High accuracy and precision at the lower end of quantification are crucial requirements of a modern HIV viral load (VL) assay, since some clinically relevant thresholds are located at 50 and 200 copies/ml. In this study, we compared the performance of two new fully automated HIV-1 VL assays, Aptima HIV-1 Quant Dx and Cobas HIV-1 (Cobas 6800), with the established RealTime m2000 assay.

In the range of clinical decision points for response-guided therapy of HCV, there is still insufficient data concerning the conformity of quantification results obtained by different assays and their correlation with the HPS/CTM v2 assay which was used for initial clinical studies. In a head-to-head comparison, assay accuracy and detection rates of six quantitative assays [artus HCV QS-RGQ, COBAS Ampliprep/COBAS TaqMan HCV v1/v2, High Pure System/COBAS TaqMan (HPS), RealTime HCV, and Versant HCV1.0] were assessed by measuring WHO and PEI standards at dilution steps near clinical decision points. Detection rates and mean differences between assays were evaluated by analyzing twenty clinical samples at 10, 100, and 1,000 IU/mL. Ten replicates from specimens with different HCV genotypes were used to analyze pan-genotypic intra-assay variation. At ≤25 IU/mL, RealTime demonstrated the highest detection rates. With 0.1 log difference when testing clinical samples, results obtained from the Versant and RealTime assays matched best with results from HPS. Mean difference analysis across all assay results revealed wide differences between 0.01 and 0.75 log IU/mL. RealTime showed the lowest intra-assay variation across genotypes 1–4 (25, 100, 1,000 IU/mL). There are substantial analytical differences between viral load assays clinicians should be aware of. These variations may have impact on clinical decisions for patients on HCV triple therapy and may argue for assay-specific decision points equivalent to reference values established in studies using HPS. A comparison of quantification is recommended prior to a switch of assays during ongoing therapy.

Proinflammatory cytokines such as tumor necrosis factor (TNF), 2 interleukin-1␤ (IL-1␤), or interleukin-6 (IL-6) have been identified as promising therapeutic targets in the treatment of chronic inflammation. A dimeric soluble TNF receptor is currently used for the treatment of inflammatory diseases caused by elevated TNF expression (1). Whereas TNF signals through a receptor homotrimer, most cytokines signal through receptor complexes consisting of two or more different receptor subunits. In this case, the respective cytokine can be inhibited by using fusion proteins composed of the different soluble receptors, as we and others showed for the inhibition of IL-6 (2-4).All cytokines signaling through the common receptor subunit gp130 belong to the family of IL-6-type cytokines (5), which includes IL-6, IL-11, IL-27, LIF, OSM, ciliary neurotrophic factor, cardiotrophin-1, cardiotophin-like cytokine, and neuropoietin. IL-6-type cytokines contain distinct receptorbinding sites that were discovered by mutagenesis studies on IL-6, ciliary neurotrophic factor, and LIF (6 -8). The IL-6-type cytokines can be subdivided into those containing three (I, II, and III) or two (II and III) receptor-binding sites. Site I determines the specificity of ␣-receptor binding. The ␣-receptor is not capable of transferring the signal into the cell but is crucial for increasing the binding affinity of the cytokine to its signaling receptors. Site II seems to be the universal gp130-binding site of all IL-6-type cytokines. Depending on the cytokine, site III is used for the recruitment of LIFR, OSMR, or a second gp130 molecule (5). The IL-6 inhibitor IL-6-RFP (2, 3) was designed to block a cytokine containing all three receptor-binding sites.However, there are also IL-6-type cytokines, which do not need to recruit an ␣-receptor analogous to human IL-6R␣, and thus do not seem to have a functional site I. One example for a cytokine belonging to this group is the leukemia inhibitory factor (LIF). In this study we present an approach to construct inhibitory receptor fusion proteins for human and murine LIF as prototypes of inhibitors targeting cytokines whose receptors only bind to the site II and III of the cytokine without occupying site I. We designate these inhibitors "site II/III inhibitors."LIF signals through a heterodimer of LIFR and gp130. Janus tyrosine kinases that are constitutively associated with the cytoplasmic parts of gp130 (9) and LIFR (10) are activated upon ligand binding and phosphorylate the receptors and the recruited transcription factor STAT3. Activated STAT3 dimerizes and translocates into the nucleus, where it induces LIF target genes (11).We wanted to integrate only those receptor domains of gp130 and LIFR into the inhibitory receptor fusion proteins that are necessary for LIF binding. For gp130, which includes six extracellular domains (D1-D6), it has been clearly shown that domains D2 and D3 forming the cytokine-binding module (CBM) are necessary and sufficient for LIF binding (12). In contrast, there are contradi...

