EU surveys insights: analytical tools, future directions, and the essential requirement for reference materials in wastewater monitoring of SARS-CoV-2, antimicrobial resistance and beyond.

BACKGROUND: Wastewater surveillance (WWS) acts as a vigilant sentinel system for communities, analysing sewage to protect public health by detecting outbreaks and monitoring trends in pathogens and contaminants. To achieve a thorough comprehension of present and upcoming practices and to identify challenges and opportunities for standardisation and improvement in WWS methodologies, two EU surveys were conducted targeting over 750 WWS laboratories across Europe and other regions. The first survey explored a diverse range of activities currently undertaken or planned by laboratories. The second survey specifically targeted methods and quality controls utilised for SARS-CoV-2 surveillance. RESULTS: The findings of the two surveys provide a comprehensive insight into the procedures and methodologies applied in WWS. In Europe, WWS primarily focuses on SARS-CoV-2 with 99% of the survey participants dedicated to this virus. However, the responses highlighted a lack of standardisation in the methodologies employed for monitoring SARS-CoV-2. The surveillance of other pathogens, including antimicrobial resistance, is currently fragmented and conducted by only a limited number of laboratories. Notably, these activities are anticipated to expand in the future. Survey replies emphasise the collective recognition of the need to enhance the accuracy of results in WWS practices, reflecting a shared commitment to advancing precision and effectiveness in WWS methodologies. CONCLUSIONS: These surveys identified a lack of standardised common procedures in WWS practices and the need for quality standards and reference materials to enhance the accuracy and reliability of WWS methods in the future. In addition, it is important to broaden surveillance efforts beyond SARS-CoV-2 to include other emerging pathogens and antimicrobial resistance to ensure a comprehensive approach to protecting public health.

Novel nuclear barcode regions for the identification of flatfish species.

The development of an efficient seafood traceability framework is crucial for the management of sustainable fisheries and the monitoring of potential substitution fraud across the food chain. Recent studies have shown the potential of DNA barcoding methods in this framework, with most of the efforts focusing on using mitochondrial targets such as the cytochrome oxidase 1 and cytochrome b genes. In this article, we show the identification of novel targets in the nuclear genome, and their associated primers, to be used for the efficient identification of flatfishes of the Pleuronectidae family. In addition, different in silico methods are described to generate a dataset of barcode reference sequences from the ever-growing wealth of publicly available sequence information, replacing, where possible, labour-intensive laboratory work. The short amplicon lengths render the analysis of these new barcode target regions ideally suited to next-generation sequencing techniques, allowing characterisation of multiple fish species in mixed and processed samples. Their location in the nucleus also improves currently used methods by allowing the identification of hybrid individuals.

Epidemiology of haemolytic uremic syndrome in children. Data from the North Italian HUS network.

UNLABELLED: Despite the severity of HUS and the fact that it represents a leading cause of acute kidney injury in children, the general epidemiology of HUS is all but well documented. The present study provides updated, population-based, purely epidemiological information on HUS in childhood from a large and densely populated area of northern Italy (9.6 million inhabitants, 1.6 million children). We systematically reviewed the files concerning patients with STEC-HUS and atypical HUS (aHUS) over a 10-year observation period (January 2003-December 2012). We included all incident cases with a documented first episode of HUS before the age of 18 years. We identified 101 cases of HUS during the 10 years. The overall mean annual incidence was 6.3 cases/million children aged <18 years (range 1.9-11.9), and 15.7/million of age-related population (MARP) among subjects aged <5 years; aHUS accounted for 11.9 % of the cases (mean incidence 0.75/MARP). The overall case fatality rate was 4.0 % (3.4 % STEC-HUS, 8.3 % aHUS). CONCLUSION: Given the public health impact of HUS, this study provides recent, population-based epidemiological data useful for healthcare planning and particularly for estimating the financial burden that healthcare providers might have to face in treating HUS, whose incidence rate seems to increase in Northern Italy. WHAT IS KNOWN: • HUS is a rare disease, but it represents the leading cause of acute kidney injury in children worldwide. • STEC-HUS (also called typical, D + HUS) is more common compared to atypical HUS, but recent, population-based epidemiological data (incidence) are scanty. What is New: • Comprehensive, population-based epidemiological data concerning both typical and atypical HUS based on a long observational period.

