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Isolation of Saint Louis Encephalitis Virus from a Horse with Neurological Disease in Brazil  [PDF]
Roberta Rosa,Erica Azevedo Costa,Rafael Elias Marques,Taismara Simas Oliveira,Ronaldo Furtini,Maria Rosa Quaresma Bomfim,Mauro Martins Teixeira,Tatiane Alves Paix?o,Renato Lima Santos
PLOS Neglected Tropical Diseases , 2013, DOI: 10.1371/journal.pntd.0002537
Abstract: St. Louis encephalitis virus (SLEV) is a causative agent of encephalitis in humans in the Western hemisphere. SLEV is a positive-sense RNA virus that belongs to the Flavivirus genus, which includes West Nile encephalitis virus, Japanese encephalitis virus, Dengue virus and other medically important viruses. Recently, we isolated a SLEV strain from the brain of a horse with neurological signs in the countryside of Minas Gerais, Brazil. The SLEV isolation was confirmed by reverse-transcription RT-PCR and sequencing of the E protein gene. Virus identity was also confirmed by indirect immunofluorescence using commercial antibodies against SLEV. To characterize this newly isolated strain in vivo, serial passages in newborn mice were performed and led to hemorrhagic manifestations associated with recruitment of inflammatory cells into the central nervous system of newborns. In summary this is the first isolation of SLEV from a horse with neurological signs in Brazil.
St. Louis encephalitis vírus: first isolation from a human in S?o Paulo state, Brasil
Rocco, Iray M.;Santos, Cecília L.S.;Bisordi, Ivani;Petrella, Selma M.C.N.;Pereira, Luiz E.;Souza, Renato P.;Coimbra, Terezinha L.M.;Bessa, Thirsa A.F.;Oshiro, Fabiola M.;Lima, Luciana B.Q.;Cerroni, Matheus P.;Marti, Antonia T.;Barbosa, Vera M.;Katz, Gizelda;Suzuki, Akemi;
Revista do Instituto de Medicina Tropical de S?o Paulo , 2005, DOI: 10.1590/S0036-46652005000500008
Abstract: this paper reports the isolation of st. louis encephalitis virus (slev) from a febrile human case suspected to be dengue, in s?o pedro, s?o paulo state. a mac-elisa done on the patient's acute and convalescent sera was inconclusive and hemagglutination inhibition test detected igg antibody for flaviviruses. an indirect immunofluorescent assay done on the c6/36 cell culture inoculated with the acute serum was positive for flaviviruses but negative when tested with dengue monoclonal antibodies. rna extracted from the infected cell culture supernatant was amplified by rt-pcr in the presence of ns5 universal flavivirus primers and directly sequenced. results of blast search indicated that this sequence shares 93% nucleotide similarity with the sequence of slev (strain-msi.7), confirmed by rt-pcr performed with slev specific primers. since slev was identified as the cause of human disease, it is necessary to improve surveillance in order to achieve early detection of this agent in the state of s?o paulo and in brazil. this finding is also an alert to health professionals about the need for more complete clinical and epidemiological investigations of febrile illnesses as in the reported case. slev infections can be unrecognized or confused with other ones caused by an arbovirus, such as dengue.
