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 2008 by the author
Postgraduate course: Update in tuberculosis:
advances in the management of drug resistant and MDR-TB cases
ERS, Vienna 12/9/2009
Resistance Pattern for
Tuberculosis in Europe:
diagnostic aspects
Daniela M. Cirillo
San Raffaele Scientific Institute
Milan Italy
Global Epidemiology of TB
•
•
•
•
1/3 of global population infected (246/ 100,000 p)
> 8.8 million new cases a year (141/ 100,000 pop)
> 2 million deaths a year
> 95% of cases and deaths occur in the developing
world
–
–
–
–
75% of cases in 15-54 age group
Devastating economic costs
> 1/$ million deaths due to TB/HIV
MDR ubiquitous
3
TBC: “good news, bad news”
Rio 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
4
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
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
TB burden, EUR, 2007
90
80
70
per 100.000 pop
60
notification rate, EUR (53
countr ie s )
e s tim ate d incide nce , EUR (53)
75,9
notification rate, EEUR (18
countr ie s )
notification rate, EU (25
countr ie s )
50
48,5
40
39,4
30
20
10
11
0
80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 00 01 02 03 04 05 06 07
Rural/ Residential versus metropolitan
areas
In Western Europe the incidence of
tuberculosis is highly increased in
Metropolitan settings
Global TB targets
• Outcome targets (World Health Assembly, Stop TB
Partnership)
– Case detection >70%
– Treatment success >85%
• 2015 Impact targets (MDGs, StopTB Partnership)
– Halt and reverse incidence of TB
– Halve prevalence and mortality levels of 1990
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)
Outcomes for those patients not successfully treated,
by WHO region, 2005 cohort
DOTS
Non - DOTS
82
• Point 5 comma II
•
•
•
•
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
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
•
•
•
•
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
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
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
•
•
•
•
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
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
19
19
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 (10–8 x 10–8 = 10–16).
– Monotherapy led to selection of drug-resistant populations in
cavitary disease more often than in cases with non-cavitary
lesions (about 103–104 organisms).
– This is the rationale for treatment regimens with more than one
20
20
drug.
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
21
MDR-TB prevalence, EUR,
2007
Estimated, among new
Estimated, among re-treatment
– 10.35% (43 600)
– 43,41%
Notified, among new
Notified, among re-treatment
– 9,59 % (7 351)
– 38,55 %
Detection rate, new
- 17%
13 settings with >30% resistance to any TB
drug among new cases 2002-2007
14 settings with ≥ 6% MDR-TB
among new cases 2002-2007
16 settings with ≥ 25% MDR-TB among
previously treated cases 2002-2007
Countries with XDR-TB confirmed cases as of May
2008
Italy
Armenia
Japan
Azerbaijan
Latvia
Australia
Lesotho
Bangladesh
Lithuania
Botswana
Mexico
Brazil
Moldova
Canada
Mozambique
Chile
Namibia
China, Hong Kong SAR
Czech Rep.
Ecuador
Nepal
Norway
France
Peru
Georgia
Philippines
Germany
Poland
India
Portugal
Rep of Korea
Islamic Rep. of Iran
Israel
Russian Fed.
Netherlands
Estonia
Ireland
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
Argentina
Romania
Slovenia
South Africa
Spain
Swaziland
Sweden
Thailand
UK
USA
Vietnam
Ukraine
Based on information provided to WHO Stop TB Department - May 2008
XDR-TB among MDR-TB cases
2002-2007
* Sub-national averages applied to Russia
< 3% or less than 3 cases in one
year of surveillance
3 - 10%
> 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
Extensively Drug‐Resistant Tuberculosis Is Worse than
Multidrug‐Resistant Tuberculosis: Different Methodology
and Settings, Same Results
Giovanni 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 2008;46:958–959
• With the multiple regression analysis, the presence of
XDR was an independent risk factor for both death (OR,
2.07; 95% CI 1.05–4.05;P V .034) and treatment failure
(OR, 2.37;95% CI, 1.14–4.89; 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)
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
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
30
rifampicin resistance detection
from TB strains
Commercial Line Probe Assays
Hain Lifescience
Innogenetics
INNO-LiPA-Rif.TB
31
Comparison GenoType® MTBDR and INNO-LiPA Rif.TB
GenoType® MTBDR
INNO-LiPA Rif.TB
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
Company
GenoType MTBDRsl (Hain Lifescience)
33
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
Available from April 14th:
GeneXpert MTB
Sensitivity and Specificity
• Sensitivity in smear negative, culture positive (S-C+) was 90.9% (70/77)
• Sensivity in smear positive, culture positive (S+C+). 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%
Xpert MTB/RIF kit
PAGE | 37
Lab-On-Chip® microfluidic technology
PCR:
• Ultra-Fast PCR:  40°C\s;  10°C\s; ± 0.1°C
• 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
Wrong DSTs have huge
impact at :
•Patient level
•Community level
•Health care costs
Leading to mismanagement of TB
cases and increasing MDR
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
Patterns of Second-line Drug Resistance in
MDR Isolates by Geographic Region
(N=3,461)
AG+CM
AG+FQ
AG+ >1 Group 4
CM+FQ
CM+ >1 Group 4
FQ+ >1 Group 4 drug
AG+CM+FQ
AG+CM+ >1 Group 4
AG+CM+FQ+1 Grp 4
Any 3 SLD classes
Latin
America
543 (%)
N Amer
320 (%)
UK /
W Euro
451 (%)
Russia /
E Euro
406 (%)
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
N Afr
Asia
Mid E 1,563 (%)
95 (%)
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
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)
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
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
MDR/TB and HIV
Existing evidences:
• Institutional outbreaks of MDR-TB have primarily affected HIVinfected 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.
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
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
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)
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