EW08 01 Cambau.ppt [Kompatibilitätsmodus]

Educational WorkshopEW08: Resistance to anti-tuberculous drugsarranged with the ESCMID Study Group for Antimicrobial Resistance Surveillance (ESGARS)

Convenor: Emmanuelle Cambau (Paris, FR)( , )

Faculty: Emmanuelle Cambau (Paris, FR)Francis Drobniewski (London, UK)Andrei Dadu (Copenhagen, DK; nopresentation submitted)Thomas Schön (Kalmar, SE)( )

Cambau - Susceptibility testing of M. tuberculosis strains

Susceptibility testing of Mycobacterium tuberculosis strains

Emmanuelle CAMBAU - ECCMID 2011- EW8

y

Emmanuelle CAMBAU, ESGARSUniversity Paris Diderot,

Saint Louis-Lariboisière Hospital, Paris, France

M b i BCG

M. tuberculosis M. canettii M. africanum M. bovisM. microtiMain

tuberculosis bacilli

Mycobacterium tuberculosis complex

+ M. caprae, M. pinnipedii


M. bovis BCGVaccinal strains

99 % DNA homology = one speciesCommon ancestor and deletions / subspecies

Identical 16S rRNA

All are tuberculous bacilli

M. bovis BCG

M. tuberculosis M. canettii M. africanum M. bovisM. microti

Main tuberculosis bacilli

Mycobacterium tuberculosis complex

+ M. caprae, M. pinnipedii


M. bovis BCGVaccinal strain

99 % DNA homology = one speciesCommon ancestor and deletions / subspecies

Identical 16S rRNA

All are tuberculous bacilliPyrazinamidePyrazinamide

resistantresistant

3


Specificities of tuberculous bacilli(M. tuberculosis complex)

Acid-fast bacilli (Ziehl-Neelsen)


Slow growth on rich medium(e.g. Löwenstein-Jensen)

Lipid rich cell wall (mycolic acids C60-C90)⇒Acids and NaOH resistance⇒Antibiotic intrinsic resistance

Requires biosafety level 3 laboratory

Cultures ofMycobacterium

tuberculosiscomplex


level 3 laboratory (BSL3)

•safety measures•safety cabinets •personal protection

World estimate of tuberculosis cases in 2008Incident cases : 9.4 Millions (139 /100 000 ha)

Prevalent cases: 11.1 Millions (164 /100 000 ha)Mortality: 1.3 Millions (28/100 000)

MDR-TB: 0.44 Millions (4 % of cases)


()

4


Objectives of performing drug susceptibility testing of M.tuberculosis

(DST-MTB)?

1. Individual treatment management of a t b l i


tuberculosis case

2. Drug-resistance surveillance at the scale of a hospital, city, region or country


(DST-MTB)?

1. Individual treatment management of a tuberculosis case


tuberculosis case – Detect resistance– Assess susceptibility– Although the treatment is standardized,

report results as soon as possible


(DST-MTB)?

2. Drug-resistance surveillance at the scale f h it l it i t


of a hospital, city, region or country– National rates of resistance– ECDC-EuroTB surveillance– WHO surveys

5


Primary and secondary resistance

• Secondary resistance or acquired resistance – Drug resistance appearing during treatment – Results of selection and multiplication of resistant

mutant isolates pre-existing in the tubercle bacillus population before therapy


p p py– Resistance rate observed in re-treatment cases

• Primary resistance– Drug resistance observed before treatment, – Consequence of exposure to a drug-resistant source

of infection– Resistance rate observed in new cases

Which drugs are tested for DST-MTB?

• First line drugs = drugs used in the standardized treatment of Tuberculosis H(6)R(6)E(2)Z(2)– Isoniazid (H) and Rifampicin (R) are the major

antituberculous drugsEthambutol (E) is a companion drug important in case


– Ethambutol (E) is a companion drug important in case of isoniazid resistance

– Streptomycin (S), is not now part of most of standard treatments but still reflect the past treatment

– Pyrazinamide (Z) is part of the standard treatment, but difficulties in obtaining DST reliable results

• Second line drugs– will be presented by Thomas Shön

When DST-TB should be done for the patient ?

