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Background: HIV-1 RNA quantification is a key component of treatment monitoring., Objectives: To assess the performance of a redesigned HIV-1 RNA quantitative assay that uses a dual-target approach: Xpert® HIV-1 Viral Load (VL) XC., Study Design: Fresh and frozen samples (N = 533) from HIV-1 positive patients tested with Abbott HIV-1 assays (Alinity m and RealTime [m2000]) were retested using the new Xpert XC assay. Three samples with known underquantification using the previous single-target Xpert assay were retested., Results: The Xpert XC assay yielded valid results in 98.5% (N = 528/536) of cases and showed high sensitivity in 80 fresh samples that had undetectable VLs or ≤1.7 log copies/mL with Alinity m. Linear regression and Bland-Altman analyses showed high concordance with the Abbott tests for quantified samples over a wide VL range (1.6-6.9 copies/mL), including non-B subtypes (mean difference=-0.1±0.23 log copies/mL). Mutations associated with integrase resistance did not impact the results. Very good linearity and reproducibility was shown for the tested subtypes B, CRF06_cpx, and CRF02_AG. Xpert XC VLs in samples that were previously underquantified using the original single-target Xpert assay were similar to those detected by the Abbott assays (±0.11 log copies/mL)., Conclusions: The Xpert XC assay showed excellent correlation with the Abbott assays for all tested HIV-1 subtypes. Sensitivity, linearity and accuracy were high in the therapeutically relevant VL range. With a time to result of only 90 min, this on-demand decentralized assay is a safe, reliable and fast option for VL monitoring in HIV-1-infected patients., (Copyright © 2022. Published by Elsevier B.V.)

Background: Detection of SARS-CoV-2 infections relies on the use of sensitive, accurate and high throughput RT-PCR assays., Objectives: We assessed the analytical performance of the Abbott RealTime SARS-CoV-2 (RT-SARS), Alinity m SARS-CoV-2 (AlinSARS) assays and compared the clinical performance of the RT-SARS, AlinSARS, and Alinity m Resp-4-Plex (Alin4Plex) assays to the Seegene Allplex assay (Allplex) and an inhouse test (Inhouse)., Results: We found 100 % positive percent agreement (PPA) and 100 % negative percent agreement (NPA) comparing RT-SARS and Allplex. RT-SARS, AlinSARS and Inhouse showed 100 % NPA and 100 % PPA across all assays, except for the RdRp target of Inhouse (PPA = 84 %). Similarly, Alin4Plex and Allplex showed high agreement with specimens containing either SARS-CoV-2, influenza A, influenza B, or RSV. Detection rates of 100 % for SARS-CoV-2 at 50 copies/mL, high precision, and no cross-reactivity with non-SARS-CoV-2 respiratory pathogens were observed for RT-SARS and AlinSARS. AlinSARS detected SARS-CoV-2 in spiked throat washes and in specimens infected with SARS-CoV-2 Alpha or Beta variants., Conclusions: The newly developed RT-SARS, AlinSARS, and Alin4Plex assays proved to be useful for detecting SARS-CoV-2 RNA in clinical samples., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

HIV-1 diversity poses major challenges to viral load assays because genetic polymorphisms can impede nucleic acid detection. In addition to the on-going viral diversification within the HIV-1 group M pandemic, HIV-1 genetic diversity is further increased by non-group M infections, such as HIV-1 groups O (HIV-1-O), N and P. We here conducted a systematic evaluation of commercially available PCR assays to detect HIV-1-O isolates. We collected 25 primary HIV-1-O isolates covering all genetic clusters within HIV-1-O. Subsequently, this panel of isolates was tested on eight commercially available quantitative and five qualitative HIV-1 PCR-based assays in serial dilutions. Sequence analyses were performed for severe cases of underquantification or lack of detection. We observed differences between the assays in quantification that depended on the HIV-1-O isolate's subgroup. All three tested HIV-1-O subgroup IV isolates were underquantified by the Roche CAP/CTM >800-fold compared to the Abbott RealTime assay. In contrast, the latter assay underquantified several subgroup I isolates >200-fold. Notably, the Xpert HIV-1 Viral Load test from Cepheid failed to detect two of the HIV-1-O isolates, whereas the Roche Cobas 8800 assay readily detected all isolates. Comparative sequence analyses identified polymorphisms in the HIV-1-O long-terminal repeat and integrase genes that likely underlie inadequate nucleic acid amplification. Potential viral load underquantification should be considered in therapeutic monitoring of HIV-1-O-infected patients. Pre-clinical assessments of HIV-1 diagnostic assays could be harmonized by establishing improved and internationally standardized panels of HIV-1 isolates that cover the dynamic diversity of circulating HIV-1 strains., (Copyright © 2020. Published by Elsevier Ltd.)

