Enhanced gonococcal antimicrobial surveillance in the era of ceftriaxone resistance: a real-time PCR assay for direct detection of the Neisseria gonorrhoeae H041 strain.
OBJECTIVE
Recent emergence of the extensively drug-resistant Neisseria gonorrhoeae H041 strain in Japan raises concerns that gonorrhoea may soon become untreatable and emphasizes the need for enhanced surveillance. In this study we developed a real-time PCR assay for direct detection of the H041 strain.


METHODS
Two real-time PCR assays for detection of the penA gene of the H041 strain, H041-PCR1 and H041-PCR2, were developed and evaluated in parallel. Assay performance was assessed using a panel of pathogenic and commensal Neisseria species (n = 167 strains) including the N. gonorrhoeae H041 strain and clinical specimens (n = 252) submitted for sexual health screening. The detection limits of the assays were compared with a standard N. gonorrhoeae real-time PCR method.


RESULTS
Both the H041-PCR1 and H041-PCR2 assays correctly detected the N. gonorrhoeae H041 strain and provided negative results for all other N. gonorrhoeae strains. However, only the H041-PCR2 assay proved to be specific when applied to the non-gonococcal Neisseria species and clinical samples. False-positive results in the H041-PCR1 included cross-reactions with two Neisseria subflava isolates and eight clinical specimens. DNA sequencing of these N. subflava strains revealed the presence of the penicillin-binding protein 2 Ala328Thr alteration previously only observed in the N. gonorrhoeae H041 strain.


CONCLUSIONS
The H041-PCR2 assay is suitable for direct detection of the N. gonorrhoeae H041 ceftriaxone-resistant strain in cultured and non-cultured samples.
Queensland Paediatric Infectious Diseases Laboratory,  Queensland Children's Medical Research Institute, Children's Health Service District, Queensland,  Australia.
Namraj
N
Goire
Makoto
M
Ohnishi
Athena E
AE
Limnios
Monica M
MM
Lahra
Stephen B
SB
Lambert
Graeme R
GR
Nimmo
Michael D
MD
Nissen
Theo P
TP
Sloots
David M
DM
Whiley
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics; D001431:Bacteriological Techniques / Q000379:methods*; D002443:Ceftriaxone / Q000494:pharmacology; D024901:Drug Resistance, Multiple, Bacterial; D006069:Gonorrhea / Q000453:epidemiology* / Q000382:microbiology; D006801:Humans; D007564:Japan / Q000453:epidemiology; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009344:Neisseria gonorrhoeae / Q000145:classification / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D011159:Population Surveillance / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002443:Ceftriaxone