Stéphane Pesce
email: stephane.pesce@inrae.fr

Laure Mamy

Anne Laure Achard

Morgane Le Gall

Sophie Le Perchec

Olivier Rechauchère

Anaïs Tibi

Sophie Leenhardt

Wilfried Sanchez

Pesce Stéphane

Mamy Laure

Achard Anne-Laure

Le Gall Morgane

Le Perchec

Réchauchère Olivier

Tibi Anaïs

Leenhardt Sophie

Sanchez Wilfried

Collective scientific assessment as a relevant tool to inform public debate and policymaking: an illustration about the effects of plant protection products on biodiversity and ecosystem services

Keywords: Expertise, Transfer of knowledge, Pesticide, Biocontrol, Ecological risk assessment

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The 2030 Agenda for Sustainable Development adopted in September 2015 sets out an ambitious action plan to end poverty, protect the planet and improve the well-being of people around the world through the implementation of 17 Sustainable Development Goals (SDGs).

Preserving or restoring environmental quality is a prerequisite to achieving most of the SDGs [START_REF] Racioppi | Reaching the sustainable development goals through healthy environments: are we on track?[END_REF]. Looking beyond these SDGs, there is growing scientific consensus that conserving terrestrial and aquatic biodiversity is a crucial objective for a wide range of sustainability programs [START_REF] Glaser | Base sustainable development goals on science[END_REF][START_REF] Bach | Soil biodiversity integrates solutions for a sustainable future[END_REF]Tickner et al. 2020). As biodiversity is fundamental for supporting and maintaining ecosystem functioning (EFSA Scientific Committee, 2016; van der Plas 2019), this consensus is in line with the notion of ecosystem goods and services that contribute to human health and well-being (Millennium Ecosystem Assessment 2005). Therefore, during The United Nations Conference on Sustainable Development that took place in June 2012 (Rio+20 Conference), and in continuation of the Convention on Biological Diversity adopted in 1992, EU Member States have explicitly recognized the intrinsic and extrinsic value of biodiversity and the need to preserve ecosystem services as critical foundations for sustainable development and human well-being (United Nations General Assembly 2012). The European Commission (2020) recently proposed a biodiversity strategy for 2030 that highlights the key role of biodiversity and associated services for Europe and the need to reduce pollution and restore ecosystems.

The EU framework programme for Research and Innovation 2021-2027 (European Commission 2018) demonstrates a clear political ambition to establish the EU as a leader in the effort to achieve the SDGs. This objective requires the implementation of an appropriate framework programme, supported by sustainability-oriented thematic agendas (Kastrinos and Weber 2020), to move towards a next generation of environmental measures purposed with supporting efforts to better preserve or restore biodiversity and ecosystem functions and services. However, understanding and addressing biodiversity loss and ecosystem functioning degradation is a challenging problem that is not solvable with current established methods [START_REF] Sharman | Wicked: the problem of biodiversity loss[END_REF][START_REF] Sarkki | Managing science-policy interfaces for impact: Interactions within the environmental governance meshwork[END_REF]. The challenge cannot, therefore, be met without integrating the best and latest scientific knowledge in the fields and disciplines related to this issue.

The role of science in informing decisionmaking and policymaking has been extensively discussed in the past decade, especially in terms of supporting global sustainability governance (e.g. [START_REF] Holmes | Enhancing the use of science in environmental policy-making and regulation[END_REF][START_REF] Likens | The role of science in decision-making: does evidence-based science drive environmental policy?[END_REF][START_REF] Van Der Hel | The authority of science in sustainability governance: A structured comparison of six science institutions engaged with the Sustainable Development Goals[END_REF]. According to the InterAcademy Partnership (IAP, 2019), effective implementation of the SDGs requires access to scientific knowledge and independent expertise. In other words, and as proposed by [START_REF] Haas | When does power listen to truth? A constructivist approach to the policy process[END_REF], there is a need for "usable knowledge" that "encompasses a substantive core that makes it usable for policymakers, and a procedural dimension that provides a mechanism for transmitting knowledge from the scientific community to the policy world". This transfer from scientists to decisionmakers and policymakers continues to prove challenging in practice [START_REF] Dale | A conceptual model to improve links between science, policy and practice in coastal management[END_REF]. However, there is a growing number of national initiatives designed to foster science-policy-practice interfaces related to biodiversity and ecosystem protection in a range of countries, such as Germany (Leibenalth et al. 2020) and the UK [START_REF] Holmes | Enhancing the use of science in environmental policy-making and regulation[END_REF].

There are several recognized methods of scientific assessment, such as meta-analysis, systematic review or participatory mapping [START_REF] Navarro | Méthodes d'expertise : comment les utiliser ? Expertise et synthèse[END_REF]. Here, we present the approach developed by the French National Research Institute for Agriculture, Food and the Environment (INRAE)'s Directorate for Scientific Assessment, Foresight and Advanced Studies (DEPE) to provide scientific expertise and inform public policy debate. Entitled "collective scientific assessment" (CSA), this kind of expertise is based on documented procedures (INRA-DEPE 2018), from the external request issued by public structures through to deliverables and dissemination.

After describing the main steps in the procedure classically followed for CSA, we illustrate the process by looking at the example of an ongoing CSA (2020-22) focused on the impacts of plant protection products (PPPs) on biodiversity and ecosystem services. PPPs are defined here as synthetic pesticides (active substances and formulations) and their transformation products, and encompassing biobased processes (biobased substances, pest control organisms, etc.) used in both agricultural and non-agricultural activities (e.g. greenspaces, gardens, public infrastructure, etc.). We then show how this kind of expertise is relevant for informing public debate and policymaking by describing the main outcomes generated by the earlier CSA published in 2005 that addressed the adverse environmental impacts of PPPs. Finally, we provide a short overview of some key expectations from the current CSA.

General description of a collective scientific assessment (CSA)

CSAs follow a documented procedure based on good-practice standards of expertise

Public research organizations can support public policy through various kinds of actions, including the provision of expertise to shed light on key issues. Decisionmakers and policymakers can consequently benefit from a critical and intelligible review of the scientific knowledge related to a complex question. This is the main objective of CSAs (abbreviated 'ESCo' in French), which are reports based on collective assessment that follow a documented procedure defined by a code of conduct established by the DEPE of INRAE, in accordance with the French National Charter on Institutional Scientific Expert Reports (Fig. 1).

Initiation, governance and monitoring

One of the specificities of CSAs is the multidisciplinary dimension of the question generally asked by public or para-public bodies including decisionmakers or policymakers (typically ministry divisions or environmental agencies), which the CSA process calls 'backers'. The project initiation phase, which lasts six to twelve months on average (Fig. 1), corresponds to a period of dialogue between the backers and one or several relevant academic research institutes (RIs) that leads to the drafting and sign-off of a mutual agreement to which the co-constructed CSA purpose, objectives and agenda are annexed. This dialogue is a co-construction process supported by a preliminary exploration of the available scientific literature in order to get an estimate of the amount of relevant source material, to assess the scope and breadth of the topic and its degree of multidisciplinarity, and to identify points in the original request made by the backers that require clarification. This phase also includes setting up the various committees that will contribute to CSA governance and monitoring (see Fig. 2 for example), such as an oversight committee and a stakeholder advisory committee. The oversight committee, which is composed of representatives of the backers, the RI and the project team, is a decisionmaking body. The stakeholder advisory committee is a consultative body that includes representatives of the main actors in society likely to be concerned by the conclusions of the CSA (e.g. environmental and consumer associations, local authorities, professional organizations, economic stakeholders in the agrifood and environmental sectors, scientific interest groups) in order to capture and integrate their reactions, comments and opinions.

Coordination and scientific experts

Each CSA is coordinated at operational and scientific levels by a project team (manager, scientific leads, librarians) (Fig. 1), following the guidelines established by INRAE (INRA-DEPE 2018). Global coordination of the process is tasked to a specific RI staff member who operates as project manager. Scientific leadership is provided by senior scientists that are appointed by the mandated RIs according to their scientific competences and their capacities for collective work. They also represent the expert panel and contribute to academic mobilization of the results.

With the support of the librarians, the scientific leads and the project manager select scientific experts based on the four core principles underpinning CSAs: competence, plurality of disciplines and approaches, impartiality, and transparency (INRA-DEPE 2018). Impartiality is assured by a declaration of interest, which both scientific leads and experts are required to submit ahead of further analysis by a dedicated review panel before final appointment. Experts almost exclusively come from academic research institutions and universities, with possible (but limited) participation by representatives of governmental agencies or offices if needed.

Experts from the private sector cannot be involved in CSAs. Declarations of interest are archived and made available in response to external request. Each final extended CSA report includes an analysis of the links and conflicts of interests to make it public.

In consultation with the scientific experts, librarians are also responsible for the bibliographic corpus (see section 2.4 below). They assist experts in producing requests for the collection of references, and they produce the final list and bibliometric analysis of the references used and cited in the CSA.

Production and use of bibliographic corpuses

CSAs are mainly based on the international academic literature, which can be supplemented as and when needed by 'grey' literature such as government reports, policy statements, thesis reports or technical publications. The use of 'grey' literature has to be justified on grounds of the specificity of the subject and its unique or recent character explaining why no equivalent source can be found in the peer-reviewed academic literature. Qualification of these sources is the responsibility of the experts who evaluate the robustness of the results and conclusions.

After the preliminary bibliographic search performed during the initiation phase, the bibliographic corpus is produced by the librarians, in collaboration with the scientific experts and the project team, throughout the assessment and write-up period (Fig. 1). Librarians and experts co-elaborate a set of bibliographic requests that are used to query and explore international bibliographical databases. The initial corpus obtained is then gradually refined by the experts to arrive at the final corpus that is exploited and referenced in the extended report. This process is based on a descriptive analysis of the corpus that provides the foundations for selecting the main results concerning established knowledge, controversies, and heterodox or innovative approaches. During this process, experts are required to explain why they have added or rejected references and which criteria they used to qualify references from the grey literature. Justifications can be integrated into the final extended report (see section 2.5). The elimination process wants special care so as not to reject certain references that serve to populate a table of current scientific controversies.

Transparency over the corpus analysis and reference selection (or rejection) process and the description of the inclusion and exclusion criteria for sources are crucial to assure the credibility of the CSA. Therefore, each CSA extended report describes the procedure used to produce the corpus together with qualitative and quantitative analyses of the corpus. To this aim, librarians conduct statistical, bibliometric and cartographic analyses of the cited bibliographic corpus.

