Comparative assessment of three commercial kits and in house optimized PCR assays for GMO screening in food and feed.

Screening strategies for GMO detection in food and feed are a crucial aspect in GMO testing laboratories for streamlining the analytical workflow and reducing turnaround time and costs. These strategies can be more or less complex or even be targeted according to the ingredients in the product, but whatever the choice, a good basic approach is generally based on the search for 35S promoter (P35S), nos-terminator (T-nos) and FMV promoter (P-FMV). In this study, we compare the singleplex real time PCR method for P35S, T-nos and P-FMV detection currently adopted by the Italian National Reference Laboratory for GM food and feed (NRL) with three commercial kits available on the market for giving a greater choice to consider the best approach suitable to the official control laboratories that are different from each other.•The NRL optimized singleplex PCR methods and the three commercial kits fully respect all the validation parameters criteria according to the minimum performance requirements (MPR) of ENGL [1]•Screening strategies for GMO detection in food and feed are a crucial aspect in GMO testing laboratories and being the commercial kits different from each other, the laboratory can choose the methods best suit their needs reducing turnaround time and costs.

Serosurveillance for Measles and Rubella.

Measles and rubella remain global health threats, despite the availability of safe and effective vaccines. Estimates of population immunity are crucial for achieving elimination goals and assessing the impact of vaccination programs, yet conducting well-designed serosurveys can be challenging, especially in resource-limited settings. In this review, we provide a comprehensive assessment of 130 measles and rubella studies published from January 2014 to January 2024. Methodologies and design aspects of serosurveys varied greatly, including sample size, assay type, and population demographics. Most studies utilized enzyme immunoassays for IgG detection. Sample sizes showed diverse sampling methods but favored convenience sampling despite its limitations. Studies spanned 59 countries, predominantly including adults, and revealed disparities in seroprevalence across demographics, regions, and notably among migrants and women. Age-related declines in antibodies were observed, particularly among infants, and correlations between vaccination status and seropositivity varied. We conclude with an outlook on measles and rubella serosurveillance, emphasizing the need for proper survey design and the advantages of standardized, multiplex serology assays.

Multiplex gradient immunochip for detection of post-vaccinal antibodies in poultry.

Multiplex analysis as an immunochip-in-a well format for simultaneous detection of post-vaccinal antibodies to three poultry infections (Newcastle disease, infectious bronchitis and bursal disease) in one chicken sera was developed. The immunochip had a microarray format printed on the bottom of a standard microtiter plate well and consisted of 36 microspots (d = 400 µm each) with three lines of viral antigens absorbed in a gradient of five decreasing concentrations. Optimization of assay conditions revealed the necessity of careful choice of the reaction buffer due to the high tendency of chicken IgY to exhibit unspecific binding. The best results were obtained for PBS buffer (pH 6.0) supplied with 0.1% Tween 20. Assay results were visualized by a number of coloured microspots that were correlated with the specific antibody titre in the analysed serum. High (> 8000), medium (3000-8000) or low (1000-3000) antibody titre level for each of three infections could be quickly assessed in one probe visually or with the help of smartphone. ELISA results (antibody titres) and visual gradient immunochip results interpretation (high, medium, low antibody level/titre) for 63 chicken sera with multiple levels of post-vaccinal antibodies against Newcastle disease, infectious bronchitis and bursal disease were in good correlation.

Multiplex Assays in Allergy Diagnosis: Allergy Explorer 2 versus ImmunoCAP ISAC E112i.

