Illegal synthetic dyes in spices: a Singapore case study.

Some synthetic dyes are fraudulently added into spices to appeal visually to consumers. Food regulations in several countries, including the United States, Australia, Japan and the European Union, strictly prohibit the use of unauthorised synthetic dyes in food. Nevertheless, illegal practices persist, where spices contaminated with potentially carcinogenic dyes have been documented, posing potential health risks to consumers. In the present study, 14 synthetic dyes were investigated through liquid chromatography/tandem mass spectrometry in 252 commercially available spices in the Singapore market. In 18 out of these (7.1%) at least 1 illegal dye was detected at concentrations ranging from 0.010 to 114 mg/kg. Besides potential health risks, presence of these adulterants also reflects the economic motivations behind their fraudulent use. Findings in the present study further emphasise the need for increased public awareness, stricter enforcement, and continuous monitoring of illegal synthetic dyes in spices to ensure Singapore's food safety.

Singapore's Total Diet Study (2021-2023): Study Design, Methodology, and Relevance to Ensuring Food Safety.

A total diet study is often used to evaluate a population's baseline dietary exposure to chemical hazards from across the diet. In 2021-2023, Singapore carried out a TDS, and this article presents an overview of the study design and methodological selections in Singapore's TDS, as well as its relevance to ensuring food safety. A food consumption survey was conducted on Singapore citizens and permanent residents, where food consumption patterns of the Singapore population were identified. The selection of chemical hazards and foods for inclusion in Singapore's TDS, as well as principal considerations on sampling, food preparation, and analytical testing are discussed. Commonly consumed foods by the Singapore population in food categories such as grain and grain-based products, meat and meat products, fish and seafood, vegetables, fruits, milk and dairy products were included in this study, and mean concentrations of chemicals tested in each food category were reported, with food categories possessing higher levels identified. Future work will include dietary exposure assessments for the population and analysis of the contributions by food and cooking method.

Determination of ethoxyquin by ultra-high performance liquid chromatography with tandem mass spectrometry and a Singapore survey of ethoxyquin residues in eggs, egg products and poultry.

In this study, an advanced ultra-high performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method was developed for quantifying ethoxyquin (EQ). The approach employed a distinctive antioxidant added extraction step designed to prevent ethoxyquin decomposition and maintain analytical precision. This method effectively determines residue levels of EQ in eggs, processed egg products, poultry muscle, salmon, and liquid milk. The method was shown to have a limit of quantitation (LOQ) for eggs, milk, salmon, and chicken muscle of 1.5 µg/kg, 1.9 µg/kg, 2.1 µg/kg, and 1.2 µg/kg, respectively. The recoveries of EQ ranged from 79.2% to 107.6%, with a relative standard deviation (RSD) below 8.4%. A surveillance study for the presence of EQ in different types of eggs and poultry muscle available in Singapore was conducted and a total of 140 samples were tested. EQ residues in all samples were found to be below the U.S. Food and Drug Administration (FDA) MRLs of 500 µg/kg. Some samples of salted and preserved eggs from China were detected with higher concentration of EQ.

Characteristics of Gastroenteritis Outbreaks Investigated in Singapore: 2018-2021.

There is a need to study the characteristics of outbreaks via Singapore's outbreak surveillance system to understand and identify the gaps in food safety for targeted policy interventions due to the increasing trend in gastroenteritis outbreaks and consequential increase in foodborne-related deaths and economic burden on public health systems worldwide. A total of 171 gastroenteritis outbreaks were investigated in Singapore from January 2018 to December 2021. This study analyzed the annual trend of investigated gastroenteritis outbreaks, the proportion of outbreaks by implicated sources of food, and the proportion of the type of pathogens identified from human cases, food samples, and environmental swabs collected from outbreak investigations. Among the foodborne gastroenteritis outbreaks (n = 121) investigated in Singapore, approximately 42.1% of the outbreaks had food prepared by caterers, 14.9% by restaurants, and 12.4% had food prepared by in-house kitchens. Clostridium perfringens and Salmonella were the most common causative pathogens in foodborne outbreaks throughout the analysis period. The food samples and environmental swabs collected were mostly detected for Bacillus cereus. Norovirus was the most common causative pathogen in non-foodborne outbreaks and was mainly attributable to preschools. This highlights the importance of monitoring and educating the catering industry and preschools to prevent future outbreaks.