Deliverables and dissemination

CSAs seek to inform public policy and foster public debate. CSA results and conclusions therefore need to be made public and freely accessible to a wide audience. First, they are presented in a final seminar that can be organized at a national or international level. To reach the widest possible audience, this seminar is freely open to the public and can be filmed to be made available online on the RI websites a few days after the event (with simultaneous translation in English and/or other languages where appropriate).

Three kinds of formal final reports are generated: i) an extended report, ii) a condensed report, and iii) a summary report (Fig. 1).

The extended report brings together all the critical contributions and analyses written by the experts on the basis of the bibliographic corpus, which is comprehensively referenced. It also describes the general framework of the CSA, the collective of experts involved, and the process and methods used throughout the exercise. Written under the responsibility of the experts and scientific leads, the extended report is posted online by the RIs. This report serves as a basis for the project team to draft a condensed report under the supervision of the experts.

The condensed report is designed to be accessible to a wide audience (e.g. scientists in other fields, teachers and students, informed public, etc.). The oversight committee is consulted to rule on whether the document is readable and relevant to the CSA specifications and whether its content is appropriate to support public decisionmaking. However, the experts group remains the final decisionmaker regarding final content and conclusions. The condensed report is then made freely available online in both French and English. Finally, a summary report of less than 10 pages (in French and in English) is freely disseminated by the RIs in the widest possible way (mailing lists, websites, social media, etc.).

Over and above these requested deliverables, it is equally important to improve the reach and impact of each CSA by disseminating its main results and conclusions within the international scientific and practitioner community. With that vision, scientific leads and experts are encouraged to publish the results of their work in peer-reviewed journals and to present them at scientific conferences. In addition, the project team needs to remain active for a few years after the CSA to communicate as much as possible to various stakeholders and the wider public in order to help share key knowledge and foster public debate, in line with the missions of public RIs.

Focus on the CSA on the effects of plant protection products on biodiversity and ecosystem services along the source-to-sea continuum

Context and objectives

In 2019, the French Ministries for Ecology, for Research and for Agriculture commissioned INRAE and Ifremer (French Research Institute for Exploitation of the Sea) RIs to conduct a CSA on the effects of PPPs (as defined in the introduction section) on biodiversity and ecosystem services along the source-to-sea (i.e. terrestrial-freshwater-marine) continuum.

More specifically, the mandated CSA concerned a critical analysis of the current science on i) contamination of terrestrial, freshwater and marine ecosystems by PPPs, ii) the resulting effects on biodiversity and ecosystem services, iii) the methods available for a priori and a posteriori environmental risk assessment of PPPs, and iv) possible strategies for prevention and mitigation of PPP contamination and ecotoxicological effects. Besides its scientific relevance (matching to both the INRAE's and Ifremer's spheres of competence), both the RIs considered the request as pivotally relevant from a societal perspective.

CSA coordination was entrusted to the INRAE's DEPE, with financial support from the French Office for Biodiversity (OFB) as part of the French ECOPHYTO plan that aims to reduce the use, risks, and impact of PPPs while sustaining economically efficient agriculture (Fig. 2). The project agreement was signed off by the different parties in early 2020.

Governance and implementation of the expert panel

The structure and governance of the CSA are illustrated in Fig. 2. The project team involved three scientific leads and three librarians coordinated by a team manager belonging to the INRAE DEPE. As indicated above (Section 2.3), the project team was in charge of identifying and recruiting the experts, mainly based on a first analysis of the scientific literature using preliminary bibliographic requests covering as of the entire CSA scope as possible. Taken together, the 46 experts, including the three scientific leads, have contributed to about 2000 publications in international journals, including a large number of co-publications (Fig. 3). This CSA also relies on two committees (Fig. 1 and2). The oversight committee is composed of representatives of the three backer ministries, the two mandated RIs, the funders, and representatives of the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) which is the national authority for marketing authorizations on PPPs. Given the large scope of the CSA and its potential impact on a wide range of environmental, economic and social sectors, the stakeholder advisory committee gathered more than 20 different public, private and associative actors.

Potential outcomes: examples from the 2005 CSA on "Pesticides, agriculture and environment", and new challenges

The earlier CSA dealing with the environmental effects of PPPs was conducted in 2004-2005.

It was entitled "Pesticides, agriculture and environment: How to reduce the use of pesticides and limit their environmental impacts" [START_REF] Aubertot | Pesticides, agriculture and environment. How to reduce the use of pesticides and to limit their environmental impacts[END_REF]. Its societal impact was assessed in 2011 [START_REF] Hocdé | ASIRPA, Socio-economic Analysis of the Impacts of Agricultural Public Research -Collective Assessment on Pesticides. Executive Summary[END_REF]. On the public action front, it was clearly established that the The 2005 CSA was dedicated to conventional pesticides used in agriculture, with a limited focus on the resulting environmental effects outside agricultural areas [START_REF] Aubertot | Pesticides, agriculture and environment. How to reduce the use of pesticides and to limit their environmental impacts[END_REF]. In comparison, the current CSA addresses a new field of investigation by considering the ecotoxicological effects of PPPs, including natural substances and biobased processes (which started to gain wider use in 2005 and so had not been high on the agenda in the previous CSA) used in both agricultural and non-agricultural activities, in both terrestrial and aquatic environments along the source to sea continuum. Moreover, and as explicitly stated in its title, the current CSA has given specific focus to biodiversity and ecosystem services. It reflects the increasing awareness among environmental managers, decisionmakers and policymakers of the importance of biodiversity and ecosystem services, which are recognized as priority goals for protection in the ecological risk assessment (ERA) on PPPs [START_REF] Maltby | Ecosystem services and the protection, restoration and management of ecosystems exposed to chemical stressors[END_REF]EFSA 2016;European Commission 2020). However, the ecosystem service approach still leaves a number of issues unresolved for ERA on PPPs. They include, but are not limited to, the need to identify quantifiable indicators and relevant endpoints for evaluating the effects of PPPs on ecosystem services (Faber and van Wesem 2012;[START_REF] Faber | Priorities and opportunities in the application of the ecosystem services concept in risk assessment for chemicals in the environment[END_REF]) and to define clear and quantifiable protection goals and restoration targets [START_REF] Maltby | Ecosystem services and the protection, restoration and management of ecosystems exposed to chemical stressors[END_REF]. In this context, the current CSA deliverables are expected to serve as an important scientific basis for further decisionmaking at French and/or EU level to better protect (or restore) biodiversity and ecosystems against the direct and indirect adverse effects of PPPs.

  2005 CSA served as a scientific support for the French Interministerial Plan for the Reduction of Pesticide Risks (adopted in June 2006) as well as for the French Grenelle de l'Environnement stakeholders forum organized from July to October 2007 by the French government, and the resulting raft of legislative measures. It further contributed to the adoption of environmental measures by the French National Assembly as well as the drafting and publication in 2008 of the first version of the French ECOPHYTO Plan to "reduce and improve the use of plant protection products", which has since been revised twice (ECOPHYTO II+ Plan 2018). This management plan has enabled to fund several research projects to improve knowledge on the fate and effects of PPPs in the environment. The dissemination to the general public was also evidenced by the publication in 2006 of 148 press articles related to the main results and conclusions of this CSA. Moreover, these results and conclusions have been used by various pro-environment non-governmental organizations to disseminate scientific knowledge and information to stakeholders and the general public. This analysis confirms the relevance of CSAs to enhance the interactions between scientific knowledge, decision and policy making, and public debate.

Figure 1 :Figure 2 :Figure 3 :

 123 Figure 1: Main steps of the procedure classically followed for Collective Scientific Assessment

Figure 3 :

 3 Figure 3:

Acknowledgments

The authors acknowledge i) the French Ministries for Ecology, for Research and for Agriculture who commissioned this collective scientific assessment, ii) the INRAE scientific directorate for science strategy and the general directorate of the Ifremer, in particular Thierry Caquet and Léa Marty, iii) Guy Richard, head of the INRAE Directorate for Collective Scientific Assessment Expertise, Foresight and Advanced Studies (DEPE) , iv) the whole INRAE task force who contributed to the writing of the Guidelines for Collective Scientific Assessments and Advanced Studies (INRA-DEPE, 2018), v) the members of the CSA Oversight Committee and the CSA Stakeholder Advisory Committee, and vi) the scientific experts who agreed to participate in this ambitious work.

Funding

This collective scientific assessment is financially supported by the French Office for Biodiversity (OFB) through the national ECOPHYTO plan.


Cyrine Ben-Hafaïedh
email: c.benhafaiedh@ieseg.fr

Claire Champenois
email: cchampenois@audencia.com

Thomas M Cooney
email: thomas.cooney@tudublin.ie

Leon Schjoedt
email: leonschjoedt@ymail.com

Entrepreneurship as collective action: The next frontier

Keywords: collective action, collective agency, collective enterprise, collective entrepreneurship, entrepreneurial teams

Collective action in entrepreneurship is lacking in the extant literature. Despite entrepreneurship research progressively moves away from the focus of a lone heroic entrepreneur, scholars have yet to absorb the full potential of entrepreneurship as collective action. Also missing is a collective stance on key entrepreneurship concepts such as opportunity discovery or construction and entrepreneurial agency. Accordingly, this article reviews and critiques five articles that constitute this Special Issue seeking to establish 'entrepreneurship as collective action' as the next frontier of entrepreneurship theory development. The articles in this Special Issue each investigate a specific instance of collective action in entrepreneurship. This article contributes to extant scholarship by highlighting transversal themes and offering further research avenues.

