The current state of knowledge:

genotypic vs phenotypic drug-susceptibility testing (DST)

Daniela M Cirillo

Emerging Bacterial Pathogens Unit,

San Raffaele Scientific Institute Milan

Outline

• Concordance between molecular based and phenotypic DST

• Rifampicin

• Fluoroquinolones

• Isoniazide

• Pyrazinamide

• SLDs

• Second line LPA preliminary data

• Conclusions

Banu S et al, JCM 2014

82% 77% 50% 51%

Complete concordance among tests:

Concordance among different tests

The use of multiple strategies to test for antibiotics susceptibilities has shown that: • Different phenotypic tests may give discordant results and discordance is drug

dependent • Strains with an mic close to the breakpoint are the most affected • Molecular tests and phenotypic tests may give discordant results and “gold

standard” is drug dependent • Different molecular tests may give discrepant results due to targets included or

not included in the tests. Their gold standard remains sequencing

Genotypic and phenotypic methods provide different pictures

Phenotypic tests :”in vitro” growth in the presence of the drug

• Drug activation/concentration

• Testing media

• Inoculum-related effects

• Reading time

Genotypic tests: identification of mutations associated to impairment of the mechanism of action

• Presence of a mixed population

• Lack of knowledge of all DR determinants

• Unclear association due to lack of gold standard or errors in the gold standard performance

• Cumulative effects

Bactericidal antibiotic that inhibits the bacterial DNA-dependent RNA polymerase.

Target: β-subunit of the RNA polymerase (encoded by rpoB), blocking elongation of the RNA chain.

Mutations in a “hot-spot” region of 81 bp of rpoB gene (Rifampin resistance-determining region) → RIF resistance (> 95%)

Rifampicin (RIF)

Updated critical concentration for first- and second-line DST (as of May 2012)

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*

Roll out of Xpert and LPA has shown discordance with MGIT results Sequencing has confirmed the presence of mutations

Liquid DST methods can miss cases of RIF-R

Adapted from Van Deun A et al, J Clin Microbiol 2009; 47(11)3501-6

Missing rate for RIF low-level R : • 81% MGIT960 • 58% Bactec radiometric • 30% agar proportion method

10% RIF-R isolates (mut. at cod. 511, 516, or 533) missed by the

Bactec radiometric method

Van Deun A et al, J Clin Microbiol 2009; 47(11)3501-6 Traore H et al, Int J Tuberc Lung Dis 2000; 4:481-84

Most of the borderline R strains are associated with bacteriologically unfavorable treatment outcomes

Non canonical rpoB mutations were identified in >10% of cases The presence of unconventional mutations correlated with a poor outcome

Silent mutations in the RRDR and mutations outside the RRDR

Silent mutations can be detected by molecular assays but do not modify the aa and the protein structure, and they are NOT relevant for drug resistance. Silent mutations may cause false positivity in molecular tests Silent mutations observed: F506, T508, Q510, L511, Q513, F514, T525, A532, L533, P535 Alonso et al, 2011: silent SNP at cod. F514 was registered in 0.8% of cases Van Deun et al 2013: silent RRDR mutations in retreatment cases occurred in <0.5% of cases Mani et al 2001: silent mutations observed in 4% of cases Yuan et al 2012: silent mutation observed in 1.5% of cases

Mutations outside the RRDR: the transformation of V146F or I572F mutated rpoB into the wild-type M. tuberculosis strains causes RIF-R phenotype Frequency of these mutations: Siu et al 2011: 4% Ahmad et al 2005: 6% Ahmad et al 2012: 11% Van Deun et al 2013: 1-2% Van Deun et al 2009: 5% Rigouts et al 2013: 5% Miotto et al in preparation: 2%

Alonso M et al, J Clin Microbiol 2011; 49(7):2688-90 Moure R et al, J Clin Microbiol 2011; 49(10):3722

Ocheretina O et al, PLoS One 2014; 9(3):e90569 Van Deun A et al, J Clin Microbiol 2013; 51(8):2633-40

Kim BJ et al, J Clin Microbiol 1997; 35(2):492-4 Yuan X et al, J Clin Microbiol 2012; 50(7):2404-13

Siu GK et al, J Antimicrob Chemother 2011; 66(4):730-3 Ahmad S et al, Int J Antimicrob Agents 2005; 26(3):205-12

