Mapping the coverage, availability and uptake of External Quality Assessment programmes across One Health sectors in Asia.

INTRODUCTION: Establishing effective external quality assessment (EQA) programmes is an important element in ensuring the quality of, and building capacity for, antimicrobial resistance (AMR) laboratory surveillance. OBJECTIVES: To understand the current coverage of, and challenges to participation in, EQAs in National Reference Laboratories (NRLs) across One Health (OH) sectors in Asia. METHODS: Current EQA coverage was evaluated through desktop review, online surveys and interviews of both EQA participants and providers. EQA coverage was mapped and summarized by laboratory type and 'readiness' level and identified challenges evaluated qualitatively. RESULTS: Of the 31 identified NRLs [16 Human Health (HH) and 15 Animal/Food Safety laboratories (A/FS)], 14 HH and 7 A/FS laboratories currently participated in international EQA schemes and several participated in two or more different schemes. Seven laboratories were currently not participating in any EQA scheme and two of these (one HH and one A/FS) do not currently perform microbiology; six HH NRLs provided national EQAs. Of the eight surveyed international EQA providers, three were based in Asia and all offered varying programmes in terms of pathogens, frequency and support mechanisms for reporting and follow-up. Only one provider currently served laboratories across all OH sectors. CONCLUSIONS: The current coverage of EQA programmes for AMR in Asia was heterogeneous across countries but especially across OH sectors. This updated overview of the coverage and challenges associated with participation in, and provision of, EQAs for AMR suggest the benefit and relevance of introducing one comprehensive and high-quality EQA programme across OH sectors in Asia.

Proficiency of WHO Global Foodborne Infections Network External Quality Assurance System Participants in Identification and Susceptibility Testing of Thermotolerant Campylobacter spp. from 2003 to 2012.

Campylobacter spp. are foodborne and waterborne pathogens. While rather accurate estimates for these pathogens are available in industrialized countries, a lack of diagnostic capacity in developing countries limits accurate assessments of prevalence in many regions. Proficiency in the identification and susceptibility testing of these organisms is critical for surveillance and control efforts. The aim of the study was to assess performance for identification and susceptibility testing of thermotolerant Campylobacter spp. among laboratories participating in the World Health Organization (WHO) Global Foodborne Infections Network (GFN) External Quality Assurance System (EQAS) over a 9-year period. Participants (primarily national-level laboratories) were encouraged to self-evaluate their performance as part of continuous quality improvement. The ability to correctly identify Campylobacter spp. varied by year and ranged from 61.9% (2008) to 90.7% (2012), and the ability to correctly perform antimicrobial susceptibility testing (AST) for Campylobacter spp. appeared to steadily increase from 91.4% to 93.6% in the test period (2009 to 2012). The poorest performance (60.0% correct identification and 86.8% correct AST results) was observed in African laboratories. Overall, approximately 10% of laboratories reported either an incorrect identification or antibiogram. As most participants were supranational reference laboratories, these data raise significant concerns regarding capacity and proficiency at the local clinical level. Addressing these diagnostic challenges is critical for both patient-level management and broader surveillance and control efforts.

Proficiency testing for bacterial whole genome sequencing: an end-user survey of current capabilities, requirements and priorities.

The advent of next-generation sequencing (NGS) has revolutionised public health microbiology. Given the potential impact of NGS, it is paramount to ensure standardisation of 'wet' laboratory and bioinformatic protocols and promote comparability of methods employed by different laboratories and their outputs. Therefore, one of the ambitious goals of the Global Microbial Identifier (GMI) initiative (http://www.globalmicrobialidentifier.org/) has been to establish a mechanism for inter-laboratory NGS proficiency testing (PT). This report presents findings from the survey recently conducted by Working Group 4 among GMI members in order to ascertain NGS end-use requirements and attitudes towards NGS PT. The survey identified the high professional diversity of laboratories engaged in NGS-based public health projects and the wide range of capabilities within institutions, at a notable range of costs. The priority pathogens reported by respondents reflected the key drivers for NGS use (high burden disease and 'high profile' pathogens). The performance of and participation in PT was perceived as important by most respondents. The wide range of sequencing and bioinformatics practices reported by end-users highlights the importance of standardisation and harmonisation of NGS in public health and underpins the use of PT as a means to assuring quality. The findings of this survey will guide the design of the GMI PT program in relation to the spectrum of pathogens included, testing frequency and volume as well as technical requirements. The PT program for external quality assurance will evolve and inform the introduction of NGS into clinical and public health microbiology practice in the post-genomic era.

Evaluation and validation of laboratory procedures for the surveillance of ESBL-, AmpC-, and carbapenemase-producing Escherichia coli from fresh meat and caecal samples.

