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.

An international inter-laboratory study to compare digital PCR with ISO standardized qPCR assays for the detection of norovirus GI and GII in oyster tissue.

An optimized digital RT-PCR (RT-dPCR) assay for the detection of human norovirus GI and GII RNA was compared with ISO 15216-conform quantitative real-time RT-PCR (RT-qPCR) assays in an interlaboratory study (ILS) among eight laboratories. A duplex GI/GII RT-dPCR assay, based on the ISO 15216-oligonucleotides, was used on a Bio-Rad QX200 platform by six laboratories. Adapted assays for Qiagen Qiacuity or ThermoFisher QuantStudio 3D were used by one laboratory each. The ILS comprised quantification of norovirus RNA in the absence of matrix and in oyster tissue samples. On average, results of the RT-dPCR assays were very similar to those obtained by RT-qPCR assays. The coefficient of variation (CV%) of norovirus GI results was, however, much lower for RT-dPCR than for RT-qPCR in intra-laboratory replicates (eight runs) and between the eight laboratories. The CV% of norovirus GII results was in the same range for both detection formats. Had in-house prepared dsDNA standards been used, the CV% of norovirus GII could have been in favor of the RT-dPCR assay. The ratio between RT-dPCR and RT-qPCR results varied per laboratory, despite using the distributed RT-qPCR dsDNA standards. The study indicates that the RT-dPCR assay is likely to increase uniformity of quantitative results between laboratories.

A survey of the representativeness and usefulness of wastewater-based surveillance systems in 10 countries across Europe in 2023.

Wastewater-based surveillance (WBS) has become a widespread method to monitor transmission of SARS-CoV-2 and other human pathogens in Europe. We conducted a survey about WBS systems' objectives, approaches, representativeness and usefulness in 10 invited European countries in 2023, i.e. Austria, Belgium, Denmark, Finland, Greece, Hungary, Italy, Luxembourg, the Netherlands and Norway. All countries completed the study questionnaire about their SARS-CoV-2 WBS systems, and shared information about WBS of other pathogens as deemed relevant. SARS-CoV-2 WBS systems primarily monitored national and subnational trends (population coverage: 25-99%), and a majority (8/10) also tracked variant distribution. Nine of 10 countries reported that their SARS-CoV-2 WBS systems were representative of their population and all countries remarked that the findings were valuable for public health decision-making. Results were shared with relevant public health authorities and published via dedicated websites and/or dashboards. WBS systems of other pathogens were mostly in the early stages, with some countries implementing pilots. Notable exceptions were the well-established poliovirus surveillance systems in Finland, Italy and the Netherlands. This study brings understanding the diverse landscape of WBS in Europe, offering insights for future developments and collaborations. Furthermore, it highlights the need for further integration of WBS into other European surveillance systems.

A Reliable Multifaceted Solution against Foodborne Viral Infections: The Case of RiLK1 Decapeptide.

Food-borne transmission is a recognized route for many viruses associated with gastrointestinal, hepatic, or neurological diseases. Therefore, it is essential to identify new bioactive compounds with broad-spectrum antiviral activity to exploit innovative solutions against these hazards. Recently, antimicrobial peptides (AMPs) have been recognized as promising antiviral agents. Indeed, while the antibacterial and antifungal effects of these molecules have been widely reported, their use as potential antiviral agents has not yet been fully investigated. Herein, the antiviral activity of previously identified or newly designed AMPs was evaluated against the non-enveloped RNA viruses, hepatitis A virus (HAV) and murine norovirus (MNV), a surrogate for human norovirus. Moreover, specific assays were performed to recognize at which stage of the viral infection cycle the peptides could function. The results showed that almost all peptides displayed virucidal effects, with about 90% of infectivity reduction in HAV or MNV. However, the decapeptide RiLK1 demonstrated, together with its antibacterial and antifungal properties, a notable reduction in viral infection for both HAV and MNV, possibly through direct interaction with viral particles causing their damage or hindering the recognition of cellular receptors. Hence, RiLK1 could represent a versatile antimicrobial agent effective against various foodborne pathogens including viruses, bacteria, and fungi.

Detection of Monkeypox Virus DNA in Airport Wastewater, Rome, Italy.

Environmental surveillance can be a complementary tool for detecting pathogens circulating in communities. We detected monkeypox virus DNA in wastewater from Italy's largest airport by using real-time PCR assays targeting the G2R region and F3L and N3R genes and sequencing. Wastewater surveillance can be quickly adapted to investigate emerging threats.

