Differences in SARS-CoV-2 antibodies depending on age, blood group, and sex in a Swedish blood donor cohort.

This study aimed to describe differences in prevalence and the long-term presence of nucleocapsid antibodies (N-antibodies) elicited by SARS-CoV-2 infection in a Swedish blood donor population not subjected to lockdown. We tested 20,651 blood donor samples for nucleocapsid antibodies from the beginning of March 2020 and 27 months onwards using the Roche Elecsys Anti-SARS-CoV-2 assay. The proportion of positive SARS-CoV-2 antibody samples was determined each week. After the exclusions of one-time donors and subjects with incomplete data, 19,726 samples from 4003 donors remained. Differences in antibody prevalences stratified for age, sex, and blood groups (ABO and RhD) were determined, as well as antibody loss and recovery. Lower antibody prevalence was seen for older donors, blood group AB, and RhD-negative subjects. A significant decrease in antibody titer between the first and the second antibody-positive donation was seen for the whole study group, females, older subjects, blood group O, AB, and RhD-positive subjects. The titer waned below the detection limit in 60 (3.0%) of 1983 N-antibody-positive donors, and for 18 of these donors, a second episode with antibodies was detected. We showed that N-antibodies persist for months or years and that surprisingly few antibody-positive donors lost their antibodies. We also conclude that antibody prevalence in a Swedish population never subject to lockdown did not apparently differ from populations that were subject to stricter regulations.

Seroprevalence of tick-borne encephalitis virus and vaccination coverage of tick-borne encephalitis, Sweden, 2018 to 2019.

BackgroundIn Sweden, information on seroprevalence of tick-borne encephalitis virus (TBEV) in the population, including vaccination coverage and infection, is scattered. This is largely due to the absence of a national tick-borne encephalitis (TBE) vaccination registry, scarcity of previous serological studies and use of serological methods not distinguishing between antibodies induced by vaccination and infection. Furthermore, the number of notified TBE cases in Sweden has continued to increase in recent years despite increased vaccination.AimThe aim was to estimate the TBEV seroprevalence in Sweden.MethodsIn 2018 and 2019, 2,700 serum samples from blood donors in nine Swedish regions were analysed using a serological method that can distinguish antibodies induced by vaccination from antibodies elicited by infection. The regions were chosen to reflect differences in notified TBE incidence.ResultsThe overall seroprevalence varied from 9.7% (95% confidence interval (CI): 6.6-13.6%) to 64.0% (95% CI: 58.3-69.4%) between regions. The proportion of vaccinated individuals ranged from 8.7% (95% CI: 5.8-12.6) to 57.0% (95% CI: 51.2-62.6) and of infected from 1.0% (95% CI: 0.2-3.0) to 7.0% (95% CI: 4.5-10.7). Thus, more than 160,000 and 1,600,000 individuals could have been infected by TBEV and vaccinated against TBE, respectively. The mean manifestation index was 3.1%.ConclusionA difference was observed between low- and high-incidence TBE regions, on the overall TBEV seroprevalence and when separated into vaccinated and infected individuals. The estimated incidence and manifestation index argue that a large proportion of TBEV infections are not diagnosed.

Detection of subarachnoid haemorrhage with spectrophotometry of cerebrospinal fluid - a comparison of two methods.

OBJECTIVES: Spectrophotometric absorption curve analysis of cerebrospinal fluid (CSF) for oxyhaemoglobin and bilirubin is necessary to accurately diagnose subarachnoid haemorrhage (SAH) in patients with typical symptoms but with negative findings on X-ray examinations. In this study, we evaluated the performance of two methods for interpreting absorption curves; one method from the United Kingdom National External Quality Assessment Service (UK-NEQAS) and the other from the national quality assurance programme in Sweden (Equalis). METHODS: Consecutive absorbance curves (n=336) were interpreted with two different methods, and their performance was compared to the diagnosis as stated in the patient records. RESULTS: The UK-NEQAS method displayed equal sensitivity to the Equalis method, but the specificity of the UK-NEQAS method was significantly higher than the Equalis method resulting in fewer false positive results. For UK-NEQAS, a positive predictive value (PPV) of 84.6% and a negative predictive value (NPV) of 99.7% were observed, whereas the Equalis method had a PPV of 27.5% and an NPV of 99.7%. CONCLUSIONS: The semi-automated method based on the guidelines from UK-NEQAS provides an efficient and correct interpretation of absorbance curves with short turn-around times. We propose using this method for the routine interpretation of CSF spectrophotometric curves.