ImmunoCAP ISAC E112i (ISAC) and Allergy Explorer 2 (ALEX2) detect specific immunoglobulin E (IgE) reactivity. Both multiplex assays contain molecular allergens and ALEX2 additionally includes allergen extracts and inhibitors that block the binding of IgE to cross-reacting carbohydrate determinants (CCDs). This study aimed to compare the performance of ISAC and ALEX2 by determining the IgE reactivity against allergen extracts and/or allergen components and by using qualitative, semiquantitative, and quantitative analyses of all comparable allergen components in sera from 216 participants recruited in South Tyrol/Italy. For extract sensitization in ALEX2, the analysis revealed negative corresponding allergen components in 18.4% and at least one positive corresponding allergen component in 81.6% of all cases. For ISAC, the corresponding results were 23.5% and 76.5% of cases, respectively. The ALEX2 CCD inhibitor eliminated CCD-positive signals detected by ISAC in 88.5% of cases. Based on sensitization values of 0.3-14.9 ISU or kUA/L, there was good agreement between ALEX2 and ISAC, although ALEX2 showed higher values than ISAC. The addition of allergen-extract tests in ALEX2 resulted in the detection of more sensitizations than with corresponding allergen components alone. In the range of <15 ISU or kUA/L, ALEX2 may be more effective in detecting sensitizations.

Pseudotyped virus infection of multiplexed ACE2 libraries reveals SARS-CoV-2 variant shifts in receptor usage.

Pairwise compatibility between virus and host proteins can dictate the outcome of infection. During transmission, both inter- and intraspecies variabilities in receptor protein sequences can impact cell susceptibility. Many viruses possess mutable viral entry proteins and the patterns of host compatibility can shift as the viral protein sequence changes. This combinatorial sequence space between virus and host is poorly understood, as traditional experimental approaches lack the throughput to simultaneously test all possible combinations of protein sequences. Here, we created a pseudotyped virus infection assay where a multiplexed target-cell library of host receptor variants can be assayed simultaneously using a DNA barcode sequencing readout. We applied this assay to test a panel of 30 ACE2 orthologs or human sequence mutants for infectability by the original SARS-CoV-2 spike protein or the Alpha, Beta, Gamma, Delta, and Omicron BA1 variant spikes. We compared these results to an analysis of the structural shifts that occurred for each variant spike's interface with human ACE2. Mutated residues were directly involved in the largest shifts, although there were also widespread indirect effects altering interface structure. The N501Y substitution in spike conferred a large structural shift for interaction with ACE2, which was partially recreated by indirect distal substitutions in Delta, which does not harbor N501Y. The structural shifts from N501Y greatly influenced the set of animal orthologs the variant spike was capable of interacting with. Out of the thirteen non-human orthologs, ten exhibited unique patterns of variant-specific compatibility, demonstrating that spike sequence changes during human transmission can toggle ACE2 compatibility and potential susceptibility of other animal species, and cumulatively increase overall compatibilities as new variants emerge. These experiments provide a blueprint for similar large-scale assessments of protein compatibility during entry by diverse viruses. This dataset demonstrates the complex compatibility relationships that occur between variable interacting host and virus proteins.

Discovery of a novel cytokine signature for the diagnosis of autism spectrum disorder in young Arab children in Qatar.

Background: Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder characterized by impaired social interaction and communication and the occurrence of stereotyped and repetitive behaviors. Several studies have reported altered cytokine profiles in ASD and hence may serve as potential diagnostic biomarkers of the disorder. This study aims to identify diagnostic biomarkers for ASD in a well-defined study cohort in Qatar. Methods: We measured the protein levels of 45 cytokines in the plasma samples of age- and gender-matched children (2-4 years) with ASD (n = 100) and controls (n = 60) using a Luminex multiplex assay. We compared the differences in the levels of these cytokines between the two study groups and then fitted the significantly altered cytokines into a logistic regression model to examine their diagnostic potential for ASD. Results: We found elevated levels of IFN-γ, FGF-2, IL-1RA, and IL-13 and reduced levels of eotaxin, HGF, IL-1 alpha, IL-22, IL-9, MCP-1, SCF, SDF-1 alpha, VEGFA, and IP-10 in the plasma of children with ASD compared to controls. Furthermore, we observed that elevated levels of IFN-γ (odds ratio (OR) = 1.823; 95% (confidence interval) CI = 1.206, 2.755; p = 0.004) and FGF-2 (OR = 2.528; 95% CI = 1.457, 4.385; p < 0.001) were significantly associated with increased odds of ASD, whereas reduced levels of eotaxin (OR = 0.350; 95% CI = 0.160, 0.765; p = 0.008) and HGF (OR = 0.220; 95% CI = 0.070, 0.696; p = 0.010) were significantly associated with lower odds of ASD relative to controls. The combination of these four cytokines revealed an area under the curve (ROC-AUC) of 0.829 (95% CI = 0.767, 0.891; p < 0.001), which demonstrates the diagnostic accuracy of the four-cytokine signature. Conclusions: Our results identified a panel of cytokines that could discriminate between children with ASD and controls in Qatar. In addition, our findings support the predominance of a Th1 immune phenotype in ASD children and emphasize the need to validate these results in larger populations.