• Definite case of tuberculosis – First positive culture for the MTBC – First specimens smear-positive for acid-fast


p pbacilli if culture is not yet available

• Should be repeated when the culture is still positive after 5 months of treatment

6


Direct and indirect testing

• Indirect testing– Cultures of M. tuberculosis complex (after

identification)– Cultures in liquid or solid media (after


Cultures in liquid or solid media (after checking purity)

• Direct testing – smear-positive specimens after classical

decontamination

Definitions• Resistance : decrease in sensitivity compared to wild type strains

(never came into contact with the drug). unlikely to show clinical responsiveness to the drug

• Susceptibility : level of sensitivity not significantly different from wild type strains

lik l t h li i l i t th d


likely to show clinical responsiveness to the drug

• Critical concentration: the lowest drug concentration at which wild type strains did not grow

• Critical proportion: The lowest percentage of the strain population that may grow at the critical concentration and which is associated with clinical ineffectiveness of that drug – 1% for first line drugs

Heifets L, ARRD 1988Mitchison DA, 1968

Performances of DST-MTB methods

• Detect resistance – No false resistant : low specificity– No false susceptibility : low sensitivity


• Assess susceptibility– No false resistant : low sensitivity– No false susceptibility : low specificity

Kim et al. 2004

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Methods for DST-MTB

• Proportion method on solid or in liquid medium– use critical concentrations and critical proportions

• Resistance Ratio method– Use MIC determination and comparison with wild type


Use MIC determination and comparison with wild type or reference strains

• Absolute concentration method– Use critical concentrations

• MIC determination– To define in vitro activity of a drug

• Many others Drobniewski, F.et al. CMI 2007.

Media and automates• Media

– egg-based solid: Löwenstein-Jensen, Ogawa, Coletsos

– synthetic solid or liquid: Middlebrook (7H9, 7H10, 7H11)


)

• Automate vs. Manual– Handling – Safety– Number of DST per month– Cost

Availability of DST results for surveillance of resistance in tuberculosis


1% new cases 3% for retreated cases(10-20% for Europe)

WHO, 2009 report

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The Proportion Method

• LJ medium, and then modified for liquid medium• calculates the proportion of resistant bacilli present in a

strain• The critical proportion was assessed by a study of a

patient cohort in the 1960s who failed tuberculosis treatment


treatment – > 1% : resistant because risk of failure and relapse with

overgrowth of resistant mutants– < 1% : sensitive because no risk of failure if at least two active

drugs are given• Growth controls: 2 to 3 inocula (10-fold diluted) growing

in absence of drug

Canetti 1965.. Am Rev Respir Dis 92:687-703

Proportion method on LJ


T 10-1 T 10-3 INH 0,2 10-1

R 10-1


100 col 1 col 0 colS S

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T 10-1 H (1mg/l)

10-1

R10-1

E10-1


100 col 200 col 1 col200 colRR S

HS R ETémoin1/100

Proportion method in liquid MGIT tubes


R SSS

Critical concentrations in mg/l

Drug LJ MGIT Versatrek

Isoniazid 0.2 0.1 0.1


Rifampicin 40 1 1

Ethambutol 2 5 5

Streptomycin 4 1 -

Pyrazinamide 200 100 300

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Endpoints for DST-TB results• Critical concentration

– MIC (or concentration inhibiting growth) is below => susceptible

– MIC is higher => resistant• Critical proportion


• Critical proportion– measured proportion below => susceptible– measured proportion higher => resistant

• Time to mature growth :– 12 days, 21 days, 28 days or 42 days depending on

the media and on the method– If the strain grows slower (such as MDR) => no result

What are MDR and XDR strains?

• Multidrug resistant M. tuberculosis (MDR): – at least resistant to isoniazid and rifampicin

• Extensively resistant M. tuberculosis


y(XDR-TB)– MDR (isoniazid and rifampicin resistant)– and– resistant to fluoroquinolones – and to the following injectable drugs (amikacin,

kanamycin, capreomycin, viomycin)

Report of results• Reporting of susceptibility results easily done for

each drug tested when the method is done correctly

• Priority reporting iti l t b l i


– smear positive pulmonary tuberculosis – cases which appear to be MDR-TB

• should be given directly to the clinician in charge of the patient

• Routine reporting should contain the start date of testing and the date of reporting

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Levels of resistance?