High-throughput immunoassays for SARS-CoV-2 - considerable differences in performance when comparing three methods.

BACKGROUND: The recently launched high-throughput assays for detecting antibodies against SARS-CoV-2 has contributed to the managing strategies for the COVID-19 pandemic. This study aimed to investigate the performance of three high-throughput assays and one rapid lateral flow test relative to regulatory authorities' recommended criteria. METHODS: A total of 315 samples, including 150 pre-pandemic samples, 152 samples from SARS-CoV-2 RT-PCR positive individuals and 13 potentially cross-reactive samples were analysed with SARS-CoV-2 IgG (Abbott, Abbott Park, IL), Elecsys Anti-SARS-CoV-2 (Roche, Solna, Sweden), LIAISON SARS-CoV-2 S1/S2 IgG (DiaSorin, Saluggia, Italy) and 2019-nCOV IgG/IgM Rapid Test (Dynamiker Biotechnology Co., Tianjin, China). RESULTS: All assays performed with a high level of specificity ranging from 96.7% to 99.3%. Sensitivity differed more between the assays, Roche exhibiting the highest sensitivity of 98.7%. The corresponding figures for Abbott, DiaSorin and Dynamiker Biotechnology were 80.9%, 89.0% and 72.4%, respectively. CONCLUSIONS: The results of the evaluated SARS-CoV-2 assays vary considerably, as well as their ability to fulfil the performance criteria proposed by regulatory authorities. Introduction into clinical use in low-prevalent settings, should, therefore, be made with caution.

Evaluation of urine dipsticks for quality control of residual erythrocytes and leukocytes in leukocyte-depleted donor plasma.

Currently used methodologies for quality control of residual leukocytes and erythrocytes in leukocyte-depleted plasma are either expensive or time-consuming. It has been proposed that urine dipsticks could be used as a screening method for residual erythrocytes. The aim was, therefore, to evaluate if urine dipsticks could be used to detect residual erythrocytes and also residual leukocytes in leukocyte-depleted plasma. Dilution series ranging over the decision limits for residual erythrocytes and leukocytes were prepared. Positive, negative and overall agreements, as well as the precision and joint frequency distributions, were calculated for five dipstick analyzers and their corresponding dipsticks. Twenty-four consecutive leukocyte-depleted donor plasma samples were also tested. None of the dipstick analyzers had both a high positive and a high negative agreement. Accordingly, none of the analyzers were able to discriminate between cell concentrations close to the decision limits. The inconsistency count revealed differences in precision between the dipstick analyzers. In the 24 consecutive donor samples, no significant correlation between the dipstick analyzers and the reference methods were found. In conclusion, urine dipsticks are not suitable for quality control of residual leukocytes and erythrocytes in leukocyte-depleted donor plasma.

Evaluation of a routine hematology analyzer for quality control of leukoreduced plasma.

BACKGROUND: Quality control of residual white blood cells (WBCs) and red blood cells (RBCs) in leukoreduced plasma is mandatory. Although technological advances have been made, analysis of quality controls using routine hematology analyzers has not generally been introduced. The aim of this study was to evaluate if the routine hematology analyzer Sysmex XN-10, (Sysmex Nordic ApS) could be used for quality control of residual WBCs and RBCs in leukoreduced plasma. STUDY DESIGN AND METHODS: Linearity, accuracy, and precision were established for two Sysmex XN-10 analyzers using spiked donor plasma. ADAM rWBC (NanoEnTek) and manual counting in the Bürker chamber (NanoEnTek) were reference methods for WBCs and RBCs, respectively. Twenty-five consecutive leukoreduced donor plasma samples were also tested. RESULTS: For WBCs, the linearity criteria were met for the ADAM rWBC, but not for the Sysmex XN-10 instruments. Precision on both Sysmex XN-10 instruments was accurate only at 6 cells/µL, and accuracy was consistently acceptable only at 5 to 6 cells/µL. The precision and accuracy of the ADAM rWBC were acceptable at 2 to 6 cells/µL. For RBCs, both Sysmex XN-10 instruments and manual counting in the Bürker chamber were linear and fulfilled the precision criteria. Accuracy was acceptable for both Sysmex instruments at 6 to 12 × 109 WBCs/L but fluctuated within the study's measuring range for the Bürker chamber. No false-positive results were seen in the 25 consecutive donor plasma samples tested. CONCLUSION: For quality control purposes of leukoreduced plasma, the Sysmex XN-10 analyzer is suitable for the enumeration of residual RBCs but not of residual WBCs.