Van Deun A et al, J Clin Microbiol 2009; 47(11)3501-6 Ahmad S et al, Indian J Med Res 2012; 135(5):756-62

Mani C et al, J Clin Microbiol 2001; 39(8):2987-90 Williamson DA et al, Diagn Microbiol Infect Dis 2011; 74(2):207-9

Kapur V et al, J Clin Microbiol 1994; 32(4):1095-8

Approx. 2%

Approx. 5%

Rif take home message

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

• 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 1mg/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

• Xpert, LPA: possible false resistance in case of silent mutations in targeted regions (no wt pattern) → sequencing-based approach to overcome the problem and possible false susceptible if the molecular assay doesn't target the mutation (Swaziland example)

FQs: mechanisms of action and resistance

Resistance: Mainly mutations on DNA gyrase disrupting the binding site for the drug

• gyrA:

─ 26 different mutations : 81% inside the QRDR (cod. 74-113)

─ 64% of FQ-R had mutations inside the QRDR of gyrA, 0.5% outside the QRDR

─ Substitutions at codon 94 most prevalent (37% of FQ-R strains, D94G/A/N/H/Y/F/V); codon 90 (13%) and 91 (4%)

─ Double mutations in gyrA frequently associated to high-level resistance

─ Frequent heteroresistance (up to 31% of isolates)

─ Polymorphisms not related to FQ-R (outside the QRDR)

• gyrB:

─ 18 different mutations of which 44% inside and 50% outside the QRDR (cod. 461-499)

─ Silent mutations

Additional mechanisms of action have been hypothesised:

• MfpA/MfpB proteins

• Efflux pumps

• Transporters Miotto et al Chest 2014

Action: Inhibition of bacterial DNA-gyrase ( and topoisomerase IV) , enzymes required for vital processes such as replication, transcription, recombination and chromosomal supercoiling.

Updated critical concentration for first- and second-line DST

P8A

R68G

H70R low-level (LEV-R) Yin 2010

A74S

T80A FQ-hyperS Aubry 2006

G88A

G88C

D89N

A90E

A90G FQ-hyperS Aubry 2006

A90L

A90V

S91A low-level (OFX-R) Chernyaeva 2013

S91P low-level (OFX-R) Chernyaeva 2013

I92M

D94A low-level (LEV-R) Yin 2010

D94N low-level (LEV-R) Yin 2010

D94G

D94H

D94F

D94T

D94V

P102H

L109V

A126R

A74S + D94G

T80A + A90E

T80A +A90G FQ-hyperS Aubry 2006

T80A + A90G + D94G

G88A + A90V

G88A + D94Y

A90V + D94A

A90V + P102H

A90V + S91P

A90V + D94N

A90V + D94G

S91P + D94G

S91P + D94G + D94A

D94A + D94Y

D94N + D94G

D94N + D94G + D94Y

D94G + D94A

R485C

S447F

D461A MOX/LEV/CIP/OFX-S Malik 2012

D461N OFX/LEV-R only Malik 2012

D461H OFX/LEV-R only Malik 2012

G470A

D494A MOX/LEV/CIP/OFX-S Malik 2012

N499D MOX/LEV/CIP/OFX-R Malik 2012

N499K MOX-R only Malik 2012

N499T

T500N low-level (OFX-R) Chernyaeva 2013

T500P unclear Malik 2012

E501D MOX-R only Malik 2012

E501V MOX/LEV/CIP/OFX-R Malik 2012

E501A low-level (OFX-R) Chernyaeva 2013

A504T

A504V low-level (OFX-R) Chernyaeva 2013

Q538H

D461H + G470A

N499T + T507M MXF/LEV/CIP/OFX-S Malik 2012

A90V + D461A

A90V + N499T

A90V + D94A + N499T

A90V + S91P + D94G + D94A + N499T

A90V + T500P

D94A + D461N

D94G + N499K

D94G + N499T

D94N + A504V

Q

R

D

R

Q

R

D

R

GyrA mutation

GyrB mutation

GyrA + gyrB mutation

• Confidence defined based on: DST results, biochemical/genetic validation, n° of studies reporting association to FQ-R • Maruri et al 2012: 42 studies 2482 clinical isolates (1220 FQ-R, 1262 FQ-S) • Total number of studies considered: gyrA 45; gyrB 16 • Few data associating gyrA-gyrB mutations and clinical outcome are available

