Comparison of single and dual latent tuberculosis screening strategies before biologic and targeted therapy in patients with rheumatic diseases: a retrospective cohort study.

PURPOSE: Before biologic and targeted synthetic disease-modifying antirheumatic drug (b/tsDMARD) treatment, latent tuberculosis infection (LTBI) screening by tuberculin skin test (TST) or interferon gamma release assay (IGRA) is recommended. However, both tests have reduced reliability in immunosuppressed patients. We investigated whether dual LTBI screening with both tests could reduce the incidence of tuberculosis. METHODS: Consecutive patients receiving b/tsDMARDs for rheumatic diseases in a regional hospital were recruited. All patients underwent either TST/IGRA or both. They were categorised into a single or dual testing group and were followed up for at least 6 months. Isoniazid was prescribed if any one test was positive. RESULTS: In total, 217 patients were included in this study; 121 underwent single LTBI testing and 96 underwent dual testing. Tuberculosis occurred in nine patients in the single testing group and one patient in the dual testing group (7.4% vs 1.0%, P=0.045). However, the difference was not statistically significant when follow-up duration was considered (log rank test). In total, 71 patients tested positive for LTBI with isoniazid treatment (28.9% in the single testing group and 45.8% in the dual testing group, P=0.007). Agreement between the IGRA and TST was 74.4% (Cohen's kappa=0.413); agreement was lower in patients receiving prednisolone. Infliximab use was independently associated with tuberculosis (P=0.032). Mild isoniazid-related side-effects occurred in seven patients. CONCLUSIONS: Dual LTBI testing with both TST and IGRA is effective and safe. It might be useful for patients receiving prednisolone at the time of LTBI screening, or if infliximab therapy is anticipated.

A bioequivalence study comparing two formulations of lopinavir/ritonavir capsules.

This investigation was carried out to evaluate the bioavailability of a new single fixed-dose combination formulation of lopinavir and ritonavir, relative to reference product, Kaletra (133.3 mg lopinavir/33.3 mg ritonavir) capsules, manufactured by Abbott Laboratories, Chicago, IL, USA. The bioavailability study was carried out on 72 healthy male and female volunteers who received a single dose of 3 capsules (133.3 mg lopinavir/33.3 mg ritonavir) of the test (T) and the reference (R) products in the fasting state, in a randomized, balanced, 2-way crossover design. After dosing, serial blood samples were collected for a period of 72 hours. Plasma harvested from blood was analyzed for lopinavir and ritonavir by a sensitive and validated simultaneous liquid-chromatographic and mass-spectrometric (LC-MS/MS) assay. Mean oral clearance (Cl/F) values of the FDC were 4.92 and 23.54 l/h for lopinavir and ritonavir, respectively, the maximum plasma concentrations (C(max)), area under the plasma concentration-time curve up to the last measurable concentration (AUC(0-t)), and to infinity (AUC(0-infinity)), were analyzed statistically under the assumption of a multiplicative model. The time to maximum concentration (t(max)) was analyzed assuming an additive model. The parametric confidence intervals (90%) were calculated by Schuirmann's two 1-sided t-test criteria. It was found that the test/reference (T/R) ratios for the pharmacokinetic parameters AUC(0-t), AUC(0-infinity) and C(max) (after initial log transformation) were well within the bioequivalence acceptance range of 80-125% as per international regulatory guidelines. Therefore, the two formulations were considered to be bioequivalent [Food and Drug Administration 2003].

High-throughput, fluorescence-based screening for soluble protein expression.

Protein expression screening methods are essential for proteomic scale characterization of gene and cDNA expression libraries. Screening methods are also important for the identification of highly expressed protein targets, for example, in quantities suitable for high-throughput screening and protein structural studies. To address these needs, we describe the implementation of several rapid, fluorescence-based protein expression screening strategies using Escherichia coli or E. coli-based in vitro transcription/translation (IVT) systems. In vitro expression screening is fast, convenient and, as we show, correlates well with in vivo expression. For screening, expressed proteins are labeled either as fusions with green fluorescent protein (GFP) or through translational incorporation of a fluorescent amino acid derivative, BODIPY-FL-Lysine. Fluorescence-based detection of GFP fusions or BODIPY-labeled proteins is considerably faster than other common expression screening methods, such as immunological detection of gels or dot blots. Furthermore, in vitro and in vivo screening used together yield a larger set of expressed proteins than either method alone. Specifically labeled proteins in cellular lysates are detected in one of three formats: a microplate using a fluorescence plate reader, a dot-blot using a fluorescence scanner or a microarray using a laser scanner. We have established a correlation among the various detection formats, which validates the use of protein microarrays for expression screening. Production of expressed proteins detected through screening can be scaled up either using IVT reactions or with in vivo expression systems in the absence of a fluorophore for subsequent characterization of protein function or interactions.

Cell mediated immunity to herpes simplex in man. IV. A correlation of lymphocyte stimulation and inhibition of leukocyte migration.

The cell mediated immune response to herpes simplex virus was assessed by a modification of the leukocyte migration inhibition test, developed to increase its sensitivity and correlated with the in vitro stimulation of lymphocytes. The introduction of a preincubation step allowed us to demonstrate significant inhibition of leukocyte migration in 15 out of 24 subjects susceptible to recurrent herpes labialis. This was not present in any of the 10 subjects without such infections. However lymphocytes from all 24 subjects demonstrated stimulation in response to herpes virus antigen. The degrees of lymphocyte stimulation and migration inhibition in the susceptible subjects were well correlated and do not support the suggestion that there is a focal defect in this aspect of the cell mediated immune response to herpes simplex virus in those patients.