Introduction of cost display reduces laboratory test utilization.

OBJECTIVES: To study the effects on the number of laboratory tests ordered after introduction of cost display (showing the cost in the computerized test ordering system at test ordering and test result delivery) and cost charge (requiring all primary healthcare centers to pay full laboratory costs of the ordered tests). STUDY DESIGN: The study included cost display for secondary healthcare centers (inpatient hospitals, emergency departments, and outpatient specialist providers) as well as publicly and privately operated primary healthcare centers (sites of nonemergency, nonspecialist healthcare). After 3 months, cost charge was introduced by management for all primary healthcare centers. METHODS: Information on laboratory test cost was appended to the laboratory test name in the test ordering system, resulting in cost display both at the moment of test ordering and at the presentation of the test result. Numbers of laboratory tests were obtained from the laboratory information system and calculated as tests per physician visit. Cost charge was managed through the established laboratory invoicing system. RESULTS: In the publicly operated primary healthcare centers, neither of the interventions had any effect on laboratory test volume, nor did cost display have an effect in the privately operated primary healthcare centers. However, introduction of cost charge significantly decreased laboratory test ordering in the privately operated primary healthcare centers. In contrast, secondary healthcare centers lowered test volumes when cost display was introduced. CONCLUSIONS: The results support cost awareness and cost charge as means of reducing laboratory utilization. However, the outcome varies with the setting.

Methods for counting residual leukocytes in leukocyte-depleted plasma-a comparison between a routine hematology instrument, the Nageotte chamber, flow cytometry, and a fluorescent microscopy analyzer.

BACKGROUND: Counting very low levels of leukocytes is technically challenging but mandatory for quality control of leukocyte-depleted plasma. Established assays, such as flow cytometry and counting in the Nageotte chamber, are laborious and expensive. The aim of this study was to test two alternative assays, the cerebrospinal fluid program in the routine hematology analyzer ADVIA 2120 and a fluorescence microscopy analyzer, the ADAM-rWBC. STUDY DESIGN AND METHODS: Linearity, accuracy, and precision were established for the ADVIA 2120, the ADAM-rWBC analyzer and the Nageotte chamber with flow cytometry as the reference method. Two hundred consecutive leukocyte-depleted donor plasma samples were also tested. RESULTS: The ADAM-rWBC analyzer and the Nageotte chamber fulfilled all quality requirements. Flow cytometry fulfilled the requirements for linearity and precision. The ADVIA 2120 analyzer did not fully reach the quality criteria, and flow cytometry did not reach quality criteria on accuracy. No false-positive results on donor plasma samples were recorded. CONCLUSION: The ADAM-rWBC is suitable for the purpose of quality control of residual leukocytes in leukocyte-depleted plasma. For the ADVIA 2120, further improvements and studies are needed to reach the quality requirements stated in this study.

A Novel Allosteric Activator of Free Fatty Acid 2 Receptor Displays Unique Gi-functional Bias.