Quantification of Pork, Chicken, Beef, and Sheep Contents in Meat Products Using Duplex Real-Time PCR.

Accurate methods for meat speciation and quantification are essential for ensuring the supply of safe and wholesome meat and composite products with animal origins to negate the potential associated hazards, aid classification of consignments at the import control system, and thwart food fraud committed for financial gain. To better enhance meat safety control and combat food fraud, this study developed two duplex real-time polymerase chain reaction (real-time PCR) systems specifically designed for chicken, pork, sheep, and beef, using single-copy, chromosomally encoded, species-specific gene sequences to accurately measure the content of each meat type in meat products. DNA extracted from the raw and boiled reference materials prepared in varying proportions (ranging from 1% to 75%) were used in the development of the duplex assay to derive calibration factors to determine the meat content in different meat products. The method was further validated using proficiency test samples and market monitoring samples. Our findings showed that this method exhibits high specificity and sensitivity, with a significant accuracy range of 0.14% to 24.07% in quantifying the four meat types in both raw and processed meat products. Validation results further confirmed the effectiveness of our method in accurately quantifying meat content. Thus, we have demonstrated the duplex qPCR assays as promising approaches for implementation in routine analysis to strengthen meat safety control systems and combat meat fraud, thereby safeguarding consumer health and trust in the meat industry.

Anti-C1s humanized monoclonal antibody SAR445088: A classical pathway complement inhibitor specific for the active form of C1s.

The objective of this study was to characterize the complement-inhibiting activity of SAR445088, a novel monoclonal antibody specific for the active form of C1s. Wieslab® and hemolytic assays were used to demonstrate that SAR445088 is a potent, selective inhibitor of the classical pathway of complement. Specificity for the active form of C1s was confirmed in a ligand binding assay. Finally, TNT010 (a precursor to SAR445088) was assessed in vitro for its ability to inhibit complement activation associated with cold agglutinin disease (CAD). TNT010 inhibited C3b/iC3b deposition on human red blood cells incubated with CAD patient serum and decreased their subsequent phagocytosis by THP-1 cells. In summary, this study identifies SAR445088 as a potential therapeutic for the treatment of classical pathway-driven diseases and supports its continued assessment in clinical trials.

Treatment burden in individuals living with and beyond cancer: A systematic review of qualitative literature.

BACKGROUND: Individuals with cancer are being given increasing responsibility for the self-management of their health and illness. In other chronic diseases, individuals who experience treatment burden are at risk of poorer health outcomes. Less is known about treatment burden and its impact on individuals with cancer. This systematic review investigated perceptions of treatment burden in individuals living with and beyond cancer. METHODS AND FINDINGS: Medline, CINAHL and EMBASE databases were searched for qualitative studies that explored treatment burden in individuals with a diagnosis of breast, prostate, colorectal, or lung cancer at any stage of their diagnostic/treatment trajectory. Descriptive and thematic analyses were conducted. Study quality was assessed using a modified CASP checklist. The review protocol was registered on PROSPERO (CRD42021145601). Forty-eight studies were included. Health management after cancer involved cognitive, practical, and relational work for patients. Individuals were motivated to perform health management work to improve life-expectancy, manage symptoms, and regain a sense of normality. Performing health care work could be empowering and gave individuals a sense of control. Treatment burden occurred when there was a mismatch between the resources needed for health management and their availability. Individuals with chronic and severe symptoms, financial challenges, language barriers, and limited social support are particularly at risk of treatment burden. For those with advanced cancer, consumption of time and energy by health care work is a significant burden. CONCLUSION: Treatment burden could be an important mediator of inequities in cancer outcomes. Many of the factors leading to treatment burden in individuals with cancer are potentially modifiable. Clinicians should consider carefully what they are asking or expecting patients to do, and the resources required, including how much patient time will be consumed.

