Laboratory preparedness and responsiveness WP7

Specific objectives

1. Improved laboratory preparedness ensuring the application of best practices and quality assured methods for detection and analyses of highly pathogenic infectious agents in SHARP partner laboratories.
2. Improved and consolidated biorisk management in SHARP partner laboratories dedicated to the diagnostics of highly pathogenic infectious agents.

Outputs & activities

Work Package (WP) 7 is structured according to the following Specific Tasks and Working Groups (WG):

• Task 7.1 – External Quality Assurance Exercises (EQAEs), including training
• Task 7.2 – Diagnostic approaches
  • WG Next Generation Sequencing (NGS)
  • WG Basic diagnostic (BD)
  • WG Antimicrobial Susceptibility Testing (AST)
• Task 7.3 – Laboratory biorisk management
  • Task 7.3.1 – Support for self-evaluation of biorisk management
  • Task 7.3.2 – Inactivation procedures

Special funding is granted to Task leaders, Co-leaders and designated WG leaders to carry out specific defined activities in cooperation with all WP7 partners, while basic funding is granted to all WP7 partners to cover essential activities, such as participation in External Quality Assurance Exercises(EQAE), meetings and training courses. Special attention is attributed to partners from low GNI Member States to allow their participation in the various tasks and Working Groups of the Work Package.

Deliverables

• Report on quality assurance of diagnostics of highly infectious pathogens (yearly EQAEs) applied in microbiological SHARP partner laboratories.
• Report on assessment and recommendations for modern molecular diagnostic methods and for Antimicrobial Susceptibility Testing (AST) for selected highly pathogenic bacteria.
• Recommendations for biorisk management in high containment facilities.

Milestones

• Implementation of EQAEs
• Specific diagnostic tasks
• Tool for self-evaluation of laboratory biosecurity

