Neisseria gonorrhoeae multi-antigen sequence typing using non-cultured clinical specimens.
OBJECTIVE
The Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) system, based on PCR amplification and sequence analysis of the gonococcal porB and tbpB genes, is widely used for molecular typing of gonococcal isolates but is not validated for non-cultured clinical samples. This study sought to examine the performance of the NG-MAST system on a range of non-cultured samples.


METHODS
Nucleic acid extracts of 73 N gonorrhoeae-positive samples, comprising eight cervical swabs, nine urethral swabs, 35 urine samples, one vaginal swab, 13 rectal swabs and seven throat swabs, were analysed by NG-MAST. For 27 specimens, corresponding gonococcal isolates were also analysed and the results compared. A panel of 44 non-gonococcal Neisseria strains and 100 N gonorrhoeae-negative clinical samples were used to investigate further the specificity of the NG-MAST PCR reactions.


RESULTS
PCR amplification and DNA sequencing of gonococcal porB and tbpB genes was successful for all N gonorrhoeae-positive urogenital specimens, 11 of 13 rectal swabs and four of seven throat swabs. For the 27 N gonorrhoeae-positive specimens with corresponding gonococcal isolates, the porB and tbpB sequences obtained from the non-cultured specimen were identical to those obtained from the isolate. Cross-reaction with non-gonococcal Neisseria species was observed for both the porB and tbpB PCR reactions, and proved to be problematical for NG-MAST typing of throat swab specimens.


CONCLUSIONS
The NG-MAST system can successfully be applied directly to non-cultured urogenital samples, but is less suitable for extragenital specimens, particularly throat swabs, due to cross-reaction with commensal Neisseria species.
Queensland Paediatric Infectious Diseases Laboratory, Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital and Health Service District, Herston Road, Herston, Queensland 4029, Australia. d.whiley@uq.edu.au
David M
DM
Whiley
Namraj
N
Goire
E Sanghamitra
ES
Ray
Athena
A
Limnios
Stephen B
SB
Lambert
Michael D
MD
Nissen
Theo P
TP
Sloots
John W
JW
Tapsall
D000942:Antigens, Bacterial / Q000235:genetics*; D015373:Bacterial Typing Techniques / Q000379:methods*; D002584:Cervix Uteri / Q000382:microbiology; D004269:DNA, Bacterial / Q000235:genetics; D005260:Female; D006069:Gonorrhea / Q000382:microbiology*; D006801:Humans; D008297:Male; D009344:Neisseria gonorrhoeae / Q000145:classification* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D010614:Pharynx / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods; D012007:Rectum / Q000382:microbiology; D012680:Sensitivity and Specificity; D014521:Urethra / Q000382:microbiology; D014556:Urine / Q000382:microbiology
D000942:Antigens, Bacterial; D004269:DNA, Bacterial