Educational Workshop

Transcription

1 Educational Workshop EW18: The basis of tuberculosis diagnosis and management of cases Arranged with the ESCMID Study Group for Mycobacterial Infections (ESGMYC) Convenor: Emmanuelle Cambau (Paris, FR) Faculty: Emmanuelle Cambau (Paris, FR) Jon Friedland (London, UK) Erik K. Böttger (Zurich, CH) Christoph Lange (Borstel, DE)

2

3 Cambau - New and old tools for tuberculosis diagnosis New and old tools for tuberculosis diagnosis Emmanuelle University Paris Diderot, APHP, Saint Louis-Lariboisière Hospital, NRC mycobacteria, Paris, France Educational Workshop - ECCMID 2012 London the basis in tuberculosis diagnosis and management of cases http//cnrmyctb.free.fr / Strategy for bacteriological diagnosis of tuberculosis Old tools minimum workflow 1 month for M+ 2 months for M0 M+ Specimen AFB Smear microscopy M0 Direct AST Culture 2ndline AST C0 C+ Identification by niacine test If Resistance Antituberculous Susceptibility Testing TST for contacts Strategy for bacteriological diagnosis of tuberculosis New tools Minimum workflow 1 day for M+ 2 weeks for M0 Specimen Smear and microscopic exam Direct amplification testing M+ M0 + = MTBC Culture mutation Rif+ Inh C+ Quick Identification C0 mutation gyra, rrs, emb, pnca, 2ndline AST Antituberculous Susceptibility Testing TST and IGRAs for contacts Genotyping if needed 1

4 Cambau - New and old tools for tuberculosis diagnosis Specificities of tuberculous bacilli (M. tuberculosis complex) Acid-fast bacilli (Ziehl-Neelsen) Pr Slow Emmanuelle growth on rich medium (e.g. Löwenstein-Jensen) Lipid rich cell wall (mycolic acids C60-C90) Acids and NaOH resistance Antibiotic intrinsic resistance Cultures of Mycobacterium tuberculosis complex Require biosafety level 3 laboratory (BSL3) safety measures safety cabinets personal protection Acid Fast bacilli smear examination Appropriate specimen Microscopy + Light-emitting diodes (LED) Auramine staining + automated stainers Ziehl Neelsen staining + automated stainers Sensitivity of 10,000 to 100,000 bacilli per ml 2

5 Cambau - New and old tools for tuberculosis diagnosis Reporting the smear results From ECDC laboratory Handbook: Primary culturing Media Smear pos Smear neg Solid LJ, Coletsos, Middlebrook Liquid* +automated systems days days 5-10 days days * Rapid detection but often required 1-2 supplementary days or secondary culturing for ID and AST testing Identification at the M. tuberculosis complex DNA / RNA Hybridization Ex. ACCUPROBE M. tuberculosis complex (GenProbe, CA, USA) Nucleic acid amplification AND ex hybridization Ex. INNOLiPA Mycobacteria (Innogenetics) GenoType Mycobacteria (Hain) 3

6 Cambau - New and old tools for tuberculosis diagnosis Rapid Identification by immunochromatography How to use 15 min incubation 100µl of liquid culture 100µl of suspension of colonies Control band only BD MGIT TBc Control band and Additional band for M. tuberculosis BIOLINE TB Ag. MPT64 15 minutes, easy and cheap (5 euros) Said JCM 2011 Mycobacterium tuberculosis complex M. tuberculosis M. canettii M. africanum M. microti M. bovis Main tuberculosis bacilli + M. caprae, M. pinnipedii All are tuberculous bacilli M. bovis BCG Vaccinal strains 99 % DNA homology = one species Common ancestor and deletions / subspecies Identical 16S rrna Speciation within the M. tuberculosis complex 1. M. tuberculosis or M. canetti or M. africanum II 2. M. africanum I 3. M. microti 4. M. bovis 5. M. bovis BCG 6. M. bovis caprae genotypemtbc (Hain Lifescience) 4

7 Cambau - New and old tools for tuberculosis diagnosis Serology for tuberculosis Meta-analysis : KR steingart et al. PLoS Medicine 2011 Pulmonary (67 studies; 5,147 patients) and extra pulmonary TB (25 studies;1,809 patients) Sensitivity : 0 to 100% Specificity : 31-59% to 100% Serology for tuberculosis - example of best results - Drug susceptibility testing (DST) for M. tuberculosis complex Control SmInhRif Emb Proportion method on solid or liquid medium Resistance Ratio method Absolute concentration method MIC microtitration Nitrate reductase Alamar Blue... 4 to 12 weeks turn-around time for results from the day of sampling Drobniewski, F.et al. CMI 2007, ECDC laboratory Handbook: 5

