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Background & Aims: Pegylated interferon alpha (pegIFNα) is commonly used for the treatment of people infected with HDV. However, its mode of action in HDV-infected cells remains elusive and only a minority of people respond to pegIFNα therapy. Herein, we aimed to assess the responsiveness of three different cloned HDV strains to pegIFNα . We used a previously cloned HDV genotype 1 strain (dubbed HDV-1a) that appeared insensitive to interferon-α in vitro , a new HDV strain (HDV-1p) we isolated from an individual achieving later sustained response to IFNα therapy, and one phylogenetically distant genotype 3 strain (HDV-3)., Methods: PegIFNα was administered to human liver chimeric mice infected with HBV and the different HDV strains or to HBV/HDV infected human hepatocytes isolated from chimeric mice. Virological parameters and host responses were analysed by qPCR, sequencing, immunoblotting, RNA in situ hybridisation and immunofluorescence staining., Results: PegIFNα treatment efficiently reduced HDV RNA viraemia (∼2-log) and intrahepatic HDV markers both in mice infected with HBV/HDV-1p and HBV/HDV-3. In contrast, HDV parameters remained unaffected by pegIFNα treatment both in mice (up to 9 weeks) and in isolated cells infected with HBV/HDV-1a. Notably, HBV viraemia was efficiently lowered (∼2-log) and human interferon-stimulated genes similarly induced in all three HBV/HDV-infected mouse groups receiving pegIFNα. Genome sequencing revealed highly conserved ribozyme and L-hepatitis D antigen post-translational modification sites among all three isolates., Conclusions: Our comparative study indicates the ability of pegIFNα to lower HDV loads in stably infected human hepatocytes in vivo and the existence of complex virus-specific determinants of IFNα responsiveness., Impact and Implications: Understanding factors counteracting HDV infections is paramount to develop curative therapies. We compared the responsiveness of three different cloned HDV strains to pegylated interferon alpha in chronically infected mice. The different responsiveness of these HDV isolates to treatment highlights a previously underestimated heterogeneity among HDV strains., Competing Interests: The authors declare no competing interests. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2023 The Author(s).)

Background & Aims: Among people living with HBV, only a subset of individuals with chronic hepatitis is in need of treatment, and this proportion varies according to the population, region, and setting. No estimates of the proportion of people who are infected with HBV and meet the treatment eligibility criteria in France are available., Methods: 552 treatment-naïve individuals with chronic HBV infection referred for the first time to a hepatology reference centre between 2008 and 2012 were prospectively included. Demographic, clinical, and laboratory data were analysed., Results: In total, 61.1% of patients were males, with a median age of 37.5 years. Moreover, 64% were born in an intermediate- or high-HBV endemicity country, and 90% were HBeAg-negative. At referral, median HBV DNA and HBsAg levels were 3.3 and 3.6 log IU/ml, respectively; 37.8% of patients had alanine aminotransferase >40 U/L, and 29.0% had moderate or severe fibrosis (≥F2), including 9.4% with cirrhosis. The most prevalent genotypes were D (34.7%), E (27.4%), and A (25.7%). Coinfections were rare: 2.4% were HIV-positive, 4.0% were HCV-positive, and 6.0% were HDV-positive. According to the 2017 EASL Clinical Practice Guidelines, using a single time point analysis, 2.7% of patients were classified as HBeAg-positive chronic infection, 6.1% as HBeAg-positive chronic hepatitis B, 26.5% as HBeAg-negative chronic hepatitis B, and 61.1% as HBeAg-negative chronic infection, whereas 3.6% patients could not be classified. The performance of HBsAg level quantification to identify individuals with HBeAg-negative chronic hepatitis B was poor. A total of 29.1% met the criteria for initiation of antiviral treatment, whereas 66.5% remained under routine clinical surveillance. Most eligible patients initiated recommended first-line therapies, including tenofovir (45.3%), entecavir (36.8%), or pegylated interferon alpha (11.6%)., Conclusions: Of all cases, 9.4% had cirrhosis at presentation and 29.1% met the 2017 EASL Clinical Practice Guidelines treatment criteria. HBsAg levels failed to accurately identify individuals with HBeAg-negative chronic infection., Lay Summary: Among French adults chronically infected with HBV referred for the first time to hepatology reference centres, about one-third had a significant liver disease. Approximately one-third of individuals met criteria for initiation of antiviral treatment based on entecavir or tenofovir or, occasionally, pegylated interferon alpha., Competing Interests: The authors declare no conflict of interest. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2022 The Author(s).)

