1. What Gold Standard for Rifampicin Testing?: the future of molecular testing TAG Meeting, Manila, 9-12 December 2014 Richard Lumb Mycobacterium Reference Laboratory, SA Pathology Adelaide, South Australia

2. Plan of Presentation Introduction Molecular concepts regarding rifampicin and resistance Does DST methodology matter? Frequency of discrepant results –false-resistance –false-susceptibility Does low-level rifampicin resistance matter? Extraordinary claims require extraordinary evidence Resistance and other gene mutations Implications for Programs Concluding comments

3. Introduction Amplification of drug resistance is caused by human activity Mycobacteria genes have a low (≈10 -6 -10 -8 ) level of spontaneous mutation –Any mycobacterial population will have a small number of organisms naturally drug resistant and usually with resistance to a single drug –Inadequate anti-TB regimen provides selection pressure for drug resistant strains to amplify and become the dominant strain MDR/XDR-TB is the step-wise accumulation of mutations; is not achieved in a single mutation

4. Molecular Concepts of Rifampicin Resistance Rifampicin resistance arises due to mutation(s) in the gene (rpoB) encoding the β-subunit of RNA polymerase –Rifampicin physically blocks RNA synthesis –Mutations reduce affinity for the rifampicin binding site ≈ 97% of mutations associated with rifampicin resistance occur within an 81bp ‘hotspot’ region –Also known as rifampicin-resistant determining region or RRDR Codons 507-533

5. Herrera et al. Int J Antimicrob Agents 2003; 21: 403-8 ‘Hot Spot’ Region of rpoB gene

6. Molecular Concepts of Rifampicin Resistance Phenotypic testing for rifampicin considered to be very reliable; until now…

7. Does DST Methodology Matter? Research revealed a subset of isolates with highly discordant RIF results Van Deun et al J. Clin. Microbiol. 2009; 47:3501-3506

9. Does DST Methodology Matter? Rigouts et al J. Clin. Microbiol. 2013; 51:2641-2645

10. Does DST Methodology Matter? Phenotypic testing misidentifies isolates with low-level RIF resistance as susceptible Xpert MTB/RIF and Line Probe Assays both detect mutations associated with RIF-resistance Associated with specific mutations –511Pro, 516Tyr, 526Asn, 533Pro, 572Phe & likely other rare mutations Liquid culture much more likely to miss such mutations than solid media DST False-susceptibility in liquid culture [Rigouts et al JCM 2013;51:2641-2645] [Van Deun et al JCM 2009;47:3501-3506] –

11. Herrera et al. Int J Antimicrob Agents 2003; 21: 403-8

12. Frequency of Discrepant Results How frequently does low-level RIF-resistance occur? Van Deun et al J. Clin. Microbiol. 2013; 51:2633-2640

13. Frequency of Discrepant Results New data shows that the proportion of RIF-resistant isolates that display low-level resistance is higher than previously thought Retreatment cases with low-level RIF-resistance –Bangladesh: 23/175 (13.1%) –Kinshasa 25/254 (10.6%) [Van Deun et al JCM 2013;51:2633-2640] New & retreatment cases –(89 RIF-resistant isolates ) Hong Kong (21%) –Counted only 511Pro, 526Leu, 533Pro [Yip et al IJTLD 2006;10:625-630]

14. Frequency of Discrepant Results Molecular False-Resistance Silent mutations –Mutation results in a base change but no change to the amino acid encoded and no change to protein structure –Change will not be detected phenotypically –Are detected by molecular testing Frequency of silent mutations varies –<0.5% by Van Deun et al 2013 –<1% in TBPANNET (Italy) Cirillo (GLI-2014)

15. Frequency of Discrepant Results Molecular False-Susceptibility Mutations occurring within the rpoB gene but outside the ‘hot spot’ –‘Hot spot’ covers codons 507-533 –Rare mutations occurring outside ‘hot spot’ 535Ser and 536Ser (<1%) 572Phe (≈2%) Likely to be others

16. Does low-level RIF-resistance matter? [Williamson et al IJTLD 2011;16:216-220] 3/3 patients with low level RIF-resistance failed Cat-1

17. Does low-level RIF-resistance matter? Pang et al IJTLD 2014;18:357-362

18. Does low-level RIF-resistance matter? 30 patients with rifampicin resistant result by molecular but phenotypic susceptibility – 9 patients treated with Cat-I 5 unfavourable outcomes – 21 patients treated with second-line regimen Only 3 had unfavourable outcomes 19 patients with rifampicin susceptible result by molecular but phenotypic resistance –13 patients treated with Cat-I 6 unfavourable outcomes –6 patients treated with second-line regimen All 6 had a favourable outcome Pang et al IJTLD 2014;18:357-362

19. Does low-level RIF-resistance matter? Globally, an additional 37,000+ MDR- TB/year go unrecognised! Williamson et al IJTLD 2011;16:216-220

20. NASA scientist 'finds alien fossils on meteorite' [1996] “ Extraordinary claims require extraordinary evidence” Carl Sagan (Cosmologist)

21. Extraordinary Claims Require Extraordinary Evidence Multiple examples where a reported ‘MDR-TB’ (or worse) actually MTB plus environmental mycobacteria or MTB plus non-mycobacteria contaminant… High income/low TB prevalent countries and with high quality laboratories –E.g-1: Thought to have XDR but only had MDR-TB… –E.g-2: Diagnosed with XDR-TB but was subsequently found to be INH-resistant MTUB plus MAC… Cryptic environmental mycobacteria a higher risk in liquid culture DST than in solid media DST MDR/XDR-TB result has potentially life-changing implications for the patient, family and community

22. False MDR-TB caused by bacterial contamination

23. Resistance and Other Gene Mutations Mutations associated with resistance incompletely understood –Mutations in pncA have strong association with pyrazinamide resistance Sensitivity 80-90% of molecular with phenotypic –InhA and katG mutations associated with 70-90% of INH resistance but varies between- and within- countries –EmbB codon 306 mutations found in 30-68% of ethambutol-resistant strains –Some mutations associated with resistance to second-line anti-TB drugs recognised but incomplete

24. Implications for Programs Molecular testing beyond Xpert and LPA is the future –More mutations associated with drug resistance are being defined –Countries need to be planning/developing extended molecular capacity for TB diagnostics –Whole Genome Sequencing (WGS) a reality and being used now as a diagnostic tool for TB Price/test falling very fast Capacity of new machines increasing quickly Bioinformatics presents a bigger challenge Multiplatform capacity –Rapid/reliable specimen/isolate shipment strategy

25. Concluding Remarks - 1 Mutations within rpoB exert variable effects upon results obtained by phenotypic DST The effect of a mutation within rpoB upon the rifampicin MIC depends upon –the amino acid change induced by the mutation –location of the mutation in the rpoB gene Low-level (borderline) resistance to rifampicin is strongly associated with treatment failure when a standard first-line treatment regimen in used Detection of mutations in the rpoB gene, and especially within the ‘hot spot’, correlate better with treatment outcome

26. Concluding Remarks - 2 Sequencing the entire rpoB gene may give the best correlation with treatment outcome Countries will require molecular capacity that goes beyond Xpert and Line Probe Assays –Technology, training, and gaining ‘hands-on’ experience must be part of National Strategic Plans Must be included in funding cycles –Will change country requirements for DST laboratories –For fast turnaround, rapid, safe, and reliable specimen/isolate shipment strategies to higher-level laboratories is vital

27. Is the sun setting on phenotypic DST testing…?