Li J et al, Emerg Microb Inf 2014; Maruri F et al, J Antimicrob Chemother 2012;

Aubry A et al, Antimicrob Agents Chemother 2006; Chernyaeva E et al, Tuberculosis 2013

Malik S et al, PLoS One 2012; Lau RW et al, Antimicrob Agents Chemother 2011;

Yin X et al, J Infect 2010 Jo KW et al, Int J Tuberc Lung Dis 2014;

Defining high and low confidence mutations for FQ-R: gyrA

Polymorphisms not related to FQ-R: T80A, A90G, E21Q, S95T, G668D, G247S, A384V Silent mutations: I614I, A830A

81% of mutations inside the QRDR 64% of FQ-R had mutations inside the QRDR of GyrA

Substitutions: cod. 94 37% cod. 90 13% cod. 91 4%

R485C

S447F

D461A MOX/LEV/CIP/OFX-S Malik 2012

D461N OFX/LEV-R only Malik 2012

D461H OFX/LEV-R only Malik 2012

G470A

D494A MOX/LEV/CIP/OFX-S Malik 2012

N499D MOX/LEV/CIP/OFX-R Malik 2012

N499K MOX-R only Malik 2012

N499T

T500N low-level (OFX-R) Chernyaeva 2013

T500P unclear Malik 2012

E501D MOX-R only Malik 2012

E501V MOX/LEV/CIP/OFX-R Malik 2012

E501A low-level (OFX-R) Chernyaeva 2013

A504T

A504V low-level (OFX-R) Chernyaeva 2013

Q538H

D461H + G470A

N499T + T507M MXF/LEV/CIP/OFX-S Malik 2012

Q

R

D

R

GyrB mutation

A90V + D461A

A90V + N499T

A90V + D94A + N499T

A90V + S91P + D94G + D94A + N499T

A90V + T500P

D94A + D461N

D94G + N499K

D94G + N499T

D94N + A504V

GyrA + gyrB mutation

461-499

44% of mutations inside the QRDR

Note: numbering system from Maruri 2012

Silent mutations: T221T, V265V, A334A Non linked to mic increase: D533A, D500A and the double mutation N538T-T546M

Defining high and low confidence mutations for FQ-R: : gyrB

Frequency and Geographic Distribution of gyrA and gyrB Mutations Associated with Fluoroquinolone Resistance in Clinical Mycobacterium Tuberculosis Isolates: A Systematic Review Avalos E, Catanzaro D, Catanzaro A,

Ganiats T, Brodine S, et al. (2015) http://127.0.0.1:8081/plosone/article?id=info:doi/10.1371/journal.pone.0120470

-gyrA mutations reported in codons 88–94 appeared to account for at least 82% of phenotypic ofloxacin resistance and 85% of moxifloxacin resistance globally -cross resistance among FQs classes -while gyrB mutations and gyrA double mutations occurred only rarely. -geographic differences in the frequencies of specific gyrA mutations between countries were observed

L.Rigouts ,A van Deun et al 2014

Gyr A mutation 94G is associated to treatment failure

data from patients in B’desh receiving the 9-month Gfx-based B’desh treatment with a Gfx-based treatment. Gfx dosage was high: 400 -800 mg based on body weight. All patients are MDR yet susceptible to kanamycin.

Mutations in gyrA QRDR are up today the major determinants for FQs resistance Most mutations outside of the gyrA and gyrB QRDR do not lead to FQ-R or only

slightly increased the MIC levels for FQs Contribution of mutations in gyrB QRDR to FQ R is 1-6% depending on studies The position and the AA substitution is relevant and needs to be identified D94G and D94N high MICs and D94G associated to treatment failure (with

newer FQ) A90V and D94A Ofloxacyn resistance Double mutations are associated to higher mic to all FQs T80A and A90G mutations are associated to FQ hyper-susceptibility Correlation between the presence of mutations and agar based phenotypic tests

for the different drugs is good. MGIT higher breakpoints may need to be revised Genetic background can influence the relevance of some mutations

FQ – take home message

GenoType MTBDRsl 1.0 vs. GenoType MTBDRsl 2.0

Conjugate Control (CC)Amplification Control (AC)

complex (TUB)M. tuberculosis

gyrA (gyrA)gyrA gyrAgyrA gyrAgyrA gyrAgyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA

Locus Control WT1)WT2)WT3)

mutation probe ( MUT )

wild type probe 1 ( wild type probe 2 ( wild type probe 3 ( mutation probe 1 ( MUT1) mutation probe 2 ( MUT2) mutation probe 3A ( MUT3A) mutation probe 3B ( MUT3B) mutation probe 3C ( MUT3C)

3D 3D

rrs (rrs)rrs rrsrrs rrsrrs rrsrrs rrs

Locus Control wild type probe 1 (

wild type probe 2 ( WT2)

mutation probe 2 (

WT1)

mutation probe 1 ( MUT1)MUT2)

embB (embB)embB embBembB embBembB embB

Locus Controlwild type probe 1 (mutation probe 1A (mutation probe 1B (

WT1)

MUT1A)MUT1B)

colored marker

Conjugate Control (CC)Amplification Control (AC)

complex (TUB)M. tuberculosisgyrA (gyrA)gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA

Locus Control wild type probe 1 ( WT1) wild type probe 2 ( WT2) wild type probe 3 ( WT3) mutation probe 1 ( MUT1) mutation probe 2 ( MUT2) mutation probe 3A ( MUT3A) mutation probe 3B ( MUT3B) mutation probe 3C ( MUT3C) mutation probe 3D ( MUT3D)

rrs (rrs)rrs rrsrrs rrsrrs rrs rrs rrs

Locus Control wild type probe 1 ( WT1) wild type probe 2 ( WT2) mutation probe 1 ( MUT1)mutation probe 2 ( MUT2)

colored marker

gyrBgyrB gyrBgyrB gyrBgyrB gyrB

Locus Control wild type probe ( WT) mutation probe 1 ( MUT1) mutation probe 2 ( MUT2)

eis (eis)eis eiseis eiseis eiseis eis

Locus Controlwild type probe 1 ( WT1)wild type probe 2 ( WT2)wild type probe 3 ( WT3)

probe 1 ( MUT1)mutation

GenoType MTBDRsl 1.0 GenoType MTBDRsl 2.0

21 positions 27 positions

Courtesy of Hain lifescences

MTBDRsl Ver 2.0 sensitivity and specificity for FQ resistance

Phenotypic culture-based DST as a reference standard

FQ Culture/DST

Ver 1.0

Ver 2.0

Theron et al compiled data

(Cochrane Rev, 2014)

(95% CI) (95% CI)

Sensitivity 83.1% (78.7-86.7) 83.6% (73.4, 90.3)

Specificity 97.7% (94.3-99.1) 100% (97.6-100)

Tagliani et al submitted to Union 2015

Global frequencies for selected mutations among phenotypically isoniazid resistant and phenotypically isoniazid sensitive isolates.

Seifert M, Catanzaro D, Catanzaro A, Rodwell TC (2015) Genetic Mutations Associated with Isoniazid Resistance in Mycobacterium tuberculosis: A Systematic Review. PLoS ONE 10(3): e0119628. doi:10.1371/journal.pone.0119628 http://127.0.0.1:8081/plosone/article?id=info:doi/10.1371/journal.pone.0119628

Pyrazinamide (PZA)

Miotto P et al, 2014. Chest

Pro-drug converted to its active form, pyrazinoic acid, by the enzyme pyrazinamidase/nicotinamidase encoded by the pncA gene

Pyrazinoic acid disrupts the bacterial membrane energetics inhibiting membrane transport.

Target: Rpsa

Large inoculum Suboptimal test media with unreliable pH Critical concentration 100 ug/mL (inconsistent results for isolates with a PZA MIC close to this

concentration)

Intermediate category?

57 isolates showed PZA-R on MGIT 960. Repeat testing of resistant isolates with the Bactec 460 reference method confirmed 33 (58%) of these isolates as resistant, and 24 (42%) were susceptible.

Are we over estimating drug resistance to PZA?

Chedore P et al, 2010. J Clin Microbiol; 48(1):300-1

• 50 codons showed a frequency of mutation over the mean value of 0.5%

• Most frequently affected regions, representing more than 70% of mutated cases are found at the promoter (-13 to -3), and at codons 6-15, 50-70, 90-100, 130-145, 170-175.