8 Cambau - New and old tools for tuberculosis diagnosis Nucleic acid amplification direct testing (NAAT) for detection of M. tuberculosis complex PCR started in 1990 using gene (Hermans JCM 1990) RNA (Boddinghaus JCM 1990) IS6110 (Thierry JCM 1990) 1996 : recommendations CDC / ATS 2008 : recommendations CDC / JAMA Pubmed More than 4400 papers, 330 review, 6 meta-analyses Sarmiento 2003: meta-analysis on smear negative specimens Greco 2009: meta-analysis on smear positive specimens ATS 1997, Ieven and Goosens 1997, Sarmiento 2003, Dinnes 2007, MMWR 2009; Sensitivity of NAAT Smear pos Smear neg N per ml specimen Sensitivity of diagnosis tools for tuberculosis Microscopy NA amplification Culture = = 10 7 M = = = = = M0 PCR+ PCR0 C+ C0 6

9 Cambau - New and old tools for tuberculosis diagnosis Specificity of NAAT No mycobacteria 100 BCG 1000 BCG Results of the interlaboratory study in detection of M.tuberculosis Noordhoek et al. JCM 1996;34: expert laboratories - 20 external quality controls - 10 = no mycobacteria - 5 = 100 BCG - 5 = 1000 BCG NAAT Performances for TB: specificity (1) We would like that only TB patients are NAAT+ Suspicion of smear positive pulmonary TB Specificity = 98% Suspicion of smear negative pulmonary TB Specificity = 86% Suspicion of extra-pulmonary TB Specificity = 74.5% (62% on CSF) 11 studies with Xpert MTB/RIF Specificity = 98.6% Laraque, CID 2009; Helb 2010; Boehme 2010 and 2011; Rachow 2011; Marlowe 2011; Armand 2011; Theron 2011 ; Bowles 2011; Scott 2011; Miller, 2011; Teo, 2011 NAAT Performances for TB: sensitivity (2) We would like that TB patients with smear negative specimens are NAAT+ Sarmiento 2003: meta-analysis for smearnegative TB cases «Sensitivity ranged from 9 to 100%» «Specificity ranged 25 to 100%» => Low positive predictive values 11 studies with Xpert MTB/RIF Range: 47% - 83% Helb 2010; Boehme 2010 and 2011; Rachow 2011; Marlowe 2011; Armand 2011 Theron 2011 ; Bowles 2011; Scott 2011; Miller, 2011; Teo,

10 Cambau - New and old tools for tuberculosis diagnosis Indications of NAAT and tuberculosis Suspicion of pulmonary tuberculosis Not for extra pulmonary TB (except if smear positive?) Not for sequellae Not for latent TB Not for mycobacteriosis (except if smear positive?) All smear positive cases=> Positive diagnosis of TB Smear negative cases if highly suspicion of TB (prevalence > 5% => PPV> 50%) => early diagnosis of TB ATS 1997, Ieven and Goosens 1997, Sarmiento 2003, Dinnes 2007, MMWR 2009; Molecular detection of Multidrug resistant-tb cases Laboratories should aim to identify TB and rifampicin resistance in over 90% of cases directly from smear + sputum where resources are available for this rapidly within 1-2 days Innogenetics (Belgium) Wild-type probes Resistant probes 8

11 Cambau - New and old tools for tuberculosis diagnosis Genotype MTBDRplus HAIN Lifescience (Germany) Gene Xpert MTB/RIF Cepheid (USA) Boehme CC et al. NEJM 2010 Performances of molecular kits for direct detection of MDR-TB Identification of M. tuberculosis complex AFB YES, all kits 98 to 100% sensitivity on smear-positive respiratory specimens Detection of drug resistance associated mutations rpob (beta subunit of RNA polymerase) mutations associated with rifampicin resistance Yes, all with a % sensitivity Isoniazid resistance associated mutations in katg and inha = GenoType MTBDRplus Traore 2000, Truffot 2002, Brossier 2006, Hillman 2005, Hillman 2007, Boehme 2010, Held

12 Cambau - New and old tools for tuberculosis diagnosis Transmission and latent TB Patient with probable tuberculosis AFB pos = risk of 90% Contact AFB neg = risk of 10% Contact person: other patient, Hospital staff, family especially children risk of infection: 10 to 30% TST? IGRA? Behr et al. 1999, Escombe et al. 2008, Aissa et al Interferon Gamma release Assays ECDC recommendations reviews and meta-analyses in 2011: CID 2011: Herrera V. et al (52: JAIDS 2011: Cattamanchi A et al. (56: ) Eur Respir 2011: Diel R.et al. (37:88-99) CMI 2011: Denkinger CM et al. (17: ) Curr Opin Rheumatol 2011: Smith R et al. (23: ) JID 2011: Metcalfe JZ et al. (204:S ) IGRA tests for TB Production of IFN by T lymphocytes after stimulation with specific antigens ESAT-6, CFP-10 (TB7,7) Sang total ou CMNS Ag M Tuberclosis ESAT 6, CFP10 Incubation O/N ou 48h Quantiferon TB-Gold in Tube (Cellestis) Sécretion IFNg T mémoires mesure IFNg SPOT-TB (Oxford immunotech) ELISA ELISpot IFNg pg/ml SFC/10 6 = in vitro TST with TB specific antigens lacking in BCG strains 10

