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Resistance Pattern for Tuberculosis in
Europediagnostic aspects
Postgraduate course Update in tuberculosis
advances in the management of drug resistant and
MDR-TB cases ERS, Vienna 12/9/2009

• Daniela M. Cirillo
• San Raffaele Scientific Institute
• Milan Italy

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Global Epidemiology of TB

• 1/3 of global population infected (246/ 100,000
  p)
• gt 8.8 million new cases a year (141/ 100,000 pop)
• gt 2 million deaths a year
• gt 95 of cases and deaths occur in the developing
  world
• 75 of cases in 15-54 age group
• Devastating economic costs
• gt 1/ million deaths due to TB/HIV
• MDR ubiquitous

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TBC good news, bad newsRio de Janeiro March
24th 09

• Good news slow decrease of incidence from 2004.
• Bad news
• 1. TB incidence is decreasing too slowly. New
  strategies are needed
• 2. 1/3rd of cases is not diagnosed by Countries
  implementing the Stop TB Strategy
• 3. MDR-TB 500.000 cases over 9.3 millions for
  2007, 150.000 deaths over 1.7 millions. 50.000
  cases of XDR per year. (55 Countries have
  reported cases)
• 4. TB/HIV 1.4 millios of cases of TB are
  associated to HIV causing half million of deaths
  (1.7 total for 2007).
• 5. Need for speeding up the implementation of
  collaborative activities

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Changing epidemiology of TB in Europe

• the white plague starting with the industrial
  revolution
• true epidemic in the 19th century
• Controlled by improving living and nutritional
  condition of the population and availability of
  drugs after the 1950
• Between1974 and 1990 notification rate declined
  of 5.4 per year inwestern Europe

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Changing epidemiology of TB in Europe (2)

• Factors reversing the trend
• HIV pandemic
• Increasing immigration from high burden Countries
• Break down of health system in Some Eastern
  European Countries
• new poverty related factors for marginalized
  population

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TB burden, EUR, 2007
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Rural/ Residential versus metropolitan areas

• In Western Europe the incidence of tuberculosis
  is highly increased in Metropolitan settings

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Global TB targets

• Outcome targets (World Health Assembly, Stop TB
  Partnership)
• Case detection gt70
• Treatment success gt85
• 2015 Impact targets (MDGs, StopTB Partnership)
• Halt and reverse incidence of TB
• Halve prevalence and mortality levels of 1990

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The European Region wont meet the MDG targets

• In 2007 reported case detection was 53
• Treatment success rate among new TB cases
  reported as 73
• Mortality rate of 1.1 (excluding HIV positive
  cases)

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Outcomes for those patients not successfully
treated, by WHO region, 2005 cohort
DOTS
Non - DOTS
82
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• Point 5 comma II

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• Increase the infectious cases detection
• Increase the number of culture confirmed cases
• Detect MDR/XDRTB cases from samples
• Perform a quality assured DST for all second and
  third line Tb drugs for the appropriate
  management of MDR/XDR TB cases

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A unique, proprietary illumination system with
high power solid-state light sources (LEDs)
replacing Mercury and Xenon arc-lamps found in
traditional epifluorescence microscopy available
as add-on kit for existing microscopes.
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• Increase the infectious cases detection
• Increase the number of culture confirmed cases
• Detect MDR/XDRTB cases from samples
• Perform a quality assured DST for all second and
  third line Tb drugs for the appropriate
  management of MDR/XDR TB cases

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Culture services

• Global effort to
• Offer to each TB patient the access to culture
• Rationalize the number of Culture services
• Improve their quality trough standard SOPs QA
  programs
• Upgrade the laboratories to liquid manual and
  liquid automated cultures systems
• European standards on biosafety in place in all
  TB laboratories

International Standards of TB care empathize the
key role of MTB detection for diagnosis and
management
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MDR-TB and XDR-TB Definitions

• Polyresistance
• strains of TB resistant to more than one anti-TB
  drug
• MDR-TB is defined as resistance to isoniazid and
  rifampin
• XDR-TB is defined as resistance to at least
  isoniazid and rifampin in addition to any
  fluoroquinolone and any of the three injectable
  anti-TB drugs
• Amikacin
• Capreomycin
• Kanamycin