BackgroundAlthough being considered as a rarely observed HIV-1 protease mutation in clinical isolates, the L76V-prevalence increased 1998-2008 in some European countries most likely due to the approval of Lopinavir, Amprenavir and Darunavir which can select L76V. Beside an enhancement of resistance, L76V is also discussed to confer hypersusceptibility to the drugs Atazanavir and Saquinavir which might enable new treatment strategies by trying to take advantage of particular mutations.ResultsBased on a cohort of 47 L76V-positive patients, we examined if there might exist a clinical advantage for L76V-positive patients concerning long-term success of PI-containing regimens in patients with limited therapy options.Genotypic- and phenotypic HIV-resistance tests from 47 mostly multi-resistant, L76V-positive patients throughout Germany were accomplished retrospectively 1999-2009. Five genotype-based drug-susceptibility predictions received from online interpretation-tools for Atazanavir, Saquinavir, Amprenavir and Lopinavir, were compared to phenotype-based predictions that were determined by using a recombinant virus assay along with a Virtual Phenotype™(Virco). The clinical outcome of the L76V-adapted follow-up therapy was determined by monitoring viral load for 96 weeks.ConclusionsIn this analysis, the mostly used interpretation systems overestimated the L76V-mutation concerning Atazanavir- and SQV resistance. In fact, a clear benefit in drug susceptibility for these drugs was observed in phenotype analysis after establishment of L76V. More importantly, long-term therapy success was significantly higher in patients receiving Atazanavir and/or Saquinavir plus one L76V-selecting drug compared to patients without L76V-selecting agents (p = 0.002).In case of L76V-occurrence ATV and/or SQV may represent encouraging options for patients in deep salvage situations.

ObjectivesAutomated molecular analyzers have accelerated diagnosis, allowing earlier intervention and better patient follow-up. A recently developed completely automated molecular analyzer, Alinity™ m (Abbott), offers consolidated, continuous, and random-access testing that may improve molecular laboratory workflow.MethodsAn international, multicenter study compared laboratory workflow metrics across various routine analyzers and Alinity m utilizing assays for human immunodeficiency virus type 1 (HIV-1), hepatitis C virus (HCV), hepatitis B virus (HBV), high-risk human papillomavirus (HR HPV), and sexually transmitted infection (STI) (Chlamydia trachomatis [CT]/Neisseria gonorrhoeae [NG]/Trichomonas vaginalis [TV]/Mycoplasma genitalium [MG]). Three turnaround times (TATs) were assessed: total TAT (sample arrival to result), sample onboard TAT (sample loading and test starting to result), and processing TAT (sample aspiration to result).ResultsTotal TAT was reduced from days with routine analyzers to hours with Alinity m, independent of requested assays. Sample onboard TATs for standard workflow using routine analyzers ranged from 7 to 32.5 h compared to 2.75–6 h for Alinity m. The mean sample onboard TAT for STAT samples on Alinity m was 2.36 h (±0.19 h). Processing TATs for Alinity m were independent of the combination of assays, with 100% of results reported within 117 min.ConclusionsThe consolidated, continuous, random-access workflow of Alinity m reduces TATs across various assays and is expected to improve both laboratory operational efficiency and patient care.