13 Cambau - New and old tools for tuberculosis diagnosis New tools versus Old tools conclusions Turn around times are shorter => Tuberculosis diagnosis is obtained earlier Main new tools Molecular detection of resistance Rapid identification of MTB cultures Still Quality and expertise : Skilled technicians Evaluation of your pratices Rush-Gerdes et al More information ESCMID group on mycobacterial infections (ESGMYC): Meeting on Sunday April 1st in Meeting room 8 from to Website : http: // Surveillance reports for TB by the European Center for Disease Prevention and Control (ECDC) : Laboratory Handbook from ECDC and European reference center network for Tb (ERLNet-TB) «Mastering the basics of TB control» 11

14 Friedland - The Host Immune Responses in Tuberculosis The Host Immune Responses in Tuberculosis Jon S. Friedland Dept of Infectious Diseases & Immunity (Hammersmith campus) Tuberculosis: the problem 1.5 million deaths Cure million prevalent cases 3.3 billion uninfected 1.7 billion infected Tissue damage in tuberculosis 12

15 Friedland - The Host Immune Responses in Tuberculosis Host Defence to Tuberculosis M. tuberculosis mrna Innate immunity - cytokines: pro- & anti-inflammatory - chemokines - MMPs Acquired immunity - CD4 cells:t H 1 v T H 2 v T H 17 responses - and T cells - CD8 T cells: MHC, CD1, Innate Immunity: first thoughts Innate Immunity Concepts 1 The whole early immune system - invertebrates Prevents infection May eliminate infection with or without interaction with acquired immune response Is an early response to infection Involves phagocytic cells 13

16 Friedland - The Host Immune Responses in Tuberculosis Innate Immunity Concepts 2 May drive adaptive immune responses Has no memory Recognises microbial conserved structures not found in host Does not react to self Often targets products necessary for microbial survival Components encoded in germline (not acquired by somatic mutation) Components of Innate Immunity Physical barriers respiratory epithelium, mucociliary escalator Cellular barriers immune active Circulating effector leucocytes Monocytes/macrophages Neutrophils NK cells Circulating proteins Complement Collectins and pentraxins (eg CRP) Antimicrobial peptides (eg defensins) Cytokines Local Enzymes The Granuloma 14

17 Friedland - The Host Immune Responses in Tuberculosis Mycobacteria escape the phagosome L = lysosome M = mitochondria * = Mtb in lysosome = Mtb in cytosol (Van der Wel Cell 2007; 129: 1287) Macrophages and M. tuberculosis: Inside the phagosome Failure of phagosome to acidify by exclusion of vesicular proton ATPase Lysosome associated membrane protein present on phagosome Lipoarabinomannan is outside phagosome Antigen presentation to T cells via CD4, CD8 & CD1 Granuloma macrophages act as incubators for mycobacteria White cells: newly arrived (Davies and Ramakrishan Cell 2009)...and macrophages disseminate mycobacterial infection (Volkman et al Science 2011;327:466-9) 15

18 Friedland - The Host Immune Responses in Tuberculosis Cytokine biology Secretion is usual transcriptionally regulated and relatively transient Cytokines are paracrine, autocrine and endocrine: local and systemic Action is via binding to specific receptors Regulation of cytokines and their receptors is complex Effector functions are multiple and often cell specific May act synergistically or antagonistically There are families of cytokines and of their receptors with redundancy in the system Innate immunity - the balance PROINFLAMMATORY DAMAGE High TNF IL MMPs CONTROL OF INFECTION Low TNF IL-1+ IL-10 TGF DESTRUCTIVE PROTECTIVE Macrophages and M. tuberculosis: Tumour necrosis factor- Monocyte secretion induced by mycobacterial lipoarabinomannan & proteins (TLR2/4/6) Apex of a cascade of pro-inflammatory cytokines Drives cachexia & metabolic disturbances modulates immune function Inhibits mycobacterial growth in murine macrophages Required for granuloma formation Co-factor in IFN mediated lethality Present in tissues of patients Anti-TNF Rx results in activation of tuberculosis 16