Correction of defective CFTR/ENaC function and tightness of cystic fibrosis airway epithelium by amniotic mesenchymal stromal (stem) cells.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with most of the mortality given by the lung disease. Human amniotic mesenchymal stromal (stem) cells (hAMSCs) hold great promise for regenerative medicine in the field of lung disease; however, their potential as therapeutics for CF lung disease has not been fully explored. In the present study, hAMSCs were analysed in co-cultures on Transwell filters with CF immortalized airway epithelial cells (CFBE41o- line) at different ratios to exploit their potency to resume basic defects associated with CF. The results show that F-actin content was increased in co-cultures as compared with CF cells and actin was reorganized to form stress fibres. Confocal microscopy studies revealed that co-cultures had a tendency of increased expression of occludin and ZO-1 at the intercellular borders, paralleled by a decrease in dextran permeability, suggestive of more organized tight junctions (TJs). Spectrofluorometric analysis of CFTR function demonstrated that hAMSC-CFBE co-cultures resumed chloride transport, in line with the appearance of the mature Band C of CFTR protein by Western blotting. Moreover, hAMSC-CFBE co-cultures, at a 1:5 ratio, showed a decrease in fluid absorption, as opposed to CFBE cell monolayers that displayed a great rate of fluid resorption from the apical side. Our data show that human amniotic MSCs can be used in co-culture with CF respiratory epithelial cells to model their engraftment into the airways and have the potential to resume a tight epithelium with partial correction of the CF phenotype.

Development of new RT-PCR assays for the specific detection of BA.2.86 SARS-CoV-2 and its descendent sublineages.

The SARS-CoV-2 BA.2.86 variant, also known as Pirola, has acquired over 30 amino acid changes in the Spike protein, evolving into >150 sublineages within ten months of its emergence. Among these, the JN.1, has been rapidly increasing globally becoming the most prevalent variant. To facilitate the identification of BA.2.86 sublineages, we designed the PiroMet-1 and PiroMet-2 assays in silico and validated them using BA.2.86 viral RNA and clinical samples to ascertain analytical specificity and sensitivity. Both assays resulted very specific with limit of detection of about 1-2 RNA copies/µL. The assays were then applied in a digital RT-PCR format to wastewater samples, combined with the OmMet assay (which identifies Omicron sublineages except BA.2.86 and its descendants) and the JRC-UCE.2 assay (which can universally recognize all SARS-CoV-2 variants). When used together with the OmMet and JRC-CoV-UCE.2 assays, the PiroMet assays accurately quantified BA.2.86 sublineages in wastewater samples. Our findings support the integration of these assays into routine SARS-CoV-2 wastewater surveillance as a timely and cost-effective complement to sequencing for monitoring the prevalence and spread of BA.2.86 sublineages within communities.

Fine characterization of the recurrent c.1584+18672A>G deep-intronic mutation in the cystic fibrosis transmembrane conductance regulator gene.

Splicing mutations account for approximately 12% of the 1,890 cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations described in cystic fibrosis (CF). However, their impact on pre-mRNA processing frequently remains unclear. An interesting opportunity to study CFTR transcripts in vivo involves the use of RNA from nasal brushings. Through this approach we previously identified a deep-intronic mutation (c.1584+18672A>G) that activates a 104-base pair (bp) out-of-frame pseudoexon by creating a donor splice site. The screening of 230 patients with CF identified c.1584+18672A>G in three additional individuals, demonstrating that it is a recurrent, and potentially overlooked, mutation among Italian patients. Haplotype analysis suggests that it originated from at least two independent events. To characterize the mutation further, a genomic region, including the activated pseudoexon and surrounding intronic sequences, was cloned into an expression vector and transfected into HeLa cells. RT-PCR analysis identified two alternative splicing products, produced by the activation of two different cryptic acceptor splice sites. One included the 104-bp pseudoexon (78.7% of transcripts), and the other led to the inclusion of a 65-bp pseudoexon (21.3% of mRNAs). The allele-specific measurement of wild-type and aberrant splicings from the nasal-brushing RNA of the three probands with genotype F508del/c.1584+18672A>G demonstrated: (1) a low level of pseudoexon inclusion in the F508del transcript (not containing the splicing mutation); (2) residual wild-type splicing in the c.1584+18672A>G mRNA; (3) the degradation of aberrant transcripts; and (4) the relative strength of the different cryptic splice sites. Interestingly, the residual wild-type splicing detected in transcripts bearing the c.1584+18672A>G mutation correlates well with the milder clinical phenotype of patients.