Background: Nucleic acid testing is essential for the detection and quantification of HCV RNA in the diagnosis of HCV infection and treatment monitoring. The Alinity m HCV assay was recently developed by Abbott Molecular for rapid detection and quantification of HCV RNA on the fully automated, continuous, random-access Alinity m analyzer., Objectives: Our study assessed the performance of the new Alinity m HCV assay for detection and quantification of HCV RNA in a large series of patient samples of various genotypes. This international, multicentric study evaluated the linearity, precision, and reproducibility of the Alinity m HCV assay and its performance in comparison to three other HCV assays currently used in clinical practice., Results: The Alinity m HCV assay demonstrated high linearity (correlation coefficient r = 1.00), precision (coefficients of variation [CV] 6.6-13.5 %) and reproducibility (CV 1.7-4.3 % across three control lots). At a concentration near the lower limit of detection, the Alinity m HCV assay exhibited >98 % detectability. The Alinity m HCV assay showed excellent correlation with comparator HCV assays in serum (n = 406) and plasma (n = 1401) samples (correlation coefficients ≥0.96, bias 0.01 to 0.14 Log 10 IU/mL). More than 95 % of the quantified results with the Alinity m HCV assay were less than mean bias ± 1.96 SD different from those of the comparator assays., Conclusions: The newly developed Alinity m HCV assay is sensitive, reproducible, and accurately quantifies HCV RNA levels in serum and plasma samples from patients with chronic HCV infection, with no impact of HCV genotype on assay performance., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Accurate molecular methods to detect and quantify hepatitis B virus (HBV) DNA are essential to diagnose chronic infections, guide treatment decisions, assess response to treatment, and determine risk of HBV-related complications. New generations of real-time HBV DNA assay platforms provide results in less than 2-3 h, with continuous loading of specimens and true random-access capability., Objectives: We examined the clinical performance of the new Alinity m HBV assay, run on the fully automated, continuous, random-access Alinity m platform, to accurately detect and quantify HBV DNA in a large series of patient samples infected with different HBV genotypes frequently encountered in clinical practice., Study Design: This international, multisite study assessed the precision and reproducibility of the Alinity m HBV assay and compared its performance to four HBV assays currently in clinical use., Results: The Alinity m HBV assay demonstrated linear quantitation of HBV DNA in plasma samples, with high precision (coefficient of variation 4.1 %-8.8 %) and reproducibility. The Alinity m HBV assay showed excellent correlation (correlation coefficients ≥0.947) with comparator HBV assays, with an overall observed bias ranging from -0.07 to 0.17 Log 10 IU/mL. 97 % of quantifiable patient results were <1 Log 10 IU/mL different than the respective comparator assays, with comparable results across HBV genotypes., Conclusions: The newly developed real-time PCR-based Alinity m HBV assay is sensitive, reproducible, and accurately quantifies HBV DNA levels from HBsAg-positive patients across the full dynamic range of quantification., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Accurate, rapid detection of HIV-1 RNA is critical for early diagnosis, treatment decision making, and long-term management of HIV-1 infection., Objective: We evaluated the diagnostic performance of the Alinity m HIV-1 assay, which uses a dual target/dual probe design against highly conserved target regions of the HIV-1 genome and is run on the fully automated Alinity m platform., Study Design: This was an international, multisite study that compared the diagnostic performance of the Alinity m HIV-1 assay to four commercially available HIV-1 assays routinely used in nine independent clinical laboratories. Alinity m HIV-1 assay precision, detectability, and reproducibility was compared across four study sites., Results: The Alinity m HIV-1 assay produced comparable results to currently available HIV-1 assays (correlation coefficient >0.995), with an overall bias of -0.1 to 0.10 Log 10 copies/mL. The Alinity m HIV-1 assay and its predecessor m2000 HIV-1 assay demonstrated comparable detection of 16 different HIV-1 subtypes (R 2  = 0.956). A high level of agreement (>88 %) between all HIV-1 assays was seen near clinical decision points of 1.7 Log 10 copies/mL (50 copies/mL) and 2.0 Log 10 copies/mL (200 copies/mL). Alinity m HIV-1 assay precision was 0.08 and 0.21 Log 10 copies/mL at VLs of 1000 and 50 copies/mL, respectively, with a high level of detectability (≥97 % hit rate) and reproducibility across sites., Conclusions: The Alinity m HIV-1 assay provides comparable diagnostic accuracy to current HIV-1 assays, and when run on the Alinity m system, has the capacity to shorten the time between diagnosis and treatment., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Although our understanding of Parkinson´s disease (PD) has improved and effective treatments are available, caring for people with PD remains a challenge. The large heterogeneity in terms of motor symptoms, nonmotor symptoms, and disease progression makes tailored individual therapy and individual timing of treatment necessary. On the other hand, only limited resources are available for a growing number of patients, and the high quality of treatment cannot be guaranteed across the board. At this point, networks can help to make better use of resources and improve care. The working group PD Networks and Integrated Care, part of the German Parkinson Society, is entrusted to convene clinicians, therapists, nurses, researchers, and patients to promote the development of PD networks. This article summarizes the work carried out by the working group PD Networks and Integrated Care in the development of standards of network care for patients with PD in Germany., Competing Interests: Tino Prell has received BMBF research grant, and honoraria for presentations/lectures AbbVie GmbH, UCB Pharma GmbH, Desitin GmbH, Licher MT GmbH, and Bayer AG Deutschland. Frank Siebecker reports no conflict of interest. Michael Lorrain has received honoraria and compensation for consultancy and lecturing from Abbvie, Afi, Bayer, Bial, Biogen, Desitin, Merck, Nordrheinische Akademie, Teva, Ucb, and Zambon. Carsten Eggers received payments as a consultant for Abbvie Inc. CE received honoraria as a speaker from Abbvie Inc., Daiichi Sankyo Inc., Bayer Vital Inc. CE received payments as a consultant for Abbvie Inc. and Philyra Inc. Stefan Lorenzl reports no conflict of interest. Jochen Klucken reports institutional research grants from Bavarian Research Foundation; Emerging Field Initiative, FAU, EIT-Health, EIT-Digital, EU (H2020), German Research Foundation (DFG), and BMBF, and industry-sponsored institutional IITs and grants from Teva GmbH, Licher MT GmbH, Astrum IT GmbH, and Alpha-Telemed AG. He is coemployed by the University Hospital Erlangen, Germany, Fraunhofer Institute for Integrated Circuits e.V., Germany, and the Medical Valley Digital Health Application Center GmbH, Bamberg, Germany. He works on advisory boards in the field of healthcare technologies and digital health of different associations of medical professionals, industries, and political authorities. He holds shares of Portabiles HealthCare Technologies GmbH, Portabiles GmbH, Alpha-Telemed AG, and received compensation and honoraria from serving on scientific advisory boards for LicherMT GmbH, Abbvie GmbH, UCB Pharma GmbH; he has lectured at UCB Pharma GmbH, TEVA Pharma GmbH, Licher MT GmbH, Desitin GmbH, Abbvie GmbH, Solvay Pharmaceuticals, Bial Deutschland GmbH; Celgene GmbH, Lundbeck-Foundation. Dr. Klucken has a patent related to gait assessments pending. Tobias Warnecke has received honoraria from AbbVie (lecture fees, consultant). AbbVie acts as coinitiator of the Parkinsonnetwork Muensterland+ (PNM+) and is cocontractor of the University Hospital of Muenster. Carsten Buhmann has received fees as speaker and/or advisor from Abbvie, Bial, Desitin, Grünenthal, Licher, Novartis, TAD Pharma, UCB, and Zambon. Lars Tönges has received travel funding and/or speaker honoraria from Abbvie, Bayer, Bial, Desitin, GE, UCB, and Zambon, and consulted for Abbvie, Bayer, Bial, Desitin, UCB, and Zambon, in the last 3 years. Reinhard Ehret reports no conflict of interests. Ingmar Wellach has received honoraria an compensation for consultancy and lecturing from AbbVie GmbH, UCB Pharma GmbH, Desitin GmbH, Bial Deutschland GmbH, Zambon Deutschland GmbH, Fagron GmbH & Co. KG, Grünenthal GmbH, and Bayer AG Deutschland. Martin Wolz has received honoraria for presentations/lectures from Zambon, Valeant, Desitin, TEVA, UCB Pharma, Abbvie, Bial, Licher, and Daiichi Sankyo.

Hepatitis C virus (HCV) antigen/antibody (Ag/Ab) assays offer the benefit of reducing the window period compared to assays that detect only HCV-Ab. In this study the performance of the Murex Ag/Ab (Murex, Abbott) and Monolisa Ag/Ab Ultra (Monolisa, Bio-Rad) ELISAs was compared for the use of filter dried serum/plasma spots (DS/PS) with a focus on the sensitivity and the percentage of correct positive test results. Correct positive ELISA results were assumed for samples that subsequently tested positive for HCV RNA by RT-qPCR, or RNA negative samples that tested positive in a Western blot (confirmed ELISA results). Sensitivity was evaluated from DS/PS eluates using HCV seroconversion panels [plasma samples of subtypes-(St) 1a, 2b)] and longitudinal HCV antibody positive serum panels (St 1b, 2b, 3a, and 4d). The proportion of correct positive test results was evaluated using 1102 newly diagnosed HIV positive clinical dried serum spots (DSS) eluates for screening of potential HCV co-infection. For the plasma HCV seroconversion samples, which were used as a reference for DSS eluates, the Murex became reactive earlier for antigen positive bleeds. However, for the HCV antibody positive eluates and dilutions thereof, the Monolisa demonstrated a superior sensitivity. Of the clinical DSS 22.8 % (28/123) of samples reactive in the Murex were negative in a subsequent RT-qPCR and Western blot, while only 1.9 % (2/105) of the samples reactive in the Monolisa were negative in these confirmatory assays. Our results indicate that the Monolisa provides fewer false positive results for HCV detection in DSS, whereas for undiluted plasma or serum samples, the Murex can serve as an additional diagnostic tool to narrow the window period., Competing Interests: Declaration of Competing Interest None, (Copyright © 2020 Elsevier B.V. All rights reserved.)

