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 2012 by the author
The new horizons of molecular
diagnosis: do we still need
conventional microbiology?
D. Cirillo
Emerging Bacterial Pathogens Unit
WHO CC for integrated laboratory strengthening on Tb
and other emerging infections
S.Raffaele Scientific Institute ,Milan
Outline
• Introduction: role of the laboratory in TB diagnosis
• Development and spreading of MDR-TB
• Challenges of conventional laboratory diagnosis of
tuberculosis
• Molecular tests for 1st line drugs resistance detection:
– LPAs; Xpert TB Rif
– Interpretation of the results
– Performance on EP samples
• WHO recommendations and diagnostic algorithms
• Conclusions
• Molecular epidemiology tools
Introduction
• Care of TB patients starts with a QA
diagnosis
• A robust network of Tb laboratories is
required:
– Adaquate biosafety
– Modern diagnostics
– SOPs
– QAs
• Integrated laboratory network
Laboratory biosafety
• Mtb is a class 3 risk pathogen
• All biosafety strategies (minimum requirements) should
be based on procedures risk assessment
• Based on:
–
–
–
–
–
Bacillary load of samples and workload
Viability of bacilli
Aerosol generation
TB local epidemiology
Fitness of the staff
Technologies and laboratory
appropriateness
Introducing new technology requires addressing of
core elements:
– Infrastructure, biosafety measures and maintenance
– Equipment validation and maintenance
– Specimen transport and referral mechanisms
– Management of laboratory commodities and supplies
– Laboratory information data and management system
– Laboratory quality management
– Strategies for HR development and retention
GLI road map at
:http://www.who.int/tb/dots/laboratory/policy/en
Role of molecular biology
• Fast detection and confirmation of pTB
cases
• Detection of MDR-TB
• Detection of EP-TB
• Performances and cost implications:
• LAMP assay has not been endorsed for
incomplete evidences
• Other PCR/molecular tests never examined by
WHO for absence of large scale evidences
Samples appropriateness and
patients selection
• Quality and amount of samples are crucial
for molecular tests as well
• Pretest probability highly influences the
parameters of molecular tests
Role of the clinical suspicion level in
the evaluation of the molecular methods
Catanzaro A. et al JAMA 2000
Gold standard: microbiology or clinical?
Development and spreading of DR-TB
MDR-TB in Europe
XDR-TB in the world
WHO, Global Report 2011
Devaux I et al 2009. Emerg Infect Dis 15(7):1052-60
18 clusters ,7 belonging to Beijing lineage
• Causes:
patient’s related
physician’s related
Drug’s/ program’s related
2007: 41 Countries
2010: 58 Countries
•Crucial health problem:
• long/expensive treatment
• second line drug
 decreased cure rate,
increased side effects
Conventional DST: technical challenges
•
•
•
•
Adequate infrastructures and biosefety levels
MGIT DST: the gold standard
MDRTB : 3-6 weeks; XDRTB : 6-9 weeks
Reproducibility and accuracy of results are drugs dependent:
– Rifampicin, isoniazid : good results
– Second-line: non raccommended in the absence of CQ (200 samplea ad high risk/year)
Standardization and correlation
with the clinical outcome is
difficult to be achieved
Van Deun A. et al 2011. IJTLD 15(1):116-124
MOLECULAR DST ON M. tuberculosis
• Based on single nucleotide mutations detection
• Data on DST from specimens not suitable for culture
• Cross-resistance prediction
•Large number of specimens analysed at the same time
• Standardization (automated systems) and TAT
• Cost-effectiveness
