serum samples. Recombinant immunoblot assays are used as supplemental tests and for the final detection and quantification of HCV RNA in confirmatory tests. In this study, we aimed to compare the HCV core antigen test with the HCV RNA assay for confirming anti-HCV results to determine whether the HCV core antigen test may be used as an alternative confirmatory test to the HCV RNA test and to assess the diagnostic values of the total HCV core antigen test by determining the diagnostic specificity and sensitivity rates compared with the HCV RNA test. Sera from a total of 212 treatment-naive patients were analyzed for anti-HCV and HCV core antigen both with the Abbott Architect test and with the molecular HCV RNA assay consisting of a reverse transcription-PCR method as a confirmatory test. The diagnostic sensitivity, specificity, and positive and negative predictive values of the HCV core antigen assay compared to the HCV RNA test were 96.3%, 100%, 100%, and 89.7%, respectively. The levels of HCV core antigen showed a good correlation with those from the HCV RNA quantification (r = 0.907). In conclusion, the Architect HCV antigen assay is highly specific, sensitive, reliable, easy to perform, reproducible, cost-effective, and applicable as a screening, supplemental, and preconfirmatory test for anti-HCV assays used in laboratory procedures for the diagnosis of hepatitis C virus infection.

  17. The relative test performance characteristics of two commercial assays for the detection of Mycobacterium tuberculosis complex in paraffin-fixed human biopsy specimens

    Directory of Open Access Journals (Sweden)

    Broukhanski George

    2008-09-01

    Full Text Available Abstract The Seeplex™ TB Detection-2 assay (Rockville, MD is a nested endpoint PCR for the Mycobacterium tuberculosis complex (MTBC targets IS6110 and MPB64 that utilizes dual priming oligonucleotide technology. When used to detect the presence of MTBC DNA in formalin-fixed paraffin-embedded tissue specimens, the sensitivity and specificity of this assay is equivalent to a labor-intensive traditional endpoint PCR assay and is more sensitive than a commercial real-time PCR assay.

  18. Comparison of interferon {gamma} release assays and conventional screening tests before tumour necrosis factor {alpha} blockade in patients with inflammatory arthritis.

    LENUS (Irish Health Repository)

    Martin, J

    2012-02-01

    OBJECTIVE: To compare the performance of two interferon gamma release assays (IGRAs) and conventional screening tests in patients with inflammatory arthritis undergoing screening for latent tuberculosis infection (LTBI) before treatment with anti-tumour necrosis factor alpha (anti-TNFalpha) compounds. METHODS: Successive patients were subjected to conventional LTBI screening, including a tuberculin skin test (TST). The T-SPOT.TB test was performed on all patients and the QuantiFERON-TB Gold test was performed on a large subset. The results of the IGRAs were compared with the results of conventional screening tests. RESULTS: A total 150 patients were evaluated. The majority (57.9%) had rheumatoid arthritis. Previous vaccination with Bacille Calmette-Guerin was confirmed in 82% of patients. No patient had received prior anti-TB treatment. A total of 57 patients (38.0%) had at least one positive conventional risk factor. In contrast, an unequivocally positive T-SPOT.TB test was seen in only 14\\/143 (9.8%). There was 98.2% agreement between the two IGRAs. Statistically significant associations were found between each of the IGRAs and both TST and risk history, but not chest x-ray (CXR). A positive IGRA result was significantly associated with increased age. TB was not reactivated in any patient during the follow-up period. Interpretation: This study suggests that IGRAs may be useful when screening for LTBI before anti-TNFalpha therapy in patients with immune-mediated inflammatory diseases. The observations reported here also highlight the inadequate performance of CXR as a marker of LTBI.

  19. Comparison of Two Assays to Determine Anti-Citrullinated Peptide Antibodies in Rheumatoid Arthritis in relation to Other Chronic Inflammatory Rheumatic Diseases: Assaying Anti-Modified Citrullinated Vimentin Antibodies Adds Value to Second-Generation Anti-Citrullinated Cyclic Peptides Testing

    Directory of Open Access Journals (Sweden)

    Miriam Lizette Díaz-Toscano

    2014-01-01

    Full Text Available Determination of anti-citrullinated peptide antibodies (ACPA plays a relevant role in the diagnosis of rheumatoid arthritis (RA. To date, it is still unclear if the use of several tests for these autoantibodies in the same patient offers additional value as compared to performing only one test. Therefore, we evaluated the performance of using two assays for ACPA: second-generation anti-citrullinated cyclic peptides antibodies (anti-CCP2 and anti-mutated citrullinated vimentin (anti-MCV antibodies for the diagnosis of RA. We compared three groups: RA (n=142, chronic inflammatory disease (CIRD, n=86, and clinically healthy subjects (CHS, n=56 to evaluate sensitivity, specificity, predictive values, and likelihood ratios (LR of these two assays for the presence of RA. A lower frequency of positivity for anti-CCP2 was found in RA (66.2% as compared with anti-MCV (81.0%. When comparing RA versus other CIRD, sensitivity increased when both assays were performed. This strategy of testing both assays had high specificity and LR+. We conclude that adding the assay of anti-MCV antibodies to the determination of anti-CCP2 increases the sensitivity for detecting seropositive RA. Therefore, we propose the use of both assays in the initial screening of RA in longitudinal studies, including early onset of undifferentiated arthritis.

  20. Standard test method for nondestructive assay of special nuclear material in low density scrap and waste by segmented passive gamma-Ray scanning

    CERN Document Server

    American Society for Testing and Materials. Philadelphia

    2010-01-01

    1.1 This test method covers the transmission-corrected nondestructive assay (NDA) of gamma-ray emitting special nuclear materials (SNMs), most commonly 235U, 239Pu, and 241Am, in low-density scrap or waste, packaged in cylindrical containers. The method can also be applied to NDA of other gamma-emitting nuclides including fission products. High-resolution gamma-ray spectroscopy is used to detect and measure the nuclides of interest and to measure and correct for gamma-ray attenuation in a series of horizontal segments (collimated gamma detector views) of the container. Corrections are also made for counting losses occasioned by signal processing limitations (1-3). 1.2 There are currently several systems in use or under development for determining the attenuation corrections for NDA of radioisotopic materials (4-8). A related technique, tomographic gamma-ray scanning (TGS), is not included in this test method (9, 10, 11). 1.2.1 This test method will cover two implementations of the Segmented Gamma Scanning ...

  1. Revision of the SNPforID 34-plex forensic ancestry test: Assay enhancements, standard reference sample genotypes and extended population studies.

    Science.gov (United States)

    Fondevila, M; Phillips, C; Santos, C; Freire Aradas, A; Vallone, P M; Butler, J M; Lareu, M V; Carracedo, A

    2013-01-01

    A revision of an established 34 SNP forensic ancestry test has been made by swapping the under-performing rs727811 component SNP with the highly informative rs3827760 that shows a near-fixed East Asian specific allele. We collated SNP variability data for the revised SNP set in 66 reference populations from 1000 Genomes and HGDP-CEPH panels and used this as reference data to analyse four U.S. populations showing a range of admixture patterns. The U.S. Hispanics sample in particular displayed heterogeneous values of co-ancestry between European, Native American and African contributors, likely to reflect in part, the way this disparate group is defined using cultural as well as population genetic parameters. The genotyping of over 700 U.S. population samples also provided the opportunity to thoroughly gauge peak mobility variation and peak height ratios observed from routine use of the single base extension chemistry of the 34-plex test. Finally, the genotyping of the widely used DNA profiling Standard Reference Material samples plus other control DNAs completes the audit of the 34-plex assay to allow forensic practitioners to apply this test more readily in their own laboratories. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  2. Avoidance Behavior against Positive Allergens Detected with a Multiple Allergen Simultaneous Test Immunoblot Assay in Patients with Urticaria: Factors Associated with Avoidance Success/Failure.

    Science.gov (United States)

    Lee, Min Kyung; Kwon, In Ho; Kim, Han Su; Kim, Heung Yeol; Cho, Eun Byul; Bae, Youin; Park, Gyeong Hun; Park, Eun Joo; Kim, Kwang Ho; Kim, Kwang Joong

    2016-02-01

    Avoidance behavior against positive allergens detected by using multiple allergen simultaneous test (MAST)-immunoblot assay in patients with urticaria has been rarely reported. We aimed to assess the avoidance behavior of patients with urticaria against positive allergens detected with a MAST. One hundred and one urticaria patients who showed positivity to at least one allergen on a MAST completed a questionnaire regarding their test results. The avoidance behavior of the patients was evaluated, and relevant determining factors of avoidance success/failure were statistically assessed. We detected 144 different data (n=51, food allergens; n=17, pollen allergens; and n=76, aeroallergens) from 101 patients with urticaria. The avoidance failure rates were 33.3% for food allergens, 70.6% for pollen allergens, and 30.3% for aeroallergens. The pollen group showed a significantly higher avoidance failure rate than the food and aeroallergen groups (psuccessfully avoid allergens (psuccess or failure against allergens in patients with urticaria when clinicians conduct allergen-specific immunoglobulin E tests.

  3. Comparison of Estrogen Receptor Assay Results from Pathology Reports with Results from Central Laboratory Testing: Implications for Population-Based Studies of Breast Cancer

    Science.gov (United States)

    Collins, LC; Marotti, J; Baer, HJ; Deitz, AC; Colditz, GA; Tamimi, RM

    2014-01-01

    Population-based studies of women with breast cancer commonly utilize information culled from pathology reports rather than central pathology review. The reliability of this information, particularly with regard to tumor biomarker results, is of concern. To address this, we evaluated the concordance between estrogen receptor (ER) results as determined from the original pathology reports and ER results obtained on the same specimens following testing in a single laboratory. Tissue microarrays (TMAs) were constructed from paraffin blocks of 3,167 breast cancers that developed in women enrolled in the Nurses’ Health Study. ER immunostains were performed on all TMA sections in single run. Results of ER immunostains performed on the TMA sections were compared with ER assay results abstracted from pathology reports. Among 1,851 cases of invasive breast cancer in which both ER results from pathology reports and central ER test results were available, the reported ER status and the ER status as determined from immunostains on TMAs were in agreement in 1,651 cases (87.3 %; kappa value 0.64, ppathology reports is a reasonable, albeit imperfect, alternative to central laboratory ER testing for large, population-based studies of patients with breast cancer. PMID:18230800

  4. Comparison of the membrane-filtration fluorescent antibody test, the enzyme-linked immunosorbent assay, and the polymerase chain reaction to detect Renibacterium salmoninarum in salmon ovarian fluid

    Science.gov (United States)

    Pascho, Ronald J.; Chase, Dorothy M.; McKibben, Constance L.

