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Eurosurveillance, Volume 11, Issue 2, 12 January 2006
Articles

Citation style for this article: Friedrich AW, Witte W, Harmsen D, de Lencastre H, Hryniewicz W, Scheres J, Westh H, the SeqNet.org participants. SeqNet.org: a European laboratory network for sequence-based typing of microbial pathogens. Euro Surveill. 2006;11(2):pii=2874. Available online: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=2874

SeqNet.org: A a European laboratory network for sequence-based typing of microbial pathogens

AW Friedrich1 (alexf@uni-muenster.de), W Witte2, D Harmsen3, H de Lencastre4,5, W Hryniewicz6, J Scheres7, H Westh8, on behalf of the SeqNet.org participants9

1Institute of Hygiene, University Hospital Münster, Germany
2Robert Koch-Institut, Wernigerode Branch, Wernigerode, Germany
3Department of Periodontology, University Hospital Münster, Germany
4Molecular Genetics Laboratory, Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa, Oeiras, Portugal
5The Rockefeller University, New York, United States of America
6Division of Microbiology, National Institute of Public Health, Warsaw, Poland
7Department of Medical Microbiology, Maastricht Infection Center (MINC), University Hospital Maastricht, The Netherlands
8Department of Clinical Microbiology, Hvidovre Hospital, Hvidovre, Denmark
930 participating European laboratories (Table)

The SeqNet.org initiative is a dynamic Europe-wide network of laboratories for DNA sequence-based typing of microbial pathogens, especially Staphylococcus aureus. SeqNet.org (http://www.SeqNet.org) aims to ensure that unambiguous, electronic, portable and easily comparable typing data for local infection control and for national and European-wide surveillance of sentinel microorganisms, such as methicillin resistant S. aureus (MRSA) are generated. Producing high quality typing data is necessary for cross-border comparisons and for relating local data to a European-wide epidemiological context.

SeqNet.org, which currently involves 30 laboratories from 20 European countries (Table), was founded in November 2004. Its main objective is to establish European standards for sequence-based typing of microbial pathogens and thereby create a common European nomenclature for identification and tracing of important pathogens.

To test the concept, sequence typing of S. aureus Protein A (spa) was chosen as a typing method. It has been shown that spa typing is stable [1, 2], fast [3, 4], and discriminatory [3, 5]. Furthermore, it is affordable for most European laboratories.

The objectives of SeqNet are:

  1. To harmonise methods for sequence-based typing, to,teach sequence typing methods and the creation and distribution of consensus documents and standard operation procedures (SOPs)
  2. To establish a quality control/quality assessment (QC/QA) programme for DNA sequencing in diagnostic microbiology through annual proficiency tests and future certification procedures. (that is, external quality control procedures) for sequence-based typing of MRSA. A ‘wet lab’ workshop on sequence-based typing of S. aureus organised by SeqNet.org and the European Antimicrobial Resistance Surveillance System (EARSS, http://www.rivm.nl/earss/) project, was held in October 2005. Twenty three European national reference and university laboratories took part in an interlaboratory spa typing certification consisting of 5 DNAs, 5 strains and 5 chromatograms. Twenty one laboratories have so far successfully completed the certification trial.
  3. To improve access to sequence-based microbial typing results via the internet and transfer of data at international level.
  4. To manage data through an internet portal. The quality of data that is synchronised for the internet-based portal is controlled using Ridom StaphType software. By quantitatively measuring the sequencing results and the logging of all editing performance, a minimum quality standard will be established by SeqNet.org, allowing sequence data to be synchronised only if it has a high quality score and has not been edited more than 3 times. The procedure is performed automatically by the software, allowing the maintenance of excellent data quality in real time.

The SeqNet.org central database server
In addition to the quality feature, 30 laboratories have been synchronising their data since 2004 within the network. This data is the basis for a virtual laboratory network for typing of MRSA. Using a special bioinformatics tool [4], the laboratory typing data are synchronised via the internet, ensuring a universal nomenclature. New spa repeats and spa types detected by one laboratory are given a preliminary name (eg, txAA or txAB) in the local database, which after synchronisation with the central database are then matched automatically with spa types found by other participants. If the repeat succession is new, the t-number is finally assigned for all future detection of this spa type. If the repeat succession has already been described and synchronised by another laboratory, the preliminary name in the local database of the inquiring laboratory is automatically changed to the pre-existing denomination. That means that the same repeat succession, provisionally designated txAA and txAB from two different laboratories, are both automatically and immediately changed during synchronisation by the StaphType software into t002 and designated as such for future identification.

Currently, the 30 SeqNet.org laboratories (Table) and 34 other submitting laboratories have synchronised more than 1040 spa types, 90 spa repeats, and 11000 S. aureus, 92% of which are MRSA. The complete dataset of the strain records is available for 8943 isolates. Currently, 69% of all submissions are sent to the central server from 13 German laboratories, but other countries including Denmark, Sweden, Norway, Austria, Belgium and the United Kingdom, also synchronise their data.

