Challenges in establishing a gold

standard for the detection of

rifampicin resistance

Daniela M Cirillo, MD, PhD

San Raffaele Scientific Institute Milan

Background:rifamycins are key drugs for today TB treatment

• RIF is one of the most potent sterilizing drugs available for TB treatment.

• Rifabutin (RFB) is a semisynthetic derivative of rifamycin S and together with RIF is part of the rifamycin family.

• Both RIF and RFB share the mechanism of action (blocking the DNA dependent RNA polymerase subunit B (RpoB).

• RFB is recommended for TB treatment in HIV-co-infected patients under anti-retroviral therapy

Background: rifampicin resistance is a proxy for MDR

• Rifamycins resistance is due mainly to mutation in an “hot spot” region of the rpoB

gene

• For a relatively low frequency of mutations in rpoB Rif resistance is considered a proxy for MDR

• Detection of rifamycins resistance requires a prompt shift to an appropriate drug regimen (use of second line drugs)

Testing for rifamycins resistance:

Gold Standard

drug diluentConcentration(µg/mL)

MGIT 960 VersaTREK L-J 7H10 7H11

Rifampicin DMSO 1,0 1,0 40 1,0 1,0

Rifabutin Methanol 0,5 ─ ─ 0,5 0,5

Gold standard

• An accepted test that is assumed to be able to determine the true disease state of a patient regardless of positive or negative test findings or sensitivities or specificities of other diagnostic tests used.

• The diagnostic test that is considered the most accurate test for a particular condition.

• In medicine and statistics, gold standard test refers to a diagnostic test or benchmark that is the best available under reasonable conditions. It does not have to be necessarily the best possible test for the condition in absolute terms.

Impact of an imperfect gold

standard on a novel test

From Rebecca A. Betensky

Conference on Statistical Issues in Clinical Trials

rpoB hot spot is the ideal target for

molecular methods

Rapid molecular tests for rifampicin resistance quickly

identify TB Rif resistant cases and the use of these tests

is recommended in high risk MDR-TB population

Even in the best performances their specificity appear

suboptimal compared to liquid cultures

Molecular detection of

Mutations

First line treatmentRefer or start MDR

treatment; DST RH, second

lineIf the test is used in a

test is recommended

If the test is used in a

Low MDR prevalence

setting a confirmatory

test is recommended

R o S?

• Discrepancies between molecular tests

and MGIT Rif sensitivity tests are

reported.

• Uncertainty of results is causing delay

in starting appropriate treatment and

undermine the confidence of clinicians

in the results of molecular

susceptibility tests.

Discrepancies between molecular

tests and MGIT as a gold standard

• Absence of both wild type

and mutated probes

indicates resistance

• Use of MGIT as

confirmatory test leads to

discrepant results

rpoB no WT, no MUT= 103 / ≈2000 cases

D516Y

H526C

H526L

H526N

H526S

L511P

L533P

L533R

S512R

S531F

S512T + M515I + H526N

S531W

L511Q

H526D

D516Y

L533P

Q513P

H526C

H526N

M515I + H526N

del. 1547→ggaccagaa

Q513L + Y526*

Q513L

H526R

L511P + D516G

S512R + H526N

H526L

H526*

H526P

del. 1448→gac (D516)

L511P

L511R + D516Y

S531L

Q510V + D516Y

M515I + D516Y

del. gac 516 + gac/ggg (E541G)

M515V + H526N

M515T + H526S

S522L + S531A

Q513H + L533P

ins. 1577 acc 1578 + ttc/ttt (F506F)

N518D + L533P

L533P + H526S

D516L

H526S

Q513P + H526S

L530M + S531P

S522L

S R

S512R 100,0 0,0

Q513L 0,0 100,0

D516Y 100,0 0,0

H526* 0,0 100,0

H526C 50,0 50,0

H526N 100,0 0,0

H526S 80,0 20,0

L533P 100,0 0,0

S531L 0,0 100,0

S531F 0,0 100,0

in/del, multiple 0,0 100,0

mutMGIT (%)