• The level of resistance may be of interest for some drugs (isoniazid, streptomycin, ethambutol) if it can be determinedExample of isoniazid: H R(0 1) H R(0 2) H R(1)


• Example of isoniazid: H-R(0.1), H-R(0.2), H-R(1)• A report of low resistance does not imply that the

drug will not be given • A report of a high level of resistance implies that

the drug is of no use to the patient

Quality and expertise

• Experience in the chosen method• Skilled technicians • Quality controls

– Internal quality control


– External quality control program• Evaluation

– Efficiency in DST-MTB (number of success/ number of tests done, number of repeated test, time to results)

– Primary and secondary resistance rates– Reproducibility– Concordance with molecular testing

Rush-Gerdes et al. 1999

Workflow and Time to Results

D0-D1 • Microscopy : M+,M0• If smear-positive M+ , PCR for MTBC

• Molecular detection of resistance if risk factors• Culture

D2-D3 if M+


D10-D30 • Culture positiviity • identification and speciation (MTBC)

• DST – 1st line drugs (H, R at least)

• DST-2nd line drugs (FQ, AGA at least) if H-R and R-R

> D30D20-D40 for DST1

D40-D60 for DST2

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Challenging issues on DST-TB

• Rapid DST for detecting resistance• Accurate DST for susceptibility results of

MDR strainsS d li d t ti


• Second line drugs testing• Concordance between phenotypic and

genotypic results,i.e. resistance and mutations

• Clinical studies for low level resistance

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Drobniewski - Is molecular testing an alternative to drug resistance testing?

Is molecular testing an alternative to drug resistance

testingFrancis DrobniewskiProfessor of International Health and TuberculosisDirector, HPA National Mycobacterium Reference Laboratory (NMRL) and Clinical TB and HIV Group, Barts and the London School of Medicine, University of [email protected]

4/26/2011

Top 19 settings with MDR among newcases > 6% (1994-2007)

Mary El Oblast, RF

Estonia

Kazakhstan*

Tashkent, Uzbekistan

Tomsk Oblast, RF

Donetsk Oblast, Ukraine

Republic of Moldova

Baku City, AzerbaijanChina, India, and the Russian Federation estimated to carry the highest number of new MDR cases.

0.0 5.0 10.0 15.0 20.0 25.0

Dominican Republic*

Georgia

Heilongjiang Province, China

Inner Mongolia Autonomous Region, China

Henan Province, China

Orel Oblast, RF

Armenia

Lithuania

Liaoning Province, China

Latvia

Ivanovo Oblast, RF*

Indicates survey data reported in an earlier phase of the project

MDR ranged from 0% in eight countries to 22.3% (in Baku, Azerbaijan and 19.4% in the Republic of Moldova.

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MDRTB and XDR-TB in the UK, 1995-2009

•Between 1995 and 2010 there were 12 confirmed XDRTB cases in the UK (one each in 2009 and 2010)

•Most were pulmonary•Most were pulmonary

•Most were male

•Most aged 20‐50

•Most had had previous TB

•Most born abroad.

•Distributed across countryThorax Jun 2009

MGIT 960

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Actions and recommendations Laboratories

Ensure swift access to rapid tests for rifampicin resistance

Laboratories should aim to identify TB and rifampicin resistance in over 90% of cases fromrifampicin resistance in over 90% of cases from smear‐positive sputum directly where resources are available for this…. ……Rapidly within 1‐2 days

Model for delivery national rapid ID and RIF/MDRTB detection

Drobniewski BMJ 1998

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Key markers for drug resistance in Mtb.

