Background: The Neisseria gonorrhoeae species presents some considerable challenges for molecular diagnostics, even a decade after N. gonorrhoeae nucleic acid amplification tests (NAATs) were first implemented. Factors that may diminish the performance of gonococcal NAATs include carry-over contamination, human error, inhibition, reagent or instrument failure, cross-reaction or absence of the target sequences in some N. gonorrhoeae strains. The first four of these factors can be controlled by good laboratory practices and effective quality control. However, sequence related problems, which may vary considerably with the patient population studied and over time, cannot be dealt with easily and require further investigation; . • False-positive results continue to be a diagnostic problem for N. gonorrhoeae NAATs, with several commercial N. gonorrhoeae NAATs found to cross-react with commensal Neisseria strains. These specificity issues continue to preclude the use of gonococcal NAATs on extragenital specimens, including pharyngeal swabs. . • False-negative N. gonorrhoeae NAAT results attributed to the absence of NAAT target sequences in some N. gonorrhoeae strains have also been reported. However, the extent of the problem is not known. . • An inability to provide antibiotic resistance data in a routine diagnostic setting remains a notable limitation of N. gonorrhoeae NAATs, given gonococci have managed to develop resistance against a broad range of antibiotics, and that the range of treatment options for gonorrhoea is being gradually diminished. .• Gonococcal subtyping methods are used by public health services to monitor disease transmission patterns. A major limitation of gonococcal genotyping methods is that they have not been evaluated for use directly on non-cultured clinical specimens. Hence, increased use of gonococcal screening NAATs has meant there are fewer isolates available for subtyping. .Methods: In this PhD study, problems affecting gonococcal NAATs were examined and possible solutions to these problems were investigated. . • The target sequence conservation and specificity of N. gonorrhoeae real-time PCR assays targeting several gonococcal sequences were investigated. . • A new N. gonorrhoeae real-time PCR assay targeting the gonococcal porA pseudogene was developed, extensively validated and adapted to a range of PCR platforms. . • The genetic basis of reduced susceptibility to ceftriaxone in N. gonorrhoeae was investigated, and a PCR assay targeting the gonococcal penA gene was designed and evaluated to screen for this phenotype. .• The N. gonorrhoeae multi-antigen sequence typing (NG-MAST) system, widely used by Neisseria gonorrhoeae reference laboratories throughout the world, was evaluated for use directly on non-cultured clinical samples. .Results: An overview of the results of this study is as follows; . • Cross-reactions are not just limited to a few gonococcal NAAT targets. Rather, cross¬reactions may occur with almost any gonococcal genetic target. . • The need for supplementary testing before reporting gonococcal NAAT positive results in clinical settings is necessary, as is the need to use different genetic targets for screening and supplementary assays. . • The results highlight that some sequence targets may not be present in all N. gonorrhoeae strains, and that sequence variation within sequence targets is a more general problem for molecular assays. . • Sequence polymorphism within primer targets may reduce the sensitivity of an assay to detect certain gonococcal strains, rather than just causing a false-negative result. . • The gonococcal porA pseudogene appears to be highly conserved and specific to N. gonorrhoeae. . • The real-time PCR assay targeting the gonococcal porA pseudogene is a very promising N. gonorrhoeae NAAT, and is suitable for use on genital and extra-genital specimens. . • N. gonorrhoeae strains exhibiting reduced sensitivity to ceftriaxone are not of a particular subtype, and various mutations in the penicillin binding protein 2 (PBP2) may contribute to this phenotype. . • The utility of screening for N. gonorrhoeae antimicrobial resistance by NAATs targeting limited regions of sequence is questioned based on the results from this study. . • NG-MAST genotyping was successfully adapted and performed directly on non-cultured clinical samples. Conclusions: In summary, I have set out to explore the problems affecting the success of gonococcal NAATs and have considered, in light of the findings of this PhD study, what amounts to best practice in performing gonococcal NAATs. The primary aims of this PhD study as well as other important questions concerning NAAT detection of N. gonorrhoeae have successfully been answered. Overall, the results of this study provide a considerable expansion on previous knowledge, and have considerable implications for the use of diagnostic gonococcal NAAT assays. In addition, I have made significant contributions to the laboratory diagnosis and typing of N. gonorrhoeae and have provided further insight into the biology of the organism.