StratoLAMP: Label-free, multiplex digital loop-mediated isothermal amplification based on visual stratification of precipitate.

Multiplex, digital nucleic acid detections have important biomedical applications, but the multiplexity of existing methods is predominantly achieved using fluorescent dyes or probes, making the detection complicated and costly. Here, we present the StratoLAMP for label-free, multiplex digital loop-mediated isothermal amplification based on visual stratification of the precipitate byproduct. The StratoLAMP designates two sets of primers with different concentrations to achieve different precipitate yields when amplifying different nucleic acid targets. In the detection, deep learning image analysis is used to stratify the precipitate within each droplet and determine the encapsulated targets for nucleic acid quantification. We investigated the effect of the amplification reagents and process on the precipitate generation and optimized the assay conditions. We then implemented a deep-learning image analysis pipeline for droplet detection, achieving an overall accuracy of 94.3%. In the application, the StratoLAMP successfully achieved the simultaneous quantification of two nucleic acid targets with high accuracy. By eliminating the need for fluorescence, StratoLAMP represents a unique concept toward label-free, multiplex nucleic acid assays and an analytical tool with great cost-effectiveness.

Quantitative assessment of multiple pathogen exposure and immune dynamics at scale.

IMPORTANCE: Serology reveals exposure to pathogens, as well as the state of autoimmune and other clinical conditions. It is used to evaluate individuals and their histories and as a public health tool to track epidemics. Employing a variety of formats, studies nearly always perform serology by testing response to only one or a few antigens. However, clinical outcomes of new infections also depend on which previous infections may have occurred. We developed a high-throughput serology method that evaluates responses to hundreds of antigens simultaneously. It can be used to evaluate thousands of samples at a time and provide a quantitative readout. This tool will enable doctors to monitor which pathogens an individual has been exposed to and how that changes in the future. Moreover, public health officials could track populations and look for infectious trends among large populations. Testing many potential antigens at a time may also aid in vaccine development.

Optimization of the 5-plex digital PCR workflow for simultaneous monitoring of SARS-CoV-2 and other pathogenic viruses in wastewater.

Wastewater-based epidemiology is a valuable tool for monitoring pathogenic viruses in the environment, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19). While quantitative polymerase chain reaction (qPCR) is widely used for pathogen surveillance in wastewater, it can be affected by inhibition and is limited to relative quantification. Digital PCR (dPCR) offers potential solutions to these limitations. In this study, a 5-plex dPCR workflow was optimized for the simultaneous detection of SARS-CoV-2, influenza A virus, enteroviruses (EnV), and noroviruses of genogroups I (NoV-GI) and GII (NoV-GII) in wastewater samples. Wastewater samples (n = 36) were collected from a wastewater treatment plant in Japan between August and October 2022. The optimization included the evaluation of singleplex and 5-plex dPCR assays, and two different concentration methods, extraction kits, and dPCR approaches. The performance of singleplex and 5-plex dPCR assays showed comparable linearity and reliability, with the 5-plex assays showing greater efficiency. The polyethylene glycol (PEG) precipitation method showed better performance over the centrifugation method, two-step reverse transcription (RT)-dPCR over the one-step RT-dPCR, and AllPrep PowerViral DNA/RNA Kit showed better performance than the QIAamp Viral RNA Mini Kit. The optimal workflow therefore included PEG precipitation, the AllPrep PowerViral DNA/RNA Kit, and two-step RT-dPCR. This workflow was selected to monitor the presence of SARS-CoV-2 and other pathogenic viruses in wastewater samples in a 5-plex dPCR approach, yielding promising results. SARS-CoV-2 RNA was detected in the majority of samples, with NoV-GI, NoV-GII, and EnV also being detected. The successful optimization and application of the 5-plex dPCR assay for pathogen surveillance in wastewater offers significant benefits, including enhanced community health assessment and more effective responses to public health threats.