• Only available for selected drugs
• Only available for selected specimens
• Low sensitivity in AFB-negative and non-respiratory samples
• Genetic diversity may influence statistical parameters of
molecular tests
TBPANNET workpackage 6
midterm meeting
Second-line drugs
First-line drugs
Genes involved in drug-resistance for major antitubercular drugs
Zhang Y et al 2009. IJTLD 13(11):1320–1330
Commercial tests for MDR-TB Diagnosis
WHO Global plan (2006-2015):development and roll out of new technologies to be adopted in
resources-limited settings
GenoType MTBDRplus, InnoLiPA Rif.TB
•Reverse hybridization, colorimetric reaction
•Results in 6-7 h
• some flexibility (n° probes/strip: 30-40)
• Technical expertise: some
Xpert MTB/RIF
•Integrated/automated qPCR
•Results in 2h
•Closed system (limited number of probes: <10)
• Technical expertise: none
LiPA Tests: work flow
1
2
3
4
5
• Decontamination, ~1 h
• DNA extraction, ~1 h
• Amplification (PCR), ~3 h
• Strip Hybridization, ~2 h
• Results interpretation and report
LPAs performance
Isoniazid
Rifampicine
Inno-LiPA Rif.TB
Morgan M et al 2005. BMC Infect Dis 5:62
GenoType MTBDRplus
Ling DI et al 2008. Eur Respir J 32:1165-1174
Ling DI et al 2008. Eur Respir J 32:1165-1174
cod. 315 gene katG
nt -8,-15,-16 gene inhA
Hot-spot gene rpoB
Sensitivity
Specificity
GenoType MTBDRplus
95-98%
98-100%
Sensitivity
Specificity
Clinical samples
Sensitivity
Specificity
95-99%
97-100%
Sensitivity
Specificity
82-93%
95-100%
95-99%
97-99%
Clinical samples
Sensitivity
Specificity
72-92%
96-99%
LPAs Performance
Few data available on the LPAs performance on smear negative samples
MTBDR vs MTBDRplus
-Improvement of performance of
20-25%
-Decreasing of indeterminates of
about 10-15%
LPAs are approved for AFB positive
respiratory samples
Miotto P et al 2008. J Clin Microbiol 46(1):393-4
TaT for LPA: 1-2 days
3° generation (GenoType MTBDRplus v. 2) commercially available since January
-AFB-negative and «scanty»
-Master mix ready and stabilized
-Easily interpretable hybridization «pattern»
Possible automation on LiPA
LiPAs require:
Level II biosafety areas
Skilled laboratory staff
Amplicon Contamination control
Xpert MTB/RIF: work flow
Boehme CC et al 2010. N Engl J Med 363(11):1005-15
Xpert MTB/RIF: performance
Hot-spot gene rpoB
Indeterminate: <2.5% (culture contamination: 4.7%)
Tb cases identification
Specificity
98-99%
Sensitivity
97-100%, AFB-pos.
75-84%, AFB-neg.
Xpert MTB/RIF, microscopy
Liquid Culture
Solid Culture
0-1 d
13-21 d
23-43 d
Rifampicin-Res Identification
Specificity
97-99%
Sensitivity
91-97%
Co-infection TB-HIV
Sensitivity
Boehme CC et al 2011. Lancet 377(9776):1495-505
86% (HIV-neg: 92%)
No differences in performances in AFB-neg. (microscopy: 47% in HIV-positive vs 65% in HIVnegative)
Theron G et al 2011. Am J Respir Crit Care Med 184:132-140
LiPA e Xpert MTB/RIF
Rifampicin resistance identification
Time to report to treatment center
Boehme CC et al 2011. Lancet 377(9776):1495-505
Xpert MTB/RIF: 0-1 d
LPA: 10-26 d*
DST in culture: 30-124 d**
Xpert MTB/RIF: 0-1 d (Microscopy: 1-2 d)
LPA: 27-53 d*
DST in culture: 38-102 d** (Culture: 42-62 d)
Some results not reported/lost
* Test on direct AFB pos sample + test on strain for culture pos smear neg samples
** DST on MGIT + DST on LJ
Xpert MTB/RIF
WHO/HTM/TB/2011.2
Potential limits of Xpert MTB/RIF
technology
• Unknown performance at a district level
• Unknown performance in children
• If RFP resistance is diagnosed in a low level MDR-TB prevalence
setting , the assay needs to be confirmed