19 Friedland - The Host Immune Responses in Tuberculosis Interferon- deficient patients Very susceptible to mycobacterial infections Susceptible to salmonella (also intra-cellular) May have deletions in the IFN Receptor Can be an acquired autoimmune antibody Lesser clinical variants have IL-12 system mutations Innate immunity and microbial killing Reactive oxygen intermediates Free radicals (eg superoxide) Chronic Granulomatous Disease Reactive nitrogen intermediates Lysosomal enzymes Phagolysosomes Proteolytic enzymes Elastase Microbiocidal peptides Microbial killing: Defensins & cathelicidins Cathelicidins Variable peptide structure with conserved N-terminal cathelin domain in phagocytic cells & epithelial cells Often undergo proteolytic cleavage; stored as proforms Vitamin D-dependent release of cathelicidins (Science 2006;311:1770-3) Defensins in phagocytic cells & epithelial cells at least 9 different human defensin genes microbiocidal at micromolar conc; stored in granules target bacteria, fungi and viruses form pores in cell membranes may be chemotactic and affect chemokine receptors (CCR6) 17

20 Friedland - The Host Immune Responses in Tuberculosis Tadpole tail degrades collagen A slice of tadpole tail on a collagen gel the clear zone shows collagenase activity Matrix metalloproteinases (MMPs) Family of zinc-containing proteases classified by substrate specificity (gelatinases, collagenases, stromelysins, MT-MMPs) Antagonised by Tissue Inhibitors of Metalloproteinases (TIMPs) Key in tissue remodelling / embryonic development Important immunological functions leukocyte migration Activate /de-activate cytokines (eg TNF-, CXCL8) Excessive MMP activity destructive (e.g. rheumatoid arthritis, emphysema) TB lymphadenitis: MMP-9,TIMP-1 and the matrix degrading phenotype (A) (B) MMP TIMP negative control 400 Confirmed in extensive cellular & human Investigations in lung & CNS TB 18

21 Friedland - The Host Immune Responses in Tuberculosis TB and monocyte-derived MMP s In vivo expression of MMP-1 in patients with pulmonary Tuberculosis Monocytic cells Epithelial cells MMP-1/3 and tissue destruction in tuberculosis patients (A) (B) (C) 19

22 Friedland - The Host Immune Responses in Tuberculosis Endogenous inhibition of tissue destruction in tuberculosis patients (No significant differences in TIMP-3/4) MMP-1 drives matrix destruction in vivo (J. Clin. Invest. 2011;121: ) MMP-1 induced collagen destruction in tuberculosis (J. Clin. Invest. 2011; 121: ) 20

23 Friedland - The Host Immune Responses in Tuberculosis A New Paradigm of Tissue Damage in Tuberculosis (Science Trans. Med. 2011;3:71 ps6) Do we already use immuno-modulation? p-aminosalicylic acid (PAS) & signalling switch points Introduced as treatment for Tb in 1947 Mechanism of action uncertain Increasingly important with the emergence of MDR-Tb Derivative of salicylic acid Salicylic acid PAS Effect of PAS on M. tb growth 21

24 Friedland - The Host Immune Responses in Tuberculosis PAS inhibits human macrophage MMP-1 but not MMP-7 gene expression & secretion M. tb TLR2 SB p38 PAS COXII PGE 2 B 2 camp MMP-1 MMP-7 MMP inhibition as a novel therapeutic approach in Tuberculosis Tetracyclines: non-specific MMP inhibitors (AJRCCM 2012 in press) Ro (Cipemastat, Trocade) - selective collagenase inhibitor - orally available - already passed phase III clinical trial in rheumatoid arthrititis 22

25 Friedland - The Host Immune Responses in Tuberculosis Team & Collaborators Paul Elkington Moerida Belton Sara Brilha Danni Kirwan Andre Kubler Rachel Moores Cecilia O Kane Catherine Ong Bernadette Pederson Lucinda Rand Tara Sathyamoorthy Shivani Singh Naomi Walker Ashley Whittington Dept Immunohistochemisty, ICL Federico Roncaroli University of Columbia, NYC, USA Dr Jeanine D Armiento Wellcome Trust Clinical Research Unit & Centre for Tropical Diseases, Vietnam Jeremy Farrar, Guy Thwaites, Hong Chau Tran & Colleagues Universidad Cayetano, Peru & Johns Hopkins, USA Bob Gilman Cesar Urgarte-Gil K-RITH, South Africa & Johns Hopkins, USA Bill Bishai 23

26 Böttger - Drug Resistance in Mycobacterium tuberculosis Molecular Mechanisms and Clinical Implications Institute of Medical Microbiology Drug Resistance in Mycobacterium tuberculosis Molecular Mechanisms and Clinical Implications Prof. Dr. Erik C. Böttger Institute of Medical Microbiology University of Zurich 3/15/2012 Page 1 Institute of Medical Microbiology Drug Resistance in Mycobacterium tuberculosis Genetic basis of resistance Laboratory drug susceptibility testing Clinical implications some complex examples for illustration ECCMID Institute of Medical Microbiology Drug Resistance in Mycobacterium tuberculosis Drug resistance in M. tuberculosis and therapy of drug-resistant TB is complex but it follows simple principles Base thinking on established mechanisms in microbiology and on common sense sapere aude! ECCMID

