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Updated project summary, January 2015

The joint research project ‘Metrology for monitoring infectious diseases, antimicrobial resistance, and harmful micro-organisms‘ is now just over half way through.

The consortium has developed a summary document that gives a concise overview of the proposed out put from the project, anticipated impact as well as information on project collaborators. Please click the link to read the summary, which also details our progress and achievements to-date or read the document below.

INFECT-MET Publishable Summary_January 2015

Metrology for monitoring infectious diseases, antimicrobial resistance, and harmful micro-organisms

Background
Infectious diseases account for over 20% of human deaths globally and 25% of all morbidity. Respiratory tract infections (RTI) including pneumonia, influenza and tuberculosis account for almost 50% of all pathogen associated deaths. Accurate and rapid diagnosis alongside methods for monitoring transmission and spread in the community and resistance to therapeutic agents are vital for public health protection. Molecular approaches, such as qPCR and sequence analysis, offer the potential to improve management of infectious diseases through increased speed, accuracy, sensitivity and information when compared to conventional microbiological methods. Consequently, the infectious disease testing market is one of the most rapidly growing segments of the in vitro diagnostics industry and is expected to reach €38 billion by 2013 with advances in molecular diagnostic technologies the main driving force behind the expected growth. However, measurement support for molecular approaches is lacking, with issues concerning quality, comparability and traceability of measurements widely highlighted.

Pathogens may be present in clinical samples at very low levels making accurate detection and measurement challenging. In many instances tests are being used in non-commercial ‘home-brew’ formats of variable and undefined quality, and even commercially available tests cannot always be directly compared.

Need for the project

Clinical reference materials which are traceable to SI or equivalent are currently lacking. Full confidence in molecular measurements can only be achieved if the appropriate metrology framework, standards and higher order reference measurement procedures are developed. Without this support healthcare providers and the biotechnology/diagnostics industry will not be able to demonstrate the reliability of their assays in a traceable and comparable manner. This is critical for implementation of assays deployed in a wide range of healthcare settings.

Higher order methods and approaches are needed for assigning traceable values to reference standards in order to improve the quality and comparability of current and emerging molecular assays. Stakeholders have identified these needs and articulated their support for this project in:

  • Developing a measurement framework to underpin infectious disease diagnosis and      monitoring
  • Assisting healthcare stakeholders to improve performance, comparability and      traceability of assays through provision of quality assurance tools and      higher order reference methods
  • Supporting effective implementation of the In Vitro Diagnostic Directive (IVDD)

Scientific and technical objectives

This project aims to develop novel measurement procedures and validation frameworks to support current and emerging molecular approaches for efficient, harmonised and rapid diagnosis, surveillance and monitoring of infectious diseases, with a particular focus on Respiratory Tract Infections (RTIs). The project’s ultimate aim is to establish routes for improving the accuracy, robustness, comparability and traceability of measurements within the metrology and diagnostics/epidemiological communities across Europe linked in to international standardisation initiatives in the area through CDC, JCTLM and WHO.

INFECT-MET addresses the following scientific and technical objectives:

  • To develop quantitative, validated and highly accurate methodologies for the measurement of infectious agents, such as viruses and bacteria
  • To develop methodologies for accurately quantifying the performance of commercially available diagnostic assays, ‘in-house’ clinical assays and novel emerging approaches
  • To quantitatively and comparatively evaluate new and emerging molecular approaches for the surveillance and monitoring of infectious disease load and detection of antimicrobial resistance mutations
  • To quantitatively and comparatively evaluate new and emerging diagnostic technologies for the rapid detection of infectious agents

The work of JRP HLT08 INFECT-MET is broken down into the following areas:

  • Development and evaluation of higher order methods based on enumeration (for example, digital PCR and single molecule counting in flow) for accurate measurement of infectious agents with known uncertainties
  • Investigation of the measurement challenges associated with emerging methodologies such as next generation sequencing and PCR for surveillance, epidemiology and antibiotic resistance screening
  • Investigation of the measurement challenges associated with emerging methodologies for rapid, near-patient testing, including DNA/microfluidic surface interactions and isothermal nucleic acid amplification evaluations
  • Development of a reference measurement framework using higher order measurement approaches in collaboration with end-user communities to improve calibration and quality assurance of current clinical PCR approaches

INFECT-MET will investigate the development of higher order reference measurement methods and procedures. It will consider the process as a whole incorporating sample extraction from different matrices (e.g. blood and saliva) and highly accurate and sensitive methods for enumerating single molecules and infectious particles and define the measurement capability of current and emerging molecular approaches. Identification of major sources of uncertainty, data interpretation and integration will assure an integral approach of the work. A measurement framework will be developed in a multidisciplinary team in close co-operation with key stakeholders including healthcare providers, public health laboratories, academics, standards bodies, biotechnology/diagnostics industries.

