Nanoluciferase-based cell fusion assay for rapid and high-throughput assessment of SARS-CoV-2-neutralizing antibodies in patient samples.

After more than two years, COVID-19 still represents a global health burden of unprecedented extent and assessing the degree of immunity of individuals against SARS-CoV-2 remains a challenge. Virus neutralization assays represent the gold standard for assessing antibody-mediated protection against SARS-CoV-2 in sera from recovered and/or vaccinated individuals. Neutralizing antibodies block the interaction of viral spike protein with human angiotensin-converting enzyme 2 (ACE2) receptor in vitro and prevent viral entry into host cells. Classical viral neutralization assays using full replication-competent viruses are restricted to specific biosafety level 3-certified laboratories, limiting their utility for routine and large-scale applications. We developed therefore a cell-fusion-based assay building on the interaction between viral spike and ACE2 receptor expressed on two different cell lines, substantially reducing biosafety risks associated with classical viral neutralization assays. This chapter describes this simple, sensitive, safe and cost-effective approach for rapid and high-throughput evaluation of SARS-CoV-2 neutralizing antibodies relying on high-affinity NanoLuc® luciferase complementation technology (HiBiT). When applied to a variety of standards and patient samples, this method yields highly reproducible results in 96-well, as well as in 384-well format. The use of novel NanoLuc® substrates with increased signal stability like Nano-Glo® Endurazine™ furthermore allows for high flexibility in assay set-up and full automatization of all reading processes. Lastly, the assay is suitable to evaluate the neutralizing capacity of sera against the existing spike variants, and potentially variants that will emerge in the future.

Whey- and Soy Protein Isolates Added to a Carrot-Tomato Juice Alter Carotenoid Bioavailability in Healthy Adults.

Recent findings suggested that proteins can differentially affect carotenoid bioaccessibility during gastro-intestinal digestion. In this crossover, randomized human trial, we aimed to confirm that proteins, specifically whey- and soy-protein isolates (WPI/SPI) impact postprandial carotenoid bioavailability. Healthy adults (n = 12 males, n = 12 females) were recruited. After 2-week washout periods, 350 g of a tomato-carrot juice mixture was served in the absence/presence of WPI or SPI (50% of the recommended dietary allowance, RDA ≈ 60 g/d). Absorption kinetics of carotenoids and triacylglycerols (TAGs) were evaluated via the triacylglycerol-rich lipoprotein (TRL) fraction response, at timed intervals up to 10 h after test meal intake, on three occasions separated by 1 week. Maximum TRL-carotenoid concentration (Cmax) and corresponding time (Tmax) were also determined. Considering both genders and carotenoids/TAGs combined, the estimated area under the curve (AUC) for WPI increased by 45% vs. the control (p = 0.018), to 92.0 ± 1.7 nmol × h/L and by 57% vs. SPI (p = 0.006). Test meal effect was significant in males (p = 0.036), but not in females (p = 0.189). In males, significant differences were found for phytoene (p = 0.026), phytofluene (p = 0.004), α-carotene (p = 0.034), and ß-carotene (p = 0.031). Cmax for total carotenoids (nmol/L ± SD) was positively influenced by WPI (135.4 ± 38.0), while significantly lowered by SPI (89.6 ± 17.3 nmol/L) vs. the control (119.6 ± 30.9, p < 0.001). Tmax did not change. The results suggest that a well-digestible protein could enhance carotenoid bioavailability, whereas the less digestible SPI results in negative effects. This is, to our knowledge, the first study finding effects of proteins on carotenoid absorption in humans.

Micronutrients and Markers of Oxidative Stress and Inflammation Related to Cardiometabolic Health: Results from the EHES-LUX Study.