Detection and Quantification of Botanical Impurities in Commercial Oregano (Origanum vulgare) Using Metabarcoding and Digital PCR.

DNA technology for food authentication is already well established, and with the advent of Next Generation Sequencing (NGS) and, more specifically, metabarcoding, compositional analysis of food at the molecular level has rapidly gained popularity. This has led to several reports in the media about the presence of foreign, non-declared species in several food commodities. As herbs and spices are attractive targets for fraudulent manipulation, a combination of digital PCR and metabarcoding by NGS was employed to check the purity of 285 oregano samples taken from the European market. By using novel primers and analytical approaches, it was possible to detect and quantify both adulterants and contaminants in these samples. The results highlight the high potential of NGS for compositional analysis, although its quantitative information (read count percentages) is unreliable, and other techniques are therefore needed to complement the sequencing information for assessing authenticity ('true to the name') of food ingredients.

Study of antibiotic resistance in freshwater ecosystems with low anthropogenic impact.

This study aimed to investigate the bacterial diversity and the background level of antibiotic resistance in two freshwater ecosystems with low anthropogenic impact in order to evaluate the presence of natural antimicrobial resistance in these areas and its potential to spread downstream. Water samples from a pre-Alpine and an Apennine river (Variola and Tiber, respectively) were collected in three different sampling campaigns and bacterial diversity was assessed by 16S sequencing, while the presence of bacteria resistant to five antibiotics was screened using a culturable approach. Overall bacterial load was higher in the Tiber River compared with the Variola River. Furthermore, the study revealed the presence of resistant bacteria, especially the Tiber River showed, for each sampling, the presence of resistance to all antibiotics tested, while for the Variola River, the detected resistance was variable, comprising two or more antibiotics. Screening of two resistance genes on a total of one hundred eighteen bacterial isolates from the two rivers showed that blaTEM, conferring resistance to ß-lactam antibiotics, was dominant and present in ~58 % of isolates compared to only ~9 % for mefA/E conferring resistance to macrolides. Moreover, ß-lactam resistance was detected in various isolates showing also resistance to additional antibiotics such as macrolides, aminoglycosides and tetracyclines. These observations would suggest the presence of co-resistant bacteria even in non-anthropogenic environments and this resistance may spread from the environment to humans and/or animals.

Amniotic mesenchymal stem cells: a new source for hepatocyte-like cells and induction of CFTR expression by coculture with cystic fibrosis airway epithelial cells.

Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with lung and liver manifestations. Because of pitfalls of gene therapy, novel approaches for reconstitution of the airway epithelium and CFTR expression should be explored. In the present study, human amniotic mesenchymal stem cells (hAMSCs) were isolated from term placentas and characterized for expression of phenotypic and pluripotency markers, and for differentiation potential towards mesoderm (osteogenic and adipogenic) lineages. Moreover, hAMSCs were induced to differentiate into hepatocyte-like cells, as demonstrated by mixed function oxidase activity and expression of albumin, alpha1-antitrypsin, and CK19. We also investigated the CFTR expression in hAMSCs upon isolation and in coculture with CF airway epithelial cells. Freshly isolated hAMSCs displayed low levels of CFTR mRNA, which even decreased with culture passages. Following staining with the vital dye CM-DiI, hAMSCs were mixed with CFBE41o- respiratory epithelial cells and seeded onto permeable filters. Flow cytometry demonstrated that 33-50% of hAMSCs acquired a detectable CFTR expression on the apical membrane, a result confirmed by confocal microscopy. Our data show that amniotic MSCs have the potential to differentiate into epithelial cells of organs relevant in CF pathogenesis and may contribute to partial correction of the CF phenotype.