Quantification and genetic diversity of Hepatitis E virus in wild boar (Sus scrofa) hunted for domestic consumption in Central Italy.

Hepatitis E is an emerging disease in industrialized countries. The food-borne transmission of hepatitis E virus (HEV) is associated principally with products derived from the domestic pig, the wild boar, and deer; however, few quantitative data are available on HEV loads in animals used in food production. This study assessed HEV occurrence, viral load and genetic variability in wild boar hunted for domestic consumption in the district of Viterbo (Central Italy) where high anti-HEV IgG seroprevalence values are reported in humans. A total of 332 liver and 69 intestine samples were obtained from wild boar hunted between 2011 and 2014. The liver tissue in 54 of the animals (16.3%) was HEV-positive. Viral loads in quantifiable liver samples (n = 29) ranged between 3.2 × 102 and 3.8 × 105 genome copies (g.c.)/g with a mean value of 1.85 × 104 g.c./g. A statistically significant positive correlation was found between viral concentration in liver and intestinal tissue, though mean viral load in the intestines was lower (3.13 × 103 g.c./g). Twenty-six samples were characterized molecularly as genotype 3 (G3) and four subtypes (a, c, f and l) were detected. Finally, twelve samples with near identical sequences were identified as G3 but could not be assigned to any of the known subtypes, and could therefore represent a potentially new subtype.

Thermal processing of live bivalve molluscs for controlling viruses: On the need for a risk-based design.

Norovirus (NoV) and Hepatitis A virus (HAV) are the most important viral hazards associated with human illness following consumption of contaminated bivalve molluscs. The effectiveness of the current EU criteria for heat processing of bivalve molluscs (i.e. raising the temperature of the internal mollusc flesh to at least 90°C for a minimum of 90 seconds) was evaluated using predictive microbiology. A HAV thermal inactivation model was developed based on literature data in mollusc matrices during isothermal heat treatment. Application of the developed model demonstrated that the 90°C-90 s requirement may lead to significantly different virus inactivation depending on the commercial process design. This shows the need for the establishment of a Performance Criterion for bivalve molluscs heat processing which will assure a common specified level of consumer protection. A risk-based approach is described that allows for an effective processing design providing a more transparent and objective relation between the thermal processing targets and public health. Model simulations demonstrate that the F-value is a more appropriate Process Criterion than a single time-temperature combination since it enables the food business operators to design a process that is compliant with the safety requirements while at the same time achieving a desired product quality.

Inhibition of the α-carbonic anhydrase from Vibrio cholerae with amides and sulfonamides incorporating imidazole moieties.

We discovered novel and selective sulfonamides/amides acting as inhibitors of the α-carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium Vibrio cholerae (VchCA). This Gram-negative bacterium is the causative agent of cholera and colonises the upper small intestine where sodium bicarbonate is present at a high concentration. The secondary sulfonamides and amides investigated here were potent, low nanomolar VchCA inhibitors whereas their inhibition of the human cytosolic isoforms CA I and II was in the micromolar range or higher. The molecules represent an interesting lead for antibacterial agents with a possibly new mechanism of action, although their CA inhibition mechanism is unknown for the moment.

Occurrence of virulence genes among Vibrio cholerae and Vibrio parahaemolyticus strains from treated wastewaters.

Pathogenic Vibrio species are an important cause of foodborne illnesses. The aim of this study was to describe the occurrence of potentially pathogenic Vibrio species in the final effluents of a wastewater treatment plant and the risk that they may pose to public health. During the 1-year monitoring, a total of 43 Vibrio strains were isolated: 23 Vibrio alginolyticus, 1 Vibrio cholerae, 4 Vibrio vulnificus, and 15 Vibrio parahaemolyticus. The PCR investigation of V. parahaemolyticus and V. cholerae virulence genes (tlh, trh, tdh, toxR, toxS, toxRS, toxT, zot, ctxAB, tcp, ace, vpi, nanH) revealed the presence of some of these genes in a significant number of strains. Intraspecies variability and genetic relationships among the environmental isolates were analyzed by random amplified polymorphic DNA-PCR (RAPD-PCR). We report the results of the first isolation and characterization of an environmental V. cholerae non-O1 non-O139 and of a toxigenic V. parahaemolyticus strain in Tunisia. We suggest that non-pathogenic Vibrio might represent a marine reservoir of virulence genes that can be transmitted between strains by horizontal transfer.

Duplex Droplet Digital PCR Assay for Quantification of Hepatitis E Virus in Food.