Background & Aims: Currently available HDV PCR assays are characterized by considerable run-to-run and inter-laboratory variability. Hence, we established a quantitative reverse transcription real-time PCR (RT-qPCR) assay on the open channel of a fully automated PCR platform (cobas6800, Roche) offering improved consistency and reliability., Methods: A primer/probe-set targeting a highly conserved region upstream of the HDV antigen was adapted for use on the cobas6800. The lower limit of detection (LLOD) was determined using a dilution panel of the HDV WHO standard (n = 21/dilution). Linearity and inclusivity were tested by preparing 10-fold dilution series of cell culture-derived virus (genotype [GT]1-8; n = 5/dilution). Patient samples containing a variety of bloodborne viral pathogens were tested to confirm exclusivity (n = 60)., Results: The LLOD of the HDV utility-channel (HDV&#95;UTC) assay was determined as 3.86 IU/ml (95% CI 2.95-5.05 IU/ml) with a linear range from 10-10ˆ8 IU/ml (GT1). Linear relationships were observed for all HDV GTs with slopes ranging from -3.481 to -4.134 cycles/log and R 2 from 0.918 to 0.994. Inter-run and intra-run variability were 0.3 and 0.6 Ct (3xLLOD), respectively. No false-positive results were observed. To evaluate clinical performance, 110 serum samples of anti-HDV-Ab+ patients were analyzed using the HDV&#95;UTC and CE-IVD RoboGene assays. 58/110 and 49/110 samples were concordant positive or negative, respectively (overall agreement 97.3%). Quantitative comparison demonstrated a strong correlation (R 2 0.8733; 95% CI 0.8914-0.9609; p value <0.0001)., Conclusion: The use of highly automated, sample-to-result solutions for molecular diagnostics holds many inherent benefits over manual workflows, including improved reliability, reproducibility and dynamic scaling of testing capacity. The assay we established showed excellent analytical and clinical performance, with inclusivity for all HDV GTs and a limit of quantification of 10 IU/ml, making it a sensitive new tool for HDV screening and viral load monitoring., Lay Summary: The hepatitis delta virus (HDV) causes a severe form of inflammation in the liver. We developed a tool for molecular diagnostics, a polymerase chain reaction HDV assay that showed great performance. It can be used to improve diagnosis of HDV, as well as for monitoring treatment responses. The assay allows for quantification of the virus in the tested samples and is performed on a fully automated platform (cobas6800), which provides various benefits including less hands-on time and excellent comparability of test results., Competing Interests: Marc Lütgehetmann has received travel expenses and speakers’ honoraria (Roche Diagnostics). The other authors declare that they have no conflict of interest. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2021 The Author(s).)

Introduction: Ravulizumab (RAVUL) is a new complement inhibitor, with a difference of 4 amino acids in the heavy chain from a predecessor compound, eculizumab (ECUL)., Objectives: First, to utilize mass spectrometry (MS) to characterize RAVUL and verify differences from its predecessor and, second, to validate and implement a lab developed test (LDT) for RAVUL that will allow for quantitative therapeutic monitoring., Methods: A time-of-flight mass spectrometer (TOF-MS) was used to characterize and differentiate the molecular weight differences between RAVUL and ECUL by both digest and reduction experiments. In parallel, an LDT for RAVUL was validated and implemented utilizing IgG4 enrichment with light chain detection and quantitation on a high throughput orbitrap MS platform., Results: The TOF-MS platform allowed for the mass difference between RAVUL and ECUL to be verified along with providing a proof of concept for a new intact protein quantitation software. An LDT on an orbitrap MS was validated and implemented using intact light chain quantitation, with the limitation that it cannot differentiate between ECUL and RAVUL. The LDT has an analytical measuring range from 5 to 600 mcg/mL, inter-assay imprecision of ≤13% CV (n = 13) and accuracy with <4% error from expected values (n = 20)., Conclusion: The TOF-MS is a versatile development platform that can be used to characterize and verify the molecular weight differences between the ECUL and RAVUL heavy chains. Routine laboratory testing for RAVUL was viable using an orbitrap-MS to quantitate using the mass of the intact light chain. These two platforms, combined, provide incomparable value in development of LDTs for the clinical laboratory., (© 2021 THE AUTHORS.)