• Different mutations at the same codon

Frequency of mutation across the pncA gene

Miotto P et al, 2014. mBio 5(5):e01819-14

PZA-R take-home messages

Need of fast resistance detection PZA DST: frequent problems of false resistance Excellent correlation between PZA resistance and pncA mutations About 85% of PZA-R strains carry mutations in pncA Possible development of rapid molecular tests to detect

resistance (sequencing-based?). No hot-spot regions.

Aminoglycosides: binding to the 30S subunit of the ribosome and misincorporation of amino acids into elongating peptides (streptomycin, kanamycin, amikacin)

Mutations in the rrs gene coding for 16S rRNA → AGs resistance Mutations in the promoter region of eis → kanamycin resistance Mutations in rpsL gene (ribosomal S12 protein) → streptomycin resistance

Kohanski M et al., Nature Reviews 2010

Eis acetylates multiple amines of many AGs. Upregulation of the eis gene (mutations in the promoter region) confers resistance to Kanamycin

Polypeptides: inhibition of the translocation of peptidyl tRNA and block of initiation of protein synthesis (capreomycin, viomycin)

Mutations in the rrs gene coding for 16S rRNA → resistance Mutations in the tlyA gene coding for a 2-O-methyltransferase→ polypeptides resistance?

Second line injectable drugs mechanism of action and resistance

rrs gene

eis promoter

gidB gene

SLIDs – take home message

Selected rrs mutations are relevant for SLIDs resistance 1401G is the most relevant mutation associated to high

resistance to all SLIDs Mutations in the eis promoter region are relevant

determinants for resistance to kanamycin. Positions and AAs substitutions are relevant

MTBDRsl Ver2.0 has highly increased his sensitivity compared to version1by the addition of eis promoter region; specificity will need to be addressed with more data on specific mutations

GenoType MTBDRsl Ver 2.0

GenoType MTBDRsl 1.0

21 positions

Courtesy of Hain lifescences

Conjugate Control (CC) Amplification Control (AC)

complex (TUB) M. tuberculosis

gyrA (gyrA) gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA

Locus Control WT1) WT2) WT3)

mutation probe ( MUT3D)

wild type probe 1 ( wild type probe 2 ( wild type probe 3 ( mutation probe 1 ( MUT1) mutation probe 2 ( MUT2) mutation probe 3A ( MUT3A) mutation probe 3B ( MUT3B) mutation probe 3C ( MUT3C)

rrs (rrs) rrs wild type probe1 (WT1)

Locus Control

rrs wild type probe 2 ( WT2)

rrs mutation probe 2 (MUT2) rrs mutation probe 1 (MUT1)

embB embB embB embB embB embB

embB Locus Control (embB) wild type probe 1 ( mutation probe 1A ( mutation probe 1B (

WT1)

MUT1A) MUT1B)

colored marker

GenoType MTBDRsl 2.0

27 positions

Conjugate Control (CC) Amplification Control (AC)

complex (TUB) M. Tuberculosis gyrA (gyrA) Locus Control

rrs (rrs) rrs rrs rrs rrs rrs rrs rrs rrs

Locus Control wild type probe 1 ( WT1) wild type probe 2 ( WT2) mutation probe 1 ( MUT1) mutation probe 2 ( MUT2)

colored marker

gyrB gyrB gyrB gyrB gyrB gyrB

Locus Control wild type probe (gyrB WT) mutation probe 1 ( MUT1) mutation probe 2 ( MUT2)

eis (eis) eis eis eis eis eis eis eis eis

Locus Control wild type probe 1( WT1) wild type probe 2( WT2) wild type probe 3( WT3)

probe 1 ( MUT1) mutation

gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA mutation probe ( MUT3D)

mutation probe 3A ( MUT3A) mutation probe 3B ( MUT3B) mutation probe 3C ( MUT3C)

gyrA gyrA gyrA gyrA gyrA gyrA

WT3) mutation probe 1 ( MUT1) mutation probe 2 ( MUT2)

wild type probe 3 (

gyrA gyrA gyrA gyrA

WT1) WT2)

wild type probe 1 ( wild type probe 2 (

Conjugate Control (CC)Amplification Control (AC)

complex (TUB)M. tuberculosisgyrA (gyrA)gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA gyrA