Hepatitis E virus (HEV) represents an emerging risk in industrialized countries where the consumption of contaminated food plays a pivotal role. Quantitative real-time RT-PCR (RT-qPCR) is one of the most suitable methods for the detection and quantification of viruses in food. Nevertheless, quantification using RT-qPCR has limitations. Droplet digital PCR (ddPCR) provides the precise quantification of nucleic acids without the need for a standard curve and a reduction in the effect on virus quantification due to the presence of inhibitors. The objectives of the present work were (i) to develop a method for the absolute quantification of HEV in swine tissues based on ddPCR technology and provide internal process control for recovery assessment and (ii) to evaluate the performance of the method by analyzing a selection of naturally contaminated wild boar muscle samples previously tested using RT-qPCR. The method was optimized using a set of in vitro synthesized HEV RNA and quantified dsDNA. The limit of detection of the developed ddPCR assay was 0.34 genome copies/µL. The analysis of the wild boar samples confirmed the validity of the ddPCR assay. The duplex ddPCR method showed no reduction in efficiency compared to individual assays. The method developed in the present study could represent a sensitive assay for the detection and absolute quantification of HEV RNA in food samples with the advantage of presenting the co-amplification of internal process control.

Droplet Digital RT-PCR (dd RT-PCR) Detection of SARS-CoV-2 in Honey Bees and Honey Collected in Apiaries across the Campania Region.

Coronaviruses (CoVs), a subfamily of Orthocoronavirinae, are viruses that sometimes present a zoonotic character. Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is responsible for the recent outbreak of COVID-19, which, since its outbreak in 2019, has caused about 774,593,066 confirmed cases and 7,028,881 deaths. Aereosols are the main route of transmission among people; however, viral droplets can contaminate surfaces and fomites as well as particulate matter (PM) in suspensions of natural and human origin. Honey bees are well known bioindicators of the presence of pollutants and PMs in the environment as they can collect a great variety of substances during their foraging activities. The aim of this study was to evaluate the possible role of honey bees as bioindicators of the prevalence SARS-CoV-2. In this regard, 91 samples of honey bees and 6 of honey were collected from different apiaries of Campania region (Southern Italy) in four time periods from September 2020 to June 2022 and were analyzed with Droplet Digital RT-PCR for SARS-CoV-2 target genes Orf1b and N. The screening revealed the presence of SARS-CoV-2 in 12/91 in honey bee samples and in 2/6 honey samples. These results suggest that honey bees could also be used as indicators of outbreaks of airborne pathogens such as SARS-CoV-2.

Detection of Hepatitis E Virus in Game Meat (Wild Boar) Supply Chain in Umbria Region, Central Italy.

Consumption of raw or undercooked wild boar (WB) meat is considered an important risk factor for hepatitis E virus (HEV) infection in humans. The possibility of HEV contamination during the slaughtering practices may pose an additional risk. Based on these assumptions, we evaluated HEV contamination of WB meat hunted in Umbria (central Italy) during the 2022-2023 hunting season by real-time RT-PCR. Herein, we show that 10.8% of livers from slaughtered WB were positive for HEV RNA, thus providing an estimate of HEV infection in WB in the Umbria region. Then, by evaluating paired liver-muscle samples from both HEV-positive and HEV-negative animals, we found evidence of muscle HEV contamination in 33% and 14% of cases, respectively. This is the first report on the detection of HEV in WB meat in Umbria, an Italian region with diffuse WB hunting and consumption. The evidence of contamination provided by our study underscores the importance of adopting good hygienic practices in the processing stages of hunted WB carcasses to significantly reduce meat contamination and the risk posed for the final consumer.

Characterisation of microbial communities and quantification of antibiotic resistance genes in Italian wastewater treatment plants using 16S rRNA sequencing and digital PCR.

The spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in humans, animals and environment is a growing threat to public health. Wastewater treatment plants (WWTPs) are crucial in mitigating the risk of environmental contamination by effectively removing contaminants before discharge. However, the persistence of ARB and ARGs even after treatment is a challenge for the management of water system. To comprehensively assess antimicrobial resistance dynamics, we conducted a one-year monitoring study in three WWTPs in central Italy, both influents and effluents. We used seasonal sampling to analyze microbial communities by 16S rRNA, as well as to determine the prevalence and behaviour of major ARGs (sul1, tetA, blaTEM, blaOXA-48, blaCTX-M-1 group, blaKPC) and the class 1 Integron (int1). Predominant genera included in order: Arcobacter, Acinetobacter, Flavobacterium, Pseudarcobacter, Bacteroides, Aeromonas, Trichococcus, Cloacibacterium, Pseudomonas and Streptococcus. A higher diversity of bacterial communities was observed in the effluents compared to the influents. Within these communities, we also identified bacteria that may be associated with antibiotic resistance and pose a significant threat to human health. The mean concentrations (in gene copies per liter, gc/L) of ARGs and int1 in untreated wastewater (absolute abundance) were as follows: sul1 (4.1 × 109), tetA (5.2 × 108), blaTEM (1.1 × 108), blaOXA-48 (2.1 × 107), blaCTX-M-1 group (1.1 × 107), blaKPC (9.4 × 105), and int1 (5.5 × 109). The mean values in treated effluents showed reductions ranging from one to three log. However, after normalizing to the 16S rRNA gene (relative abundance), it was observed that in 37.5 % (42/112) of measurements, the relative abundance of ARGs increased in effluents compared to influents. Furthermore, correlations were identified between ARGs and bacterial genera including priority pathogens. This study improves our understanding of the dynamics of ARGs and provides insights to develop more effective strategies to reduce their spread, protecting public health and preserving the future efficacy of antibiotics.

Presence of Zonula Occludens Toxin-Coding Genes among Vibrio parahaemolyticus Isolates of Clinical and Environmental Origin.

In recent studies, emphasis has been placed on the zonula occludens toxin (Zot) from the non-toxigenic Vibrio parahaemolyticus strain PMC53.7 as an agent inducing alterations in the actin cytoskeleton of infected Caco-2 cells and which appears as a relevant virulence factor. Universal zot primers were designed by the alignment of different types of zot gene and identification of conserved sequences to investigate the presence in diverse environmental and clinical V. parahaemolyticus isolates, in co-occurrence with virulence factors, such a hemolysins and secretion systems. The study screened a total of 390 isolates from environmental sources from Chile and Italy and 95 Chilean clinical isolates. The results revealed that around 37.2% of Chilean environmental strains and 25.9% of Italian strains, and 24.2% of clinical isolates carried the zot gene. The Zot-C2 cluster was present in 71.4% of Chilean environmental strains but absent in clinical isolates, while the Zot-C4 cluster was identified in 28.6% of environmental and 100% of clinical isolates. Understanding the role of zot in V. parahaemolyticus virulence is crucial, especially considering the risk associated with consuming diverse isolates from bivalves and the co-occurrence with virulence factors such as TDH, TRH or T3SS2.

Evaluation of alternative methods of tunnel composting (submitted by the European Composting Network) II.

Two alternative methods for producing compost in a tunnel, from certain category (Cat.) 3 animal by-products (ABP) and other non-ABP material, were assessed. The first method proposed a minimum temperature of 55°C for 72 h and the second 60°C for 48 h, both with a maximum particle size of 200 mm. The assessment of the Panel on Biological Hazards (BIOHAZ) exclusively focused on Cat. 3 ABP materials (catering waste and processed foodstuffs of animal origin no longer intended for human consumption). The proposed composting processes were evaluated for their efficacy to achieve a reduction of at least 5 log10 of Enterococcus faecalis and Salmonella Senftenberg (775W, H2S negative) and at least 3 log10 of relevant thermoresistant viruses. The applicant provided a list of biological hazards that may enter the composting process and selected parvoviruses as the indicator of the thermoresistant viruses. The evidence provided by the applicant included: (a) literature data on thermal inactivation of biological hazards; (b) results from validation studies on the reduction of E. faecalis, Salmonella Senftenberg 775W H2S negative and canine parvovirus carried out in composting plants across Europe; (c) and experimental data from direct measurements of reduction of infectivity of murine parvovirus in compost material applying the time/temperature conditions of the two alternative methods. The evidence provided showed the capacity of the proposed alternative methods to reduce E. faecalis and Salmonella Senftenberg 775W H2S negative by at least 5 log10, and parvoviruses by at least 3 log10. The BIOHAZ Panel concluded that the two alternative methods under assessment can be considered to be equivalent to the processing method currently approved in the Commission Regulation (EU) No 142/2011.

BSE risk posed by ruminant collagen and gelatine derived from bones.