Monitoring recency of infection helps to identify current transmission in vulnerable populations for effective disease control. We have established an in-house avidity based hepatitis C virus (HCV) recency assay based on the Monolisa Anti-HCV PLUS Version 3 ELISA kit for use of dried serum/plasma spots (DS/PS) in order to distinguish recent and long-term infections. A first panel of DS/PS (n = 218; genotype 1 n = 170 and non-genotype 1 n = 48) consisting of primary and at least one follow up sample was used to analyze the temporal changes of the Avidity Index (AI) over time. Sub-panels of longitudinal DS/PS (n = 66) and acute cases (<26 weeks; n = 34) were taken to calculate the Mean Duration of Recent Infection (MDRI) and the False Long-term Rate (FLTR), respectively. A second panel of DS/PS >104 weeks (n = 132) and a third panel of DS/PS prepared from resolved infections (≥180 days since last positive; n = 32) were used to calculate the False Recent Rate (FRR). For all genotypes, the optimal AI cut-off was determined to be 40% resulting in an MDRI of 364 days (95% CI: 223-485). FLTR was 5.9% (95% CI: 0.7-19.7), 8.3% (95% CI: 1-27), and 0% (-) and FRR was 13.6% (95% CI: 8.3-20.7), 11.7% (95% CI: 6.6-19), and 30.6% (95% CI: 9.1-61.4) for all genotypes, genotype 1, and non-genotype 1 infections, respectively. For resolved infections, the FRR was 53.1% (95% CI: 35.8-70.4). Thus, this assay performs particularly well for genotype 1 reaching a high rate of correct discriminations between infections acquired less than a year before diagnosis and those acquired earlier by applying an AI cut-off of 40%. Due to a rapid decline in avidity post resolution of an HCV infection this assay is not recommended to be used in HCV RNA negative patients., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 RealTi m e m 2000 assay. Assay precision and accuracy were evaluated in a retrospective evaluation out of excess plasma material from four HIV-1+ individuals (subtypes B, C, CRF01&#95;AE, and CRF02&#95;AG). Native plasma samples were diluted to nominal concentrations at 50 and 200 copies/ml (according to the RealTi m e m 2000 assay). All dilutions were tested in triplicate in five independent runs over 5 days and in three labs per system. Assay concordance was determined using 1,011 surplus clinical routine samples, as well as selected retrospective longitudinal samples from 7 patients on treatment. The three assays yielded highly concordant results for individual clinical samples ( R 2 > 0.98; average difference, ≤0.2 log copies/ml) and retrospective longitudinal samples from patients on treatment. The Aptima and RealTi m e assays showed similar high precision, meeting the 5σ criterion for the majority of samples across all labs and subtypes. The Cobas assay was less precise, missing the 5σ criterion for the majority of samples at low concentrations. In this analysis, results from the Cobas assay appeared less reliable near the clinically relevant cutoff and should be interpreted with more caution in this context. Due to high precision, full automation, and high concordance with the RealTi m e assay, the Aptima assay represents a good alternative in routine VL monitoring., (Copyright © 2018 Wiesmann et al.)

Background: The treatment of levodopa-induced dyskinesia in Parkinson's disease (PD) is an unmet need with no approved drug therapy., Objective: The purpose of this study was to investigate the efficacy and safety of 274 mg ADS-5102 (amantadine) extended-release capsules (equivalent to 340-mg amantadine HCl) for levodopa-induced dyskinesia in a randomized controlled trial., Methods: PD patients with ≥1 hour of troublesome dyskinesia and at least mild functional impact were randomized to placebo or ADS-5102 once daily at bedtime for 13 weeks. The primary efficacy analysis was based on change from baseline to week 12 on the Unified Dyskinesia Rating Scale total score in the modified intent-to-treat population. OFF time was a key secondary measure., Results: At week 12, least-squares mean change in the Unified Dyskinesia Rating Scale was -20.7 (standard error 2.2) for ADS-5102 (n = 37) and -6.3 (standard error 2.1) for placebo (n = 38; treatment difference -14.4, 95% confidence interval -20.4 to -8.3, P < .0001), indicating improvement in levodopa-induced dyskinesia. OFF time decreased 0.5 hours (standard error 0.3) for ADS-5102 from a baseline mean of 2.6 hours and increased 0.6 hours (standard error 0.3) for placebo from a baseline mean of 2.0 hours (treatment difference -1.1 hours, 95% confidence interval -2.0 to -0.2, P = .0199). The most common adverse events (ADS-5102 versus placebo) included dry mouth (13.5% versus 2.6%), nausea (13.5% versus 2.6%), decreased appetite (10.8% versus 0%), insomnia (10.8% versus 0%), orthostatic hypotension (10.8% versus 0%), constipation (8.1% versus 0%), falls (8.1% versus 5.3%), and visual hallucinations (8.1% versus 5.3%). Adverse events led to treatment discontinuation in 19% versus 8%, respectively., Conclusion: ADS-5102 274 mg is an oral pharmacotherapy demonstrating a significant decrease in levodopa-induced dyskinesia and improving OFF time. © 2017 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society., (© 2017 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.)

In distinction to idiopathic Parkinson's disease (PD), the diagnosis of atypical Parkinson syndromes comprises dementia with Lewy bodies (DLB), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). We set out to write a state-of-the-art guideline as to which investigations and examinations help to differentiate PD vs. atypical Parkinson syndromes in clinical routine.

After the start of antiretroviral therapy (ART), plasma HIV-RNA levels should fall below the limit of detection (LOD) within 24 weeks. Hence, the prolonged decline of HIV-RNA after ART initiation is defined as persistent viremia (PV). In this retrospective study, we analyzed factors associated with PV. Next-generation sequencing of viral RNA/DNA was performed to study viral evolution and the emergence of drug-resistance mutations in HIV-infected patients with PV (n = 20). In addition, HIV-DNA species, immunological parameters, and clinical data of the patients were analyzed. We found that the possible causes for PV were divers, and both virologic and host parameters of this particular cohort were heterogeneous. We identified viruses with therapy-associated DRMs in six patients (30%); two of these were detected as minority variants. Five patients had sub-optimal drug levels (25%) and the baseline plasma viral loads were relatively high. Strikingly, we observed that >40% of the PV patients finally reaching HIV levels below the LOD later on showed up with episodes of low-level viremia (LLV). However, the amount of PBMC derived HIV-DNA species was not correlated with the likelihood of LLV after PV. According to our data, we conclude that drug-resistant viruses, sub-optimal drug level, and high baseline viral loads might be probable reasons for the prolonged RNA decline only in a sub-set of patients. In the absence of emerging DRMs and/or compliance issues, the clinical implications of PV remain unclear; however, PV appears to be a risk factor for episodes of LLV.

The ambulatory care of patients with Parkinson's disease (PD) in Germany has been established for a long time. As the prevalence of Parkinson's disease continues to increase, the outpatient neurological sector is becoming more and more important and needs to adapt itself to current needs. This includes an optimization of the care structures for Parkinson's patients as well as adequate concepts for the execution of differentiated diagnostics and therapy. For many patients care is provided by non-specialized neurological practices or general practitioners, without exchange of views with neurologists or a specialized university outpatient clinic for movement disorders. A connective link between these care structures could be provided by a "practice with focus on Parkinson's disease", whose idea and conception is presented in this article. In addition to the necessity and usefulness of such an institution, structural prerequisites and basic principles for the treatment of Parkinsonian patients in a disease state-centered manner will be presented but also current limitations of the concept are pointed out. This article presents the results of an expert workshop on Parkinson's disease, which took place in Frankfurt am Main on 21 November 2015., Competing Interests: Interessenkonflikt: Lars Tönges: Der Autor hat Honorare für Teilnahme an Advisory Boards, Vorträge oder Artikelerstellung erhalten von: UCB, AbbVie, Bayer.Reinhard Ehret: Der Autor hat Honorare für Teilnahme an Advisory Boards, Vorträge oder Artikelerstellung erhalten von: Bial, Desitin, Global kinetics, Mundipharma, Novartis, Roche, Sanofi, Teva, UCB, AbbVie, Desitin, GE Healthcare, Medtronics, Mundipharma, Novartis, Zambon.Michael Lorrain: Der Autor hat Honorare für Teilnahme an Advisory Boards, Vorträge oder Artikelerstellung erhalten von: AbbVie, Desitin, UCB, Medtronic, TEVA, Gruenenthal, Bial.Peter Riederer: Der Autor hat keine Interessenkonflikte. Martina Müngersdorf: Die Autorin hat keine Interessenkonflikte., (© Georg Thieme Verlag KG Stuttgart · New York.)