Serologic evidence of the recent circulation of Saint Louis encephalitis virus and high prevalence of equine encephalitis viruses in horses in the Nhecolandia sub-region in South Pantanal, Central-West Brazil
Pauvolid-Corrêa, Alex;Tavares, Fernando Neto;Costa, Eliane Veiga da;Burlandy, Fernanda Marcicano;Murta, Michele;Pellegrin, Aiesca Oliveira;Nogueira, Márcia Furlan;Silva, Edson Elias da;
Memórias do Instituto Oswaldo Cruz , 2010, DOI: 10.1590/S0074-02762010000600017
Abstract: as in humans, sub-clinical infection by arboviruses in domestic animals is common; however, its detection only occurs during epizootics and the silent circulation of some arboviruses may remain undetected. the objective of the present paper was to assess the current circulation of arboviruses in the nhecolandia sub-region of south pantanal, brazil. sera from a total of 135 horses, of which 75 were immunized with bivalent vaccine composed of inactive eastern equine encephalitis virus (eeev) and western equine encephalitis virus(weev) and 60 were unvaccinated, were submitted to thorough viral isolation, reverse transcriptase polymerase chain reaction (rt-pcr) and neutralization tests for saint louis encephalitis virus (slev), eeev, weev and mayaro virus (mayv). no virus was isolated and viral nucleic-acid detection by rt-pcr was also negative. nevertheless, the prevalence of neutralizing antibodies in horses older than seven months was 43.7% for slev in equines regardless of vaccine status, and 36.4% for weev and 47.7% for eeev in unvaccinated horses. there was no evidence of mayv infections. the serologic evidence of circulation of arboviruses responsible for equine and human encephalitis, without recent official reports of clinical infections in the area, suggests that the nhecolandia sub-region in south pantanal is an important area for detection of silent activity of arboviruses in brazil.
Comparison of Argentinean Saint Louis Encephalitis Virus Non-Epidemic and Epidemic Strain Infections in an Avian Model  [PDF]
Luis Adrián Diaz ,Nicole M. Nemeth,Richard A. Bowen,Walter R. Almiron,Marta S. Contigiani
PLOS Neglected Tropical Diseases , 2011, DOI: 10.1371/journal.pntd.0001177
Abstract: St. Louis encephalitis virus (SLEV, Flavivirus, Flaviviridae) is an emerging mosquito-borne pathogen in South America, with human SLEV encephalitis cases reported in Argentina and Brazil. Genotype III strains of SLEV were isolated from Culex quinquefasciatus mosquitoes in Cordoba, Argentina in 2005, during the largest SLEV outbreak ever reported in South America. The present study tested the hypothesis that the recent, epidemic SLEV strain exhibits greater virulence in birds as compared with a non-epidemic genotype III strain isolated from mosquitoes in Santa Fe Province 27 years earlier. The observed differences in infection parameters between adult House sparrows (Passer domesticus) that were needle-inoculated with either the epidemic or historic SLEV strain were not statistically significant. However, only the House sparrows that were infected with the epidemic strain achieved infectious-level viremia titers sufficient to infect Cx. spp. mosquitoes vectors. Furthermore, the vertebrate reservoir competence index values indicated an approximately 3-fold increase in amplification potential of House sparrows infected with the epidemic strain when pre-existing flavivirus-reactive antibodies were present, suggesting the possibility that antibody-dependent enhancement may increase the risk of avian-amplified transmission of SLEV in South America.
Confirmation of Chlamydophila abortus in infected cell culture using Indirect Immunofluorescence technique
Sreeja R Nair,Mini M,Krishnan Nair G,Jayaprakasan V
Veterinary World , 2011,
Abstract: Chlamydophila abortus (C. abortus) is an important abortifacient agent in bovines and ovines. Clinical diagnosis of the disease is often difficult. An early diagnosis can be achieved based on direct demonstration of the organism in clinical material and through the cultural recovery of the organism in embryonated chicken egg. For confirmatory diagnosis antigen detection methods or serological techniques can be adopted. The present study is aimed at the confirmatory diagnosis of C. abortus infection by indirect immunofluorescence technique following the isolation of the organism in cell culture. Specific apple green fluorescing inclusions of C. abortus in McCoy cell lines was detected from 72 h to 96 h post infection employing anti-chlamydial group specific monoclonal antibodies. Thus, a confirmatory diagnosis of the infection was possible with this study. [Vet. World 2011; 4(10.000): 473-474]
Original Approach for Automated Quantification of Antinuclear Autoantibodies by Indirect Immunofluorescence  [PDF]
Daniel Bertin,Noémie Jourde-Chiche,Pierre Bongrand,Nathalie Bardin
Journal of Immunology Research , 2013, DOI: 10.1155/2013/182172
