RESUMEN
Emergence and spread of Neisseria gonorrhoeae resistant to extended spectrum cephalosporins is a major problem threatening treatment of gonorrhoea and is further highlighted by the recent report of a second ceftriaxone-resistant N. gonorrhoeae strain (F89) in Europe, initially observed in France and subsequently identified in Spain. N. gonorrhoeae antimicrobial resistance (AMR) surveillance has acquired new importance and molecular tools have the potential to enhance bacterial culture-based methods. In this study, we established a polymerase chain reaction (PCR) protocol for direct detection of the F89 strain. A key component of this screening protocol was the development of a hybridisation probe-based melting curve analysis assay (mosaic501-hybPCR) to detect the presence of an A501P substitution on the N. gonorrhoeae mosaic penicillin binding protein 2 (PBP2) sequence, an important characteristic of the F89 strain. The mosaic501-hybPCR was evaluated using plasmid-derived positive controls (n=3) and characterised gonococcal (n=33) and non-gonococcal (n=58) isolates. The protocol was then applied to 159 clinical specimens from Sydney, Australia, collected during the first half of the year 2012 that were N. gonorrhoeae PCR-positive. Overall, the results indicate that the PCR-based protocol is suitable for direct detection of the N. gonorrhoeae F89 strain in non-cultured clinical samples. It therefore provides an additional tool to aid investigations into the potential spread of F89 strain throughout Europe and elsewhere.
Asunto(s)
Ceftriaxona/farmacología , Neisseria gonorrhoeae/efectos de los fármacos , Proteínas de Unión a las Penicilinas/genética , Antibacterianos , Farmacorresistencia Bacteriana , Europa (Continente) , Humanos , Pruebas de Sensibilidad Microbiana , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/patogenicidad , Reacción en Cadena de la PolimerasaRESUMEN
The gonococcal porA pseudogene is a popular target for in-house Neisseria gonorrhoeae PCR methods. With this study we present two novel findings: the first case of an N. gonorrhoeae porA pseudogene PCR false-negative result caused by sequence variation, and in the same organism, the first description of a clinical N. gonorrhoeae strain harbouring an N. meningitidis porA sequence.
Asunto(s)
Gonorrea/genética , Gonorrea/microbiología , Neisseria gonorrhoeae/genética , Reacción en Cadena de la Polimerasa/métodos , Porinas/genética , Secuencia de Bases , Reacciones Falso Negativas , Gonorrea/diagnóstico , Humanos , Datos de Secuencia Molecular , Mutación , Adulto JovenRESUMEN
Nucleic acid amplification tests (NAATs) have numerous advantages over traditional diagnostic techniques and so are now widely used by diagnostic laboratories for routine detection of infectious agents. However, there is some concern over the increasing numbers of reports of NAAT false-negative results caused by sequence variation. Highly conserved NAAT target sequences have been reported for many organisms, yet sequence-related problems continue to be observed in commercial and in-house assays targeting a broad range of microbial pathogens. In light of these ongoing problems, it may be time to consider the use of two genetic targets in NAAT methods to reduce the potential for sequence-related false-negative results.