Drivers and Constraints in the Use of Modern Typing Tools to Trace Foodborne Disease

Mats Lindblad, National Food Agency, mats.lindblad@slv.se
Catarina Flink, National Food Agency
Cecilia Jernberg, Public Health Agency of Sweden
Ann Lindberg, National Veterinary Institute

The aim of this workshop is to stimulate differ-ent stakeholders and disciplines to realize the benefits of data sharing and the opportunities the new molecular surveillance tool, Whole Genome Sequencing (WGS), is providing in a One Health perspective. What are the national and international drivers and constraints in regard to data sharing, joint analysis and communication in outbreak investigations?

Globally, infectious diseases account for about 22 percent of all human deaths, and are also a major burden on animal health. In addition, they also increase the financial burden on health systems and society at large. The longer it takes before the causative agents are detected, the greater the consequences for the individual or the population. The (often international) epidemiology of foodborne infections can also have significant implications for trade. Therefore, rapid national and international surveillance systems and methodologies for the exchange and comparison of information on the worldwide spread of food-borne zoonotic pathogens are greatly needed to trace the origin of the source and to investigate complex outbreaks.

The focus areas of the workshop examine the different challenges of data sharing by means of Whole Genome Sequencing:

•   What are the legal constraints between both sectors and countries?
•   The technical challenges and analysis normalization where different platforms and analysis software are used.
•   In the transition from old to new technology, will backward compatibility and thereby the link to historical data be lost?
•   What is needed to reach the goal of real-time surveillance?

New possibilities provided by Whole Genome Sequencing (WGS)

Molecular typing methods for foodborne pathogens are beginning to be routinely applied worldwide for public health protection, e.g. investigating foodborne outbreaks, identifying strains of foodborne bacteria with high virulence potential or resistance to antimicrobials. This development stems from continuous advances in the understanding of the molecular charac-teristics of bacteria and their genetics linked to technological developments. Over time, a range of molecular methods has evolved, such as pulsed field gel electrophoresis (PFGE), multiple locus variable number of tandem repeats analysis (MLVA) and multilocus sequence typing (MLST), which are all commonly applied to foodborne pathogens, with applications in outbreak investigations as well as source attribution. All of these methods have some limitations; e.g. they only provide one type of characterization of the genome, older methods are not easily replaced with more modern methods due to a lack of backwards compatibility and they are not suitable for all purposes due to differences in phylogenetic resolution.

However, the evolution of rapid sequencing technology as well as an increased capacity in bioinformatics has led to bacterial whole genome sequencing (WGS) methods becoming more and more feasible. The potential of WGS is now actively being considered in several areas including: pathogen characterization and typing, outbreak detection and investigation, risk assessment and high-resolution epidemiology. Using WGS will also improve the accuracy and effectiveness of disease surveillance and the evaluation of prevention policies by enhanced assessment of disease and drug resistance transmission dynamics with the final goal to make a difference for public health intervention. WGS data make it possible to assess the molecular diversity and circulation of strains within the food chain and could be useful for source attribution studies when estimating the contribution of different food categories or animal species as sources of human infections.

Inter-agency and inter-lab co-operation assists cluster detection

In the United States, agencies like the Centers for Disease Control and Prevention (CDC) and the Food and Drug Administration (FDA) have taken initiatives to facilitate a more widespread application of WGS. Building upon existing data sharing networks among laboratories in addition to tighter cooperation between the food and public health sector in regard to data sharing have proven successful in solving an array of different foodborne outbreaks. In Europe, the European Centre for Disease Prevention and Control (ECDC) started a two-year molecular surveillance pilot project in 2012, linking public health reference laboratories across Europe for real-time data sharing from traditional molecular typing techniques for selected foodborne bacterial pathogens. In addition, limited additional information about the isolates (metadata) can be shared, such as country of origin, age and gender, depending on the ability of each country. Today, the European Surveillance System (TESSy)’s molecular surveillance service (MSS) is used by ECDC for routine molecular surveil-lance. If multi-country clusters are detected, the countries concerned are informed and, when needed, ECDC can also provide support during the outbreak investigations.

A joint database for molecular surveillance in Europe

The European Food Safety Authority (EFSA), whose mission is to improve EU food safety and ensure a high level of consumer protection, recommended at their 10th Anniversary Con-ference in 2012 the building of a centralized WGS database for foodborne pathogens. Since then, EFSA, together with the European Union Reference Laboratories (EURLs), and ECDC, in partnership with the public health agencies, have successively strengthened their collaboration in the field of molecular surveillance of foodborne diseases. A first step has been to initiate the sharing of data generated using traditional molecular typing tools within the ECDC platform. The purpose of the joint ECDC-EFSA molecular typing database is to share comparable typing data in a common repository so that microbiological data from humans can be linked to similar data from the food chain (1). In other words, at EU level there is a clear need and willingness to foster a multidisciplinary interpretation of the information arising from the combination of epidemiological and molecular pathogen characterization data to guide public health action.

