Comparison of three different sensitive assays for hepatitis B virus DNA in monitoring of responses to antiviral therapy

The aim of our study was to compare the performances of two new hepatitis B virus (HBV) DNA assays, a cross-linking assay (NAXCOR) and a hybrid-capture amplification assay (Digene), versus the widely used branched-DNA (bDNA) assay (Chiron) in the monitoring of HBV DNA levels during antiviral treatment. Serial serum samples from 12 chronically HBV infected patients undergoing a phase II trial of an antiviral drug, 2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC), were studied. A total of 96 serum samples were tested for HBV DNA using the cross-linking, hybrid-capture amplification, and bDNA assays. In the comparison of the cross-linking and bDNA assays, concordant results were found in 77 (80.3%) samples, no significant difference was found between the median log(10) HBV DNA levels (6.66 versus 7. 17 meq/ml), and the results of the two assays were closely correlated (r = 0.95). In the comparison of the hybrid-capture amplification and bDNA assays, concordant results were found in 79 (82.3%) samples, no significant difference was found between the median log(10) HBV DNA levels (6.98 versus 6.99 meq/ml), and the results of the two assays were closely correlated (r = 0.99). Six (6. 3%) samples by the cross-linking assay and 10 (10.4%) samples by the bDNA assay required retesting because of unacceptably high within-run coefficients of variance. No sample required retesting in the hybrid-capture amplification assay according to the internal validation. In conclusion, the cross-linking and hybrid-capture amplification assays were as sensitive as the bDNA assay for HBV DNA detection and can be recommended for monitoring of HBV DNA levels during antiviral treatment.     

Detection of highly pathogenic and low pathogenic avian influenza subtype H5 (Eurasian lineage) using NASBA

Nucleic acid sequence-based amplification (NASBA) is a technique that allows the rapid amplification of specific regions of nucleic acid obtained from a diverse range of sources. It is especially suitable for amplifying RNA sequences. A NASBA technique has been developed that allows the detection of avian influenza A subtype H5 from allantoic fluid harvested from inoculated chick embryos. The amplified viral RNA is detected by electrochemiluminescence. The NASBA technique described below is rapid and specific for the identification of influenza A subtype H5 viruses of the Eurasian lineage. More importantly, it can be used to distinguish highly pathogenic and low pathogenic strains of the H5 subtype.     

Identification of semen in 500 patients seen because of rape

Of 500 patients seen because of rape, semen was identified in vaginal secretions by the identification of spermatozoa in 61%, by an acid phosphatase value of 50 units or more in 40%, and by the identification of a foreign blood group substance or a high titer of own blood group substance in 16%. The addition of the determination of the acid phosphatase to the search for spermatozoa identified semen in only 1.4% more patients, or a total of 62.4%. Identification and titers of blood group substance were confirmatory only, but further characterized the source of the semen in 25% of those patients with spermatozoa. Spermatozoa were identified for as long as 48 hours, and elevated acid phosphatase was not found after 18 hours. Acid phosphatase was elevated in only 62% of patients with spermatozoa.     

Impaired release of a T-cell specific suppressor factor in rheumatoid arthritis.

Peripheral blood T cells from patients with rheumatoid arthritis (RA) and scleroderma (PSS) were assessed for their ability to release T-cell-specific suppressor activity (TRSA) upon incubation with a suppressor activating factor (SAF) derived from a human lymphoblastoid cell line (CEM). T cells from 11/20 (55%) RA patients exhibited impaired TRSA release in contrast to 1/12 (8%) of PSS patients. RA patients demonstrating impaired TRSA release exhibited more active arthritis than patients demonstrating normal TRSA release.     

Comparison of different PCR approaches for characterization of Burkholderia (Pseudomonas) cepacia isolates.

In this study, we evaluated three PCR methods for epidemiological typing of Burkholderia (Pseudomonas) cepacia--PCR-ribotyping, arbitrarily primed PCR (AP-PCR) and enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR)--and compared them with pulsed-field gel electrophoresis. The analysis was performed with 31 isolates of B. cepacia, comprising 23 epidemiologically unrelated isolates and 8 isolates collected from the same patient during two episodes of bacteremia. Pulsed-field gel electrophoresis, ERIC-PCR, and AP-PCR identified 23 distinct types among the 23 unrelated isolates, while PCR-ribotyping only identified 12 strain types, even after AluI digestion of the amplification products. Among the eight isolates collected from the same patient, all typing techniques revealed two clones of strains. The day-to-day reproducibilities of PCR-ribotyping and ERIC-PCR were good, while greater day-to-day variations were noted in the fingerprints obtained by AP-PCR. We conclude that all three PCR techniques are useful for rapid epidemiological typing of B. cepacia, but ERIC-PCR seems to be more reproducible and discriminative.     