• Testing for Rif-R only
• Need to perform a culture for DST to evaluate other drug resistance
• Need to perform a culture for monitoring issue (culture conversion)
• It requires uninterrupted and stable electronic power supplies and
yearly calibration
• Storage of reagents
Cost consideration
• Cost–effectiveness modeling:
– Increase of 30% of case finding if
replacement or add-on to microscopy
• Cost-comparison
– Current cost (16.86$) per test higher than
microscopy, lower than culture/DST on solid
and liquid media may drop to 10$ in the future
– Initial capital cost: higher that microscope
lower than a biosafe culture laboratory
Cost effectiveness
Courtesy of C Boheme
WHO recommended policy
• Strains or AFB positive respiratory samples
• Adequate infrastructures (biosafety, molecolar
biology)
• Tecnical capacities (supervision, QC)
• Appropriate transport and storage of reagents
• Central or Regional level
• INH drug-sensitive cases needs to be confermed by
culture
 Test to be adopted in settings with
adequate capacity and resources in
agreement with local NTP and WHO
reccomandations
•
•
•
•
•
•
•
•
Approved for smear-negative cases
Biosafety at microscopy level
No technical skill required
Annual module’s calibration
Distrect peripheral labs
Appropriate transport and storage of reagents
High NPV (99%)
Rif-RES cases to be reconfermed by LiA /colture if
prevalence of RIF-R è <10%
 Reference test for MDR suspects ,TB/HIV
Performance of Xpert on extra-pulmonary
specimens (adults and pediatrics)*
* Tortoli et. Al ERJ 2012
Comparison GenoType® MTBDRplus and XpertTB/MDR
GenoType® MTBDRplus
XpertTB-MDRplus
Hain Lifescience
Cepheid
M. tuberculosis detection
Yes
Yes
Detection of RMP Resistance in M. tb Complex
Yes
Yes
Detection INH Resistance in M. tb Complex
Intermediate Reference labs
Yes
No
Company
Fully automated /training
DNA tech
Patients testing from Sm/ C+ (Rif)
No/Yes
Fast tool Surveillance purposes
Yes/No
PCR
Mol Beacon
From liquid or solid culture
Yes
NA
Direct assay
Yes
Yes
Level of biosafety
Time to results
Potential to District IIlevel as fast
patients diagnostic tool, needs
Same day
evaluation at district
level
microscopy
2h
Low/Mod
Mod/High
Universal control
Yes
Yes
Extraction control
No
Yes
Cost of Maintenance
Contamination control
Low
High
Yes
Cost per test
No
Heteroresistance: mixed population of sensitive and
resistant bacteria
“…Our data show that cultures are not necessarily representative of what is in the clinical
specimen. Out of 54 PCR products amplified from DNA isolated from sputum samples of 48
patients in whom drug resistances were known or suspected, a large proportion (17%) showed
more than one genotype by RFLP analysis…” [Rinder H et al 2001. IJTLD 5:339-345]
Mixed bacterial population cannot be identified by sequencing but can be risolved
LPAs.
by
Molecolar diagnosis of resistance to drugs other than R and
H: the GenoType MTBDRsl test (Hain Lifescience)
Analyzed
codons:
gyrA : 85-97
rrs:1401,
1402,1484
embB: 306
GenoType MTBDRsl: performance
 High PPV and specificity  rapid identification of resistant cases
 Low sensitivity and NPV  need to confirm SENSITIVE cases by conventional DST
 Can be used for screening MDR-TB cases at high risk to develop XDR-TB
 For ETB sensitivity is increased (15-20%) when using the presence of mutations as
marker for resistance
 Overall diagnosis of XDR-TB: 44.4%  additional studies and markers are needed
Miotto P et al. ERJ 2012
Ethambutol resistance: is molecular detection of
resistance better than MGIT DST?
