High-throughput informative single nucleotide polymorphism-based typing of Neisseria gonorrhoeae using the Sequenom MassARRAY iPLEX platform.
OBJECTIVE
Neisseria gonorrhoeae antimicrobial resistance (AMR) is a global problem heightened by emerging resistance to ceftriaxone. Appropriate molecular typing methods are important for understanding the emergence and spread of N. gonorrhoeae AMR. We report on the development, validation and testing of a Sequenom MassARRAY iPLEX method for multilocus sequence typing (MLST)-style genotyping of N. gonorrhoeae isolates.


METHODS
An iPLEX MassARRAY method (iPLEX14SNP) was developed targeting 14 informative gonococcal single nucleotide polymorphisms (SNPs) previously shown to predict MLST types. The method was initially validated using 24 N. gonorrhoeae control isolates and was then applied to 397 test isolates collected throughout Queensland, Australia in the first half of 2012.


RESULTS
The iPLEX14SNP method provided 100% accuracy for the control isolates, correctly identifying all 14 SNPs for all 24 isolates (336/336). For the 397 test isolates, the iPLEX14SNP assigned results for 5461 of the possible 5558 SNPs (SNP call rate 98.25%), with complete 14 SNP profiles obtained for 364 isolates. Based on the complete SNP profile data, there were 49 different sequence types identified in Queensland, with 11 of the 49 SNP profiles accounting for the majority (n = 280; 77%) of isolates. AMR was dominated by several geographically clustered sequence types. Using the iPLEX14SNP method, up to 384 isolates could be tested within 1 working day for less than Aus$10 per isolate.


CONCLUSIONS
The iPLEX14SNP offers an accurate and high-throughput method for the MLST-style genotyping of N. gonorrhoeae and may prove particularly useful for large-scale studies investigating the emergence and spread of gonococcal AMR.
Queensland Paediatric Infectious Diseases Laboratory, Royal Children's Hospital, Brisbane, Queensland 4029, Australia Queensland Children's Medical Research Institute, Royal Children's Hospital, The University of Queensland, Brisbane, Queensland 4029, Australia.
Ella
E
Trembizki
Public Health Microbiology, Communicable Disease, Queensland Health Forensic and Scientific Services, Archerfield, Queensland, Australia.
Helen
H
Smith
WHO Collaborating Centre for STD and HIV, Microbiology Department, South Eastern Area Laboratory Services, Prince of Wales Hospital, Sydney, New South Wales 2031, Australia.
Monica M
MM
Lahra
Melbourne Sexual Health Centre, Alfred Health, Carlton, Victoria 3053, Australia Melbourne School of Population Health, University of Melbourne, Victoria 3010, Australia.
Marcus
M
Chen
Kirby Institute, University of New South Wales, Sydney, New South Wales 2052, Australia Sydney Sexual Health Centre, Sydney Hospital, Sydney, New South Wales 2000, Australia.
Basil
B
Donovan
WHO Collaborating Centre for STD and HIV, Microbiology Department, South Eastern Area Laboratory Services, Prince of Wales Hospital, Sydney, New South Wales 2031, Australia Melbourne Sexual Health Centre, Alfred Health, Carlton, Victoria 3053, Australia.
Christopher K
CK
Fairley
Kirby Institute, University of New South Wales, Sydney, New South Wales 2052, Australia.
Rebecca
R
Guy
Kirby Institute, University of New South Wales, Sydney, New South Wales 2052, Australia.
John
J
Kaldor
Kirby Institute, University of New South Wales, Sydney, New South Wales 2052, Australia.
David
D
Regan
BakerIDI Central Australia, Alice Springs, Northern Territory 0870, Australia.
James
J
Ward
Queensland Paediatric Infectious Diseases Laboratory, Royal Children's Hospital, Brisbane, Queensland 4029, Australia Queensland Children's Medical Research Institute, Royal Children's Hospital, The University of Queensland, Brisbane, Queensland 4029, Australia Microbiology Division, Pathology Queensland Central, Royal Brisbane and Women's Hospital Campus, Queensland, Australia.
Michael D
MD
Nissen
Queensland Paediatric Infectious Diseases Laboratory, Royal Children's Hospital, Brisbane, Queensland 4029, Australia Queensland Children's Medical Research Institute, Royal Children's Hospital, The University of Queensland, Brisbane, Queensland 4029, Australia Microbiology Division, Pathology Queensland Central, Royal Brisbane and Women's Hospital Campus, Queensland, Australia.
Theo P
TP
Sloots
Queensland Paediatric Infectious Diseases Laboratory, Royal Children's Hospital, Brisbane, Queensland 4029, Australia Queensland Children's Medical Research Institute, Royal Children's Hospital, The University of Queensland, Brisbane, Queensland 4029, Australia d.whiley@uq.edu.au.
David M
DM
Whiley
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D019295:Computational Biology; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005838:Genotype; D006069:Gonorrhea / Q000382:microbiology; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008826:Microbial Sensitivity Tests; D058885:Multilocus Sequence Typing; D009344:Neisseria gonorrhoeae / Q000145:classification* / Q000187:drug effects / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D011793:Queensland
D000900:Anti-Bacterial Agents
SNP; gonorrhoea; resistance; sequenom; typing