Introduction

Entrepreneurship research is progressively moving away from the focus on a heroic individual [START_REF] Dodd | Mumpsimus and the Mything of the Individualistic Entrepreneur[END_REF][START_REF] Ogbor | Mythicizing and reification in entrepreneurial discourse: ideology-critique of entrepreneurial studies[END_REF] towards an interest in the collective dimensions of entrepreneurship. Over the last two decades, a growing research stream has highlighted the importance of entrepreneurial teams and of their collective dynamics [START_REF] Ben-Hafaïedh | Entrepreneurial Teams Research in Movement[END_REF][START_REF] Cooney | Editorial: What is an Entrepreneurial Team?[END_REF][START_REF] Piva | Should I sell my shares to an external buyer? The role of the entrepreneurial team in entrepreneurial exit[END_REF][START_REF] Preller | Entrepreneurial visions in founding teams: Conceptualization, emergence, and effects on opportunity development[END_REF][START_REF] Schjoedt | Entrepreneurial teams: definition and performance factors[END_REF][START_REF] Schjoedt | Teams[END_REF]. At present, a collective perspective on entrepreneurship as prosocial organizing is gaining traction [START_REF] Branzei | Going pro-social: Extending the individual-venture nexus to the collective level[END_REF][START_REF] Castellanza | Discipline, abjection, and poverty alleviation through entrepreneurship: A constitutive perspective[END_REF][START_REF] Doh | Adapting to grand environmental challenges through collective entrepreneurship[END_REF][START_REF] Hertel | Getting more from many-A framework of community resourcefulness in new venture creation[END_REF][START_REF] Wigger | We're All in the Same Boat: A Collective Model of Preserving and Accessing Nature-Based Opportunities[END_REF]. Under various labels such as 'social entrepreneurship' [START_REF] Chatterjee | Social entrepreneurship and values work: The role of practices in shaping values and negotiating change[END_REF][START_REF] Kimmitt | Sensemaking the 'social' in social entrepreneurship[END_REF], 'community-based entrepreneurship' [START_REF] Murphy | From principles to action: Community-based entrepreneurship in the Toquaht Nation[END_REF][START_REF] Seyb | Exoskeletons, entrepreneurs, and communities: A model of co-constructing a potential opportunity[END_REF], 'collective entrepreneurship' [START_REF] Dufays | Where do hybrids come from? Entrepreneurial team heterogeneity as an avenue for the emergence of hybrid organizations[END_REF][START_REF] Meyer | The commons: A model for understanding collective action and entrepreneurship in communities[END_REF], 'impact entrepreneurship' [START_REF] Markman | E Pluribus Unum: Impact Entrepreneurship as a Solution to Grand Challenges[END_REF], or 'activist entrepreneuring' [START_REF] Dey | Overcoming constraints of collective imagination: An inquiry into activist entrepreneuring, disruptive truth-telling and the creation of 'possible worlds[END_REF], an emerging array of contributions has started to challenge the dominant understanding of entrepreneurship by arguing that entrepreneurship may also be considered as a social force of change instigated by the collective action of individual persons pursuing a common purpose.

As a result of these developments, vibrant, yet disconnected, conversations about entrepreneurship as a collective action have begun. However, these discussions have left many questions open such as how to conceive entrepreneurial collective action beyond the politicaleconomic perspective [START_REF] Olson | The logic of collective action: public goods and the theory of groups[END_REF] reinterpreted by [START_REF] Ostrom | A behavioral approach to the rational choice theory of collective action[END_REF][START_REF] Ostrom | Collective action and the evolution of social norms[END_REF] that specifies collective action as a pursuit of a common group interest by people that goes beyond individual self-interests.

Several questions remain unexplored regarding entrepreneurship as collective action including are there any common features in heterogeneous entrepreneurial collective endeavours; and, when considering different stakeholders' involvement in the entrepreneurial process [START_REF] Sarasvathy | Causation and effectuation: Toward a theoretical shift from economic inevitability to entrepreneurial contingency[END_REF][START_REF] Shane | The Promise of Entrepreneurship as a Field of Research[END_REF], why does entrepreneurship require collectives to occur? Despite such issues and questions, the entrepreneurship conceptualization commonly relies on individual agentic behaviour as a paradigmatic foundation. Even when collective action is explicitly mentioned, the individual entrepreneur often remains the key unit of analysis (e.g., [START_REF] Sarasvathy | An effectual model of collective action for addressing sustainability challenges[END_REF]. This means that scholars have yet to realize the full potential of entrepreneurship as a collective action; and few scholars have adopted a collective stance on key entrepreneurship concepts such as opportunity discovery or construction, effectuation, agency, or entrepreneurial alertness.

Purpose of the Special Issue

With this Special Issue (SI) we seek to establish 'entrepreneurship as collective action' as the next frontier of entrepreneurship research. Our ambition is to stimulate research and theory building on entrepreneurship as a collective action by linking extant recent research on this topic, by empirically illustrating it, and by conceptualising it. Departing from an individualistic perspective [START_REF] Dimov | Beyond the Single-Person, Single-Insight Attribution in Understanding Entrepreneurial Opportunities[END_REF] for this SI, we sought contributions that instead focus on collective action in entrepreneurship to offer new insights regarding how to conceive entrepreneurship as a collective action may challenge, renew, or enrich the entrepreneurship literature. In our efforts, we recognise that entrepreneurship as collective action requires further conceptual development and the conversation in the literature to advance to define the concept and process of entrepreneurship as collective action [START_REF] Schad | Quo vadis, paradox? Centripetal and centrifugal forces in theory development[END_REF]. We encourage such development and advancement of the literature to unfold the multiple meanings of the concept by questioning the premises and assumptions regarding entrepreneurship as collective action. With the SI we also sought research that focuses on entrepreneurship as collective action that would "challenge the core and extend

[the] boundaries" (Schad et al., 2019: 107) of entrepreneurship research. It is arguable that approaching entrepreneurship as a collective practice -that is as organized patterns of activity involving shared rules, principles, tasks, and projects, and that include both "doings" and "sayings" [START_REF] Schatzki | Social Practices: A Wittgensteinian Approach to Human Activity and the Social[END_REF][START_REF] Champenois | Entrepreneurship as practice: systematic literature review of a nascent field[END_REF] -offers opportunities to advance greater understanding of the topic.

In line with the considerations above, we encouraged empirical and conceptual submissions for this SI, particularly research advancing understanding and conceptualization of "collective action" in entrepreneurship. We specifically sought research on the following themes and research questions:

1. Linkages between existing research streams offering neighbouring notions: social entrepreneurship, community entrepreneurship, impact entrepreneurship, collective enterprise, and entrepreneurial teams.

Special issue process and overview of selected papers

The initial call for papers for our SI was published in 2021. The submission deadline for full papers was 31 st of May 2022. The Call for Papers generated 32 submissions. Of these, 16 papers were selected to enter a double-blind peer review process. On completion of the review process, five papers were selected for publication in this SI. The five selected papers in this SI exemplify the processes and practices of entrepreneurship as collective action. The studies span geographical contexts of Belgium, France, Italy, Norway, and Scotland. The contributing authors adopted various qualitative approaches to explore the emerging theme of entrepreneurship as collective action including longitudinal comparative as well as individual studies, enactive research, in-depth interviews, detached observation methodologies, plus secondary data analysed through grounded theory building, thematic analysis, and temporal bracketing methods. The research in this SI encompasses and connects various levels of analysis (micro, meso, and macro) and each study draws from rich and deep empirical data. The theoretical stances encompass social enterprise and community-based enterprise research crossed with an agency perspective; collective action and entrepreneurial ecosystems considered through a critical realist epistemology; collective entrepreneurial identity narratives approached through a practice theory perspective; Transactive Memory Systems combined with new venture teams' framework; and art collectives as public entrepreneurs.

The first three articles study entrepreneurship as a collective action at the meso level of a region. In their article, titled "The creation of collective enterprises for social impact: An agency perspective", Deschamps and Slitine investigate the creation process of collective enterprises that wash and reuse drinking bottles of glass for the purpose of reducing waste, promoting local business, and creating local jobs. Drawing on a longitudinal empirical study of two cases in France, with privileged access to participant and non-participant observations, interviews (n=40), and secondary data, the researchers examine the issue of entrepreneurship as collective action as collective agency.

Specifically, Deschamps and Slitine ask: What form of agency develops and sustains the emergence of collective enterprises for social impact? These scholars offer a theoretical model of collective agency that sustains the emergence of collective enterprises. The key finding in this study is that the process of creating collective enterprises for social impact requires a specific form of collective agency by combining three interrelated dimensions (i.e., temporal stages): 'collective entrepreneurship' (i.e., practices laying the foundations of collective action); 'collective animatorship' (i.e., practices supporting the collective action, by embedding the project locally and by engaging new partners); and 'collective organising' (i.e., ensuring the continuity of collective action). Each of these stages involves different actors. Collective entrepreneurship is driven by a group of actors involved in the company's development who seek to engage collective action and hold a strong set of shared values including a desire to change the world. Collective animatorship refers to actors who are keen to contribute to local development. And, collective organising encompasses multiple stakeholders who are being mobilised. The study by Deschamps and Slitine offers two key contributions. First, through a theoretical model of collective agency, the authors provide new theoretical insights on the notion of entrepreneurial agency --key in the field of entrepreneurship [START_REF] Mcmullen | What Makes an Entrepreneurship Study Entrepreneurial? Toward A Unified Theory of Entrepreneurial Agency[END_REF], but is often conceived as an individual agency even if studied in a collective context [START_REF] Mcmullen | What Makes an Entrepreneurship Study Entrepreneurial? Toward A Unified Theory of Entrepreneurial Agency[END_REF][START_REF] Dacin | Social entrepreneurship: A critique and future directions[END_REF][START_REF] Gupta | Social entrepreneurship research: A review and future research agenda[END_REF], or 'distributed' [START_REF] Enfield | Distributed agency[END_REF][START_REF] Garud | Bricolage versus breakthrough: distributed and embedded agency in technology entrepreneurship[END_REF]. Deschamps and Slitine show that collective agency is, and subsequently, should be examined as a sum of practices, a process, [START_REF] Caldwell | Things fall apart? Discourses on agency and change in organizations[END_REF] involving multiple practitioners [START_REF] Champenois | Entrepreneurship as practice: systematic literature review of a nascent field[END_REF]. With their conceptualisation of collective agency, these authors go beyond the focus on individual self-interest to emphasize that convergence among various practitioners around a common objective plays a key role in entrepreneurship as a collection action.