    1998-01-01

    Ovarian fluid samples from naturally infected chinook salmon (Oncorhynchus tshawytscha) were examined for the presence of Renibacterium salmoninarum by the membrane-filtration fluorescent antibody test (MF-FAT), an antigen capture enzyme-linked immunosorbent assay (ELISA), and a nested polymerase chain reaction (PCR). On the basis of the MF-FAT, 64% (66/103) samples contained detectable levels of R. salmoninarum cells. Among the positive fish, the R. salmoninarum concentrations ranged from 25 cells/ml to 4.3 × 109cells/ml. A soluble antigenic fraction of R. salmoninarum was detected in 39% of the fish (40/103) by the ELISA. The ELISA is considered one of the most sensitive detection methods for bacterial kidney disease in tissues, yet it did not detect R. salmoninarum antigen consistently at bacterial cell concentrations below about 1.3 × 104cells/ml according to the MF-FAT counts. When total DNA was extracted and tested in a nested PCR designed to amplify a 320-base-pair region of the gene encoding a soluble 57-kD protein of R. salmoninarum, 100% of the 100 samples tested were positive. The results provided strong evidence that R. salmoninarum may be present in ovarian fluids thought to be free of the bacterium on the basis of standard diagnostic methods.

  5. Effective implementation of novel MET pharmacodynamic assays in translational studies.

    Science.gov (United States)

    Srivastava, Apurva K; Navas, Tony; Herrick, William G; Hollingshead, Melinda G; Bottaro, Donald P; Doroshow, James H; Parchment, Ralph E

    2017-01-01

    MET tyrosine kinase (TK) dysregulation is significantly implicated in many types of cancer. Despite over 20 years of drug development to target MET in cancers, a pure anti-MET therapeutic has not yet received market approval. The failure of two recently concluded phase III trials point to a major weakness in biomarker strategies to identify patients who will benefit most from MET therapies. The capability to interrogate oncogenic mutations in MET via circulating tumor DNA (ctDNA) provides an important advancement in identification and stratification of patients for MET therapy. However, a wide range in type and frequency of these mutations suggest there is a need to carefully link these mutations to MET dysregulation, at least in proof-of-concept studies. In this review, we elaborate how we can utilize recently developed and validated pharmacodynamic biomarkers of MET not only to show target engagement, but more importantly to quantitatively measure MET dysregulation in tumor tissues. The MET assay endpoints provide evidence of both canonical and non-canonical MET signaling, can be used as "effect markers" to define biologically effective doses (BEDs) for molecularly targeted drugs, confirm mechanism-of-action in testing combination of drugs, and establish whether a diagnostic test is reporting MET dysregulation. We have established standard operating procedures for tumor biopsy collections to control pre-analytical variables that have produced valid results in proof-of-concept studies. The reagents and procedures are made available to the research community for potential implementation on multiple platforms such as ELISA, quantitative immunofluorescence assay (qIFA), and immuno-MRM assays.

  6. Utility of a Multiplex PCR Assay for Detecting Herpesvirus DNA in Clinical Samples

    Science.gov (United States)

    Druce, Julian; Catton, Mike; Chibo, Doris; Minerds, Kirsty; Tyssen, David; Kostecki, Renata; Maskill, Bill; Leong-Shaw, Wendy; Gerrard, Marie; Birch, Chris

    2002-01-01

    A multiplex PCR was designed to amplify herpes simplex virus types 1 and 2, cytomegalovirus, and varicella-zoster virus DNA present in a diverse range of clinical material. The susceptibility of these viruses to in vivo inhibition by at least one antiviral drug was an important consideration in their inclusion in the multiplex detection system. An aliquot of equine herpesvirus was introduced into each specimen prior to extraction and served as an indicator of potential inhibitors of the PCR and a detector of suboptimal PCR conditions. Compared to virus isolation and immunofluorescence-based antigen detection, the multiplex assay yielded higher detection rates for all viruses represented in the assay. The turnaround time for performance of the assay was markedly reduced compared to those for the other techniques used to identify these viruses. More than 21,000 tests have been performed using the assay. Overall, the multiplex PCR enabled the detection of substantially increased numbers of herpesviruses, in some cases in specimens or anatomical sites where previously they were rarely if ever identified using traditional detection methods. PMID:11980951

  7. Development of immunofluorescence colony staining (IFC) for detection of Xanthomonas campestris pv. vesicatoria and Clavibacter michiganensis subsp michiganensis in tomato seeds

    NARCIS (Netherlands)

    Nemeth, J.; Vuurde, van J.W.L.

    2006-01-01

    Immunofluorescence colony-staining (IFC) is based on sample pour plating in combination with immunofluorescence staining for recognition of the target colony. IFC was optimised for detecting Xanthomonas campestris pv. vesicatoria (Xcv) and Clavibacter michiganensis subsp. michiganensis (Cmm) in

  8. Nail unit in collagen vascular diseases: A clinical, histopathological and direct immunofluorescence study

    Directory of Open Access Journals (Sweden)

    Nabil P

    2006-01-01

    Full Text Available Background: Abnormalities of the nail unit are common in patients with connective tissue diseases. Clinical examination of the nail unit, coupled with biopsy of proximal nail fold offers an additional advantage in the diagnosis. Purpose: Our aim was to record clinical changes of the nail unit in connective tissue diseases and to study the histopathological (both H and E and periodic acid Schiff and direct immunofluorescence (DIF findings of nail-fold biopsy. Materials and Methods: Thirty-eight confirmed cases connective tissue diseases attending skin OPD were enrolled in the study. After detailed clinical examination of the nail unit, a crescentric biopsy was taken from the proximal nail fold (PNF. Histopathological and DIF studies were was carried out. Findings: Nail changes could be demonstrated in 65% connective tissue diseases. Specific histopathological (H and E and immunofluorescence findings were also encountered in many patients. Conclusion: Clinical examination of the nail unit offers additional clue in the diagnosis of connective tissue diseases. Though DIF of PNF biopsy is useful in the diagnosis, it is not an ideal site for H and E study, as the yield is very low. Limitations: Lack of adequate comparison group and non-utilization of capillary microscopy for the detection of nail fold capillary abnormalities.

  9. Kidney lesions in Rocky Mountain spotted fever: a light-, immunofluorescence-, and electron-microscopic study.

    Science.gov (United States)

    Bradford, W. D.; Croker, B. P.; Tisher, C. C.

    1979-01-01

    The essential pathologic lesion in Rocky Mountain spotted fever (RMSF) is a vasculitis that may involve the kidneys as well as the heart, brain, skin, and subcutaneous tissues. Histopathologic information concerning the response of the kidneys in RMSF is rather limited, however. In this study renal tissue from 17 children who died of RMSF was examined by light, electron, and immunofluorescence microscopy. A lymphocytic or mixed inflammation, or both, involving vessels and interstitium of the kidney was found in all patients. In addition, 10 patients had histologic evidence of acute tubular necrosis, and another 3 had glomerular lesions consisting of focal segmental tuft necrosis or increased cellularity secondary to neutophilic infiltration, or both. Immunofluorescence- and electron-microscopic studies failed to demonstrate immune-complex deposition within glomeruli, a finding that suggests that immunoglobulin and classic immune complexes were not involved in the pathogenesis of the renal lesions at the time of death. These findings suggest the possibility that the pathogenesis of the renal lesion in RMSF may be due to a direct action of the organism (Rickettsia rickettsii) on the vessel wall. Images Figure 2 Figure 1 PMID:525676

  10. Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections.

    Directory of Open Access Journals (Sweden)

    Sandrine Prost

    Full Text Available The recent availability of novel dyes and alternative light sources to facilitate complex tissue immunofluorescence studies such as multiplex labelling has not been matched by reports critically evaluating the considerations and relative benefits of these new tools, particularly in combination. Product information is often limited to wavelengths used for older fluorophores (FITC, TRITC & corresponding Alexa dyes family. Consequently, novel agents such as Quantum dots are not widely appreciated or used, despite highly favourable properties including extremely bright emission, stability and potentially reduced tissue autofluorescence at the excitation wavelength. Using spectral analysis, we report here a detailed critical appraisal and comparative evaluation of different light sources and fluorophores in multiplex immunofluorescence of clinical biopsy sections. The comparison includes mercury light, metal halide and 3 different LED-based systems, using 7 Qdots (525, 565, 585, 605, 625, 705, Cy3 and Cy5. We discuss the considerations relevant to achieving the best combination of light source and fluorophore for accurate multiplex fluorescence quantitation. We highlight practical limitations and confounders to quantitation with filter-based approaches.

  11. Original Approach for Automated Quantification of Antinuclear Autoantibodies by Indirect Immunofluorescence

    Directory of Open Access Journals (Sweden)

    Daniel Bertin

    2013-01-01

    Full Text Available Introduction. Indirect immunofluorescence (IIF is the gold standard method for the detection of antinuclear antibodies (ANA which are essential markers for the diagnosis of systemic autoimmune rheumatic diseases. For the discrimination of positive and negative samples, we propose here an original approach named Immunofluorescence for Computed Antinuclear antibody Rational Evaluation (ICARE based on the calculation of a fluorescence index (FI. Methods. We made comparison between FI and visual evaluations on 237 consecutive samples and on a cohort of 25 patients with SLE. Results. We obtained very good technical performance of FI (95% sensitivity, 98% specificity, and a kappa of 0.92, even in a subgroup of weakly positive samples. A significant correlation between quantification of FI and IIF ANA titers was found (Spearman's ρ=0.80, P<0.0001. Clinical performance of ICARE was validated on a cohort of patients with SLE corroborating the fact that FI could represent an attractive alternative for the evaluation of antibody titer. Conclusion. Our results represent a major step for automated quantification of IIF ANA, opening attractive perspectives such as rapid sample screening and laboratory standardization.

  12. Comparing an in vivo egg reduction test and in vitro egg hatching assay for different anthelmintics against Fasciola species, in cattle.

    Science.gov (United States)

    Arafa, Waleed M; Shokeir, Khalid M; Khateib, Abdelrahman M

    2015-11-30

    This study aimed to compare between the efficiency of in vivo fecal egg reduction test (FERT) and in vitro egg hatching assay (EHA) in evaluating of the anti-Fasciola activity of albendazole, triclabendazole, oxyclozanide and praziquantel. A field trial was carried out on fifty naturally Fasciola infected cattle that were divided equally into 5 groups (A-E). On day zero; groups A-D were drenched with albendazole, triclabendazole, oxyclozanide or praziquantel, respectively, while the remaining one, group E, was kept as untreated control. Fecal egg counts of the different groups were conducted weekly over a period of one month post-treatment. In vitro, commercial albendazole and oxyclozanide were diluted to 0.0002, 0.002, 0.02, 0.2 and 2.0 μg/ml, while commercial triclabendazole and praziquantel were diluted to concentrations of 25, 50, 75 and 100 μg/ml with dimethyl sulfoxide (DMSO). In vivo, at the 2nd week post-treatment, triclabendazole and oxyclozanide showed 100% fecal egg reduction (FER), and albendazole had a maximum of 73.7% reduction (P egg counts. In vitro, triclabendazole treated Fasciola gigantica eggs showed early embryonic lysis with zero% hatching at the different concentrations (P egg development and hatching percentage of oxyclozanide or praziquantel treated groups. In conclusion, the efficacy of triclabendazole and albendazole as fasciolicdes could be predicted by Egg Hatching Assay (EHA). Meanwhile fasciolicide activity of oxyclozanide could not be assessed with EHA. Based on in vivo and in vitro findings, paraziquantel did not show any fasciolicide effect. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Scintillation proximity assay

    International Nuclear Information System (INIS)

    Hart, H.

