Background
Challenges in the management of infectious diseases include treatment failure due to antimicrobial resistance (AMR) and lack of a reliable test-of-cure (TOC). Whilst culture is a trusted method it is slow and insensitive, thus diagnosis is through nucleic acid amplification tests (NAATs). NAATSs can also assess AMR by detecting mutations associated with resistance. However, this approach requires knowledge of the molecular mechanism(s), and as these evolve, tests require reconfiguration. Use of NAATs for TOC is problematic since residual nucleic acids have been reported following effective therapy. InSignia is a new approach, enabling simultaneous detection of the pathogen, assertation of viability and antibiotic susceptibility. The technology combines genotypic and phenotypic determinations within a single culture-independent test, filling the gap of current methods.
Methods
InSignia requires a 10-min exposure to antibiotic prior to amplification of responsive transcripts by Reverse Transcriptase-qPCR using extracted Total Nucleic Acid. Results, measured as a VITA score, reflect the level of transcription, elucidating whether antibiotics induce changes compared to untreated controls. Analysis of untreated controls alone allows assertation of pathogen viability at the time of sample collection.
Results
InSignia allowed pan-antibiotic susceptibility profiling in a panel of isolates, in response to commonly used antibiotics. Susceptibility corresponded to greater transcriptional differences compared to non-treated controls, while resistance was characterized by minimal change. Concordance of VITA scores and breakpoints elucidated a correlation between antibiotic susceptibility and transcriptional response to antibiotic exposure. For viability assessment, InSignia displayed similar pathogen detection sensitivity to a commercial NAAT yet provided greater information regarding organism viability compared with culture.
Conclusion
An informative molecular test is needed in this fast-evolving AMR landscape. InSignia displays application for infection diagnosis, antibiotic susceptibility and confirmation of successful treatment. It is suitable for rapid point of care devices, allowing timely targeted treatment for symptomatic and asymptomatic patients alike. The pan-antibiotic response provides a powerful and universal approach which can be employed to safeguard the current arsenal of effective antibiotics.