Biomolecular diagnostics, gene sequencing and whole genome sequencing. Rob Warren

Transcription

1 Biomolecular diagnostics, gene sequencing and whole genome sequencing Rob Warren

2 Classic molecular epidemiology IS6110 DNA fingerprinting (between 0 and 26 loci) Spoligotyping (1 locus) MIRU-VNTR (24 loci)

3 The Road Map 4,411,529 base pairs 4,000 genes Two new families of glycinerich proteins with a repetitive structure that may represent a source of antigenic variation. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence Nature 393, (11 June 1998)

4 Whole genome sequencing (Illumina) DNA extraction Sample preparation Cluster generation on flow cell Shear DNA Ligate adapters Data analysis Sequencing and imaging

5 Mapping/Aligning Reads to a Reference Reference genome X X X X X Trimmed Reads X100 X X

6 USAP BWA Alignment Smalt NOVOAlign Variant calling SAMTools GATK Filtered Variants used in downstream applications

7 Phylogeny

8 Global Phylogeny of Mycobacterium tuberculosis Out-of-Africa migration and Neolithic coexpansion of Mycobacterium tuberculosis with modern humans. Nat Genet Oct;45(10):

9 Transmission

10 House hold contacts IS6110 RFLP 100% transmission (ngd < 2) WGS 6/12 < 10 SNVs 50% transmission 0 band differences 6/12 > 45 SNVs 2 band differences J Clin Microbiol Apr;40(4): Evolution of the IS6110-based restriction fragment length polymorphism pattern during the transmission of Mycobacterium tuberculosis.

11 Relapse / Reinfection 1 IS6110 RFLP 25/25 relapse (ngd < 2) 2 WGS 22/25 relapse (< 5 SNVs) Clin Infect Dis Jun;58(12): The temporal dynamics of relapse and reinfection tuberculosis after successful treatment: a retrospective cohort study.

12 Relapse / Reinfection WGS 2/25 reinfection (mixed infection) First episode isolate Second episode isolate Patient Isolate number Isolate number IS6110 evolution Total variation no no 833 (rpob S531L) (katg S315T) Heterogeneous / underlying population

13 WGS Relapse / Reinfection 1/25 20 SNVs 3667 (0) 2626 (0) (41) 3773 (46) 298 (1) (2) 770 (0) 2167 (5) 2621 (3) 644 (0) 1485 (1) 1457 (4) 1454 (3) M. tuberculosis L4 M. tuberculosis H37Rv M. tuberculosis K37 L (1) 903 (2) 5084 (0) 5946 (0) 2069 (0) 5181 (0) 4913 (0) 4224 (0) 7484 (1) 6314 (1) M. tuberculosis K93 L1 M. tuberculosis T17 L1 M. tuberculosis T92 L1 M. tuberculosis M4100A L2 M. tuberculosis T85 L2 318 (2) 1696 (0) 5557 (1) 4664 (0) 5408 (0) 3954 (0) M. tuberculosis T67 L (4) 3752 (0) 1994 (1) 1522 (0) 1389 (0) 1492 (0) 5577 (1) 3835 (0) 6420 (0) 5159 (0) 1411 (2) 1376 (3) (0) 3327 (0) 3726 (0) (0) M. tuberculosis L3 M. tuberculosis K49 L (35) 4954 (31) (1) 3857 (0) (1) 3778 (1) 3205 (2) 3901 (0) 7403 (1) 6434 (0) 340 (0) 123 (0) 2415 (0) 2778 (1) 682 (1) 4885 (1) 3667 (0) 2626 (0) (41) 3773 (46) 298 (1) (2) 770 (0) 2167 (5) 2621 (3) 644 (0) 1485 (1) 1457 (4) 1454 (3) M. tuberculosis L4 M. tuberculosis H37Rv M. tuberculosis K37 L (1) 903 (2) 5084 (0) 5946 (0) 2069 (0) 5181 (0) 4913 (0) (0) 7484 (1) 6314 (1) (2) 6798 (0) (0) 4759 (0) 7182 (7) 5130 (4) 7793 (1) 6558 (0) 6072 (0) 4402 (0) M. canettii 8156 (2) 6798 (0) (1) 3840 (0) 4759 (0) 7182 (7) 5130 (4) 7793 (1) 6558 (0)