In/del and multiple mutations � MIC ≥ 20 µg/mL

MIC evaluation on 7H10

WT

MUT

WT MUT

WT

MUT

D516VS531L

H526Y

SNPs in the rpoB gene and RIF-R: high confidence mutations

Adapted from Campbell EA et al. Cell 2001; 104:901-912

Structure impairment is observed in mutations

MGIT sensitive

MUT MGIT Delta-day 7H10 mic rpoAC

H526S S 7,3 ± 6,7 I ≤1,0 WT

H526S S 7,0 R 4 WT

H526S S 3,0 ± 2,1 R ≥20,0 rpoCG594E

H526S R - R ≥20,0 WT

H526S S 7,5 ± 5,7 R 1,0 WT

WT

MUT

SNPs in the rpoB gene and phenotypic DST

Adapted from Campbell EA et al. Cell 2001; 104:901-912

Probes position in the “Hot Spot” of rpoB and

mutations non detected by MGIT

507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533

511 Pro°

522 Glu/Gln 526 Asn°/Leu°531 Trp

533 Pro 572 Phe

511 Gln

531 Phe/Tyr

533 Arg/Pro

526 Cys/Asn/Ser

512 Arg

516 Tyr°

Miotto et al submitted

Rigouts t al. JCM 2013

°Williamson IJTLD 2011

*YuanJCM2012

972Thr*

Discrepancies between molecular

tests and MGIT as a gold standard

• Approximately 10% discordant results on selected

cases with MGIT test using a breakpoint of

1µg/ml (considered the “gold standard”)

• Previously reported as “false positives

(negatives)” by molecular tests

• Absolute number of reports are increasing with

the roll out of Xpert and LPA

• “False positive” in culture are true positive by

sequencing

With low prevalence, maintaining high specificity is more

important than high sensitivity.

From Rebecca A. Betensky

Conference on Statistical Issues in Clinical Trials

‘‘Borderline’’

resistance to RIF has been strongly

associated with treatment

failure

Non canonical rpoB mutations were

identified in >10% of cases

The presence of unconventional

mutations correlated with a poor

outcome

Borderline mic

Applying a 0.0625 µg/ml susceptibility breakpoint to the isolates

of the present study would change susceptibility status of at least

7 of 16 discrepant cases to ‘‘resistant’’ and so increase the

specificity of molecular tests for RIF susceptibility from 89.5% to

94.1%.

16 isolates (10.5%) tested RIF-sens by phenotypic assay. Five

strains with discordant genotypic and phenotypic

susceptibility results had RIF minimal inhibitory

concentration (MIC) close to the cut-off value of 1 µg/ml

used in phenotypic susceptibility assays and were confirmed

as resistant by DST on solid media.

rpoB mutations and their correlation to (A)

RIF and (B) RFB resistance levels in MGIT

Jamieson FB, J. Clin. Microbiol. doi:10.1128/JCM.00691-14

“true” false-positives in molecular

tests

Silent mutations in rpoB

Silent mutations can be detected by molecular assays but by

definition do not modify the aa and the protein structure, NOT

relevant for DR. SM may cause false positivity in molecular tests

Silent

mutation

Country Reference Frequency LPA/Xpert

P535 Italy TBPANNET

database<1:100 -

L511

Q513

Korea Kim BJ JCM1997 nd No WT2/PA

No WT3/PB

T524 China Yuan JCM 2012 2.5% (77) No WT6/PD

F514 US

Spain

Kapur V JCM 1994

Alonso M JCM

2011

Moure JCM 2011

0.8% (1450) No WT3/PB

A532

L533

India JCM 2001 4%( 50) No WT8/PE

Q510 New Zeeland Williamson DMID

2011No WT1-2/PA

T508 Haiti Ocheretina PLoS

ONE 9(3): e90569. 1.3%(150) No WT1/PA

Additional considerations

Reasons for success –

compensatory evolution

The study supports a role for rpoC mutations in the transmission of multidrug-resistant

tuberculosis and illustrates how epistatic interactions between drug resistance-conferring

mutations, compensatory mutations, and different strain genetic backgrounds might

influence compensatory evolution in drug-resistant M. tuberculosis.

Conclusions

• Not every genotypic modification of rpoB gene affects phenotypic resistance to RIF equally

• The value of the RIF MIC correlates with the position and nature of the amino-acid substitution in rpoB RRDR

• Correlation between MGIT resistance and mutations is high for rpoB codon 531 and for 526D

• Resistance associated to other mutations at codon 526 are not detected by MGIT at CC of 1 μg/ml but is detected by proportion methods on LJ or 7H11

• ‘‘Borderline’’ or resistance to RIF associated to unconventional mutations has been strongly associated with treatment failure

• MGIT test for RIF breakpoint may needs to be revaluated

Conclusions: Which Gold Standard for Rif?

• Presence of mutations in rpoB hot spot show the best correlation with the clinical outcome

• Discrepancies between results obtained by MGIT and molecular tests should be sorted out by sequencing considering sequencing as the GOLDstandard for a molecular test

• It is possible that not only the presence of the

mutation but the type of mutation will be

relevant for patient’s management and clinicians

will need to be educated in this sense

Riccardo Alagna

Emanuele Borroni

Andrea M. Cabibbe

Lucinda Furci

Paola Mantegani

Paolo Miotto

Luca Norbis

Elisa Schena

Enrico Tortoli

Elisa Tagliani

Ilaria Valente

Emerging Bacterial Pathogens Unit

San Raffaele Scientific Institute

Milano, ITALY

AcknowledgmentsAcknowledgments