Diagnosi genotipica delle resistenze

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Diagnosi genotipica delle resistenze

• DM Cirillo
• Unità Patogeni Batterici Emergenti, HSR Milano

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TBC MDR nuovi casi, 1994-2007
TBC MDR ritrattamenti, 1994-2007
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Diagnosi di labortorio
Nat Med 13 2007

• Verso lo sviluppo di test
• RAPIDI
• Sensibili
• Specifici
• Precoce diagnosi di infezione attiva
• Precoce identificazione di MDR/XDR
• Precoce identificazione di particolari genotipi

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DST in M. tuberculosis

• Metodi Fenotipici (valutazione dela crescita in
  terreno solido/liquido in presenza del farmaco)
• Costo-efficace
• Semplice da eseguire più complessa da
  standardizzare
• Risultati disponibili in settimane/mesi
• Metodi moleculari (identificazione delle
  mutazioni responsabili di resistenza)
• (generalmente) costosi
• Difficoltà di esecuzione, limitato ad alcuni
  targets
• Risultati disponibili in ore
• Non richiede il ceppo vitale, indipendente da
  inqiuinmento del campione

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Limiti del test fenotipico

• Un Gene che non è Espresso In Vitro può Essere
  Espresso In Vivo

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APPROCCIO CHEMIOTERAPICO ALLA TUBERCOLOSI

• Diverso dalle altre malattie batteriche per
• Lungo tempo di replicazione dei micobatteri
• Fase di quiescenza
• Crescita in situazioni metaboliche molto diverse
• Crescita in ambienti molto diversi (presenza di
  ossigeno, microarofilia, basso pH)
• Necessità di più farmaci attivi contemporaneamente

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Farmaci antitubercolari di prima scelta

• Isoniazide
• Rifampicina
• Etambutolo
• Pirazinamide
• Streptomicina

NUMERO LIMITATO
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Resistance in M. tuberculosis

• Due exclusively to chromosomal mutations
• Mutations responsible of drug resistance occur
  spontaneously with variable frequencies
  (1/106-1/108)
• Resistance is the results of the selection of
  resistant mutants due to inadequate therapy
• The use of at least two active drugs decreases
  the occurrence of resistances
• DST must be reliable and rapid to perform

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DST molecolare in M. tuberculosis

• Basato sulla analisi di singole mutazioni
  nucleotidiche
• permette di ottenere dati indipendentemente
  dalla coltura
• Predice cross-resistenze
• Consente lanalisi simultanea di molti campioni.
• Standardizzazione (automzione) e TAT

• Costo-efficacia
• Solo per pochi farmaci
• Bassa sensibilità per alcuni campioni

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Molecular techniques used to detect
drug-resistances

• There is not a universal technique
• The choice among the different available
  techniques depends on the information to be
  collected, and on the considered target
• Technology is on continuous up-grading the
  application depends on the capability of the
  laboratory
• The majority of molecular tests are able to
  identify only known mutations
• Opportunity of automation
• (decreased risk of contaminations reduced
  hand-time increased biosafety)

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Metodi molecolari Overview
Sequencing/Pyrosequencing PCR Restriction
Fragment Length Polymorphism (PCR-FRLP)
Time-consuming expensive
Not specific for M. tuberculosis (not applicable
in clinical samples)
PCR Single Strand Conformation Polymorphism
(PCR-SSCP)
Highly specific and sensitive but expensive
Real Time PCR Molecular beacons
Expensive not easily available
Peptide Nucleic Acid Probe (PNA)
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Metodi molecolari Overview
Line Probe Assay (LiPA)
Advantages - Easy to perform and easy read-out
cost-effective
Disadvantages - Limited number of probes that
can be used - Fails to distinguish insertions
mutations
Microarray
Advantages - High throughput for screening due
to the high number of probes - High automation
(high standardization)

• Disadvantages
• Higher number of probes ? higher complex ity in
  results interpretation
• Standardization requires reproducibility of
  data
• Cost-effectiveness? To be evaluated

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Home-made or commercial, the choice

• Home- made
• Cost-contained
• Protocols may be modified/upgraded
• Technical capability (HR and equipment)
• Lack of controls and QA

• Commercial
• Expensive
• Provided protocols often difficult to be modified
• Minimal technical capability required
• Equipment could be provided
• Controls usually provided
• QA in place

