Modified MLVA for Genotyping Queensland Invasive Streptococcus pneumoniae.
BACKGROUND
Globally, over 800 000 children under five die each year from infectious diseases caused by Streptococcus pneumoniae. To understand genetic relatedness between isolates, study transmission routes, assess the impact of human interventions e.g. vaccines, and determine infection sources, genotyping methods are required. The 'gold standard' genotyping method, Multi-Locus Sequence Typing (MLST), is useful for long-term and global studies. Another genotyping method, Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA), has emerged as a more discriminatory, inexpensive and faster technique; however there is no universally accepted method and it is currently suitable for short-term and localised epidemiology studies. Currently Australia has no national MLST database, nor has it adopted any MLVA method for short-term or localised studies. This study aims to improve S. pneumoniae genotyping methods by modifying the existing MLVA techniques to be more discriminatory, faster, cheaper and technically less demanding than previously published MLVA methods and MLST.


METHODS
Four different MLVA protocols, including a modified method, were applied to 317 isolates of serotyped invasive S. pneumoniae isolated from sterile body sites of Queensland children under 15 years from 2007-2012. MLST was applied to 202 isolates for comparison.


RESULTS
The modified MLVA4 is significantly more discriminatory than the 'gold standard' MLST method. MLVA4 has similar discrimination compared to other MLVA techniques in this study). The failure to amplify particular loci in previous MLVA methods were minimised in MLVA4. Failure to amplify BOX-13 and Spneu19 were found to be serotype specific.


CONCLUSIONS
We have modified a highly discriminatory MLVA technique for genotyping Queensland invasive S. pneumoniae. MLVA4 has the ability to enhance our understanding of the pneumococcal epidemiology and the changing genetics of the pneumococcus in localised and short-term studies.
School of Biomedical Sciences, Queensland University of Technology, Brisbane, Australia; Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia.
Rachael E
RE
Rayner
Public Health Microbiology Laboratory, Queensland Health Forensic and Scientific Services, Brisbane, Australia.
John
J
Savill
School of Biomedical Sciences, Queensland University of Technology, Brisbane, Australia; Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia.
Louise M
LM
Hafner
School of Biomedical Sciences, Queensland University of Technology, Brisbane, Australia; Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia.
Flavia
F
Huygens
D015373:Bacterial Typing Techniques / Q000379:methods*; D004269:DNA, Bacterial; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D058885:Multilocus Sequence Typing / Q000379:methods*; D013296:Streptococcus pneumoniae / Q000235:genetics* / Q000302:isolation & purification; D020080:Tandem Repeat Sequences
D004269:DNA, Bacterial