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#29053403   2017/10/20 Save this To Up

Human Cytomegalovirus vaccine development: Immune responses to look into vaccine strategy.

Human cytomegalovirus (HCMV) causes considerable morbidity and disability in high risk, immunocompromised populations including recipients of solid organ transplants, and fetuses whose immune systems are not yet mature. Vaccines aimed at ameliorating the severity of disease and preventing HCMV infection can be categorized into two main approaches of vaccine design, with one focusing on virus modification and the other on individual antigens. However, no candidates in either class have been successful in achieving durable and protective immunity. Recent studies on the natural immune response provide new insight into HCMV vaccine strategy. In particular, studies have demonstrated that the incorporation of a pentameric complex is necessary for a vaccine to generate the potent neutralizing antibodies often seen in seropositive individuals. This review summarizes recent findings in the development of HCMV vaccines and key considerations that should be taken into vaccine design based on improved understanding of natural HCMV immunity.

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#29053397   2017/10/20 Save this To Up

Combined Intravitreal and Systemic Antibiotic Therapy in a Patient with Syphilitic Uveitis.

To report the novel use of combined intravitreal and systemic antibiotic therapy in a patient with syphilitic panuveitis and discuss the management of ocular syphilis.

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#29046410   2017/10/19 Save this To Up

Correlation of Treponemal Immunoassay Signal Strength Values With Reactivity of Confirmatory Treponemal Testing.

Automated treponemal immunoassays are used for syphilis screening with the reverse sequence algorithm; discordant results (e.g., enzyme immunoassay [EIA]-reactive, reactive plasma reagin [RPR]-non-reactive) are resolved with a second treponemal test. We conducted a study to determine automated immunoassay signal strength values consistently correlating with reactive confirmatory treponemal testing.We conducted a cross-sectional analysis of four automated immunoassays: BioPlex 2200 microbead immunoassay (MBIA), LIAISON chemiluminesence immunoassay (CIA), ADVIA-Centaur CIA, and TrepSure EIA, and three manual assays: Treponema Pallidum Particle Agglutination (TP-PA), Fluorescent Treponemal Antibody-Absorption (FTA-ABS) test, INNO-LIA line immunoassay. We compared signal strength values of automated immunoassays and positive and negative agreement. Among 1995 specimens, 908 (45.5%) were true positives (≥4/7 tests reactive) and 1087 (54.5%) were true negatives (≥4/7 tests non-reactive). Positive agreement ranged from 86.1% (83.7-88.2%) for FTA-ABS to 99.7% (99.0-99.9%) for ADVIA-Centaur CIA; negative agreement ranged from 86.3% (84.1-88.2%) for TrepSure EIA to 100% for TP-PA (99.6-100%). Increasing signal strength values correlated with increasing reactivity of confirmatory testing (ptrend <0.0001 for all automated immunoassay). All automated immunoassays had signal strength cutoffs corresponding to ≥4/7 reactive treponemal tests. Bioplex MBIA and LIAISON CIA had signal strength cutoffs correlating with ≥99% and 100% TP-PA reactivity, respectively. ADVIA-Centaur CIA and TrepSure EIA had signal strength cutoffs correlating with at least 95%TP-PA reactivity. All automated immunoassays had signal strength cutoffs correlating with at least 95% FTA-ABS reactivity. Assuming that a 95% level of confirmation is adequate, these signal strength values can be used in lieu of confirmatory testing with TP-PA and FTA-ABS.

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#29039100   2017/10/17 Save this To Up

Analysis of Complement Activation by Nanoparticles.

The complement system is a group of proteins, which function in plasma to assist the innate immunity in rapid clearance of pathogens. The complement system also contributes to coordination of the adaptive immune response. Complement Activation Related Pseudo Allergy or CARPA is a life-threatening condition commonly reported with certain types of drugs and nanotechnology-based combination products. While CARPA symptoms are similar to that of anaphylaxis, the mechanism behind this pathology does not involve IgE and is mediated by the complement system. In vitro assays using serum or plasma derived from healthy donor volunteers correlate with the in vivo complement-mediated reactions, and therefore are helpful in understanding the propensity of a given drug formulation to cause CARPA in patients. In the first edition of this book, we have described an in vitro method for qualitative assessment of the complement activation by nanomaterials using western blotting. Herein, we present a similar method utilizing enzyme-linked immunoassay for quantitative analysis of the complement activation, and we compare the performance of this approach to that of the qualitative western blotting technique. The revised chapter also includes new details about nanoparticle sample preparation.

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#29031156   2017/10/14 Save this To Up

HIV misdiagnosis: A root cause analysis leading to improvements in HIV diagnosis and patient care.

Standard diagnostic testing for HIV infection has traditionally relied on a high sensitivity HIV antibody screening test using an enzyme-linked immunosorbent assay (ELISA) followed by a high specificity antibody confirmatory test such as a Western Blot. Recently several of the screening assays have been enhanced with an ability to identify p24 antigen thereby narrowing the diagnostic window.

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#29020213   2017/10/11 Save this To Up

Improvement in diagnosis of Histoplasma meningitis by combined testing for Histoplasma antigen and IgG and IgM anti-Histoplasma antibody in cerebrospinal fluid.