Ultrafiltration versus Diuretics on Prognostic Cardiac and Renal Biomarkers in Acute Decompensated Heart Failure: A Systematic Review and Meta-Analysis.

Existing systematic reviews have insufficiently delineated the differing cardiac and renal profile of ultrafiltration compared to diuretics as a method of decongestion in acute decompensated heart failure. This meta-analysis will investigate the impact of ultrafiltration compared to diuretics on prognostic cardiac and renal biomarkers. We searched PubMed Central, Ovid MEDLINE®, Ovid Embase, all EBM reviews, and Web of Science Core Collection for randomised controlled trials published before 21 July 2022. Our main outcome measures were cardiac (brain natriuretic peptide and N-terminal pro-brain natriuretic peptide) and renal biomarkers (serum creatinine, serum sodium, and blood urea nitrogen). A total of 10 randomised trials were included in our analysis after screening. An inverse-variance random effects meta-analysis of the pooled results demonstrated no significant difference between ultrafiltration and diuretics for brain natriuretic peptide, N-terminal pro-brain natriuretic peptide, creatinine, sodium and long-term blood urea nitrogen. However, ultrafiltration produced statistically greater increases in blood urea nitrogen in the short-term (mean difference, 3.88; 95% confidence interval 0.59-7.17 mg/dL). Overall, ultrafiltration produces a similar impact on prognostic cardiac and renal biomarkers when compared to diuretic therapy. We highlight ultrafiltration's significant impact on short-term BUN and recommend further research to investigate more optimal protocols of ultrafiltration administration.

A Real-Time PCR Approach for Rapid Detection of Viable Salmonella Enteritidis in Shell Eggs.

Rapid and robust detection assays for Salmonella Enteritidis (SE) in shell eggs are essential to enable a quick testing turnaround time (TAT) at the earliest checkpoint and to ensure effective food safety control. Real-time polymerase chain reaction (qPCR) assays provide a workaround for the protracted lead times associated with conventional Salmonella diagnostic testing. However, DNA-based analysis cannot reliably discriminate between signals from viable and dead bacteria. We developed a strategy based on an SE qPCR assay that can be integrated into system testing to accelerate the detection of viable SE in egg-enriched cultures and verify the yielded SE isolates. The specificity of the assay was evaluated against 89 Salmonella strains, and SE was accurately identified in every instance. To define the indicator for a viable bacteria readout, viable or heat-inactivated SE were spiked into shell egg contents to generate post-enriched, artificially contaminated cultures to establish the quantification cycle (Cq) for viable SE. Our study has demonstrated that this technique could potentially be applied to accurately identify viable SE during the screening stage of naturally contaminated shell eggs following enrichment to provide an early alert, and that it consistently identified the serotypes of SE isolates in a shorter time than conventional testing.

SAR443809: a selective inhibitor of the complement alternative pathway, targeting complement factor Bb.

Dysregulated activation of the complement system is implicated in the onset or progression of several diseases. Most clinical-stage complement inhibitors target the inactive complement proteins present at high concentrations in plasma, which increases target-mediated drug disposition and necessitates high drug levels to sustain therapeutic inhibition. Furthermore, many efforts are aimed at inhibiting only terminal pathway activity, which leaves opsonin-mediated effector functions intact. We describe the discovery of SAR443809, a specific inhibitor of the alternative pathway C3/C5 convertase (C3bBb). SAR443809 selectively binds to the activated form of factor B (factor Bb) and inhibits alternative pathway activity by blocking the cleavage of C3, leaving the initiation of classical and lectin complement pathways unaffected. Ex vivo experiments with patient-derived paroxysmal nocturnal hemoglobinuria erythrocytes show that, although terminal pathway inhibition via C5 blockade can effectively inhibit hemolysis, proximal complement inhibition with SAR443809 inhibits both hemolysis and C3b deposition, abrogating the propensity for extravascular hemolysis. Finally, intravenous and subcutaneous administration of the antibody in nonhuman primates demonstrated sustained inhibition of complement activity for several weeks after injection. Overall, SAR443809 shows strong potential for treatment of alternative pathway-mediated disorders.