Background: Pain is a common non-motor symptom of Parkinson's disease. We investigated the analgesic efficacy of prolonged-release oxycodone-naloxone (OXN PR) in patients with Parkinson's disease and chronic, severe pain., Methods: We did this phase 2 study in 47 secondary care centres in the Czech Republic, Germany, Hungary, Poland, Romania, Spain, and the UK. We enrolled patients with Hoehn and Yahr Stage II-IV Parkinson's disease, at least one type of severe pain, and an average 24-h pain score of at least 6 (assessed on an 11-point rating scale from 0=no pain to 10=pain as bad as you can imagine). Participants were randomly assigned (1:1) with a validated automated system (block size four) to either oral OXN PR or placebo for 16 weeks (starting dose oxycodone 5 mg, naloxone 2·5 mg, twice daily). Patients and investigators were masked to treatment assignment. The primary endpoint was average 24-h pain score at 16 weeks in the full analysis population. This study is registered with EudraCT (2011-002901-31) and ClinicalTrials.gov (NCT01439100)., Findings: We enrolled 202 patients; 93 were assigned to OXN PR and 109 to placebo; the full analysis population consisted of 88 patients versus 106 patients. Least squares mean average 24-h pain score at 16 weeks in the full analysis population was 5·0 (95% CI 4·5 to 5·5) in the OXN PR group versus 5·6 (5·1 to 6·0) in the placebo group (difference -0·6, 95% CI -1·3 to 0·0; p=0·058). Similar proportions of patients in each group had adverse events (60/92 [65%] vs 76/109 [70%]), treatment-related adverse events (52/92 [57%] vs 62/109 [57%]), and serious adverse events (5/92 [5%] vs 7/109 [6%]). Treatment-related nausea was more common in the OXN PR group than in the placebo group (16/92 [17%] vs 10/109 [9%]), as was treatment-related constipation (16/92 [17%] vs 6/109 [6%])., Interpretation: The primary endpoint, based on the full analysis population at week 16, was not significant. Nonetheless, the results of this study highlight the potential efficacy of OXN PR for patients with Parkinson's disease-related pain and might warrant further research on OXN PR in this setting., Funding: Mundipharma Research., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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.

Introduction: The most recently approved antiretroviral, the integrase inhibitor dolutegravir (DTG), is described to be a very potent drug with a unique resistance profile, but a certain degree of cross-resistance to RAL or EVG induced drug resistance, which is mediated mainly by integrase mutations at positions 140 and 148. The impact of a single N155H mutation to DTG resistance is described to be minor. However, there is only rare data available about the impact of N155H in the context of secondary site integrase mutations. Here, we present a case of virological failure in a DTG treated patient based on N155H mutation background., Methods: Therapy monitoring of an HIV-HCV co-infected patient harbouring already an omni-drug-class resistant HIV-1 in consequence of more than 20 years ART history. Drug susceptibility testing was performed by RT-PCR from plasma and subsequent Sanger sequencing. Tropism testing was done from proviral DNA with FPR cut-offs according to the German recommendations., Results: In 2013, the patient harbouring a virus with high level resistance to all RTI and PI received a regimen containing FTC, TDF, DRV/r, RPV, T20, and RAL to handle a viral load of 5000 RNA copies/mL, but never achieved fully suppressed viral load. In June 2013, after S119R, N155H and E157Q mutations in viral integrase were detected, the patient received DTG, and RAL was stopped. One month later, when viral load was undetectable for the first time since 2007, ART was de-escalated by removing T20. Since February 2014, low-level viral load was re-detectable. Two new mutations T97A and S147G in the integrase and no other new resistance associated mutations in protease and reverse transcriptase in comparison to the sample analyzed in June 2013 were detected., Conclusions: Highly resistant HIV-1 strains have been a common problem in the past. Their frequencies were pushed back by highly potent ART, but the virus is still able to become resistant against all available antiretrovirals at once. The here documented strain became resistant to DTG without carrying mutations at the described positions 140 and 148, but in the context of integrase N155H. Since N155H alone is described not to contribute sufficient resistance to DTG, there seems to be a need to re-check viruses with N155H plus minor mutations (like T97A, S119R, S147G and E157Q) potentially contributing to DTG resistance.

Introduction: Due to drug-drug interactions of HIV- and HCV-specific antivirals when initiating an HCV-therapy, the antiretroviral therapy (ART) often has to be changed. The spectrum of applicable antiretrovirals is small, therefore many patients were switched to raltegravir/truvada (RAL/TVD) in our cohort. Due to the relatively low genetic barrier of RAL, this regimen may be endangered to fail, if the NRTI backbone is not fully active because of pre-existing NRTI resistance. We investigated the long-term follow-up and safety of RAL/TVD in co-infected patients after hepatitis C virus (HCV) therapy was stopped and the protective antiretroviral effect of interferon ended., Materials and Methods: Twenty patients initiated a direct-acting antiviral (DAA) containing HCV therapy (8x faldaprevir, 6x telaprevir, 2x daclatasvir and 4x simeprevir) between 11/2011 and 01/2013. Seventeen were switched to RAL/TVD, three patients were not treated before, but started with the regimen. Diagnosis of HIV infection was dated between 1985 and 2010. The HI-viral suppression was monitored retrospectively to date., Results: Thirteen of the twenty patients (65%) remained on RAL/TVD after finishing HCV treatment, for seven patients, no data about their ART continuation was available, after HCV therapy had stopped. All remaining thirteen patients showed an HI-viral load below detection limit up to date (for 15 to 22 months, median 20 months). Only for four patients, historic resistance data were available but none showed NRTI mutations., Conclusions: Switch to RAL/TVD as HIV ART due to initiating HCV therapy was safe for the observed small cohort even in long-term follow-up without switchback or a second ART switch. However, resistance data for the cohort was little, showing no NRTI mutations, indicating a relatively safe setting. Since no further data is available, physicians should keep in mind ART history, historical therapy failure and HIV-resistance while switching ART to treat HCV in co-infected patients. Further investigation in larger cohorts is needed, especially thinking of upcoming interferon-free HCV regimen in heavily pre-treated co-infected patients.

Introduction: Proven resistance against HIV drugs, either by phenotyping or genotyping is a rare event in clinical trials. The overall assumption of drug resistance disappearing is additionally driven by the recommendations to screen for transmitted drug resistance, leading to large numbers of examinations with relatively low rates of resistance. Goal of our analysis was to assess if drug resistance in treatment failure is also decreasing outside of clinical trials., Materials and Methods: The MIB database at timepoint of analysis consists of data from 2876 HIV infected patients. Besides various laboratory parameters, clinical data and treatment history is included. HIV-1 protease and reverse transcriptase sequences were analyzed using the HIV-GRADE drug resistance algorithm. As in only a small number of patients genotypic resistance testing for integrase inhibitors was performed, mainly due to reimbursement reasons, it was assumed that failing treatment on a previous integrase inhibitor containing regimen is equate to resistance., Results: Of the 2876 patients in the database, 220 had a treatment change due to treatment failure between 2009 and 2012, a genotypic resistance testing at an appropriate timepoint of maximum four weeks before treatment change and a treatment duration of at least six months before treatment failure. In 2009, 61% of patients showed no drug resistance while 39% showed resistance against one or more drug classes (two or more drug classes: 19.5%; three or more drug classes: 2.4%, four drug classes 2.4%). In 2012, no resistance was found in 52% of patients while resistance against three or more drug classes was found in nearly 14% of patients (one or more: 48%; two or more 23%; four classes: 4.5%)., Conclusions: Treatment failure with viral load sufficiently high for drug resistance testing was not frequently observed in our database. Nevertheless, treatment failure was often associated with drug resistance against at least one drug class. With more use of the newer drug classes, resistance against those new classes will become more common and rates of multiclass resistance will be increasing.