Scientific journal publications from work package 7

• Pappa, S., and Papa, A. (2023). Evaluation of six molecular assays for the detection of Aigai virus. Population Medicine, 5(October), 26. https://doi.org/10.18332/popmed/172259
• Mulders Daphne G. J. C., Beerkens Rob J. J. Standardization of SARS-CoV-2 Nucleic Acid Amplification Techniques by Calibration and Quantification to the First WHO International Standard for SARS-CoV-2 RNA. International Journal of Microbiology Volume Feb 2023 | Article ID 7803864 | https://doi.org/10.1155/2023/7803864. 270 Views and 200 Downloads by October 2023.
• Bettini A, Lapa D, Garbuglia AR. Diagnostics of Ebola virus.Front Public Health. 2023 Feb 23;11:1123024. doi: 10.3389/fpubh.2023.1123024. eCollection 2023.PMID: 3690845
• Bordi L, Sberna G, Lalle E, Fabeni L, Mazzotta V, Lanini S, Corpolongo A, Garbuglia AR, Nicastri E, Girardi E, Vaia F, Antinori A, Maggi F.I Comparison of SARS-CoV-2 Detection in Nasopharyngeal Swab and Saliva Samples from Patients Infected with Omicron Variant. Int J Mol Sci. 2023 Mar 2;24(5):4847. doi: 10.3390/ijms24054847.PMID: 36902277
• Burton JE, Easterbrook L, Pitman J, Anderson D, Roddy S, Bailey D, Vipond R, Bruce CB, Roberts AD. The effect of a non-denaturing detergent and a guanidinium-based inactivation agent on the viability of Ebola virus in mock clinical serum samples. J Virol Methods. 2017 Dec;250:34-40. doi: 10.1016/j.jviromet.2017.09.020. Epub 2017 Sep 20. PMID: 28941617
• Davies K, Arnold U, Buczkowski H et al. Virucidal efficacy of guanidine-free inactivants and rapid test buffers against SARS-CoV-2. Sci Rep 11, 23379 (2021). https://doi.org/10.1038/s41598-021-02942-4
• Elveborg S, Monteil VM, Mirazimi A. Methods of Inactivation of Highly Pathogenic Viruses for Molecular, Serology or Vaccine Development Purposes. Pathogens. 2022 Feb 19;11(2):271. doi: 10.3390/pathogens11020271. PMID: 35215213; PMCID: PMC8879476.
• Haddock E, Feldmann F, Feldmann H. Effective Chemical Inactivation of Ebola Virus. Emerg Infect Dis. 2016 Jul;22(7):1292-4. doi: 10.3201/eid2207.160233. Epub 2016 Jul 15. PMID: 27070504; PMCID: PMC4918181
• Lapa D, Specchiarello E, Francalancia M, Girardi E, Maggi F, Garbuglia AR. Detection of Anti-Rift Valley Fever Virus Antibodies in Serum Samples of Patients with Suspected Arbovirus Infection. Microorganisms. 2023 Aug 14;11(8):2081. doi: 10.3390/microorganisms11082081.PMID: 3763064
• Llorente MT, Escudero R, Ramiro R, Remacha MA, Martínez-Ruiz R, Galán-Sánchez F, de Frutos M, Elía M, Onrubia I and Sánchez S (2023) Enteroaggregative Escherichia coli as etiological agent of endemic diarrhea in Spain: A prospective multicenter prevalence study with molecular characterization of isolates. Front. Microbiol. 14:1120285. doi: 10.3389/fmicb.2023.1120285
• Mazzotta V, Scorzolini L, Falasca L, Lionetti R, Aguglia C, Kontogiannis D, Colombo D, Colavita F, De Palo MG, Carletti F, Mondi A, Pinnetti C, Maffongelli G, Garbuglia AR, Baldini F, Corpolongo A, Maggi F, D’Offizi G, Girardi E, Vaia F, Nicastri E, Del Nonno F, Antinori A. Lymphofollicular lesions associated with monkeypox (Mpox) virus proctitis.Int J Infect Dis. 2023 May;130:48-51. doi: 10.1016/j.ijid.2023.02.021. Epub 2023 Feb 27.PMID: 36858309
• Mondi A, Mastrorosa I, Piselli P, Cimaglia C, Matusali G, Carletti F, Giannico G, Milozzi E, Biliotti E, Di Bari S, Chinello P, Beccacece A, Faraglia F, Vittozzi P, Mosti S, Tetaj N, Stazi GV, Pinnetti C, Camici M, D’Annunzio A, Marani A, Fabeni L, Specchiarello E, Gruber CEM, Villanacci A, Minicucci S, Garbuglia AR, Ianniello S, Marchioni L, Taglietti F, D’Offizi G, Palmieri F, Nicastri E, Maggi F, Vaia F, Girardi E, Antinori A. Evolution of SARS-CoV-2 variants of concern over a period of Delta and Omicron cocirculation, among patients hospitalized for COVID-19 in an Italian reference hospital: Impact on clinical outcomes.J Med Virol. 2023 Jun;95(6):e28831. doi: 10.1002/jmv.28831.PMID: 37246793
• Oude Munnink BB, Nieuwenhuijse DF, Stein M et al. Rapid SARS-CoV-2 whole-genome sequencing and analysis for informed public health decision-making in the Netherlands. Nat Med 26, 1405–1410 (2020). https://doi.org/10.1038/s41591-020-0997-y
• Oude Munnink BB, Sikkema RS, Nieuwenhuijse DF, Molenaar RJ, Munger E, Molenkamp R, van der Spek A, Tolsma P, Rietveld A, Brouwer M, Bouwmeester-Vincken N, Harders F, Hakze-van der Honing R, Wegdam-Blans MCA, Bouwstra RJ, GeurtsvanKessel C, van der Eijk AA, Velkers FC, Smit LAM, Stegeman A, van der Poel WHM, Koopmans MPG. Transmission of SARS-CoV-2 on mink farms between humans and mink and back to humans. Science. 2021 Jan 8;371(6525):172-177. doi: 10.1126/science.abe5901. Epub 2020 Nov 10. PMID: 33172935; PMCID: PMC7857398
• Paltansing S, Sikkema RS, de Man SJ, Koopmans MPG, Oude Munnink BB, de Man P, Transmission of SARS-CoV-2 among healthcare workers and patients in a teaching hospital in the Netherlands confirmed by whole-genome sequencing, Journal of Hospital Infection, Volume 110, 2021, Pages 178-183, ISSN 0195-6701,    https://doi.org/10.1016/j.jhin.2021.02.005.
• Ruggieri S, Aiello A, Tortorella C, Navarra A, Vanini V, Meschi S, Lapa D, Haggiag S, Prosperini L, Cuzzi G, Salmi A, Quartuccio ME, Altera AMG, Garbuglia AR, Ascoli Bartoli T, Galgani S, Notari S, Agrati C, Puro V, Nicastri E, Gasperini C, Goletti D. Dynamic Evolution of Humoral and T-Cell Specific Immune Response to COVID-19 mRNA Vaccine in Patients with Multiple Sclerosis Followed until the Booster Dose. Int J Mol Sci. 2023 May 10;24(10):8525. doi: 10.3390/ijms24108525.PMID: 37239872
• Specchiarello E, Matusali G, Carletti F, Gruber CEM, Fabeni L, Minosse C, Giombini E, Rueca M, Maggi F, Amendola A, Garbuglia AR. Detection of SARS-CoV-2 Variants via Different Diagnostics Assays Based on Single-Nucleotide Polymorphism Analysis.Diagnostics (Basel). 2023 Apr 27;13(9):1573. doi: 10.3390/diagnostics13091573.PMID: 37174964
• Tscherne A, Mantel E, Boskani T, Budniak S, Elschner M, Fasanella A, Feruglio SL, Galante D, Giske CG, Grunow R, Henczko J, Hinz C, Iwaniak W, Jacob D, Kedrak-Jablonska A, Jensen VK, Johansen TB, Kahlmeter G, Manzulli V, Matuschek E, Melzer F, Nuncio MS, Papaparaskevas J, Pelerito A, Solheim M, Thomann S, Tsakris A, Wahab T, Weiner M, Zoeller L, Zange S, on behalf of the EMERGE AST Working Group. Adaptation of Brucella melitensis Antimicrobial Susceptibility Testing to the ISO 20776 Standard and Validation of the Method. Microorganisms 2022, 10, 1470. https://doi.org/10.3390/microorganisms10071470. 1962 Article Views. 3 Citations at October 2023.

