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PPT Cirillo "I Stage: from Koch to Xpert"
1. TUBERCOLOSI:UNA
MALATTIA
SOCIALE
21-‐22
SETTEMBRE
2012
Ia
tappa:
da
Kock
a
Xpert
D
Cirillo
San
Raffaele
Scien/fic
Ins/tute
Milan
Italy
2. Outline
• Urgent
need
for
an
improved
diagnosis
• Microscopy
• Culture
and
DST
needs
for
standards
and
EQAs
• Molecular
diagnosis
is
a
reality
• What
can
be
achieved
with
current
molecular
tools
• What
cannot
be
achieved
with
the
current
molecular
tools
• Global
prospec/ve
3. Urgent
need
for
new
diagnosGcs
• TB
case
detec/on
gaps:
– cases
undiagnosed
• Inaccessible
facili/es
• Not
self
repor/ng,
not
returning
• Wrong
diagnosis
– Cases
diagnosed
in
private
care
and
not
reported
• Infec/on
control
(Stop
TB/MDR-‐TB
transmission)
• Guide
treatment
• Monitoring
treatment
4. Microscopy
:
a
century
old
procedure
Rapid test
Inexpensive
n Does not allow species identification
n Not applicable to all samples
n Specificity for Mycobacterium spp:
>95%
Sensitivity: 25-65% (90 % of higly
n
infectious cases) Fluorescence Ziehl-Neelsen staining
Positive Predictive Value for TB
n 1st AFB smear 80-82 %
depends on epidemiological situation
2nd AFB smear 10-14 %
LED
microscopy
recommended
over
light
and
fluorescent
3rd AFB smear 5-8 %
microscopy
5. TB
Culture
Advantages Disadvantages
• Definitive diagnosis of TB • Complex and expensive
compared to microscopy
• Increases case finding of • Requires complex handling of
30-50% specimens
• Early detection of cases • Skilled technicians
• Provide strains for DST • Appropriate infrastructure and
biosafety levels
and epidemiological
studies
LIMITATIONS: need for decontamination and identification
*coverage 500.000/1000000
6. Culture:
solid/
liquid
solid liquid
• Low cost for reagents, not • Complex and expensive can be
automated automated (MGIT)
• Highest infrastructure and
• Culture level infrastructure biosafety levels
• Low contamination rate • Case finding increased 10% over
• Long time to positivity solid
• Colony morphology • Diagnostic delay reduced to days
• ID required • ID required
• DST only for selected drugs
• DST only for selected drugs
Strip speciation tests for fast ID of Tbcomplex
Molecular test for speciation of most common mycobacteria
7. M. tuberculosis identification
Morphology/ Molecular
Immuno-‐
Biochemical
tests
cromatographic
tests
LPAs
test
Probes
on
liquid
phase
Sequencing
EQA
and
standardiza/on
Spoligotyping
Enzyme
7
restric/on
8. DST
• Definitive diagnosis of DRTB
3
main
methods
Absolute
Propor/on
concentra/on
Resistant
Ra/o
methods
method
method
9. DST:
Liquid/solid
mtedia
comparison
Liquid
media
compared
o
solid
media
Advantages
compared
to
solid
media:
•
more
rapid
•
high
quality
of
media
•
fully
automated
system
•
tes/ng
of
1st,
2nd
,
and
new
drugs
(Linezolid)
•
safety:
plas/c
tubes
•
pyrazinamide
sensi/vity
test
Break
points
for
2nd
line
drugs
Disadvantages:
recently
revised
•
expensive
S/ll
poor
correla/on
with
clinical
•
higher
contamina/on
rate
outcome,
some
tes/ng
not
fully
•
dependency
on
a
company
reliable
•
no
DST
for
Cycloserine
12. Is
new
real-‐Gme
technology
improving
the
sensiGvity
of
molecular
tests?
13. New
generaGon
of
tests
does
not
improve
sensiGvity
Tortoli
et
al
2012
JCM
Methods
Amplicor
n
on
NAs
amples
Taqman
vs
based
oon
15000
s detec/on
have
intrinsic
limita/ons:
• Absence
on
specificity
ubop/mal
specimen
selec/on,
quan/ty,
Improvement
i f
target:
s
quality,..
Decrease
in
invalid
results
Improvement
in
post
test
probability
• Subop/mal
sample
prepara/on
14. Are
molecular
tests
tools
for
difficult
to
diagnose
cases?
• TB
in
Children
• Extrapulmonary
Tuberculosis
17. Diagnosis
in
difficult
cases
• Children
diagnosis
is
not
microbiologically
confirmed
in
40-‐60%
of
cases,
current
molecular
tools
s/ll
subop/mal.