Past and present seroprevalence and disease burden estimates of Toxoplasma gondii infections in Germany: An appreciation of the role of serodiagnostics.

Toxoplasmosis is one of the major foodborne parasitic diseases in Germany, with 49% of its population chronically infected with its causative agent, Toxoplasma gondii. Although the acute disease is usually benign in immunocompetent individuals, it is a threat for immunocompromised patients as well as for fetuses of seronegative mothers. As a result of infection, congenital and ocular toxoplasmosis can have serious lifelong consequences. Here I will highlight the epidemiologic situation, from its past in the two separate parts of Germany, to its unification 30 years ago and up to the present day. The main identified risk factor for infection in Germany is thought to be the consumption of undercooked or raw meat or sausages. However, the relative impact of this risky eating habit as well as that of other risk factors are changing and are discussed and compared to the situation in the Netherlands. Finally, the importance of robust and efficient high-throughput serological assays for obtaining reliable epidemiological data, on which public health decisions can be made, is highlighted. The potential of bead-based multiplex assays, which allow the incorporation of multiple antigens with different analytical properties and thus yield additional information, are described in this context. It illustrates the interdependence of new analytic assay developments and sound epidemiology, a foundation that decades-old data from Germany did not have.

Multi-center clinical evaluation of the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay in nasopharyngeal swab specimens from symptomatic individuals.

The symptomology is overlapping for respiratory infections due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), influenza A/B viruses, and respiratory syncytial virus (RSV). Accurate detection is essential for proper medical management decisions. This study evaluated the clinical performance of the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay in nasopharyngeal swab (NPS) specimens from individuals of all ages with signs and symptoms of respiratory infection consistent with COVID-19, influenza, or RSV. Retrospective known-positive and prospectively obtained residual NPS specimens were collected during two respiratory seasons in the USA. Clinical performance was established by comparing Panther Fusion SARS-CoV-2/Flu assay results to a three-molecular assay composite comparator interpretation for SARS-CoV-2 and to the FDA-cleared Panther Fusion Flu A/B/RSV assay results for all non-SARS-CoV-2 targets. A total of 1,900 prospective and 95 retrospective NPS specimens were included in the analyses. The overall prevalence in prospectively obtained specimens was 20.7% for SARS-CoV-2, 6.7% for influenza A, and 0.7% for RSV; all influenza B-positive specimens were retrospective specimens. The positive percent agreement of the Panther Fusion assay was 96.9% (378/390) for SARS-CoV-2, 98.0% (121/123) for influenza A virus, 95.2% (20/21) for influenza B virus, and 96.6% (57/59) for RSV. The negative percent agreement was ≥98.5% for all target viruses. Specimens with discordant Panther Fusion SARS/Flu/RSV assay results all had cycle threshold values of ≥32.4 (by comparator or by Panther Fusion SARS/Flu/RSV assay). Only five co-infections were detected in the study specimens. The Panther Fusion SARS-CoV-2/Flu/RSV assay provides highly sensitive and specific detection of SARS-CoV-2, influenza A virus, influenza B virus, and RSV in NPS specimens.

Identification of Mixtures of Two Types of Body Fluids Using the Multiplex Methylation System and Random Forest Models.

