Diagnosi genotipica delle
resistenze
DM Cirillo
Unità Patogeni Batterici Emergenti, HSR Milano
TBC MDR – nuovi casi, 1994-2007
TBC MDR – ritrattamenti, 1994-2007
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
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
Limiti del test fenotipico
Un Gene che non è
Espresso “In Vitro” può
Essere Espresso “In
Vivo”
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
Farmaci antitubercolari di prima scelta
•
•
•
•
Isoniazide
Rifampicina
Etambutolo
Pirazinamide
• Streptomicina
NUMERO LIMITATO
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
DST molecolare in M. tuberculosis
Basato sulla analisi di singole mutazioni
nucleotidiche
permette di ottenere dati indipendentemente dalla
coltura
Predice cross-resistenze
Consente l’analisi simultanea di molti campioni.
Standardizzazione (automzione) e TAT
Costo-efficacia
Solo per pochi farmaci
Bassa sensibilità per alcuni campioni
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…)
Metodi molecolari – Overview
Sequencing/Pyrosequencing
PCR Restriction Fragment Length
Polymorphism (PCR-FRLP)
Time-consuming; expensive
PCR Single Strand Conformation
Polymorphism (PCR-SSCP)
Not specific for M. tuberculosis
Real Time PCR
Highly specific and sensitive but
expensive
Molecular beacons
Peptide Nucleic Acid Probe (PNA)
(not applicable in clinical samples)
Expensive; not easily available
Metodi molecolari– Overview
Line Probe Assay (LiPA)
Advantages:
Disadvantages:
- Easy to perform and easy read-out; costeffective
- Limited number of probes that can be used;
- Fails to distinguish insertions mutations
Microarray
Advantages:
Disadvantages:
- High throughput for screening due to the
high number of probes;
- Higher number of probes  higher complex
ity in results interpretation;
- High automation (high standardization)
- Standardization requires reproducibility of
data;
- Cost-effectiveness? To be evaluated
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
Geni coinvolti nella resistanza ai
maggiori anti-tuberculari
Drug
Streptomycin
Gene
rpsL
Mutations
12S ribosomal protein
Coding region (60%)
16S rRNA
Reg. 530 and reg. 915 (8%)
katG
Catalase-peroxidase
coding region (cod. 315 - 60-80%)
inhA
NADH-dep enoyl-ACP red
promoter reg. (Ribosome binding site - 15%); coding region
ndh
NADH dehydrogenase
coding region
rrs
Isoniazid
Gene product
ahpC-OxyR regulon (controls katG and several other genes) promoter region (mutations relatively rare)
Rifampin
rpoB
RNA pol (β subunit)
hot spot region (cod. from 508 to 535 - 98%); N-term region
Ethambutol
embB
Arabinosyl transferase
ERDR (cod. 306 - 70%)
embC
Arabinosyl transferase
coding region
inhA
NADH-dep enoyl-ACP red
promoter reg. (Ribosome binding site); coding region
ethA
Monooxygenase
coding region
ethR
Monooxygenase repressor
coding region
ndh
NADH dehydrogenase
coding region
Pyrazinamide
pncA
Pyrazinamidase
coding region (70%)
Fluoroquinolones
gyrA
DNA gyrase (sub. A)
QRDR (70%)
gyrB
DNA gyrase (sub. B)
QRDR
rpoB
RNA pol (β subunit)
coding region
rrs
16S rRNA
coding region
tlyA
rRNA methyltransferase
coding region
Viomycin
rrs
16S rRNA
coding region
Kanamycin
rrs
16S rRNA
coding region
Amikacin
rrs
16S rRNA
coding region
D-Cycloserine
alr
D-Ala racemase
promoter region
T hymidylate synthase
coding region
Ethionamide
Rifabutin
Capreomycin
Para-salicylic acid
thyA
but we could obtain information about cross-resistance
Cross-resistances
CAP-VIO
Mutations in tlyA or rrs C to T 1402, G to T 1484
AMK-KAN
Mutations in tlyA and rrs A to G 1401
KAN-CAP
Mutations in rrs: A to G 1401, G to T 1484
KAN-VIO
Mutations in rrs: A to G 705, A to G 1400, G to A/T 1483;
Mutations in rpsL: Lys43Arg (cross-R to STR)
KAN-CAP-VIO
Mutations in rrs: C to T 1402, G to T 1484
RFB-RIF
Cross-resistance rate: 90%.