27 Böttger - Drug Resistance in Mycobacterium tuberculosis Molecular Mechanisms and Clinical Implications Adequate Treatment of Tuberculosis Disease Requires Combination Drug Therapy Frequency of drug resistant mutants in the bacterial population Isoniazid 10-6 Rifampicin 10-7 Streptomycin 10-7 Monotherapy invariably results in early (3-6 months) clinical failure following instalment of therapy due to rapid development of resistance Probability Relapse of strains simultaneous are drug resistance resistant towards Isoniazid, Rifampicin, Streptomycin is Combination therapy to prevent resistance development ECCMID Adequate Treatment of Tuberculosis Disease Requires Drug Therapy for 6 Months Standard Short Course Therapy (SSC) month 1 and 2 Isoniazid, Rifampicin, Ethambutol, Pyrazinamid month 3 to 6 Isoniazid, Rifampicin shortening treatment length results in high rates of late (6-12 months) clinical relapse following completion of therapy due to failure of eradication Basel TB ECCMID

28 Böttger - Drug Resistance in Mycobacterium tuberculosis Molecular Mechanisms and Clinical Implications Prevalence of any drug resistance among new TB cases, Prevalence of MDR-TB among new TB cases, Anti-Tuberculosis Drug Resistance in the World; Third Global Report WHO, 2004 ECCMID Drug resistance in Mycobacterium tuberculosis is exclusively due to chromosomal alterations, e.g. mutations, deletions drug target drug activating enzymes Multi-drug resistance is due to accumulation of chromosomal alterations Plasmid- or transposon-mediated mechanisms of resistance are absent ECCMID Mycobacterial Drug Resistance Mechanisms Drug Resistance Mechanism Isoniazid katg inha (target) Rifampicin rpob (target) Ethambutol embb (target) Pyrazinamid pnca Streptomycin rpsl (target) rrs (target) Aminoglycosides rrs (target) Quinolones gyra (target) Ethionamid etha inha (target) ECCMID

29 Böttger - Drug Resistance in Mycobacterium tuberculosis Molecular Mechanisms and Clinical Implications Institute of Medical Microbiology Mutation and Resistance Drug Resistance in Mycobacterium tuberculosis Molecular Mechanisms and Clinical Implications The finding of a mutation per se does not imply drug resistance strain-specific genetic polymorphism irrelevant spontaneous mutation not connected to drug resistance affect susceptibility but not confer clinical resistance Correlate genetic sequences with phenotypic levels of drug susceptibility ECCMID Resistance is defined as a decrease in sensitivity of sufficient degree to be reasonably certain that the strain concerned is different from a sample of wild strains of human type that have never come into contact with the drug. Mitchison, 1962 ECCMID Institute of Medical Microbiology Peculiarities of Mycobacterial Drug Susceptibility Testing (DST) 1. Critical proportion (proportion method) 2. Critical concentration (drug concentration) ECCMID

30 Böttger - Drug Resistance in Mycobacterium tuberculosis Molecular Mechanisms and Clinical Implications Institute of Medical Microbiology The Proportion Method Principle of the method "All strains of tuberculosis contain some bacilli that are resistant to antibacillary drugs - in resistant strains, the proportion of such bacilli is considerably higher than in sensitive strains." Drug Isoniazid Rifampicin Pyrazinamid Ethambutol Streptomycin Canetti et al. Bull. WHO 1969, 41: Concentration ( g/ml) Critical Proportion for Resistance % ECCMID Institute of Medical Microbiology The Critical Concentration mostly a single drug concentration termed critical concentration) is used to define susceptibility vs. resistance Antimicrobial agent MIC (µg/ml) of susceptible M. tuberculosis Conc (µg/ml) in serum BACTEC 12B low Conc (µg/ml) BACTEC 12B high INH RMP PZA EMB SM ECCMID Institute of Medical Microbiology Proportion and Critical Concentration Wild Type Population ECCMID