Network of typing networks
Recently, a BURP (‘based upon repeat patterns’) algorithm module (Ridom StaphType) was released, making it possible to group the spa types by means of their relatedness. Preliminary results show a correlation of spa clonal complexes (CC) with MLST CC and PFGE groups of about 95% [Witte W, personal communication, 12 October 2005], so that spa typing generated within the SeqNet.org network seems to be highly concordant with PFGE and MLST results. A direct link to the mlst.net database has been established. For example, MRSA isolates belonging to MLST type ST80 are often associated in Europe with the Panton-Valentine Leukocidin (PVL) coding gene (lukS-lukF) [6]. These isolates so far correspond to spa type t044 and its variants t376 and t455. Broader use of spa typing could make associations between specific virulence markers and spa types apparent, which might be then useful as surrogate markers for virulence and could be used for risk assessment and early-warning algorithms [4]. The SeqNet.org laboratory network delivers the tools to detect the cross-border spreading of MRSA and enables participating countries to establish local, national and international early warning systems. Moreover, the SeqNet.org typing data can already be used for trend analysis and early warning algorithms.

Other laboratories are welcome to join this initiative. The results of the certification trial and the common standard for external and internal quality control will be published in the near future. As the participating laboratories are able to store very detailed information about the isolates and their origin, SeqNet.org will decide, in collaboration with other networks and institutions interested in the SeqNet.org data (eg EARSS, national reference laboratories), how the data should flow nationally and internationally (eg, via the national reference laboratories) and if further data should be synchronised (MLST types, specimen, source, etc.).

Table. The 30 laboratories and institutions in Europe participating in SeqNet.org

Organisation Contact City Country
Institut für Hygiene, Mikrobiologie und Tropenmedizin H Mittermayer Linz Austria
Österreichische Agentur für Gesundheit und Ernährungssicherheit F Allerberger Vienna Austria
Hôpital Erasme - Centre for Molecular Diagnostic (CMD) M Struelens Brussels Belgium
National Institute of Public Health H Zemlickova Prague Czech Republic
Laboratory of Veterinary Medicine (LABVET) O Melter Prague Czech Republic
Hvidovre Hospital H Westh (advisory board) Hvidovre Denmark
Statens Seruminstitut R Skov Copenhagen Denmark
National Public Health Institute J Vuopio-Varkila Helsinki Finland
Hôpital Edouard Herriot J Etienne Lyon France
Institute of Hygiene of the University of Muenster AW Friedrich (coordinator) Muenster Germany
Institute of Medical Microbiology of the University Hospital Muenster K Becker, B Kahl Muenster Germany
University of Wuerzburg U Vogel Wuerzburg Germany
Robert Koch-Institut W Witte (coordinator) Wernigerode Germany
University of Applied Sciences/Public Health R Reintjes Hamburg Germany
University of Athens N Legakis Athens Greece
"Johan Bela" National Center for Epidemiology M Fuzi Budapest Hungary
Istituto Superiore di Sanità A Pantosti Rome Italy
P. Stradins Clinical University Hospital E Miklasevicz Riga Latvia
University of Malta A McElhatton Malta Malta
Laboratorium Microbiologie Twente Achterhoek R Hendrix Enschede The Netherlands
National Institute of Public Health and the Environment X Huijsdens Bilthoven The Netherlands
University Hospital Maastricht E Stobberingh Maastricht The Netherlands
Akershus University Hospital G Bukholm Lørenskog Norway
Telelab Y Tveten Skien Norway
National Institute of Public Health W Hryniewicz (advisory board) Warsaw Poland
Instituto de Tecnologia Quimica e Biologica (ITQB) H de Lencastre (advisory board) Oeiras Portugal
National Intitute for Research and Development for Microbiology and Immunology I Codita Bucharest Romania
Scottish MRSA Reference Laboratory D Morrison Glasgow Scotland (UK)
University of Ljubljana, Medical Faculty, Institute of Microbiology and Immunology M Mueller-Premru Ljubljana Slovenia
Lund University Hospital A-C Petersson Lund Sweden
Swedish Institute for Infectious Disease Control (Smittskyddsinstitutet) S Haeggman Solna Sweden

 

References:
  1. Frenay HM, Bunschoten AE, Schouls LM, van Leeuwen WJ, Vandenbroucke-Grauls CM, Verhoef J et al. 1996. Molecular typing of methicillin-resistant Staphylococcus aureus on the basis of protein A gene polymorphism. Eur J Clin Microbiol Infect Dis 1996 Jan;15(1):60-64.
  2. Shopsin B, Gomez M, Montgomery SO, Smith DH, Waddington M, Dodge DE, et al. Evaluation of protein A gene polymorphic region DNA sequencing for typing of Staphylococcus aureus strains. J Clin Microbiol. 1999 Nov;37(11):3556-3563.
  3. Mellmann A, Friedrich, AW, Rosenkötter, N, Rothgänger, J, Karch, H, Reintjes, R, et al. Automated DNA sequence-based early-warning system for the detection of MRSA outbreaks in hospitals. PLoS Med 2006; 3(3): e33. (http://medicine.plosjournals.org/archive/1549-1676/3/3/pdf/10.1371_journal.pmed.0030033-p-S.pdf)
  4. Harmsen D, Claus H, Witte W, Rothganger J, Claus H, Turnwald D, et al. Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management. J Clin Microbiol. 2003 Dec;41(12):5442-5448.
  5. Aires de Sousa M, Conceicao T, Simas C, de Lencastre H. Comparison of genetic backgrounds of methicillin-resistant and -susceptible Staphylococcus aureus isolates from Portuguese hospitals and the community. J Clin Microbiol. 2005 Oct;43(10):5150-5157.
  6. Witte W, Braulke C, Cuny C, Strommenger B, Werner G, Heuck D, et al. Emergence of methicillin-resistant Staphylococcus aureus with Panton-Valentine leukocidin genes in central Europe. Eur J Clin Microbiol Infect Dis. 2005 Jan;24(1):1-5.

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