Use of parthenogenetic activation of human oocytes as an experimental model for evaluation of polar body based PGD assay performance.

PURPOSE: To develop an experimental model to assess the feasibility of polar body preimplantation genetic diagnosis without requiring oocyte fertilization. METHODS: First polar body was removed from donated oocytes and second polar body was biopsied after parthenogenetic activation. Molecular analysis on both polar bodies involved a fluorescent multiplex polymerase chain reaction of short-tandem repeat markers, closely linked to genes of interest. Main outcome measures were: allele segregation through polar bodies and haploid nucleus, recombination rates between alleles and frequency of Allele Drop Out. RESULTS: Twenty-six out of 39 oocytes extruded a second polar body after activation. Ninety-two percent of the first polar bodies and 20 out of 26 (77%) second polar bodies were successfully amplified. Eighty percent of first polar bodies were heterozygous for CFTR and 55% for HBB. Analysis of second polar bodies predicted the genotype of the oocytes in case of heterozygous first polar body, and validated results in homozygous cases. Frequency of allele drop out was 4%. CONCLUSIONS: Our model confirms that polar body preimplantation genetic diagnosis for single gene disorders can be evaluated using parthenogenetic oocytes and offers an option to set up procedures without requiring oocyte fertilization.

DNA Accounting: Tallying Genomes to Detect Adulterated Saffron.

The EU General Food Law not only aims at ensuring food safety but also to 'prevent fraudulent or deceptive practices; the adulteration of food; and any other practices which may mislead the consumer'. Especially the partial or complete, deliberate, and intentional substitution of valuable ingredients (e.g., Saffron) for less valuable ones is of concern. Due to the variety of products on the market an approach to detect food adulteration that works well for one species may not be easily applicable to another. Here we present a broadly applicable approach for the detection of substitution of biological materials based on digital PCR. By simultaneously measuring and forecasting the number of genome copies in a sample, fraud is detectable as a discrepancy between these two values. Apart from the choice of target gene, the procedure is identical across all species. It is scalable, rapid, and has a high dynamic range. We provide proof of concept by presenting the analysis of 141 samples of Saffron (Crocus sativus) from across the European market by DNA accounting and the verification of these results by NGS analysis.

A roadmap for the generation of benchmarking resources for antimicrobial resistance detection using next generation sequencing.

Next Generation Sequencing technologies significantly impact the field of Antimicrobial Resistance (AMR) detection and monitoring, with immediate uses in diagnosis and risk assessment. For this application and in general, considerable challenges remain in demonstrating sufficient trust to act upon the meaningful information produced from raw data, partly because of the reliance on bioinformatics pipelines, which can produce different results and therefore lead to different interpretations. With the constant evolution of the field, it is difficult to identify, harmonise and recommend specific methods for large-scale implementations over time. In this article, we propose to address this challenge through establishing a transparent, performance-based, evaluation approach to provide flexibility in the bioinformatics tools of choice, while demonstrating proficiency in meeting common performance standards. The approach is two-fold: first, a community-driven effort to establish and maintain "live" (dynamic) benchmarking platforms to provide relevant performance metrics, based on different use-cases, that would evolve together with the AMR field; second, agreed and defined datasets to allow the pipelines' implementation, validation, and quality-control over time. Following previous discussions on the main challenges linked to this approach, we provide concrete recommendations and future steps, related to different aspects of the design of benchmarks, such as the selection and the characteristics of the datasets (quality, choice of pathogens and resistances, etc.), the evaluation criteria of the pipelines, and the way these resources should be deployed in the community.

The EU one-stop-shop collection of publicly available information on COVID-19 in vitro diagnostic medical devices.