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• Increase the infectious cases detection
• Increase the number of culture confirmed cases
• Detect MDR/XDRTB cases from samples
• Perform a quality assured DST for all second and
  third line Tb drugs for the appropriate
  management of MDR/XDR TB cases

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Background on drug resistance in the MTB complex

• Natural resistance to antibiotics
• hydrophobic cell envelope (permeability barrier)
  
• drug efflux systems and drug-modifying enzymes
• Resistance is due to chromosomal point mutations
  leading to amino acid substitution
• Mutations occur spontaneously with different
  frequency for different drugs
• Resistance emergence is linked to a large
  bacterial population

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Selection of drug-resistant mutants

• Spontaneous mutations occur in the DNA of all
  cells
• Mutations can change the structure of a protein
  that is a drug target.
• Protein still functions, but is no longer
  inactivated by the drug.
• TB can therefore grow in the presence of the
  drug.
• Resistance is linked to large bacterial
  populations
• Mutants resistant to any drug occur on average
  once in every 100 million (108) cells .
• In TB in the lung, cavities often contain 107
  109 organisms.
• By using two antibiotics, the chance of both
  targets mutating is extremely small (108 x 108
  1016).
• Monotherapy led to selection of drug-resistant
  populations in cavitary disease more often than
  in cases with non-cavitary lesions (about 103104
  organisms).
• This is the rationale for treatment regimens with
  more than one drug.

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MDR-TB is spreading worldwide

• The spread of drug-resistant M. tuberculosis
  strains compromises the positive clinical outcome
  and the efficiency of TB control programmes
• MDR-TB outbreaks are associated with fatal
  outcome (90 in HIV pos.)
• The treatment and the isolation of infected
  patients increase public health expenses
• MDR-TB incidence is increasing in Eastern Europe
  (10 )
• Developed by selective pressure caused by
• Inadequate treatment
• Low compliance
• Intermittent therapy
• OR
• Primary infection by MDR strains

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MDR-TB prevalence, EUR, 2007
Estimated, among new 10.35 (43
600) Estimated, among re-treatment
43,41 Notified, among new 9,59 (7
351) Notified, among re-treatment 38,55
Detection rate, new - 17
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13 settings with gt30 resistance to any TB drug
among new cases 2002-2007
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14 settings with 6 MDR-TB among new cases
2002-2007
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16 settings with 25 MDR-TB among previously
treated cases 2002-2007
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Countries with XDR-TB confirmed cases as of May
2008
Argentina
Italy
Armenia
Japan
Latvia
Azerbaijan
Australia
Lesotho
Bangladesh
Lithuania
Mexico
Botswana
The boundaries and names shown and the
designations used on this map do not imply the
expression of any opinion whatsoever on the part
of the WHO concerning the legal status of any
country, territory, city or area or of its
authorities, or concerning the delimitation of
its frontiers or boundaries. Dotted lines on
maps represent approximate border lines for which
there may not yet be full agreement. ? WHO 2005.
All rights reserved
Brazil
Moldova
Canada
Mozambique
Chile
Namibia
Russian Fed.
China, Hong Kong SAR
Czech Rep.
Netherlands
Ecuador
Slovenia
Nepal
South Africa
Norway
Estonia
Peru
Spain
France
Georgia
Philippines
Swaziland
Germany
Sweden
Poland
Portugal
Thailand
Ireland
Rep of Korea
UK
India
USA
Ukraine
Islamic Rep. of Iran
Romania
Israel
Vietnam
Based on information provided to WHO Stop TB
Department - May 2008
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XDR-TB among MDR-TB cases 2002-2007
Sub-national averages applied to Russia
lt 3 or less than 3 cases in one
year of surveillance
3 - 10
gt 10
Report of at least one case
No data
The boundaries and names shown and the
designations used on this map do not imply the
expression of any opinion whatsoever on the part
of the World Health Organization concerning the
legal status of any country, territory, city or
area or of its authorities, or concerning the
delimitation of its frontiers or boundaries.
Dotted lines on maps represent approximate border
lines for which there may not yet be full
agreement. ? WHO 2006. All rights reserved
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Extensively Drug-Resistant Tuberculosis Is Worse
than Multidrug-Resistant Tuberculosis Different
Methodology and Settings, Same ResultsGiovanni
Battista Migliori,Christoph Lange,Enrico
Girardi,Rosella Centis,Giorgio Besozzi,Kai
Kliiman,Johannes Ortmann,Alberto
Matteelli,Antonio Spanevello, and Daniela M.
Cirillo Clinical Infectious Diseases
200846958959