• Isoniazid katG, inhA• rifampicin rpoB• ethambutol embB• pyrazinamide pncA• streptomycin rrsstreptomycin rrs• capreomycin tlyA• amikacin rrs• ofloxacin gyrA• moxifloxacin gyrA• ethionamide ?inhA, ethA, ethR• clarithromycin ?erm• PAS ?thyA

Different molecular strategies

• Cobas Amplicor TB Roche‐ PCR 16sRNA

• MTD Gen‐Probe TMA of rRNA

• BD ProbeTec SDA IS6110, 16sRNA

• Eiken LAMP Isothermal amplification +uv fluorescence

• Artus Realart Real time PCR

• +DST• +DST

• Innolipa RifTB

• Hain Lifesciences MTBDRPlus

• Cepheid GeneXpert

Molecular methods of drug susceptibility testing – PCR+hybridization

Based on amplification of fragments of genes responsible for drug resistance development followed by hybridization with oligonucleotide y gprobes immobilized on membranes;

Commercial kits and in‐house macro‐arrays have been reported to demonstrate high sensitivity and specificity

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Fastrack: Analysis UK National Fastrackfor primary specimens UK and Latvia

UK Jan 1999‐Dec 2002: n=1997 patient specimens.

Detection of rifampicin resistance in specimens yielding MTBC on culture:

Concordance, Sensitivity, Specificity, PPV, NPV:99 1% 95 0% 99 6% 92 7% 99 7%99.1%; 95.0%; 99.6%; 92.7%, 99.7% Sam et al. EID 2006 12(5) 752‐759

Latvia: Jan 2003‐Mar 2004: n=89 smear positive respiratory specimens

Concordance, Sensitivity, Specificity, PPV, NPV:

91%; 96%; 94% 96%Skenders et al EID 2005 11 1461‐3

HAIN GenoType® MTBDR Plus-S Africa

Rapid detection RIF and INH directly on 536 consecutive smear‐positive sputum specimens Compared with conventional DST.

97% smear‐pos specimens interpretable results in 1‐2 days.

Assay performed well on specimens contaminated onAssay performed well on specimens contaminated on culture

Sensitivity, specificity, PPV and NPV were:98.9%, 99.4%, 97.9% and 99.7% for RIF resistance;94.2%, 99.7%, 99.1% and 97.9% for INH resistance; 98.8%, 100%, 100% and 99.7% for MDRTBBarnard et al Am J Respir Crit Care Med. 2008

WHO Expert Committee 2008 (1) Recommendations

•“The expert committee considers that there is sufficient generalisable evidence to justify a recommendation of the use of line‐probe assays for rapid screening for MDR TB within country‐for rapid screening for MDR TB within countryspecific settings”

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Molecular methods of diagnosis/drug susceptibility testing – PCR-basedmethods

Molecular Beacons Piatek et al; Alland et al.

Torres, M.J., et al. 2000. J Clin Microbiol 38:3194-3199

Cepheid GeneExpert

New Eng J Medicine 1 Sept 2010

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Cepheid GeneExpert NEJM

2/3 samples randomly processed with NALC and NaOH before microscopy, solid and liquid culture, and the MTB/RIF test, and one specimen used for direct testing with microscopy and the MTB/RIF test.py /Among culture‐positive patients, single, direct MTB/RIF test identified 551/561 patients with smear‐positive TB (98.2%) and 124/171 with smear‐negative TB (72.5%).

Cepheid GeneExpert NEJM

Test specific in 604/609 patients without TB (99.2%).Addition of second MTB/RIF test increased sensitivity by 12.6% and a third by 5.1 % to a total of 90.2%.MTB/RIF testing correctly identified 200/205 patients (97.6%) with rifampicin‐resist TB and 504/514 (98.1%) with rifampin‐sensitive bacteria. Sequencing resolved all but two cases in favor of the MTB/RIF assay. Boehme et al NEJM 2010

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Background material

Barnard M, et al Rapid molecular screening for multidrug‐resistant tuberculosis in a high‐volume public health laboratory in South Africa. Am J Respir Crit Care Med. 2008 Apr 1;177(7):787‐92. Epub 2008 Jan 17.

Boehme, CC, et al Rapid molecular detection of tuberculosis and rifampin resistance. N Engl J Med. 2010 Sep 9;363(11):1005‐15.

Drobniewski F A. Diagnosing MDRTB in Britain. BMJ 1998. 317:1263‐1264.