The JRC COVID-19 In Vitro Diagnostic Devices and Test Methods Database, aimed to collect in a single place all publicly available information on performance of CE-marked in vitro diagnostic medical devices (IVDs) as well as in house laboratory-developed devices and related test methods for COVID-19, is here presented. The database, manually curated and regularly updated, has been developed as a follow-up to the Communication from the European Commission "Guidelines on in vitro diagnostic tests and their performance" of 15 April 2020 and is freely accessible at https://covid-19-diagnostics.jrc.ec.europa.eu/.

Nuclear DNA barcodes for cod identification in mildly-treated and processed food products.

Gadoids are a group of fish with historical importance in the fishing industry. The high demand for cod is one of the reasons why cod products are often mislabelled, and numerous observations have been made on the replacement of Atlantic cod (Gadus morhua) by cheaper species or its illegal capture in contravention of fish quotas. Fish species identification is traditionally based on morphological features, but this may be difficult in case of heat-treated or processed products, or where the species look similar, as in the Gadoid group. DNA-based approaches (using either nuclear or mitochondrial DNA) are most commonly used in this case, due to their high specificity and to the high resilience of the target molecules to food processing techniques. In this article, we identified, using an automated screening approach, novel barcode regions and their associated primers in the nuclear genome, to be used for the efficient identification of Gadoids. The barcode regions were tested on official and commercial samples, raw or mildly treated products, like frozen, or salted, as well as pre-cooked complex mixtures and processed samples, using next-generation sequencing (NGS) technique. The method proposed could complement existing fish identification strategies in establishing an efficient framework to detect and prevent frauds along the food chain.

The challenges of designing a benchmark strategy for bioinformatics pipelines in the identification of antimicrobial resistance determinants using next generation sequencing technologies.

Next-Generation Sequencing (NGS) technologies are expected to play a crucial role in the surveillance of infectious diseases, with their unprecedented capabilities for the characterisation of genetic information underlying the virulence and antimicrobial resistance (AMR) properties of microorganisms.  In the implementation of any novel technology for regulatory purposes, important considerations such as harmonisation, validation and quality assurance need to be addressed.  NGS technologies pose unique challenges in these regards, in part due to their reliance on bioinformatics for the processing and proper interpretation of the data produced.  Well-designed benchmark resources are thus needed to evaluate, validate and ensure continued quality control over the bioinformatics component of the process.  This concept was explored as part of a workshop on "Next-generation sequencing technologies and antimicrobial resistance" held October 4-5 2017.   Challenges involved in the development of such a benchmark resource, with a specific focus on identifying the molecular determinants of AMR, were identified. For each of the challenges, sets of unsolved questions that will need to be tackled for them to be properly addressed were compiled. These take into consideration the requirement for monitoring of AMR bacteria in humans, animals, food and the environment, which is aligned with the principles of a "One Health" approach.

Effects of metabolic genotypes on intermediary biomarkers in subjects exposed to PAHS: results from the EXPAH study.

Data from the EXPAH project on PAH exposure and intermediary biomarkers were analyzed with respect to individual genotypes at seven metabolic gene loci. The GSTM1 null allele was associated with significantly higher levels of two biomarkers, malondialdehyde-2'-deoxyguanosine and benzo[a]pyrene DNA adducts in the total population from three Central and Eastern European countries. The CYP1B1 Leu/Val variant demonstrated effects on both markers of oxidative DNA damage in opposite directions, producing a higher level of M(1)dG with a trend from wild type (Leu/Leu) to heterozygotes to homozygous (Val/Val) variants, whereas the effects of these variants were reversed for 8-oxodG. Cluster Analysis was used to group composite genotypes in order to determine if combined genotypes of multiple loci could explain some of the variation seen with the biomarkers, expressed per unit of exposure, referred to as a sensitivity index. This analysis revealed two closely related genotypes each involving four of the loci (GSTM1*0/*0, CYP1A1*1*1, CYP1B1*1/*2, GSTP1*1/*1 and GSTT1*0/*0, CYP1A1*1*1, CYP1B1*1/*2, GSTP1*1/*1.) that conferred significant resistance to the DNA damaging effects of benzo[a]pyrene, measured as the level of a benzo[a]pyrene-like adduct per unit of benzo[a]pyrene exposed.