Ethambutol in MGIT:
-Decreaesed sensitivity and specificity
- Reduced riproducibility
-False-sensitive using 5 μg/mL as break point
Scarparo C et al 2008. J Clin Microbiol 42(3):1109-1114
Van Deun A et al 2011. IJTLD 15(1):116-124
70-80% of ETB-resistant isolates are mutated in the codon 306 of embB
Gene
84 clinical isolates ETB-R (MGIT, 5 μg/mL)
M306V
42,9%
Other mutations cod. 306 13,1%
Non mutated
44,0%
91 clinical isolatesi ETB-S (MGIT, 5 μg/mL)
>95% results ETB-resistant if
M306V
18,6%
MIC is determined
Other mutations cod. 306 24,3%
Non mutated
57,1%
Miotto P et al. Manuscript submitted 2011
Genetic diversity in M. tuberculosis
Mutation rate is different in different geografic areas:
Phiilogeographic distribution in M. tuberculosis:
Gagneux S et al 2006. PNAS 103:2869-2873
Genetic diversity in M. tuberculosis and implications on
genotypic detection of Drug Resistance
Experimental evidences show the association of specific mutations responsable for MDR phenotype to
selected genotypes (eg. Beijing)
Little is known for second line drugs?
Miotto P et al ERJ 2012
Common problems in interpreting
molecular results
• Discrepancies genotype/phenotype:
• False negative due to duplication
• Double pattern
Common problems in interpreting molecular
results: INH, E
• Is therapy modified based
on resistance data?
• inhA - 15 alone:
increased mic, needs to
follow closely over time
• Resistance to
Ethionamide
• Eth 306: main
mechanism fot ETH
resistance
A common problem: the “double pattern”
•
Hetero-resistance = equal
representation of susceptible and
resistant mutants of the same strain
•
Mixed pattern = mutual presence of
a resistant strain and a second,
susceptible strain
•
Not pure culture
•
Carry-over contamination
Further research is needed to clarify the clinical
role of selected mutations or mixed infections
Propidium MonoazideTM (PMA)
Codons
analysed:
• PMA is a membrane
impermeant
intercalating into free
extracellular DNA
and DNA from
nonviable bacteria
• PMA is excluded
from viable bacteria.
• Exposure to a light
source makes the
PMA-DNA complex
not amplifiable.
PMA pretreatment of clinical samples allows selective amplification of the DNA
derived from live bacteria
Comparison between DNA amplified from PMA treated (- -) and untreated ( ) sputum
samples collected at diagnosis (t0) and at 14 days from beginning of antitubercular therapy
Not treated
PMA treated
Miotto P et al. ERJ 2012
Non-tuberculous mycobacteria
(NTM)
Most mycobacteria are saprophytic bacteria but some NTM
are occasionally pathogenic to both humans and animals,
causing pulmonary, skin diseases, lymphadenitis and
disseminated infections.
• Increase
in infections caused by NTM
• Diseases caused by NTM are often associated with various
forms of immunosuppression, particularly HIV infection
Mycobacterium avium complex (MAC)
M. avium (subsp paratuberculosis, lepraemurium, and silvaticum), M. intracellulare
Mycobacterium ulcerans (skin infections)
Mycobacterium marinum
Mycobacterium xenopi, Mycobacterium malmoense (lung disease)
Mycobacterium kansasii
Molecular DST in TB: clinical impact
The use of MTBDRplus test, even in the absence of culture and
DST, allowed readjustment of patients’ treatment in Burkina
Faso:
- Patients who were classified and treated as MDR cases harbouring RIF- and
INH-S strains (n 26);
- Patients negative for MTB complex DNA (n 18);
- Patients with a non-tuberculous mycobacteria (NTM) infection (n 14).
Conclusions
1. Smear microscopy is not sufficient for management of chronic
patients (NTM infections)
2. Molecular assays can identify MDR-TB cases in the absence of
culture facilities.
3. Molecular assay for second-line drug resistance identification
allowed identification of XDR cases if mutations are present but can’t
exclude resistance
Miotto P et al. BMC Infect. Dis. 2009; 9:142
Badoum et al. ERJ 2011
41
Drug resistance: what’s new?