In the article by Hruskova, titled "Ecosystem pipelines: collective action in entrepreneurial ecosystems", the focus is on entrepreneurial ecosystems (and on their governance) at a regional level unit of analysis. Stressing that "we still know little about how ecosystem actors work collectively to assist entrepreneurs with starting and scaling their ventures" (p. XX), the author investigates how actors in an ecosystem engage in collective action, and how this collective action unfolds. Hruskova employs a case study approach using a Scottish entrepreneurial ecosystem and encompassing 51 semi-structured interviews with representatives of key ecosystem actors such as entrepreneurs, advisors, investors, entrepreneurship support organisations, professional service providers, corporate organisations, universities, and representatives from the local and national government. The author draws from [START_REF] Ostrom | Governing the commons: The evolution of institutions for collective action[END_REF] to consider collective action as a situation in which "diverse actors work together to achieve a common objective" (p. XX). A key contribution of the paper is the coining of the concept of 'ecosystem pipelines' to describe a novel ecosystem governance mechanism that results from collective action. Specifically, ecosystem pipeline refers to "logical pathways between ecosystem actors developed through collective action that enable entrepreneurs to secure resources as they launch, develop, and grow their venture" (p. XX). These ecosystem pipelines are relational structures formed through relationship building among actors via referrals and signposting allowing entrepreneurs to access necessary resources, including advice, support, and more, as the new ventures develop. Considering an entrepreneurial ecosystem as a result of collective action means that recognizing their structural dimensions (such as the number and nature of participants) and their relational resources such as trust, reputation, reciprocity, and a shared purpose. This contribution renews research on entrepreneurial ecosystems by proposing a horizontal governance, rather than a top-down or a bottom-up approach, in entrepreneurship as collective action.

In the third article, titled "Scratching the surface of urban change: art collectives as public entrepreneurs", Morea and Dalla Chiesa examine how artists in Venice, Italy, engage with the city to collectively meet local demands. As such, the artists act as public entrepreneurs seeking to achieve societal change [START_REF] Beyes | Fictions of the possible: Art, the city, and public entrepreneurship[END_REF]. These researchers interviewed representatives of 11 art collectives to examine the intended outcomes of various initiatives. Based on semi-structured interviews, their analysis took an interpretative turn. The authors examine how the art collectives address institutional voids, that is a lack of governmental or institutional support for overcoming social challenges [START_REF] Stephan | Institutions and social entrepreneurship: The role of institutional voids, institutional support, and institutional configurations[END_REF], and try "to fill identified gaps in the public agenda (public policy, urban planning and tourism strategies)" (p. XX) in the sense of provoking change [START_REF] Steyaert | Entrepreneurship as social change: A third new movements in entrepreneurship book[END_REF]. The main tension is that between a tourism-led urban ecosystem versus a city focused on locals. Three themes are discussed, tracing how the Venetian art collectives became public entrepreneurs: a locally grounded raison d'être, the translation of core values into artistic proposals, and the alignment of the collectives' private goals with a public service ethos.

The findings provided by Morea and Dalla Chiesa reveal that, through their collective actions, the artists' initiatives convey a message of urban change, contributing both discursively and materially, but do not have a substantial impact at the policy level as the artists' initiatives is merely scratch the surface by making proposals for interventions. The authors argue that art collectives are a different form of public entrepreneurship as their actions are frequently quite ephemeral and therefore provoke or raise awareness rather than directly confront policymakers. According to Morea and Dalla Chiesa, art collectives "are not institutional changemakers; they try to upset the institutional arrangement by mediating local demands and offering sporadic and unsystematic cultural projects" (p.XX). Their work suggests that while art collectives may not be viewed as typical public entrepreneurs, it opens "a possible avenue of research for another type of urban actor, one caught up in the middle ground between grassroots artistic creation and impact-driven work within institutional contexts" (p.XX).

The last two articles take a micro level perspective in studying collective action within entrepreneurial teams in different contexts. In the article, titled "It all starts with a story: questioning dominant entrepreneurial identities through collective narrative practices", Solbreux, Hermans, Pondeville, and Dufays adopt an entrepreneurship-as-practice [START_REF] Champenois | Entrepreneurship as practice: systematic literature review of a nascent field[END_REF] perspective to entrepreneurship as collective action. Developing a theoretical framework on collective entrepreneurial identity narratives, they consider entrepreneurial identity as something that is 'done' and 'said' [START_REF] Gherardi | Authoring the female entrepreneur while talking the discourse of work-family life balance[END_REF] and, that is fluid and ever-changing. They offer a collective perspective on narrative identity construction. Solbreux et al. explore how collective narrative practices provide individual people with opportunities to depart from dominant entrepreneurial identities and to coconstruct new, socially anchored identities. Using an original research method -scaffolding conversations [START_REF] White | Narrative means to therapeutic ends[END_REF], they study teams of students engaged in a 10-week social entrepreneurship course at a business school in Belgium. The data are gathered from reflective journals written by 68 students and are analysed to identify four archetypes (Witnesses, Resisters, Victims and Perpetrators). Solbreux et al. illustrate how identities evolve, as students incorporate other possible viewpoints that refer to, and emerge from, collective endeavours through the process of "thinning the plot" of dominant identities and "thickening the counterplot". Interestingly, they show through collective narrative practices, how practitioners may disrupt the dominant individual heroic entrepreneur myth to develop identities that align with an understanding of entrepreneurship as collective action. The study contributes to entrepreneurship-as-practice by offering a multiplepractitioner perspective on entrepreneurial identity construction. Notably, it shows how collective narratives provide opportunities to reflect on the role of teams and ecosystems in the process of entrepreneurial identity construction; conversely, the role of the protagonists as influencers in teams and ecosystems. Collective narratives support "attuning" with peers (i. 1987) as their theoretical framework, Georgiadou et al. approach entrepreneurship as collective action as the dynamics of working together within entrepreneurial teams. Transactive memory systems (TMS) refers to "the mechanisms that help to integrate and coordinate the expertise NVT members collectively possess" (p. XX). Building on the idea that individual persons may serve as external memory support to each other, TMS are conceptualized as emerging when individual people search for help by contacting others (to solve problems that they cannot address alone), and by remembering these contacts as well as their contributions [START_REF] Nebus | Building collegial information networks: A theory of advice network generation[END_REF]. Georgiadou et al. explore how TMS emerge within entrepreneurial teams, including how expertise is identified, shared, and used by NVT members. Specifically, they investigate the process of collectivisation of individual members' expertise through integration and coordination of their expertise at the team level. The study by Georgiadou et al. advances our appreciation of entrepreneurship as collective action by identifying four processes through which TMS develop (enabling, encoding, storage, and retrieval) that are placed in a process model. By doing so, the authors reveal how TMS processes unfold in NVTs, and how entrepreneurial teams develop specialisation, credibility, and coordination that are considered indicators of TMS (Lewis, 2003). The strength of the process model provided is that it encompasses three stages: (i) pre-formation stage when entrepreneurial teams are just formed; (ii) formation stage when teams begin to develop their business idea, and (iii) collaboration stage when teams further advance their business idea. In addition, the authors provide three contributions pertaining to entrepreneurial teams and their work. First, the study furnishes evidence of the many efforts required by all team members in order to develop TMS such as collective expertise coordination and integration mechanisms. Second, it highlights the fluidity of knowledge circulation and use within NVTs as members continuously reassess each other's expertise revealing a need of constantly updating and refining TMS to be effective. Third, it offers new foundations for NVTs' efficiency through specialisation, credibility of others' expertise, and coordination among members.

Like the other four contributions in this SI, the fifth contribution reveals insights into entrepreneurship as collective action.

Reflections and Insights

Our intent with this SI was to provide research contributions that depart from the conventional individualistic perspective in the entrepreneurship literature [START_REF] Dimov | Beyond the Single-Person, Single-Insight Attribution in Understanding Entrepreneurial Opportunities[END_REF] by focusing on entrepreneurship as collective action by showing how entrepreneurship as collective action challenges, renews, and enriches our collective understanding of entrepreneurship. Based on the studies in this SI, we obtained a richer and more diverse understanding of the concept of entrepreneurship as collective action.

Firstly, several articles explore the notion of collective agency. When [START_REF] Mcmullen | What Makes an Entrepreneurship Study Entrepreneurial? Toward A Unified Theory of Entrepreneurial Agency[END_REF] observe that entrepreneurial agency is not limited to individual people, they, in effect, propose distributed agency, and discuss the issues of communication and coordination costs as well as conflicting interests. The study by Deschamps and Slitine in this SI provides conceptual clarification of collective agency in entrepreneurship; an issue that deserves more attention from researchers. Thus, these scholars with their contribution open avenues for future research that further conceptualize 'collective agency'. The findings by Deschamps and Slitine also highlight the value of research that combine the concept of collective agency with existing entrepreneurship concepts such as 'entrepreneurial ecosystems', which is furthered and confirmed by Hruskova's contribution in this SI. Further, Solbreux et al., in this SI, point to the discursive dimension of entrepreneurship as collective action. They focus on collective narratives that "reveal an increased sense of collective agency for nascent entrepreneurs, as they realise that the team members and the community share a desired social change and a motivation to act on it" (p. XX). Future research adopting a language-centred perspective of entrepreneurship as collective action holds potential to shed new informative insights.

Secondly, additional new insights that may be gained by considering the contributions in this SI collectively is the notion of self-organizing. Giorgiadou et al. discuss self-organizing teams and horizontal leadership within entrepreneurial teams. Hruskova observes relational organizing [START_REF] Powell | Neither market nor hierarchy: Network forms of organization[END_REF] Thirdly, our SI with its research contributions is suggestive of a strong link between entrepreneurship as collective action and the possibility to address the grand challenges in our World, sustainability [START_REF] Markman | E Pluribus Unum: Impact Entrepreneurship as a Solution to Grand Challenges[END_REF][START_REF] Doh | Adapting to grand environmental challenges through collective entrepreneurship[END_REF], rather than growth per se. Such research holds potential to further the claim "that enterprises run by collectives and communities are agents for social change" (Meyer, 2020: 2). Solbreux et al. point out that the co-construction of emboldened yet realistic collective identities in an entrepreneurship context is needed to face the grand challenges of this world. Morea and Della Chiesa show that the art collectives via entrepreneurship as collective action mediate local demands and act for a better world. These findings encourage further investigation of entrepreneurship as collective action as the best-suited form to address Anthropocene [START_REF] Parrique | Degrowth[END_REF]; the contemporary challenges of whether human civilization and its ability to meet and overcome a dramatically and turbulent world. Again, future research on entrepreneurship as collective action holds potential to assist with these challenges.

