Determination of Neisseria gonorrhoeae susceptibility to ciprofloxacin in clinical specimens from men using a real-time PCR assay

Abstract A real-time PCR (RT-PCR) assay was modified to simultaneously detect Neisseria gonorrhoeae and to determine gonococcal susceptibility to ciprofloxacin using clinical samples. The modified RT-PCR assay was validated using DNA extracted from 40 linked isolates and urethral swabs, 24 of which...

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Bibliographic Details
Published in:	International journal of antimicrobial agents Vol. 42; no. 1; pp. 63 - 67
Main Authors:	Magooa, M.P, Müller, E.E, Gumede, L, Lewis, D.A
Format:	Journal Article
Language:	English
Published:	Netherlands Elsevier B.V 01-07-2013
Subjects:	Anti-Bacterial Agents - pharmacology Ciprofloxacin Ciprofloxacin - pharmacology DNA, Bacterial - genetics Drug Resistance, Bacterial Genes, Bacterial Genotype Gonorrhea - microbiology Humans Infectious Disease Male Microbial Sensitivity Tests - methods Molecular Diagnostic Techniques - methods Mutation Neisseria gonorrhoeae Neisseria gonorrhoeae - drug effects Neisseria gonorrhoeae - isolation & purification Quinolone Real-time PCR Real-Time Polymerase Chain Reaction - methods Resistance Sequence Analysis, DNA Susceptibility Resistance Quinolone Neisseria gonorrhoeae Real-time PCR Ciprofloxacin Susceptibility
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Summary:	Abstract A real-time PCR (RT-PCR) assay was modified to simultaneously detect Neisseria gonorrhoeae and to determine gonococcal susceptibility to ciprofloxacin using clinical samples. The modified RT-PCR assay was validated using DNA extracted from 40 linked isolates and urethral swabs, 24 of which had linked first-pass urine samples, obtained from men presenting with urethral gonorrhoea. The RT-PCR assay enabled amplification of N. gonorrhoeae dcmH , gyrA and parC genes. The quinolone resistance-determining regions (QRDRs) of the isolates’ gyrA and parC genes were sequenced. Following successful validation, 33 first-pass urine-derived DNA extracts, obtained from men with gonorrhoea, were tested with the assay and results were compared with blinded ciprofloxacin susceptibility data. Gonococcal susceptibility to ciprofloxacin correlated perfectly with gyrA amplicon generation. No gyrA amplicons were detected for gonococcal infections due to ciprofloxacin-intermediate/resistant organisms. Amplification of parC correlated less well with ciprofloxacin susceptibility phenotypes. Simultaneous non-generation of gyrA and parC amplicons consistently predicted the presence of ciprofloxacin-resistant gonococci. Characteristic point mutations in the gyrA / parC QRDRs were found in DNA amplified from those extracts that failed to produce gyrA / parC amplicons. The RT-PCR assay performed well with DNA extracted from first-pass urine specimens and results correlated perfectly with ciprofloxacin susceptibility phenotypes. In conclusion, the modified RT-PCR assay can detect N. gonorrhoeae in DNA extracted from first-pass urine specimens of men with urethral gonorrhoea and accurately predicts gonococcal susceptibility to ciprofloxacin. This molecular assay provides a useful tool for surveillance and patient management in settings where fluoroquinolones can still be used for treatment of gonorrhoea.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0924-8579 1872-7913
DOI:	10.1016/j.ijantimicag.2013.02.026