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International consensus achieved on development, validation, nomenclature and quality control of multiple-loci variable-number tandem repeat analysis (MLVA) method for molecular typing of Salmonella Typhimurium: does it matter for public health?

02 Sep 2013
​An international consensus study on standardisation of a widely developed molecular subtyping method for Salmonella Typhimurium, multiple loci variable-number tandem repeat analysis (MLVA), was published by Nadon et al. in Eurosurveillance on 29 August 2013 [1]. The paper describes in details all necessary steps to develop and validate a MLVA typing protocol for S. Typhimurium, including nomenclature, internal and external validation. The reproducibility of the method across laboratories was further demonstrated through a proof-of-concept inter-laboratory study, published by Larsson et al. in the same Eurosurveillance issue [2]. The inter-laboratory comparison highlights the need for a calibration of the equipment that is used for the analyses so that possible systematic deviations can be properly corrected with compensation factors. The authors investigated in details all discrepancies detected in the inter-laboratory study providing thus valuable insight to possible problems in the implementation of the method. The consensus meeting in Denmark in 2011, co-organised by United States (US) Centers for Disease Control and Prevention (CDC), the European Centre for Disease Prevention and Control (ECDC), the Association of Public Health Laboratories in United States, the Public Health Agency of Canada and the Statens Serum Institut, Denmark, resulted in a historical step in the global surveillance of foodborne pathogens by producing this global agreement on a molecular typing method, demonstrating the crucial role of collaboration across public health institutions, and providing a solid basis for the surveillance and subsequent detection and investigation of international foodborne outbreaks.
ECDC comment 02-09-2013:
Salmonella Typhimurium infection in humans is, in spite of a declining trend, the second most commonly reported Salmonella serotype after S. Enteritidis with around 20 000 human salmonellosis cases (about 25% of all cases) reported to the European Surveillance System annually [3]. Salmonella infections cause regularly local, national, multi-country and international foodborne outbreaks [3,4]. Due to the high outbreak potential, one of the core needs in surveillance of salmonellosis is to have a globally agreed and well validated standard molecular typing method so that geographically dispersed cases can be linked to each other and further investigated epidemiologically in a timely manner. So far, pulsed-field-gel-electrophoresis (PFGE)-based genome fingerprinting as developed by CDC, USA has been the best standardised and epidemiologically validated molecular typing method used internationally for surveillance of bacterial foodborne diseases, demonstrating its added value in numerous outbreak investigations. Although PFGE has been the gold standard for several years it has limited discrimination for several common Salmonella serotypes and remains an expensive and laborious method.
This faster and reproducible MLVA typing method, based on a protocol published by Lindstedt et al. 2004 [5] and nomenclature further developed by Larsson et al 2009 [6], has been well studied and is increasingly used in Europe. In 2010-2011 ECDC supported the validation studies, production of a standard protocol, and formation of a strain collection used for reference and method validation purposes [7]. In addition, ECDC facilitated the implementation of the method in 11 national public health laboratories and four veterinary reference laboratories that occasionally type also human isolates. Since 2012, ECDC has funded external quality assessment schemes for this standard MLVA method with the aim to enhance laboratory capabilities to produce valid and comparable results as part of  the piloting of molecular surveillance for three foodborne pathogens; Salmonella, Shigatoxin/verocytotoxin –producing Escherichia coli and Listeria monocytogenes [8].  ECDC also ensures that the set of reference strains as well as molecular typing services using , globally standardised methods, currently PFGE and MLVA (S. Typhimurium) are available for EU Member States.
It is crucial that molecular typing results from different laboratories in diverse geographical locations and with different skill levels are comparable and reproducible. Without this, efficient international surveillance for detection and investigation of multi-country outbreaks is impossible. However, there is a huge variation in the capacity of national public health laboratories to perform molecular characterisation of human pathogens [9]. ECDC is committed to support the Member States work towards global consensus on as well as standardisation of  methods that fulfil the criteria for integration of molecular typing methods into EU-level surveillance [10]. This work is done in close collaboration with the food and veterinary sector.
1. Nadon CA, Trees E, Ng LK, Møller Nielsen E, Reimer A, Maxwell N, et al. Development and application of MLVA methods as a tool for inter-laboratory surveillance. Eurosurveillance [Internet]. 2013; 18(35):[pii=20565 p.]. Available from:
2. Larsson JT, Torpdahl M, MLVA working group, Møller Nielsen E. Proof-of-concept study for successful inter-laboratory comparison of MLVA results. Eurosurveillance [Internet]. 2013; 18(35):[pii=20566 p.]. Available from:
3. European Centre for Disease Prevention and Control. Annual Epidemiological Report 2011. Reporting on 2009 surveillance data and 2010 epidemic intelligence data. Stockholm: ECDC; 2011. Available from:
4. EFSA (European Food Safety Authority), ECDC (European Centre for Disease Prevention and Control), 2013. The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-borne Outbreaks in 2011. EFSA Journal [Internet]. 2013; 11(4):[250 p.]. Available from:
5. Lindstedt BA, Vardund T, Aas L, Kapperud G. Multiple-locus variable-number tandem-repeats analysis of Salmonella enterica subsp. enterica serovar Typhimurium using PCR multiplexing and multicolor capillary electrophoresis. Journal of microbiological methods. 2004 Nov;59(2):163-72. PubMed PMID: 15369852. Epub 2004/09/17. eng.
6. Larsson JT, Torpdahl M, Petersen RF, Sorensen G, Lindstedt BA, Nielsen EM. Development of a new nomenclature for Salmonella typhimurium multilocus variable number of tandem repeats analysis (MLVA). Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin. 2009;14(15). PubMed PMID: 19371515. Epub 2009/04/18. eng.
7. European Centre for Disease Prevention and Control. Laboratory standard operating procedure for MLVA of Salmonella enterica serotype Typhimurium. Stockholm: ECDC; 2011. Available from:
8. van Walle I. ECDC starts pilot phase for collection of molecular typing data. Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin. 2013;18(3). PubMed PMID: 23351656. Epub 2013/01/29. eng.
9. Control ECfDPa. Survey of National Reference Laboratory (NRL) capacity for six food-and waterborne diseases in EU/EEA countries. Stockholm: ECDC; 2012. Available from:
10. Control ECfDPa. Surveillance of communicable diseases in Europe – a concept to integrate molecular typing data into EU-level surveillance. Stockholm: ECDC; 2013. Available from:
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