Lastly, [START_REF] Dufays | Where do hybrids come from? Entrepreneurial team heterogeneity as an avenue for the emergence of hybrid organizations[END_REF] note that there is a need for identifying antecedents and outcomes of entrepreneurship as collective action. The contributions in this SI focus on the antecedents while adopting a processual lens. Specifically, Deschamps and Slitine study the process of creating collective enterprises for social impact to find that such venture creation requires a specific form of collective agency. Adding to this, Hruskova studies the formation of ecosystem Additional research is needed on the roles and specificities of entrepreneurship as collective action.

Concluding Thoughts

We, the guest editors, all share an interest in entrepreneurship as collective action. We acknowledge that entrepreneurship is traditionally perceived as a process in which an individual pioneer, or, on occasion, a small group-an entrepreneurial team, brings a new venture into existence. This body of research is undergoing a profound transformation to become more inclusive of actors who have not traditionally been considered in entrepreneurship research. This development and an acknowledgement that individual entrepreneurs or entrepreneurial teams do not create their new ventures in isolation; instead, actors outside the nascent or new venture also make important contributions such as, but are not limited to, crowdfunding in which the public make monetary and other forms of contributions (e.g., information, feedback) to a new venture (Troise et al., Forthcoming). This means that our collective knowledge about entrepreneurship will be enriched by going beyond focusing on actions, decisions, or characteristics of an individual entrepreneur or an entrepreneurial team to entrepreneurship as collective action in which entrepreneurship includes actors, processes, and outcomes external yet impacting to the new venture and its performance.

These considerations form the basis for our primary goal for this SI. We had a second goal with the SI. This was to provide examples of novel or, at least, different research methods in line with recent calls made in our field [START_REF] Van Burg | Advancing qualitative entrepreneurship research: Leveraging methodological plurality for achieving scholarly impact[END_REF]. In other words, we sought to move beyond correlational and positivist research approaches, specifically the widespread use of regression analysis, which traditionally dominates the entrepreneurship literature [START_REF] Schjoedt | Control variables: Use, misuse, and recommended use[END_REF].

The five contributions that make up this SI advance the entrepreneurship literature by investigating entrepreneurship as collective action and by employing non-conventional research methods. Collectively, the five studies that make up this SI fulfil the two goals we set out to achieve for the SI. Furthermore as highlighted previously in this SI, entrepreneurship as collective action may be characterized by diverse practices situated in specific places, both geographical and virtual, and groups of actors, whom with their distinct strengths converge into pools of resources for the new venture creation or to craft solutions to foster economic or societal change such as obtaining resources for basic needs of the new venture such as capital or information via crowdfunding, or fulfilling any of the United Nations' Sustainable Development Goals. Our editorial shows that there is substantial opportunities for future research to advance our collective knowledge in the area of entrepreneurship as collective action.

2.

  New insights on collective processes and practices of entrepreneurship: how do they unfold over time? With what outcome? What stabilised patterns or institutionalised forms emerge from collective entrepreneurial actions? How are they maintained or challenged over time? How to contextualize them? 3. Methodological contributions and critical perspectives: the different research approaches that might be employed to study entrepreneurship as collective action, and consideration of the possible dark sides of entrepreneurial collective action. 4. Contributions to core concepts of entrepreneurship: these might include concepts such as opportunity discovery or construction, effectuation, agency, or entrepreneurial alertness. Our Call for Papers was kept deliberately open to encourage innovative or alternative approaches to the topic of entrepreneurship as collective action. This offered potential contributors and us, the Guest Editors, the possibility of receiving imaginative or visionary interpretations to entrepreneurship as collective action.

  e., articulating and feeling what matters and what people care about in the collective) which is a key element in the formation of collective agency. Solbreux et al. also offers methodological contributions by proposing a method that grasps practices at the group level, rather than at the individual one, to further advance research on entrepreneurship as collective action. In the final article, titled 'Development of transactive memory systems in new venture teams', Georgiadou, Steinmo, and Lauvås investigate the dynamics taking place within technology-based entrepreneurial teams founded in a venture creation program at a Norwegian university. Their findings are rooted in a longitudinal study that encompasses 52 semi-structured interviews with individual participants and groups of participants from five new venture teams (NVTs) over three rounds of data collection, as well as secondary data. Using transactive memory systems (Wegner,

  in purpose-driven networks; while Morea and Della Chiesa examine bottom-up collectives. Drawing on the findings from these three studies collectively, future research may provide new insights by investigating the potential and limits of self-organizing and its dynamics. Such research may also integrate the conceptual foundations employed in other contributions to this SI: actors' awareness of partner activities (Hruskova), Transactive Memory Systems (Giogiadou et al.), and collective animatorship (Deschamps and Slitine) to generate new insights into entrepreneurship as collective action.

  pipelines; while Solbreux et al. investigate the emergence of collective entrepreneurial identities. By exploring the emergence and stabilisation dynamics of entrepreneurship as collective action, as done in this SI, future research holds potential to provide new insights on antecedents and outcomes of entrepreneurship as collective action. The role(s) and specificities of entrepreneurship as collective action are also shown in this SI by Deschamps and Slitine, Hruskova, and Morea and Dalla Chiesa.

Acknowledgements

The Guest Editors would like to thank ISBJ for accepting this Special Issue. We are especially grateful to Professor Susan Marlow for her guidance and to Ms. Valerie Thorne for supporting us through the process. We are also grateful to the many anonymous reviewers who provided detailed and constructive feedback on the manuscripts that ultimately helped us decide on the final selection.

Finally, we thank the authors of the five papers that make up this Special Issue for choosing to share their valuable and insightful research work with us.


Aurélie Hennebique

Fabienne Gas

Hélène Batina

Cécilia De Araujo

Karine Bizet

Max Maurin
email: mmaurin@chu-grenoble.fr.

Evaluation of the Biotoxis qPCR Detection Kit for Francisella tularensis Detection in Clinical and Environmental Samples

Keywords:

Rapid and reliable detection and identification of Francisella tularensis (a tier 1 select agent) are of primary interest for both medical and biological threat surveillance purposes. The Biotoxis qPCR detection kit is a real-time quantitative PCR (qPCR) assay designed for the detection of Bacillus anthracis, Yersinia pestis, and F. tularensis in environmental or biological samples. Here, we evaluated its performance for detecting F. tularensis in comparison to previously validated qPCR assays. The Biotoxis qPCR was positive for 87/87 F. tularensis subsp. holarctica (type B) strains but also for F. tularensis subsp. novicida. It was negative for Francisella philomiragia and 24/24 strains belonging to other bacterial species. For 31 tularemia clinical specimens, the Biotoxis qPCR displayed a sensitivity between 90.32% and 96.55%, compared to qPCR tests targeting ISFtu2 or a type B-specific DNA sequence, respectively. All 30 nontularemia clinical specimens were Biotoxis qPCR negative. For water samples, the Biotoxis qPCR limit of detection was 1,000 CFU/liter of F. tularensis. For 57 environmental water samples collected in France, the Biotoxis qPCR was positive for 6/15 samples positive for ISFtu2 qPCR and 4/4 positive for type B qPCR. In conclusion, the Biotoxis qPCR detection kit demonstrated good performances for F. tularensis detection in various biological and environmental samples, although crossamplification of F. tularensis subsp. novicida must be considered. This plate format assay could be useful to test a large number of clinical or environmental specimens, especially in the context of natural or intentional tularemia outbreaks.

nated food or water [START_REF] Sjöstedt | Tularemia: history, epidemiology, pathogen physiology, and clinical manifestations[END_REF][START_REF] Maurin | Tularaemia: clinical aspects in Europe[END_REF]. Several clinical forms of tularemia exist, mainly depending on the portal of entry of the bacteria. The ulceroglandular and glandular forms correspond to localized lymphadenopathy with or without a skin ulcer, respectively. The oropharyngeal form combines pharyngitis and cervical lymphadenopathy. The oculoglandular form usually corresponds to conjunctivitis with periauricular lymphadenopathy. The pneumonic form (pneumonia or pleuropneumonia) usually occurs through the inhalation of contaminated aerosols. The typhoidal form is severe sepsis, often with confusion, but with no inoculation lesion or lymphadenopathy [START_REF] Sjöstedt | Tularemia: history, epidemiology, pathogen physiology, and clinical manifestations[END_REF][START_REF] Maurin | Tularaemia: clinical aspects in Europe[END_REF]. Because of its ability to be spread by aerosols, its low infectious dose, and high virulence, F. tularensis is classified in category A of potential biological threat agents by the U.S. Centers for Disease Control and Prevention (CDC) [START_REF] Dennis | Tularemia as a biological weapon: medical and public health management[END_REF]. Pneumonic tularemia cases caused by the most virulent type A strains are associated with up to 30% mortality rates [START_REF] Sjöstedt | Tularemia: history, epidemiology, pathogen physiology, and clinical manifestations[END_REF][START_REF] Maurin | Tularaemia: clinical aspects in Europe[END_REF][START_REF] Dennis | Tularemia as a biological weapon: medical and public health management[END_REF].

Tularemia diagnosis is currently based on serological methods, and F. tularensis detection is performed by culture or PCR-based methods [START_REF] Maurin | Tularaemia: clinical aspects in Europe[END_REF]. However, this fastidious and slow-growing bacterium is usually isolated from less than 10% of tularemia patients [START_REF] Maurin | Tularaemia: clinical aspects in Europe[END_REF]. Serological methods can detect significant antibody titers only 2 to 3 weeks after disease onset [START_REF] Maurin | Tularaemia: clinical aspects in Europe[END_REF][START_REF] Yanes | Evaluation of in-house and commercial serological tests for diagnosis of human tularemia[END_REF]. Therefore, real-time PCR tests are currently the most effective method for detecting F. tularensis in human, animal, or environmental samples [START_REF] Maurin | Tularaemia: clinical aspects in Europe[END_REF]. Although many in-house qPCR tests have been developed, a user-friendly commercial test allowing rapid, accurate, and standardized detection of F. tularensis in various types of samples is a high priority.

The Biotoxis quantitative PCR (qPCR) detection kit is a plate-format TaqMan probebased real-time PCR assay designed for the combined detection of Bacillus anthracis, Yersinia pestis, and F. tularensis DNA in various sample types. In the present study, we evaluated this kit's performance for detection and identification of F. tularensis in DNA extracts from bacterial strains, clinical samples, and environmental water samples, in comparison to previously validated PCR tests used routinely in our laboratory.