Expected results and potential impact

The main output from the JRP HLT08 INFECT-MET will be the development of quantitative, validated and highly accurate methodologies to support current and emerging molecular approaches for diagnosis, surveillance and monitoring of infectious diseases. The findings of the project will be disseminated at conferences, stakeholder workshops, training courses, through the website and via standardisation activities.

The project outputs have the potential to make a key contribution to significantly improve the accuracy, reliability and comparability of respiratory pathogen diagnostics, thus contributing to greater confidence in public health monitoring and improved healthcare.

The methodology being developed in this project will provide:

  • Proof of concept and creation of a reference measurement framework to enable National Metrology Institutes (NMIs) to perform traceable and accurate measurement of infectious agents
  • A move from arbitrary consensus values towards SI traceable measurement for the bacterial & viral model systems employed, therefore supporting diagnostic measurement comparability
  • Input to documentary standards guidance (specifically ISO 17511 revision, CLSI metrological traceability) for clinical laboratory testing and IVD manufacturers enabling clearer regulatory compliance and faster IVD      approval

To date, INFECT-MET has addressed the scientific and technical objectives as follows:

To develop quantitative, validated and highly accurate methodologies for the measurement of infectious agents, such as viruses and bacteria

Model systems of infectious agents for the development and evaluation of higher order measurement methods were selected in consultation with key stakeholders. Tuberculosis was selected as a bacterial model, Cytomegalovirus (CMV) as a viral DNA model and Influenza as a viral RNA model. Work to compare the commonly used nucleic acid (DNA and RNA) extraction methods for the chosen model systems was completed highlighting differences in yield and precision between the approaches. Higher order measurement procedures (digital PCR and real-time quantitative PCR) for the identification and quantification of the chosen model systems were developed and a comprehensive assessment of the relative performance of these procedures was completed. Well-characterised test materials of differing levels of complexity (Nucleic acid molecule, Nucleic acid extract and Whole microbe preparation) were prepared to facilitate these evaluations. A series of inter-laboratory studies were completed to determine the reproducibility of the higher order measurement procedures developed and to assign values to the corresponding panels of test materials.

To develop methodologies for accurately quantifying the performance of commercially available diagnostic assays, ‘in-house’ clinical assays and novel emerging approaches

Wider inter-laboratory comparison studies for Tuberculosis and CMV, involving end-user laboratories are currently being carried out which aim to evaluate the suitability of the high order methods developed for the analysis of clinical samples and the commutability of the test materials developed. These studies are a means of highlighting the materials and higher order methods developed in the project to end-user laboratories/stakeholders and demonstrate value for, and so encourage uptake of, the adoption of prototype reference systems for increased standardisation of molecular diagnostics.

To quantitatively and comparatively evaluate new and emerging molecular approaches for the surveillance and monitoring of infectious disease load and detection of antimicrobial resistance mutations

Model systems to develop and evaluate methodologies to monitor antimicrobial resistance mutations in infectious diseases were selected. Digital PCR methods to detect rare single RNA mutations using Influenza oseltamivir (Tamiflu) resistance as a model system are currently being developed. Work has focussed on assay optimisation to improve discrimination between wild-type (sensitive) and mutant (resistant) sequences. Development of novel sequencing approaches to detect known DNA mutations using Multi-drug-resistant tuberculosis (MDR-TB) as a model system has been completed. Results show that this sequencing approach is able to detect drug resistant genotypes as minority targets. Work to develop Next Generation Sequencing approaches for the surveillance and monitoring of the pathogens involved in Chronic obstructive pulmonary disease (COPD) has started. Assays to target the common causative pathogens of COPD have been optimised and working ranges established. A whole microbe control material containing a mixture of 6 COPD-causing bacterial species (H. influenza, M. catarrhalis, S. pneumoniae, E. coli, K. pneumonia & P. aeruginosa) at clinically relevant levels has been prepared and is being characterised prior to sequencing analysis.