Hillemann D et al Evaluation of the GenoType MTBDRplus assay for rifampin and isoniazid susceptibility testing of Mycobacterium tuberculosis strains and clinical specimens. J Clin Microbiol. 2007 Aug;45(8):2635‐40. Epub 2007 May 30Epub 2007 May 30

Ling DI, et al GenoType MTBDR assays for the diagnosis of multidrug‐resistant tuberculosis: a meta‐analysis. Eur Respir J. 2008;32:1165–74.

Morgan M, Kalantri S, Flores L, Pai M. A commercial line probe assay for the rapid detection of rifampicin resistance in Mycobacterium tuberculosis: a systematic review and meta‐analysis. BMC Infect Dis. 2005; 5:62.

Sam, IC, Drobniewski, F, More, P, Kemp, M and Brown, T. Mycobacterium tuberculosis and rifampin resistance, in the United Kingdom. Emerging Infectious Diseases 2006; 12:(5):752‐9

WHO Expert Group Report. Molecular line ning of patients at risk of multi‐drug resistant tuberculosis (MDR‐TB). Geneva: World Health Organization; 2008

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Schön - Susceptibility to second line drugs

Susceptibility to second line drugs

• Which are the second line drugs ?

Thomas Schön, MD PhD Dept of Clinical Microbiology and Infectious Diseases

Kalmar County Hospital, Sweden.

– WHO Classification• How do the second line drugs act ?

– Implications for molecular based testing– MIC-distributions– Cross resistance

• When and how to test them ?

Drugs used against tuberculosis• Group I, first line drugs

– Rifampicin, Isoniazid, Pyrazinamide, Ethambutol

• Group II, injectables– Amikacin, Kanamycin, Streptomycin, Capreomycin, Viomycin

1st Line

2nd Line

• Group III, fluoroquinolones– Levofloxacin, Moxifloxacin, (Ofloxacin)

• Group IV, oral bacteriostatic drugs– Etionamide, Protionamide, Cycloserine, PAS

• Group V, agents with unclear role– Linezolide, Clofazimine, Thioacetazone, high-dose Isoniazid

“-cidal”

3rd Line“-static”

2nd line versus 1st line drugs

• Less effective – Increases treatment duration from 6 to at least 18 months

• More side effects C li bl– Compliance problems

• Susceptibility testing not standardized– Correlation to outcome ?

• Less accessible

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Current TB treatment –mechanisms of action

Cell wall inhibitors - Isoniazid, Ethambutol, Ethionamide, Cycloserine

Inhibition of membrane energy metabolism - Pyrazinamide

Protein synthesis inhibitors - Aminoglycosides

Folate synthesis - PAS

Rifampicin, Rifabutin, Fluoroquinolones,Linezolide - Nucleic acid synthesis inhibitors

Resistance genes for 1st and 2nd line drugs against TB

WHO Drug Gene target Encoded Protein or RNA product Relative frequency Most commonGroup in resistant strains* mutationI Rifampicin rpo B Beta-subunit of RNA polymerase 90-95 % Codon 531

Isoniazid (INH) kat G Catalase/peroxidase (activator) 50-95 % Codon 315Isoniazid ahpC Alkyl hyroxyperoxide C reductase 10-15 %Ehtionamide/INH inh A Enoyl-acyl carrier protein reductase 6-30 % Nucleotide 15Pyrazinamide pnc A Pyrazinamidase 62-97 % NoneEthambutol emb B Arabinosyltransferase 47-89 %** Codon 306**

II Streptomycin rps L Ribosomal protein s12 40-95 % Codon 43Streptomycin rrs 16s rRNA 5-20 %Kanamycin/Amikacin rrs 16s rRNA (1401 or 1484)Capreomycin/Viomycin rrs 16s rRNA (1401 or 1484)Capreomycin/Viomycin tly A Cytotoxin/hemolysin homologue

III Fluoroquinolones gyr A A subunit of DNA gyraseFluoroquinolones gyr B A subunit of DNA gyrase

IV Ethionamide eth A Monooxygenase (activator)Cycloserine alrA Peptidoglycan synthesisPAS fol P (?) dihydropteroatsyntase

*Highly variable depending on region**Using DST at current critical concentration of questionable validity

What is not detected in the rapid molecular tests for 2nd line drugs ?