31 Böttger - Drug Resistance in Mycobacterium tuberculosis Molecular Mechanisms and Clinical Implications Institute of Medical Microbiology Mutation and Resistance Drug Resistance in Mycobacterium tuberculosis Molecular Mechanisms and Clinical Implications The finding of a mutation per se does not imply drug resistance strain-specific genetic polymorphism irrelevant spontaneous mutation not connected to drug resistance affect susceptibility but not confer clinical resistance Correlate genetic sequences with phenotypic levels of drug susceptibility ECCMID RIFAMPICIN wt population high level resistance, various rpob mutations e.g. rpob S531L, H526Y, H526D Schematized changes in drug susceptibility upon mutational alterations exemplary Gaussian distributions of a population s drug susceptibility % Population Rifampicin: predominantly high-level drug resistance associated with mutations in rpob Drug Concentration mg/l STREPTOMYCIN low-level resistance, various mutations moderate resistance high level resistance, wt e.g. Δ gldb e.g. rrs, rpsl K87 mut i.e. rpsl K42L population % Population Drug Concentration mg/l ISONIAZID low-level resistance, moderate and high level resistance, various mutations various katg mutations wt e.g. inha e.g. katg S315T population Streptomycin: various levels of phenotypic resistance low-, moderate-, and high-level drug resistance; different phenotypic resistance levels are associated with distinct chromosomal mutations. Isoniazid: various levels of phenotypic resistance low-, moderate-, and high-level drug resistance; different chromosomal mutations are associated with distinct phenotypic resistance levels; in addition, a given resistance mutation may be associated with variable phenotypic expression of drug resistance. = critical concentration = drug serum level % Population "critical concentrations" drug serum level Drug Concentration mg/l from Böttger, E.C. and Springer, B. 2009, Mycobacterium tuberculosis: drug resistance and genetic mechanisms facts, artifacts and fallacies. In: HIV and tuberculosis: a deadly liaison, eds. S.H.E. Kaufmann and B. Walker, Wiley VCH, p ECCMID Institute of Medical Microbiology Drug Resistance in Mycobacterium tuberculosis Molecular Mechanisms and Clinical Implications Low-Level Drug Resistance in M. tuberculosis Drug Genetic Frequency in locus clinical isolates Isoniazid inha 20-30% Streptomycin gldb 20-30% Capreomycin tlya >30% Kanamycin eis 20-30% Ethambutol embb >50% Low-level drug resistance makes up a significant part of drug resistant M. tuberculosis Banerjee et al. Science 1994, 263: 227 Johansen et al. Mol. Cell 2006, 23: 173 Meier et al. Antimicrob. Agents Chemother. 1996, 40:2452 Zaunbrecher et al. Proc. Natl. Acad. Sci. USA 2009, 106: Okamoto et al. Mol. Microbiol. 2007, 63: 1096 Telenti et al. Nat. Med. 1997, 3: 567 ECCMID

32 Böttger - Drug Resistance in Mycobacterium tuberculosis Molecular Mechanisms and Clinical Implications Institute of Medical Microbiology Mycobacterial Drug Resistance and Susceptibility Testing Drug Resistance in M. tuberculosis is a mixed bag: significant heterogeneity is present, i.e., low-, moderate- and high-level drug resistance. ECCMID Institute of Medical Microbiology Some Complex Examples for Illustration Ethambutol Fluoroquinolones Capreomycin ECCMID Ethambutol Drug Susceptibility Testing and Resistance Mechanisms MIC wild-type mg/l Critical Concentration mg/l Drug Serum Levels mg/l the only small difference between MIC of susceptible wild-type bacteria and the drug concentration used for in-vitro testing makes in-vitro test results unreliable MIC embb mutants mg/l mutations in embb (aa 306) confer low-level drug resistance, i.e., a 3-4 fold increase in MIC overlapping MIC distributions for wt and embb mutant strains dependent on the critical concentration chosen, susceptible isolates may carry an embb mutation and resistant isolates may show a wild-type embb Starks et al. Antimicrob. Agents Chemother. 2009, 53: Plinke et al. Antimicrob. Agents Chemother. 2011, Safi et al. Antimicrob. Agents Chemother. 2010, 54: Sirgel et al. submitted for publication no data exist correlating in-vitro test results with in-vivo outcome ECCMID

