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Veracode Challenge Winner
Jimmy Twin
Royal Women’s Hospital
Melbourne, Australia
Development of a rapid diagnostic method for infectious urethritis that will identify the causative microorganisms and predict antibiotic resistance enabling a more effective treatment strategy.
Application Abstract
Infectious urethritis is an inflammation of the urethra caused by a variety of microorganisms. It is a common condition that causes significant morbidity to both men and women and can lead to serious complications such as chronic persistent prostatitis and sterility for men and cervicitis, pelvic inflammatory disease and complications during pregnancy for women. An assay is to be developed using the Illumina VeraCode technology to simultaneously detect the aetiological agents for infectious urethritis and predict whether any potential macrolide resistance is involved. The targets in this proof of concept project will be Neisseria gonorrhoeae, LGV and non-LGV serovars of Chlamydia trachomatis, and Mycoplasma genitalium. The treatment for infectious urethritis is dependent upon the causative agent and whether macrolide antibiotic resistant occurs. A rapid assay such as the one in this study will enable a more direct and effect treatment strategy and promote the screening for emerging pathogens such as Mycoplasma genitalium in cases of nongonococcal and nonchlamydial urethritis.
Biological Question
Infectious urethritis is an inflammation of the urethra caused primarily by the bacterium Neisseria gonorrhoeae. In cases of non-gonococcal urethritis (NGU), the causative organism is predominantly the bacterium Chlamydia trachomatis, but can also be caused by the bacteria Mycoplasma genitalium, Ureaplasma urealyticum, Haemophilus vaginalis, and Trichomonas vaginalis. In more rare cases, the causative agent can be viral such as Herpes simplex virus or Adenovirus, or by a parasite such as Trichomonas vaginalis. Depending on the causative organism and severity of infection, the symptoms and severity of urethritis can vary, and can often be asymptomatic leading to the spread of infection primarily through sexual contact. Prolonged or persistent infection can lead to conditions such as chronic persistent prostatitis and infertility for men and pelvic inflammatory disease and complications during pregnancy for women. Infection with different strains of a particular pathogen can lead to different disease and treatment outcomes, such as with C. trachomatis, where some serovars are associated with a serious condition known as
lymphogranuloma venereum (LGV) where the infection crosses to the lymphatic system requiring a more comprehensive antibiotic treatment. Recent findings suggest the current macrolide-based antibiotic treatments for NGU are not completely effective, particularly in cases where the emerging pathogen M. genitalium is detected. The ability to predict the most effective treatment regime for infectious urethritis will reduce the morbidity for those infected and cost for the community in treatment, and so also decrease the incidence of serious progression of disease.
Experimental Outline
For the initial 6 month proof of concept project, existing described gene targets will be utilised for the VeraCode assay to simultaneously identify the bacteria Neisseria gonorrhoeae, Chlamydia trachomatis, and Mycoplasma genitalium from clinical samples. In addition, work in our laboratory has identified genomic regions differentiating C. trachomatis LGV and non-LGV serovars that will also be incorporated in this assay. To complement this, single-base pair mutations of the 23S rRNA gene known to confer macrolide resistance will also be included. For the initial validation of the VeraCode assay and to determine its sensitivity, mock samples will be created with known concentrations of the targeted genomic regions in question. To assess the robustness of this assay with real samples, a panel will be screened consisting of known N. gonorrhoea positive samples, C. trachomatis positive samples from LGV and non-LGV serovars, and M. genitalium cases where the initial macrolide antibiotic treatment was successful and a selection of treatment failures. The Women's Centre for Infectious Diseases carries out a diagnostic service as well as research, and so we are fortunate to have access to large number of clinical samples. DNA from each of these samples has been extracted using the MagNA Pure LC DNA Isolation Kit I with the MagNA Pure LC DNA extraction system (Roche Diagnostics, Mannheim, Germany) and are currently archived at -30°C. For each sample, qPCR will be used to identify the bacterial load for each of the pathogens to be tested, and work is currently underway sequencing a selection of M. genitalium positive treatment failure cases to identify the macrolide resistant mutation involved.
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