Introduction: Extended-spectrum ß-lactamase- (ESBL) and AmpC- ß-lactamase-producing Enterobacterales are widely distributed and emerging in both human and animal reservoirs worldwide. A growing concern has emerged in Europe following the appearance of carbapenemase-producing Escherichia coli (E. coli) in the primary production of food animals. In 2013, the European Commission (EC) issued the Implementing Decision on the monitoring and reporting of antimicrobial resistance in zoonotic and commensal bacteria. The European Union Reference Laboratory for Antimicrobial Resistance (EURL-AR) was tasked with providing two laboratory protocols for samples derived from meat and caecal content, respectively, for the isolation of ESBL- and AmpC-producing E. coli (part 1) and carbapenemase-producing (CP) E. coli (part 2). In this study, we describe the current protocols, including the preparatory work for the development. Methods: Up to nine laboratory procedures were tested using minced meat as the matrix from beef, pork, and chicken as well as six procedures for the caecal content of cattle, pigs, and chicken. Variables included sample volume, pre-enrichment volume, pre-enrichment broth with and without antimicrobial supplementation, and incubation time/temperature. The procedures were evaluated against up to nine E. coli strains harboring different AMR genes and belonging to the three ß-lactamase groups. Results and discussion: The laboratory procedures tested revealed that the most sensitive and specific methodologies were based on a Buffered Peptone Water pre-enrichment of 225 ml to 25 g or 9 ml to 1 g for minced meat and caecal content, respectively, incubated at 37°C overnight, followed by inoculation onto MacConkey agar supplemented with 1 mg/L cefotaxime for detecting ESBL- and AmpC-producing E. coli and Chrom ID SMART (Chrom ID CARBA and OXA) for CP E. coli, incubated overnight at 37 and 44°C, respectively. We provided two isolation protocols for the EU-specific monitoring of ESBL- and AmpC- producing E. coli (part 1) and CP E. coli (part 2) from fresh meat (protocol 1) and caecal (protocol 2) samples, which have been successfully implemented by all EU Member States for the monitoring period 2014-2027 (EU 2020/1729).

European Union Reference Laboratories support the National food, feed and veterinary Reference Laboratories with rolling out whole genome sequencing in Europe.

The Inter European Union Reference Laboratories (EURLs) Working Group on Next Generation Sequencing (NGS) involves eight EURLs for microbiological food and feed hazards and has been working since 2017 to promote the adoption of NGS by the National Reference Laboratories (NRLs) in the European Union. This work illustrates the results of the first 5 years of activity. By working together, the EURLs involved have released guidance documents for assisting NRLs in all the steps of NGS, helping the transition from classical molecular methods towards whole genome sequencing while ensuring harmonization, with the final aim of improving preparedness in the use of NGS to characterize microbial hazards and trace the sources of infection.

Results of the 2020 Genomic Proficiency Test for the network of European Union Reference Laboratory for Antimicrobial Resistance assessing whole-genome-sequencing capacities.

The global surveillance and outbreak investigation of antimicrobial resistance (AMR) is amidst a paradigm shift from traditional biology to bioinformatics. This is due to developments in whole-genome-sequencing (WGS) technologies, bioinformatics tools, and reduced costs. The increased use of WGS is accompanied by challenges such as standardization, quality control (QC), and data sharing. Thus, there is global need for inter-laboratory WGS proficiency test (PT) schemes to evaluate laboratories' capacity to produce reliable genomic data. Here, we present the results of the first iteration of the Genomic PT (GPT) organized by the Global Capacity Building Group at the Technical University of Denmark in 2020. Participating laboratories sequenced two isolates and corresponding DNA of Salmonella enterica, Escherichia coli and Campylobacter coli, using WGS methodologies routinely employed at their laboratories. The participants' ability to obtain consistently good-quality WGS data was assessed based on several QC WGS metrics. A total of 21 laboratories from 21 European countries submitted WGS and meta-data. Most delivered high-quality sequence data with only two laboratories identified as overall underperforming. The QC metrics, N50 and number of contigs, were identified as good indicators for high-sequencing quality. We propose QC thresholds for N50 greater than 20 000 and 25 000 for Campylobacter coli and Escherichia coli, respectively, and number of contigs >200 bp greater than 225, 265 and 100 for Salmonella enterica, Escherichia coli and Campylobacter coli, respectively. The GPT2020 results confirm the importance of systematic QC procedures, ensuring the submission of reliable WGS data for surveillance and outbreak investigation to meet the requirements of the paradigm shift in methodology.

Global monitoring of antimicrobial resistance based on metagenomics analyses of urban sewage.

Antimicrobial resistance (AMR) is a serious threat to global public health, but obtaining representative data on AMR for healthy human populations is difficult. Here, we use metagenomic analysis of untreated sewage to characterize the bacterial resistome from 79 sites in 60 countries. We find systematic differences in abundance and diversity of AMR genes between Europe/North-America/Oceania and Africa/Asia/South-America. Antimicrobial use data and bacterial taxonomy only explains a minor part of the AMR variation that we observe. We find no evidence for cross-selection between antimicrobial classes, or for effect of air travel between sites. However, AMR gene abundance strongly correlates with socio-economic, health and environmental factors, which we use to predict AMR gene abundances in all countries in the world. Our findings suggest that global AMR gene diversity and abundance vary by region, and that improving sanitation and health could potentially limit the global burden of AMR. We propose metagenomic analysis of sewage as an ethically acceptable and economically feasible approach for continuous global surveillance and prediction of AMR.