• With the multiple regression analysis, the
  presence of XDR was an independent risk factor
  for both death (OR, 2.07 95 CI 1.054.05P V
  .034) and treatment failure (OR, 2.3795 CI,
  1.144.89 P V .02).
• XDR TB has a negative clinical and prognostic
  significance, even in patients with different
  susceptibility profiles and from different
  setting (e.g., Korea and Eastern and Western
  Europe)

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Priority Countries for MDR/XDR TB response

• 18 Countries in the European Region mostly from
  the former Soviet Union
• From some countries data are based on modeling
  programs
• Big differences in the need to respond to MDR-XDR
  TB
• Common the need to
• Improve case detection
• Address a quality assured prompt diagnosis of the
  resistance pattern of the strain

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Molecular line probe assays for rapid screening
of patients at risk of MDR-TB

• Policy statement by WHO and Partners
• June 27, 2008

Endorsement of the two commercial line probe
assays for rifampicin resistance
detection Tests are CE marked and meet
predefined performance targets in controlled
evaluation studies Both tests are highly
sensitive and specific for rifampicin resistance
detection from TB strains
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Commercial Line Probe Assays
Hain Lifescience
Innogenetics INNO-LiPA-Rif.TB
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Comparison GenoType MTBDR and INNO-LiPA Rif.TB
GenoType MTBDR INNO-LiPA Rif.TB
Company Hain Lifescience Innogenetics
M. tuberculosis detection Yes Yes
Detection of RMP Resistance in M. tb Complex Yes Yes
Detection INH Resistance in M. tb Complex Yes No
Strip Assay Yes Yes
DNA-Basis PCR Yes Yes
Culture requested Yes Yes
Direct assay No Yes (modified version)
TB-complex Detection 23S-rRNA/16S-rRNA Yes Yes
RMP-Resistance rpoB gene Yes Yes
INH-Resistance katG gene/inhA gene Yes No
Universalcontrol Yes No
rpoB unicontrol Yes No
kat G unicontrol Yes No
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GenoType MTBDRsl (Hain Lifescience)
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The GenoType MTBDR platform

• Targets the most common mutations conferring
  resistance to 1st and 2nd line drugs
• Allows to meet MDR and XDR definition
• Test is simple to be performed
• Detects mixed infections
• But ( areas for improvement)
• DNA extraction needs to be improved
• Not automated
• Some possibility of misinterpretation (double
  patterns or faint bands)
• Limited in the number of probes (absence on
  probes for NTMs)
• P2 biosafety required
• Possibility of cross contamination

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Available from April 14th GeneXpert MTB
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Sensitivity and Specificity

• Sensitivity in smear negative, culture positive
  (S-C) was 90.9 (70/77)
• Sensivity in smear positive, culture positive
  (SC). was 100 (275/275)
• Specificity of the assay was 98.3
• Sensitivity observed for Rifampicin resistance
  was 96.7
• Specificity observed for Rifampicin resistance
  was 98.6

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Xpert MTB/RIF kit
PAGE 37
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Lab-On-Chip microfluidic technology

• PCR
• Ultra-Fast PCR ? 40C\s ? 10C\s 0.1C
• PCR strategy Asymmetric Cy5-dCTP multiple
  incorporation
• Microarray
• Orientation probes Pre-labeled oligo capture
  probes to correctly align the grids onto the
  array during data analysis using MAT software
• PCR Control probes un-labeled oligo capture
  probes that assure the correct functionality of
  the PCR module
• Hybridization Control probes un-labeled oligo
  capture probes that assure the correct
  functionality of the microarray module (3
  different sequences at equal concentration the
  hybridization buffer carry out the complementary
  labeled targets at different concentrations)
• Hybridization Negative Controls probes
  un-labeled oligo capture probes containing
  randomized sequence probes used to estimate the
  level of non-specific binding on the array