• March 2012, WHO:
RIF-R is not equivalent to MDR for surveillance purposes
• There is an increasing need for DST testing on anti-TB
drugs:
Moving forward to personalized therapeutic regimens
Characterization of novel mutations involved in drugresistant phenotype and virulence markers
• AMK/KAN/CAP: Rv3919c (gidB), Rv2416 (eis)
• Characterization of mutations occurring in genes encoding putative
targets for new drugs (nitroimidazopyran, linezolid)
• Compensatory mutations in MDR-TB strains (Comas et al. 2011, Nat
Genetics )
• Better understanding of genetic diversity and drug resistance
relationships
Molecular DST in TB: wrap up
2/2
 Clinically relevant when/where culture facilities are not easily
available
 Possibility/need to perform molecular DST on an increasing number
of drugs
 Increasing possibility to use molecular approaches not only in case
detection step, but also in further steps (e.g. treatment monitoring)
 Increasing need to better understand infection aethiology and/or
mixed infection (e.g. NTM)
However…
• Sensitivity affected by MTB epidemiology
• Limited number of targets that are detectable per assay (thus limiting
molecular approach potential usefulness)
• cannot exclude phenotypic resistance (wild-type result)
Molecular DST in TB: wrap up
2/2
 Clinically relevant when/where culture facilities are not easily
available
 Possibility/need to perform molecular DST on an increasing number
of drugs
 Increasing possibility to use molecular approaches not only in case
detection step, but also in further steps (e.g. treatment monitoring)
 Increasing need to better understand infection aethiology and/or
mixed infection (e.g. NTM)
However…
• Sensitivity affected by MTB epidemiology
• Limited number of targets that are detectable per assay (thus limiting
molecular approach potential usefulness)
• cannot exclude phenotypic resistance (wild-type result)
Lab-on-Chip (LoC) platforms: a possible
answer?
Tuberculosis
Rapid identification
complex
Malaria
of
MTB
Rapid diagnosis of MDR cases
Rapid genotyping of MTBC
Rapid identification of Plasmodium
pathogenic species
Rapid diagnosis of malaria drug
resistant cases
Rapid identification of clinically
relevant NTMs
The integrated PCR and Microarray lab-on-chip tool should represent a clear
innovation over the conventional molecular diagnostics for its robustness,
simplicity of use and low-cost.
Lab-on-Chip for molecular diagnostics
PCR:
• Ultra-Fast PCR
• Asymmetric Cy-5 PCR strategy
Microarray:
• Orientation probes
• Hybridization Control probes
• Hybridization Negative Controls probes
Lab-on-chip architecture
2 PCR reactors of 12.5 uL volume each (Total 25 ul)
1 Hybridization chamber of 30 uL
A 126 spots DNA microarray
2 in-let ports compatible with standard micro-pipettor tips
Integrated Heaters and Sensors
All the reaction modules are
fluidically integrated
Statistical analysis on clinical isolates
Drug resistance
Species identification
Detection limit:
Species identified:




M. tuberculosis complex 
M. avium
M. intracellulare

M. simiae, M. kansasii, 
M. scrofulaceum
M. abscessus, M.
chelonae
M. xenopi
M. fortuitum
 rpoB target: 104/mL (AFB: 1+ / scanty)
 All other targets: < 102/mL (AFB: scanty)
E@syCheck software
Chip
information
Summary of
results
Automatic generation
of final report
TB LoC: adds on
Genotyping by spoligotyping
•Directly used for specimens
• “Real-time” typing (nosocomial transmission, prisons, community etc…)
• Laboratory cross-contaminations
• M. tuberculosis complex genotype identification (family)
• TaT: 6-7 h, up to 43 samples per run
nnnnnnnooooonnnnnn…. (binary-code
translation)
Automatic report
Geographical distribution: the advantages of multi-purposes platform
TB
TB/HIV
Malaria
Neglected tropical
diseases
Dengue
Buruli ulcer
treponematoses
Leprosy
cysticercosis
dracunculiasis
lymphatic filariasis
human rabies
Trachoma
…
Algorithm for rapid DST testing: one doasn’t fitt all!