Metabolic syndrome (MetS) characteristics include chronic inflammation and elevated oxidative stress. This study assessed associations between circulating concentrations of micronutrients/phytochemicals and inflammatory/oxidative stress markers with MetS and MetS components. Adults (N = 606) from the European Health Examination Survey in Luxembourg (2013-2015) were randomly selected. We performed a multivariable logistic regression model using the least absolute shrinkage and selection operator to identify MetS-associated variables. Participants with MetS had higher concentrations of C-reactive protein (CRP), 8-iso-prostaglandin F2α, leptin, insulin, and vitamins E/A, but lower concentrations of adiponectin, beta-carotene, and oxidized low-density lipoprotein. A one-unit increase in log-CRP was associated with 51% greater odds of MetS (OR = 1.51 (95% CI: 1.16, 1.98)). Adults with a one-unit increase in log-leptin were 3.1 times more likely to have MetS (3.10 (2.10, 4.72)). Women with a one-unit increase in vitamin A were associated with 3% increased odds of MetS (1.03 (1.01, 1.05)), while those with a one-unit increase in log-adiponectin were associated with 82% decreased odds (0.18 (0.07, 0.46)). Chronic inflammation best characterized adults with MetS, as CRP, adiponectin, and leptin were selected as the main MetS determinants. Micronutrients did not seem to affect MetS, except for vitamin A in women.

Relationship of oxidative stress to visceral adiposity in youth and role played by vitamin D.

BACKGROUND: Visceral adipose tissue (VAT) accumulation is a major cardiometabolic risk factor, associated with increased inflammation. Oxidative stress (OS) is also associated with inflammation and cardiometabolic issues, yet mainly through general obesity. Both OS and obesity were linked to vitamin D deficiency. OBJECTIVES: To investigate whether OS increase is associated with VAT accumulation in youth, and whether in the presence of VAT accumulation, a higher vitamin D status is associated with lower OS. METHODS: One hundred and fifty-eight youth with overweight/obesity, 7 to 17 years old, were recruited (Pediatric Clinic, Luxembourg). We assessed visceral and subcutaneous abdominal adipose tissues by magnetic resonance imaging, OS by DNA/RNA oxidative damage with ELISA and vitamin D by high-performance liquid chromatography. RESULTS: VAT was the body fat compartment the most strongly associated with OS (RPearson : 0.298; P < 10-4 ). The general linear (GLM) models assessing the relationship between OS, VAT and vitamin D concentrations showed that "Log10 OS = (0.003 × VAT) + 3.911 (R2adjusted : 0.083; P-value < 10-4 )"; "Log10 OS = (0.003 × VAT) - (0.156 × log10 vitamin D) + 4.110 (R2adjusted : 0.101; P-value < 10-4 )". After back-transformation of the log-values into normal values, the GLM showed that, for a person with an average value of VAT (40.7 cm2 ), a 10 cm2 increase in VAT would increase OS by approx. 771.833 pg/mL, after age, gender, Tanner stage and physical activity adjustment. An approximate increase of 9 ng/mL of vitamin D would counterbalance this negative effect of increased VAT. CONCLUSION: Dietary strategies improving vitamin D status should be investigated to tackle VAT and OS increase.

Etiology of viral respiratory infections in Northern Lao People's Democratic Republic.

In Lao People's Democratic Republic (PDR), acute respiratory infections overburden the health care system, but viral etiology, genetic diversity, and seasonality, especially in light of the introduction of influenza vaccination in the country, are poorly understood. From August 2010 to April 2011, 309 outpatients were recruited at the Luang Prabang Provincial Hospital covering highland Lao communities. Nasopharyngeal swabs were screened for the presence of 13 respiratory viruses. At least one virus was detected in 69.6% and dual/triple viral infections in 12.9%/1.9% of the patients. Influenza A and B viruses combined were the most frequently detected pathogens, followed by human adenovirus and respiratory syncytial virus (RSV). The other viruses were detected in less than 10% of the patients. Phylogenetic analyses on a representative set of RSV strains revealed that, while otherwise very rare, the RSV-B CB1/THB genotype cocirculated with other common genotypes. A single wave of influenza virus and RSV activity was observed during the rainy season, providing further support to influenza vaccination before the onset of the rains. This study provides recommendations for influenza vaccination that still needs optimization and highlights the need for revised guidelines for treatment and prevention of respiratory infections in Lao PDR, as well as for increased surveillance efforts.

No influence of supplemental dietary calcium intake on the bioavailability of spinach carotenoids in humans.