People and partners

Work package lead: Robert Koch Institute (RKI), Germany (contact: EMERGE-Coordination(at)rki.de)

The EMERGE Laboratory Network, comprising 40 diagnostic laboratories across 25 European countries, focuses on high-risk pathogens. It provides surge capacity and expertise during outbreaks and conducts annual External Quality Assurance Exercises (EQAEs) to ensure readiness and best practices.

Co-lead: National Institute of Infectious Diseases Lazzaro Spallanzani (INMI), Italy

Activities

30 October 2023, Laboratory biorisk management. The “The European Tool for Enhancing Laboratory Biorisk Management” has been completed by the Public Health Agency of Sweden (FoHM) and available to the scientific community for evaluating activities with the aim to continuously improve biorisk management activities at the individual laboratory, for laboratory management and laboratory personnel, helping to raise awareness providing an overview of to what extent national and international requirement for biosafety and biosecurity are met.  The Tool was presented at the final WP7 meeting and evaluated by a number of EMERGE laboratory network members in terms of its usefulness and applicability. The Tool covers biosafety and biosecurity aspects of laboratory biorisk management and includes information to be used by organisations within the SHARP consortium in order to enhance a systematic approach to laboratory biorisk management in BSL3 and BSL4 laboratories. The format for the Tool is a document with up-to-date information and web link for detailed information. The Tool also includes an easy to use checklist to be used for self-assessment awareness within the organisation. Download the EBRM guidance tool

30 October 2023, the article, Evaluation of six molecular assays for the detection of Aigai virus, authored by Styliani Pappa and Anna Papa published by Population Medicine.