Mutations in rpoB (cod. 526, 531).
Mutations at cod. 516 and 522 in RIF-R predict RFB-S (Sintchenko 1999, Pathology).
INH-ETH
Mutations in inhA: Ser94Ala; Ribosome binding site;
Mutations in ndh: coding region
DCS-VCM
Mutations in ddl
Resistenza alla Rifampicina: il “gold
target”
• Key-drug per il regime di trattamento
• Le mutazioni sono concentrate in un hotspot del gene rpoB
Candidato ottimale per la diagnosi
molecolare
Metodi:
-“Home made”
- Commerciali
Hot Spot Region di rpoB (RIF-R)
10.1%
65.2%
15.2%
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
MTB / Rif-resistance test
Workflow
•sputum
•simple 1-step external sample prep.
procedure
•time-to-result < 2 h
•throughput: > 16 tests / day / module
•no need for biosafety cabinet
•integrated controls
Sample
Prep
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 nonmycobacteriologists
<10 minutes
GeneXpert
Amplification
and Detection
< 1 hour
Test commerciali per la farmacoresistenza
Hain Lifescience
Innogenetics
INNO-LiPA-Rif.TB
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
katG (INH-R)
+ + – + – +
+ – – + –
+ – + + –
+ + – – –
+ + – – +
+ – – – +
rpoB (RIF-R)
+
+
+
+
+
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
Sample spotting and labelling
Inactivation
Transport
Spot collection with the
washable punch
puncher cleaning between
samples
Test Genotype MTBDRplus: molecular DST in
clinical isolates
> sensitivity
> Concordance vs. DST
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 Positive+False Negative)
RIF-R: rpoB gene
INH-R: katG gene and inhA promoter region
Promoter inhA (associated
with katG mutations)
I : +5.0%
MOC: +7.2%
BF: +35.0%
MDR associated mutations
Comparison between the results obtained with traditional
DST, sequencing analysis and Genotype MTBDR (RIF-R)
Sequencing data hot-spot rpoB
Mutation/site of mutation
No. strains
Concordance
MTBDR/sequence
–
Dup514
D516V
D516V+S531L
531
533
S531L
526
H526Y
522
Del516
515+516
513
511+512+516
516+533
512+515+526
4
1
8
1
6
2
83
11
9
4
1
1
3
2
1
1
4/4
0/1
8/8
1/1
6/6
2/2
83/83
11/11
9/9
4/4
1/1
1/1
3/3
2/2
1/1
1/1
Tot.
138
137/138
Comparison between the results obtained with traditional
DST, sequencing analysis and Genotype MTBDR (INH-R)
Sequencing data
Mutation
Reg. katG
S315T
S315N
E261Stop-T262K-A264G-T271N-A348S
–
tot.
Reg. inhA
tot.
C-15T
–
No.
strain (%)
Concordance
MTBDR/sequence
115 (67.3)
1 (0.6)
1 (0.6)
54 (31.6)
115/115
1/1
0/1
54/54
171
170/171
20 (11.7)
151 (88.3)
nd
nd
171
nd
InnoLiPA RIF.TB: meta-analysis
BMC Infectious Dis ,Pai 2005
14 pubblished studies:
12/14 sensitivity >95% specificity
100%
(for clinical samples are excluded
indeterminate results)
GenoType MTBDR assays for the diagnosis
of MDR-TB: meta-analysis
Ling et al (2008). Eur Respir J (32):1165-1174
Multiplex PCR to detect INH-R
Promoter region inhA
mabAF
5’-TCGAAGTGTG CTGAGTCACA CCGACAAACG TCACGAGCGT AACCCCAGTG..