Molecular characterization of an unauthorized genetically modified Bacillus subtilis production strain identified in a vitamin B2 feed additive.

Many food and feed additives result from fermentation of genetically modified (GM) microorganisms. For vitamin B2 (riboflavin), GM Bacillus subtilis production strains have been developed and are often used. The presence of neither the GM strain nor its recombinant DNA is allowed for fermentation products placed on the EU market as food or feed additive. A vitamin B2 product (80% feed grade) imported from China was analysed. Viable B. subtilis cells were identified and DNAs of two bacterial isolates (LHL and LGL) were subjected to three whole genome sequencing (WGS) runs with different devices (MiSeq, 454 or HiSeq system). WGS data revealed the integration of a chloramphenicol resistance gene, the deletion of the endogenous riboflavin (rib) operon and presence of four putative plasmids harbouring rib operons. Event- and construct-specific real-time PCR methods for detection of the GM strain and its putative plasmids in food and feed products have been developed.

Review of prevalence of Simian Virus 40 (SV40) genomic infection in healthy subjects.

Because of the carcinogenicity of SV40 in rodents, and its possible distribution through the polio vaccine, many studies have been conducted to determine if there is an association between SV40 genomic infection and different types of cancer; sometimes, these studies included data on the prevalence of genomic infection in healthy subjects as secondary information. We reviewed all the studies that reported the prevalence of SV40 genomic infection in healthy subjects, tested by PCR based methods. The 20 articles considered here included 1103 samples from healthy subjects, with a prevalence of infection ranging from 0 to 25.6%, with high heterogeneity, and no association with the type of sample analyzed (Mantel-Haenszel OR: 0.74; 95% CI: 0.44-1.23). The wide variation in frequency pose problems in terms of study design; in fact, the representativeness of the samples used as controls in the published studies may be very limited. Larger studies on healthy subjects, tested for SV40 genomic infection at various genomic regions, conducted in different geographic areas, are needed.

Towards Plant Species Identification in Complex Samples: A Bioinformatics Pipeline for the Identification of Novel Nuclear Barcode Candidates.

Monitoring of the food chain to fight fraud and protect consumer health relies on the availability of methods to correctly identify the species present in samples, for which DNA barcoding is a promising candidate. The nuclear genome is a rich potential source of barcode targets, but has been relatively unexploited until now. Here, we show the development and use of a bioinformatics pipeline that processes available genome sequences to automatically screen large numbers of input candidates, identifies novel nuclear barcode targets and designs associated primer pairs, according to a specific set of requirements. We applied this pipeline to identify novel barcodes for plant species, a kingdom for which the currently available solutions are known to be insufficient. We tested one of the identified primer pairs and show its capability to correctly identify the plant species in simple and complex samples, validating the output of our approach.

GSTM1 [corrected] deletion modifies the levels of polycyclic aromatic hydrocarbon-DNA adducts in human sperm.

DNA adducts measured in tissues are promising markers for identifying damage in organs that could be a target for carcinogens. Polymorphisms in genes involved in polycyclic aromatic hydrocarbons (PAHs) metabolism have been shown to modify the levels of PAH-DNA adducts in target tissues. In order to study the role of metabolic gene polymorphisms on DNA-adduct formation in sperm, we determined the GSTM1 genotype in a group of men in whom PAH-DNA adducts in sperm had been previously measured by immunofluorescence. The mean level of adducts in sperm was significantly higher in subjects carrying the homozygous deletion variant of GSTM1 than in subjects with a functional GSTM1 (mean fluorescence staining intensity: 1.62+/-0.62 versus 1.33+/-0.55; p=0.02). With respect to environmental factors, subjects who reported occupational exposure to PAHs and who carried the GSTM1 deletion had a significant increase in PAH-DNA adducts in sperm in comparison with subjects who were not exposed and had a functional GSTM1 (mean staining intensity: 1.83+/-0.67 versus 1.30+/-0.53; p=0.05), although among GSTM1-null subjects there was no significant difference with or without occupational exposure. This study presents for the first time the effect of a common polymorphism in a gene that metabolizes PAHs on DNA-adduct levels in sperm.