XpertTB/
MDR
Expected impact of rolling out molecular
technology for MDR-TB detection
WHO RECOMMENDATION, December 8th, 2010
Conclusions
•Appropriate lab data guided TB therapy is often still a challenge
• New diagnostics and supporting policies are available, globally the next major step is
translation of policy into practice.
Molecular tests for DR TB:
•Fast diagnosis of DR in risk groups
•Performance may be affected by genotypic background
•Identification of “special cases” mixed infections…
•Conventional methods STILL needed for :
• confirming molecular results
• Additional drugs
• Therapy monitoring
• Xpert MTB/RIF is the new standard for HIV infected TB suspects.
The impact of new tests will depend on:
– Appropriate use (pretest probability, sufficient
sample, samples selection)
– Availability of new generation of tests
covering more targets
– Extent of their introduction and capacity to
interpret the results and translate results into
clinical practice
– Use of the test for more than diagnosis alone
– Combination of different tests in an
appropriate diagnostic algorithms
Molecular Typing Tools for Surveillance
• To identify epidemiological links between TB patients to detect and control
outbreaks early and rapidly
• Rule out suspected outbreaks and confirm transmission has NOT occurred
• To identify incorrect TB diagnosis based on false-positive cultures and thus
avoid unnecessary investigation and treatment
• To distinguish exogenous re-infection from endogenous reactivation in
patients with a past history of TB
• Discover unusual transmission settings and transmission between different
regions
• Monitor the size of clusters and thus monitor progress towards TB
elimination
Molecular strain typing methods
Repetitive DNA sequences were first described in
Mycobacteria in 1988 (Eisenach et al., 1988).
Restriction fragment length polymorphism (RFLP)
IS6110 used as a probe
Copy numbers and the position on the
genome varies between strains
Standardised procedure so compare
nationally and internationally
Disadvantages
Requires viable culture, large amount of DNA, labour intensive,
comparative analysis of patterns tedious.
Mycobacterial Interspersed Repetitive Units -Variable Number of
Tandem Repeats (MIRU-VNTR)
Based on tandem repeats in
mini-satellite regions of the
genome.
The original 15 loci consisted of 5
exact tandem repeats (ETR A-E)
followed by 10 MIRUs. Described
by Supply et al., 2006.
MIRU 24 is now considered the
gold standard
VNTR- Identical pieces of DNA repeated a variable number of
times, next to each other (in tandem) at specific positions
(loci) on the genome.
2
3
2
4
2
PCR is used to determine the
number of repeats at each loci
(24 in total)
Strain type profile = 232425673216524316425375
5
SPOLIGOTYPING
• The DR sequences (direct repeat) are
repeated sequences of 36 bp in only one
locus of the MTB chromosome, separated by
sequences of 34 to 41 bp.
• The technique is based on a PCR and
hybridization
• The presence or absence of different DR
allows a specific pattern for each strain.
• Advantages: Few DNA is required, easy
interpretation
• Disadvantages: Lesser discriminative power
than the RFLP.
Acknowledgements
WHO Collaborating Centre for Integrated Lab. strengthening on TB and other Emerging
Infections
hSR Scientific Park
San Raffaele Scientific Institute
Paolo Miotto
Andrea M. Cabibbe
Ilaria C. Valente
Emanuele Borroni
Paola Mantegani
Enrico Tortoli
Queen Mary and Westfield College, University of London, United Kingdom
Forschungszentrum Borstel, Leibniz-Zentrum für Medizin und Biowissenschaften, Germany
Foundation for Innovative New Diagnostics (FIND), Switzerland
Università degli Studi di Siena, Italy
National Tuberculosis and Infectious Diseases University Hospital, Lithuania
University of Vilnius, Institute of Biotechnology, Lithuania
Scientific Institute of Public Health – Institut Pasteur, Belgium
University of Glasgow, United Kingdom
Institute of Infectious and Tropical Diseases, University of Brescia, Italy
Dept. of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità, Italy
Fondazione Maugeri ,Tradate
Università di Brescia…
Hain Lifescience , Germany
Cepheid, USA
ST Microelectronics, Italy
Veredus Laboratories, Singapore