MATERIALS AND METHODS

Biotoxis qPCR assay. The Biotoxis qPCR detection kit (Bertin Bioreagent, Montigny-le-Bretonneux, France) was carried out according to the manufacturer's instructions. Briefly, each of the PCR mixtures (25 l) contained 12.5 l of the qPCR mix, 3.75 l of primer and probe mix, 3.75 l of water, and 5 l of DNA sample (at various concentrations according to sample type; see below). The PCR was performed on a LightCycler 480 instrument II (Roche) with an initial enzyme activation step of 95°C for 3 min, followed by 45 cycles of denaturation at 95°C for 15 s and annealing and extension at 60°C for 30 s. The results were analyzed at the endpoint.

Francisella tularensis reference qPCR assays. We compared results obtained with the Biotoxis qPCR detection kit with those obtained with three previously validated qPCR methods, which are routinely used at the French National Reference Center for Francisella (French NRCF) (Grenoble University Hospital, Grenoble, France). One qPCR test (referred to as ISFtu2 qPCR) targets the ISFtu2 insertion sequence present in multiple copies in the F. tularensis genome [START_REF] Versage | Development of a multitarget real-time TaqMan PCR assay for enhanced detection of Francisella tularensis in complex specimens[END_REF]. The second one (Tul4 qPCR) targets the gene encoding the single-copy Tul4 surface protein-encoding gene [START_REF] Versage | Development of a multitarget real-time TaqMan PCR assay for enhanced detection of Francisella tularensis in complex specimens[END_REF]. The last one (type B qPCR) targets a DNA fragment located between ISFtu2 and a flanking 3= region and is specific for F. tularensis subsp. holarctica [START_REF] Kugeler | Real-time PCR for Francisella tularensis types A and B[END_REF].

These three qPCR tests were performed using the same protocol. Each of the PCR mixtures (20 l) contained 10 l of 2ϫ TaqMan Fast Advanced PCR master mix (Applied Biosystems, Thermo Fisher Scientific, Vilnius, Lithuania), 0.4 l of each primer at 10 M, 0.4 l of 2 M probe, 3.8 l of water, and 5 l of DNA sample (at various concentrations according to sample type; see below). The PCRs were run on a LightCycler 480 instrument II (Roche) with an initial enzyme activation step of 50°C for 2 min and 95°C for 2 min, followed by 45 cycles of denaturation at 95°C for 3 s and annealing and extension at 60°C for 30 s. The results were analyzed at the endpoint.

DNA extraction methods. For bacterial strains and clinical samples, DNA extraction was obtained using the QIAamp DNA minikit (Qiagen, Courtaboeuf, France), according to the manufacturer's recommendations.

For artificial and environmental water samples, 1 liter of water was filtered through 0.22-m or 0.45-m filters, and DNA was extracted from the filter using the NucleoMag DNA/RNA water kit (Macherey-Nagel, Düren, Germany), according to the manufacturer's recommendations.

Concentrations of bacterial DNA extracts and environmental water DNA extracts were then assessed using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific).

Bacterial strains. We tested 90 Francisella sp. strains, including 86 clinical strains of F. tularensis subsp. holarctica, one clinical strain of F. philomiragia, and the reference strains F. philomiragia ATCC 25015, F. novicida CIP 56.12, and F. tularensis subsp. holarctica LVS NCTC 10857 (Table 1). Clinical strains of Francisella sp. were previously identified at the French NRCF. F. philomiragia was identified by whole 16S rRNA gene sequencing [START_REF] Kreitmann | Disseminated infection caused by Francisella philomiragia, France, 2014[END_REF]. F. tularensis subsp. holarctica was identified at the subspecies level by combining a positive ISFtu2 or Tul4 qPCR test and either a positive type B qPCR or PCR amplification and sequencing of the expected 16S-23S intergenic region [START_REF] Maurin | Human tularemia in France, 2006 -2010[END_REF]. The French NRCF owns all the above strains, and specific authorizations were obtained from the Agence Nationale de Sécurité du Médicament et des Produits de Santé (ANSM; authorization number ADE-103892019-7). In addition, 24 reference or clinical strains belonging to bacterial species other than Francisella sp. were used (Table 1).

Bacteria were grown on chocolate agar medium supplemented with PolyViteX (bioMérieux, Marcyl'Etoile, France) or on sheep blood agar medium (bioMérieux, Marcy-l'Etoile, France) at 35°C in a 5% CO 2 -enriched atmosphere for 1 or 2 days. Francisella tularensis strains were grown in a biosafety level 3 laboratory.

DNA extracts from Francisella sp. strains were prepared at 10 ng/l for analysis with the Biotoxis qPCR detection kit. DNA extracts from four clinical strains of F. tularensis subsp. holarctica, one clinical strain of F. philomiragia (F. philomiragia ATCC 25015), and F. novicida CIP 56.12 were used to compare cycle threshold (C T ) values obtained with the Biotoxis qPCR to those of the ISFtu2, Tul4, and type B qPCR tests (Table 2). For non-Francisella strains, three DNA extract concentrations were used (0.1, 1, and 10 ng/l).

Clinical specimens. For sensitivity purposes, we tested 31 clinical samples (mainly lymph node and respiratory samples) collected for routine medical care in 30 tularemia patients, including 29 confirmed cases and one probable case (Table 3). These samples were received at the French NRCF between 2018 and 2019 for tularemia diagnostic confirmation. A tularemia confirmed case was defined as an instance of clinically compatible disease with detection of F. tularensis from any clinical specimen by culture or PCR (ISFtu2 or type B qPCR, C T Յ 35), seroconversion, or a 4-fold (or greater) change in titers of serum antibody to F. tularensis antigen between acute-and convalescent-phase sera. A probable case was defined as an instance of a clinically compatible disease with a single positive serum sample. According to our laboratory's routine procedure, all clinical samples were first tested using ISFtu2 qPCR for F. tularensis detection [START_REF] Versage | Development of a multitarget real-time TaqMan PCR assay for enhanced detection of Francisella tularensis in complex specimens[END_REF]. Positive samples were further tested using type B qPCR for the identification of subspecies F. tularensis subsp. holarctica [START_REF] Kugeler | Real-time PCR for Francisella tularensis types A and B[END_REF]. When possible, F. tularensis culture was also performed. These samples were analyzed by the Biotoxis qPCR detection kit retrospectively. DNA extracts were analyzed pure and diluted to 1/10 and 1/100. Samples were considered positive if at least one of the three DNA dilutions tested was positive. Negative results were duplicated for confirmation.

For specificity purposes, we also tested 30 clinical samples from patients for which the French NRCF excluded tularemia diagnosis because of negative diagnostic tests for F. tularensis, including eight samples positive for Bartonella henselae by PCR. All these samples were collected as part of routine patient care and analyzed retrospectively using the Biotoxis qPCR detection kit.

Environmental samples. We first determined the limit of detection (LOD) of the Biotoxis qPCR using F. tularensis-spiked water samples. For this purpose, a water sample collected in the environment was first sterilized by filtration on a 0.22-m filter. Several aliquots were then inoculated with F. tularensis subsp. holarctica LVS NCTC 10857 strain at concentrations ranging from 0.1 to 10,000 CFU/liter. One liter of each aliquot was filtered through a 0.22-m filter, and DNA was extracted from the filter. The DNA extracts were tested using the Biotoxis qPCR detection kit (in triplicate) and by ISFtu2, Tul4, and type B qPCRs, which were used as controls.

We then tested 57 surface water samples collected in France in 2019 from various aquatic environments. One liter of each sample was passed through a 0.45-or 0.22-m filter, from which DNA was extracted. DNA extracts were then adjusted to 10 ng/l and tested in duplicate by the ISFtu2, Tul4, and type B qPCR tests. These qPCR tests were considered positive only when both duplicate tests displayed a C T of Յ36 for ISFtu2 qPCR or a C T of Ͻ40 for Tul4 and type B qPCRs. However, the presence of F. tularensis DNA was considered confirmed only for water samples with a positive type B qPCR test, which can specifically detect F. tularensis subsp. holarctica strains found in France. Water samples were then tested using the Biotoxis qPCR detection kit, and results were compared to those of the three previous qPCR tests.

RESULTS

Bacterial strains. The Biotoxis qPCR gave a strongly positive signal (C T values between 15 and 22) for the 86 clinical strains of F. tularensis subsp. holarctica and for the reference strain F. tularensis subsp. holarctica LVS NCTC 10857, corresponding to a 100% sensitivity for the tested type B tularemia strains. DNA extracts from four clinical strains of F. tularensis subsp. holarctica were tested to determine the analytical sensitivity of the Biotoxis qPCR, compared to those of the ISFtu2, Tul4, and type B qPCR tests. The C T ranges for these four samples were 18 to 20 for the Biotoxis qPCR, 12 to 14 for ISFtu2 qPCR, 15 to 19 for Tul4 qPCR, and 16 to 19 for type B qPCR (Table 2).

The Biotoxis qPCR also gave a strongly positive signal for F. novicida CIP 56.12, like the ISFtu2 and Tul4 qPCR tests [START_REF] Versage | Development of a multitarget real-time TaqMan PCR assay for enhanced detection of Francisella tularensis in complex specimens[END_REF]. As expected, type B qPCR was negative for this strain (Table 2). Biotoxis qPCR did not amplify the two F. philomiragia strains tested. The ISFtu2 qPCR gave a weak signal for this species, while Tul4 and type B qPCRs were negative (Table 2) [START_REF] Versage | Development of a multitarget real-time TaqMan PCR assay for enhanced detection of Francisella tularensis in complex specimens[END_REF].

The Biotoxis qPCR was negative for 21 non-Francisella strains but gave a weak signal (C T between 34 and 37 at 10 ng/l of DNA) for Streptococcus salivarius, Pseudomonas aeruginosa CIP 5933, and Neisseria elongata strains. For these three strains, testing lower DNA concentrations (0.1 to 1 ng/l) abolished the amplification signal. Overall, the Biotoxis qPCR displayed a 96.30% specificity for the 27 non-type B strains tested owing to the cross-amplification of F. novicida.

Clinical samples. The 31 clinical samples collected from 30 tularemia patients included 24 lymph nodes, four respiratory samples, two peritoneal samples, and one osteoarticular sample (Table 3).

