Rapid Molecular Diagnostics of Antimicrobial Resistance

1. Rapid Molecular Diagnostics of Antimicrobial Resistance Dr. Vicky Enne Centre for Clinical Microbiology Co-ordinator FP7 RiD-RTI PI Prof. Ali Zumla Co-I Dr.Vanya Gant

2. Talk Outline • Resistance problem & need for rapid diagnostics • Complexity of resistance • Potential benefit of rapid molecular diagnostics • Potential problems of rapid molecular diagnostics • Type of technologies • Examples of point of care systems on/near market • Technology in development at UCL – RiD-RTI • Knowledge gaps & future need

3. Penicillin-resistant pneumococci vs. outpatient beta-lactam sales (From: Livermore. 2005. Lancet Infectious Diseases 5:450-459) The Problem of Antibiotic Resistance • Antibiotics used heavily in human & veterinary medicine • Use undoubtedly selects resistance • Multi-resistant organisms e.g. MRSA,VRE & C. difficile cause serious problems in hospital setting • Biggest current & future threat thought to come from Gram-negatives

4. Complexity of Resistance Genetics in Gram-ve rods • Enormous complexity complicates both treatment and diagnosis • Resistance generally transmitted on mobile genetic elements – plasmids & transposons • Infection control needs to prevent not only spread of individual organisms but also genetic elements • Several different mechanisms for one resistance phenotype • e.g. at least 4 known for fluoroquinolones, can all be present at one • One bacterium / plasmid can carry a large number of different resistance genes – >10 commonplace

5. Many variants of a single gene, sometimes with different phenotypes. (From: Cornagliaet al. Lancet Infect Dis 2011) There are approx. 30 distinct acquired beta-lactamase families, among these there can be up to 370 variants of a single type e.g. TEM, OXA (From: Canton et al. CMI 2012)

6. The Need for Rapid Diagnostics • Conventional methods > 48h to identify antibiotic susceptibility • Reference laboratories often required to identify specific resistance gene – slows down outbreak tracing • Importance recognised in recent reports & policy documents (CMO Report, UK Strategy etc.)

7. Potential Benefits of Rapid Diagnostics • Improved treatment outcome for patient • Improved infection control and outbreak monitoring achieved by acquiring more precise information sooner • Reduction in empirical antimicrobial prescriptions • Preservation of broad spectrum antimicrobials • Reduction in duration of treatment • Reduction in cost of treatment • Overall reduction antimicrobial consumption • Potential reduction in levels of resistance

8. 8 years 3 years 2 years 8 years 7 months Does reduced prescribing reduce resistance? From: Enne 2010 JAC 65: 179-182

9. Problems Associated with Molecular Diagnostics of Resistance • Discrepancy between genotype and phenotype • The potential presence of unknown resistance mechanisms leading to treatment failure • Genotypic markers of resistance not resulting in clinically significant resistance. Patient may be denied therapeutic drug needlessly. • Rapid molecular methods should be supplemented with culture-based techniques

10. Sources of Pitfalls • Resistance gene not detected by test • PCR based technologies may not be able to include all possibilities – constantly changing epidemiology • New genes/mechanisms of resistance • Resistance genes not conferring resistance • Poorly expressed genes • Non-functional mutated genes • Silent genes • Difficulty in predicting exact resistance phenotype from genotype e.g. interplay of multiple simultaneous mechanisms e.g. low level efflux + mutations

11. Technologies for Rapid Diagnostics • Point of care – near-patient systems requiring minimal user expertise • Kit-based – provides reagents but skilled operator & specialist equipment still required • PCR Based • Real-time/qPCR • Droplet PCR • Microarray – usually for 2ndary detection only • MALDI-TOF MS + PCR • Whole genome/next generation sequencing • Transcriptomics

12. Point of Care Products on/near Market • Cepheid GeneXpert – Known for TB diagnostics, sample in answer out • CuretisUnyvero – sample in/answer out, pneumonia cartridge • NanosphereVerigene – sample in/answer out blood culture • Biofire (Biomerieux) FilmArray – blood culture • EpistemGenedrive – small, low cost. TB diagnostics.

13. Cepheid GeneXpert • 1st point of care system introduced to market • MTB/RIF test, WHO endorsed • Good evaluation data • Integrated sample prep & PCR; cartridge based, < 1 hour • Low number of targets per test • Strong focus on colonisation/infection control • SA/MRSA testing from swabs or positive blood cultures • Carbapenemase assay from rectal swabs in pipeline (KPC, VIM, NDM)

14. CuretisUnyVero Point of Care PCR based instrument. Test time < 4h Separate lysis unit First cartridge for pneumonia Comprehensive detection of bacterial pathogens , including those implicated in HAP & atypical infections Diverse list of resistance genes – mecA, macrolides, ESBLs, KPC, OXA-51, integron markers, FQR mutations Validation data not yet available

15. NanosphereVerigene • Point of Care, PCR- based instrument • Works on positive blood cultures, < 2.5 h • Gram positive test • mecA, vanA, vanB • FDA cleared, generally good evaluation results but mis-ID of streptococci • Gram-negative test • KPC, NDM, IMP, VIM, CTX-M, OXA • No evaluation available yet

16. In development at UCL – RiD-RTI • EU FP7 funded project to develop rapid diagnostics for respiratory tract infections • Low cost, point of care device • Integrated sample prep, cartridge based • PCR & microarray detection • Answer in < 2 hours • Ability to scale up

17. Unique partnership of SMEs, Universities and Hospitals Sample Preparation & Real Time PCR assays Microarray chip design Clinical evaluation using patient samples Instrument & Software design

18. 3 RTI Diagnostic products • Community acquired pneumonia • Bacteria vs. virus • Eliminates unnecessary empirical prescriptions • Resistance markers in phase 2 product • Hospital acquired pneumonia • S. aureus& enteric pathogens & non-fermenters • Comprehensive ESBL and carbapenamase • Opportunistic respiratory tract infections • Fungi, bacteria and virus

19. What is Needed in the Future ? • Still in early stages of commercialisation – many tests not comprehensively validated yet • Many systems require regulatory approval - CE-IVD (Europe) & FDA (US) • Clinical evaluation of rapid diagnostics to guide treatment – will potential benefits be realised? • More comprehensive tests & scaleable devices • More focus on global problems, current devices heavily US based • More tests simultaneously • Ability of technology to adapt rapidly to evolving pathogens & emerging resistances

20. Acknowledgements – RiD-RTI Consortium NUIG, Ireland Tom Barry Nina Tuite Kate Reddington Mobidiag, Finland JuhaKirveskari Genewave, France Francis Domain Yann Marcy Claude Weisbuch www.rid-rti.eu • UCL Ali Zumla (PI) LjubanGrgic Gareth Platt Stefan Schwenk YoannPersonne Mary Lenahan • UCLH Vanya Gant