Abstract: 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 , ). 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. 1. Introduction Antinuclear antibodies (ANA) are essential biological markers for the diagnosis [1], classification, and disease activity monitoring [2] of systemic autoimmune rheumatic diseases. Given this central role, ANA screening should be accurate and reproducible. For several decades, indirect immunofluorescence (IIF) on HEp-2 cells has been the reference technique for ANA testing. Although new available techniques [3, 4] such as ELISA or multiplexing solid phase technologies have been proposed to replace IIF, the American College of Rheumatology (ACR) still recommends IIF as the gold standard method for ANA detection [5]. The main drawback of this technique is IIF reading subjectivity, intra- and interlaboratory variabilities complicating the standardization expected in modern laboratories. Recently, commercial automated systems for ANA IIF reading and interpretation have become available and were described in the literature [6–11]. Most of them are based on data mining and supervised machine learning methods [12]. In addition to their complexity, they share a common weakness in the detection of weak positivity. In this work, we describe an original algorithm named Immunofluorescence for Computed Antinuclear antibody Rational Evaluation (ICARE) for automation of IIF ANA evaluation offering excellent analytical performance and an attractive quantitative
A contribution to the diagnosis of Capillaria hepatica infection by indirect immunofluorescence test
Assis, Bárbara CA;Cunha, Liliane M;Baptista, Ana Paula;Andrade, Zilton A;
Memórias do Instituto Oswaldo Cruz , 2004, DOI: 10.1590/S0074-02762004000200010
Abstract: a highly specific pattern of immunofluorescence was noted when sera from capillaria hepatica-infected rats were tested against the homologous worms and eggs present either in paraffin or cryostat sections from mouse liver. the pattern was represented by a combined apple green fluorescence of the internal contents of worms and eggs, which persisted in serum-dilutions of 1:400 up to 1:1600. unequivocal fluorescent pattern was observed from 15 days up to 3 months following inoculation of rats with embryonated c. hepatica eggs and such result was confirmed by the elisa. after the 4th month of infection, the indirect immunofluorescence test turned negative, probably revealing the extinction of parasitism, however the elisa was contradictory, disclosing high levels of antibodies in this period . the iif was also negative when control normal rat sera and sera from rats administered by gavage with immature c. hepatica eggs (spurious infection), or for reactions made against schistosoma mansoni eggs, although a weakly positive pattern occurred with fasciola hepatica eggs. the indirect immunofluorescence test may be recommended for use with human sera to detect early c. hepatica infection in special clinical instances and in epidemiological surveys, since it is a simple, inexpensive, and reliable test, presenting excellent sensitivity and specificity. although the diagnosis is positive only during early infection, this is the period when the symptoms are usually more severe and the need for differential diagnosis is greater.
Evaluation of Serological Diagnostic Test Systems Assessing the Immune Response to Japanese Encephalitis Vaccination  [PDF]
Nadine Litzba ,Christoph S. Klade,Sabine Lederer,Matthias Niedrig
PLOS Neglected Tropical Diseases , 2010, DOI: 10.1371/journal.pntd.0000883
Abstract: A new commercial anti-Japanese encephalitis virus IgM and IgG indirect immunofluorescence test (IIFT) was evaluated for the detection of the humoral immune response after Japanese encephalitis vaccination. The IgM IIFT was compared to two IgM capture ELISAs and the IgG IIFT was analysed in comparison to a plaque reduction neutralization test (PRNT50) and an IgG ELISA. Moreover, the course of the immune reaction after vaccination with an inactivated JEV vaccine was examined. For the present study 300 serum samples from different blood withdrawals from 100 persons vaccinated against Japanese encephalitis were used. For the IgM evaluation, altogether 78 PRNT50 positive samples taken 7 to 56 days after vaccination and 78 PRNT50 negative sera were analyzed with the Euroimmun anti-JEV IgM IIFT, the Panbio Japanese Encephalitis – Dengue IgM Combo ELISA and the InBios JE Detect IgM capture ELISA. For the IgG evaluation, 100 sera taken 56 days after vaccination and 100 corresponding sera taken before vaccination were tested in the PRNT50, the Euroimmun anti-JEV IgG IIFT, and the InBios JE Detect IgG ELISA. The Euroimmun IgM IIFT showed in comparison to the Panbio ELISA a specificity of 95% and a sensitivity of 86%. With respect to the InBios ELISA, the values were 100% and 83.9%, respectively. The analysis of the Euroimmun IgG IIFT performance and the PRNT50 results demonstrated a specificity of 100% and a sensitivity of 93.8%, whereas it was not possible to detect more than 6.6% of the PRNT50 positive sera as positive with the InBios JE Detect IgG ELISA. Thus, the IIFT is a valuable alternative to the established methods in detecting anti-JEV antibodies after vaccination in travellers and it might prove useful for the diagnosis of acutely infected persons.