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Geni coinvolti nella resistanza ai maggiori
anti-tuberculari
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but we could obtain information about
cross-resistance
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Resistenza alla Rifampicina il gold target

• Key-drug per il regime di trattamento
• Le mutazioni sono concentrate in un hot-spot del
  gene rpoB
• Candidato ottimale per la diagnosi molecolare

• Metodi
• Home made
• - Commerciali

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Hot Spot Region di rpoB (RIF-R)
65.2
10.1
15.2
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Identificazione delle mutazioni che conferiscono
resistenza a Isoniazide i problemi

• Mutazioni in più geni strutturali e regolatori
  (inhA) o mutazioni multiple nello stesso gene
  (katG, ahpC)
• Solo alcune mut (katG) correlanano con il
  fenotipo di resistenza ad alta concentrazione,
  altre non hanno significato clinico (?)
• Frequenza di mutazione diversa su base geografica
• Relazione tra over-expressione di ahpC e fenotipo
  resistente non chiara

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MTB / Rif-resistance test

• Workflow
• sputum
• simple 1-step external sample prep. procedure
• time-to-result lt 2 h
• throughput gt 16 tests / day / module
• no need for biosafety cabinet
• integrated controls
• Performance
• specific for MTB
• sensitivity similar to culture
• detection of rif-resistance via rpoB gene
• Product and system design
• test cartridges for GeneXpert System
• modular expansion and swap replacement of
  detection unit
• 1 day technician training for non-mycobacteriolog
  ists

GeneXpert
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Test commerciali per la farmacoresistenza
Hain Lifescience
Innogenetics INNO-LiPA-Rif.TB
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Read-out of the test Drug-resistance is
detected by the lack of hybridization of one or
more wt probes with or without the hybridization
of mutated probes
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GenoCard a tool for transport and storage of
samples for tuberculosis molecular drug
susceptibility testing
1 drop of cell suspension or culture-positive MGIT
1 drop of clinical specimens
Paper-like support that retains PCR inhibitors
Dry-out (Room Temperature for 2 hour)
Inactivation of M. tuberculosis (incubation at
110 C for 15 min)
A spot (Ø 1 mm) of the GenoCard was used
directly as DNA template in the amplification
reaction
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Sample spotting and labelling
Inactivation Transport
Spot collection with the washable punch
puncher cleaning between samples
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Test Genotype MTBDRplus molecular DST in
clinical isolates
gt sensitivity
gt Concordance vs. DST
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Test commerciali per la farmacoresistenza
Expected sensitivity for INNOLiPA, MTBDR, and
MTBDRplus based on frequency of targeted mutations
RIF
INH
Limiting factor INH sensitivity
MDR
Real sensitivity for MTBDR and MTBDRplus on
Italian strains
Sensitivity True Positive/(True PositiveFalse
Negative)
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RIF-R rpoB gene
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INH-R katG gene and inhA promoter region
Promoter inhA (associated with katG mutations) I
5.0 MOC 7.2 BF 35.0
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MDR associated mutations
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Comparison between the results obtained with
traditional DST, sequencing analysis and Genotype
MTBDR (RIF-R)
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Comparison between the results obtained with
traditional DST, sequencing analysis and Genotype
MTBDR (INH-R)
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InnoLiPA RIF.TB meta-analysis

• BMC Infectious Dis ,Pai 2005
• 14 pubblished studies
• 12/14 sensitivity gt95 specificity 100
• (for clinical samples are excluded indeterminate
  results)

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GenoType MTBDR assays for the diagnosis of
MDR-TB meta-analysis
Ling et al (2008). Eur Respir J (32)1165-1174
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Multiplex PCR to detect INH-R
Promoter region inhA
Mut nt C-15T
Cod. 315 katG
Mut aa S315T
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Multiplex PCR results (INH-R)
Controllo amplificazione
Mut aa S315T
Mut nt C-15T
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Multiplex PCR for XDR-TB rapid detection
rrs a1401g
650 bp
gyrA A90V
300 bp
rpsL K43R
200 bp
Improvement set of primers TB specific
amplification-positive control to be added

• Other targeted mutations with alternative sets of
  primers
• gyrA D94V
• rpsL K88R

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Evaluation of the TB-Biochip oligonucleotide
microarray system for rapid detection of rifampin
resistance in Mycobacterium tuberculosis Caoili
et al. 2006 JCM 44 2378-2381

• Suitable for use in clinical laboratories (little
  hands-on time specialized training not
  required).
• Affordable (microarrays are anticipated to cost
  5 to 10 dollars each. Much of the cost of the
  system lies in the PCR amplification steps.
• Excellent specificity and good sensitivity
  discrepancies between the results of conventional
  DST and the TB-Biochip system result from the
  limited range of mutations included on the
  biochip.
• Inclusion of probes for additional mutations
  could, in principle, further increase the overall
  sensitivity of the system. However, the
  associated increase in production costs and
  potential for compromised specificity must be
  weighed against any gain in sensitivity.