• Hain MTBDRsl– Fluoroquinolones: gyrA ~75-90 %*– Ethambutol: emb306 ~30-60 %*– Aminoglykosides: rrs ~75-85 %*

*Uncertain estimates of sensitivity to detect phenotypic resistance due to small number of resistant strains tested in published studies Variability depending onnumber of resistant strains tested in published studies. Variability depending onregion and prevalence of resistance mutations

• Additional known resistance mutations (and new will be found ?)– Fluoroquinolones: gyr B, D472 and N510, gyrA G88– Ethambutol: 406 and 497 and problems with reference method– Aminoglykocides: tlyA, rrs 1402 (C1402T: AMK S, low level KAN R)

• Genotyping of known resistance mutations vs phenotype testing– Rapid results (high clinical relevance !) – Phenotype resistance with mutation in unknown resistance genes not detected – Phenotypic screening using refined breakpoints useful for epidemiological survey

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Group II, InjectablesBactericidal key drugs in treating MDR TB

Group II Usual daily dose PK MIC range ECOFFInjectable drugs (70 kg) fCmax 7H10* 7H10* 7H10 MGIT LJ

Amikacin 1g (15 mg/kg) 21.6 0.25-1 1 ND 1 40Kanamycin 1g (15 mg/kg) 19.8 0.5-4 4 5 - 30Capreomycin 1g (15 mg/kg) 20.0 1.0-4.0 4 10 2.5 40Streptomycin 1g (15 mg/kg) 22.5 0.125-2 2 2 1 4Viomycin 1g (15 mg/kg) 20.0 0.5-2 2 - - -

Critical concentration

*Juréen P, et al J Clin Microbiol. 2010, 48(5):1853-8.

• To be used at least 6 months after negative cultures• Variation in critical concentrations for some drugs (Capreomycin)• PK/PD

– fCmax/MIC >10 for all group II drugs against the majority of WT isolates• Side effects

– Ototoxicity, renal damage• Cross resistance

– Amikacin S can be Kanamycin R– Capreomycin or Viomycin can be S if Amikacin/Kanamycin R

Group III, FluoroquinolonesEssential bactericidal drugs for treating MDR TB

Group III Usual daily dose PK MIC range ECOFFFluroquinolones (70 kg) fAUC 7H10* 7H10* 7H10 MGIT LJ

Levofloxacin 750 mg 45 0.125-0.5 0.5 2 2 -Moxifloxacin 400 mg 18 0.03-0.5 0.5 - 0.25 -Ofloxacin 800 mg 39 0.25-1 1 2 2 2


• Early bactericidal activity (EBA)** comparable to isoniazid• PK/PD

– fAUC/MIC> 90 for majority of isolates below ECOFF for Levofloxacin/Moxifloxacin• Ciprofloxacin is no longer recommended (WHO)• Cross resistance

– Unclear clinical relevance of ofloxacin R and moxifloxacin/levofloxacin S – Moxifloxacin and levofloxacin probably more effective than ofloxacin

*Ängeby K, et al J Antimicrob Chemother. 2010 65(5):946-52.**EBA=The fall in counts/mL sputum/day during the first 2 days of treatment

Group IV, Oral bacteriostatic drugsGroup IV Usual daily dose PK MIC range ECOFFOral bacteriostatic (70 kg) fAUC 7H10* 7H10* 7H10 MGIT LJ Ethionamide 750mg-1000mg 5.5 0.5-2 2 5 5 40PAS 4gx2 - - - 5 - 1Cycloserine 500mg-1000mg - 8-32 - - - 40Protionamide 750mg-1000mg - 0.125-1 1 - 2.5 40


• Not as effective as group I-III• Side effects

– Gastrointestinal side effects common (PAS, Ethionamide)– CNS (Cykloserine)

• Unclear relevance of cross resistance – Isoniazid (inhA) R, usually ethionamide/protionamide R

*Schön T, et al. Int J Tuberc Lung Dis. 2011;15(4):502-9.

