Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2018

Whole genome sequencing as an improved means of identifying Neisseria gonorrhoeae treatment failures (#219)

Cameron Buckley 1 , Brian M Forde 2 3 4 , Tiffany Hogan 5 , Monica M Lahra 5 6 , David M Whiley 1 2 7 , Scott A Beatson 2 3 4
  1. Faculty of Medicine, UQ Centre for Clinical Research, The University of Queensland, Herston, QLD, Australia
  2. Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, QLD
  3. School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD
  4. Australian Centre for Ecogenomics, The University of Queensland, Brisbane, QLD
  5. WHO Collaborating Centre for STD, Microbiology Department, South Eastern Area Laboratory Services, Prince of Wales Hospital, Sydney, NSW
  6. School of Medical Sciences, The University of New South Wales, Sydney, NSW
  7. Pathology Queensland, Microbiology Department, Herston, QLD

Ceftriaxone is the mainstay of treatment as part of a dual therapy approach for treating gonorrhoea, however, treatment failures associated with ceftriaxone have been reported. The World Health Organisation (WHO) recommend the development of standardised protocols to verify gonorrhoea treatment failures. Currently, N. gonorrhoeae multi-antigen sequence typing (NG-MAST) which targets two highly variable regions (porB and tbpB), has been used extensively to examine these cases. However, these regions may mutate during therapy, inappropriately distinguishing isolates of the same strain. Two cases from Australia were previously investigated using NG-MAST; isolates from Case 1 were indistinguishable; whereas Case 2 isolates were distinguished based on an 18bp deletion in porB. The patient from Case 2 denied sexual contact in the follow-up period, raising questions over reliability of the NG-MAST results. Here we used whole genome sequencing (WGS) to reinvestigate Cases 1 and 2 above, with a view to examine WGS for assessing treatment failures.

Both pre- and post-treatment isolates for each Case underwent short-read Illumina sequencing, and the two post-treatment isolates underwent additional long-read PacBio sequencing. Sequence data was interrogated to determine SNP, indel and structural variation differences between pre- and post-treatment isolates.

WGS did not identify differences for isolates in Case 1. WGS confirmed the 18bp deletion for Case 2, but otherwise isolates were identical. Isolates from each Case displayed a non-mosaic penA, a single nucleotide (A) deletion in the mtrR promoter and key alterations in porB (G120K and A121D).

Case 2 highlights the benefits of WGS and its enhanced resolution over NG-MAST which only targets two highly variable genes. Sequencing both porB and tbpB via traditional Sanger sequencing costs approximately AU$40, whereas Illumina WGS currently costs approximately AU$100 per isolate. Although PacBio can resolve complex regions, the current cost for PacBio sequencing may prohibit its routine use for verifying treatment failures.

The WHO recommends standardised protocols for verifying N. gonorrhoeae treatment failures. Here, we highlight the immediate impact that WGS can have in an important and direct clinical application for N. gonorrhoeae. Assessing the whole genome compared to two highly variable regions provides a more confident predictor for determining treatment failure and facilitates rapid comparisons of these cases in the future.