The current state of knowledge: genotypic vs phenotypic drug-susceptibility testing (DST)

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1 The current state of knowledge: genotypic vs phenotypic drug-susceptibility testing (DST) Daniela M Cirillo Emerging Bacterial Pathogens Unit, San Raffaele Scientific Institute Milan

2 Outline Concordance between molecular based and phenotypic DST Rifampicin Fluoroquinolones Isoniazide Pyrazinamide SLDs Second line LPA preliminary data Conclusions

Concordance among different tests Complete concordance among tests: The use of multiple 82% strategies 77% to test 50% for antibiotics 51% susceptibilities has shown that: Different phenotypic tests

3 Concordance among different tests Complete concordance among tests: The use of multiple 82% strategies 77% to test 50% for antibiotics 51% 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 Banu S et al, JCM 2014

4 Genotypic and phenotypic methods provide different pictures Phenotypic tests : in vitro growth in the presence of the drug Genotypic tests: identification of mutations associated to impairment of the mechanism of action Drug activation/concentration Testing media Inoculum-related effects Reading time 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

5 Rifampicin (RIF) 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%)

6 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

530 531 532 533 Miotto et al submitted Rigouts t al.

of Xpert and LPA has shown discordance with MGIT results Sequencing has confirmed

7 Probes position in the Hot Spot of rpob and mutations non detected by MGIT Miotto et al submitted Rigouts t al. JCM 2013 Williamson IJTLD 2011 *YuanJCM Pro 511 Gln 512 Arg 516 Tyr Roll out of Xpert and LPA has shown discordance with MGIT results Sequencing has confirmed the presence of mutations 522 Glu/Gln 526 Asn /Leu 526 Cys/Asn/Ser 533 Pro 572 Phe 533 Arg/Pro 972Thr* 531 Trp 531 Phe/Tyr

8 Liquid DST methods can miss cases of RIF-R 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 Adapted from Van Deun A et al, J Clin Microbiol 2009; 47(11) Most of the borderline R strains are associated with bacteriologically unfavorable treatment outcomes Van Deun A et al, J Clin Microbiol 2009; 47(11) Traore H et al, Int J Tuberc Lung Dis 2000; 4:481-84

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

10 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: Van Deun et al 2013: Mani et al 2001: Yuan et al 2012: silent SNP at cod. F514 was registered in 0.8% of cases silent RRDR mutations in retreatment cases occurred in <0.5% of cases silent mutations observed in 4% of cases silent mutation observed in 1.5% of cases Approx. 2% 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): 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): Kim BJ et al, J Clin Microbiol 1997; 35(2):492-4 Yuan X et al, J Clin Microbiol 2012; 50(7): Approx. 5% Siu GK et al, J Antimicrob Chemother 2011; 66(4):730-3 Ahmad S et al, Int J Antimicrob Agents 2005; 26(3): Van Deun A et al, J Clin Microbiol 2009; 47(11) Ahmad S et al, Indian J Med Res 2012; 135(5): Mani C et al, J Clin Microbiol 2001; 39(8): 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

11 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 Action: Inhibition of bacterial DNAgyrase ( and topoisomerase IV), enzymes required for vital processes such as replication, transcription, recombination and

12 FQs: mechanisms of action and resistance Action: Inhibition of bacterial DNAgyrase ( and topoisomerase IV), enzymes required for vital processes such as replication, transcription, recombination and chromosomal supercoiling. Miotto et al Chest 2014 Resistance: Mainly mutations on DNA gyrase disrupting the binding site for the drug gyra: 26 different mutations : 81% inside the QRDR (cod ) 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%) gyrb: 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) 18 different mutations of which 44% inside and 50% outside the QRDR (cod ) Silent mutations Additional mechanisms of action have been hypothesised: MfpA/MfpB proteins Efflux pumps Transporters

13 Updated critical concentration for first- and second-line DST

Defining high and low confidence mutations for FQ-R: gyra Confidence defined based on: DST results, biochemical/genetic validation, n of studies reporting association to FQ-R Maruri et al 2012: 42

14 Defining high and low confidence mutations for FQ-R: gyra 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 GyrA mutation Q R D R 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 81% of mutations inside the QRDR 64% of FQ-R had mutations inside the QRDR of GyrA Polymorphisms not related to FQ-R: T80A, A90G, E21Q, S95T, G668D, G247S, A384V Silent mutations: I614I, A830A Substitutions: cod % cod % cod. 91 4% 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;

15 Defining high and low confidence mutations for FQ-R: : gyrb 44% of mutations inside the QRDR GyrB mutation Q R D R 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 GyrA + gyrb mutation A90V + D461A A90V + N499T A90V + D94A + N499T A90V + S91P + D94G + D94A + N499T A90V + T500P D94A + D461N D94G + N499K D94G + N499T D94N + A504V Silent mutations: T221T, V265V, A334A Non linked to mic increase: D533A, D500A and the double mutation N538T-T546M Note: numbering system from Maruri 2012