Tests were positive for 31/31 clinical samples for ISFtu2 qPCR, 29/31 for type B qPCR, and 28/31 for Biotoxis qPCR (Table 3). Therefore, the Biotoxis qPCR test's sensitivity could be evaluated at 90.32% and 96.55% compared to ISFtu2 and type B qPCR tests, respectively. The C T ranged from 20.47 to 36.61 for ISFtu2 qPCR (with 30/31 tests with a C T of Յ35), 28.89 to 39.71 (20/31 tests with a C T of Յ35) for type B qPCR, and 27.37 to 39.06 (15/31 tests with a C T of Յ35) for Biotoxis qPCR (Table 3).

The Biotoxis qPCR was positive for 27/31 pure DNA extracts (with C T values ranging from 27.37 to 39.06), 19/31 DNA extracts diluted 1/10 (C T , 29.85 to 39.43), and 13/31 DNA extracts diluted 1/100 (C T , 33.26 to 39.26). Interestingly, sample S4 was positive by ISFtu2 qPCR, type B qPCR, and Biotoxis qPCR only when diluted 1/100, suggesting the presence of PCR inhibitors.

The 30 clinical samples from patients for which tularemia diagnosis was excluded corresponded to 14 lymph nodes, six blood samples, five abscess samples, one respiratory sample, and four biopsy samples. The Biotoxis qPCR gave negative results for these 30 clinical samples, including the eight patients with bartonellosis, corresponding to a 100% specificity.

Environmental samples. Water samples artificially inoculated with F. tularensis subsp. holarctica LVS NCTC 10857 from 0.1 to 10,000 CFU/liter were analyzed by Biotoxis qPCR and by ISFtu2, Tul4, and type B qPCRs. Biotoxis qPCR detected F. tularensis from 1,000 CFU/liter (with a C T at 34.47). It was less sensitive than type B qPCR (positive from 100 CFU/liter with a C T at 38.23), Tul4 qPCR (positive from 10 CFU/liter with a C T at 36.96), and ISFtu2 qPCR (positive from 0.1 CFU/liter with a C T at 37.57).

The 57 environmental water samples collected in France were previously tested using the ISFtu2, Tul4, and type B qPCR tests. Fifteen were positive by ISFtu2 qPCR, nine were positive by both ISFtu2 qPCR and Tul4 qPCR, and four were positive by ISFtu2 qPCR, Tul4 qPCR, and type B qPCRs. The Biotoxis qPCR was positive for six (40%) of the 15 samples that were positive by ISFtu2 qPCR, five of the nine samples that were positive by both ISFtu2 and Tul4 qPCR, and the four samples that were positive by ISFtu2, Tul4, and type B qPCRs (Table 4). The Biotoxis qPCR was negative for all 42 samples that were negative by the ISFtu2, Tul4, and type B qPCRs (Table 4).

DISCUSSION

In this study, we evaluated the commercial Biotoxis qPCR detection kit's performances for the detection of F. tularensis in clinical and water samples, compared to previously validated qPCR tests recommended by the World Health Organization for tularemia diagnosis [START_REF]WHO guidelines on tularaemia[END_REF].

We first evaluated this kit's ability to detect clinical and reference strains of F. tularensis previously identified in our laboratory. The 86 clinical strains and the reference LVS strain of F. tularensis subsp. holarctica gave strong amplification signals with the Biotoxis qPCR (C T between 15 and 22 for a DNA extract standardized at 10 ng/l). Similar C T values were obtained with the qPCR tests targeting the single-copy Tul4 or type B-specific DNA sequences. In contrast, lower C T values were obtained with the multicopy ISFtu2 target (26 to 30 copies in F. tularensis subsp. holarctica genome [START_REF] Versage | Development of a multitarget real-time TaqMan PCR assay for enhanced detection of Francisella tularensis in complex specimens[END_REF]) (Table 2). To evaluate the specificity of the Biotoxis qPCR, we tested two F. philomiragia strains, one F. novicida strain, and 24 strains not belonging to Francisella species. The Biotoxis qPCR gave a strongly positive signal for the reference strain of F. novicida. This result was not unexpected, since F. novicida (also referred to as F. tularensis subsp. novicida) has Ն97.7% similarity at the genome level to F. tularensis [START_REF] Larsson | Molecular evolutionary consequences of niche restriction in Francisella tularensis, a facultative intracellular pathogen[END_REF]. The same cross-amplification has been described for the ISFtu2 and Tul4 qPCR tests [START_REF] Versage | Development of a multitarget real-time TaqMan PCR assay for enhanced detection of Francisella tularensis in complex specimens[END_REF]. Differentiating these two closely related microorganisms using a qPCR test remains highly challenging. However, such cross-amplification currently has little impact on tularemia diagnosis, because human infections with F. novicida are rare and associated with clinical and epidemiological contexts different from those of tularemia (4). Additional diagnostic tests are usually performed for tularemia diagnostic confirmation and differentiation of type A and type B infections [START_REF]WHO guidelines on tularaemia[END_REF]. Because F. novicida is an aquatic bacterium, the situation is different when environmental water samples are tested. In this case, the Biotoxis qPCR test is not sufficiently discriminating. A more specific test must be performed for accurate differentiation between F. novicida and F. tularensis. Therefore, in the context of bioterrorism, the Biotoxis qPCR kit is currently not specific enough for rapid and accurate confirmation of the presence of F. tularensis in environmental samples.

The Biotoxis qPCR was negative for F. philomiragia, confirming the absence of cross-amplification of this other aquatic bacterium. The Biotoxis qPCR was also negative for 21 non-Francisella species tested, representing common human pathogens. When testing high DNA concentrations (10 ng/l), we observed a weak qPCR signal (C T Ն 34) for the S. salivarius, P. aeruginosa, and N. elongata strains. Using lower DNA concentrations could eliminate such nonspecific signals. It should be recommended that DNA concentrations of 0.1 ng/l be used when DNA extracts from bacterial strains are being tested, to avoid nonspecific amplification.

We did not include F. tularensis subsp. tularensis (type A) strains because we do not possess such strains in the French NRCF. The genes classically used to target F. tularensis (i.e., the ISFtu2 element, the 23-kDa-protein gene, fopA, and tul4) detect all four F. tularensis subspecies [START_REF] Versage | Development of a multitarget real-time TaqMan PCR assay for enhanced detection of Francisella tularensis in complex specimens[END_REF]. Although the identity of the Biotoxis qPCR detection kit's targeted genes is proprietary, the manufacturer validated during the development process that this test can detect both type A and type B strains. This kit's performance for type A strains in different sample types will have to be checked by further studies.

As for clinical specimens, we tested 31 samples previously collected from 30 tularemia patients. At the time of diagnosis, all samples were positive for the ISFtu2 qPCR (C T range of 20.47 to 36.61) and 29/31 for type B qPCR (C T range of 28.89 to 39.71). The Biotoxis qPCR was positive for 28/31 clinical samples, with C T values ranging from 27.37 to 39.06 (Table 3). Therefore, the Biotoxis qPCR test's sensitivity could be evaluated at 90.32% and 96.55% compared to ISFtu2 and type B qPCR tests, respectively. The F. tularensis subsp. holarctica genome contains 26 to 30 copies of ISFtu2 [START_REF] Versage | Development of a multitarget real-time TaqMan PCR assay for enhanced detection of Francisella tularensis in complex specimens[END_REF], explaining the higher sensitivity of the ISFtu2 qPCR compared to the Tul4 and type B qPCR tests, which target a single DNA copy. However, the ISFtu2 insertion sequence is also found in the genomes of other Francisella species, including F. novicida and F. philomiragia [START_REF] Versage | Development of a multitarget real-time TaqMan PCR assay for enhanced detection of Francisella tularensis in complex specimens[END_REF]. The Biotoxis qPCR displayed sensitivity similar to that of the type B qPCR. C T values higher than 35 were found for 9/29 samples for type B qPCR and 13/28 for Biotoxis qPCR. Such high C T values likely reflected the low F. tularensis inoculum and the presence of PCR inhibitors in the tested clinical samples. However, for the Biotoxis qPCR test, no C T is specified by the manufacturer. Overall, this test's sensitivity could be considered very satisfactory, taking into account its multiplex nature. In addition, clinical samples were stored at Ϫ80°C for 1 to 2 years before Biotoxis qPCR testing, which could have slightly altered DNA quality. Regarding specificity, no amplification was observed for the 30 F. tularensis-free clinical specimens with this kit.

When testing water samples artificially contaminated with F. tularensis subsp. holarctica, we found LODs of 1,000 CFU/liter for the Biotoxis qPCR, 100 CFU/liter for type B qPCR, 10 CFU/liter for Tul4 qPCR, and 0.1 CFU/liter for ISFtu2 qPCR. Here again, the lower LOD of ISFtu2 qPCR could be explained by this target's multicopy nature [START_REF] Versage | Development of a multitarget real-time TaqMan PCR assay for enhanced detection of Francisella tularensis in complex specimens[END_REF]. The higher LOD of the Biotoxis qPCR compared to those of the Tul4 and type B qPCR assays could be related to the multiplex nature of this commercial test. Multiplexed qPCR tests usually display lower analytical sensitivities than their simplex counterparts do [START_REF] Elnifro | Multiplex PCR: optimization and application in diagnostic virology[END_REF].

We then tested 57 environmental water samples collected from natural aquatic environments. The Biotoxis qPCR was positive for 6/15 samples that were positive by ISFtu2 qPCR, 5/9 Tul4 qPCR-positive samples, and 4/4 type B qPCR-positive samples (Table 4). As expected, the Biotoxis qPCR did not detect some samples that were positive in the ISFtu2 and Tul4 qPCR tests. However, only samples positive in the highly specific type B qPCR test could be considered genuinely contaminated with F. tularensis (type B strain). The same samples tested positive with the Biotoxis qPCR test. This result likely indicates the higher specificity of these two tests to detect F. tularensis DNA in aquatic environments. It should be noted that high C T values were found for most qPCR-positive water samples, reflecting low bacterial loads, as previously reported in other environmental studies [START_REF] Janse | Environmental surveillance of zoonotic Francisella tularensis in the Netherlands[END_REF]. A much higher bacterial load would be expected in a bioterrorist attack context. It would have been interesting to culture these environmental water samples to correlate PCR and culture results. However, we did not try to culture them, since it would be tedious because of (i) the fastidious nature of F. tularensis and (ii) the high quantity of contaminant bacteria in these samples.