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Specificity Mutations (control wt)
Probes repeated at least twice
Aragón et al. J Antimicrob Chemother (2006)
57825-831
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Rapid diagnosis of DR TB using LiPA from
evidence to policy
Ling et al (2008). Expert Rev Resp Med 2583-588
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MOLECULAR LiPAs FOR RAPID SCREENING OF PATIENTS
AT RISK OF MDR-TB WHO POLICY STATEMENT

• LiPAs are highly sensitive (gt97) and specific
  (gt99) for the detection of RIF-R, alone or in
  combination with INH (sensitivity gt90
  specificity gt99), on isolates of M.
  tuberculosis and on smear-positive sputum
  specimens.
• Overall accuracy for detection of MDR was
  equally high at 99, and retained when RIF-R
  alone was used as a marker for MDR.
• Current WHO recommendations
• Specimen processing for mycobacterial culture
  BSC under at least BSL2 conditions
• Procedures involving manipulation of M.
  tuberculosis cultures laboratories complying
  with BSL3 standards
• Applying these recommendations to LiPAs,
  processing of smear-positive specimens for direct
  testing should be performed in a BSL2 level
  laboratory, whereas performing the assay on
  positive cultures would require BSL3 facilities.

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LiPAs dati mancanti

• The evaluation of LiPAs in screening and
  diagnostic algorithms in different
  epidemiological settings
• The cost-effectiveness and cost-benefit of LiPAs
  in different programmatic settings
• The role of LiPAs in combination with
  conventional culture in smear-negative specimens
• The impact of specimen inactivation/disinfection
  procedures on LiPA performance
• Methods to optimize DNA extraction, especially
  from specimens with low numbers of organisms.

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Resistance to second-line injectable anti-TB
drugs and treatment outcomes in MDR-TB and XDR-TB
casesGiovanni Battista Migliori, Christoph
Lange, Rosella Centis, Giovanni Sotgiu, Ralf
Mütterlein, Harald Hoffmann, Kai Kliiman,
Giuseppina De Iaco, Francesco Lauria, M.
D'Arcy Richardson, Antonio Spanevello,
Daniela M. Cirillo and TBNET ( ERJ 2008)
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Altri Farmaci diagnosi di XDR-TB
58,7
69,5

• PZA-R
• 58,5 of strains carrying mutations in pncA no
  hotspot or specific frequency of mutations
  available

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Frequenze di mutazioni osservate sui ceppi XDR
italiani
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• Reverse line blot assay for detecting OFL
  resistance derived from point mutations in the
  gyrA gene.
• Specific oligonucleotide probes spanning the QRDR
  of the gyrA gene immobilized on nitrocellulose
  strips and hybridized with digoxigenin-labeled
  PCR products
• Region 1 H37Rv wild-type (WT1)
• amino acid substitutions in pos. 90 (A/V) and
  91 (S/P).
• Region 2 H37Rv wild-type (WT2)
• amino acid substitutions in pos. 94 (D/A, D/N,
  D/H, D/G)
• polymorphism S95T.
• Hybridizations detected colorimetrically.

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• The assay correctly identified all OFL-S and 17
  out of 19 (89.5) OFL-R strains results were
  100 concordant with those of nucleotide
  sequencing.
• A nested-PCR protocol was also set up for the
  line probe assay to amplify DNA extracted from
  sputum samples, with a sensitivity of 2x103 M.
  tuberculosis CFU/ml of sputum. This value is
  lower than that required for recognition of
  acid-fast smear positivity by Ziehl-Neelsen
  staining (0.5x104 to 1x104 CFU/ml of sputum).
• 75 to 94 of FQ-resistant isolates had gyrA
  mutations in the QRDR. Other mechanisms of
  resistance include mutations in regions of gyrA
  and gyrB outside the QRDR, decreased cell wall
  permeability, active drug efflux pump mechanisms,
  sequestration of drug, and drug inactivation.