To quantitatively and comparatively evaluate new and emerging diagnostic technologies for the rapid detection of infectious agents

Emerging isothermal approaches such as loop-mediated isothermal amplification (LAMP) were evaluated (and published) for near-patient testing alongside qPCR based near-patient instrumentation. In parallel, X-ray photoelectron spectroscopy (XPS) and time of flight secondary mass spectrometry (ToF-SIMS) are being used to characterise DNA binding to disposable PCR microfluidic chips. Methods to investigate the adhesion loss of DNA fragments and selected PCR reagents on different surface materials and coatings used to manufacture PCR devices such as digital PCR chips are currently being developed.

So far, the project’s impact and dissemination activities have included:

  • Ongoing contribution to documentary standards development. Specifically, input to      ISO TC 212 plenary and WG2 meeting to continue with the revision of ISO      17511 of key importance for IVD calibration including IVDs for pathogens
  • Scientific dissemination via publications and presentations:

1. Scientific publications

  •  “Comparative study of sensitivity, linearity, and resistance to inhibition of digital and non-digital polymerase chain reaction and loop mediated isothermal amplification assays for quantification of human cytomegalovirus”. Nixon G, Garson J, Nastouli E, Foy C & Huggett J. Anal Chem. 2014 May 6;86(9):4387-94
  • “The Molecular Bacterial Load Assay replaces solid culture for measuring early bactericidal response to anti-tuberculosis treatment”. I. Honeybourne. J. Clin. Microbiol. 2014, 52(8):3064.
  • “Standardising Clinical Measurements of Bacteria and Viruses Using Nucleic Acid Tests”. Pavšič J, Devonshire A, Foy C, Gutierrez Aguirre I, Honeybourne I, Huggett J, McHugh T, Milavec M, Zeichhardt H, Žel J. J Clin. Microbiol. 2014 Nov 12. pii: JCM.02136-14. [Epub ahead of print].

2. 20 Presentations at relevant international professional and clinical symposiums including:

  • SoGAT(Standardisation of Genome Amplification Techniques) – Joint Blood Virology and Clinical Diagnostics meeting, 2013 & 2014
  • Digital PCR Conference: Technologies and Tools for Precision Diagnostics, San Diego, 2013 & 2014
  • IFCC supported INFECT-MET/Bio-SITrace workshop “Clinical Biomeasurement Standardisation and Traceability” at IFCC Worldlab congress, Istanbul 2014
  • Next Generation Dx Summit, Molecular Diagnostics for Infectious Disease, Washington 2014

3. 7 Poster exhibits at European conferences.

4. Workshops/Training:

  • “Digital PCR” at the ESCMID (European Society Clinical Microbiology & Infectious Diseases) Post-graduate Education “Principles of Molecular Diagnostics” course, Maastricht, March 2014
  • “Pathogenic microbes sequencing workshop”, LGC, May 2014
  • “Digital PCR Experiment Design and Primer-Digital PCR Conference: Technologies and Tools for Precision Diagnostics”, October 2014

 

JRP start date and duration: 1 June 2012, 36 months
JRP-Coordinator:

Dr Carole Foy, LGC, UK                          Tel:  +44 (0) 208 943 7000                         E-mail:  carole.foy@lgcgroup.com

JRP website   address: http://infectmet.lgcgroup.com/

JRP-Partners:

JRP-Partner 1 LGC, UK

JRP-Partner 2 BAM, Germany

JRP-Partner 3 JRC, EU

 

JRP-Partner 4 NIB, Slovenia

JRP-Partner 5 PTB, Germany

JRP-Partner 6 TUBITAK, Turkey

REG-Researcher
(associated Home Organisation):
Isobella Honeyborne, UK

UCL, UK

REG-Researcher
(associated Home Organisation):
Heinz Zeichhardt, Germany

Charité, Germany

REG-Researcher
(associated Home Organisation):
Viktorija Tomic, Slovenia

UCG, Slovenia

 

The EMRP is jointly funded by the EMRP participating countries within EURAMET and the European Union