Dietary carotenoid intake, especially from fruits and vegetables, has been associated with a reduced incidence of several chronic diseases. However, its bioavailability can vary, depending on the food matrix and host factors. Recently, it has been suggested that divalent minerals negatively impinge on carotenoid bioavailability by reducing bile-salt and non-esterified fatty-acid levels in the gut, which normally aid in emulsifying carotenoids. The aim of the present study was to investigate whether supplemental Ca would negatively influence carotenoid absorption in humans. A total of twenty-five healthy, non-obese men (age: 20-46 years, BMI<30 kg/m2) were recruited for this postprandial, randomised, crossover, double-blinded trial. Following a randomised block design, each participant received (after 2-week washout periods), on three occasions separated by 1 week, 270 g of spinach-based meals (8·61 (sd 1·08) mg carotenoids/100 g fresh weight), supplemented with 0, 500 or 1000 mg of Ca (as calcium carbonate), with each participant acting as his or her own control. Blood samples were collected at regular postprandial intervals for up to 10 h following test meal intake, and standardised lunches were served. TAG-rich lipoprotein fractions were separated and carotenoid concentrations determined. AUC for meals without supplemented Ca were 22·72 (sem 2·78) nmol×h/l (lutein), 0·19 (sem 3·90) nmol×h/l (ß-carotene) and 2·80 (sem 1·75) nmol×h/l (ß-cryptoxanthin). No significant influence of supplementation with either 500 or 1000 mg of supplemental Ca was found. In conclusion, Ca - the most abundant divalent mineral in the diet - given at high but physiological concentrations, does not appear to have repercussions on the bioavailability of carotenoids from a spinach-based meal.

Role of genetic polymorphism in nutritional supplementation therapy in personalized medicine.

Genetic-guided nutritional supplementation therapy in personalized medicine is the type of treatment that prevents and acts against errors during the copying process of a cell's deoxyribonucleic acid (DNA), mistakes that lead to diversification in the DNA sequence at certain locations, called single nucleotide polymorphisms (SNPs). Positive results are quickly achieved using one of the four types of therapy. These types are: personalized, when individual human genetic variations drive individual treatment, preventive, with a tailored healthcare strategy and therapeutic preventive drugs and vaccines, participatory, when empowered patients make informed choices and take responsibility of their own health and predictive, using a proactive approach to health and medicine.

The distribution of hepatitis B virus genotypes in Thailand.

Phylogenetic analysis was performed on hepatitis B virus (HBV) strains obtained from 86 hepatitis B surface antigen (HBsAg) positive donors from Thailand originating throughout the country. Based on the S gene, 87.5% of strains were of genotype C while 10.5% were of genotype B, with all genotype B strains obtained from patients originating from the central or the south Thailand. No genotype B strains were found in the north of Thailand. Surprisingly, one patient was infected with a genotype H strain while another patient was infected with a genotype G strain. Complete genome sequencing and recombination analysis identified the latter as being a genotype G and C2 recombinant with the breakpoint around nucleotide position 700. The origin of the genotype G fragment was not identifiable while the genotype C2 fragment most likely came from strains circulating in Laos or Malaysia. The performance of different HBsAg diagnostic kits and HBV nucleic acid amplification technology (NAT) was evaluated. The genotype H and G/C2 recombination did not interfere with HBV detection.

Regional hyperthermia combined with chemoradiotherapy in primary or recurrent locally advanced pancreatic cancer : an open-label comparative cohort trial.

PURPOSE: To evaluate the therapeutic effect of delivering regional hyperthermia (HT) plus chemoradiotherapy (CRT) in patients suffering from locally advanced unresectable pancreatic cancer (LAPC). METHODS: Between January 2000 and December 2008, 68 patients affected by primary (56/68) or recurrent (12/68) LAPC were treated either with CRT alone or CRT plus HT. Radiotherapy (RT) consisted of 3D conformal irradiation of tumor and regional lymph nodes (dose ranged from 30 Gy/10 fractions to 66 Gy/33 fractions). Chemotherapy (CT) consisted of gemcitabine (GEM) alone or in association with either oxaliplatin, cisplatin, or 5-FU. HT was delivered twice a week, concomitant with RT. RESULTS: In the current study, 60 of the original 68 patients were included. Median overall survival (OS) was 15 months in the HT group versus 11 months in the control group (log-rank test: p = 0.025). HT did not increase CRT toxicity. CONCLUSION: HT can be added safely to CRT in LAPC, thus, resulting in slightly prolonged survival in certain cases.

