Adviescollege Verloftoetsing TBS

MLVA

Haemophilus influenzae

H. influenzae is a respiratory pathogen which causes a wide spectrum of infections in humans. Strains carrying a polysaccharide capsule have been an important cause of serious invasive diseases like meningitis and septicemia in children. The majority of cases of invasive disease are caused by H. influenzae serotype b (Hib), and this has led to the introduction of nationwide vaccination with Hib polysaccharide conjugate vaccines in many developed countries. Vaccination has resulted in impressive reductions in Hib disease and reduced carriage of Hib among both vaccinated and non-vaccinated individuals. Despite nearly complete vaccine coverage a small number of fully vaccinated children in the Netherlands have experienced invasive H. influenzae type b infection. A more impressive rise has been observed in the UK where incidence among children in the age group of 0-4 years started to rise in 1999 to reach 3.7 per 100,000 in 2002.

Multiple-Locus Variable number of tandem repeat Analysis (MLVA) of H. influenzae

The increase of invasive Hib in the UK and the Netherlands demonstrate that changes in the composition of bacterial population that are under selective pressure due to national immunization programs need to be monitored. A multi-locus sequence typing (MLST) to perform genotyping of H. influenzae has been developed. However, this technique has low discriminatory power for Hib. Furthermore, MLST is expensive, labor intensive and therefore not the method of choice for high throughput typing for most laboratories. Therefore, we developed a MLVA for H. influenzae. The MLVA used here is based on accurate band sizing using an automated DNA sequencer.

MLVA for H. influenzae 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).
Schouls, L. M., A. van der Ende, I. van de Pol, C. Schot, L. Spanjaard, P. Vauterin, D. Wilderbeek, and S. Witteveen. 2005. Increase in genetic diversity of Haemophilus influenzae serotype b (Hib) strains after introduction of Hib vaccination in The Netherlands. J Clin Microbiol 43:2741-9.

Profiles and types

In the MLVA of H. influenzae 4 VNTR loci are used. The H. influenzae MLVA type list contains more than 100 distinct allelic profiles and their corresponding MLVA type designation. These profiles were obtained from approximately 1,100 H. influenzae isolates obtained from patients with invasive H. influenzae disease. Approximately 95% of these isolates H. influenzae serotype b, the remaining isolates were predominantly serotype f and unencapsulated H. influenzae.

Since the publication of the original MLVA for H. influenzae the MLVA scheme has been expanded to include 2 more VNTR loci. For this reason the profile and type table includes these additional loci. The typing tool allows you to choose between the MLVA with 4 or 5 VNTR loci. MLVA with the 5 VNTR loci has been performed on 1000 H. influenzae isolates and yielded more than 275 distinct MLVA profiles.

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.

  • Protocol_MLVA_Hinfluenzae.pdf A detailed protocol for the MLVA scheme containing details on the reagents, primers
  • 4locus | 5 locus A file containing all currently known MLVA types and profiles - will open in csv format.
  • Hinfluenzae_MLVA_allele_size_table.xls A file containing the theoretical size of all currently known alleles.
  • Hinfluenzae_MLVA_mix1.xml Panel file for use with the GeneMarker software. This file contains all allele sizes and their bin sizes and is used to assign the number of repeats in the VNTRs amplified in multiplex 1.
  • Hinfluenzae_MLVA_mix2.xml Panel file for use with the GeneMarker software. This file contains all allele sizes and their bin sizes and is used to assign the number of repeats in the VNTRs amplified in multiplex 2.
  • LIZ1200.xml Size standard file for use with the GeneMarker software.
  • Hinfluenzae_MST.pdf Minimum spanning tree based on MLVA profiles.
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 Leo Schouls. 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.

V05_02
V12_01
V06_01
V12_02

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 Leo Schouls. 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.

 
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 Leo Schouls. 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.

V05_02
V12_01
V06_01
V12_02
V06_10

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 Leo Schouls. 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

17/10/2017

4 locus Total - 116 profiles

6 locus Total - 157 profiles

Contact:
Leo Schouls