The Biotoxis qPCR detection kit is designed to perform multiple tests in a 96-wellplate format. Unlike other commercial products, such as the BioFire FilmArray Biothreat panel, it is not suitable for unit sample analysis. Consequently, the Biotoxis qPCR detection kit appears to be more appropriate for testing many biological or environmental samples at a moderate cost. In our hands, this kit displayed equivalent specificity but a slightly lower sensitivity compared to F. tularensis reference qPCR assays. However, the purpose of the Biotoxis kit is to simultaneously detect the presence of F. tularensis, Y. pestis, and B. anthracis in a bioterrorism context. It is a ready-to-use kit that can be handled in all laboratories equipped with a qPCR apparatus, without need for specific expertise. We did not evaluate the Biotoxis qPCR detection kit's performances for the detection of B. anthracis and Y. pestis. The kit was validated for all three pathogens by the manufacturer during the development process. However, further studies will be needed to assess the sensitivity and specificity of the Biotoxis kit for detection of Y. pestis and B. anthracis. Consequently, our study does not enable us to state the usefulness of the Biotoxis qPCR kit in the context of bioterrorism. Apart from the investigation of a suspected bioterrorist attack, we believe that this test could be useful for rapid exploration of the potential sources of human infections during natural tularemia outbreaks.

In conclusion, the Biotoxis qPCR detection kit displayed good performance for detecting F. tularensis in clinical specimens and environmental water samples. However, the cross-amplification of F. novicida should be taken into account. This kit was easy to use, and results were available within 60 min. It can be useful for the rapid detection of F. tularensis DNA in many clinical samples, especially in the context of a tularemia outbreak. It can also be used to detect F. tularensis in a large number of environmental samples. The Biotoxis qPCR detection kit was designed primarily for simultaneous detection of F. tularensis, B. anthracis, and Y. pestis in a bioterrorism context. Further studies are needed to assess the performance of this kit for detection of the two latter pathogens.

TABLE 1

 1 Bacterial strains used in this study

	Species	Strain
	F. tularensis subsp. holarctica	Clinical strains (86 strains, including Ft5 to -46,
		Ft48 to -65, Ft67 to -74, Ft76 to -80,
		Ft83 to -89, Ft91, and Ft92 to -96)
		LVS NCTC 10857
	F. novicida	CIP 56.12
	F. philomiragia	Clinical strain Ft47
		ATCC 25015
	Corynebacterium jeikeium	CIP 8251
	Staphylococcus epidermidis	CIP 103627
	Staphylococcus aureus	ATCC 6538
	Streptococcus agalactiae	ATCC 12400
	Streptococcus pneumoniae	ATCC 49619
	Streptococcus uberis	ATCC 9727
	Streptococcus salivarius	Clinical strain
	Streptococcus mitis	CIP 103335
	Streptococcus oralis	Clinical strain
	Enterococcus faecalis	ATCC 29212
	Enterococcus faecium	CIP 5432
	Bacillus subtilis	ATCC 6633
	Haemophilus influenzae	ATCC 49766
	Acinetobacter baumannii	ATCC 19606
	Escherichia coli	ATCC 25922
	Enterobacter cloacae	ATCC 13047
	Klebsiella pneumoniae	ATCC 35657
	Staphylococcus sciuri	ATCC 29061
	Stenotrophomonas maltophilia	ATCC 17666
	Pseudomonas aeruginosa	CIP 5933
	Streptococcus pyogenes	CIP 104226
	Neisseria elongata	Clinical strain
	Moraxella catarrhalis	Clinical strain
	Serratia marcescens	CIP 103551

TABLE 2

 2 Biotoxis qPCR testing of bacterial strains

		Result (C T ) in qPCR
	Strain a	Biotoxis	ISFtu2	Tul4	Type B
	F. tularensis type B Ft41	Positive (20)	Positive (14)	Positive (19)	Positive (19)
	F. tularensis type B Ft54	Positive (20)	Positive (13)	Positive (17)	Positive (18)
	F. tularensis type B Ft72	Positive (19)	Positive (13)	Positive (17)	Positive (18)
	F. tularensis type B Ft92	Positive (18)	Positive (12)	Positive (15)	Positive (16)
	F. novicida CIP 56.12	Positive (18)	Positive (12)	Positive (16)	Negative
	F. philomiragia Ft47	Negative	Positive (26)	Negative	Negative
	F. philomiragia ATCC 25015	Negative	Positive (36)	Negative	Negative

a DNA extracts were standardized at 10 ng/l.

TABLE 3

 3 Biotoxis qPCR testing of 31 clinical samples from 30 tularemia patients a

				Result of:
					qPCR (C T ) b
	Sample	Type	Clinical form	Serology/culture	ISFtu2	Type B	Biotoxis
	S1	LN	GL	POS/NA	POS (27.97)	POS (31.96)	POS (34.50)
	S2 c	Peritoneal tissue	INT	POS/NEG	POS (29.35)	POS (35.06)	POS (32.59)
	S3 c	Peritoneal liquid	INT	POS/NEG	POS (28.22)	POS (32.57)	POS (35.05)
	S4	LN	GL	POS/NEG	POS, 1/100 (31.98)	POS, 1/100 (39.71)	POS, 1/100 (38.06)
	S5	LN	PNE	POS/NEG	POS (20.47)	POS (28.89)	POS (27.37)
	S6	BAL	PNE	POS/NA	POS (30.48)	POS (34.65)	POS (37.29)
	S7	BAL	PNE	POS/NA	POS (28.64)	POS (33.06)	POS (35.66)
	S8	LN	GL	POS/NEG	POS (27.35)	POS (34.82)	POS (32.60)
	S9	LN	PNE	POS/NEG	POS (30.62)	POS (35.17)	POS (39.06)
	S10	Tracheal aspirate	PNE	NA/NEG	POS (30.88)	POS (37.02)	POS (37.63)
	S11	LN	GL	NA/NEG	POS (30.55)	POS (34.66)	POS (36.23)
	S12	LN	GL	POS/NEG	POS (23.56)	POS (32.92)	POS (30.22)
	S13	LN	GL	NEG/NEG	POS (32.14)	POS (36.43)	POS (38.76)
	S14	LN	GL	SCV/NEG	POS (33.85)	POS (38.25)	NEG
	S15	LN	GL	SCV/NEG	POS (30.56)	POS (35.02)	POS (34.90)
	S16	Sputum	PNE	NA/NA	POS (29.64)	POS (36.09)	POS (36.04)
	S17	LN	UG	POS/NEG	POS (27.10)	POS (33.76)	POS (32.23)
	S18	LN	GL	NA/NEG	POS (26.24)	POS (30.64)	POS (30.09)
	S19	LN	GL	POS/NEG	POS (28.50)	POS (34.76)	POS (31.90)
	S20	LN	PNE	POS/NA	POS (36.61)	NEG	NEG
	S21	Knee prosthesis	OA	POS/POS	POS (30.94)	POS (35.89)	POS (37.19)
	S22	LN	UG	POS/NEG	POS (23.82)	POS (29.68)	POS (30.79)
	S23	LN	UG	NA/NEG	POS (25.29)	POS (29.53)	POS (28.71)
	S24	LN	OG	NA/NEG	POS (28.70)	POS (35.76)	POS (35.80)
	S25	LN	OP	POS/NEG	POS (24.18)	POS (30.14)	POS (31.69)
	S26	LN	UG	POS/NEG	POS (30.11)	POS (35.57)	POS (35.77)
	S27	LN	GL	FFR/NEG	POS (34.85)	NEG	NEG
	S28	LN	GL	POS/NEG	POS (25.50)	POS (30.33)	POS (31.91)
	S29	LN	GL	POS/NEG	POS (29.46)	POS (33.56)	POS (35.86)
	S30	LN	PNE	NA/NEG	POS (32.82)	POS (35.83)	POS (37.96)
	S31	LN	UG	NA/NEG	POS (24.72)	POS (31.98)	POS (31.25)

a UG, ulceroglandular; GL, glandular; PNE, pneumonic; OG, oculoglandular; OP, oropharyngeal; INT, intestinal; OA, osteoarticular; LN, lymph node; BAL, bronchoalveolar lavage; NA, not available; NEG, negative; POS, positive; SCV, seroconversion; FFR, a fourfold rise in antibody titers. b DNA extracts were tested pure or at the specified dilution. c Samples from the same patient.

TABLE 4

 4 Biotoxis qPCR testing of 57 environmental water samples

	Result of qPCR

Downloaded from https://journals.asm.org/journal/jcm on 15 February 2024 by 132.168.4.38.

January 2021 Volume 59 Issue 1 e01434-20 jcm.asm.org 2 Downloaded from https://journals.asm.org/journal/jcm on 15 February 2024 by 132.168.4.38.

January 2021 Volume 59 Issue 1 e01434-20 jcm.asm.org 3 Downloaded from https://journals.asm.org/journal/jcm on 15 February 2024 by 132.168.4.38.

January 2021 Volume 59 Issue 1 e01434-20 jcm.asm.org 4 Downloaded from https://journals.asm.org/journal/jcm on 15 February 2024 by 132.168.4.38.

January 2021 Volume 59 Issue 1 e01434-20 jcm.asm.org 5 Downloaded from https://journals.asm.org/journal/jcm on 15 February 2024 by 132.168.4.38.

January 2021 Volume 59 Issue 1 e01434-20 jcm.asm.org 6 Downloaded from https://journals.asm.org/journal/jcm on 15 February 2024 by 132.168.4.38.

January 2021 Volume 59 Issue 1 e01434-20 jcm.asm.org 7 Downloaded from https://journals.asm.org/journal/jcm on 15 February 2024 by 132.168.4.38.

January 2021 Volume 59 Issue 1 e01434-20 jcm.asm.org 8 Downloaded from https://journals.asm.org/journal/jcm on 15 February 2024 by 132.168.4.38.

ACKNOWLEDGMENTS

We declare that we have no conflicts of interest. Bertin Bioreagent provided the Biotoxis qPCR detection kit used in this study but was not involved in the study design, result interpretation, or the article content.

We thank the Direction Generale de l'Armement (ANR-17-ASTR-0024) and the Programme Interministériel de R&D NRBC for funding.