19 – 21 September 2023, Aristotle University of Thessaloniki Greece – affiliated entity of SHARP Joint Action – organised the 3rd International Conference on Crimean-Congo Haemorrhagic Fever (CCHF), at the Makedonia Palace Hotel in Thessaloniki, Greece. Aristotle University, Thessaloniki is an EMERGE laboratory network partner in the Network of Infectious Viruses (NIV). The EMERGE maximum biocontainment laboratory network, headquartered at the Robert Koch Institute, leads Laboratory preparedness and responsiveness for the SHARP Joint Action. The conference gave scientists the chance to exchange information on all aspects of the virus and the disease and report new findings. Plus, the opportunity to compare CCHF with other viral haemorrhagic fevers and discuss common approaches for treatment and prevention – of particular interest to SHARP partners in Spain and the Balkan region. Click on the image to view a poster presentation delivered by Professor Anna Papa, Aristotle University of Thessaloniki | AUTH · Department of Microbiology on the Evaluation of six molecular assays for the detection of Aigai virus. Presented at the conference on Crimean-Congo Haemorrhagic Fever.

17-18 October 2022 Online course on Viral Haemorrhagic Fevers, organized by INMI L Spallanzani IRCCS, Rome – Italy. Read the agenda (PDF 469 KB).

External Quality Assurance Exercises (EQAEs)
EQAEs: Three rounds of bacterial and viral EQAEs have been carried out by RKI and Philipps-Universität Marburg (UMR), Germany, together with the participating WP7 partner laboratories located in Austria, Belgium, Croatia, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Lithuania, Netherlands, Norway, Poland, Portugal, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. These laboratories are specialized in identifying highly pathogenic bacteria of RG 3 and highly pathogenic viruses of RG 4.

Different bacterial sample sets, mimicking clinical, environmental and food samples, included living bacteria and/or inactivated bacteria and/or serum samples were prepared for the EQAEs and sent to the participants for laboratory analyses. The target bacteria comprised Bacillus anthracis, Yersinia pestis, Francisella tularensis (different subspecies), Burkholderia pseudomallei, Burkholderia mallei, Brucella species, and Coxiella burnetii. The tasks of the exercises were the identification and exclusion of these bacteria and their further characterization (e.g. number of genome equivalents, plasmid content, antimicrobial susceptibility testing) and antibody detection against selected bacteria (see above).

The viral sample sets comprised clinical samples that were spiked with different concentrations of non-infectious viral material of Ebolavirus, Marburgvirus, Lassavirus, Henipavirus, Crimean-Congo hemorrhagic fever virus or New World and Old World Arenaviruses, respectively.

The EQAEs showed that the participating laboratories are operating at a very high quality and biosafety/biosecurity level. Yet, diagnostic gaps could be identified during the exercises and were discussed at the yearly WP7 meetings. The exchange of knowledge among the partners is an essential part of the network activities aiming to maintain and increase the diagnostic capacities and capabilities of the involved labs, especially in low GNI countries.

Training

After an initial evaluation of the training needs at WP7 partner laboratories, a request for training offers was circulated among all partners. The feedback obtained was used to develop a regularly updated booklet of finally 25 courses offered by 13 partner labs. The topics ranged from antimicrobial susceptibility testing to biosafety and next generation sequencing, from virus isolation to bacterial isolation and cultivation. Due to the pandemic, a number of these training courses were carried out as online courses, if possible.

Diagnostic approaches

Working Group on Next Generation Sequencing (NGS)

A survey circulated by the Erasmus Medical Center (EMC), The Netherlands, among WP7 partner laboratories to assess the availability and type of NGS platforms, work processes and bioinformatics support demonstrated a good level of capacity and knowledge of laboratory processes and a wide variety of platforms being used. Availability and level of bioinformatics support differ and range up to the availability of specialized bioinformatics department.