OBJECTIVE: Body fluid mixtures are complex biological samples that frequently occur in crime scenes, and can provide important clues for criminal case analysis. DNA methylation assay has been applied in the identification of human body fluids, and has exhibited excellent performance in predicting single-source body fluids. The present study aims to develop a methylation SNaPshot multiplex system for body fluid identification, and accurately predict the mixture samples. In addition, the value of DNA methylation in the prediction of body fluid mixtures was further explored. METHODS: In the present study, 420 samples of body fluid mixtures and 250 samples of single body fluids were tested using an optimized multiplex methylation system. Each kind of body fluid sample presented the specific methylation profiles of the 10 markers. RESULTS: Significant differences in methylation levels were observed between the mixtures and single body fluids. For all kinds of mixtures, the Spearman's correlation analysis revealed a significantly strong correlation between the methylation levels and component proportions (1:20, 1:10, 1:5, 1:1, 5:1, 10:1 and 20:1). Two random forest classification models were trained for the prediction of mixture types and the prediction of the mixture proportion of 2 components, based on the methylation levels of 10 markers. For the mixture prediction, Model-1 presented outstanding prediction accuracy, which reached up to 99.3% in 427 training samples, and had a remarkable accuracy of 100% in 243 independent test samples. For the mixture proportion prediction, Model-2 demonstrated an excellent accuracy of 98.8% in 252 training samples, and 98.2% in 168 independent test samples. The total prediction accuracy reached 99.3% for body fluid mixtures and 98.6% for the mixture proportions. CONCLUSION: These results indicate the excellent capability and powerful value of the multiplex methylation system in the identification of forensic body fluid mixtures.

Identification of novel clusters of co-expressing cytokines in a diagnostic cytokine multiplex test.

Introduction: Cytokines are mediators of the immune system that are essential for the maintenance, development and resolution of immune responses. Beneficial immune responses depend on complex, interdependent networks of signaling and regulatory events in which individual cytokines influence the production and release of others. Since disruptions in these signaling networks are associated with a wide spectrum of diseases, cytokines have gained considerable interest as diagnostic, prognostic and precision therapy-relevant biomarkers. However, currently individual cytokines testing has limited value because the wider immune response context is often overlooked. The aim of this study was to identify specific cytokine signaling patterns associated with different diseases. Methods: Unbiased clustering analyses were performed on a clinical cytokine multiplex test using a cohort of human plasma specimens drawn from individuals with known or suspected diseases for which cytokine profiling was considered clinically indicated by the attending physician. Results and discussion: Seven clusters of co-expressing cytokines were identified, representing common patterns of immune activation. Common expression profiles of the cytokine clusters and preliminary associations of these profiles with specific diseases or disease categories were also identified. These findings increase our understanding of the immune environments underlying the clinical presentations of patients of inflammatory, autoimmune and neoplastic diseases, which could then improve diagnoses and the identification of evidence-based treatment targets.

Circulation of SARS-CoV-2 Omicron sub-lineages revealed by multiplex genotyping RT-qPCR assays for sewage surveillance.

Sewage surveillance has proven to be an essential complementary tool to clinical diagnosis in combating the COVID-19 pandemic by tracking the spread of the SARS-CoV-2 virus and evaluating infection levels in populations. With the striking spreading and continuous evolution of SARS-CoV-2 Omicron VOC that characterized with higher transmissibility and potential immune evasion, there is an urgent need for the rapid surveillance of this prevalent strain and its sub-lineages in sewage. In this study, based on three multiplex allele-specific (AS) RT-qPCR assays, we established a rapid and high-throughput detection workflow for the simultaneous discrimination of Omicron sub-lineages BA.2.2, BA.2.12.1, BA.4 and BA.5 (hereafter referred to as BA.4/BA.5) to track their community circulation in Hong Kong. All primer-probe sets in the multiplex assays could correctly discriminate and quantitate their target genotypes with high sensitivity and specificity, even when multiple variants co-existed in the sewage samples. Using the established multiplex assays, the trends of SARS-CoV-2 total viral load and variant dynamics in influent samples collected from 11 wastewater treatment plants (WWTPs) during June 2022 and September 2022, aligned with the clinical data, successfully unveiling the swift emergence and predominance of Omicron BA.4/BA.5 in Hong Kong. The study highlights the feasibility and applicability of multiplex RT-qPCR assays for monitoring epidemic trends and tracking variant displacement dynamics in sewage samples, providing a more rapid, high-throughput and cost-effective alternative to enhance the current sewage surveillance system.

Facile Method for High-throughput Identification of Stabilizing Mutations.