Introduction: Simeprevir, a new oral NS3/4A protease inhibitor, was recently approved by the FDA and the EMA for the treatment of patients with chronic HCV genotype 1, 4, 5 and 6 infection l. It has been recommended in the 2014 UK Consensus Guidelines as a possible treatment of previously untreated genotype 1a-infected patients. The antiviral efficacy of simeprevir is adversely affected by the mutation at the Q80K loci. There is controversial discussion that the incidence of Q80K in the European HCV 1a-infected community is very low and therefore testing of Q80K before starting a therapy including simeprevir is not necessary. We analyzed the appearance of Q80K in all sequenced HCV NS3A samples in 2014 in our laboratory., Materials and Methods: All in 2014 received orders for HCV resistance tests were analyzed with an in-house bulk sequencing method analyzing NS3A amino acids 1-181. Analysis was performed using geno2pheno HCV. The genotype 1a samples were selected, Q80K status and data of HIV co-infection were collected., Results: Forty-two HCV 1a samples were sent to us for resistance analyses from nine different medical centres in Berlin and Hannover, Germany. Nineteen (or 45%) of the sequences showed a Q80K mutation. Six extra clade I viruses had no Q80K mutation. Comparison between mono- and HIV-1 co-infected patients showed no difference in frequency of Q80K (mono-infected: 8 out of 19 patients; co-infected: 9 out of 23). For two 80K-positive patients, the HIV-status was not available., Conclusions: The incidence for Q80K mutation in HCV genotype 1a with overall 45% is substantially high in our cohort and does not differ between mono- and HIV-1 co-infected patients. Response to simeprevir is affected by the presence of viral Q80K. When treating HCV-infected patients with a simeprevir containing regimen, it is therefore important that HCV does not contain the Q80K mutation.

Objectives: The aim of this study was to investigate the effects of piribedil on vigilance and cognitive performance in patients with Parkinson disease experiencing excessive daytime sleepiness on pramipexole or ropinirole., Methods: In this 11-week randomized, active-controlled, rater-blinded phase III study, eligible patients were randomly assigned to either receive piribedil or to continue on pramipexole or ropinirole. The primary outcome was the median reaction times during the second 15 minutes of the subtest "vigilance" of the Test battery for Attention Performances (TAP). Secondary outcomes included the Epworth Sleepiness Scale, Unified Parkinson's Disease Rating Scale, neuropsychological testing, and items of the Clinical Global Impression., Results: Forty-four patients received piribedil; 36 continued on either pramipexole or ropinirole. There was no difference in the primary end point reaction time of the TAP subtest vigilance between piribedil and the comparator (996 vs 954 milliseconds, P = 0.68). Piribedil reduced daytime sleepiness with lower Epworth Sleepiness Scale scores at the end of treatment compared with the comparator (-4 vs -2 points; P = 0.01). The median Unified Parkinson's Disease Rating Scale III score at the end of treatment was comparable between the 2 groups. Neuropsychological tests revealed no significant between-treatment differences. A higher therapeutic effect and global improvement were shown by the Clinical Global Impression of piribedil-treated patients., Conclusions: This study shows that switching from pramipexole or ropinirole to piribedil has no effect on the reaction time of the TAP subtest vigilance but upholds the same therapeutic motor effect and reduces daytime sleepiness to a clinically relevant degree in patients with excessive daytime sleepiness.

We present a novel, multiparametric microphysiometry system for the dynamic online monitoring of human cancer cell metabolism. The optically transparent, modular, hybrid microsystem is based on a glass chip and combines a cell cultivation chamber, microfluidics and metabolic monitoring with fully integrated chemo- and biosensors. pH and oxygen are measured in the cell culture area, and biosensors for lactate and glucose are connected downstream by microfluidics. The wafer-level fabrication features thin-film platinum and iridium oxide microelectrodes on a glass chip, microfluidics in an epoxy resist, a hybrid assembly and an on-chip reference electrode. The reliable analytical performance of the sensors in cell culture medium was demonstrated. The pH sensors exhibit a long-term stable, linear response. The oxygen sensors show a linear behaviour, which is also observed for low oxygen concentrations. Glucose and lactate measurements show a linear, long-term stable, selective and reversible behaviour in the desired range. T98G human brain cancer cells were cultivated and cell culture metabolism was measured on-chip. Stop/flow cycles were applied and extracellular acidification, respiration, glucose consumption and lactate production were quantified. Long-term metabolic rates were determined and all parameters could be measured in the outlet channel. A placement downstream of the cell cultivation area for biosensors was realised. A highly effective medium exchange and undiluted sampling from the cell culture chamber with low flow rates (2 μl min(-1)) and low volumes (15 μl per cycle) were achieved. The drug screening application was demonstrated by detecting alteration and recovery effects of cellular metabolism induced by the addition of substances to the medium.

The goal of antiretroviral therapy is reduction in morbidity and mortality via suppression of human immunodeficiency virus (HIV) viral load (VL) to undetectable levels. VL assay sensitivity has improved over time, but the reproducibility and clinical importance of VL results marginally higher than the limit of detection (LoD) are uncertain. We assessed the reproducibility and concordance of low VL results obtained with the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 version 2.0 (CAP-CTM) and the Abbott RealTime (m2000) HIV-1 assays, using longitudinal specimens from HIV-1-infected patients with low VL (<300 copies/ml) and stable CD4+ cell counts. Based on replicate testing of 3 specimens, coefficients of variation for log-transformed VL results were 5-8 % for m2000 and 9-10 % for CAP-CTM. The concordance between assays in specimens from patients with previously undetectable, detectable but not quantifiable VL, or variable (undetectable/detectable but not quantifiable VL) results over time was 90, 56, and 56 %, respectively. Correlation between results for specimens with quantifiable VL (initially 40-300 copies/ml) was moderate (R (2) = 0.48) with significantly higher results for CAP-CTM and a mean difference (CAP-CTM minus m2000) of 0.10 log(10) copies/ml. T-cell activation (CD8+/CD38+ percentage) in patients with low VL was initially higher than in patients with undetectable VL, and then decreased to equivalent levels over time. These results indicate that residual viremia at levels slightly above the LoD have no negative effect on T-cell activation.

Background: Genotypic drug resistance testing provides essential information for guiding treatment in HIV-infected patients. It may either be used for identifying patients with transmitted drug resistance or to clarify reasons for treatment failure and to check for remaining treatment options. While different approaches for the interpretation of HIV sequence information are already available, no other available rules-based systems specifically have looked into the effects of combinations of drugs. HIV-GRADE (Genotypischer Resistenz Algorithmus Deutschland) was planned as a countrywide approach to establish standardized drug resistance interpretation in Germany and also to introduce rules for estimating the influence of mutations on drug combinations. The rules for HIV-GRADE are taken from the literature, clinical follow-up data and from a bioinformatics-driven interpretation system (geno2pheno([resistance])). HIV-GRADE presents the option of seeing the rules and results of other drug resistance algorithms for a given sequence simultaneously., Methods: The HIV-GRADE rules-based interpretation system was developed by the members of the HIV-GRADE registered society. For continuous updates, this expert committee meets twice a year to analyze data from various sources. Besides data from clinical studies and the centers involved, published correlations for mutations with drug resistance and genotype-phenotype correlation data information from the bioinformatic models of geno2pheno are used to generate the rules for the HIV-GRADE interpretation system. A freely available online tool was developed on the basis of the Stanford HIVdb rules interpretation tool using the algorithm specification interface. Clinical validation of the interpretation system was performed on the data of treatment episodes consisting of sequence information, antiretroviral treatment and viral load, before and 3 months after treatment change. Data were analyzed using multiple linear regression., Results: As the developed online tool allows easy comparison of different drug resistance interpretation systems, coefficients of determination (R(2)) were compared for the freely available rules-based systems. HIV-GRADE (R(2) = 0.40), Stanford HIVdb (R(2) = 0.40), REGA algorithm (R(2) = 0.36) and ANRS (R(2) = 0.35) had a very similar performance using this multiple linear regression model., Conclusion: The performance of HIV-GRADE is comparable to alternative rules-based interpretation systems. While there is still room for improvement, HIV-GRADE has been made publicly available to allow access to our approach regarding the interpretation of resistance against single drugs and drug combinations., (Copyright © 2012 S. Karger AG, Basel.)