Consumption Patterns of Processed Foods in Singapore-A Cross-Sectional Study.

The consumption of processed foods is increasingly widespread and could have an impact on diet quality and health. Understanding the factors influencing people's eating habits is useful for assessing such impact. There are limited data on the consumption patterns of processed foods and associated factors influencing the dietary patterns in Singapore. This cross-sectional study based on a food frequency questionnaire aimed to examine how the consumption of processed foods among 2079 Singapore residents aged 18 to 89 years varies with sociodemographic factors. The analysis of the consumption by processed food groups showed that the studied factors, i.e., age, gender, ethnicity, housing and health status, all contributed to differences in processed food consumption to varying extents, with ethnicity being the key factor driving the variation. Such differences were also confirmed to a limited degree by determining another measure of consumption, i.e., a processed food variety score. The findings in this study could inform further work in relation to dietary risks.

Age-dependent changes in circulating Tfh cells influence development of functional malaria antibodies in children.

T-follicular helper (Tfh) cells are key drivers of antibodies that protect from malaria. However, little is known regarding the host and parasite factors that influence Tfh and functional antibody development. Here, we use samples from a large cross-sectional study of children residing in an area of high malaria transmission in Uganda to characterize Tfh cells and functional antibodies to multiple parasites stages. We identify a dramatic re-distribution of the Tfh cell compartment with age that is independent of malaria exposure, with Th2-Tfh cells predominating in early childhood, while Th1-Tfh cell gradually increase to adult levels over the first decade of life. Functional antibody acquisition is age-dependent and hierarchical acquired based on parasite stage, with merozoite responses followed by sporozoite and gametocyte antibodies. Antibodies are boosted in children with current infection, and are higher in females. The children with the very highest antibody levels have increased Tfh cell activation and proliferation, consistent with a key role of Tfh cells in antibody development. Together, these data reveal a complex relationship between the circulating Tfh compartment, antibody development and protection from malaria.

Anti-Gametocyte Antigen Humoral Immunity and Gametocytemia During Treatment of Uncomplicated Falciparum Malaria: A Multi-National Study.

Introduction: Understanding the human immune response to Plasmodium falciparum gametocytes and its association with gametocytemia is essential for understanding the transmission of malaria as well as progressing transmission blocking vaccine candidates. Methods: In a multi-national clinical efficacy trial of artemisinin therapies (13 sites of varying transmission over South-East Asia and Africa), we measured Immunoglobulin G (IgG) responses to recombinant P. falciparum gametocyte antigens expressed on the gametocyte plasma membrane and leading transmission blocking vaccine candidates Pfs230 (Pfs230c and Pfs230D1M) and Pfs48/45 at enrolment in 1,114 participants with clinical falciparum malaria. Mixed effects linear and logistic regression were used to determine the association between gametocyte measures (gametocytemia and gametocyte density) and antibody outcomes at enrolment. Results: Microscopy detectable gametocytemia was observed in 11% (127/1,114) of participants at enrolment, and an additional 9% (95/1,114) over the follow-up period (up to day 42) (total 20% of participants [222/1,114]). IgG levels in response to Pfs230c, Pfs48/45 and Pfs230D1M varied across study sites at enrolment (p < 0.001), as did IgG seroprevalence for anti-Pfs230c and D1M IgG (p < 0.001), but not for anti-Pfs48/45 IgG (p = 0.159). In adjusted analyses, microscopy detectable gametocytemia at enrolment was associated with an increase in the odds of IgG seropositivity to the three gametocyte antigens (Pfs230c OR [95% CI], p: 1.70 [1.10, 2.62], 0.017; Pfs48/45: 1.45 [0.85, 2.46], 0.174; Pfs230D1M: 1.70 [1.03, 2.80], 0.037), as was higher gametocyte density at enrolment (per two-fold change in gametocyte density Pfs230c OR [95% CI], p: 1.09 [1.02, 1.17], 0.008; Pfs48/45: 1.05 [0.98, 1.13], 0.185; Pfs230D1M: 1.07 [0.99, 1.14], 0.071). Conclusion: Pfs230 and Pfs48/45 antibodies are naturally immunogenic targets associated with patent gametocytemia and increasing gametocyte density across multiple malaria endemic settings, including regions with emerging artemisinin-resistant P. falciparum.