Characterizing the effects of mutations on stability is critical for understanding the function and evolution of proteins and improving their biophysical properties. High throughput folding and abundance assays have been successfully used to characterize missense mutations associated with reduced stability. However, screening for increased thermodynamic stability is more challenging since such mutations are rarer and their impact on assay readout is more subtle. Here, a multiplex assay for high throughput screening of protein folding was developed by combining deep mutational scanning, fluorescence-activated cell sorting, and deep sequencing. By analyzing a library of 2000 variants of Adenylate kinase we demonstrate that the readout of the method correlates with stability and that mutants with up to 13 °C increase in thermal melting temperature could be identified with low false positive rate. The discovery of many stabilizing mutations also enabled the analysis of general substitution patterns associated with increased stability in Adenylate kinase. This high throughput method to identify stabilizing mutations can be combined with functional screens to identify mutations that improve both stability and activity.

Proximity Extension Assay (PEA) Platform to Detect Vitreous Biomarkers of Diabetic Retinopathy.

Diabetic retinopathy (DR) is one of the leading causes of blindness, affecting more than 100 million people worldwide. Currently, DR prognosis and management are based mainly on biomarkers identified by direct retinal fundus observation or by imaging devices. The use of molecular biology to discover biomarkers of DR has great potential to impact the standard of care, and the vitreous humor can serve as an indirect source for those molecular biomarkers because it is rich in proteins secreted by the retina. Proximity extension assay (PEA) is a technology that combines antibody-based immunoassays with DNA-coupled methodology to obtain information on the abundance of multiple proteins while using minimal sample volume, with high specificity and sensitivity. Matched antibodies labelled with a complementary sequence of oligonucleotides are used to simultaneously bind a target protein in solution, and when in proximity, the complementary sequences on each antibody hybridize, serving as template for DNA polymerase-dependent extension and the generation of a unique double-stranded DNA "barcode." PEA works well with vitreous matrix and has great potential to support the identification of novel predictive and prognostic biomarkers of DR.

One assay to test them all: Multiplex assays for expansion of respiratory virus surveillance.

Molecular multiplex assays (MPAs) for simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza and respiratory syncytial virus (RSV) in a single RT-PCR reaction reduce time and increase efficiency to identify multiple pathogens with overlapping clinical presentation but different treatments or public health implications. Clinical performance of XpertXpress® SARS-CoV-2/Flu/RSV (Cepheid, GX), TaqPath™ COVID-19, FluA/B, RSV Combo kit (Thermo Fisher Scientific, TP), and PowerChek™ SARS-CoV-2/Influenza A&B/RSV Multiplex RT-PCR kit II (KogeneBiotech, PC) was compared to individual Standards of Care (SoC). Thirteen isolates of SARS-CoV-2, human seasonal influenza, and avian influenza served to assess limit of detection (LoD). Then, positive and negative residual nasopharyngeal specimens, collected under public health surveillance and pandemic response served for evaluation. Subsequently, comparison of effectiveness was assessed. The three MPAs confidently detect all lineages of SARS-CoV-2 and influenza viruses. MPA-LoDs vary from 1 to 2 Log10 differences from SoC depending on assay and strain. Clinical evaluation resulted in overall agreement between 97 and 100%, demonstrating a high accuracy to detect all targets. Existing differences in costs, testing burden and implementation constraints influence the choice in primary or community settings. TP, PC and GX, reliably detect SARS-CoV-2, influenza and RSV simultaneously, with reduced time-to-results and simplified workflows. MPAs have the potential to enhance diagnostics, surveillance system, and epidemic response to drive policy on prevention and control of viral respiratory infections.

A Duplex Polymerization Strategy for General, Programmable and High-Resolution Nanopore-Based Sensing.

Nanopore sensing is highly promising in single molecular analysis but their broad applications have been challenged by the limited strategies that can transduce a target-of-interest into a specific and anti-false/inference signal, especially for solid-state nanopores with relatively lower resolution and higher noise. Here we report a high-resolution signal-production concept named target-induced duplex polymerization strategy (DPS). Through linking the same or different duplex substrates (DSs) with a special linker (L) and an optional structure tag (ST), the DPS can generate target-specific DS polymers with highly controllable duration times, duration intervals and even distinguished secondary tagging currents. Experimentally, DPS mono-polymerization of single DS and co-polymerization of multiple DSs has verified the duration time of a DPS product is the sum of those for each DS monomer. Tetrahedron-DNA structures with different sizes are used as the STs to provide needle-like secondary peaks for further resolution enhancement and multiplex assay. With these examples DPS represents a general, programmable and advanced strategy that may simultaneously provide size-amplification, concentration amplification, and signal-specificity for molecular recognition. It is also promisingly in various applications regarding to single molecular investigation, such as polymerization degree, structure/side chain conformation, programmable multiplex decoding and information index.