Inborn errors of propionate, cobalamin and methionine metabolism are targets for Newborn Screening (NBS) in most programs world-wide, and are primarily screened by analyzing for propionyl carnitine (C3) and methionine in dried blood spot (DBS) cards using tandem mass spectrometry (MS/MS). Single-tier NBS approaches using C3 and methionine alone lack specificity, which can lead to an increased false-positive rate if conservative cut-offs are applied to minimize the risk of missing cases. Implementation of liquid chromatography tandem mass spectrometry (LC-MS/MS) second-tier testing for 2-methylcitric acid (MCA), methylmalonic acid (MMA), and homocysteine (HCY) from the same DBS card can improve disease screening performance by reducing the false-positive rate and eliminating the need for repeat specimen collection. However, DBS analysis of MCA, MMA, and HCY by LC-MS/MS is challenging due to limited specimen size and analyte characteristics leading to a combination of low MS/MS sensitivity and poor reverse-phase chromatographic retention. Sufficient MS response and analytical performance can be achieved for MCA by amidation using DAABD-AE and by butylation for MMA and HCY. Herein we describe the validation of a second-tier dual derivatization LC-MS/MS approach to detect elevated MCA, MMA, and HCY in DBS cards for NBS. Clinical utility was demonstrated by retrospective analysis of specimens, an interlaboratory method comparison, and assessment of external proficiency samples. Imprecision was <10.8% CV, with analyte recoveries between 90.2 and 109.4%. Workflows and analytical performance characteristics of this second-tier LC-MS/MS approach are amenable to implementation in the NBS laboratory., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 THE AUTHORS. Publishing services by ELSEVIER B.V. on behalf of MSACL.)

Background & Aims: Health-related quality of life (HRQoL) determined by patient-reported outcomes (PROs) is impaired in chronic hepatitis B (CHB) and C patients, but there are no data regarding patients with chronic hepatitis D (CHD). The aim of this study was to assess PRO scores in untreated patients with CHD and compare them with those obtained for patients with CHB., Methods: Patients with CHD completed 3 PRO instruments (Chronic Liver Disease Questionnaire [CLDQ], Functional Assessment of Chronic Illness Therapy-Fatigue [FACIT-F], and Work Productivity and Activity Impairment [WPAI]), and the results were compared with those of patients mono-infected with CHB., Results: In total, 125 patients were included: 43 with CHD and 82 with CHB. Overall, baseline PROs showed differences between both groups. Several assessments, such as the worry score from CLDQ ( p  = 0.0118), functional well-being from FACIT-F ( p  = 0.0281), and activity impairment from WPAI ( p  = 0.0029) showed a significant trend to worse scores in patients with CHD than with CHB. In addition, the linear regression model supports the finding that having CHD as opposed to having CHB was a predictor of a higher worry score (CLDQ) and a higher activity impairment (WPAI)., Conclusions: In this first assessment in CHD, PROs recorded in patients with CHD showed a significant impairment in some domains of HRQoL questionnaires in comparison with those with CHB. Studies in larger cohorts with lengthier follow-up are needed to fully assess patient-reported quality of life over the course of CHD., Lay Summary: Chronic hepatitis D (CHD) is a viral disease that causes rapid evolution to liver cirrhosis, amongst other severe complications, when compared to patients with chronic hepatitis B (CHB). Health-related quality of life in chronic hepatitis C and CHB has been reported widely, but no studies have been performed on patient-reported outcomes in patients with CHD. Results showed that CHD patients reported worse outcomes in psychological domains such as worry and emotional well-being, as well as in physical domains such as abdominal symptoms, physical well-being, and activity impairment in comparison with patients with CHB., Competing Interests: MB and RE have served as a speaker and advisory board member for Gilead, Roche, and Arbutus. MRB has served as a speaker for AbbVie and Gilead. ZY has received research funds from Intercept, 10.13039/100004334Merck, 10.13039/100002491BMS, and 10.13039/100004340Siemens, and has served as consultant for Gilead, Terns, Viking, Intercept, Merck, Abbvie, Novartis, BMS, Shionogi, and Siemens. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2021 The Authors.)