The Plasmodium falciparum parasitophorous vacuole protein P113 interacts with the parasite protein export machinery and maintains normal vacuole architecture.

Infection with Plasmodium falciparum parasites results in approximately 627,000 deaths from malaria annually. Key to the parasite's success is their ability to invade and subsequently grow within human erythrocytes. Parasite proteins involved in parasite invasion and proliferation are therefore intrinsically of great interest, as targeting these proteins could provide novel means of therapeutic intervention. One such protein is P113 which has been reported to be both an invasion protein and an intracellular protein located within the parasitophorous vacuole (PV). The PV is delimited by a membrane (PVM) across which a plethora of parasite-specific proteins are exported via the Plasmodium Translocon of Exported proteins (PTEX) into the erythrocyte to enact various immune evasion functions. To better understand the role of P113 we isolated its binding partners from in vitro cultures of P. falciparum. We detected interactions with the protein export machinery (PTEX and exported protein-interacting complex) and a variety of proteins that either transit through the PV or reside on the parasite plasma membrane. Genetic knockdown or partial deletion of P113 did not significantly reduce parasite growth or protein export but did disrupt the morphology of the PVM, suggesting that P113 may play a role in maintaining normal PVM architecture.

Development of reconstructed intestinal micronucleus cytome (RICyt) assay in 3D human gut model for genotoxicity assessment of orally ingested substances.

The micronucleus (MN) assay is widely used as part of a battery of tests applied to evaluate the genotoxic potential of chemicals, including new food additives and novel food ingredients. Micronucleus assays typically utilise homogenous in vitro cell lines which poorly recapitulate the physiology, biochemistry and genomic events in the gut, the site of first contact for ingested materials. Here we have adapted and validated the MN endpoint assay protocol for use with complex 3D reconstructed intestinal microtissues; we have named this new protocol the reconstructed intestine micronucleus cytome (RICyt) assay. Our data suggest the commercial 3D microtissues replicate the physiological, biochemical and genomic responses of native human small intestine to exogenous compounds. Tissues were shown to maintain log-phase proliferation throughout the period of exposure and expressed low background MN. Analysis using the RICyt assay protocol revealed the presence of diverse cell types and nuclear anomalies (cytome) in addition to MN, indicating evidence for comprehensive DNA damage and mode(s) of cell death reported by the assay. The assay correctly identified and discriminated direct-acting clastogen, aneugen and clastogen requiring exogenous metabolic activation, and a non-genotoxic chemical. We are confident that the genotoxic response in the 3D microtissues more closely resembles the native tissues due to the inherent tissue architecture, surface area, barrier effects and tissue matrix interactions. This proof-of-concept study highlights the RICyt MN cytome assay in 3D reconstructed intestinal microtissues is a promising tool for applications in predictive toxicology.

Adults with Plasmodium falciparum malaria have higher magnitude and quality of circulating T-follicular helper cells compared to children.

BACKGROUND: Protective malarial antibodies are acquired more rapidly in adults than children, independently of cumulative exposure, however the cellular responses mediating these differences are unknown. CD4 T-follicular helper (Tfh) cells have key roles in inducing antibodies, with Th2-Tfh cell activation associated with antibody development in malaria. Whether Tfh cell activation in malaria is age dependent is unknown and no studies have compared Tfh cell activation in children and adults with malaria. METHODS: We undertook a comprehensive study of Tfh cells, along with B cells and antibody induction in children and adults with malaria. Activation and proliferation of circulating Tfh (cTfh) cell subsets was measured ex vivo and parasite-specific Tfh cell frequencies and functions studied with Activation Induced Marker (AIM) assays and intracellular cytokine staining. FINDINGS: During acute malaria, the magnitude of cTfh cell activation was higher in adults than in children and occurred across all cTfh cell subsets in adults but was restricted only to the Th1-cTfh subset in children. Further, adults had higher levels of parasite-specific cTfh cells, and cTfh cells which produced more Th2-Tfh associated cytokine IL-4. Consistent with a role of higher Tfh cell activation in rapid immune development in adults, adults had higher activation of B cells during infection and higher induction of antibodies 7 and 28 days after malaria compared to children. INTERPRETATION: Our data provide evidence that age impacts Tfh cell activation during malaria, and that these differences may influence antibody induction after treatment. Findings have important implications for vaccine development in children. FUNDING: This word was supported by the National Health and Medical Research Council of Australia, Wellcome Trust, Charles Darwin University Menzies School of Health Research, Channel 7 Children's Research Foundation, and National Health Institute.