    1980-01-01

    In a method of immunological assay two different classes of particles which interact at short distances to produce characteristic detectable signals are employed in a modification of the usual latex fixation test. In one embodiment an aqueous suspension of antigen coated tritiated latex particles (LH) and antigen coated polystyrene scintillant particles (L*) is employed to assay antibody in the aqueous medium. The amount of (LH) (L*) dimer formation and higher order aggregation induced and therefore the concentration of antibody (or antigen) present which caused the aggregation can be determined by using standard liquid scintillation counting equipment. (author)

  14. Performance evaluation of cobas HBV real-time PCR assay on Roche cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test.

    Science.gov (United States)

    Kim, Hanah; Hur, Mina; Bae, Eunsin; Lee, Kyung-A; Lee, Woo-In

    2018-02-19

    Hepatitis B virus (HBV) nucleic acid amplification testing (NAAT) is important for the diagnosis and management of HBV infection. We evaluated the analytical performance of the cobas HBV NAAT (Roche Diagnostics GmbH, Mannheim, Germany) on the cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test (CAP/CTM HBV). Precision was evaluated using three levels of cobas HBV/HCV/HIV-1 Control Kit, and linearity was evaluated across the anticipated measuring range (10.0-1.0×109 IU/mL) at seven levels using clinical samples. Detection capability, including limit of blank (LOB), limit of detection (LOD) and limit of quantitation (LOQ), was verified using the 4th WHO International Standard for HBV DNA for NAT (NIBSC code: 10/266). Correlation between the two systems was compared using 205 clinical samples (102 sera and 103 EDTA plasma). Repeatability and total imprecision (coefficient of variation) ranged from 0.5% to 3.8% and from 0.5% to 3.5%, respectively. Linearity (coefficient of determination, R2) was 0.999. LOB, LOD and LOQ were all acceptable within the observed proportion rate (85%). Correlation was very high between the two systems in both serum and plasma samples (correlation coefficient [r]=0.995). The new cobas HBV real-time PCR assay on the cobas 4800 System showed reliable analytical performances.

  15. Skin sensitization potency of isoeugenol and its dimers evaluated by a non-radioisotopic modification of the local lymph node assay and guinea pig maximization test.

    Science.gov (United States)

    Takeyoshi, Masahiro; Iida, Kenji; Suzuki, Keiko; Yamazaki, Shunsuke

    2008-05-01

    Allergic contact dermatitis is the serious unwanted effect arising from the use of consumer products such as cosmetics. Isoeugenol is a fragrance chemical with spicy, carnation-like scent, is used in many kinds of cosmetics and is a well-known moderate human sensitizer. It was previously reported that the dimerization of eugenol yielded two types of dimer possessing different sensitization potencies. This study reports the differences in skin sensitization potencies for isoeugenol and two types of dimer, beta-O-4-dilignol and dehydrodiisoeugenol (DIEG), as evaluated by the non-radioisotopic local lymph node assay (non-RI LLNA) and guinea pig maximization test. In the guinea pig maximization test, isoeugenol, beta-O-4-dilignol and DIEG were classified as extreme, weak and moderate sensitizers, respectively. As for the results of non-RI LLNA, the EC3 for isoeugenol, beta-O-4-dilignol and DIEG were calculated as 12.7%, >30% and 9.4%, respectively. The two types of isoeugenol dimer showed different sensitizing activities similar to the case for eugenol dimers. A reduction of sensitization potency achieved by dimerization may lead to developing safer cosmetic ingredients. Isoeugenol dimers are not currently used for fragrance chemicals. However, the dimerization of isoeugenol may yield a promising candidate as a cosmetic ingredient with low sensitization risk. The data may also provide useful information for the structure-activity relationship (SAR) in skin sensitization. Copyright (c) 2007 John Wiley & Sons, Ltd.

  16. Differences in detection of Aeromonas salmonicida in covertly infected salmonid fishes by the stress-inducible furunculosis test and culture-based assays

    Science.gov (United States)

    Cipriano, R.C.; Ford, L.A.; Smith, D.R.; Schachte, J.H.; Petrie, C.J.

    1997-01-01

    Accurate detection of Aeromonas salmonicida subsp. salmonicida (the cause of furunculosis disease) in covertly infected salmonids is difficult and is a cause of concern for those involved in fish health inspection and resource management programs. In this study, we examined populations of brook trout Salvelinus fontinalis, Atlantic salmon Salmo salar, and lake trout Salvelinus namaycush that previously sustained natural episodes of furunculosis. Consequently, the sampled fish were presumed to harbor latent infections. Mucus, gill, liver, kidney, heart, spleen, and intestine samples (N = 100 fish per group sampled) were processed and examined by (1) direct dilution counts and (2) quadrant streaking after a 48-h pre-enrichment in trypticase soy broth (TSB). Another subsample of fish from each group was then subjected to stress-inducible furunculosis tests. Stress tests detected A. salmonicida in three of four groups of fish that were examined whereas the pathogen was detected in only two of the groups analyzed with culture-based assays. Although pre-enrichment in TSB enhanced detection within internal sampling sites including the liver, heart, spleen, and kidney, enrichment did not enhance detection from mucus, gill, or intestinal samples.

  17. Rapid identification and susceptibility testing of uropathogenic microbes via immunosorbent ATP-bioluminescence assay on a microfluidic simulator for antibiotic therapy.

    Science.gov (United States)

    Dong, Tao; Zhao, Xinyan

    2015-02-17

    The incorporation of pathogen identification with antimicrobial susceptibility testing (AST) was implemented on a concept microfluidic simulator, which is well suited for personalizing antibiotic treatment of urinary tract infections (UTIs). The microfluidic device employs a fiberglass membrane sandwiched between two polypropylene components, with capture antibodies immobilized on the membrane. The chambers in the microfluidic device share the same geometric distribution as the wells in a standard 384-well microplate, resulting in compatibility with common microplate readers. Thirteen types of common uropathogenic microbes were selected as the analytes in this study. The microbes can be specifically captured by various capture antibodies and then quantified via an ATP bioluminescence assay (ATP-BLA) either directly or after a variety of follow-up tests, including urine culture, antibiotic treatment, and personalized antibiotic therapy simulation. Owing to the design of the microfluidic device, as well as the antibody specificity and the ATP-BLA sensitivity, the simulator was proven to be able to identify UTI pathogen species in artificial urine samples within 20 min and to reliably and simultaneously verify the antiseptic effects of eight antibiotic drugs within 3-6 h. The measurement range of the device spreads from 1 × 10(3) to 1 × 10(5) cells/mL in urine samples. We envision that the medical simulator might be broadly employed in UTI treatment and could serve as a model for the diagnosis and treatment of other diseases.

  18. Evaluation of the Aptima HBV Quant assay vs. the COBAS TaqMan HBV test using the high pure system for the quantitation of HBV DNA in plasma and serum samples.

    Science.gov (United States)

    Schalasta, Gunnar; Börner, Anna; Speicher, Andrea; Enders, Martin

    2018-03-28

    Proper management of patients with chronic hepatitis B virus (HBV) infection requires monitoring of plasma or serum HBV DNA levels using a highly sensitive nucleic acid amplification test. Because commercially available assays differ in performance, we compared herein the performance of the Hologic Aptima HBV Quant assay (Aptima) to that of the Roche Cobas TaqMan HBV test for use with the high pure system (HPS/CTM). Assay performance was assessed using HBV reference panels as well as plasma and serum samples from chronically HBV-infected patients. Method correlation, analytical sensitivity, precision/reproducibility, linearity, bias and influence of genotype were evaluated. Data analysis was performed using linear regression, Deming correlation analysis and Bland-Altman analysis. Agreement between the assays for the two reference panels was good, with a difference in assay values vs. target 0.98). The two assays had similar bias and precision across the different genotypes tested at low viral loads (25-1000 IU/mL). Aptima has a performance comparable with that of HPS/CTM, making it suitable for use for HBV infection monitoring. Aptima runs on a fully automated platform (the Panther system) and therefore offers a significantly improved workflow compared with HPS/CTM.

  19. A reassessment of the in vitro RBC haemolysis assay with defibrinated sheep blood for the determination of the ocular irritation potential of cosmetic products: comparison with the in vivo Draize rabbit test.

    Science.gov (United States)

    Alves, Eloísa Nunes; Presgrave, Rosaura de Farias; Presgrave, Octávio Augusto França; Sabagh, Fernanda Peres; de Freitas, João Carlos Borges Rolim; Corrado, Alexandre P

    2008-07-01

    We examined the correlation between results obtained from the in vivo Draize test for ocular irritation and in vitro results obtained from the sheep red blood cell (RBC) haemolytic assay, which assesses haemolysis and protein denaturation in erythrocytes, induced by cosmetic products. We sought to validate the haemolytic assay as a preliminary test for identifying highly-irritative products, and also to evaluate the in vitro test as alternative assay for replacement of the in vivo test. In vitro and in vivo analyses were carried out on 19 cosmetic products, in order to correlate the lesions in the ocular structures with three in vitro parameters: (i) the extent of haemolysis (H50); (ii) the protein denaturation index (DI); and (iii) the H50/DI ratio, which reflects the irritation potential (IP). There was significant correlation between maximum average scores (MAS) and the parameters determined in vitro (r = 0.752-0.764). These results indicate that the RBC assay is a useful and rapid test for use as a screening method to assess the IP of cosmetic products, and for predicting the IP value with a high level of concordance (94.7%). The assay showed high sensitivity and specificity rates of 91.6% and 100%, respectively.

  20. Use of an Interferon Gamma Release Assay (IGRA) to test T-cell responsiveness to soluble Leishmania infantum antigen in whole blood of dogs from endemic areas.