European collaborative evaluation of the Enzygnost HBsAg 6.0 assay: performance on hepatitis B virus surface antigen variants.

Amino acid changes within the major antigenic determinant of the hepatitis B virus (HBV) surface antigen (HBsAg) may modify eventually the antigenic properties of the protein and may have impact on the sensitivity of diagnostic assays. Modifications in the design of an assay can, however, improve significantly its ability to detect HBV mutants. One hundred forty-seven clinical samples containing HBsAg variants, and 54 supernatants of cells expressing recombinant HBsAg mutants were tested by two generations of a commercial HBsAg test (Enzygnost® HBsAg 5.0 and 6.0, Siemens Healthcare Diagnostics Products, Marburg, Germany), and the results were compared. A significant improvement was demonstrated for the second test by comparing the mean and individual sample/cut-off values, as well as by the detection of several samples displaying amino acid changes in residues 120 and 145 of the HBsAg which were recorded as negative by the former test. The results showed that modifications in design of the assay improved considerably the ability of the test to detect HBsAg mutants, and that difficulties in detecting such HBV variants should not be expected with the routine use of the test in diagnostic laboratories and in blood transfusion centers.

Study of intra-subject random variations of stabilometric parameters.

This study of intra-subject random variations of stabilometric parameters was achieved in the context of the standardized clinical stabilometry, in use in France and Southern Europe since 1985. The outstanding interest of stabilometry to follow up patients makes the results of this study indispensable for clinicians and their international publication is particularly important as, these days, the standardization Committee for clinical stabilometry resumes its work within the International Society for Postural and Gait Research. Such a study is possible only on the topological stabilometric parameters because of the stroboscopic effect on the dynamic parameters of the sampling rate of our computerized measuring chains.

Avaliação comparativa entre o desempenho dos reagentes paratoxoplasmose AxSYM, VIDAS e VIDIA em um país latinoamericano com alta soroprevalência / Comparative evaluation of AxSYM, VIDAS and VIDIA toxoplasmosis reagent performance in a high seroprevalence Latin American country

Aims: The purpose of this study was to compare the performance of three automated immunoassays for the detection of IgM and IgG Toxoplasma gondii antibodies using sera of pregnant women living in Colombia, a Latin American country with a high seroprevalence. Methods: A total of 905 sera were tested for IgM antibodies and 914 for IgG antibodies with AxSYM, VIDAS and VIDIA immunoassays. Discrepancies were resolved by using the dye test for IgG antibodies, and the ISAGA test for IgM. Results: The overall agreement between AxSYM, VIDAS and VIDIA assays was excellent for detection of IgG and IgM antibodies, and discrepancies were relatively rare (3.6% and 5.5% of sera for IgG and IgM antibodies, respectively). The performance of the three immunoassays was similar for the detection of IgG antibodies with high sensitivity (100.00% for VIDIA, 99.59% for VIDAS, 99.38% for AxSYM) and specificity (99.04% for VIDIA, 98.82% for AxSYM, 98.57% for VIDAS). The specificity for IgM antibodies was excellent for the three immunassays (99.88% for VIDIA, 99.76% for AxSYM and VIDAS). The sensitivity of the detection of IgM antibodies was higher with VIDIA (95.12%) than with VIDAS (76.74%) and AxSYM (61.90%) assays. The correlation between IgG titers was limited between AxSYM and VIDAS assays and between AxSYM and VIDIA assays, but was excellent between VIDIA and VIDAS assays. Conclusions: Our study performed with Latin American sera confirmed the excellent specificity of AxSYM, VIDAS and VIDIA assays for the detection of IgG and IgM antibodies already reported in other countries. The sensitivity of the detection of IgG antibodies was slightly higher with VIDIA than with VIDAS and AxSYM assays. The sensitivity of the detection of IgM antibodies was higher with VIDIA than with VIDAS and AxSYM assays.

Hepatitis B virus genotype E surface antigen detection with different immunoassays and diagnostic impact of mutations in the preS/S gene.

