Comparable MLVA results achieved between labs

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Internationally comparable Salmonella Enteritidis MLVA results could be seen between labs often using different equipment, according to a validation study.

Participants were asked to analyse the same five loci, in the same order and report the number of tandem repeats found at each locus.

More than 90% reported less than 5% discrepant Multilocus variable-number tandem repeat analysis (MLVA) profiles for the blinded set of validation strains.

The most critical phase was conversion of raw fragment length data to repeat numbers, according to the study.

Preventing more infections

A total of 14 labs (12 from EU/EEA and two from North America) did an inter-laboratory validation of MLVA for S. Enteritidis using a blinded set of 24 strains with known allele sizes.

Researchers said the ability to rapidly identify primary sources of bacterial contamination using genetic subtyping is critical in the investigation of foodborne infections as if common outbreak sources can be determined further infections can be prevented.

MLVA is a high-resolution typing method based on multiplex PCR amplification of repetitive DNA elements in tandem within the genome (tandem repeats) followed by fragment size analysis of the resulting amplified polymorphic regions.

The latter are detected using capillary electrophoresis (CE) where an internal size standard is included for each sample.

Eight labs reported expected profiles for all 22 validation strains and their 110 loci. Five had expected profiles for 21 out of 22 strains and 109 of their 110 loci, although one lab reported all loci as expected when using another sequencing platform.

Another lab reported expected profiles for 20 out of 22 validation strains and 108 of their 110 loci.

S. Enteritidis MLVA data collection for EU/EEA countries started in The European Surveillance System (TESSy) in June 2016.

Serovar specific so part of toolbox

Due to the lack of genetic variation within the serovar Enteritidis, previous molecular methods, such as pulsed-field gel electrophoresis (PFGE), lack discrimination for informing outbreak investigations.

Production of comparable data between labs is crucial for the usefulness of typing foodborne pathogens, said the researchers.

All countries used a single multiplex PCR except three that used two separate multiplex PCR, two used the PulseNet protocol and one an in-house protocol targeting five loci.

Annealing temperatures ranged from 55 °C to 60 °C and were individually optimised for each lab. Primer concentrations were also individually optimised as recommended by Nadon et al.

Twelve labs used Applied Biosystems Genetic Analyzer (ABI) platforms for CE, one used the Beckman Coulter platform and the remaining lab used both systems.

Researchers said regular External Quality Assessments (EQAs) for MLVA for S. Enteritidis at EU/EEA level would ensure data remain comparable and consistent.

Since MLVA schemes for Salmonella are serovar-specific, it cannot fully replace PFGE, they added.

“Subtyping methods based on next generation sequencing technologies show enormous potential.

“Until then, MLVA could have a role in providing a common international strain nomenclature and providing an adequate typing method for laboratories that do not foresee moving to whole genome sequencing technology in the near future.”

Source: Eurosurveillance, Volume 22, Issue 9, 2 March 2017

DOI: 10.2807/1560-7917.ES.2017.22.9.30477

“Multi-laboratory validation study of multilocus variable-number tandem repeat analysis (MLVA) for Salmonella enterica serovar Enteritidis, 2015”

Authors: T Peters, S Bertrand, JT Björkman, LT Brandal, DJ Brown, T Erdõsi, M Heck, S Ibrahem, K Johansson, C Kornschober, SM Kotila, S Le Hello, T Lienemann, W Mattheus, EM Nielsen, C Ragimbeau, J Rumore, A Sabol, M Torpdahl, E Trees, A Tuohy, E de Pinna

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