33 Böttger - Drug Resistance in Mycobacterium tuberculosis Molecular Mechanisms and Clinical Implications Institute of Medical Microbiology Can we Explain how Quinolone Resistance Rapidly Developed in MDR, Despite DOTS Plus? frequent observation in Eastern Europe and South Africa numerous hypotheses have been put forward: - induction of efflux pumps - antagonistic drug interactions - mixed infections and re-infections MIC mg/l a,b,c wt A90V D94A D94N D94G C max (mg/l) Ofloxacin Moxifloxacin a Kam et al. Microb. Drug Res. 2000, 12: 7-11 b Poissy et al. Antimicrob. Agents Chemother. 2010, 54: c Sirgel et al. J. Antimicrob. Chemother. 2012, in press Ofloxacin will result in rapid resistance development as MIC of wild-type is close to in-vivo drug concentrations use Moxifloxacin for treatment of MDR-TB ECCMID Ribosomal Binding Sites: Tuberactinomycin Capreomycin and Aminoglycoside Kanamycin Binding site of capreomycin. Capreomycin binds in the ribosomal interface and forms hydrogen-bond interactions with h44 of the 16S rrna (orange) and H69 of the 23S rrna (blue). Binding site of kanamycin. Kanamycin binds to the ribosomal A-site and makes hydrogen-bond interactions with h44 of the 16S rrna. ECCMID Institute of Medical Microbiology Mutations in 16S rrna A1408G and Resistance to Amikacin and Capreomycin in M. tuberculosis Amikacin, Kanamycin: MIC wt ~1.0 mg/l Critical concentration: 2 mg/l Drug serum concentrations: mg/l Mutation A1408G confers high-level resistance: MIC >512 mg/l Sander et al. Mol. Microbiol. 1996, 22:841 Capreomycin: MIC wt ~2.5 mg/l Critical concentration: 5 mg/l Drug serum concentrations: mg/l Mutation A1408G confers low-level resistance: MIC mg/l Maur et al. Antimicrob. Agents Chemother. 2005, 49:3192 Feuerriegel et al. Antimicrob. Agents Chemother. 2009, 53:3356 Jugheli et al. Antimicrob. Agents Chemother. 2009, 53:5064 Sirgel et al. Microb. Drug Res. 2012, in press ECCMID

34 Böttger - Drug Resistance in Mycobacterium tuberculosis Molecular Mechanisms and Clinical Implications Institute of Medical Microbiology Genetic Analyses to Identify Drug Resistance - Limitations Mutations need to be correlated to quantitative levels of drug resistance to allow for meaningful conclusions regarding resistance and implementation of treatment regimens ECCMID Institute of Medical Microbiology Mycobacterial Drug Susceptibility Testing The Limitations of Current Procedures Drug Resistance in M. tuberculosis is a mixed bag: significant heterogeneity is present, i.e., low-, moderate- and high-level drug resistance. Regardless of low-, moderate-, or high-level drug resistance any corresponding clinical isolate is categorized as resistant in the diagnostic laboratory due to the procedure of critical concentration testing. However, the biological impact of low- versus high-level drug resistance is to be different. drug concentrations present in-vivo tuberculosis chemotherapy is a combination therapy of compounds which act in part in an additive / synergistic fashion and which includes agents targeting cell wall synthesis The critical concentration corresponds to the ECOFF (epidemiological cut-off) value, rather than a clinical breakpoint. ECCMID Institute of Medical Microbiology Mycobacterial Drug Resistance and Susceptibility Testing The clinical implications of any laboratory resistance are to a large extent dependent on quantitative aspects of resistance, i.e., low-level drug resistance may not correspond to clinical resistance Implement semi-quantitative measures of drugsusceptibility testing. Prospective studies are required to correlate the results of semi-quantitative AST with clinical outcome. ECCMID

35 Böttger - Drug Resistance in Mycobacterium tuberculosis Molecular Mechanisms and Clinical Implications Institute of Medical Microbiology Acknowledgements A European Study Group to establish standards for semi-quantitative drug susceptibility testing in Mycobacterium tuberculosis Laboratory Staff (Academics, Technicians) Support Home Institutions Erik C. Böttger Emmanuelle Cambau Maryse Fauville-Dufaux Sven Hoffner Maria Luisa Perez del Molino Sabine Rüsch-Gerdes Dick van Soolingen Enrico Tortoli Miguel Viveiros ECCMID Institute of Medical Microbiology Thank you for your attention. ECCMID

36 Lange - Treatment of susceptible and drug-resistant cases of tuberculosis Treatment of susceptible and drug resistant cases of tuberculosis Christoph Christoph Lange Lange Tuberculosis 8. position of leading causes of deaths 1/3 of the world s population could be infected > 80% can be cured prevention can be > 90% effective WHO 2012 Estimated tuberculosis incidence rates 2009 WHO

37 Lange - Treatment of susceptible and drug-resistant cases of tuberculosis Standard treatment of tuberculosis: 2HRZE/4HR Yew WW et al., EurRespirJ 2011 Emerging M. tuberculosis drug resistance Definition of M. tuberculosis drug resistance Monodrug-resistance Polydrug-resistance Multidrug-resistance MDR Extensively drug-resistance XDR Resistance against one (first-line) drug, Resistance against > 1 (firstline) drugs, but sensitivity to INH and/or RMP Resistance against at least INH and RMP MDR plus resistance to - any fluoroquinolone - amikacin, capreomycin or kanamycin Uncomplicated treatment. Duration of treatment may be prolonged Usually uncomplicated treatment. Duration of treatment is is prolonged Complicated treatment. Duration of treatment is prolonged to > 18 months Outcome depends on level of drug resistance Complicated treatment. Duration of treatment is prolonged to > 24 months Outcome depends on level of drug resistance 35