A
host/
pathogens
biomarkers
approach
is
probably
needed
• Extrapulmonary
TB:
the
performance
of
molecular
tools
varies
and
should
be
considered
separately
for
each
specific
specimen
type.
18. Unsolved
Problems
of
Molecular
Tests
for
Direct
Diagnosis
of
Tuberculosis
• Subop/mal
sensi/vity/specificity
versus
a
gold
standard:
– Compared
to
culture
in
liquid
media
– Compared
to
a
combined
standard
based
on
the
“inten/on
to
treat”/
response
to
treatment
Decreased
confidence
in
the
test
19. 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:
fluoroquinolones
and
injectables
CorrelaGon
of
sensiGvity
test
results
and
clinical
outcome
is
difficult
to
evaluate
and
we
have
very
limited
or
no
evidence
for
Pyr,E,
and
2nd
line
drugs
other
than
INJ
and
FQs
on
MDR
cases
In
addi/on:
Capital
cost
of
facili/es
Cost
of
maintenance
Cost
of
staff
Van
Deun
A.
et
al
2011.
IJTLD
15(1):116-‐124
20. MECHANISMS
OF
DRUG
RESISTANCE
IN
M.
tuberculosis
Zhang
Y
and
Yew
W,
Int
J
Tuberc
Lung
Dis
2009
21. Commercial
Molecular
tests
for
e
Tdopted
in
WHO
Global
plan
(2006-‐2015):development
and
roll
out
of
new
technologies
to
b a
B/
MDR
TB
detecGon
endorsed
by
WHO
resources-‐limited
senngs
GenoType
MTBDRplus,
InnoLiPA
Rif.TB
• Reverse
hybridiza/on,
colorimetric
reac/on
• Results
in
6-‐7
h
•
some
flexibility
(n
probes/strip:
30-‐40)
•
Technical
exper/se:
some
• Biosafety
lev
2
Xpert
MTB/RIF
• Integrated/automated
qPCR
• Results
in
2h
• Closed
system
(limited
number
of
probes:
<10)
•
Technical
exper/se:
none
22. LPA
performance
i(n
isolates
and
clinical
LPAs:
performances
*based
on
2°
generaGon)
Rifampicin
samples
Isoniazid
Inno-‐LiPA
Rif.TB
GenoType
MTBDRplus*
GenoType
MTBDRplus
Hot-‐spot
rpoB
gene
cod.
315
katG
gene
nt
-‐8,-‐15,-‐16
inhA
gene
Morgan
M
et
al
2005.
BMC
Infect
Dis
5:62
Ling
DI
et
al
2008.
Eur
Respir
J
32:1165-‐1174
Ling
DI
et
al
2008.
Eur
Respir
J
32:1165-‐1174
Sensi.vity
95-‐98%
Sensi.vity
95-‐99%
Sensi.vity
82-‐93%
Specificity
98-‐100%
Specificity
97-‐100%
Specificity
95-‐100%
Decontaminated
clinical
specimens
(AFB-‐posi?ve)
Dec.
clin.
spec.
(AFB-‐pos)
Sensi.vity
95-‐99%
Sensi.vity
72-‐92%
Specificity
97-‐99%
Specificity
96-‐99%
23. TAT
to
Rif
–R
detecGon
and
reporGng
RIF-‐R
detecGon
Time
to
report
to
treatment
center
Boehme
CC
et
al
2011.
Lancet
377(9776):1495-‐505
Xpert
MTB/RIF:
0-‐1
d
Xpert
MTB/RIF:
0-‐1
d
(Microscopy:
1-‐2
d)
LPA:
10-‐26
d*
LPA:
27-‐53
d*
Culture
DST:
30-‐124
d**
Culture
DST:
38-‐102
d**
(culture:
42-‐62
d)
Some
results
not
reported/lost
*
test
on
AFB-‐pos
clinical
specimen
+
test
on
clinical
isolate
for
AFB-‐neg
cases
**
DST
performed
by
MGIT
+
DST
performed
on
LJ
24. What
can
be
achieved
or
parGally
achieve
with
the
current
molecular
tools
• Diagnosis
of
TB,
rifampicin
resistant
TB,
MDR-‐TB
in
smear
posi/ve/nega/ve
samples
• Iden/fica/on
of
up
to
80%
fluoroquinolones
and
up
to
40-‐80%
of
injectable
resistant
cases
among
MDR-‐
TB
cases
• Improvement
of
diagnosis
of
TB
in
extrapulmonary
samples
• Support
to
diagnosis
of
TB
in
children
• Diagnosis
of
NTMs
infec/on
26. DR
tesGng:
Rifampicin
Bactericidal
an/bio/c
that
inhibits
the
bacterial
DNA-‐
dependent
RNA
polymerase.