The short chain fatty acid receptor FFA2 is able to stimulate signaling via both Gi- and Gq/G11-promoted pathways. These pathways are believed to control distinct physiological end points but FFA2 receptor ligands appropriate to test this hypothesis have been lacking. Herein, we characterize AZ1729, a novel FFA2 regulator that acts as a direct allosteric agonist and as a positive allosteric modulator, increasing the activity of the endogenously produced short chain fatty acid propionate in Gi-mediated pathways, but not at those transduced by Gq/G11 Using AZ1729 in combination with direct inhibitors of Gi and Gq/G11 family G proteins demonstrated that although both arms contribute to propionate-mediated regulation of phospho-ERK1/2 MAP kinase signaling in FFA2-expressing 293 cells, the Gq/G11-mediated pathway is predominant. We extend these studies by employing AZ1729 to dissect physiological FFA2 signaling pathways. The capacity of AZ1729 to act at FFA2 receptors to inhibit ß-adrenoreceptor agonist-promoted lipolysis in primary mouse adipocytes and to promote chemotaxis of isolated human neutrophils confirmed these as FFA2 processes mediated by Gi signaling, whereas, in concert with blockade by the Gq/G11 inhibitor FR900359, the inability of AZ1729 to mimic or regulate propionate-mediated release of GLP-1 from mouse colonic preparations defined this physiological response as an end point transduced via activation of Gq/G11.

GPR103 antagonists demonstrating anorexigenic activity in vivo: design and development of pyrrolo[2,3-c]pyridines that mimic the C-terminal Arg-Phe motif of QRFP26.

GPR103, a G-protein coupled receptor, has been reported to have orexigenic properties through activation by the endogenous neuropeptide ligands QRFP26 and QRFP43. Recognizing that central administration of QRFP26 and QRFP43 increases high fat food intake in rats, we decided to investigate if antagonists of GPR103 could play a role in managing feeding behaviors. Here we present the development of a new series of pyrrolo[2,3-c]pyridines as GPR103 small molecule antagonists with GPR103 affinity, drug metabolism and pharmacokinetics and safety parameters suitable for drug development. In a preclinical obesity model measuring food intake, the anorexigenic effect of a pyrrolo[2,3-c]pyridine GPR103 antagonist was demonstrated. In addition, the dynamic 3D solution structure of the C-terminal heptapeptide of the endogenous agonist QRFP26(20-26) was determined using NMR. The synthetic pyrrolo[2,3-c]pyridine antagonists were compared to this experimental structure, which displayed a possible overlay of pharmacophore features supportive for further design of GPR103 antagonists.

Identification and design of a novel series of MGAT2 inhibitors.

[Acyl CoA]monoacylglycerol acyltransferase 2 (MGAT2) is of interest as a target for therapeutic treatment of diabetes, obesity and other diseases which together constitute the metabolic syndrome. In this Letter we report our discovery and optimisation of a novel series of MGAT2 inhibitors. The development of the SAR of the series and a detailed discussion around some key parameters monitored and addressed during the lead generation phase will be given. The in vivo results from an oral lipid tolerance test (OLTT) using the MGAT2 inhibitor (S)-10, shows a significant reduction (68% inhibition relative to naїve, p<0.01) in plasma triacylglycerol (TAG) concentration.

Design and optimization of pyrazinecarboxamide-based inhibitors of diacylglycerol acyltransferase 1 (DGAT1) leading to a clinical candidate dimethylpyrazinecarboxamide phenylcyclohexylacetic acid (AZD7687).

A new series of pyrazinecarboxamide DGAT1 inhibitors was designed to address the need for a candidate drug with good potency, selectivity, and physical and DMPK properties combined with a low predicted dose in man. Rational design and optimization of this series led to the discovery of compound 30 (AZD7687), which met the project objectives for potency, selectivity, in particular over ACAT1, solubility, and preclinical PK profiles. This compound showed the anticipated excellent pharmacokinetic properties in human volunteers.

A novel series of piperazinyl-pyridine ureas as antagonists of the purinergic P2Y12 receptor.

A novel series of P2Y(12) antagonists for development of drugs within the antiplatelet area is presented. The synthesis of the piperazinyl-pyridine urea derivatives and their structure-activity relationships (SAR) are described. Several compounds showed P2Y(12) antagonistic activities in the sub-micromolar range.

Diastereoselective intramolecular SmI2-H2O-amine mediated couplings.

The SmI2-H2O-amine mixture has been shown to be effective for intramolecular couplings providing diastereoselectivities of up to 100% de in the coupling of O-cyclohexenyliodophenol derivatives into heterocycles.