Adviescollege Verloftoetsing TBS

MLVA

Streptococcus pneumoniae

S. pneumoniae is a major human pathogen causing considerable morbidity and mortality throughout the world. The pathogen has been shown to carry a large number of virulence factors, but its polysaccharide capsule is still considered the most important factor. The capsule provides resistance to opsonophagocytosis and reactivity of the capsular polysaccharide with specific antisera is the basis of the classical serotyping technique. Currently, 92 pneumococcal serotypes are recognized and approximately a quarter of these serotypes are responsible for the majority of cases of invasive pneumococcal disease. Immunization against the pneumococcus with a 7-, 10- or 13-valent polysaccharide conjugate vaccine has been introduced in many different countries in the world leading to a considerable reduction of the incidence of invasive disease caused by the vaccine serotypes.

Multiple-Locus Variable number of tandem repeat Analysis (MLVA) of Streptococcus pneumoniae
Since vaccination against the pneumococcus is based on capsular polysaccharides, which are also the main serotype specific virulence factors, immunization will put pressure upon the bacterial population. Important vaccine effects following immunization could be serotype replacement and capsule switch. Serotype replacement is the replacement of vaccine types by non-vaccine types. Capsule switch is the ability to transfer capsule genes, by which the bacteria will change its serotype but will keep its genetic background. Changes in genotype and serotype may have considerable consequences for future vaccination strategies.
To monitor alterations in the pneumococcal population, both serotyping and genotyping methods are required. Many typing techniques have been employed for the analysis of S. pneumoniae of which multi-locus sequence typing (MLST) is considered to be the current gold standard. However, MLST is expensive, labor intensive and therefore not the method of choice for high throughput typing for many laboratories. For these reasons we developed a typing technique based on the composition of genomic loci containing tandem repeats. Already a MLVA scheme for S. pneumoniae has been designed and used to type this pathogen. However, this MLVA scheme relies on analysis in agarose gels making it less suitable for high throughput molecular typing. The MLVA used here is based on accurate band sizing using an automated DNA sequencer.

MLVA for Streptococcus pneumoniae has been developed in the Laboratory for Infectious Diseases and Perinatal Screening (LIS) of the Centre for Infectious Disease Control Netherlands (CIb) at the National Institute for Public Health and the Environment (RIVM).
Multiple-Locus Variable Number Tandem Repeat Analysis for Streptococcus pneumoniae: Comparison with PFGE and MLST. 2011. Karin E. M. Elberse, Sónia Nunes, Raquel Sá-Leão, Han G. J. van der Heide, M. Schouls. PLoS ONE: 10.1371/journal.pone.0019668

Profiles and types
In the MLVA of S. pneumoniae 8 VNTR loci are used. The S. pneumoniae MLVA type list contains over 1,100 distinct allelic profiles and their corresponding MLVA type designation. These profiles were obtained from over 3,400 S. pneumoniae isolates. Approximately 85% of these isolates were obtained from Dutch patients with invasive pneumococcal disease; the remaining 15% were carriage isolates.

How to ensure accurate sizing of the PCR products
Standardizing typing techniques that are not sequence based has proven to be difficult. Although the separation of the PCR products obtained in MLVA is performed on a DNA sequencer standardization may pose a problem for MLVA as well. The results of sizing of PCR products performed in one lab may differ from the results obtained in other labs. Such differences may have been caused by the use of different sequencers, different buffers, different capillaries etc. In order to eliminate such problems a calibration set may used which we may provide if you want to implement the MLVA in your lab. This calibration set is composed of a number of allelic ladders for each of the 8 BOX loci used. These allelic ladders contain each allele that we currently have identified. If obtain the allelic ladders from us you can perform the MLVA PCRs and separate the PCR products on you sequencer. This will reveal the positions to which the alleles will migrate on you sequencer. Once you have established this, you can use this to define the bin positions for your own sequencing system. Although we do not recommend this, this would in principle allow for the use of other fluorescent labels on the primers. A detailed protocol how to use this calibration set can be found on the Protocols and Tables’ page. If you require such a calibration set please contact us.

Capsular Sequence Typing (CST) of Streptococcus pneumoniae
All information about the CST Typing has been moved to http://www.rivm.nl/mpf/spn/cst. The new site includes a typing tool for automatic assignment of your capsular type.

The combined use of MLVA and CST provides insights in the genetic background of the pneumococcus and the serotype specific capsular genes and can be used to observe changes in the pneumococcal population, including serotype replacement and capsule switch.

Protocol and tables for data analysis.

A number of files that may be useful if you want to perform MLVA can be downloaded here. These files may be updated if alterations in the protocol or allele tables have been made.

MLVA

MLVA ready made PCR mixes are available at Eurogentec Dispensing service: dispensing@eurogentec.com

Please enter your MLVA profile in the following text boxes and click submit.

If you discover a new allele or MLVA type please which you would like to be added to the list please contact Hester Bootsma. You probably will need to send either the .fsa trace files or DNA/lysate for confirmation and official assignment of a new allele or MLVA type.

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Please enter MLVA profiles with an isolate identifier in TAB delimited format.
(Click here for example data)

If you discover a new allele or MLVA type please which you would like to be added to the list please contact Hester Bootsma. You probably will need to send either the .fsa trace files or DNA/lysate for confirmation and official assignment of a new allele or MLVA type.

 
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Statistics

24/08/2017

Total - 3144 profiles

Total - 56 complexes

Contact:
Hester Bootsma