Limited reliability of the indirect immunofluorescence technique for the detection of anti-Rib-P antibodies
Michael Mahler, Jennifer T Ngo, Johannes Schulte-Pelkum, Tanja Luettich, Marvin J Fritzler
Arthritis Research & Therapy , 2008, DOI: 10.1186/ar2548
Abstract: Anti-ribosomal P-positive sera (n = 345) as detected by an addressable laser bead immunoassay were collected between 2003 and 2007 and analysed by indirect immunofluorescence. Furthermore, 51 anti-ribosomal P-positive samples from an unselected systemic lupus erythematosus cohort (n = 100) and the Centers for Disease Control and Prevention (CDC) anti-nuclear antibody (ANA) reference sera were tested for anti-ribosomal P reactivity.In the cohort of 345 anti-ribosomal P-positive samples identified by addressable laser bead immunoassay, a low sensitivity (<30%) of indirect immunofluorescence on HEp-2 cell substrates was observed. Although the degree of sensitivity varied among different manufacturers, all immunofluorescence substrates exhibited limited sensitivity and false-negative results were not restricted to samples with low anti-ribosomal P titers. Even the anti-ribosomal P reactivity of CDC ANA reference serum number 12 was not clearly predictable by indirect immunofluorescence. Comparison of five different methods for the detection of anti-ribosomal P found moderate qualitative agreements.Based on our data, we conclude that indirect immunofluorescence on HEp-2 cells is not a reliable screening test for the prediction of ribosomal P antibodies. As this method is widely used as a first-line screening test for anti-nuclear and other autoantibodies, special considerations for the detection of ribosomal P antibodies are needed. As with many other autoantibodies, further effort is required for the standardisation of ribosomal P immunoassays.Although more than 25 years have passed since their first description as a highly specific biomarker for systemic lupus erythematosus (SLE) [1], autoantibodies (aab) to the ribosomal P proteins (referred to as Rib-P) have not achieved the attention or clinical utility that anti-Sm, anti-dsDNA (anti-double-stranded DNA), or anti-cardiolipin antibodies have. This might be attributed to the limited reliability of indirect immunofluor
Quantile Representation for Indirect Immunofluorescence Image Classification  [PDF]
David M. J. Tax,Veronika Cheplygina,Marco Loog
Computer Science , 2014,
Abstract: In the diagnosis of autoimmune diseases, an important task is to classify images of slides containing several HEp-2 cells. All cells from one slide share the same label, and by classifying cells from one slide independently, some information on the global image quality and intensity is lost. Considering one whole slide as a collection (a bag) of feature vectors, however, poses the problem of how to handle this bag. A simple, and surprisingly effective, approach is to summarize the bag of feature vectors by a few quantile values per feature. This characterizes the full distribution of all instances, thereby assuming that all instances in a bag are informative. This representation is particularly useful when each bag contains many feature vectors, which is the case in the classification of the immunofluorescence images. Experiments on the classification of indirect immunofluorescence images show the usefulness of this approach.
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