typing using non-cultured clinical specimens multi-antigen sequence Neisseria gonorrhoeae

Objectives The Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) system, based on PCR amplification and sequence analysis of the gonococcal porB and tbpB genes, is widely used for molecular typing of gonococcal isolates but is not validated for noncultured clinical samples. This study sought to examine the performance of the NG-MAST system on a range of non-cultured samples. Methods Nucleic acid extracts of 73 N gonorrhoeaepositive samples, comprising eight cervical swabs, nine urethral swabs, 35 urine samples, one vaginal swab, 13 rectal swabs and seven throat swabs, were analysed by NG-MAST. For 27 specimens, corresponding gonococcal isolates were also analysed and the results compared. A panel of 44 non-gonococcal Neisseria strains and 100 N gonorrhoeae-negative clinical samples were used to investigate further the specificity of the NG-MAST PCR reactions. Results PCR amplification and DNA sequencing of gonococcal porB and tbpB genes was successful for all N gonorrhoeae-positive urogenital specimens, 11 of 13 rectal swabs and four of seven throat swabs. For the 27 N gonorrhoeae-positive specimens with corresponding gonococcal isolates, the porB and tbpB sequences obtained from the non-cultured specimen were identical to those obtained from the isolate. Cross-reaction with non-gonococcal Neisseria species was observed for both the porB and tbpB PCR reactions, and proved to be problematical for NG-MAST typing of throat swab specimens. Conclusions The NG-MAST system can successfully be applied directly to non-cultured urogenital samples, but is less suitable for extragenital specimens, particularly throat swabs, due to cross-reaction with commensal Neisseria species. Typing of Neisseria gonorrhoeae has many applications including the definition of sexual networks, interventions to arrest disease outbreaks by defining the contacts of outbreak strains, confirmation or exclusion of possible treatment failures, 4 monitoring the spread of subtypes of gonococci with altered diagnostic features, 6 and the detection of the emergence and subsequent spread of antibioticresistant gonococci. Gonococcal typing methods have progressed from single (auxotyping) to combined (auxotyping and serovar determination) phenotypic systems, but genotyping systems are now generally regarded as being more sensitive than phenotypic examination. Of these, the most widely used in Australia and elsewhere is the N gonorrhoeae multi-antigen sequence typing (NG-MAST) system. Briefly, this system involves DNA sequencing of variable regions of the gonococcal porB and tbpB genes and utilises an openaccess website (http://www.ng-mast.net/) for data analysis. It thus offers good discriminatory power and can be readily used for comparative international studies. With the increasing use of nucleic acid amplification tests (NAAT) for the detection of gonorrhoea, the proportion of cultured isolates to reported cases is steadily decreasing. This has implications for epidemiological investigations and public health interventions that rely on organism isolation. In this respect, an emerging limitation of the NG-MAST system is that it is primarily restricted for use on gonococcal isolates and has not been extensively evaluated for use on non-cultured clinical specimens. Preliminary studies undertaken to ascertain the possible application of NG-MAST typing to non-cultured clinical samples obtained in two different settings have had mixed results. Martin et al successfully applied the NG-MAST method to gonococcal DNA obtained from a single piece of clothing in a medicolegal case, whereas Ng et al found that the NG-MAST system had poor sensitivity when applied directly to non-cultured urogenital samples submitted for sexual health screen. In the current study, we examined the suitability of the NG-MAST system for use on a range of non-cultured clinical samples, including urogenital and extragenital specimens. MATERIALS AND METHODS Testing centres Testing was performed in two Australian centres: the Queensland Paediatric Infectious Diseases Laboratory located in Brisbane, Queensland (QPIDBris) and the Neisseria Reference Laboratory, South Eastern Area Laboratory Services in Sydney, New South Wales (SEALS-Syd). N gonorrhoeae-positive clinical samples A total of 73 N gonorrhoeae-positive samples was used in the study, comprising 27 culture-positive samples (table 1) and 46 samples analysed by PCR only (table 2). All cultured isolates were grown and identified by standard methods, and national recommendations for the detection of gonorrhoea by nucleic acid amplification assays were followed for the detection of gonococcal-specific nucleic acids that included the use of supplemental assays. For the 27 culture-positive samples, NG-MAST analysis was performed on both a DNA extract of the neat clinical specimen (the non-cultured clinical sample) as well as the corresponding gonococcal Queensland Paediatric Infectious Diseases Laboratory, Sir Albert Sakzewski Virus Research Centre, Queensland Children’s Medical Research Institute, Children’s Health Service District, Queensland, Australia Clinical Medical Virology Centre, University of Queensland, Queensland, Australia WHO Collaborating Centre for STD and HIV, Microbiology Department, South Eastern Area Laboratory Services, Prince of Wales Hospital, Sydney, New South Wales, Australia Microbiology Division, Pathology Queensland Central, Royal Brisbane and Women’s Hospital Campus, Queensland, Australia Correspondence to Dr David M Whiley, Queensland Paediatric Infectious Diseases Laboratory, Sir Albert Sakzewski Virus Research Centre, Royal Children’s Hospital and Health Service District, Herston Road, Herston, Queensland 4029, Australia; d.whiley@uq.edu. au Accepted 8 August 2009 Published Online First 19 October 2009 Sex Transm Infect 2010;86:51e55. doi:10.1136/sti.2009. 037689 51 Basic science group.bmj. com on March 12, 2010 Published by sti.bmj. com Downloaded from