14 Transmission chain: Beijing outbreak N = 16 (1993 to 1997) katg 315 (AGC to ACC) rpob 531 (TCG to TTG) embb 306 (ATG to ATA) rrs 513 (CAG to CCG)

15 Transmission chain: Beijing outbreak J Infect Dis Nov;180(5): Transmission of a multidrug-resistant Mycobacterium tuberculosis strain resembling "strain W" among noninstitutionalized, human immunodeficiency virus-seronegative patients.

16 Transmission = 84% N Engl J Med Jan 19;376(3): doi: /NEJMoa Transmission of Extensively Drug-Resistant Tuberculosis in South Africa. Shah NS 1, Auld SC 1, Brust JC 1, Mathema B 1, Ismail N 1, Moodley P 1, Mlisana K 1, Allana S 1, Campbell A 1, Mthiyane T 1, Morris N 1, Mpangase P 1, van der Meulen H 1, Omar SV 1, Brown TS 1, Narechania A 1, Shaskina E 1, Kapwata T 1, Kreiswirth B 1, Gandhi NR 1.

17 Social networks 30%

18 Transmission through Migration: Atypical Beijing Atypical Beijing Other Atypical Beijing J Clin Microbiol Nov;53(11): Molecular Epidemiological Interpretation of the Epidemic of Extensively Drug- Resistant Tuberculosis in South Africa.