The challenges to achieving EU-wide use of WGS

In 2016, ECDC published a strategy, according to which WGS will be the method of choice for typing of bacterial pathogen across the EU by 2021. The role of the EU authorities will be to enable and facilitate the EU-wide use of this universal typing technique. Already today, WGS is the tool used in many international investiga-tions, although it is only used after the outbreak signal has been identified using existing, ‘old’, typing methodology.

To reach this goal – EU-wide use of WGS in routine surveillance – there are a number of challenges that have to be addressed, such as:
(i) technical constraints in the laboratories;
(ii) different national and sectoral legal frameworks;
(iii) the need for agreement and understanding on sharing data among countries and sectors;
(iv) to jointly interpret and communicate the data (WGS and epidemiological).

The agreement about sharing molecular typing data among countries and sectors described above is a large step forward ‘politically’. One of the challenges that now lie ahead concerns the sharing of both sequence data, or interpretation thereof, and more specifically epidemiological data that is connected to each and every isolate.

Legal obstacles: the conflict between openness and privacy

Today the epidemiological data shared is either non-existent or very limited. There could be sensitive epidemiological data that will have to remain available only to the competent authorities, so releasing this sensitive data will not easily, or even necessarily, become a routine procedure. There could be several impediments for the free sharing of sequencing data. By contrast, the deposition of the microbial genomic sequence data in databases for public access beyond the control of the owner of the data is common practice. Legal obstacles are to be expected and a careful balance must be struck between desirable complete openness from a food safety point of view and the privacy and related concerns that necessitate confidentiality. A standard for encryption may well need to be developed to allow exchange of data to be limited to authorized parties only. Ignoring these issues is likely to considerably delay the successful large-scale implementation of WGS for public health at international level.

Technical challenges

Currently, there are different commercially available, high-throughput next generation sequencing (NGS) platforms. In addition, there are different bioinformatics pipelines for sequence data analysis, both in-house pipelines as well as commercial and free software. A laboratory for routine WGS application that aims to share data requires the following:

(i) the adoption of appropriate quality assur-ance/quality control (QA/QC) measures;
(ii) the development and harmonization of standard operating procedures, SOPs;
(iii) the establishment of database infrastructure; and
(iv) the generation and dissemination of appropriate genomic reference datasets.

Transition from old to new techniques and linkage to historical data

There is a need to link these data to previous isolate characterization schemes and nomenclatures as well as to the phenotypic properties of the isolates from which the genomic data are obtained. A major difference between WGS and the traditional typing methods is that WGS allows all genes to be included in the analysis, instead of a well-defined subset of genes or variable intergenic regions. Therefore, the analysis of WGS data will yield new types of insight. With the sequencing technique in place, this is still a financial limitation for many laboratories due to the high costs of the new technology. The costs for the NGS platforms and the sequencing per se are continuously dropping; however the transition from the old typing techniques to WGS will probably take a number of years before most laboratories have reached this goal.

Real-time surveillance

To be able to upload the data in a timely manner, it is important to have a chance to act upon multinational clusters that are identified, break the chain and stop the spread. Usually in out-break scenarios, the authorities are always one step behind, timewise. After a case has been exposed to a pathogen, the incubation time can be anything from a few days up to a week before illness onset. Then the pathogen has to be identified and reported to a surveillance system and typed. It is important to upload the information and data to the surveillance system more or less in real time in order not to lose more time before food no longer can be sampled, or other information can be lost and the transmission chain can be a challenge to identify and break. Consequently, reporting systems that are easy to use and that can link isolate data with case data must be in place, which can also be a challenge on a national level.

In conclusion, in order to have an efficient, multi-national molecular surveillance system based on WGS, three major issues must be fulfilled: to be able to legally share the data; to have a common database platform; and to be able to analyse and interpret the molecular typing tool in a normalized way in a timely manner.

The workshop will start with an introduction highlighting the work done on EU level to facilitate cross-border sharing and communication and to trace back investigations. In addition, an illustration will be given of a recent multinational outbreak investigation where WGS was the pathogen typing tool. The workshop participants will work to collectively identify existing barriers that prevent the sharing of molecular and epidemiological data, and then solutions for sharing and tools that will allow the full potential use of WGS will be discussed.

References

Rizzi, V, Da Silvia Felico, T, Felix, B, Gossner, CM, Jacobs,W, Johansson, K, Kotila, S, Michelon, D, Monduidi, M, Mooijman, K, Morabito, S, Pasinato, L, Björkman, JT, Torpdahl, M, Tozzoli, R and Van Walle, I. 2017. The ECDC-EFSA molecular typing database for European Union public health protection. Eurofererence 2, March 2017.