38 Lange - Treatment of susceptible and drug-resistant cases of tuberculosis Treatment of mono and poly drug resistant TB M/XDR TB global numbers new cases of MDR TB in deaths due to MDR TB in % of cases globally are MDR TB XDR TB confirmed in 58 countries WHO 2011 Notified MDR TB cases 2010 WHO

39 Lange - Treatment of susceptible and drug-resistant cases of tuberculosis Estimated MDR TB prevalence rates 2010 WHO 2011 MDR TB in the European region ECDC, WHO 2011 M. tuberculosis drug resistance European numbers cases of confirmed MDR TB in the EU/EEA in % of TB cases are MDR TB % in Baltic countries 11.2 % in Romania 19.8 % deaths attributed to MDR TB in 2009 XDR TB in 15/28 states 7.1 % of MDR TB are XDR TB 37

40 Lange - Treatment of susceptible and drug-resistant cases of tuberculosis Treatment outcome of MDR TB in Europe 2006 and % 35% 30% 25% 2007 cohort (15 reporting countries) 2006 cohort (15 reporting countries) 20% 15% 10% 5% 0% Success Died Failed Defaulted Still on treatment Transferred or unknown ECDC, WHO 2011 Groups of drugs for M/XDR TB treatment 1. Orally available 1st line drugs Ethambutol Pyrazinamide 2. Injectible drugs Amikacin Capreomycin Kanamycin Streptomycin 3. Fluoroquinolones Levofloxacin Moxifloxacin 4. Orally available drugs (2nd line) Cycloserine (Terizidon) P Aminosalicylic Acid Etionamide/Protionamide 5. Additional drugs with unproven Amoxicillin/Clavulanic Acid efficacy against M. tuberculosis Clarithromycin Clofazimine Imipinem/Cilastin Isoniazid (high dose) Meropenem/Clavulanic Acid Linezolid WHO 38

41 Lange - Treatment of susceptible and drug-resistant cases of tuberculosis 2011 WHO recommendations: FLQs In the treatment of patients with MDR TB, a fluoroquinolone should be used In the treatment of patients with MDR TB, a latergeneration fluoroquinolone rather than an earlier generation fluoroquinolone should be used. WHO Moxifloxacin for the treatment against tuberculosis n = 74 n = 72 Conde et al. Lancet

42 Lange - Treatment of susceptible and drug-resistant cases of tuberculosis Moxifloxacin for the treatment against tuberculosis Dormann et al. AJRCCM 2009 FLQ-drug resistance of M. tuberculosis in Mumbai, India Agrawal et al. IJTLD WHO recommendations: 4 +PZA In the treatment of patients with MDR TB, ethionamide (or prothionamide) should be used In the treatment of patients with MDR TB, four second line anti TB drugs likely to be effective (including a parenteral agent from among the second line injectables kanamycin, amikacin or capreomycin), as well as pyrazinamide, should be included in the intensive phase (the initial part of a course of treatment during which a parenteral agent is used) WHO 40

43 Lange - Treatment of susceptible and drug-resistant cases of tuberculosis 2011 WHO recommendations: coctail recipe In the treatment of patients with MDR TB, regimens should include at least pyrazinamide, a fluoroquinolone, a parenteral agent (kanamycin, amikacin or capreomycin), ethionamide (or prothionamide), and either cycloserine or p aminosalicylic acid (PAS) if cycloserine cannot be used. WHO 2011 WHO recommendations: duration In the treatment of patients with MDR TB, an intensive phase of at least 8 months duration is recommended In the treatment of patients with MDR TB, a total treatment duration of at least 20 months is recommended in patients without any previous MDR TB treatment. WHO Designing a M/XDR drugregimen 1. Orally available 1st line drugs Ethambutol Pyrazinamide 2. Injectible drugs Amikacin Capreomycin Kanamycin Streptomycin 3. Fluoroquinolones Levofloxacin Moxifloxacin 4. Orally available drugs (2nd line) Cycloserine (Terizidon) P Aminosalicylic Acid Etionamide/Protionamide 5. Additional drugs with unproven Amoxicillin/Clavulanic Acid efficacy against M. tuberculosis Clarithromycin Clofazimine Imipinem/Cilastin Isoniazid (high dose) Meropenem/Clavulanic Acid Linezolid WHO 41

44 Lange - Treatment of susceptible and drug-resistant cases of tuberculosis Diarylquinoline TMC207 for the treatment against MDR-tuberculosis Diacon et al. NEJM 2009 TB drug pipeline (March 2012) Treatment Action Group Never add to a failing a single drug regimen 42

45 Lange - Treatment of susceptible and drug-resistant cases of tuberculosis Grzybowski and Enarson 1978 High prevalence M/XDR TB Grzybowski and Enarson 1978, ammended