Modified MLVA for Genotyping Queensland Invasive Streptococcus pneumoniae

Abstract

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.

Conclusion: 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.

Fig 1. MLVA4 eBurst of Queensland invasive S. pneumoniae isolates from 2007 to 2012 (n = 317).

MLST eBurst of invasive S. pneumoniae isolates from 2007–2012 (n = 202). MLST results are overlayed (coloured circles) to allow ease of comparison against MLVA4 results. Grey circles represent isolates that have not been assigned a MLST type. The size of the isolate circles corresponds to the number of isolates. Clonal clusters are identified in black ovals, and those that contain predominantly one colour indicate that the isolates have a single MLST type. Isolates are labelled with MLVA4 type (MT).

Fig 2. The MLVA1 eBurst diagram depicting invasive S. pneumoniae isolates from 2007 to 2012 (n = 317).

MLST results are overlayed (coloured circles) to allow ease of comparison against MLVA1 results. Grey circles represent isolates that have not been assigned a MLST type. CC are surrounded by a black oval and contain SLV and DLV. The size of the dots corresponds to the number of isolates.

Fig 3. The MLVA2 eBurst diagram depicting invasive S. pneumoniae isolates from 2007 to2012 (n = 317).

MLST results are overlayed (coloured circles) to allow ease of comparison against MLVA1 results. Grey circles represent isolates that have not been assigned a MLST type. CC are surrounded by a black oval and contain SLV and DLV. The size of the dots correspond to the number of isolates.

Fig 4. Comparison of discriminatory power for MLST, MLVA1, MLVA2 and MLVA4 when genotyping serotype 3 and serotype 7F in Queensland, Australia.

The population structure based on the respective genotypes determined by each genotyping method varies. MLST is considered the least discriminatory as only one genotype is assigned to serotype 3 and serotype 7F, whereas MLVA4 provides increased discrimination by identifying a number of genetically related but different genotypes.