3’-AGCTTCACAC GACTCAGTGT GGCTGTTTGC AGTGCTCGCA TTGGGGTCAC..
..ATTTCGGCCC GGCCGCGGCG AGACGATAGG TTGTCGGGGT GACTGCCACA-3’
..TAAAGCCGGG CCGGCGCCGC TCTGCTATCC AACAGCCCCA CTGACGGTGT-5’
AT
inhARMut
Mut nt C-15T
Cod. 315 katG
katG_0F
5’-GAACCCGCTG GCCGCGGTGC AGATGGGGCT GATCTACGTG AACCCGGAGG..
3’-CTTGGGCGAC CGGCGCCACG TCTACCCCGA CTAGATGCAC TTGGGCCTCC..
..GACGCGATCA CCAGCGGCAT CGAGGTCGTA TGGACGAACA CCCCGACGAA-3’
..CTGCGCTAGT GGTCGCCGTA GCTCCAGCAT ACCTGCTTGT GGGGCTGCTT-5’
GA
katG_R315Mut2
Mut aa S315T
Multiplex PCR results (INH-R)
1
gyrB
Mut aa S315T
katG
Mut nt C-15T
mabAF-inhA
Mutations
Reg. katG
S315T
S315N
Several
–
tot.
Reg. inhA
tot.
C-15T
–
No.
strains (%)
3
4
1020 bp
Controllo amplificazione
Sequencing data
2
300 bp
120 bp
Concordance
MTBDR/sequence
Concordance
PCR Multiplex
115 (67.3)
1 (0.6)
1 (0.6)
54 (31.6)
115/115
1/1
0/1
54/54
115/115
0/1
0/1
54/54
171
170/171
169/171
20 (11.7)
151 (88.3)
nd
nd
20/20
151/151
171
nd
171/171
Multiplex PCR for XDR-TB rapid detection
650 bp
300 bp
200 bp
rrs a1401g
gyrA A90V
rpsL K43R
Improvement: set of primers TB specific amplification-positive control to be added
Other targeted mutations with alternative sets of primers:
- gyrA D94V
- rpsL K88R
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
1.Suitable for use in clinical laboratories (little hands-on time; specialized training not
required).
2.Affordable (microarrays are anticipated to cost 5 to 10 dollars each. Much of the cost
of the system lies in the PCR amplification steps.
3.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.
Specificity
Probes repeated at
least twice
Mutations
(control: wt)
Aragón et al. J Antimicrob Chemother (2006) 57:825-831
Rapid diagnosis of DR TB using LiPA: from
evidence to policy
Ling et al (2008). Expert Rev Resp Med 2:583-588
MOLECULAR LiPAs FOR RAPID SCREENING
OF PATIENTS AT RISK OF MDR-TB: WHO
POLICY STATEMENT
• LiPAs are highly sensitive (>=97%) and specific (>=99%) for the
detection of RIF-R, alone or in combination with INH (sensitivity
>=90%; specificity >=99%), on isolates of M. tuberculosis and on smearpositive 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.
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.
Resistance to second-line injectable anti-TB drugs and treatment outcomes in MDR-TB and XDR-TB cases
Giovanni 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)
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
Frequenze di mutazioni osservate sui ceppi
XDR italiani
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.
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.
Alla ricerca di nuove mutazioni
Preliminary data on methyltransferases-coding genes from secondline 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%)
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
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: M515I+H526N, L533P, and H526N respectively.
Further analysis allowed to detect increased MICs in these 3 strains
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.
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)
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 patient’s
mismanagement and new cases of MDR
Advantages of extensive use
• Centralization and extensive on
selected patient’s groups use may
improve technical capacity and reduce
costs
• Prompt availability of data on drug
sensitivity for correct patient’s
management
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).
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
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