Project Lead(s): Jo-Anne Dillon
Antibiotic resistance is one of the most serious public health threats worldwide, threatening to reverse a multitude of advances in treating bacterial infections and also dangerously reducing therapeutic options.
Neisseria gonorrhoeae (Ng), a sexually transmitted pathogen that was estimated to cause 106 million new gonorrhea infections annually during the 2000s, has the potential to become untreatable due to antibiotic resistance.
In low- and middle-income settings, the antibiotic susceptibility of Ng is monitored through time-consuming methods, requiring isolation and culture of the pathogen followed by culture-based antimicrobial susceptibility testing. Such tests are often not completed because human and physical resources are not available.
In an effort to overcome the problem of antimicrobial resistance, and using N. gonorrhoeae as a model organism, the project team developed a prototype rapid point-of-care test (POCT) that will facilitate treatment of patients, while minimizing the risk of antibiotic resistance.
The approach used involved identification of unique molecular targets that can be used for the determination of antimicrobial susceptibility, coupled with a diagnosis of the pathogen that causes gonorrhea. These targets were then used in different test formats.
The team developed a unique test using the technology of Aquila Diagnostics, a portable platform that delivers low-cost, high-speed nucleic acid-based tests. This test comprises a plastic disposable test device and a portable detection instrument that can detect multiple genetic traits simultaneously (for pathogen identity and antibiotic resistance).
Each test device compartment performs a separate reaction to amplify DNA from raw, unprocessed clinical samples (such as urine) using the polymerase chain reaction (PCR).
The Aquila test system allows multiple tests on a single sample (with results in about an hour) and potential application at the point of care.
In Phase 1 of the project, the team’s goals were to:
1) Design specific primers for PCR for identification of Ng isolates
2) Identify DNA sequences and design primers for PCR, to diagnose Ng ciprofloxacin susceptibility and resistance
3) Design POCT device primer sets specific for both Ng diagnosis and ciprofloxacin susceptibility
4) Standardize PCR conditions and evaluate the sensitivity and specificity of the test
5) Evaluate the prototype test for sensitivity and specificity by comparison with ‘gold standard’ culture, identification and in vitro antimicrobial susceptibility testing.
Numerous primers were designed to specifically identify N. gonorrhoeae, and their specificity was evaluated in silico using public genomic databases.
The sensitivity of the primers for detection of Ng was validated on a test panel comprising the DNA from 300 gonococcal strains from North America, Latin America, China and the World Health Organization (WHO) reference strains, using real-time PCR. The selected primers were 100% sensitive and specific using this panel of strains.
The team also developed a method for the simultaneous detection of Ng identification and the determination of its ciprofloxacin susceptibility status.
The knowledge gathered through these investigations was used to advance two additional projects: a Natural Sciences and Engineering Research Council of Canada (NSERC) Engage project and a Saskatchewan Health Research Foundation (SHRF) project.
Knowledge about the projects was also disseminated through conference presentations and a manuscript describing the project is under preparation.