    Science.gov (United States)

    Zribi, Lilia; El-Goulli, Amel F; Ben-Abid, Meriem; Gharbi, Mohamed; Ben-Sghaier, Ines; Boufaden, Imed; Aoun, Karim; Bouratbine, Aïda

    2017-11-15

    Interferon-Gamma (IFN-γ) Release Assays (IGRAs) are easy tests that allow rapid screening of primed memory T-cells immunity in response to antigen. The aim of this study was to use IGRA to assess IFN-γ release in response to Soluble Leishmania infantum antigen (SLA) in whole blood of dogs living in endemic area of visceral leishmaniasis and to interpret IGRA results according to clinical examination, specific anti-Leishmania humoral response and presence of L. infantum DNA in blood. The study was carried out on 56 dogs living in greater Tunis area. Physical examination, quantitative serology and PCR on blood were used to characterize dogs' status in relation to Leishmania infection and disease. IGRA consisted on testing by ELISA for IFN-γ-secretion in whole blood after a 20-h challenge with SLA. PBS and Phytohemagglutinin (PHA) stimulations were used as controls. Four groups of dogs were characterized: 31 were negative by both serology and PCR, two had doubtful serology, 10 presented no to mild clinical signs but low antibodies levels and 13 were affected by Canine Leishmaniasis (CanL). In seronegative dogs, IGRA was little contributory in 4 puppies (age dogs (median age=72months, IQR: 45-84 months) that didn't respond to PHA stimulation, IGRA was negative in 19 and positive in three animals with lymph node enlargement. In dogs with doubtful serology, IGRA was positive in one dog and negative in the other. In infected dogs with no to mild clinical signs, one dog exhibited high level of IFN-γ in absence of antigenic stimulation and all the other were positive by IGRA. CanL dogs showed variable IGRA results. Negative IGRAs (n=4) were shown in animals with the highest parasitic burden whereas positive IGRAs (n=5) were shown in dogs with negative PCR or low parasitic load. The 4 remaining dogs either didn't respond to PHA (n=2) or showed non-specific secretion in PBS tube (n=2). The results of this study showed that IGRA is a useful new tool that can assess

  1. A multiplexed reverse transcriptase PCR assay for identification of viral respiratory pathogens at point-of-care

    Energy Technology Data Exchange (ETDEWEB)

    Letant, S E; .Ortiz, J I; Tammero, L; Birch, J M; Derlet, R W; Cohen, S; Manning, D; McBride, M T

    2007-04-11

    We have developed a nucleic acid-based assay that is rapid, sensitive, specific, and can be used for the simultaneous detection of 5 common human respiratory pathogens including influenza A, influenza B, parainfluenza type 1 and 3, respiratory syncytial virus, and adenovirus group B, C, and E. Typically, diagnosis on an un-extracted clinical sample can be provided in less than 3 hours, including sample collection, preparation, and processing, as well as data analysis. Such a multiplexed panel would enable rapid broad-spectrum pathogen testing on nasal swabs, and therefore allow implementation of infection control measures, and timely administration of antiviral therapies. This article presents a summary of the assay performance in terms of sensitivity and specificity. Limits of detection are provided for each targeted respiratory pathogen, and result comparisons are performed on clinical samples, our goal being to compare the sensitivity and specificity of the multiplexed assay to the combination of immunofluorescence and shell vial culture currently implemented at the UCDMC hospital. Overall, the use of the multiplexed RT-PCR assay reduced the rate of false negatives by 4% and reduced the rate of false positives by up to 10%. The assay correctly identified 99.3% of the clinical negatives, 97% of adenovirus, 95% of RSV, 92% of influenza B, and 77% of influenza A without any extraction performed on the clinical samples. The data also showed that extraction will be needed for parainfluenza virus, which was only identified correctly 24% of the time on un-extracted samples.

  2. [CLINICAL UTILITY OF T-SPOT.TB ASSAY WITH T-Cell Xtend REAGENT FOR ACTIVE TUBERCULOSIS DIAGNOSIS IN THE FIELD TEST AT OUR HOSPITAL].

    Science.gov (United States)

    Nemoto, Kenji; Oh-ishi, Shuji; Taguchi, Masato; Hyodo, Kentaro; Kanazawa, Jun; Miura, Yukiko; Takaku, Takio; Usui, Shingo; Hayashihara, Kenji; Saito, Takefumi

    2016-04-01

    T-SPOT.TB (T-SPOT), an interferon-gamma release assay, has shown promise as a diagnostic tool for active tuberculosis (TB), and its use is expanding. Addition of the T-Cell Xtend (TCX) reagent may allow delayed processing, and this characteristic is important for using this test in the field. However, limited data is available on the usefulness of T-SPOT with TCX as a field test for diagnosing active TB. To investigate the clinical utility of T-SPOT with TCX and the risk factors for a false-negative result in patients with active TB. A total of 57 patients with active TB who underwent the T-SPOT test with TCX prior to treatment were enrolled between May 2013 and May 2015. One patient with an indeterminate result for T-SPOT was excluded; therefore, the data of 56 patients were eventually included in the final analysis. The basic characteristics and clinical findings were compared between the true-positive and false-negative T-SPOT groups. Of the 56 patients, 40 (71.4%), 13 (23.2%), 3 (5.4%) had true-positive, false-negative, and borderline T-SPOT results, respectively. This study did not reveal any significant risk factors for a false-negative T-SPOT result. In this clinical study, the proportion of patients with a false-negative result for T-SPOT with TCX for active TB was higher than that reported previously. Therefore, careful interpretation of a negative result for T-SPOT with TCX is necessary, regardless of the patient's background.

  3. Six rapid tests for direct detection of Clostridium difficile and its toxins in fecal samples compared with the fibroblast cytotoxicity assay.

    Science.gov (United States)

    Turgeon, David K; Novicki, Thomas J; Quick, John; Carlson, LaDonna; Miller, Pat; Ulness, Bruce; Cent, Anne; Ashley, Rhoda; Larson, Ann; Coyle, Marie; Limaye, Ajit P; Cookson, Brad T; Fritsche, Thomas R

    2003-02-01

    Clostridium difficile is one of the most frequent causes of nosocomial gastrointestinal disease. Risk factors include prior antibiotic therapy, bowel surgery, and the immunocompromised state. Direct fecal analysis for C. difficile toxin B by tissue culture cytotoxin B assay (CBA), while only 60 to 85% sensitive overall, is a common laboratory method. We have used 1,003 consecutive, nonduplicate fecal samples to compare six commercially available immunoassays (IA) for C. difficile detection with CBA: Prima System Clostridium difficile Tox A and VIDAS Clostridium difficile Tox A II, which detect C. difficile toxin A; Premier Cytoclone A/B and Techlab Clostridium difficile Tox A/B, which detect toxins A and B; and ImmunoCard Clostridium difficile and Triage Micro C. difficile panels, which detect toxin A and a species-specific antigen. For all tests, Triage antigen was most sensitive (89.1%; negative predictive value [NPV] = 98.7%) while ImmunoCard was most specific (99.7%; positive predictive value [PPV] = 95.0%). For toxin tests only, Prima System had the highest sensitivity (82.2%; NPV = 98.0%) while ImmunoCard had the highest specificity (99.7%; PPV = 95.0%). Hematopoietic stem cell transplant (HSCT) patients contributed 44.7% of all samples tested, and no significant differences in sensitivity or specificity were noted between HSCT and non-HSCT patients. IAs, while not as sensitive as direct fecal CBA, produce reasonable predictive values, especially when both antigen and toxin are detected. They also offer significant advantages over CBA in terms of turnaround time and ease of use.

  4. Development and testing of species-specific ELISA assays to measure IFN-gamma and TNF-alpha in bottlenose dolphins (Tursiops truncatus)

    Science.gov (United States)

    Monitoring the immune status of cetaceans is important for a variety of health conditions. Assays to quantify cytokines, especially pro-inflammatory cytokines, could be employed, in addition to currently available diagnostic assays, to screen for alterations in the health status of an animal. Thou...

  5. Multiplexed Immunofluorescence Reveals Potential PD-1/PD-L1 Pathway Vulnerabilities in Craniopharyngioma.

    Science.gov (United States)

    Coy, Shannon; Rashid, Rumana; Lin, Jia-Ren; Du, Ziming; Donson, Andrew M; Hankinson, Todd C; Foreman, Nicholas K; Manley, Peter E; Kieran, Mark W; Reardon, David A; Sorger, Peter K; Santagata, Sandro

    2018-03-02

    Craniopharyngiomas are neoplasms of the sellar/parasellar region that are classified into adamantinomatous (ACP) and papillary (PCP) subtypes. Surgical resection of craniopharyngiomas is challenging, and recurrence is common, frequently leading to profound morbidity. BRAF V600E mutations render PCP susceptible to BRAF/MEK inhibitors, but effective targeted therapies are needed for ACP. We explored the feasibility of targeting the PD-1/PD-L1 immune checkpoint pathway in ACP and PCP. We mapped and quantified PD-L1 and PD-1 expression in ACP and PCP resections using immunohistochemistry, immunofluorescence, and RNA in situ hybridization. We used tissue-based cyclic immunofluorescence (t-CyCIF) to map the spatial distribution of immune cells and characterize cell cycle and signaling pathways in ACP tumor cells which intrinsically express PD-1. All ACP (15±14% of cells, n=23, average±S.D.) and PCP (35±22% of cells, n=18) resections expressed PD-L1. In ACP, PD-L1 was predominantly expressed by tumor cells comprising the cyst-lining. In PCP, PD-L1 was highly-expressed by tumor cells surrounding the stromal fibrovascular cores. ACP also exhibited tumor cell-intrinsic PD-1 expression in whorled epithelial cells with nuclear-localized beta-catenin. These cells exhibited evidence of elevated mTOR and MAPK signaling. Profiling of immune populations in ACP and PCP showed a modest density of CD8+ T-cells. ACP exhibit PD-L1 expression in the tumor cyst-lining and intrinsic PD-1 expression in cells proposed to comprise an oncogenic stem-like population. In PCP, proliferative tumor cells express PD-L1 in a continuous band at the stromal-epithelial interface. Targeting PD-L1 and/or PD-1 in both subtypes of craniopharyngioma might therefore be an effective therapeutic strategy.

  6. An expert protocol for immunofluorescent detection of calcium channels in tsA-201 cells.

    Science.gov (United States)

    Koch, Peter; Herzig, Stefan; Matthes, Jan

    Pore-forming subunits of voltage gated calcium channels (VGCC) are large membrane proteins (260kDa) containing 24 transmembrane domains. Despite transfection with viral promoter driven vectors, biochemical analysis of VGCC is often hampered by rather low expression levels in heterologous systems rendering VGCC challenging targets. Especially in immunofluorescent detection, calcium channels are demanding proteins. We provide an expert step-by-step protocol with adapted conditions for handling procedures (tsA-201 cell culture, transient transfection, incubation time and temperature at 28°C or 37°C and immunostaining) to address the L-type calcium-channel pore Ca v 1.2 in an immunofluorescent approach. We performed immunocytochemical analysis of Ca v 1.2 expression at single-cell level in combination with detection of different markers for cellular organelles. We show confluency levels and shapes of tsA-201 cells at different time points during an experiment. Our experiments reveal sufficient levels of Ca v 1.2 protein and a correct Ca v 1.2 expression pattern in polygonal shaped cells already 12h after transfection. A sequence of elaborated protocol modifications allows subcellular localization analysis of Ca v 1.2 in an immunocytochemical approach. We provide a protocol that may be used to achieve insights into physiological and pathophysiological processes involving voltage gated calcium channels. Our protocol may be used for expression analysis of other challenging proteins and efficient overexpression may be exploited in related biochemical techniques requiring immunolabels. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. The lethality test used for estimating the potency of antivenoms against Bothrops asper snake venom: pathophysiological mechanisms, prophylactic analgesia, and a surrogate in vitro assay.