Central nervous system (CNS) histoplasmosis is a life-threatening condition and represents a diagnostic and therapeutic challenge. Isolation of H. capsulatum from cerebrospinal fluid (CSF) or brain tissue is diagnostic; however, culture is insensitive and slow growth may result in significant treatment delay. We performed a retrospective multicenter study to evaluate the sensitivity and specificity of a new anti-Histoplasma antibody enzyme immunoassay (EIA) for the detection of IgG and IgM antibody in the CSF for diagnosis of CNS histoplasmosis, the primary objective of the study. The secondary objective was to determine the effect of improvements in the Histoplasma galactomannan antigen detection EIA on the diagnosis of Histoplasma meningitis.

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#29020212   2017/10/11 Save this To Up

Screening Veterans for Syphilis: Implementation of the Reverse Sequence Algorithm.

We evaluated the syphilis reverse sequence algorithm (RSA) in a Veteran Affairs facility, finding 5.5% reactive Treponema pallidum enzyme immunoassay (EIA) tests. In a subset of EIA+/VDRL-/TP-PA+ cases, 48% were previously treated. Of veterans with unknown/no prior therapy, only 45% had documentation of subsequent treatment, suggesting suboptimal interpretation of RSA results.

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#28986292   2017/10/07 Save this To Up

Validation of an indirect ELISA employing a chimeric recombinant gag and env peptide for the serological diagnosis of equine infectious anemia.

The National Reference Center for equine infectious anemia (EIA) validated a commercial ELISA (Eradikit(®) EIAV Indirect ELISA, In3diagnostic(®), Turin, Italy) employing a chimeric recombinant gag and env peptide for the detection of EIA virus antibodies, following the guidelines of the World Organization for Animal Health. The validation parameters evaluated were: analytical sensitivity (Se) and specificity (Sp); diagnostic Se and Sp; precision, based on repeatability and reproducibility through the estimation of the standard deviation (SD) and the coefficient of variation (CV); accuracy, estimated from a multiple K and relative Sp and Se with respect to those of the agar gel immunodiffusion test (AGIDT). Positive and negative predictive values were also defined. The assay showed a high specificity and a limit of detection of 1.43 log10 major than AGIDT. Diagnostic Se was 100% and Sp was 99.3%, while SD values ranged from 1.58 to 5.01 with a CV between 2.8% and 28.8%. Multiple K was 0.98 and relative Se and Sp were respectively 99.1% and 100%. The assay proved to be robust and to possess a high sensitivity in detecting first antibodies produced at onset of infection as well as high analytic and diagnostic Se and Sp values, confirming it as a serological assay fit for purpose within EIA surveillance programs.

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#28983237   2017/10/06 Save this To Up

Validation of a Commercially Available Enzyme ImmunoAssay for the Determination of Oxytocin in Plasma Samples from Seven Domestic Animal Species.

The neurohormone oxytocin (OT) has a broad range of behavioral effects in mammals. It modulates a multitude of social behaviors, e.g., affiliative and sexual interactions. Consequently, the OT role in various animal species is increasingly explored. However, several issues have been raised regarding the peripheral OT measurement. Indeed, various methods have been described, leading to assay discrepancies and inconsistent results. This highlights the need for a recognized and reliable method to measure peripheral OT. Our aim was to validate a method combining a pre-extraction step, previously demonstrated as essential by several authors, and a commercially available enzyme immunoassay (EIA) for OT measurement, using plasma from seven domestic species (cat, dog, horse, cow, pig, sheep, and goat). The Oxytocin EIA kit (EnzoLifeSciences) was used to assay the solid-phase extracted samples following the manufacturer's instructions with slight modifications. For all species except dogs and cats, concentration factors were applied to work above the kit's sensitivity (15 pg/ml). To validate the method, the following performance characteristics were evaluated using Validation Samples (VS) at various concentrations in each species: extraction efficiency via spiking tests and intra- and inter-assay precision, allowing for the calculation of total errors. Parallelism studies to assess matrix effects could not be performed because of too low basal concentrations. Quantification ranges and associated precision profiles were established to account for the various OT plasma concentrations in each species. According to guidelines for bioanalytical validation of immunoassays, the measurements were sufficiently precise and accurate in each species to achieve a total error ≤30% in each VS sample. In each species, the inter-assay precision after 3 runs was acceptable, except in low concentration samples. The linearity under dilution of dogs and cats' samples was verified. Although matrix effects assessments are lacking, our results indicate that OT plasma levels can reliably be measured in several domestic animal species by the method described here. Studies involving samples with low OT plasma concentrations should pay attention to reproducibility issues. This work opens new perspectives to reliably study peripheral OT in a substantial number of domestic animal species in various behavioral contexts.

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#28970055   2017/10/03 Save this To Up

A two-step approach improves the diagnosis of Clostridium difficile infection.

Clostridium difficile infection (CDI) is a major cause of health care-associated diarrhea. The aim of the present study was to evaluate a two-step approach for the diagnosis of CDI. The two-step procedure consisted of GDH-toxin A/B EIA (Enzyme immunoassay targeting enterotoxin A and Cytotoxin B), followed by PCR detecting toxigenic C. difficile. Results indicate that EIAs provide a rapid screening assay for the laboratory diagnosis of CDI but, in GDH-positive and toxins-negative samples, EIA should be always followed by PCR to distinguish toxigenic vs nontoxigenic strains. GDH-toxin A/B EIA-rapid test has high specificity but low sensitivity to detect CDI. The implementation of a two-step procedure significantly increases the diagnostic accuracy to detect CDI and provides a toxigenic type characterization of C. difficile isolates.

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