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Alla ricerca di nuove mutazioni

• Preliminary data on methyltransferases-coding
  genes from second-line injectable drug-resistant
  strains
• Mutations found in gidB gene
• Nucleotidic polymorphism found in tlyA gene
• Preliminary data on ETH-R
• No mutation found in ndh gene
• Mutations found in ethA gene (33)

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Molecular DRS in Burkina Faso (Dec 2006 Oct
2008)

• Background
• No culture/culture DST facilities availability
• HIV-TB co-infection high prevalence

Population
108 chronic patients enrolled at 15 sites from
Miotto et al EID 09 submitted
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DRS perfomed by molecular assay on decontaminated
sputum specimens

• 3 MDR by MTBDRplus assay were not confirmed to
  be RIF-R on culture DST.
• Sequence analysis of the rpoB gene M515IH526N,
  L533P, and H526N respectively.
• Further analysis allowed to detect increased MICs
  in these 3 strains

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Discrepancies
Discrepant sputum smear microscopy results could
be due to the different samples examined in
Burkina Faso and in Italy. Sub-optimal transport
conditions this may have affected culture
results Most of the culture-negative samples
harboured DNA from MTB sensitive strains by
MTBDRplus treatment was effective against those
bacteria and category IV regimen was
unnecessary. One smear-positive culture-negative
resulted MDR at the molecular assay may be due to
the fact that this case was sampled at M6 after
the beginning of the cat. IV regimen we observed
during treatment follow-up that the molecular
assay became negative between M3 and M6 in
patients under effective treatment Two sputum
smear-negative samples harbouring NTM (M.
intracellulare, M. avium) have been identified as
MTB complex by the MTBDRplus sub-optimal
transport conditions and successful treatment
that selected the microorganisms not belonging to
the MTB complex.
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Concluding remarks

• The use of the molecular assay, with further
  confirmation from culture and DST when available,
  suggested readdressing classification and
  reconsidering treatment of different groups of
  patients
• Patients that were classified and treated as MDR
  cases harbouring RIF- and INH-S strains (n 24)
• Patients negative for MTB complex DNA (n 29)
• Patients considered chronic due to smear
  positivity but instead carrying NTM (n 15)

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Risks of extensive use

• Increasing costs for the TB programme due to
  reagents, disposable material equipment,
  personnel
• Sub optimal performance in non adequately
  equipped/maintained laboratory
• High risks of false positive/cross contamination
• Misinterpretation of results may lead to
  patients mismanagement and new cases of MDR

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Advantages of extensive use

• Centralization and extensive on selected
  patients groups use may improve technical
  capacity and reduce costs
• Prompt availability of data on drug sensitivity
  for correct patients management

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Molecular DST conclusions

• Usefulness of molecular techniques in DST is
  still limited by the lacks of knowledge of all
  molecular mechanisms of resistance.
• Negative results from genotypic tests do not
  exclude a resistant phenotype
• Although these assays cannot replace conventional
  DST, the high sensitivity and specificity for
  RIF-R and INH-R can facilitate the early
  diagnosis and treatment of MDR-TB, particularly
  for patients with a history of prior TB
  treatment.
• Sensitivity of commercial tests could be
  influenced by geographic regions
• Identification of mutations by molecular DST
  allows to predict cross-resistances among drugs
  (e.g. aminoglycosids, cyclic peptides).

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Di cosa abbiamo bisogno

• Chiarire i meccanismi di resistenza per
  aumentare il numero delle mutazioni note
  coinvolte nei fenotipi di resistenza.
• Miglioramento della performance negli
  smear-negative
• DST molecolare per i farmaci di 2ndalinea per
  migliorare la capacita diagnostica di MDR-XDRTB
• DST molecolare per i farmaci di 2ndalinea per
  migliorare i regimi terapeutici

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Future perspectives
hSR, ITALY - FZB, GERMANY - UNISI, ITALY -
NCIPD-NRL, BULGARIA - ST srl, ITALY - UHLD,
ALBANIA - UNIG, UK - FIND, SWITZERLAND -
HPA-MRU-QM, UK

• Rapid identification of MTC
• Rapid diagnosis of MDR/XDR-TB cases
• Rapid epidemiological analysis

Better and earlier patients management