Molecular susceptibility testing

Transcription

1 Molecular susceptibility testing Dr Andrew Ginn Supervising Scientist Antimicrobial Resistance Reference Laboratory ICPMR, Westmead Hospital

2 Resistance genes Gram negatives Transmissible; e.g. ESBLs, MBLs, carbapenemases Chromosomal; e.g. colistin (until recently), quinolones Gram positives meca in MRSA vana/b in VRE

3 Antifungal resistance Echinocandin resistance Mutation hotspots Fluconazole resistance SNPs Require sequencing No PCR offerings

4 Molecular methods Polymerase chain reaction (PCR) Microarray PCR + electrospray ionisation (ESI) MALDI-TOF (DNA based) Sequence based Pyrosequencing Whole genome sequencing

5 In-house vs Commercial In-house Requires extensive validation Requires high level bioinformatics skills Tailored to epidemiology Low-cost

6 In-house vs Commercial Commercial Requires minimal validation Many seeking FDA clearance No bioinformatics skills required Higher cost Some require specialist infrastructure

7 Whole genome sequencing Gives entire picture Requires expert bioinformatics knowledge Analysis of large multi-resistance plasmids Association of phenotypic variants High cost (> 10X $ of PCR)

8 β-lactams ESBLs carbapenemases plasmid AmpCs SHV > 180 OXA > 400 TEM > 200 CTX-M > 150 IMP, VIM, NDM, KPC, CMY, DHA, VEB, GES, bla BLAH BLAH

9 Aminoglycosides Partridge et al. (2009) FEMS Micro Rev

10 Aminoglycosides Partridge et al. (2009) FEMS Micro Rev

11 Aminoglycosides Partridge et al. (2009) FEMS Micro Rev 33:

12 Choose the targets Third-generation cephalosporins (3GC) ESBL ampc MBL Aminoglycosides Modifying enzymes 16S methylases

13 Local epidemiology is essential

14 E. coli MICs (µg/ml) Cefotaxime Ceftazidime WT E. coli CTX-M-3 > CTX-M-15 (CTX-M-3 D240G) > CTX-M-1 >128 6 CTX-M-32 (CTX-M-1 D240G) >128 >256 CTX-M CTX-M-16 (CTX-M-9 D240G) 16 8 Bonnet (2004) Antimicrob. Agents Chemother. 48:1 Cartelle et al. (2004) Antimicrob. Agents Chemother. 48:2308

15 Commercial PCR Manufacturer Targets Carba-R assay Cepheid CPE Check-Direct assays Verigene Gram-negative blood culture nucleic acid test Checkpoints Nanosphere CPE, KPC, ESBL, MDR meca, vana/b, KPC FilmArray (BioFire) biomerieux meca, vana/b, KPC MT-PCR asays Ausdiagnostics CPE, ESBL, MRSA, VRE Sepsis Flow Chip Master Diagnostica ESBL, CPE, MRSA, VRE

16 Commercial PCR Targeting often US or Europe driven Focus on KPC (US/Italy CPMase) Focus on OXA-48 (UK/Europe CPMase) Primer designs may miss locally important variants Cepheid -> IMP-4

17 Principle of test design Know locally important targets Develop bioinformatics database Design primers Optimise assay Perform validation

18 Principle of test design Know locally important targets Develop bioinformatics database Design primers Optimise assay Perform validation

19 Practicality of molecular testing Commercial assays Remove need for bioinformatics Increase cost/assay Relatively easy to implement Targeting may not be appropriate

20 Practicality of molecular testing In-house assays Significant cost saving Tailor to suit local epidemiology Requires bioinformatics investment

21 Practicality of molecular testing Reality = combination of in-house and commercial Most major centres offer PCR for R genes Westmead MRGNR In-house PCR bla and 16S RMTase MRSA BD Max Staph SR

22 Practicality of molecular testing Reality = combination of in-house and commercial Most major centres offer PCR for R genes Predictive power of targets known for some species and agents MRSA, VRE, E. coli, K. pneumoniae

23 Phenotype inference meca = MRSA Not all MRSA have typical Class A meca cassette Potential for assay failure needs to be recognised and monitored vana/b = VRE E. coli + bla (or Gm/Amk R gene) = resistant K. pneumoniae + bla (or Gm/Amk R gene) = resistant

24 Local epidemiology is essential

25 3GC Resistant E. coli (2000) EARS-Net

26 3GC Resistant E. coli (2006) EARS-Net

27 3GC Resistant E. coli (2012) EARS-Net

28 Distribution of bla CTX-M Hawkey and Jones (2009) J Antimicrob Chemother

29 Success stories Global bla CTX-M bla NDM

30 Success stories Global bla CTX-M bla NDM Local (Sydney) bla CTX-M bla IMP

31 Success stories Global bla CTX-M bla NDM Local (Sydney) bla CTX-M bla IMP Epidemiology important

32 Co-selection bla CTX-M-15 -> bla OXA-30 + aac(6')-ib-cr, aac(3)-ii, bla TEM bla IMP-4 -> aaca4, aac(3)-ii, bla TEM, qnrb2 Predictable patterns in local settings Espedido (2008) Antimicrob. Agents Chemother. Zong (2008) Antimicrob. Agents Chemother.

33 bla NDM-1 Partridge SR, Iredell JR (2012) Antimicrob Agents Chemother 56: bla IMP-4 Partridge SR et al. (2012) Antimicrob Agents Chemother 56: G

34 horizontal/lateral gene transfer plasmid chromosome R

35 horizontal/lateral gene transfer plasmid chromosome R R

36 horizontal/lateral gene transfer plasmid chromosome R R R S

37 Phenotypes Acquired genes simple to target Sequencing required to detect Chromosomal mutation (SNPs) Colistin LPS Quinolones gyra, parc

38 Colistin Previously chromosomal Modification of LPS Typically arnbcadtef, pmrab, mgrb, lpxm and phopq* *Olaitan (2014) Front Microbiol.

39 Colistin Now transmissible mcr genes modify LPS Captured from Moraxella (mcr-1; other species identified as origin for other variants) Identified in Sydney/NSW Ellem et al 2017 Emerg Inf Dis Have not become endemic Detected in bacteria isolated in 1980s

40 Phenotypes Acquired genes simple to target Sequencing required to detect Chromosomal mutation Porins/pumps

41 E. coli Acquisition of genetic material is most important K. pneumoniae P. aeruginosa Acinetobacter spp. Ability to manage the cell membrane is most important Adapted from Iredell (2016) BMC

42 E. coli Acquisition of genetic material is most important K. pneumoniae P. aeruginosa Acinetobacter spp. Ability to manage the cell membrane is most important Adapted from Iredell (2016) BMC

43 Other molecular methods MALDI-TOF (DNA) Agena Bioscience Multiplex-PCR coupled with MALDI detection Highly specific, SNP-based detection High infrastructure cost PCR-ESI Abbott IRIDICA Now defunct

44 Conclusions Advantages Rapid TAT High-throughput Multiple targets and samples High specificity Low cost of consumables Certain species direct association to phenotype Many commercial options

45 Conclusions Disadvantages Can only detect what you target No novel gene detection Need to monitor epidemiology regularly Infrastructure can be expensive Genotype doesn t always equal phenotype Not all commercial assays are useful/appropriate