Levodopa/carbidopa (LD/CD) application elevates total plasma homocysteine (thcys). We determined thcys-, LD- and 3-O-methyldopa (3-OMD) concentrations in 28 patients with Parkinson's disease (PD) on a LD/CD duodenal gel treatment. We found a distinct thcys increase (29.52 ± 28.98 μmol/l [median ± SD]) above the 15 μmol/l threshold and a significant (R = 0.7) correlation between LD and 3-OMD. thcys ascent was observed in relation with the onset of atherosclerosis, non-motor symptoms and polyneuropathy in PD patients in the long term.

Early pre-motor symptoms (also frequently termed "non-motor" symptoms) in Parkinson's disease (PD), which precede the onset of motor symptoms, are being increasingly recognized by clinicians. Non-motor symptoms in the pre-motor phase of PD include impaired olfaction (hyposmia), sleep disturbances (i.e., radid eye movement sleep behavior disorder, daytime sleepiness), behavioral/emotional dysfunction (i.e., change of personality or change of core personal characteristics), dysautonomia (i.e., constipation, urinary dysfunction, orthostatic hypotension), depressive symptoms (i.e., fatigue, apathy, anxiety), and chronic pain (joint and muscle). The pre-motor phase of PD is based on current pathophysiological concepts that relate these symptoms to early structural changes within lower brainstem nuclei and the peripheral nervous system including the autonomic and enteric ganglia. The perspective to identify these symptoms as early as possible will enable neurologists to make a diagnosis at the pre-motor stage of PD. Thus, the development of a PD risk score will be the first means to identify individuals at risk who are most likely to develop the prototypical motor symptoms of PD later in life. More importantly, these individuals at risk will be the first to benefit from disease-modifying strategies. In this workshop report, the elements of a PD risk score are proposed, including the stepwise sequence of escalating diagnostic measures to diagnose the pre-motor stage in PD.

Background: Although 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., Results: Based 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., Conclusions: In 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.

Background and Objectives: Minority drug-resistant HIV-1 variants, undetected by conventional genotyping, may impair the outcome of antiretroviral therapy (ART). Thus, we retrospectively analyzed the prevalence of minority drug-resistant HIV-1 variants before ART in chronically HIV-1 infected patients initiating first-line therapy and assessed the impact on clinical outcome in the prospective German Truvada cohort., Study Design: Samples from 146 antiretroviral treatment-naïve patients were collected between April 2005 and August 2006. K65R, K103N, and M184V variants at low frequencies were detected by allele-specific real-time PCR., Results: Minority drug-resistant HIV-1 variants were detected in 20/146 patients (13.7%): the M184V mutation in 12/146 patients (8.2%), the K103N mutation in 8/146 patients (5.5%), and the K65R mutation in 4/146 patients (2.7%). Four patients with the M184V mutation also harbored the K65R or the K103N mutation. The 12- and 24 months virological efficacy data revealed that the rate of treatment failure was not increased in the group of patients harboring minority drug-resistant HIV-1 variants prior to ART., Conclusions: Minority drug-resistant HIV-1 variants can be frequently detected in treatment-naïve, chronically HIV-1 infected patients. Despite the presence of those mutations as minority variants before initiating ART, most of the patients were successfully treated., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: Phytoestrogens are naturally occurring, plant-derived, nonsteroidal phytochemicals with anticarcinogenic potential. The aim of this study was to isolate phytoestrogens from the flax root of Linum usitatissimum and to test their effect on cellular metabolism in the human mammalian carcinoma cell line MCF-7 using the Bionas 2500 analysis system., Materials and Methods: Metabolically relevant parameters such as acidification, oxygen consumption and cell adhesion were registered continuously over 8 and 24 hours on six sensor chips in parallel at different concentrations of flax root extracts., Results: The extracts from flax roots of L. usitatissimum reduced extracellular acidification, respiration and adhesion in a concentration-dependent manner., Conclusion: The Bionas 2500 analysis system allows multiparametric online monitoring of cellular processes and can be used to detect the mode of action of anticarcinogenic compounds in cellular metabolism.

Background: Aim of this study was to assess the direct costs of Parkinson's disease (PD) within a 3-month period (i.e. the accounting period for the German statutory health insurance) in 12 neurological outpatient practices in Berlin during 2006., Material and Methods: A total of 425 patients (age 69.1+/-9.3 years, 185 females) were recruited, and sociodemographic and clinical data were obtained by a specific questionnaire. The distribution of costs was analyzed based on several clinical and patient parameters. The costs were calculated with different approaches: (1) prospectively, with the practices' accounting according to German uniform scales (GoA, EbM) and (2) retrospectively, with questionnaires for the Parkinson's patients. Costs were calculated according to current German guidelines of the statutory health insurance. Clinical parameters were assessed with a questionnaire for physicians., Results: The direct medical costs totaled 1,667 EUR (range 1,436-1,995 EUR, CI 95%) per patient per 3 months. Charges by physicians were 42 EUR (39-45 EUR, CI 95%) for patients with statutory health insurance and 135 EUR (106-177 EUR, CI 95%) for those with private insurance. Disease severity and disease duration correlated with higher direct medical costs. Motor fluctuations and depression also were major factors influencing cost., Conclusion: Our study emphasizes the large economic burden caused mainly by PD medication and hospitalization. For the first time a direct comparison between costs and actual physicians' reimbursement was possible. In combination with further economic studies, this comparison will help to define shortcomings and excesses in PD health care services.

A multiparametric chip-based system was employed to measure cell adhesion, metabolism, and response to metal compounds previously classified as cytotoxic in immortalized mouse fibroblasts (BALB/3T3 cell line). The system measures in parallel, online, and in label-free conditions the extracellular acidification rates (with pH-sensitive field effect transistors [ISFETs]), the cellular oxygen consumption (with amperometric electrode structures [Clark-type sensors]), and cell adhesion (with impedimetric interdigitated electrode structures [IDESs]). The experimental protocol was optimized to monitor metabolism and adhesion of the BALB/3T3 cell line. A total of 70,000 cells and a bicarbonate buffer-free running low-glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal clone serum III and 1mM Hepes were selected to maintain cells in good conditions on the chip during the measurements performed under perfusion conditions. Cells were exposed to sodium arsenite, cadmium chloride, and cis-platinum at concentrations ranging from 1 to 100 microM. The kinetics of cell response to these compounds was analyzed and suggests that the Clark-type sensors can be more sensitive than IDESs and ISFETs in detecting the presence of high chemical concentration when short exposure times (i.e., 2h) are considered. The cytotoxicity data obtained from the online measurements of acidification, respiration, and adhesion at 24h compare well, in terms of half-inhibition concentration values (IC(50)), with the ones obtained using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and colony-forming efficiency (CFE) assay. The results show a good sensitivity of the system combined with the advantages of the online and label-free detection methods that allow following cell status before, during, and after the treatment in the same experiment.

Objective: Drug resistance interpretation systems are used to select the optimal antiretroviral therapy in HIV-infected patients. It is unclear how the systems perform in predicting therapy success and failure and in how far the interpretations are affected by insufficient drug levels., Methods: The accuracy of nine different interpretation systems in predicting therapy outcomes was evaluated using virological, immunological, pharmacological, and clinical data of 130 patients treated at 13 outpatient centers. Individual susceptibility scores of the interpretation systems were converted into active drug scores (ADS) and correlated with therapy success and failure, defined as viral load reduction of equal to or more (n=66) and less than 1 log10 copies/ml (n=64) at three months after drug resistance testing., Results: Three interpretation systems considered the respective therapies as more active compared to the other interpretation systems (p<0.01). These systems predicted therapy success better than the other systems, while the others performed better in predicting therapy failure. Thus, the overall rate of correctly predicted treatment outcomes was comparable between the different systems (73.1-80.0 %). Univariate and multivariate regression analysis revealed significant correlations between the ADS of all interpretation systems and virological therapy outcomes (p<0.0001). In contrast, only three interpretation systems were significantly correlated with immunological therapy outcomes in univariate and just one in multivariate models (p<0.05). Among 128 determinations of drug levels in 64 patient samples, 19.4 % revealed no detectable drug levels. The consideration of insufficient drug levels significantly improved the prediction accuracy of all interpretation systems (p<0.005)., Conclusion: Differences between interpretation systems in predicting therapy failures and success need to be considered for future consensus algorithms. The prediction accuracy of interpretation systems can be improved by consideration of plasma drug levels.

