title: Evolution of Neisseria gonorrhoeae is a continuing challenge for molecular detection of gonorrhoea: false negative gonococcal porA mutants are spreading internationally.
abstract: OBJECTIVE
  Identification of genetic targets specific to Neisseria gonorrhoeae for use in molecular detection methods has been a challenge. The porA pseudogene in N gonorrhoeae has 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 and 16S rRNA gene sequencing. Molecular typing using the N gonorrhoeae multi-antigen sequence typing (NG-MAST) and multilocus sequence typing (MLST) was performed. The DNA sequence of the full-length gonococcal porA pseudogene was determined and compared with published sequences.
  
  
  RESULTS
  Both isolates were confirmed, biochemically and immunologically as N gonorrhoeae, but repeatedly gave negative results with two in house real-time PCR assays for the porA pseudogene. Further characterisation of these isolates identified the presence of a meningococcal porA sequence 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 with porA pseudogene 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 of N gonorrhoeae, which remains a challenge for the molecular detection methods.
authors.affiliation: Sexually Transmitted Bacteria Reference Unit, Health Protection Agency, 61 Colindale Avenue, Colindale, London NW9 5HT, UK. catherine.ison@hpa.org.uk
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Ison
authors.forename: Daniel
authors.initials: D
authors.lastname: Golparian
authors.forename: Pamela
authors.initials: P
authors.lastname: Saunders
authors.forename: Stephanie
authors.initials: S
authors.lastname: Chisholm
authors.forename: Magnus
authors.initials: M
authors.lastname: Unemo
mesh_terms: D000328:Adult; D015373:Bacterial Typing Techniques; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D005188:False Negative Reactions; D014644:Genetic Variation; D006069:Gonorrhea / Q000175:diagnosis* / Q000382:microbiology*; D006801:Humans; D008131:London; D008297:Male; D008826:Microbial Sensitivity Tests; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D058889:Molecular Typing; D009344:Neisseria gonorrhoeae / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000379:methods; D018272:Porins / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D012703:Serotyping; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D018272:Porins; D012336:RNA, Ribosomal, 16S; C057699:porin protein, Neisseria