19 Precision Medicine

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21 H37Rv gene afta,embc ahpc ahpc upstream ald ald upstream alr alr upstream atpe atpe upstream blac blac upstream cyca cyca upstream dacb2 dacb2 upstream ddla ddla upstream ddn ddn upstream dfra dfra upstream eccb5 upstream eccb5,eccc5 eis eis upstream emba upstream emba,embb embr embr upstream erm(37) erm(37) upstream etha etha upstream ethr fabg1 fabg1 upstream fbia upstream fbia,b fbic fbic upstream fgd1 fgd1 upstream folc folp1 folp1 upstream folp2 folp2 upstream fura fura upstream gidb gidb upstream gpsi gpsi upstream gyra associated antibiotic resistance ethambutol compensates for KatG deficit compensates for KatG deficit cycloserine cycloserine cycloserine cycloserine bedaquiline bedaquiline beta-lactams beta-lactams cycloserine cycloserine beta-lactams beta-lactams cycloserine cycloserine delamanid, PA-824 delamanid, PA-824 PAS, co-trimoxazole PAS, co-trimoxazole moxifloxacin, gatifloxacin, ofloxacin, levofloxacin moxifloxacin, gatifloxacin, ofloxacin, levofloxacin kanamycin kanamycin ethambutol ethambutol ethambutol ethambutol clarithromycin clarithromycin ethionamide, prothionamide, thioacetazone ethionamide, prothionamide, thioacetazone ethionamide, prothionamide, thioacetazone isoniazid, ethionamide, prothionamide isoniazid, ethionamide, prothionamide delamanid, PA-824 delamanid, PA-824 delamanid, PA-824 delamanid, PA-824 delamanid, PA-824 delamanid, PA-824 PAS PAS, co-trimoxazole PAS, co-trimoxazole co-trimoxazole co-trimoxazole isoniazid isoniazid streptomycin streptomycin pyrazinamide pyrazinamide moxifloxacin, gatifloxacin, ofloxacin, levofloxacin gyra gyra upstream gyrb gyrb upstream hada upstream hada,hadb,hadc inbr inbr upstream inha inha upstream kasa katg katg upstream ldta ldta upstream ldtb ldtb upstream ldtd ldtd upstream ldte ldte upstream lprq lprq upstream mmaa2 mmaa2 upstream mmaa3 mmaa3 upstream mmaa4 mmaa4 upstream mmpl5,mmps5 mmpr mmpr upstream msha msha upstream mshb mshb upstream mshc mshc upstream myma myma upstream nat nat upstream ndh ndh upstream nudc nudc upstream pand pbpa pepq pepq upstream pnca pnca upstream pona1 pona1 upstream pyka pyka upstream rpfb upstream moxifloxacin, gatifloxacin, ofloxacin, levofloxacin moxifloxacin, gatifloxacin, ofloxacin, levofloxacin moxifloxacin, gatifloxacin, ofloxacin, levofloxacin moxifloxacin, gatifloxacin, ofloxacin, levofloxacin thioacetazone thioacetazone isoniazid isoniazid isoniazid, ethionamide, prothionamide isoniazid, ethionamide, prothionamide isoniazid isoniazid isoniazid beta-lactams beta-lactams beta-lactams beta-lactams beta-lactams beta-lactams beta-lactams beta-lactams beta-lactams beta-lactams thioacetazone thioacetazone isoniazid, ethionamide, prothionamide isoniazid, ethionamide, prothionamide thioacetazone thioacetazone bedaquiline, clofazimine bedaquiline, clofazimine bedaquiline, clofazimine isoniazid, ethionamide, prothionamide isoniazid, ethionamide, prothionamide isoniazid isoniazid isoniazid, ethionamide, prothionamide isoniazid, ethionamide, prothionamide isoniazid, ethionamide, prothionamide isoniazid, ethionamide, prothionamide isoniazid isoniazid isoniazid, ethionamide, prothionamide, clofazimine, thioridazine isoniazid, ethionamide, prothionamide, clofazimine, thioridazine isoniazid, ethionamide, prothionamide isoniazid, ethionamide, prothionamide pyrazinamide beta-lactams bedaquiline, clofazimine bedaquiline, clofazimine pyrazinamide pyrazinamide rifampicin, rifabutin, beta-lactams rifampicin, rifabutin, beta-lactams cycloserine cycloserine clarithromycin pona1 upstream pyka pyka upstream rpfb upstream rpfb, ksga rplc rplc upstream rpoa rpoa upstream rpob rpob upstream rpoc rpoc upstream rpsa rpsa upstream rpsl rpsl upstream rrl rrl upstream rrs rrs upstream Rv0565c Rv0565c upstream Rv1979c Rv1979c upstream Rv2671 Rv2671 upstream Rv2731 Rv3169 serb2 serb2 upstream sigi sigi upstream tap tap upstream thya thya upstream thyx thyx upstream tlya ubia ubia upstream whib7 whib7 upstream rifampicin, rifabutin, beta-lactams cycloserine cycloserine clarithromycin clarithromycin linezolid linezolid compensates for rpob mutations compensates for rpob mutations rifampicin, rifabutin rifampicin, rifabutin compensates for rpob mutations compensates for rpob mutations pyrazinamide pyrazinamide streptomycin streptomycin linezolid, capreomycin, clarithromycin linezolid, capreomycin, clarithromycin streptomycin, amikacin, kanamycin, capreomycin streptomycin, amikacin, kanamycin, capreomycin ethionamide, prothionamide ethionamide, prothionamide bedaquiline, clofazimine bedaquiline, clofazimine PAS PAS pyrazinamide pyrazinamide clofazimine clofazimine isoniazid isoniazid streptomycin, PAS streptomycin, PAS PAS PAS compensates for ThyA deficit compensates for ThyA deficit capreomycin ethambutol ethambutol streptomycin, kanamycin, clarithromycin, PAS streptomycin, kanamycin, clarithromycin, PAS