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Wrong DSTs have huge impact at

• Patient level
• Community level
• Health care costs
• Leading to mismanagement of TB cases and
  increasing MDR

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Global WHO/IUATLD/CDC Survey

• Convenience sample (17,690 isolates) submitted to
  participating international SRL network,
  2000-2004
• 3520 (20) of isolates MDR TB
• 347 ( 2) of isolates XDR TB
• XDRTB in all regions, more common FSU and Asia
  (Republic of Korea)
• Denominator information unavailable

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Patterns of Second-line Drug Resistance in MDR
Isolates by Geographic Region (N3,461)
Latin America 543 () N Amer 320 () UK / W Euro 451 () Russia / E Euro 406 () N Afr Mid E 95 () Asia 1,563 ()
AGCM AGFQ AG gt1 Group 4 CMFQ CM gt1 Group 4 FQ gt1 Group 4 drug AGCMFQ AGCM gt1 Group 4 AGCMFQ1 Grp 4 Any 3 SLD classes 6.6 6.1 6.6 1.8 2.2 5.0 1.8 2.2 0.9 5.9 6.2 3.1 5.3 2.2 4.1 4.7 2.2 4.1 2.2 4.7 4.0 4.0 5.8 2.4 3.5 9.3 1.5 2.7 1.3 7.8 20.9 5.7 17.7 1.2 7.6 6.6 1.2 7.6 0.5 13.5 0 0 9.5 0 0 2.1 0 0 0 0 5.6 8.1 6.4 4.7 4.0 17.7 3.8 3.0 2.4 13.0
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MOLECULAR TYPING and MDR-TB

• Methods should be standardized and QA
• MIRU 24 may replace IS6110 RLFP
• Role in patient management ( failure/
  reinfection/ changing in sensitivity
  pattern/laboratory cross contamination)
• Epidemiological studies
• Monitoring recent transmission (contact tracing
  and contact management)

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Risk Factors for Clustering

• 521 cases
• 250 (48) belonged to European clusters
• 271 (52) unique fingerprinting
• Multivariate analysis
• Association between origin from Baltic states or
  FSU countries and clustering
• Clustering and Beijing genotype strongly
  associated
• High proportion of MDR among clustered strains

Devaux et al. EID 2009
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MDR/TB and HIV

• Data are controversial
• Data from 5 different countries on DR stratified
  by HIV status showed no association
• Data from Latvia and Ukraine supported the
  association
• Not clear if association is related to
  acquisition or transmission

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MDR/TB and HIV

• Existing evidences
• Institutional outbreaks of MDR-TB have primarily
  affected HIV-infected persons.
• Highly fatal MDR-TB appears not to cause
  infection or disease more readily than
  drug-susceptible TB in HIV-infected persons.
• HIV infection may lead to malabsorption of
  anti-TB drugs and acquired rifamycin resistance.

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Problems related to TB diagnosis in Europe

• Inconsistent DOTS coverage
• Concentration of TB cases among marginalized
  population
• Western Europe decreased interest in TB among
  health professionals
• Eastern Europe quality assurance of laboratory
  structure

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Improving capacity to diagnose MDRTB and collect
quality data

• Prompt access to diagnostic facilities
• Quality assured laboratories
• Implementation of liquid cultures
• Implementation of molecular methods for MDR/XDR
  TB detection

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Ongoing programs supporting the MDR-TB control in
Europe

• WHO Euro support and technical assistance to Tb
  programs
• ECDC
• Molecular surveillance of MDR-TB and database
• ERLN-TB network
• Analysis of TB case management in the EU and
  EEA/EFTA countries with special focus on MDR/XDR
  TB
• EU DG research (TBPANNET)

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Conclusion

• National and regional variation in DR TB
  (possible undereporting)
• Absence of data on DR or questionable quality
• FSU Countries are facing an MDRTB epidemic
• Half of TB cases in FSU are resistant to at least
  one TB drug with the Baltic countries
  representing the best scenario for the region
• XDR is emerging where 2nd line drugs are used
• Data on XDR are mined by shortage in laboratory
  capacity
• New methods for gathering resistance data are
  neded
• Better understanding on the association between
  TB and HIV is needed