    Science.gov (United States)

    Chacón, Francisco; Oviedo, Andrea; Escalante, Teresa; Solano, Gabriela; Rucavado, Alexandra; Gutiérrez, José María

    2015-01-01

    The potency of antivenoms is assessed by analyzing the neutralization of venom-induced lethality, and is expressed as the Median Effective Dose (ED50). The present study was designed to investigate the pathophysiological mechanisms responsible for lethality induced by the venom of Bothrops asper, in the experimental conditions used for the evaluation of the neutralizing potency of antivenoms. Mice injected with 4 LD50s of venom by the intraperitoneal route died within ∼25 min with drastic alterations in the abdominal organs, characterized by hemorrhage, increment in plasma extravasation, and hemoconcentration, thus leading to hypovolemia and cardiovascular collapse. Snake venom metalloproteinases (SVMPs) play a predominat role in lethality, as judged by partial inhibition by the chelating agent CaNa2EDTA. When venom was mixed with antivenom, there was a venom/antivenom ratio at which hemorrhage was significantly reduced, but mice died at later time intervals with evident hemoconcentration, indicating that other components in addition to SVMPs also contribute to plasma extravasation and lethality. Pretreatment with the analgesic tramadol did not affect the outcome of the neutralization test, thus suggesting that prophylactic (precautionary) analgesia can be introduced in this assay. Neutralization of lethality in mice correlated with neutralization of in vitro coagulant activity in human plasma. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Evaluation of the prognostic value of IFN-gamma release assay and tuberculin skin test in household contacts of infectious tuberculosis cases in Senegal.

    Directory of Open Access Journals (Sweden)

    Christian Lienhardt

    2010-05-01

    Full Text Available Chemoprophylaxis of contacts of infectious tuberculosis (TB cases is recommended for TB control, particularly in endemic countries, but is hampered by the difficulty to diagnose latent TB infection (LTBI, classically assessed through response to the Tuberculin Skin Test (TST. Interferon-gamma release assays (IGRA are proposed new tools to diagnose LTBI, but there are limited data on their ability to predict the development of active TB disease. To address this, we investigated the response to TST and IGRA in household contacts of infectious TB cases in a TB high-burden country and the potential correlation with development of TB.Prospective household contacts study conducted in two health centres in Dakar, Senegal. A total of 2679 household contacts of 206 newly detected smear and/or culture positive index TB cases aged 18 years or greater were identified A TST was performed in each contact and an ESAT6/CFP10 ELISPOT assay performed in a random sample of those. Contacts were followed-up for 24 months. TB was diagnosed in 52 contacts, an incidence rate of 9.27/1000 person-years. In univariable analysis, the presence of positive TST (> or = 10 mm and ELISPOT (>32 SFC/million PBMC responses at baseline were associated with active TB during follow-up: Rate Ratio [RR] = 2.32 (95%CI:1.12-4.84 and RR = 2.09 (95%CI:0.83-5.31, respectively. After adjustment for age, sex and proximity to index case, adjusted RRs were 1.51 (95%CI:0.71-3.19 and 1.98 (95%CI:0.77-5.09, respectively. Restricting analysis to the 40 microbiologically confirmed cases, the adjusted RR for positive ELISPOT was 3.61 (95%CI:1.03-12.65. The median ELISPOT response in contacts who developed TB was 5-fold greater than in those who did not develop TB (p = 0.02.TST and IGRAs are markers of a contact of the immune system with tubercle bacilli. In a TB endemic area, a high ELISPOT response may reflect increased bacterial replication that may subsequently be associated with development of TB

  9. Management of bacterial kidney disease in Chinook Salmon hatcheries based on broodstock testing by enzyme-linked immunosorbent assay: A multiyear study

    Science.gov (United States)

    Munson, A. Douglas; Elliott, Diane G.; Johnson, Keith

    2010-01-01

    From the mid-1980s through the early 1990s, outbreaks of bacterial kidney disease (BKD) caused by Renibacterium salmoninarum continued in Chinook salmon Oncorhynchus tshawytscha in Idaho Department of Fish and Game (IDFG) hatcheries despite the use of three control methods: (1) injection of returning adult fish with erythromycin to reduce prespawning BKD mortality and limit vertical transmission of R. salmoninarum, (2) topical disinfection of green eggs with iodophor, and (3) prophylactic treatments of juvenile fish with erythromycin-medicated feed. In addition, programs to manage BKD through measurement of R. salmoninarum antigen levels in kidney tissues from spawning female Chinook salmon by an enzyme-linked immunosorbent assay (ELISA) were tested over 13–15 brood years at three IDFG hatcheries. The ELISA results were used for either (1) segregated rearing of progeny from females with high ELISA optical density (OD) values (usually ≥0.25), which are indicative of high R. salmoninarum antigen levels, or (2) culling of eggs from females with high ELISA OD values. The ELISA-based culling program had the most profound positive effects on the study populations. Mortality of juvenile fish during rearing was significantly lower at each hatchery for brood years derived from culling compared with brood years for which culling was not practiced. The prevalence of R. salmoninarum in juvenile fish, as evidenced by detection of the bacterium in kidney smears by the direct fluorescent antibody test, also decreased significantly at each hatchery. In addition, the proportions of returning adult females with kidney ELISA OD values of 0.25 or more decreased 56–85% for fish reared in brood years during which culling was practiced, whereas the proportions of ELISA-negative adults increased 55–58%. This management strategy may allow IDFG Chinook salmon hatcheries to reduce or eliminate prophylactic erythromycin-medicated feed treatments. We recommend using ELISA

  10. In vitro versus in vivo concordance: a case study of the replacement of an animal potency test with an immunochemical assay.

    Science.gov (United States)

    Schofield, T

    2002-01-01

    Early in its development, the potency of Merck's recombinant hepatitis B vaccine, RECOMBIVAX HB, was monitored using an assay performed in mice. A specification was determined to be the lowest potency which induced acceptable response in clinical trials. As a post-licensing commitment, Merck was asked to replace its mouse potency assay with an in vitro procedure for product release in the US market. Early studies with a commercial enzyme immunoassay (EIA) yielded highly variable results. That assay, combined with a sample pretreatment step, proved more dependable and predictive of potency in the mouse assay. Based on measurements made on manufactured materials, combined with experiments contrived to yield a wide range of reactivity in the two assays, concordance was established between the EIA and the mouse potency assay. This concordance was used to calibrate a specification for the in vitro assay that is predictive of a satisfactory response in vivo. Data from clinical trials established a correspondence between human immunogenicity and these potency markers.

  11. Interferon Gamma Release Assay versus Tuberculin Skin Testing among Healthcare Workers of Highly Diverse Origin in a Moderate Tuberculosis Burden Country.

    Directory of Open Access Journals (Sweden)

    Sahal Al Hajoj

    Full Text Available Health care workers (HCW's are always at an increased risk of contracting tuberculosis (TB infection. In Saudi Arabia, Interferon Gamma Release Assay (IGRA has not been evaluated as a screening tool for latent TB infection (LTBI among HCW's considering their high demographic diversity. During February 2012 to January 2015 a cross sectional study has been conducted in a tertiary care center with maximum demographically diverse staff population in the capital city-Riyadh. After a short interview and consenting, all the candidates were subjected to tuberculin skin test (TST and QuantiFERON TB gold In-tube test (QFT. A logistic regression analysis was carried out for establishing the associations between putative risk factors and the diagnostic tests. The candidates were classified according to geographical origin and a detailed analysis was conducted on the impact of their origin towards the results of TST and QFT. Of the 1595 candidates enrolled, 90.6% were BCG vaccinated, female (67.9% and mainly nurses (53.2%. Candidates with high risk of suspected or confirmed TB patient exposure were 56.1% and 76.5% of them had <10 year's work experience. TST positivity was observed in 503 (31.5% candidates, while QFT was positive among 399 (25%. Majority of the candidates were non-Saudi (83% and predominantly (52.4% from Western Pacific region. Concordant results were obtained in 14.2% of positive cases and 57.7% negative cases. The disagreements between the two tests were relatively high (kappa co-efficient-0.312±0.026, p value- <0.00001 as TST positive/QFT negative discordance was 54.8% while TST negative/QFT positive discordance was 15.7%. Age of the candidates, BCG vaccination, and South East Asian origin were associated with TST positivity while Occupational TB exposure and geographical origin of the candidates were associated with QFT positivity. A regular follow up on recently TST converted candidates showed no progression to active TB. The putative

  12. Use of a standardized JaCVAM in vivo rat comet assay protocol to assess the genotoxicity of three coded test compounds; ampicillin trihydrate, 1,2-dimethylhydrazine dihydrochloride, and N-nitrosodimethylamine.

    Science.gov (United States)

    McNamee, J P; Bellier, P V

    2015-07-01

    As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay (comet assay), our laboratory examined ampicillin trihydrate (AMP), 1,2-dimethylhydrazine dihydrochloride (DMH), and N-nitrosodimethylamine (NDA) using a standard comet assay validation protocol (v14.2) developed by the JaCVAM validation management team (VMT). Coded samples were received by our laboratory along with basic MSDS information. Solubility analysis and range-finding experiments of the coded test compounds were conducted for dose selection. Animal dosing schedules, the comet assay processing and analysis, and statistical analysis were conducted in accordance with the standard protocol. Based upon our blinded evaluation, AMP was not found to exhibit evidence of genotoxicity in either the rat liver or stomach. However, both NDA and DMH were observed to cause a significant increase in % tail DNA in the rat liver at all dose levels tested. While acute hepatoxicity was observed for these compounds in the high dose group, in the investigators opinion there were a sufficient number of consistently damaged/measurable cells at the medium and low dose groups to judge these compounds as genotoxic. There was no evidence of genotoxicity from either NDA or DMH in the rat stomach. In conclusion, our laboratory observed increased DNA damage from two blinded test compounds in rat liver (later identified as genotoxic carcinogens), while no evidence of genotoxicity was observed for the third blinded test compound (later identified as a non-genotoxic, non-carcinogen). This data supports the use of a standardized protocol of the in vivo comet assay as a cost-effective alternative genotoxicity assay for regulatory testing purposes. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

  13. Comparison of interferon-γ release assay to two cut-off points of tuberculin skin test to detect latent Mycobacterium tuberculosis infection in primary health care workers.

    Directory of Open Access Journals (Sweden)

    Fernanda Mattos de Souza

    Full Text Available An interferon-γ release assay, QuantiFERON-TB (QFT test, has been introduced an alternative test for the diagnosis of latent Mycobacterium tuberculosis infection (LTBI. Here, we compared the performance of QFT with tuberculin skin test (TST measured at two different cut-off points among primary health care work (HCW in Brazil.A cross-sectional study was carried out among HCWs in four Brazilian cities with a known history of high incidence of TB. Results of the QFT were compared to TST results based on both ≥5 mm and ≥10 mm as cut-off points.We enrolled 632 HCWs. When the cut-off value of ≥10 mm was used, agreement between QFT and TST was 69% (k = 0.31, and when the cut-off of ≥5 mm was chosen, the agreement was 57% (k = 0.22. We investigated possible factors of discordance of TST vs QFT. Compared to the TST-/QFT- group, risk factors for discordance in the TST+/QFT- group with TST cut-off of ≥5 mm included age between 41-45 years [OR = 2.70; CI 95%: 1.32-5.51] and 46-64 years [OR = 2.04; CI 95%: 1.05-3.93], BCG scar [OR = 2.72; CI 95%: 1.40-5.25], and having worked only in primary health care [OR = 2.30; CI 95%: 1.09-4.86]. On the other hand, for the cut-off of ≥10 mm, BCG scar [OR = 2.26; CI 95%: 1.03-4.91], being a household contact of a TB patient [OR = 1.72; CI 95%: 1.01-2.92] and having had a previous TST [OR = 1.66; CI 95%: 1.05-2.62], were significantly associated with the TST+/QFT- group. No statistically significant associations were found among the TST-/QFT+ discordant group with either TST cut-off value.Although we identified BCG vaccination to contribute to the discordance at both TST cut-off measures, the current Brazilian recommendation for the initiation of LTBI treatment, based on information gathered from medical history, TST, chest radiograph and physical examination, should not be changed.