Safety, infectivity and immunogenicity of a genetically attenuated blood-stage malaria vaccine.

BACKGROUND: There is a clear need for novel approaches to malaria vaccine development. We aimed to develop a genetically attenuated blood-stage vaccine and test its safety, infectivity, and immunogenicity in healthy volunteers. Our approach was to target the gene encoding the knob-associated histidine-rich protein (KAHRP), which is responsible for the assembly of knob structures at the infected erythrocyte surface. Knobs are required for correct display of the polymorphic adhesion ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1), a key virulence determinant encoded by a repertoire of var genes. METHODS: The gene encoding KAHRP was deleted from P. falciparum 3D7 and a master cell bank was produced in accordance with Good Manufacturing Practice. Eight malaria naïve males were intravenously inoculated (day 0) with 1800 (2 subjects), 1.8 × 105 (2 subjects), or 3 × 106 viable parasites (4 subjects). Parasitemia was measured using qPCR; immunogenicity was determined using standard assays. Parasites were rescued into culture for in vitro analyses (genome sequencing, cytoadhesion assays, scanning electron microscopy, var gene expression). RESULTS: None of the subjects who were administered with 1800 or 1.8 × 105 parasites developed parasitemia; 3/4 subjects administered 3× 106 parasites developed significant parasitemia, first detected on days 13, 18, and 22. One of these three subjects developed symptoms of malaria simultaneously with influenza B (day 17; 14,022 parasites/mL); one subject developed mild symptoms on day 28 (19,956 parasites/mL); and one subject remained asymptomatic up to day 35 (5046 parasites/mL). Parasitemia rapidly cleared with artemether/lumefantrine. Parasitemia induced a parasite-specific antibody and cell-mediated immune response. Parasites cultured ex vivo exhibited genotypic and phenotypic properties similar to inoculated parasites, although the var gene expression profile changed during growth in vivo. CONCLUSIONS: This study represents the first clinical investigation of a genetically attenuated blood-stage human malaria vaccine. A P. falciparum 3D7 kahrp- strain was tested in vivo and found to be immunogenic but can lead to patent parasitemia at high doses. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (number: ACTRN12617000824369 ; date: 06 June 2017).

Multi-functional antibody profiling for malaria vaccine development and evaluation.

INTRODUCTION: A vaccine would greatly accelerate current global efforts toward malaria elimination. While a partially efficacious vaccine has been achieved for Plasmodium falciparum, a major bottleneck in developing highly efficacious vaccines is a lack of reliable correlates of protection, and the limited application of assays that quantify functional immune responses to evaluate and down-select vaccine candidates in pre-clinical studies and clinical trials. AREAS COVERED: In this review, we describe the important role of antibodies in immunity against malaria and detail the nature and functional activities of antibodies against the malaria-causing parasite. We highlight the growing understanding of antibody effector functions against malaria and in vitro assays to measure these functional antibody responses. We discuss the application of these assays to quantify antibody functions in vaccine development and evaluation. EXPERT OPINION: It is becoming increasingly clear that multiple antibody effector functions are involved in immunity to malaria. Therefore, we propose that evaluating vaccine candidates needs to move beyond individual assays or measuring IgG magnitude alone. Instead, vaccine evaluation should incorporate the full breadth of antibody response types and harness a wider range of assays measuring functional antibody responses. We propose a 3-tier approach to implementing assays to inform vaccine evaluation.