For SARS-CoV-2, genomic surveillance was continuously performed in the Netherlands. A SARS-CoV-2 specific sequencing protocol was set up and used for rapid informed public health decision making in the Netherlands and to investigate several outbreaks. An automated data analysis tool was developed to enable real-time data analysis to generate consensus sequences. View the Pipeline for mapping SARS-CoV2 nanopore reads using nextflow.

Working Group on Basic Diagnostic (BD)

In order to exchange best practices and to identify needs for support, detailed information on all diagnostic procedures (including in-house and commercial kits) targeting RG3 bacteria and RG4 viruses available at WP7 partner laboratories was collected. The diagnostic capabilities have been surveyed by circulating a detailed questionnaire: Bacterial diagnostics is performed at a very high level by the participating labs showing a broad capacity for detection and quantification of bacteria based on a number of different detection methods, among which whole genome sequencing and NGS. Viral diagnostics are widely using molecular methods. NGS technology is broadly distributed and increasingly applied. Further exchange of technical approaches for diagnostics and mutual support in case of requests is planned. 

Working Group on Antimicrobial Susceptibility Testing (AST)

Since 2011, the AST Working Group (lead: Bundeswehr Institute of Microbiology (BwIM), Germany) has been working on the determination of guidelines and epidemiological cut-off values for antimicrobial susceptibility testing of highly pathogenic bacteria within the EMERGE Joint Action / Laboratory Network as an important milestone. Until today, the guidelines for antimicrobial susceptibility testing of highly pathogenic bacteria only available at the American Clinical Standard Laboratory Institute (CLSI), but not at the European Committee of Antimicrobial Susceptibility Testing (EUCAST). For Burkholderia pseudomallei, it has been achieved, that, in 2021, EUCAST published epidemiological cut-off values, based, among others, on data produced by participants of the EMERGE Lab Network AST group. Reference: Burkholderia pseudomallei multi-centre study to establish EUCAST MIC and zone diameter distributions and epidemiological cut-off values; O. Karatuna et al., Clinical Microbiology and Infection 27 (2021) 736e741.

The testing of strain collections with two AST methods in parallel was finalized by the AST Working Group members and results regarding Brucella melitensis and Bacillus anthracis AST were analysed at BwIM. A total of 499 B. melitensis strains and 336 B. anthracis strains were tested by the partners.

20 July 2022 The work from the previous project concerning AST of Brucella melitensis, which was finalised during SHARP, was successfully published in the peer reviewed journal “Microorganisms 2022 Jul 20;10(7):1470. doi: 10.3390/microorganisms10071470 Adaptation of Brucella melitensis Antimicrobial Susceptibility Testing to the ISO 20776 Standard and Validation of the Method. Therne, A. et al. WP7 provided antimicrobial susceptibility testing (AST) utilising the EMERGE laboratory network who specialize in highly pathogenic infectious agents. Their contribution was important, as improvement to testing standards for Brucella melitensis is an urgent need.

Data on wild type distribution for Brucella melitensis and Bacillus anthracis is now available at EUCAST.

Maintenance of the EMERGE Laboratory Network

Based on previous EU funded projects and Joint Actions (EQADeBa, QUANDHIP and EMERGE), the EMERGE Laboratory Network is currently embedded in the JA SHARP. The fact that the network partners mostly know each other for many years considerably contributes to a vivid and trustful interaction for the benefit of all. To keep the functionality of this grown structure, existing since 2008, a number of options are currently pursued. The partner laboratories have repeatedly underlined the benefits and added value gained by the network activities with regard to diagnostic quality assurance and self-evaluation. In 2022, a detailed Pilot Plan has been developed in close cooperation with colleagues of WP4 who considerably promoted the development of this plan. It will be used to persuade political decision-makers to support the efforts to obtain permanent funding and to set the network on stable pillars. A very concrete perspective for the maintenance of the EMERGE Laboratory Network was opened with the new “Regulation (EU) 2022/2371 of the European Parliament and of the Council of 23 November 2022 on serious cross-border threats to health and repealing Decision No 1082/2013/EU”. Here, the need for European diagnostic laboratory networks and approaches for their implementation are formulated. 

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