Assessment of Enterovirus Antibodies during Early Childhood Using a Multiplex Immunoassay.

Enteroviruses are a group of positive single-stranded viruses that belong to the Picornaviridae family. They regularly infect humans and cause symptoms ranging from the common cold and hand-foot-and-mouth disease to life-threatening conditions, such as dilated cardiomyopathy and poliomyelitis. Enteroviruses have also been associated with chronic immune-mediated diseases, such as type 1 diabetes, celiac disease, and asthma. Studying these disease-pathogen connections is challenging due to the high prevalence of enterovirus infections in the population and the transient appearance of the virus during the acute infection phase, which limit the identification of the causative agent via methods based on the virus genome. Serological assays can detect the antibodies induced by acute and past infections, which is useful when direct virus detection is not possible. We describe in this immuno-epidemiological study how the antibody levels against VP1 proteins from eight different enterovirus types, representing all seven of the human infecting enterovirus species, vary over time. VP1 responses first significantly (P < 0.001) decline until 6 months of age, reflecting maternal antibodies, and they then start to increase as the infections accumulate and the immune system develops. All 58 children in this study were selected from the DiabImmnune cohort for having PCR-confirmed enterovirus infections. Additionally, we show that there is great, although not complete, cross-reactivity of VP1 proteins from different enteroviruses and that the response against 3C-pro could reasonably well reflect the recent Enterovirus infection history (ρ = 0.94, P = 0.017). The serological analysis of enterovirus antibodies in sera from children paves the way for the development of tools for monitoring the Enterovirus epidemics and associated diseases. IMPORTANCE Enteroviruses cause a wide variety of symptoms ranging from a mild rash and the common cold to paralyzing poliomyelitis. While enteroviruses are among the most common human pathogens, there is a need for new, affordable serological assays with which to study pathogen-disease connections in large cohorts, as enteroviruses have been linked to several chronic illnesses, such as type 1 diabetes mellitus and asthma exacerbations. However, proving causality remains an issue. In this study, we describe the use of an easily customizable multiplexed assay that is based on structural and nonstructural enterovirus proteins to study antibody responses in a cohort of 58 children from birth to 3 years of age. We demonstrate how declining maternal antibody levels can obscure the serological detection of enteroviruses before the age of six months and how antibody responses to nonstructural enterovirus proteins could be interesting targets for serodiagnosis.

Estimating the prevalence of two or more diseases using outcomes from multiplex group testing.

When screening a population for infectious diseases, pooling individual specimens (e.g., blood, swabs, urine, etc.) can provide enormous cost savings when compared to testing specimens individually. In the biostatistics literature, testing pools of specimens is commonly known as group testing or pooled testing. Although estimating a population-level prevalence with group testing data has received a large amount of attention, most of this work has focused on applications involving a single disease, such as human immunodeficiency virus. Modern methods of screening now involve testing pools and individuals for multiple diseases simultaneously through the use of multiplex assays. Hou et al. (2017, Biometrics, 73, 656-665) and Hou et al. (2020, Biostatistics, 21, 417-431) recently proposed group testing protocols for multiplex assays and derived relevant case identification characteristics, including the expected number of tests and those which quantify classification accuracy. In this article, we describe Bayesian methods to estimate population-level disease probabilities from implementing these protocols or any other multiplex group testing protocol which might be carried out in practice. Our estimation methods can be used with multiplex assays for two or more diseases while incorporating the possibility of test misclassification for each disease. We use chlamydia and gonorrhea testing data collected at the State Hygienic Laboratory at the University of Iowa to illustrate our work. We also provide an online R resource practitioners can use to implement the methods in this article.