  14. Comparison of interferon-γ release assay to two cut-off points of tuberculin skin test to detect latent Mycobacterium tuberculosis infection in primary health care workers.

    Science.gov (United States)

    de Souza, Fernanda Mattos; do Prado, Thiago Nascimento; Pinheiro, Jair dos Santos; Peres, Renata Lyrio; Lacerda, Thamy Carvalho; Loureiro, Rafaela Borge; Carvalho, Jose Américo; Fregona, Geisa; Dias, Elias Santos; Cosme, Lorrayne Beliqui; Rodrigues, Rodrigo Ribeiro; Riley, Lee Wood; Maciel, Ethel Leonor Noia

    2014-01-01

    An interferon-γ release assay, QuantiFERON-TB (QFT) test, has been introduced an alternative test for the diagnosis of latent Mycobacterium tuberculosis infection (LTBI). Here, we compared the performance of QFT with tuberculin skin test (TST) measured at two different cut-off points among primary health care work (HCW) in Brazil. A cross-sectional study was carried out among HCWs in four Brazilian cities with a known history of high incidence of TB. Results of the QFT were compared to TST results based on both ≥5 mm and ≥10 mm as cut-off points. We enrolled 632 HCWs. When the cut-off value of ≥10 mm was used, agreement between QFT and TST was 69% (k = 0.31), and when the cut-off of ≥5 mm was chosen, the agreement was 57% (k = 0.22). We investigated possible factors of discordance of TST vs QFT. Compared to the TST-/QFT- group, risk factors for discordance in the TST+/QFT- group with TST cut-off of ≥5 mm included age between 41-45 years [OR = 2.70; CI 95%: 1.32-5.51] and 46-64 years [OR = 2.04; CI 95%: 1.05-3.93], BCG scar [OR = 2.72; CI 95%: 1.40-5.25], and having worked only in primary health care [OR = 2.30; CI 95%: 1.09-4.86]. On the other hand, for the cut-off of ≥10 mm, BCG scar [OR = 2.26; CI 95%: 1.03-4.91], being a household contact of a TB patient [OR = 1.72; CI 95%: 1.01-2.92] and having had a previous TST [OR = 1.66; CI 95%: 1.05-2.62], were significantly associated with the TST+/QFT- group. No statistically significant associations were found among the TST-/QFT+ discordant group with either TST cut-off value. Although we identified BCG vaccination to contribute to the discordance at both TST cut-off measures, the current Brazilian recommendation for the initiation of LTBI treatment, based on information gathered from medical history, TST, chest radiograph and physical examination, should not be changed.

  15. In vitro effects of piracetam on the radiosensitivity of hypoxic cells (adaptation of MTT assay to hypoxic conditions); Effets in vitro du piracetam sur la radiosensibilite des cellules hypoxiques (adapatation du test au MTT aux conditions d`hypoxie)

    Energy Technology Data Exchange (ETDEWEB)

    Gheuens, E.E.O.; Bruijn, E.A. de; Van der Heyden, S.; Van Oosterom, A.T. [Universitaire Instelling Antwerpen, Antwerp (Belgium); Lagarde, P. [Universitaire Instelling Antwerpen, Antwerp (Belgium)]|[Institut Bergonie, 33 - Bordeaux (France); Pooter, C.M.J. de [Universitaire Instelling Antwerpen, Antwerp (Belgium)]|[Hopital de Middelheim, Anvers (Belgium); Chomy, F. [Institut Bergonie, 33 - Bordeaux (France)

    1995-12-31

    This paper describes the adaptation of the MTT assay to hypoxic conditions in order to test the in vitro effect of piracetam on hypoxic cells and particularly on the radiosensitivity of hypoxic cells since this drug has shown clinical effect on acute and chronic hypoxia. The V79 cell line was selected by reference to preliminary hypoxic experiments using clonogenic assay and euoxic experiments using clonogenic and MTT assays. Cell growth and survival in our hypoxic conditions were assessed using MTT assay with an enclosure and special 48-well plates both made of glass. Growth curves on glass plates after 1-hour exposure to nitrogen versus air were comparable, so there is no bias effect due to gas composition. Survival curves using MTT versus reference clonogenic assay were comparable after radiation exposure in eu- and hypoxic conditions, and confirm the validity of our original technique for creating hypoxia. The Oxygen Enhancement Ratio was of about 3 for 1-hour hypoxic exposure. Piracetam gave no cytotoxic effect up to 10 mM of piracetam. Growth curves after continuous drug exposure and 1-hour euoxic versus hypoxic exposure gave no cytotoxic effect up to 10 mM of piracetam. Survival curves after continuous drug exposure to 10 mM of piracetam gave no significant effect on the radiosensitivity of hypoxic V79 cells using MTT or clonogenic assay. (author). 32 refs., 6 figs.

  16. The Role of Glutamate Dehydrogenase (GDH Testing Assay in the Diagnosis of Clostridium difficile Infections: A High Sensitive Screening Test and an Essential Step in the Proposed Laboratory Diagnosis Workflow for Developing Countries like China.

    Directory of Open Access Journals (Sweden)

    Jing-Wei Cheng

    Full Text Available The incidence and severity of Clostridium difficile infection (CDI in North America and Europe has increased significantly since the 2000s. However, CDI is not widely recognized in China and other developing countries due to limited laboratory diagnostic capacity and low awareness. Most published studies on laboratory workflows for CDI diagnosis are from developed countries, and thus may not be suitable for most developing countries. Therefore, an alternative strategy for developing countries is needed. In this study, we evaluated the performance of the Glutamate Dehydrogenase (GDH test and its associated workflow on 416 fecal specimens from suspected CDI cases. The assay exhibited excellent sensitivity (100.0% and specificity (92.8%, compared to culture based method, and thus could be a good screening marker for C. difficile but not for indication of toxin production. The VIDAS CDAB assay, which can detect toxin A/B directly from fecal specimens, showed good specificity (99.7% and positive predictive value (97.2%, but low sensitivity (45.0% and negative predictive value (88.3%, compared with PCR-based toxin gene detection. Therefore, we propose a practical and efficient GDH test based workflow strategy for the laboratory diagnosis of CDI in developing countries like China. By applying this new workflow, the CDI laboratory diagnosis rate was notably improved in our center, yet the increasing cost was kept at a minimum level. Furthermore, to gain some insights into the genetic population structure of C. difficile isolates from our hospital, we performed MLST and PCR toxin gene typing.

  17. Results of the Abbott RealTime HIV-1 assay for specimens yielding "target not detected" results by the Cobas AmpliPrep/Cobas TaqMan HIV-1 Test.

    Science.gov (United States)

    Babady, N Esther; Germer, Jeffrey J; Yao, Joseph D C

    2010-03-01

    No significantly discordant results were observed between the Abbott RealTime HIV-1 assay and the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test (CTM) among 1,190 unique clinical plasma specimens obtained from laboratories located in 40 states representing all nine U.S. geographic regions and previously yielding "target not detected" results by CTM.

  18. Comparison of the Hologic Aptima HIV-1 Quant Dx Assay to the Roche COBAS Ampliprep/COBAS TaqMan HIV-1 Test v2.0 for the quantification of HIV-1 RNA in plasma samples

    DEFF Research Database (Denmark)

    Schønning, Kristian; Johansen, Kim; Landt, Bodil

    2017-01-01

    load compared to the CAPCTMv2 was observed in Bland-Altman analysis, by Deming regression (Slope 1.13) and in dilution series of clinical samples. Precision evaluated using the Acrometrix Positive Controls was similar for the High Control (CV: 1.2% vs. 1.3%; Aptima assay vs. CAPCTMv2 test, respectively...

  19. Evaluation of serological tests for the diagnosis of rickettsiosis in Denmark

    DEFF Research Database (Denmark)

    Kantsø, Bjørn; Svendsen, Claus Bo; Jørgensen, Charlotte Svaerke

    2009-01-01

    Two commercially available immunofluorescence assays (IFA) were compared using historical sera evaluated for rickettsial antibodies by the Weil-Felix test. An IFA test produced by Focus Diagnostics prepared with Rickettsia rickettsii and R. typhi antigens was compared with a custom made kit from....... When analyzing the data using the manufacturers' cut-off values, 41% of samples from presumably healthy blood donors were found positive for spotted fever group Rickettsia antibodies. This does not correlate to the general picture of rickettsiosis in Denmark. Furthermore, sera with Coxiella burnetii...

  20. Antibody class capture assay (ACCA) for rubella-specific IgM antibody.

    Science.gov (United States)

    Isaac, M; Payne, R A

    1982-01-01

    Enzyme-linked immunosorbent assays for IgM antirubella were carried out on 1,546 sera, using an IgM capture method with a F (ab')2 conjugate (ACCA). Under the conditions described, sera containing IgM antirubella bound up to 15 times as much enzyme activity as negative specimens. Paired serum specimens from 27 patients, serial serum specimens from 6 patients, and single serum specimens from 15 patients who had had recent rubella were examined by the haemagglutination inhibition test (HAI) in the presence and absence of 2-mercaptoethanol following sucrose density gradient centrifugation (SDGC). ACCA confirmed all the results found with HAI following SDGC. Specimens were examined from ten patients with congenital rubella; ACCA confirmed the results found with both immunofluorescence following SDGC and radioimmunoassay. Pre- and post-vaccination specimens from 123 patients who had been vaccinated against rubella were examined. An IgM response could only be demonstrated in the 57 cases when IgG was absent in the first specimen. The specificity of the assay was confirmed by testing 31 serum specimens from rubella immune patients that also contained rheumatoid factor, 163 serum specimens from patients with acute infections other than rubella, and 12 serum specimens from infants with miscellaneous neonatal abnormalities other than congenital rubella. The ACCA proved a simple, sensitive, and specific test for IgM antirubella and the results compared favourably with those obtained by the SDGC technique.

  1. Development of an artificial neural network model for risk assessment of skin sensitization using human cell line activation test, direct peptide reactivity assay, KeratinoSens™ and in silico structure alert parameter.

    Science.gov (United States)

    Hirota, Morihiko; Ashikaga, Takao; Kouzuki, Hirokazu

    2018-04-01

    It is important to predict the potential of cosmetic ingredients to cause skin sensitization, and in accordance with the European Union cosmetic directive for the replacement of animal tests, several in vitro tests based on the adverse outcome pathway have been developed for hazard identification, such as the direct peptide reactivity assay, KeratinoSens™ and the human cell line activation test. Here, we describe the development of an artificial neural network (ANN) prediction model for skin sensitization risk assessment based on the integrated testing strategy concept, using direct peptide reactivity assay, KeratinoSens™, human cell line activation test and an in silico or structure alert parameter. We first investigated the relationship between published murine local lymph node assay EC3 values, which represent skin sensitization potency, and in vitro test results using a panel of about 134 chemicals for which all the required data were available. Predictions based on ANN analysis using combinations of parameters from all three in vitro tests showed a good correlation with local lymph node assay EC3 values. However, when the ANN model was applied to a testing set of 28 chemicals that had not been included in the training set, predicted EC3s were overestimated for some chemicals. Incorporation of an additional in silico or structure alert descriptor (obtained with TIMES-M or Toxtree software) in the ANN model improved the results. Our findings suggest that the ANN model based on the integrated testing strategy concept could be useful for evaluating the skin sensitization potential. Copyright © 2017 John Wiley & Sons, Ltd.