The major neutralizing epitope, the "a" determinant of the hepatitis B virus (HBV) genotype E surface antigen (HBsAg) is most divergent from that of genotype A, which is used for preparing monoclonal antibodies used in commercially available HBV reagents. To evaluate the performance of the latest generation of HBsAg detection assays with respect to genotype E HBsAg. Three commercial assays were evaluated using sera from 200 Nigerian patients compared to the preS/S sequence of DNA positive samples. Out of 200 samples, 61 and 103 gave concordant positive and negative results between the three HBsAg assays. Of 36 samples with discordant results, 35 were confirmed negative by neutralisation. One of the three assays showed significantly high rate of false positives (29 of 35). DNA positive samples with no detectable HBsAg or reduced HBsAg detection signals (<75% of mean signal obtained with HBsAg positive samples) revealed several mutations (V14A, F46S, N48T, L49R, I49T, D51G, A53V, P54L, Q82P, F83C, L127P, A184V, T189I, S204N, V224A), mostly outside the a-determinant. Several of these mutations are found as wild type nucleotides normally in genotype A and only exceptionally in genotype E. All three assays showed comparable sensitivities for genotype E HBsAg detection (98.4-100%) but differed considerably in specificity (84-99%). Failure to detect HBsAg antigen and differences in signal intensity were mainly associated with mutations in the preS/S gene outside the "a" determinant.

Screening of HIV infection: role of molecular and immunological assays.

Due to technical improvements and new developments of immunological assays, the reliability of serological laboratory diagnosis of HIV infection has improved considerably and the residual risk, due to the diagnostic window for transfusion-transmitted HIV, has been reduced significantly. Through the addition of nucleic acid amplification tests (NAT) to blood donor screening, the residual risk can de further decreased by up to 50%, depending on the sensitivity of the NAT protocol and whether individual or pooled blood donations are screened. In-house and commercially available NAT have been implemented in blood banks as HIV only or multiplexed HIV and hepatitis B or C virus assays. As an alternative to separate antigen and antibody screening, combined fourth-generation assays have been developed in 1997, and have achieved a high degree of sensitivity and specificity. Thus, they can replace stand-alone antigen and third-generation antibody assays. While they are used in the routine diagnostics of HIV infection in many countries throughout the world, they probably represent no alternative for NAT in blood-donor screening in industrialized countries. In the next few years, technical improvements will further simplify NAT screening. While there is still some potential to improve the detection threshold of NAT, the sensitivity of the antigen module of fourth-generation assays (a lowest concentration of 3-5 pg of p24 antigen) is probably very close to its technical limit.

Diagnostic impact of the genetic variability of the hepatitis B virus surface antigen gene.

The genetic variability of hepatitis B virus (HBV) represents a challenge for the sensitivity of immunologic and molecular based assays. Genotyping studies show that the genetic diversity of HBV is very high even in industrialized countries. The analytical sensitivity of HBsAg and anti-HBs assays may be dependent on HBV genotype or subtype and could possibly lead to false negative results in samples with low-level HBsAg. It is possible that the recognition of genotypes E and F may be impaired. Immunoassays based on polyclonal capture antibody show the highest sensitivity for the recognition of recombinant mutants or serum samples harboring mutant forms of HBsAg. However, they do not guarantee full sensitivity, especially for the detection of the G145R mutation and amino acid insertions or substitutions in positions 120-123. Detection of HBsAg needs to be improved by the introduction of new HBsAg assays able to recognize so far described S gene mutants and with a lower detection threshold than current immunoassays in order to detect smallest amounts of HBsAg in low level carriers. There is also a need for more complete epidemiological data on the prevalence of HBsAg mutants especially for G145R and assays for the (differential) screening of mutants need to be developed and evaluated.

Evaluation of a new automated assay for hepatitis B surface antigen (HBsAg) detection VIDAS HBsAg Ultra.

