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What Gold Standard for
Rifampicin Testing?:
the future of molecular testing
Richard Lumb
Mycobacterium Reference Laboratory, SA Pathology
Adelaide, South Australia
TAG Meeting, Manila, 9-12 December 2014
Plan of Presentation
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Introduction
Molecular concepts regarding rifampicin and resistance
Does DST methodology matter?
Frequency of discrepant results
– false-resistance
– false-susceptibility
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Does low-level rifampicin resistance matter?
Extraordinary claims require extraordinary evidence
Resistance and other gene mutations
Implications for Programs
Concluding comments
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
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
‘Hot Spot’ Region of rpoB gene
Herrera et al. Int J Antimicrob Agents 2003; 21: 403-8
Molecular Concepts of Rifampicin Resistance
• Phenotypic testing for rifampicin
considered to be very reliable; until now…
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
Van Deun et al J. Clin. Microbiol. 2009; 47:3501-3506
Does DST Methodology Matter?
Rigouts et al J. Clin. Microbiol. 2013; 51:2641-2645
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]
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Herrera et al. Int J Antimicrob Agents 2003; 21: 403-8
Frequency of Discrepant Results
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How frequently does low-level RIF-resistance occur?
Van Deun et al J. Clin. Microbiol. 2013; 51:2633-2640
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]
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)
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
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
Does low-level RIF-resistance matter?
Pang et al IJTLD 2014;18:357-362
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
Does low-level RIF-resistance matter?
• Globally, an additional 37,000+ MDRTB/year go unrecognised!
Williamson et al IJTLD 2011;16:216-220
NASA scientist 'finds alien fossils on meteorite' [1996]
“Extraordinary claims require
extraordinary evidence”
Carl Sagan (Cosmologist)
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
False MDR-TB caused by bacterial contamination
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 withincountries
– 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
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
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Price/test falling very fast
Capacity of new machines increasing quickly
Bioinformatics presents a bigger challenge
Multiplatform capacity
– Rapid/reliable specimen/isolate shipment strategy
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
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
Is the sun setting on phenotypic DST testing…?