Evolution ofNeisseria gonorrhoeaeis a continuing challenge for molecular detection of gonorrhoea: false negative gonococcalporAmutants are spreading internationally

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Abstract
Objectives: Identification of genetic targets specific toNeisseria gonorrhoeaefor use in molecular detection methods has been a challenge. TheporApseudogene inN gonorrhoeaehas been commonly used but recently gonococcal isolates giving a negative result in these PCRs have been reported. Here we describe the characterisation of two such gonococcal isolates received by the reference service at the Health Protection Agency, London, England.Methods: Phenotypic characterisation was achieved using conventional biochemical and immunological tests, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), antimicrobial susceptibility testing, serovar determination and detection of meningococcal PorA using monoclonal antibody 4BG4-E7. Genetic species confirmation was determined using commercial and in house PCRs and16S rRNAgene sequencing. Molecular typing using theN gonorrhoeaemulti-antigen sequence typing (NG-MAST) and multilocus sequence typing (MLST) was performed. The DNA sequence of the full-length gonococcalporApseudogene was determined and compared with published sequences.Results: Both isolates were confirmed, biochemically and immunologically asN gonorrhoeae, but repeatedly gave negative results with two in house real-time PCR assays for theporApseudogene. Further characterisation of these isolates identified the presence of a meningococcalporAsequence and showed these isolates belong to serovar Bropyst, and to NG-MAST sequence type (ST) 5967 and MLST ST1901.Conclusions: Gonococcal isolates that give false negative results withporApseudogene PCR assays have now been identified in four countries, three of which are in Europe, and do not appear clonal. This report highlights the genetic diversity ofN gonorrhoeae, which remains a challenge for the molecular detection methods.

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