In a multicenter study a new automated screening assay, VIDAS HBsAg Ultra (long (L) and short (S) incubation protocol (Biomérieux, Marcy l'Etoile, France), was compared to a well established test (AxSYM HBsAg v2, Abbott Diagnostics, Wiesbaden, Germany) for the detection of hepatitis B virus (HBV) surface antigen (HBsAg). A total of 32 seroconversion panels, sera from the chronic phase of infection, dilution series of the WHO standard, S gene mutants (recombinant mutants and diluted and undiluted sera harbouring mutants with single or multiple amino acid (aa) substitutions, n = 40) and isolated anti-HBc positive samples were tested for the evaluation of sensitivity. Sera from HBsAg negative blood donors, pregnant women, hospitalized patients and potentially cross-reactive samples were investigated to determine the specificity of the new assay. The VIDAS HBsAg Ultra (L+S) had a higher sensitivity than the alternative assay for the detection of acute hepatitis B in seroconversion panels. The mean time of the diagnostic window was shortened with the VIDAS HBsAg Ultra (L) and (S) in comparison with the AxSYM HBsAg v2 by 1.06 and 0.66 days, respectively. The VIDAS HBsAg Ultra (L) did not detect one diluted sample out of six bearing the single aa G145R substitution, and two out of 12 diluted samples harbouring multiple aa substitutions. The analytical sensitivity of the assays varied from one surface mutant to another. While no false positive results were obtained with the VIDAS HBsAg Ultra (L+S) among potentially interfering samples, four false positives were detected with the AxSYM HBsAg v2. The respective values for sensitivity for the VIDAS HBsAg Ultra (L), (S) and the AxSYM HBsAg v2 were 99.07%, 97.87% and 94.14%. The specificities were 100% (VIDAS HBsAg Ultra L and S) and 99.6% (AxSYM HBsAg v2). In conclusion, the VIDAS HBsAg Ultra is highly sensitive and specific and represents an improvement for the detection of HBsAg in routine diagnostic laboratories.

A new automated fourth-generation HIV screening assay with sensitive antigen detection module and high specificity.

New screening enzyme immunoassays, which permit the simultaneous detection of HIV antigens reduce the diagnostic window period between the time of immunodeficiency virus (HIV) infection and seroconversion. The VIDAS HIV DUO Ultra is an enzyme-linked fluorescent assay (ELFA) for the screening of HIV infection. It is performed with the fully automated VIDAS or mini-VIDAS instruments, which are so-called walk away systems. The detection limit is 3 pg of HIV-1 p24 Ag/mL serum. HIV antibody is detected with the same sensitivity as stand-alone third-generation antibody tests. The total incubation time is about 2 h. Results are calculated, interpreted, and printed by the VIDAS instrument. Usually, fourth-generation assays demand a special algorithm for the analysis of reactive samples. For the anti-HIV part of the assay, confirmation of reactivity should be done with an assay that lacks the p24-antigen detection module and when reactivity persists subsequently by immunoblot. For the p24-antigen part, confirmation of reactivity should be analyzed in an assay that lacks the anti-HIV detection part.

Recent developments in the diagnosis and monitoring of HBV infection and role of the genetic variability of the S gene.

Recent developments in the laboratory diagnosis of hepatitis B virus infection include the optimization of key serologic markers, including hepatitis B virus surface antigen and antihepatitis B virus core antibody, as well as the development of automated nucleic acid amplification assays. There is still a lack of standardization for nucleic acid amplification assays that are used for the monitoring of antiviral therapy and follow-up of chronic infection and the clinical significance of hepatitis B virus DNA levels need to be clarified. Although highly sensitive automated nucleic acid amplification assays for blood donor screening are available, their implementation is still subject to discussion and certain countries rejected hepatitis B virus DNA testing for blood donation due to poor cost effectiveness. Genetic variability of hepatitis B virus constitutes a major challenge for diagnosis of hepatitis B virus infection, particularly with regard to hepatitis B virus surface antigen detection, antihepatitis B virus surface antigen quantification and nucleic acid amplification assays. The performances of hepatitis B virus surface antigen enzyme immunoassays in regard to genotype and surface antigen variability need to be further improved. Polyclonal antibody-based hepatitis B virus surface antigen enzyme immunoassays, although they cannot guarantee 100% sensitivity, demonstrate superior S gene mutant recognition to assays using monoclonal capture and tracer antibodies. Isolated antihepatitis B virus core reactivity is an unusual but frequent result, which requires a test algorithm for resolution and hepatitis B virus DNA detection with sensitive nucleic acid amplification assays in order to exclude occult hepatitis B virus infection.