  2. Fluorescence Adherence Inhibition Assay: A Novel Functional Assessment of Blocking Virus Attachment by Vaccine-Induced Antibodies.

    Directory of Open Access Journals (Sweden)

    Atul Asati

    Full Text Available Neutralizing antibodies induced by vaccination or natural infection play a critically important role in protection against the viral diseases. In general, neutralization of the viral infection occurs via two major pathways: pre- and post-attachment modes, the first being the most important for such infections as influenza and polio, the latter being significant for filoviruses. Neutralizing capacity of antibodies is typically evaluated by virus neutralization assays that assess reduction of viral infectivity to the target cells in the presence of functional antibodies. Plaque reduction neutralization test, microneutralization and immunofluorescent assays are often used as gold standard virus neutralization assays. However, these methods are associated with several important prerequisites such as use of live virus requiring safety precautions, tedious evaluation procedure and long assessment time. Hence, there is a need for a robust, inexpensive high throughput functional assay that can be performed rapidly using inactivated virus, without extensive safety precautions. Herein, we report a novel high throughput Fluorescence Adherence Inhibition assay (fADI using inactivated virus labeled with fluorescent secondary antibodies virus and Vero cells or erythrocytes as targets. It requires only few hours to assess pre-attachment neutralizing capacity of donor sera. fADI assay was tested successfully on donors immunized with polio, yellow fever and influenza vaccines. To further simplify and improve the throughput of the assay, we have developed a mathematical approach for calculating the 50% titers from a single sample dilution, without the need to analyze multi-point titration curves. Assessment of pre- and post-vaccination human sera from subjects immunized with IPOL®, YF-VAX® and 2013-2014 Fluzone® vaccines demonstrated high efficiency of the assay. The results correlated very well with microneutralization assay performed independently by the FDA

  3. Safety and efficacy of the C-Tb skin test to diagnose Mycobacterium tuberculosis infection, compared with an interferon γ release assay and the tuberculin skin test: a phase 3, double-blind, randomised, controlled trial.

    Science.gov (United States)

    Ruhwald, Morten; Aggerbeck, Henrik; Gallardo, Rafael Vázquez; Hoff, Søren T; Villate, José I; Borregaard, Bettine; Martinez, José A; Kromann, Ingrid; Penas, Antón; Anibarro, Luis L; de Souza-Galvão, Maria Luiza; Sánchez, Francisca; Rodrigo-Pendás, Jose Ángel; Noguera-Julian, Antoni; Martínez-Lacasa, Xavier; Tuñez, Maria Victoria; Fernández, Virginia Leiro; Millet, Joan P; Moreno, Antonio; Cobos, Nazaret; Miró, José M; Roldan, Llanos; Orcau, Angels; Andersen, Peter; Caylá, Joan A

    2017-04-01

    Targeted screening and treatment of Mycobacterium tuberculosis infection substantially reduces the risk of developing active tuberculosis. C-Tb (Statens Serum Institute, Copenhagen, Denmark) is a novel specific skin test based on ESAT-6 and CFP10 antigens. We investigated the safety and diagnostic potential of C-Tb compared with established tests in the contact-tracing setting. Negative controls, close contacts, occasional contacts, and patients with active pulmonary tuberculosis were enrolled at 13 centres in Spain. We compared C-Tb with the QuantiFERON-TB Gold In-Tube ([QFT] Qiagen, Hilden, Germany) interferon γ release assay (IGRA) and the purified protein derivative (PPD) RT 23 tuberculin skin test ([TST] Statens Serum Institute). All participants older than 5 years were tested with QFT. Some participants in the negative control group received C-Tb without the TST to test for potential interactions between C-Tb and PPD RT 23. The rest were randomly assigned in blocks of ten and tested with both C-Tb and TST, with five in each block receiving injection of C-Tb in the right arm and the TST in the left arm and five vice versa. The primary and safety analyses were done in all participants randomly assigned to a group who received any test. This trial is registered with ClinicalTrials.gov, number NCT01631266, and with EudraCT, number 2011-005617-36. From July 24, 2012, to Oct 2, 2014, 979 participants were enrolled, of whom 263 were negative controls, 299 were occasional contacts, 316 were close contacts, and 101 were patients with tuberculosis. 970 (99%) participants completed the trial. Induration sizes were similar for C-Tb and TST, but TST positivity was affected by BCG vaccination status. We found a strong positive trend towards C-Tb test positivity with increasing risk of infection, from 3% in negative controls to 16% in occasional contacts, to 43% in close contacts. C-Tb and QFT results were concordant in 785 (94%) of 834 participants aged 5 years and older

  4. Comparison of central HER2 testing with quantitative total HER2 expression and HER2 homodimer measurements using a novel proximity-based assay.

    Science.gov (United States)

    Huang, Weidong; Reinholz, Monica; Weidler, Jodi; Yolanda, Lie; Paquet, Agnes; Whitcomb, Jeannette; Lingle, Wilma; Jenkins, Robert B; Chen, Beiyun; Larson, Jeffrey S; Tan, Yuping; Sherwood, Thomas; Bates, Michael; Perez, Edith A

    2010-08-01

    The accuracy and reliability of immunohistochemical analysis and in situ hybridization for the assessment of HER2 status remains a subject of debate. We developed a novel assay (HERmark Breast Cancer Assay, Monogram Biosciences, South San Francisco, CA) that provides precise quantification of total HER2 protein expression (H2T) and HER2 homodimers (H2D) in formalin-fixed, paraffin-embedded tissue specimens. H2T and H2D results of 237 breast cancers were compared with those of immunohistochemical studies and fluorescence in situ hybridization (FISH) centrally performed at the Mayo Clinic, Rochester, MN. H2T described a continuum across a wide dynamic range ( approximately 2.5 log). Excluding the equivocal cases, HERmark showed 98% concordance with immunohistochemical studies for positive and negative assay values. For the 94 immunohistochemically equivocal cases, 67% and 39% concordance values were observed between HERmark and FISH for positive and negative assay values, respectively. Polysomy 17 in the absence of HER2 gene amplification did not result in HER2 overexpression as evaluated quantitatively using the HERmark assay.

  5. Segment and fit thresholding: a new method for image analysis applied to microarray and immunofluorescence data.

    Science.gov (United States)

    Ensink, Elliot; Sinha, Jessica; Sinha, Arkadeep; Tang, Huiyuan; Calderone, Heather M; Hostetter, Galen; Winter, Jordan; Cherba, David; Brand, Randall E; Allen, Peter J; Sempere, Lorenzo F; Haab, Brian B

    2015-10-06

    Experiments involving the high-throughput quantification of image data require algorithms for automation. A challenge in the development of such algorithms is to properly interpret signals over a broad range of image characteristics, without the need for manual adjustment of parameters. Here we present a new approach for locating signals in image data, called Segment and Fit Thresholding (SFT). The method assesses statistical characteristics of small segments of the image and determines the best-fit trends between the statistics. Based on the relationships, SFT identifies segments belonging to background regions; analyzes the background to determine optimal thresholds; and analyzes all segments to identify signal pixels. We optimized the initial settings for locating background and signal in antibody microarray and immunofluorescence data and found that SFT performed well over multiple, diverse image characteristics without readjustment of settings. When used for the automated analysis of multicolor, tissue-microarray images, SFT correctly found the overlap of markers with known subcellular localization, and it performed better than a fixed threshold and Otsu's method for selected images. SFT promises to advance the goal of full automation in image analysis.

  6. AMP deaminase histochemical activity and immunofluorescent isozyme localization in rat skeletal muscle

    Science.gov (United States)

    Thompson, J. L.; Sabina, R. L.; Ogasawara, N.; Riley, D. A.

    1992-01-01

    The cellular distribution of AMP deaminase (AMPda) isozymes was documented for rat soleus and plantaris muscles, utilizing immunofluorescence microscopy and immunoprecipitation methods. AMPda is a ubiquitous enzyme existing as three distinct isozymes, A, B and C, which were initially purified from skeletal muscle, liver (and kidney), and heart, respectively. AMPda-A is primarily concentrated subsarcolemmally and intermyofibrillarly within muscle cells, while isozymes B and C are concentrated within non-myofiber elements of muscle tissue. AMPda-B is principally associated with connective tissues surrounding neural elements and the muscle spindle capsule, and AMPda-C is predominantly associated with circulatory elements, such as arterial and venous walls, capillary endothelium, and red blood cells. These specific localizations, combined with documented differences in kinetic properties, suggest multiple functional roles for the AMPda isozymes or temporal segregation of similar AMPda functions. Linkage of the AMPda substrate with adenosine production pathways at the AMP level and the localization of isozyme-C in vascular tissue suggest a regulatory role in the microcirculation.

  7. Semi-automated, occupationally safe immunofluorescence microtip sensor for rapid detection of Mycobacterium cells in sputum.

    Directory of Open Access Journals (Sweden)

    Shinnosuke Inoue

    Full Text Available An occupationally safe (biosafe sputum liquefaction protocol was developed for use with a semi-automated antibody-based microtip immunofluorescence sensor. The protocol effectively liquefied sputum and inactivated microorganisms including Mycobacterium tuberculosis, while preserving the antibody-binding activity of Mycobacterium cell surface antigens. Sputum was treated with a synergistic chemical-thermal protocol that included moderate concentrations of NaOH and detergent at 60°C for 5 to 10 min. Samples spiked with M. tuberculosis complex cells showed approximately 10(6-fold inactivation of the pathogen after treatment. Antibody binding was retained post-treatment, as determined by analysis with a microtip immunosensor. The sensor correctly distinguished between Mycobacterium species and other cell types naturally present in biosafe-treated sputum, with a detection limit of 100 CFU/mL for M. tuberculosis, in a 30-minute sample-to-result process. The microtip device was also semi-automated and shown to be compatible with low-cost, LED-powered fluorescence microscopy. The device and biosafe sputum liquefaction method opens the door to rapid detection of tuberculosis in settings with limited laboratory infrastructure.

  8. Spatial Mixture Modelling for Unobserved Point Processes: Examples in Immunofluorescence Histology.

    Science.gov (United States)

    Ji, Chunlin; Merl, Daniel; Kepler, Thomas B; West, Mike

    2009-12-04

    We discuss Bayesian modelling and computational methods in analysis of indirectly observed spatial point processes. The context involves noisy measurements on an underlying point process that provide indirect and noisy data on locations of point outcomes. We are interested in problems in which the spatial intensity function may be highly heterogenous, and so is modelled via flexible nonparametric Bayesian mixture models. Analysis aims to estimate the underlying intensity function and the abundance of realized but unobserved points. Our motivating applications involve immunological studies of multiple fluorescent intensity images in sections of lymphatic tissue where the point processes represent geographical configurations of cells. We are interested in estimating intensity functions and cell abundance for each of a series of such data sets to facilitate comparisons of outcomes at different times and with respect to differing experimental conditions. The analysis is heavily computational, utilizing recently introduced MCMC approaches for spatial point process mixtures and extending them to the broader new context here of unobserved outcomes. Further, our example applications are problems in which the individual objects of interest are not simply points, but rather small groups of pixels; this implies a need to work at an aggregate pixel region level and we develop the resulting novel methodology for this. Two examples with with immunofluorescence histology data demonstrate the models and computational methodology.