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,

16 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) -gyra mutations reported in codons 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

18 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: mg based on body weight. All patients are MDR yet susceptible to kanamycin. L.Rigouts,A van Deun et al 2014

19 FQ take home message 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

20 GenoType MTBDRsl 1.0 vs. GenoType MTBDRsl 2.0 Conjugate Control (CC) Amplification Control (AC) M. tuberculosis complex (TUB) gyra Locus Control (gyra) gyra wild type probe 1 ( gyra WT1) gyra wild type probe 2 ( gyra WT2) gyra wild type probe 3 ( gyra WT3) gyra mutation probe 1 ( gyra MUT1) gyra mutation probe 2 ( gyra MUT2) gyra mutation probe 3A ( gyra MUT3A) gyra mutation probe 3B ( gyra MUT3B) gyra mutation probe 3C ( gyra MUT3C) gyra mutation probe 3D ( gyra MUT 3D) rrs Locus Control (rrs) rrs wild type probe 1 ( rrs WT1) rrs wild type probe 2 ( rrs WT2) rrs mutation probe 1 ( rrs MUT1) rrs mutation probe 2 ( rrs MUT2) embb Locus Control (embb) embb wild type probe 1 ( embb WT1) embb mutation probe 1A ( embb MUT1A) embb mutation probe 1B ( embb MUT1B) colored marker Conjugate Control (CC) Amplification Control (AC) M. tuberculosis complex (TUB) gyra Locus Control (gyra) gyra wild type probe 1 ( gyra WT1) gyra wild type probe 2 ( gyra WT2) gyra wild type probe 3 ( gyra WT3) gyra mutation probe 1 ( gyra MUT1) gyra mutation probe 2 ( gyra MUT2) gyra mutation probe 3A ( gyra MUT3A) gyra mutation probe 3B ( gyra MUT3B) gyra mutation probe 3C ( gyra MUT3C) gyra mutation probe 3D ( gyra MUT3D) gyrb Locus Control gyrb wild type probe ( gyrb WT) gyrb mutation probe 1 ( gyrb MUT1) gyrb mutation probe 2 ( gyrb MUT2) rrs Locus Control (rrs) rrs wild type probe 1 ( rrs WT1) rrs wild type probe 2 ( rrs WT2) rrs mutation probe 1 ( rrs MUT1) rrs mutation probe 2 ( rrs MUT2) eis Locus Control (eis) eis wild type probe 1 ( eis WT1) eis wild type probe 2 ( eis WT2) eis wild type probe 3 ( eis WT3) eis mutation probe 1 ( eis MUT1) colored marker 21 positions 27 positions GenoType MTBDRsl 1.0 GenoType MTBDRsl 2.0 Courtesy of Hain lifescences

21 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% ( ) 83.6% (73.4, 90.3) Specificity 97.7% ( ) 100% ( ) Tagliani et al submitted to Union 2015

22 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): e doi: /journal.pone

23 Pyrazinamide (PZA) Pro-drug converted to its active form, pyrazinoic acid, by the enzyme pyrazinamidase/nicotinamidase encoded by the pnca gene Target: Rpsa Pyrazinoic acid disrupts the bacterial membrane energetics inhibiting membrane transport. Miotto P et al, Chest

resistant, and 24 (42%) were susceptible.

24 Are we over estimating drug resistance to PZA? 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. 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? Chedore P et al, J Clin Microbiol; 48(1):300-1

25 Frequency of mutation across the pnca gene 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, , , Different mutations at the same codon Miotto P et al, mbio 5(5):e

26 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.

27 Second line injectable drugs mechanism of action and resistance Kohanski M et al., Nature Reviews 2010 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 Polypeptides: inhibition of the translocation of peptidyl trna and block of initiation of protein synthesis (capreomycin, viomycin) Aminoglycosides: binding to the 30S subunit of the ribosome and misincorporation of amino acids into elongating peptides (streptomycin, kanamycin, amikacin) Eis acetylates multiple amines of many AGs. Upregulation of the eis gene (mutations in the promoter region) confers resistance to Kanamycin Mutations in the rrs gene coding for 16S rrna resistance Mutations in the tlya gene coding for a 2-O-methyltransferase polypeptides resistance?