There is mounting evidence of systemic inflammation in post-traumatic stress disorder (PTSD) and Parkinson's disease (PD), yet inconsistency and a lack of replicability in findings of putative biological markers have delayed progress in this space. Variability in performance between platforms may contribute to the lack of consensus in the biomarker literature, as has been seen for a number of psychiatric disorders, including PTSD. Thus, there is a need for high-performance, scalable, and validated platforms for the discovery and development of biomarkers of inflammation for use in drug development and as clinical diagnostics. To identify the best platform for use in future biomarker discovery efforts, we conducted a comprehensive cross-platform and cross-assay evaluation across five leading platform technologies. This initial assessment focused on four cytokines that have been implicated PTSD - interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ. To assess platform performance and understand likely measurements in individuals with brain disorders, serum and plasma samples were obtained from individuals with PTSD (n = 13) or Parkinson's Disease (n = 14) as well as healthy controls (n = 5). We compared platform performance across a number of common analytic parameters, including assay precision, sensitivity, frequency of endogenous analyte detection (FEAD), correlation between platforms, and parallelism in measurement of cytokines using a serial dilution series. The single molecule array (Simoa™) ultra-sensitive platform (Quanterix), MESO V-Plex (Mesoscale Discovery), and Luminex xMAP® (Myriad) were conducted by their respective vendors, while Luminex® and Quantikine® high-sensitivity ELISA assays were evaluated by R&D System's Biomarker Testing Services. The assay with the highest sensitivity in detecting endogenous analytes across all analytes and clinical populations (i.e. the highest FEAD), was the Simoa™ platform. In contrast, more variable performance was observed for MESO V-plex, R&D Luminex® and Quantikine®, while Myriad's Luminex xMAP® exhibited low FEAD across all analytes and samples. Simoa™ also demonstrated high precision in detecting endogenous cytokines, as reflected in < 20 percent coefficient of variance (%CV) across replicate runs for samples from the healthy controls, PTSD patients, and PD patients. In contrast, MESO V-Plex, R&D Luminex® and Quantikine® had variable performance in terms of precision across cytokines. Myriad Luminex xMAP® could not be included in precision estimates because the vendor did not run samples in duplicate. For cross-platform performance comparisons, the highest cross-platform correlations were observed for IL-6 such that all platforms - except for Myriad's Luminex xMAP® - had strong correlations with one another in measurements of IL-6 (r range = 0.59 - 0.86). For the other cytokines, there was low to no correlation across platforms, such that reported measurements of IL-1β, TNF-α, and IFN-γ varied across assays. Taken together, these findings provide novel evidence that the choice of immunoassay could greatly impact reported cytokine findings. The current study provides crucial information on the variability in performance between platforms and across immunoassays that may help inform the selection of assay in future research studies. Further, the results emphasize the need for performing comparative evaluations of immunoassays as new technologies emerge over time, particularly given the lack of reference standards for the quantitative assessments of cytokines., Competing Interests: AJ has been an advisor to Quanterix, Inc and holds stock options. The other authors (HCL, ACP, LC, NPD, and MH) declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Author(s).)

Background: Therapeutic drug monitoring (TDM) of antidepressants is important to ensure compliance and to rule out pharmacokinetic abnormalities. Therefore, reliable methods for quantification are important for clinical laboratories. Most of the currently used mass spectrometry methods use triple quadrupoles as mass analyzers. We aimed to develop a method using high-resolution mass spectrometry (HRMS) and wanted to test the suitability of this analyzer for quantitative TDM assays. This would be beneficial since HRMS instruments can also be used for metabolomics and protein analysis and, thus, many different analyses could be run on one instrument., Methods: After manual protein precipitation of serum samples, further sample clean-up was achieved using a Turbo Flow column preconnected to the analytical LC column. Stable isotope-labelled counterparts of the target analytes were used as internal standards. For detection, we used a Q Exactive Focus Orbitrap mass spectrometer operating in full-scan mode. Ionization was performed in positive ESI., Results: Accuracy, recovery, and matrix effect were acceptable for all analytes. The method demonstrated outstanding precision (within-run imprecision <4.5%, between-run imprecision <7.5%). The selectivity of the method was ensured by chromatographic separation of all isobaric compounds. Close agreement between Orbitrap and triple stage based quantification was observed in a comparison measurement of leftover patient samples., Conclusions: We have established a selective method for the quantification of antidepressants with outstanding precision using a high-resolution Orbitrap mass spectrometer. The applicability of HRMS instruments to TDM was demonstrated., Competing Interests: The work was supported by Thermo Fisher Scientific providing the instrument for the duration of the study and by RECIPE providing the internal standard mixes; no financial grants or honoraria were provided., (© 2019 The Association for Mass Spectrometry: Applications to the Clinical Lab (MSACL). Published by Elsevier B.V. All rights reserved.)