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Group V, Drugs with unclear clinical effect against TB

• Linezolid

Group V Usual daily dose PK MIC range ECOFFUncertain effect (70 kg) fAUC 7H10* 7H10* 7H10 MGIT LJ

Linezolid 600mg 56 0.125-0.5 0.5 - 1 -Clofazimin 100-300mg ND 0.064-0.125 - - - -Thiacetazone 150mg ND 0.125-2 2 - - -


– Hematological side effects, expensive– fAUC/MIC > 100 for most WT strains, bactericidal

• Thioacetazone – Dermatological side effects, can not be used in HIV/TB-patients

• Highly uncertain effect– Amoxicillin/clavulanate, Meropenem/clav., Imipenem,Claritromycin

• “New” drugs – Sulfametoxazole, Thioridazine

*Schön T, et al. Int J Tuberc Lung Dis. 2011;15(4):502-9.

When and how should 2nd line drugs be tested ?

• When ?– Confirmed or high suspicion of MDR/XDR TB– Previous treatment with 2nd line drugs

H ?• How ?– Indirect proportion method

• Slow but validated– Liquid methods

• BACTEC 960 MGIT– Solid based methods

• 7H10 • Löwenstein Jensen

Drug resistant TB - definitions• Drug resistant tuberculosis

– TB resistant to any first line antituberculosis drug

• Multidrug resistant tuberculosis (MDR-TB)– TB resistant to at least isoniazid and rifampicinTB resistant to at least isoniazid and rifampicin

• Extensively drug resistant tuberculosis (XDR-TB)– MDR TB + resistance to a fluoroquinolone and a

second line injectable agent (amikacin, capreomycin, or kanamycin)

24


In total: ~ 500 000 MDR TB and ~50 000 XDR TB cases !MDR TB, 15 % in previously treated and 3 % in new patients

Basic treatment strategy for MDR TB

• At least 5 drugs– A Fluoroquinolone – An Injectable j– Ethambutol and/or Pyrazinamide if susceptible

• If DST result not present - consult surveillance data– 1-3 Group IV-drugs– Ethionamide/Protionamide, Cycloserine, PAS

25


Which 2nd line drugs should be tested?

• MDR-TB strains should be tested for 2nd line drugs– Internal and external control programs (QC)– Primarily Group II and III drugs

• High reliability and reproducibility of DSTR i t t th d d fi XDR• Resistance to these drugs defines XDR

– Rapid molecular testing is recommended (MTBDRsl) – Identifying MDR TB (RIF R) directly on sputum samples

• Increase diagnostic speed, further 2nd line testing• Line probe assay, Hain MTBDR+, GeneXpert

• In reference laboratories– Group IV (LJ) and V drugs: Ethionamide, Cycloserine, PAS

18

26


Challenges for 2nd line DST• Second-line DST has not been standardized internationally

• The drug critical concentration is close to the minimal inhibitory concentration (MIC) required for antimycobacterial activity– Increased misclassification of susceptibility or resistance

• Lack of correlation to clinical response

• Varation in testing systems and critical concentrations– Bactec 960 MGIT, LJ, 7H10

• Systematic evaluation of all available DST methods simultaneously for all available SLDs ?

Policy guidance on drug-susceptibility testing (DST) of second-lineantituberculosis drugs. WHO 2008.WHO/HTM/TB/2008.392

• Low resistance level for many years (rapidly changing !) • Current “critical concentrations” – clinical correlation ?• Lack of published wild type distributions

Problems in susceptibility testing against Mycobacterium

tuberculosis

• Lack of published wild type distributions • Drug combinations used • No clearly defined PD targets (AUC/MIC, Cmax/MIC)• Bactericidal AND sterilizing effect needed

WHO/HTM/TB/2009.420 (2010): “Susceptibility testing for isoniazid, rifampicin, thefluoroquinolones, and the injectable agents is fairly reliable. For other agents it isless reliable, and basing individualized treatments on DST for these agentsshould be avoided. The clinical effectiveness or ineffectiveness of a drugcannot be predicted by DST with 100% certainty.”

Can we be sure about the critical concentrations for 2nd line drugs?