Reliable, language-independent, short screening instruments to test for cognitive function in patients with multiple sclerosis (MS) remain rare, despite the high number of patients affected by cognitive decline. We developed a new, short screening instrument, the Faces Symbol Test (FST), and compared its diagnostic test characteristics with a composite of the Digit Symbol Substitution Test (DSST) and the Paced Auditory Serial Addition Test (PASAT), in 108 MS patients and 33 healthy controls. An Informant-Report Questionnaire, a Self-Report Questionnaire, and a neurologist's estimation of the Every Day Life Cognitive Status were also applied to the MS patients. The statistical analyses comprised of a receiver operating characteristic analysis for test accuracy and for confounding variables. The PASAT and DSST composite score estimated that 36.5% of the MS patients had cognitive impairment. The FST estimated that 40.7% of the MS patients were cognitively impaired (sensitivity 84%; specificity 85%). The FST, DSST and PASAT results were significantly correlated with the patients' physical impairment, as measured by the Expanded Disability Status Scale (EDSS). The results suggest that the FST might be a culture-free, sensitive, and practical short screening instrument for the detection of cognitive decline in patients with MS, including those in the early stages.

To characterize modes of action of substances and their cytotoxic effects Bionas GmbH has developed a new screening system to allow the continuous recording of how an active substance can act (Bionas 2500 analyzing system). In the pharmaceutical industry it is important to acquire as much information as possible about the metabolic effects of an active substance. Most classical pre-clinical studies are very expensive and time-consuming. Often they are so-called end-point tests which require many individual tests before approximate statements can be made about how an effect takes its course. With the Bionas 2500 analyzing system metabolically relevant data including oxygen consumption, acidification rate and the adhesion (cell impedance) of cells can be measured in parallel, online and label-free. Using e.g. ion-sensitive field effect-transistors (ISFET) and electrode structures it is possible to observe metabolic parameters non-invasively and continuously over longer periods of time. The system has already been established for several cell models, cell lines as well as primary cells. It also offers the advantage that regenerative effects can be observed during the same test run.

Background: Quantification of viral load (VL) is standard for monitoring HIV-1 therapy and is crucial before deciding whether to switch or to continue a current antiretroviral regimen., Methods: We compared the performance of the four most widely used commercial viral-load assays, COBAS Amplicor Monitor v1.5, Versant HIV-1 RNA 3.0, Abbott RealTime HIV-1 and Cobas AmpliPrep/Cobas TaqMan HIV-1 (CAP/CTM), in terms of intra- and inter-assay variability, as well as hands-on-time, specificity and ability to quantify group M subtypes., Results: Although linearity and correlation were confirmed for the assays and comparable sensitivity and specificity were verified for genetically diverse HIV-1 subtypes, demonstrating suitability for monitoring of HIV group M isolates, the viral loads obtained showed variations, with a mean difference of 0.1-0.4 log, depending on the system used., Conclusions: Although sensitivity and precision were confirmed for all the systems, differences between them should be taken into account when viral load monitoring of the same person is performed using different systems.

Prediction of liver toxicity and compound responses continues to be a major challenge for the pharmaceutical industry. In vitro studies on liver cells have been developed to reduce or replace animal experiments. However, most of the tests in use are based on cell lines which do not necessarily represent normal cell physiology. We compared the response of primary human hepatocytes from two donors with primary rat hepatocytes and the cell line HepG2 to the test compound acetaminophen (AAP) by measuring oxygen consumption, extracellular acidification and cell adhesion as dynamic parameters of cell metabolism. Primary human hepatocytes were cultured on collagen pre-coated sensor chips or in conventional two-dimensional cultures in chemically defined Human Hepatocyte Maintenance Medium. This medium allows cultivation of functionally differentiated hepatocytes for several weeks. Sensor chip based results were compared with conventional assays for hepatocytes like albumin release and urea release. The hepatocytes were exposed to AAP (50-2815 mg/l) for 24 h. Cell respiration was inhibited by AAP concentrations of 500 mg/l and more in all three cell types, whereas only the cellular acidification rates and cell adhesion of the rat hepatocytes and the HepG2 cells were affected by AAP. In conventional cultures of human hepatocytes, AAP had no effect on cellular viability. Whereas high doses of AAP (2815 mg/l) diminished albumin secretion by 70-80%.

There are a large variety of dopamine agonists available. Especially de novo patients are treated with dopamine agonists to avoid dyskinesia. Dopamine agonists can be subdivided into ergoline and non-ergoline derivatives. This distinction raises the question whether there are differences in the effects to treat symptoms, not only in the side effects between the individual dopamine agonists but also between these two groups. Pergolide is now considered a second line drug because of its particularly high tendency towards valvular heart disease. Some authors claim that all ergoline-derivatives may cause this problem, while own results do not necessarily support this view. We recommend performing echocardiography on those patients being treated with an ergot-derivative. New data support the view that all dopaminergic drugs may cause somnolence and that there is no preference for non-ergots. It may be that the number of gamblers is slightly higher among patients treated with pramipexole than in others. Dopamine agonists with a high affinity to D3 receptors have a good anti-anhedonic potency. In cell culture all dopamine agonists studied so far show neuroprotective properties in cell culture. The introduction of a slow-release formulation for ropinirole and the rotigotine and lisuride patches have opened new ways of continuous dopamine receptor stimulation. Taken together, dopamine agonists show individual properties and there are differences between ergot and non-ergot derivatives.

In vivo, the pH value and oxygen partial pressure are the most important physico-chemical parameters in the microenvironment of human tissues. In vitro, the extracellular acidification rate of cell cultures is an indicator of global cellular metabolism, while the rate of oxygen consumption is a measure of mitochondrial activity. Earlier approaches had the disadvantage that these two values had to be measured with two separate sensors at different loci within the tissue or cell culture. Furthermore, conventional Clark-type oxygen sensors are not very compatible for miniaturisation, making it impossible to measure at small cell volumes or even at the single cell level. We have, therefore, developed an ISFET based sensor structure which is able to measure both pH and oxygen partial pressure. This sensor structure was tested in vitro for simultaneous records of cellular acidification and respiration rates at the same site within the cell culture. This sensor is manufactured by a CMOS-process.

Although not widely practiced by oncologists, in vitro tumor chemosensitivity assays (TCA) have proved to increase the lifetime of tumor patients in prospective clinical trials. By individualizing cancer therapy, they can support the clinician's decision which is usually based on empirically retrieved data and thereby prevent inadequate chemotherapy. We present the first results of a new sensor-chip-based technology which might be useful for a multiparametric TCA. In particular, the aspect of dynamic on-line data generation on intact cellular specimens is a major difference to alternative assays. A series of experiments has been performed on cell lines and human tumor explants. Cell cultures and tumor tissue explants were placed on miniaturized silicon and glass sensor chips. The sensor data currently analyze metabolic profiles (rates of extracellular acidification and cellular oxygen consumption) and changes in cell morphology (monitoring of electric impedance). With the cell lines, drug-associated cellular signals have been detected with all three parameters, while primary explants so far caused metabolic responses only. In particular, cellular respiration or mitochondrial activity seems to be a most sensitive indicator of acute cytotoxic effects. The experimental results were achieved using different test versions. Besides giving a status report, the theoretical potential and current problems of sensor chip technology in TCA is discussed.