Locus Control wild type probe 1 ( WT1) wild type probe 2 ( WT2) wild type probe 3 ( WT3) mutation probe 1 ( MUT1) mutation probe 2 ( MUT2) mutation probe 3A ( MUT3A) mutation probe 3B ( MUT3B) mutation probe 3C ( MUT3C) mutation probe 3D ( MUT3D)

rrs (rrs)rrs rrsrrs rrsrrs rrs rrs rrs

Locus Control wild type probe 1 ( WT1) wild type probe 2 ( WT2) mutation probe 1 ( MUT1)mutation probe 2 ( MUT2)

colored marker

gyrBgyrB gyrBgyrB gyrBgyrB gyrB

Locus Control wild type probe ( WT) mutation probe 1 ( MUT1) mutation probe 2 ( MUT2)

eis (eis)eis eiseis eiseis eiseis eis

Locus Controlwild type probe 1 ( WT1)wild type probe 2 ( WT2)wild type probe 3 ( WT3)

probe 1 ( MUT1)mutation

First Data on the new MTBDRsl VER2.0 eis target

MTBDRsl Ver 2.0 sensitivity and specificity for SLID resistance

Phenotypic culture-based DST as a reference standard

Kanamycin Culture/DST

Ver 1.0

Ver 2.0

Theron et al compiled data

(Cochrane Rev, 2014)

(95% CI) (95% CI)

Sensitivity 66.9% (44.1-83.8) 95.5% (90.6, 97.9)

Specificity 98.6% (96.1-99.5) 91.4% (83.9, 95.6)

SLID Culture/DST

Ver 1.0

Ver 2.0

Theron et al compiled data

(Cochrane Rev, 2014)

(95% CI) (95% CI)

Sensitivity 76.9% (61.1-87.6) 86.4% (81.7, 94.9)

Specificity 99.5% (97.1-99.9) 90.1% (81.7, 94.9)

Tagliani et al submitted to Union 2015

Performance characteristics of Genotype®MTBDRsl V2.0 for detection of FQ resistance is comparable to Genotype®MTBDRsl V1.0.

MTBDRsl test can correctly ID the relevant mutations in gyr A (and gyr B).

Contribution of mutations in gyrB QRDR to FQ R is 1-6% depending on studies.

MTBDRsl Ver2.0 has an increased sensitivity for SLID resistance compared to Ver 1.0 due to the addition of eis promoter region; specificity will need to be addressed with more data on specific mutation.

Mutations in the eis promoter region are relevant determinants for resistance to kanamycin. Positions and Ns substitutions are relevant.

Use of Hain sl– take home message

Conclusion • The identification of the nature of the mutations is needed for accurate diagnosis of

resistance

• There are high confidence genetic markers of resistance that can replace conventional DST

• RIF, rpoB gene: mutations at cod. 531 and specific mutations at codons 513 and 526; multiple mutations

• INH, katG gene: mutations at cod. 315 • FQ, gyrA-gyrB genes: specific mutations within the QRDRs • PZA, pncA gene: specific mutations (85%) • SLID, rrs gene: a1401g

• There are genetic markers for low-level resistance that can be used to improve clinical management of the patients • RIF, rpoB gene: 511P, 515I, 516Y, 526L, 526N, 526C, 526S, 532V, 533P, and 572F • INH, inhA gene: c-15t • FQ, gyrA gene: T80A, A90G, A90V, D94A

• Identification of the type of mutation is relevant for patient management

A common platform to investigate the relationship between mutations, phenotypic, surveillance and clinical data If interested please contact:

Dr David Dolinger: David.Dolinger@finddx.org Dr Paolo Miotto: miotto.paolo@hsr.it

A joint effort toward a common goal: providing effective diagnostic tools where are mostly needed

More data are needed

IRCCS San Raffaele

R Alagna

B Asimewe

R Baldan

S.Battaglia

E Borroni

AM Cabibbe

L Furci

P Miotto

E Schena

E Tagliani

E Tortoli

A Trovato

Acknowledgements

Hain lifesciences

FZB Borstel:

S. RueshGerdes

D. Hilleman

SRL Stockolm:

Sven Hoffner

NTP/NRL Belarus

Alena Skrahina

Aksana Zalutskaya

SRLN and TBPANNET Consortium