• Determined by consensus when 2nd line drug resistance was very low– In other bacterial and fungal pathogens 3 basic

components are used to determine clinicalcomponents are used to determine clinical breakpoints

• Wild type distributions• PK/PD determination• Clinical outcome data

– Clinical outcome data for individual 2nd line drugs very difficult to achieve for TB

27


WHO/HTM/TB/2009.420 (p 90):

“DST results should complement, rather than invalidate, other sources of data on the likely effectiveness of a specific drug.

For example, if a history of prior anti-TB drug use suggests that a drug is likely to be ineffective, that drug should not be relied on as one of the four core drugs in the regimen, even if DST shows the strain to be susceptible.

Alternatively, if the patient has never taken a particular drug and resistance to that drug is extremely uncommon in the community, DST results that indicate resistance g y y,may be the result case of a laboratory error or of the limited specificity of DST for some second-line drugs.

Since a fluoroquinolone and an injectable agent form the backbone of MDR treatment, they should be used even if the patient’s M. tuberculosis demonstrates resistance to all available drugs in these two classes.”

-> The development of a harmonized, reproducible and quality controlled 2nd line DST is urgently needed !

What is the usefulness of MIC-determinations of M. tuberculosis ?

• To identify the wild type distribution for TB drugs– Determine ECOFF (epidemiological wild type cut off)– Isolates below ECOFF probably susceptible – ECOFF for epidemiological screening

• Optimized dosing strategy in MDR and XDR TB– Determine MIC and drug concentration - TDM – Minimize side effects and maximize the effect– PK/PD calculations

Wild type distributions - exampleWild type distributions and mutations in

embB

2

4

6

8

10

12

No

of s

trai

ns

20

30

40

50

60

70

80

Num

ber o

f iso

late

s (n

)

Wild type EMB-resistantBACTEC460 R, 7H10 S BACTEC460 S, 7H10 R

Breakpoint5mg/L

00,25 0,5 1 2 4 8

MIC

wt Glu378Ala Met306Val Met306Ile

Tyr334His Glu378Ala,Leu504Arg Gly406Asp Gln497Gly

Ethambutol

Isolate WHO SRL % Consensus Consensus Bactec 460 7H10

MIC (mg/L) 7H10

1 26/26 S 100.0 S S S 4 2 16R/9S 64.0 R S R 8 3 21R/4S 84.0 R R R 8 4 22R/3S 88.0 R S S 4 5 26/26 R 100.0 R R R 8 6 22R/4S 84.6 R R R 8 7 25/26S 100.0 S S S 2

Breakpoint too close to WT cut off ?

S≤2, I=4 mg/L, R≥8 ?

0

10

20

0,125 0,25 0,5 1 2 4 8 16 32

MIC (mg/L)

Schön T et al JAC 2009, July

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References – suggested readingDST - methodology

• Policy guidance on drug-susceptibility testing (DST) of second-line antituberculosis drugs. WHO/HTM/TB/2008.392

• American (CLSI) and ECDC guidelines (published 2011 ?) • Kim SJ. Eur Respir J 2005; 25: 564–569

Clinical treatment guidelines and resistance epidemiology• Global Resistance Epidemiology: Wright A et al. Lancet 2009; 373:1861-73• Management of MDR-TB: A field guide. WHO/HTM/TB/2008.402a (2010) • Multidrug and extensively drug-resistant TB. WHO/HTM/TB/2010.3• MDR/XDR treatment: Caminero JA et al. Lancet inf dis 2010; 10: 621-29 • Treatment of Tuberculosis 4th Ed. WHO. WHO/HTM/TB/2009.420 (2010)• ATS http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5211a1.htm• BTS www.nice.org.uk/guidance/CG117

PK/PD, EBA, Overview 2nd line drugs• 2nd line drugs: Tuberculosis 2008; 88(2):85-165.• PK/PD: Pasipanodya J and Gumbo T. AAC 2011; (55): 24-34.• PK/PD: Nuermberger E. Eur J Clin Microbiol Inf Dis 2004; 23: 243-255• Early bactericidal activity: Donald PR. Tuberculosis 2008; (88):S75-S83

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EW08 01 Cambau.ppt [Kompatibilitätsmodus]

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