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23 Disputed rpob mutations rpob codon rpob base change No of isolates RIF MIC range RBT MIC range Xpert 1 WT WT 27 <1 - <100 <0.125 NONE bp deletion 2 <1 <0.125 Probe A T>C 14 <1 - >1 <0.125 Probe A bp deletion 2 > C>A 3 >20 - >100 >2 Probe B A>C 2 >50 - > Probe B A>T 2 >50 - >100 >2 Probe B bp deletion 2 >100 >2 Probe B bp deletion 4 >1 - < Probe B G>T 15 <1 - <100 < Probe B GA>TG 1 <1 <0.125 Probe B GA>TT 1 >1 <5 <0.125 Probe B A>C 2 <1 <0.125 MISSED?? A>T 81 >1 - <100 < >2 Probe B bp deletion 3 <1 - <10 <0.125 Mixed B + C bp deletion 1 >1 <5 <0.125 Probe B C>T 2 > Probe C C>A 3 <1 <0.125 Probe D C>G 19 >50 - >100 >2 Probe D C>T 13 >50 - >100 >2 Probe D CA>TG 1 >1 <5 <0.125 Probe D A>G 3 >20 - >100 >2 Probe D A>T 4 <1 - <10 < <2 Probe D C>G 4 > C>T 146 <1 - < >2 Probe E CG>AC 1 > CG>TT 2 >100 >2 Probe E T>C 10 <1 - <50 < Probe E T>G + G>T 1 >5 <10 >1<2 Probe A only G>A + G>T 1 > C>G + A>T 1 >100 >2 Probe B only C>G + A>G 1 >1 <5 <0.125 Probe B only T>C + C>T 1 >1 <5 <0.125 Probe A only

24 Pyrazinamide genotypic DST 608 Mutations (SNPs and in/dels) at 397 positions A Global Perspective on Pyrazinamide Resistance: Systematic Review and Meta-Analysis. PLoS One Jul 28;10(7):e

25 Terizidone resistance

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27 Heteroresistance Drug-susceptible population Mixed population Mixed population Resistant population Drug pressure Spontaneous mutation x Drug pressure Mutant grows better than progenitor x x x x x x Mutant outcompetes progenitor

28 Detecting Heteroresistance Gold standard Limited number of Accurate markers detected in Lengthy 1 reaction Detects some INH and RIF Standard of care Detects RIF only Sequencing of the entire genome of the organism Expensive Requires culture Detects any known resistance marker

29 Clinical Example (Day 1) PCR/Line Probe Assay (MTBDRplus) Isoniazid (INH) Sensitive

30 Follow up culture based DST (Day 24) First Line Drugs Agar Culture Based: Isoniazid (INH) Resistant

31 Additional Tests NHLS Results: LPA: Susceptible Culture: Resistant Requested DNA sequencing of KatG gene and inha promoter (Stellenbosch University) Sanger sequencing Susceptible

32 Whole Genome Sequencing Automated pipeline analysis Susceptible katg Resistant population Susceptible population

33 When does heteroresistance become clinically relevant? Mixed population < 1% Mixed population 1% Mixed population 50% Mutant population 90% Phenotypically Suceptible Phenotypically Resistant???? Phenotypically Resistant Phenotypically Resistant

34 Targeted deep sequencing Based on WGS technology Amplification of a target region of the genome X >50,000 x Error rate <0.1% X Reference

35 Allele percentage (%) A lle le p e r c e n ta g e (% ) Allele percentage (%) A lle le p e r c e n ta g e (% ) Allele percentage (%) 1.0 R 6747_Q RDR of g y r A R _ Q R D R o f g y r A 0.9 DST = S 28/08/ /10/ DST = R A A C G T A lle le p e r c e n ta g e (% ) "T" mutant (68,18%) codon 90 (position 7570) C G T "T" mutant (0,17%) codon 90 (position 7570) Allele position in Q RDR of gyra Allele positions in the QRDR of gyra Allele position in Q RDR of gyra Allele positions in the QRDR of gyra R 7515_Q RDR of g y r A "T" mutant (86,14%) codon 90 A C G T DST = R 27/10/ Allele position in Q RDR of gyra Allele positions in the QRDR of gyra

36 gyra 90 T 99.91% gyra 90 T 98.6% gyra 94 C 0.07% gyra 90 T 41.59% gyra 91 C 9.07% gyra 94 C 1.33%

37 Limitations Time to result Data analysis Cost Lack of knowledge on drug-resistance mutations Ability to detect heteroresistance 10 to 15% of the genome is not analyses

38 Acknowledgements