28 rrs gene

29 eis promoter

30 gidb gene

31 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

mutation probe 2 ( gyra MUT2) gyra mutation probe 3A ( gyra MUT3A) gyra mutation probe 3B ( gyra MUT3B) gyra mutation probe 3C ( gyra MUT3C) gyra mutation probe ( gyra MUT3D) rrs Locus Control (rrs)

32 GenoType MTBDRsl Ver 2.0 GenoType MTBDRsl 1.0 Conjugate Control (CC) Amplification Control (AC) M. tuberculosis complex (TUB) gyra Locus Control (gyra) gyra wild type probe 1 ( gyra WT1) gyra wild type probe 2 ( gyra WT2) gyra wild type probe 3 ( gyra WT3) gyra mutation probe 1 ( gyra MUT1) gyra mutation probe 2 ( gyra MUT2) gyra mutation probe 3A ( gyra MUT3A) gyra mutation probe 3B ( gyra MUT3B) gyra mutation probe 3C ( gyra MUT3C) gyra mutation probe ( gyra MUT3D) rrs Locus Control (rrs) rrs wild type probe1 (WT1) rrs wild type probe 2 ( WT2) rrs mutation probe 1 (MUT1) rrs mutation probe 2 (MUT2) embb Locus Control (embb) embb wild type probe 1 ( embb WT1) embb mutation probe 1A ( embb embb mutation probe 1B ( embb colored marker MUT1A) MUT1B) GenoType MTBDRsl 2.0 Conjugate Control (CC) Amplification Control (AC) M. Tuberculosis complex (TUB) gyra Locus Control (gyra) gyra wild type probe 1 ( gyra gyra wild type probe 2 ( gyra WT1) WT2) gyra wild type probe 3 ( gyra WT3) gyra mutation probe 1 ( gyra MUT1) gyra mutation probe 2 ( gyra MUT2) gyra mutation probe 3A ( gyra MUT3A) gyra mutation probe 3B ( gyra MUT3B) gyra mutation probe 3C ( gyra MUT3C) gyra mutation probe ( gyra MUT3D) gyrb Locus Control gyrb wild type probe (gyrb WT) gyrb mutation probe 1 ( gyrb MUT1) gyrb mutation probe 2 ( gyrb MUT2) rrs Locus Control (rrs) rrs wild type probe 1 ( rrs WT1) rrs wild type probe 2 ( rrs WT2) rrs mutation probe 1 ( rrs MUT1) rrs mutation probe 2 ( rrs MUT2) eis Locus Control (eis) eis wild type probe 1( eis WT1) eis wild type probe 2( eis WT2) eis wild type probe 3( eis WT3) eis mutation probe 1 ( eis MUT1) 21 positions colored marker 27 positions Courtesy of Hain lifescences

33 First Data on the new MTBDRsl VER2.0 eis target Conjugate Control (CC) Amplification Control (AC) M. tuberculosis complex (TUB) gyra Locus Control (gyra) gyra wild type probe 1 ( gyra WT1) gyra wild type probe 2 ( gyra WT2) gyra wild type probe 3 ( gyra WT3) gyra mutation probe 1 ( gyra MUT1) gyra mutation probe 2 ( gyra MUT2) gyra mutation probe 3A ( gyra MUT3A) gyra mutation probe 3B ( gyra MUT3B) gyra mutation probe 3C ( gyra MUT3C) gyra mutation probe 3D ( gyra MUT3D) gyrb Locus Control gyrb wild type probe ( gyrb WT) gyrb mutation probe 1 ( gyrb MUT1) gyrb mutation probe 2 ( gyrb MUT2) rrs Locus Control (rrs) rrs wild type probe 1 ( rrs WT1) rrs wild type probe 2 ( rrs WT2) rrs mutation probe 1 ( rrs MUT1) rrs mutation probe 2 ( rrs MUT2) eis Locus Control (eis) eis wild type probe 1 ( eis WT1) eis wild type probe 2 ( eis WT2) eis wild type probe 3 ( eis WT3) eis mutation probe 1 ( eis MUT1) colored mar ker

34 MTBDRsl Ver 2.0 sensitivity and specificity for SLID resistance Kanamycin Phenotypic culture-based DST as Culture/DST a reference standard Ver 1.0 Ver 2.0 Theron et al compiled data (Cochrane Rev, 2014) (95% CI) (95% CI) Sensitivity 66.9% ( ) 95.5% (90.6, 97.9) Specificity 98.6% ( ) 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% ( ) 86.4% (81.7, 94.9) Specificity 99.5% ( ) 90.1% (81.7, 94.9) Tagliani et al submitted to Union 2015

35 Use of Hain sl take home message 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.

36 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

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

38 Acknowledgements 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 Hain lifesciences FZB Borstel: S. RueshGerdes D. Hilleman SRL Stockolm: Sven Hoffner NTP/NRL Belarus Alena Skrahina Aksana Zalutskaya SRLN and TBPANNET Consortium A Trovato

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