The European Commission requested an estimation of the BSE risk (C-, L- and H-BSE) from gelatine and collagen derived from ovine, caprine or bovine bones, and produced in accordance with Regulation (EC) No 853/2004, or Regulation (EC) No 1069/2009 and its implementing Regulation (EU) No 142/2011. A quantitative risk assessment was developed to estimate the BSE infectivity, measured in cattle oral infectious dose 50 (CoID50), in a small size batch of gelatine including one BSE-infected bovine or ovine animal at the clinical stage. The model was built on a scenario where all ruminant bones could be used for the production of gelatine and high-infectivity tissues remained attached to the skull (brain) and vertebral column (spinal cord). The risk and exposure pathways defined for humans and animals, respectively, were identified. Exposure routes other than oral via food and feed were considered and discussed but not assessed quantitatively. Other aspects were also considered as integrating evidence, like the epidemiological situation of the disease, the species barrier, the susceptibility of species to BSE and the assumption of an exponential dose-response relationship to determine the probability of BSE infection in ruminants. Exposure to infectivity in humans cannot be directly translated to risk of disease because the transmission barrier has not yet been quantified, although it is considered to be substantial, i.e. much greater amounts of infectivity would be needed to successfully infect a human and greater in the oral than in the parenteral route of exposure. The probability that no new case of BSE in the cattle or small ruminant population would be generated through oral exposure to gelatine made of ruminant bones is 99%-100% (almost certain) This conclusion is based on the current state of knowledge, the epidemiological situation of the disease and the current practices, and is also valid for collagen.

Update of the list of qualified presumption of safety (QPS) recommended microbiological agents intentionally added to food or feed as notified to EFSA 20: Suitability of taxonomic units notified to EFSA until March 2024.

The qualified presumption of safety (QPS) process was developed to provide a safety assessment approach for microorganisms intended for use in food or feed chains. In the period covered by this statement, no new information was found that would change the status of previously recommended QPS TUs. The TUs in the QPS list were updated based on a verification, against their respective authoritative databases, of the correctness of the names and completeness of synonyms. A new procedure has been established to ensure the TUs are kept up to date in relation to recent taxonomical insights. Of 83 microorganisms notified to EFSA between October 2023 and March 2024 (47 as feed additives, 25 as food enzymes or additives, 11 as novel foods), 75 were not evaluated because: 15 were filamentous fungi, 1 was Enterococcus faecium, 10 were Escherichia coli, 1 was a Streptomyces (all excluded from the QPS evaluation) and 48 were TUs that already have a QPS status. Two of the other eight notifications were already evaluated for a possible QPS status in the previous Panel Statement: Heyndrickxia faecalis (previously Weizmannia faecalis) and Serratia marcescens. One was notified at genus level so could not be assessed for QPS status. The other five notifications belonging to five TUs were assessed for possible QPS status. Akkermansia muciniphila and Actinomadura roseirufa were still not recommended for QPS status due to safety concerns. Rhizobium radiobacter can be recommended for QPS status with the qualification for production purposes. Microbacterium arborescens and Burkholderia stagnalis cannot be included in the QPS list due to a lack of body of knowledge for its use in the food and feed chain and for B. stagnalis also due to safety concerns. A. roseirufa and B. stagnalis have been excluded from further QPS assessment.

Public health aspects of Vibrio spp. related to the consumption of seafood in the EU.

Vibrio parahaemolyticus, Vibrio vulnificus and non-O1/non-O139 Vibrio cholerae are the Vibrio spp. of highest relevance for public health in the EU through seafood consumption. Infection with V. parahaemolyticus is associated with the haemolysins thermostable direct haemolysin (TDH) and TDH-related haemolysin (TRH) and mainly leads to acute gastroenteritis. V. vulnificus infections can lead to sepsis and death in susceptible individuals. V. cholerae non-O1/non-O139 can cause mild gastroenteritis or lead to severe infections, including sepsis, in susceptible individuals. The pooled prevalence estimate in seafood is 19.6% (95% CI 13.7-27.4), 6.1% (95% CI 3.0-11.8) and 4.1% (95% CI 2.4-6.9) for V. parahaemolyticus, V. vulnificus and non-choleragenic V. cholerae, respectively. Approximately one out of five V. parahaemolyticus-positive samples contain pathogenic strains. A large spectrum of antimicrobial resistances, some of which are intrinsic, has been found in vibrios isolated from seafood or food-borne infections in Europe. Genes conferring resistance to medically important antimicrobials and associated with mobile genetic elements are increasingly detected in vibrios. Temperature and salinity are the most relevant drivers for Vibrio abundance in the aquatic environment. It is anticipated that the occurrence and levels of the relevant Vibrio spp. in seafood will increase in response to coastal warming and extreme weather events, especially in low-salinity/brackish waters. While some measures, like high-pressure processing, irradiation or depuration reduce the levels of Vibrio spp. in seafood, maintaining the cold chain is important to prevent their growth. Available risk assessments addressed V. parahaemolyticus in various types of seafood and V. vulnificus in raw oysters and octopus. A quantitative microbiological risk assessment relevant in an EU context would be V. parahaemolyticus in bivalve molluscs (oysters), evaluating the effect of mitigations, especially in a climate change scenario. Knowledge gaps related to Vibrio spp. in seafood and aquatic environments are identified and future research needs are prioritised.