  9. Pattern of glomerular diseases in oman: A study based on light microscopy and immunofluorescence

    Directory of Open Access Journals (Sweden)

    Nasar Yousuf Alwahaibi

    2013-01-01

    Full Text Available Light microscopy and immunofluorescence play an important part in the final diagnosis of renal biopsy. The aim of this study was to analyze the pattern of various glomerular diseases in Oman. A total of 424 renal biopsies were retrospectively analyzed at the Sultan Qaboos University Hospital between 1999 and 2010. Focal and segmental glomerulosclerosis (FSGS, minimal change disease (MCD, membranous glomerulopathy (MGN and IgA nephropathy were the most common primary glomerular diseases encountered, accounting for 21.2%, 17%, 12.3% and 8.3%, respectively, of all cases. Lupus nephritis was the most common secondary glomerular disease and was the most prevalent among all biopsies, accounting for 30.4% of all biopsies. Amyloidosis was seen in only two cases. The presence of fluorescein isothiocyanatefibrin in all renal cases was low when compared with IgG, IgA, IgM, C3 and C1q markers. In conclusion, based on the findings of this study, lupus nephritis was the most common of all glomerular diseases and FSGS was the most common primary glomerular disease. The importance of fluorescein isothiocyanate-fibrin in the diagnosis of renal biopsy needs to be further investigated.

  10. Review on the acute Daphnia magna toxicity test – Evaluation of the sensitivity and the precision of assays performed with organisms from laboratory cultures or hatched from dormant eggs

    Directory of Open Access Journals (Sweden)

    G. Persoone

    2009-08-01

    Full Text Available One of the most internationally used bioassays for toxicity screening of chemicals and for toxicity monitoring of effluents and contaminated waters is the acute toxicity test with daphnid crustaceans, and in particular that performed with Daphnia magna.Standard methods have been developed for this assay that were gradually endorsed by national and international organisations dealing with toxicity testing procedures, in view of its application within a regulatory framework. As for all toxicity tests, the organisms used for the acute D. magna assay have to be obtained from live stocks which are cultured in the laboratory on live food (micro-algae.Unsurprisingly the various standard protocols of this particular assay differ – at least to a certain extent – with regard to the test organism culturing conditions. In addition, some technical aspects of the toxicity test such as the effect criterion (mortality of immobility, the exposure time, the type of dilution water, etc., also vary from one standard to another.Although this particular assay is currently used in many countries, the technical and biological problems inherent in year-round culturing and availability of the biological material and the culturing/maintenance costs of live stocks restrict its application to a limited number of highly specialised laboratories.This fundamental bottleneck in toxicity testing triggered investigations which brought forward the concept of “microbiotests” or “small-scale” toxicity tests. “Culture/maintenance free” aquatic microbiotests with species of different phylogenetic groups were developed in the early 1990s at the Laboratory for Environmental Toxicology and Aquatic Ecology at the Ghent University in Belgium.These assays which were given the generic name “Toxkits”, are unique in that they employ dormant stages (“cryptobiotic eggs” of the test species, which can be stored for long periods of time and “hatched” at the time of

  11. Review on the acute Daphnia magna toxicity test – Evaluation of the sensitivity and the precision of assays performed with organisms from laboratory cultures or hatched from dormant eggs

    Directory of Open Access Journals (Sweden)

    Persoone G.

    2009-08-01

    “Culture/maintenance free” aquatic microbiotests with species of different phylogenetic groups were developed in the early 1990s at the Laboratory for Environmental Toxicology and Aquatic Ecology at the Ghent University in Belgium. These assays which were given the generic name “Toxkits”, are unique in that they employ dormant stages (“cryptobiotic eggs” of the test species, which can be stored for long periods of time and “hatched” at the time of performance of the assays. One of these microbiotests is the Daphtoxkit F magna, which is currently used in many laboratories worldwide for research as well as for toxicity monitoring purposes. The microbiotest technology has several advantages in comparison to the “traditional” tests based on laboratory cultures, especially its independence of the stock culturing burden. However, the acceptance (or possible non-acceptance of performing assays with test organisms obtained from “dormant eggs” should be clearly dictated by the “sensitivity” and “precision” criteria of the former assays in comparison to the latter. The first part of this review therefore thoroughly reviews the scientific literature and of data obtained from various laboratories for assays performed with either D. magna test organisms obtained from lab cultures or hatched from dormant eggs. Attention has focused on data of quality control tests performed on reference chemicals, and in particular on potassium dichromate (K2Cr2O7 for which an acceptability range of 0.6–2.1 mg·L–1 has been set in ISO standard 6341 for the 24 h EC50 of the acute D. magna assay. Mean EC50s, standard deviations and variation coefficients were calculated from the collected data, all of which are presented in tables and figures and discussed in detail. The major conclusions drawn from the analysis of the large number of quality control (QC data on the acute D. magna toxicity test are that : (1 Virtually all results from assays performed with

  12. The comet assay as a rapid test in biomonitoring occupational exposure to DNA-damaging agents and effect of confounding factors

    DEFF Research Database (Denmark)

    Møller, P; Knudsen, Lisbeth E.; Loft, S

    2000-01-01

    appeared to have less power than the positive studies. Also, there were poor dose-response relationships in many of the biomonitoring studies. Many factors have been reported to produce effects by the comet assay, e.g., age, air pollution exposure, diet, exercise, gender, infection, residential radon...... be used as criteria for the selection of populations and that data on exercise, diet, and recent infections be registered before blood sampling. Samples from exposed and unexposed populations should be collected at the same time to avoid seasonal variation. In general, the comet assay is considered...... exposure, smoking, and season. Until now, the use of the comet assay has been hampered by the uncertainty of the influence of confounding factors. We argue that none of the confounding factors are unequivocally positive in the majority of the studies. We recommend that age, gender, and smoking status...

  13. Validation of a single nucleotide polymorphism (SNP) typing assay with 49 SNPs for forensic genetic testing in a laboratory accredited according to the ISO 17025 standard

    DEFF Research Database (Denmark)

    Børsting, Claus; Rockenbauer, Eszter; Morling, Niels

    2009-01-01

    cases and 33 twin cases were typed at least twice for the 49 SNPs. All electropherograms were analysed independently by two expert analysts prior to approval. Based on these results, detailed guidelines for analysis of the SBE products were developed. With these guidelines, the peak height ratio...... of a heterozygous allele call or the signal to noise ratio of a homozygous allele call is compared with previously obtained ratios. A laboratory protocol for analysis of SBE products was developed where allele calls with unusual ratios were highlighted to facilitate the analysis of difficult allele calls......A multiplex assay with 49 autosomal single nucleotide polymorphisms (SNPs) developed for human identification was validated for forensic genetic casework and accredited according to the ISO 17025 standard. The multiplex assay was based on the SNPforID 52plex SNP assay [J.J. Sanchez, C. Phillips, C...

  14. A rapid and cell-free assay to test the activity of lynch syndrome-associated MSH2 and MSH6 missense variants

    DEFF Research Database (Denmark)

    Drost, Mark; Zonneveld, José B M; van Hees, Sandrine

    2012-01-01

    amino acid alterations. The pathogenicity of these variants of uncertain significance (VUS) is difficult to assess, precluding diagnosis of carriers and their relatives. Here we present a rapid cell-free assay to investigate MMR activity of MSH2 or MSH6 VUS. We used this assay to analyze a series of MSH......2 and MSH6 VUS, selected from the Leiden Open Variation Database. Whereas a significant fraction of the MSH2 VUS has lost MMR activity, suggesting pathogenicity, the large majority of the MSH6 VUS appears MMR proficient. We anticipate that this assay will be an important tool in the development...... of a comprehensive and widely applicable diagnostic procedure for LS-associated VUS....

  15. Combination of physico-chemical analysis, Allium cepa test system and Oreochromis niloticus erythrocyte based comet assay/nuclear abnormalities tests for cyto-genotoxicity assessments of treated effluents discharged from textile industries.

    Science.gov (United States)

    Hemachandra, Chamini K; Pathiratne, Asoka

    2016-09-01

    Bioassays for cyto-genotoxicity assessments are generally not required in current textile industry effluent discharge management regulations. The present study applied in vivo plant and fish based toxicity tests viz. Allium cepa test system and Oreochromis niloticus erythrocyte based comet assay and nuclear abnormalities tests in combination with physico-chemical analysis for assessing potential cytotoxic/genotoxic impacts of treated textile industry effluents reaching a major river (Kelani River) in Sri Lanka. Of the treated effluents tested from two textile industries, color in the Textile industry 1 effluents occasionally and color, biochemical oxygen demand and chemical oxygen demand in the Textile industry 2 effluents frequently exceeded the specified Sri Lankan tolerance limits for discharge of industrial effluents into inland surface waters. Exposure of A. cepa bulbs to 100% and 12.5% treated effluents from both industries resulted in statistically significant root growth retardation, mito-depression, and induction of chromosomal abnormalities in root meristematic cells in comparison to the dilution water in all cases demonstrating cyto-genotoxicity associated with the treated effluents. Exposure of O. niloticus to the 100% and 12.5% effluents, resulted in erythrocytic genetic damage as shown by elevated total comet scores and induction of nuclear abnormalities confirming the genotoxicity of the treated effluents even with 1:8 dilution. The results provide strong scientific evidence for the crucial necessity of incorporating cyto-genotoxicity impact assessment tools in textile industry effluent management regulations considering human health and ecological health of the receiving water course under chronic exposure. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Characterization of pars intermedia connections in amphibians by biocytin tract tracing and immunofluorescence aided by confocal microscopy

    NARCIS (Netherlands)

    Jansen, K; Fabro, C; Artero, C; Feuilloley, M; Vaudry, H; Fasolo, A; Franzoni, MF

    Biocytin, recently introduced in neuroanatomical studies, was used as a retrograde tract tracer in combination with immunofluorescence in order to analyse the neurochemical characters of some central neuronal projections to the pars intermedia in two amphibian species, the anuran Rana esculenta and

  17. Evaluation of the Xpert Flu test and comparison with in-house real-time RT-PCR assays for detection of influenza virus from 2008 to 2011 in Marseille, France.

    Science.gov (United States)

    Salez, N; Ninove, L; Thirion, L; Gazin, C; Zandotti, C; de Lamballerie, X; Charrel, R N

    2012-04-01

    Rapid documentation of respiratory specimens can have an impact on the management of patients and their relatives in terms of preventive and curative measures. We compared the results of the Xpert(®) Flu assay (Cepheid) with three real-time RT-PCR assays</