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<jats:p><jats:bold>Background</jats:bold>. Empirical gonorrhoea treatment at initial diagnosis reduces onward transmission. However, increasing resistance to multiple antibiotics may necessitate waiting for culture-based diagnostics to select an effective treatment. There is a need for same-day culture-free diagnostics that identify infection and detect antimicrobial resistance.</jats:p> <jats:p><jats:bold>Methods</jats:bold>. We investigated if Nanopore sequencing can detect sufficient <jats:italic>N. gonorrhoeae</jats:italic> DNA to reconstruct whole genomes directly from urine samples. We used <jats:italic>N. gonorrhoeae</jats:italic> spiked urine samples and samples from gonorrhoea infections to determine optimal DNA extraction methods that maximize the amount of <jats:italic>N. gonorrhoeae</jats:italic> DNA sequenced whilst minimizing contaminating host DNA.</jats:p> <jats:p><jats:bold>Results</jats:bold>. In simulated infections the Qiagen UCP Pathogen Mini kit provided the highest ratio <jats:italic>N. gonorrhoeae</jats:italic> to human DNA and the most consistent results. Depletion of human DNA with saponin increased <jats:italic>N. gonorrhoeae</jats:italic> yields in simulated infections, but decreased yields in clinical samples. In ten urine samples from men with symptomatic urethral gonorrhoea, ≥92.8% coverage of an <jats:italic>N. gonorrhoeae</jats:italic> reference genome was achieved in all samples, with ≥93.8% coverage breath at ≥10-fold depth in 7 (70%) samples. In simulated infections if ≥10<jats:sup>4</jats:sup> CFU/ml of <jats:italic>N. gonorrhoeae</jats:italic> was present, sequencing of the large majority of the genome was frequently achieved. <jats:italic>N. gonorrhoeae</jats:italic> could also be detected from urine in cobas PCR Media tubes and from urethral swabs, and in the presence of simulated <jats:italic>Chlamydia</jats:italic> co-infection.</jats:p> <jats:p><jats:bold>Conclusion</jats:bold>. Using Nanopore sequencing of urine samples from men with urethral gonorrhoea sufficient data can be obtained to reconstruct whole genomes in the majority of samples without the need for culture.</jats:p>

Original publication

DOI

10.1128/jcm.01822-19

Type

Journal article

Journal

Journal of Clinical Microbiology

Publisher

American Society for Microbiology

Publication Date

18/12/2019