The identification of drug targets for pharmaceutical screening can be greatly accelerated by gene databases and expression studies. The identification of leading compounds from growing libraries is realized by high throughput screening platforms. Subsequently, for optimization and validation of identified leading compounds studies of their functionality have to be carried out, and just these functionality tests are a limiting factor. A rigorous preselection of identified compounds by in vitro cellular screening is necessary prior to using the drug candidates for the further time consuming and expensive stage, e.g. in animal models. Our efforts are focused to the parallel development, adaptation and integration of different microelectronic sensors into miniaturized biochips for a multiparametric, functional on-line analysis of living cells in physiologically environments. Parallel and on-line acquisition of data related to different cellular targets is required for advanced stages of drug screening and for economizing animal tests.

Due to their amphiphilic properties, detergents readily disrupt cellular membranes and cause rapid cytolysis. In this study we demonstrate that treatment of cells with sublytic concentrations of detergents such as Triton X-100, Nonidet P-40, n-octylglucoside and the bile salt sodium deoxycholate induce typical signs of apoptosis including DNA fragmentation and cleavage of poly(ADP-ribose) polymerase molecules. The detergent concentration required for apoptosis was below the critical micellar concentration. Induction of apoptosis was not restricted to human cells but similarly occurred in a variety of other vertebrate cell lines. Unstimulated peripheral blood mononuclear cells were susceptible to apoptosis induction by detergent suggesting that apoptosis in this circumstance is not mediated by CD95. Cell death was not due to influx of calcium from the medium. Apoptosis was blocked and cytolysis prevented by treatment with peptide inhibitors of caspases. These findings suggest a process of apoptosis that is initiated upon nonspecific alterations at the cell membrane level. Physiologic correlates of this process still have to be defined.

The pH in the cellular microenvironment (pH(M)) is an important regulator of cell-to-cell and cell-to-host interactions. Additionally the extracellular acidification rate of a cell culture is an important indicator of global cellular metabolism. In a new approach a biocompatible ion-sensitive field effect transistor (ISFET)-array was developed to measure the pH(M) close to a surface and the global extracellular acidification rate at the same time. This ISFET-array is part of a new multiparametric microsensor chip. The paper highlights some basic applications of this method for in-vitro measurements. Using a fluid perfusion system for cell culture media, it is possible to measure the pH(M) of few (five to ten) adherent tumor cells in a distance of 10-100 nm from the cell plasma membrane. Experiments showed a pH(M)-value of 6.68 +/- 0.06 pH. Further experiments suggest that both the low pH, and the extracellular acidification rate of the examined tumor cell line are mainly built up by glycolysis.

An initial event in colon cancer progression is the migration of epithelial cells through the basement membrane (BM) and the invasion of the colon submucosa, where tumour cells enter blood and lymph vessels to spread throughout the body. To interrupt this process would mean the prevention of metastasis. In order to investigate tumour cell invasion orthotopically in the human system, we established novel in vitro models which mimic normal human colon tissue (colon reproductions, CoRes) and primary colon carcinomas (artificial tumours, ArTs). These models are based on the isolated extracellular matrix (iECM) of the respective human tissues. Two isolation methods were established, the Digestion Method and the Lysis Method neither of which destroyed the characteristic architecture of the ECM found in the original tissues. BM components, i.e. laminin, fibronectin and collagen IV, were detectable in the iECM isolated with the Lysis Method but not those isolated with the Digestion Method. Scanning electron microscopic analysis of the normal colon iECM demonstrated that even if the BM was missing, the luminal surface consisted of densely packed ECM filaments which do not allow cell infiltration without degradation of the iECM. Furthermore, we demonstrated that iECM can be separately supplemented with different cell types, i.e. colorectal carcinoma cells, normal fibroblasts and immune cells at any desired concentration, combination and localisation. Therefore, these models could be used to determine the role of the BM and of the tumour cell/normal cell crosstalk in the infiltration process of human colorectal carcinoma cells.

Critical parameters to be assessed in cell culture are the number of viable cells and cell viability. Growth, product formation, toxicity effects and the overall success of cell culture can depend largely on these. With interdigitated electrode structures (IDES) adherent cells are cultured directly on a pair of interdigitated electrodes, and the impedance of the system gives insight into the adhesive behaviour of the cells. The signal is influenced by the changes in number, growth and morphological behaviour of adherently growing cells, mainly owing to the insulating effects of the cell membranes. Five different cell lines are used, and their divergent behaviour is monitored over a period of four days, from inoculation of the cells to killing of the cells at the end of the experiments. Even when the cells from close monolayers, great fluctuations in the impedance signal can be observed. Nevertheless, for a more complete description of cellular systems, other parameters, such as acidification and respiration, have to be included in the measuring system.

Microsensors provide instruments particularly suited for the noninvasive analysis of cell and tissue cultures. The outstanding benefit lies in the passive behaviour of continuously working transducers, which in turn allows the dynamic recording of function-specific cellular processes. The microsensor system presented in this paper is a modular arrangement of various planar and non-planar sensor elements surrounding small cell culture chambers. An optic access to the cultures (e.g. for high resolution light microscopy and spectro-photometric techniques) enables a parallel and comparative data acquisition. The system was originally designed for biomedical research in chemotherapy and pharmacology but it proved to be an effective device both for toxicological and environmental research.

Microsensors provide instruments particularly suited for the rapid, noninvasive and on-line analysis of cell and tissue cultures. The microsensor system presented in this paper is a modular arrangement of various planar and nonplanar sensor elements for the measurement of physiological parameters of cell cultures. An optic access to the cultures (e.g. for light microscopy and spectrophotometric techniques) is also provided for a parallel and comparative data acquisition. The system was originally designed for biomedical research in chemotherapy (predicative chemotherapy assays) and pharmacology but it turned out to be also an effective tool for toxicological and environmental research.

Human immunodeficiency virus (HIV) glycoprotein-specific CD4+ cytotoxic T lymphocytes (CTLs) lyse target cells in an MHC-restricted calcium-dependent fashion similar to the mechanism used by CD8+ CTLs. However, contact of unprimed peripheral blood CD4+ T cells with HIV glycoprotein-expressing cells has been shown to cause, in addition to cell-cell fusion, rapid cytolysis that may resemble antigen-specific cytotoxicity in the chromium release assay. In this study, the ability of glycoprotein-specific CD4+ CTLs to undergo similar fusion-related cytolysis was examined. The data obtained demonstrate that in addition to antigen-specific calcium-dependent cytotoxicity, envelope-specific CD4+ CTLs are involved in fusion-related, calcium-independent cytolysis.

The vaccinia virus (VV) strain Western Reserve B13R gene encodes a 38.5 kDa intracellular polypeptide that is non-essential for virus replication in vitro and does not affect virus virulence in a murine intranasal model. The protein has 92% amino acid identity with the cowpox virus cytokine response modifier A (crmA) protein which inhibits the interleukin (IL)-1beta converting enzyme (ICE). Here, we show that extracts from THP-1 cells infected with VV strains expressing B13R prevent the cleavage of in vitro transcribed and translated pro-IL-1beta into mature IL-1beta. Similarly, THP-1 cells infected with VVs expressing B13R process pro-IL-1beta into mature IL-1beta inefficiently in situ. Despite its inhibition of ICE, B13R does not prevent fever in infected mice, a systemic effect mediated by IL-1beta. Instead, fever is controlled by the VV IL-1beta receptor, encoded by gene B15R, and deletion of both the B13R and B15R genes did not increase the febrile response compared to deletion of B15R alone. The B13R protein does, however, block apoptosis mediated by anti-Fas antibodies or by tumour necrosis factor (TNF) and cycloheximide. Using DNA fragmentation, chromium release and microscopic analyses it was shown that cells infected with wild-type VV strain WR, or a revertant virus in which the B13R gene had been re-inserted into the B13R deletion mutant, are more resistant than uninfected cells or deletion mutant-infected cells to apoptosis mediated by anti-Fas and TNF.