Re-evaluation of certain aspects of the EFSA Scientific Opinion of April 2010 on risk assessment of parasites in fishery products, based on new scientific data. Part 1: ToRs1-3.

Surveillance data published since 2010, although limited, showed that there is no evidence of zoonotic parasite infection in market quality Atlantic salmon, marine rainbow trout, gilthead seabream, turbot, meagre, Atlantic halibut, common carp and European catfish. No studies were found for greater amberjack, brown trout, African catfish, European eel and pikeperch. Anisakis pegreffii, A. simplex (s. s.) and Cryptocotyle lingua were found in European seabass, Atlantic bluefin tuna and/or cod, and Pseudamphistomum truncatum and Paracoenogonimus ovatus in tench, produced in open offshore cages or flow-through ponds or tanks. It is almost certain that fish produced in closed recirculating aquaculture systems (RAS) or flow-through facilities with filtered water intake and exclusively fed heat-treated feed are free of zoonotic parasites. Since the last EFSA opinion, the UV-press and artificial digestion methods have been developed into ISO standards to detect parasites in fish, while new UV-scanning, optical, molecular and OMICs technologies and methodologies have been developed for the detection, visualisation, isolation and/or identification of zoonotic parasites in fish. Freezing and heating continue to be the most efficient methods to kill parasites in fishery products. High-pressure processing may be suitable for some specific products. Pulsed electric field is a promising technology although further development is needed. Ultrasound treatments were not effective. Traditional dry salting of anchovies successfully inactivated Anisakis. Studies on other traditional processes - air-drying and double salting (brine salting plus dry salting) - suggest that anisakids are successfully inactivated, but more data covering these and other parasites in more fish species and products is required to determine if these processes are always effective. Marinade combinations with anchovies have not effectively inactivated anisakids. Natural products, essential oils and plant extracts, may kill parasites but safety and organoleptic data are lacking. Advanced processing techniques for intelligent gutting and trimming are being developed to remove parasites from fish.

Update of the list of qualified presumption of safety (QPS) recommended microbiological agents intentionally added to food or feed as notified to EFSA 19: Suitability of taxonomic units notified to EFSA until September 2023.

The qualified presumption of safety (QPS) process was developed to provide a safety assessment approach for microorganisms intended for use in food or feed chains. The QPS approach is based on an assessment of published data for each taxonomic unit (TU), with respect to its taxonomic identity, the body of relevant knowledge and safety concerns. Safety concerns identified for a TU are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications'. In the period covered by this Statement, no new information was found that would change the status of previously recommended QPS TUs. Of 71 microorganisms notified to EFSA between April and September 2023 (30 as feed additives, 22 as food enzymes or additives, 7 as novel foods and 12 from plant protection products [PPP]), 61 were not evaluated because: 26 were filamentous fungi, 1 was Enterococcus faecium, 5 were Escherichia coli, 1 was a bacteriophage (all excluded from the QPS evaluation) and 28 were TUs that already have a QPS status. The other 10 notifications belonged to 9 TUs which were evaluated for a possible QPS status: Ensifer adhaerens and Heyndrickxia faecalis did not get the QPS recommendation due to the limited body of knowledge about their occurrence in the food and/or feed chains and Burkholderia ubonensis also due to its ability to generate biologically active compounds with antimicrobial activity; Klebsiella pneumoniae, Serratia marcescens and Pseudomonas putida due to safety concerns. K. pneumoniae is excluded from future QPS evaluations. Chlamydomonas reinhardtii is recommended for QPS status with the qualification 'for production purposes only'; Clostridium tyrobutyricum is recommended for QPS status with the qualification 'absence of genetic determinants for toxigenic activity'; Candida oleophila has been added as a synonym of Yarrowia lipolytica. The Panel clarifies the extension of the QPS status for genetically modified strains.