Objectives: Lysyl oxidase like 2 (LOXL2) is associated with poor prognosis in idiopathic pulmonary disease (IPF) and cancer. We developed an Enzyme-linked immunosorbent assay (ELISA) targeting the LOXL2 neo-epitope generated through the release of the signal peptide during LOXL2 maturation., Design and Methods: An ELISA targeting the N-terminal site of the human LOXL2 was developed including technical optimization and validation steps. Serum LOXL2 was measured in patients with breast, colorectal, lung, ovarian, pancreatic and prostate cancer, melanoma, IPF and in healthy controls (n = 16)., Results: A technically robust and specific assay was developed. LOXL2 was detectable in serum from healthy controls and showed reactivity towards recombinant LOXL2. Compared to controls, LOXL2 levels were significantly (p < 0.001-0.05) elevated in serum from patients with breast, colerectal, lung, ovarian and pancreatic cancer (mean range: 49-84 ng/mL), but not in prostate cancer (mean: 36 ng/mL) and malignant melanoma patients (41 ng/mL). Serum LOXL2 was elevated in IPF patients compared to healthy controls (mean: 76.5 vs 46.8 ng/mL; p > 0.001)., Conclusions: A specific ELISA towards the N-terminal neo-epitope site in LOXL2 was developed which detected significantly elevated serum levels from patients with above-mentioned cancer types or IPF compared to healthy controls.

We present a simple and fast high-performance liquid chromatography method with fluorescence detection for the determination of the antiepileptic drug perampanel in human plasma. The chromatographic separation was performed on a Kinetex PFP (100 × 2.6 mm, 4.6 µm) column, using a mobile phase of sodium acetate 0.03 M pH 3.7 and acetonitrile (40/60, v/v), at a flow rate of 0.8 mL/min. Total chromatography time for each run was 5 min. Sample preparation (250 µL) involved only one simple precipitation step by acetonitrile spiked with mirtazapine as internal standard. The method was validated over a concentration range of 20-1000 ng/mL and successfully applied to measure perampanel concentrations in plasma samples obtained from patients with epilepsy. This assay combines the high specificity of fluorescence detection with a very simple and fast sample pretreatment and can offer real advantages over existing methods in terms of simplicity and transferability to a therapeutic drug monitoring setting.

Lactic acid is a valuable and fully degradable organic acid with promising applications in poly-lactic acid production (Taskila S and Ojamo, 2013 [1]). Despite their efficiency, the cost of the current lactic acid bio-processes is still an obstacle to this application (Miller et al., 2011 [2]). To ameliorate lactic acid producing strains, researchers are using mutations and metabolic engineering techniques, as well as medium optimization. All these studies necessitate a good and high throughput screening method. Currently, researchers mostly use HPLC methods which often necessitate sample preparation, are not stereospecific and do not allow high throughput. To help optimizing l-lactic acid production, we developed a high throughput colorimetric method inspired by the blood l-lactic acid detection method used for diagnosis (Lin et al., 1999 [3]).•Two sequential enzymatic reactions using l-lactate oxidase, peroxidase and ABTS (2,2'-azino-di-[3-ethylbenzthiazoine-sulfonate]), a chromogenic peroxidase substrate, are used to quantify l-lactate between 13.8 and 90 mg/l.•The accuracy of the method was ascertained before automation.•The method was successfully applied for the direct determination of l-lactate content in fungal culture supernatants.

The glutathione S-transferase (GST)-L1 multiplex serology assay has favorable properties for use in clinical trials and epidemiologic studies, including low cost, high throughput capacity, and low serum volume requirement. Therefore, we evaluated the GST-L1 assay as a measure of HPV16/18 vaccine immunogenicity. Our study population included 65 women selected from the Costa Rica Vaccine Trial who received the bivalent HPV16/18 virus-like particle (VLP) vaccine at the recommended 0/1/6-month schedule. We tested replicate serum samples from months 0/1/12 (i.e., after 0/1/3 doses) by GST-L1 and 3 other commonly used serology assays, VLP-ELISA, SEAP-NA, and cLIA. We calculated the percentage of women seropositive by GST-L1 by time point and HPV type (14 HPV types), and compared GST-L1 to other assays using Spearman rank correlation coefficients. After 1 vaccine dose, seropositivity by GST-L1 was 40% each for HPV16 and HPV18, increasing to 100% and 98%, respectively, after 3 doses. Seropositivity after 3 doses ranged from 32% to 69% for HPV types 31/33/45, for which partial vaccine efficacy is reported, though increases also occurred for types with no evidence for cross-protection (e.g., HPV77). GST-L1 correlated best after 3 doses with VLP-ELISA (HPV16 and HPV18 each ρ = 0.72) and SEAP-NA (HPV16 ρ = 0.65, HPV18 ρ = 0.71) (all P < 0.001); correlation was lower with cLIA. The GST-L1 is suitable for evaluating HPV16/18 vaccine immunogenicity after 3 vaccine doses, although in contrast to other assays it may classify some samples as HPV16/18 seronegative. The assay's utility is limited for lower antibody levels such as after receipt of 1 dose.