Effects of acrivastine,azelastine, cetirizine and noberastine on rat peritoneal mast cells

Inflammation Research -     

Intracellular T lymphocyte cytokine profiles in the aqueous humour of patients with uveitis and correlation with clinical phenotype

This study aimed to investigate whether T cells in aqueous humour are different in different types of uveitis and correlate with clinical phenotype. Patients with clinically different types of uveitis, but all displaying active anterior uveitis, were phenotyped and samples of aqueous humour (AH) and peripheral blood (PB) collected. Cells from AH and PB were separated by centrifugation and by density gradient centrifugation (to obtain mononuclear cells PBMC), respectively. Cells were activated with PMA and ionomycin in the presence of Brefeldin A, stained for surface markers and intracellular cytokines, and analysed by flow cytometry. The cytokine profile was correlated with the clinical phenotype. Increased percentages of interleukin (IL)-10+-, but not interferon (IFN)-gamma+ T lymphocytes were found in AH compared with PB in patients with acute anterior uveitis (AAU), FHC or chronic panuveitis (PU). There was a trend towards elevated levels of IL-10+ T cells in AH from patients with FHC compared with AH from acute uveitis and panuveitis patients. Increased levels of IL-10+ T cells in AH compared with PB were also found in samples from patients with isolated uveitis, but not those with associated systemic disease. Levels of cytokine-positive T cells were not associated with the use of topical steroids or to the severity of the anterior uveitis. While type I cytokine-producing T lymphocytes are present in AH during AU, the presence of increased proportions of IL-10+ T lymphocytes in AH from patients with uveitis may be indicative of an anti-inflammatory mechanism that may influence the type and course of ocular inflammation in these patients.     

Internet teleconferencing method for telepathology consultations from lung and heart transplant patients

Current Internet-based teleconferencing techniques allow a referring pathologist to transmit real-time images from a microscope to a consultant, while maintaining a verbal conversation using Internet telephony. In our study, 50 randomly selected transbronchial biopsies from lung allograft recipients and 58 randomly selected endomyocardial biopsies from heart transplant patients were diagnosed by consultant pathologists using Internet-based teleconferencing methods. The referring pathologists acquired the real-time video images from the biopsies using a light microscope equipped with a phototube adapter and a video camera. The consultant pathologists viewed the processed images on a video monitor at 800 x 600 resolution, using a standard microcomputer equipped with Netmeeting software, and directed the referring pathologist to move the slide under the microscopy and/or change image magnification. The validity of telepathology diagnoses was assessed with kappa coefficients. Consultations were completed in 5 to 15 minutes per case. Sound transmission was unreliable, and in approximately 25% of consultations the referring pathologist needed to "call back" to reestablish verbal communication. In all but 2 transbronchial biopsies there was agreement between the original diagnosis and the diagnosis by telepathology (kappa = 0.92). In 48 of 58 endomyocardial biopsies there was concordance between the 2 diagnoses (kappa = 0.692). Only 3 out of 10 of these discrepancies were clinically significant (kappa = 0.897). Internet-based teleconferencing techniques provide effective and relatively inexpensive tools for real time telepathology consultations. The technology is probably best suited for the study of small specimens from patients that require rapid diagnosis by a consultant.     

Differential sensitivities of severe acute respiratory syndrome (SARS) coronavirus spike polypeptide enzyme-linked immunosorbent assay (ELISA) and SARS coronavirus nucleocapsid protein ELISA for serodiagnosis of SARS coronavirus pneumonia

The use of recombinant severe acute respiratory syndrome-coronavirus (SARS-CoV) nucleocapsid protein (N) enzyme-linked immunosorbent assay (ELISA)-based antibody and antigen tests for diagnosis of SARS-CoV infections have been widely reported. However, no recombinant SARS-CoV spike protein (S)-based ELISA is currently available. In this article, we describe the problems and solutions of setting up the recombinant SARS-CoV S-based ELISA for antibody detection. The SARS-CoV S-based immunoglobulin M (IgM) and IgG ELISAs were evaluated and compared with the corresponding N-based ELISA for serodiagnosis of SARS-CoV pneumonia, using sera from 148 healthy blood donors who donated blood 3 years ago as controls and 95 SARS-CoV pneumonia patients in Hong Kong. Results obtained by the recombinant S (rS)-based IgG ELISA using the regenerated S prepared by dialysis with decreasing concentrations of urea or direct addition of different coating buffers, followed by addition of different regeneration buffer, identified 4 M urea and 1 M sarcosine for plate coating and no regeneration buffer as the most optimal conditions for antibody detection. The specificities of the S-based ELISA for IgG and IgM detection were 98.6% and 93.9%, with corresponding sensitivities of 58.9% and 74.7%, respectively. The sensitivity of the rN IgG ELISA (94.7%) is significantly higher than that of the rS IgG ELISA (P < 0.001), whereas the sensitivity of the rS IgM ELISA is significantly higher than that of the rN IgM ELISA (55.2%) (P < 0.01). An ELISA for detection of IgM against S and N could be more sensitive than one that detects IgM against N alone for serodiagnosis of SARS-CoV pneumonia.