Target:
β-‐subunit
of
the
RNA
polymerase
(encoded
by
rpoB),
blocking
elonga/on
of
the
RNA
chain.
codon
526/
531
high
level
resistance
to
rifampicin,
MutaGons
in
a
“hot-‐spot”
region
of
81
bp
of
rifabu/n
e
rifapen/n
rpoB
gene
(Rifampin
resistance-‐determining
516
e
522
high
PPV
for
region)
→
RIF
resistance
(>
95%)
rifabu/n
res
27. ISONIAZID
ISONIAZID
INH:
targeGng
mycolic
acid
biosysthesis
MutaGons
in
KatG
gene
prevent
INH
acGvaGon
(cod.
315,
60-‐90%)
MutaGons
in
the
direct
target
inhA
(inhA
belongs
to
the
family
of
short-‐chain
dehydrogenases/reductases.
It
is
essenGal
in
MTB)
MutaGons
in
the
promoter
of
inhA
gene
leading
to
drug
tritraGon
(direct
target
over-‐producGon)
Only
Kat
G
315
and
inhA
are
included
in
LPA
Ratan
A
et
al.
EID
1998
28. PPV
and
NPV
for
Rif
resistance
at
different
prevalence
of
Rif
resistance
WHO/HTM/TB/2011.2
29. Discrepancies
with
MGIT
DST
• In vitro growth of Rif sensitive strains from
samples identified as Rif res or MDR by LPA
• Few cases from strains tested by LPA
• rpoB absence of WT8 (codo530-533) and absence of
rpoB MUT3
• Strains bearing the mutations grow slowly in liquid
culture and are identified as Rif sensitive
• inhA mut strains sensitive to INH by MGIT DST
Discordant
reports
are
confusing
for
pa/ents
Management
if
not
appropriately
explained
30. Heteroresistance
Real
heteroresistance
False
heteroresistance
• Co-‐presence
of
mutated
• Laboratory
cross
and
wild
type
popula/on
contamina/on
due
to
• Two
different
strains
insufficient
control
of
amplicons
LPAs
can
detect
heteroresistance
from
clinical
strains:
clinical
role?
31. Role
of
point
mutaGons
in
predicGng
clinical
outcome:
embB
Codon
306
• Low
sensi/vity
of
embB306
locus
for
predic/ng
Eth
Res
and
MDR-‐TB
(35.5%),
High
specificity
for
iden/fying
MDR-‐TB
(92.6%;
87/94).
The
posi/ve
predic/ve
value
(77.4%;
24/31)
and
the
nega/ve
predic/ve
value
(66.4%;
87/131)
of
this
locus
are
moderate.
Xin
Shen,
AAC2007
• Plinke
et
al
AAC
2006,
Mioxo
et
al
ERJ
2012:
higher
PPV
for
ETH
resistance
32. Commercial
tests
for
Fluoroquinolones
and
injectables
• Commercial
LPA
tests
for
detec/on
of
resistance
to
second
line
drugs
show
a
high
PPV
and
a
low
NPV
due
to
the
limited
number
of
muta/ons
included
in
the
tests
• PPV
and
NPV
may
vary
with
the
genotypic
background
of
the
strains
and/or
with
the
prevalence
of
specific
genotypes
in
the
target
popula/on
Mioxo
et
al.
ERJ
2012
33. Molecular
test
performances
should
be
evaluated
in
the
epidemiological
contests
• PPV
and
NPV
may
vary
in
different
genotypic
backgrounds
• High
prevalence
of
specific
clones
bearing
selected
muta/ons
not
included
in
current
diagnos/c
assays
may
modify
PPV
and
NPV
34. Role
of
Molecular
typing
• To
iden/fy
epidemiological
links
between
TB
pa/ents
to
detect
and
control
outbreaks
early
and
rapidly
• Rule
out
suspected
outbreaks
and
confirm
transmission
has
NOT
occurred
• To
iden/fy
incorrect
TB
diagnosis
based
on
false-‐posi/ve
cultures
and
thus
avoid
unnecessary
inves/ga/on
and
treatment
• To
dis/nguish
exogenous
re-‐infec/on
from
endogenous
reac/va/on
in
pa/ents
with
a
past
history
of
TB
• Discover
unusual
transmission
senngs
and
transmission
between
different
regions
• Monitor
the
size
of
clusters
and
thus
monitor
progress
towards
TB
elimina/on
• Vaccine
and
DR
detec/on
implica/ons
35. Needs
for
berer
tools
Among
3037
pa/ents
with
new
cases
of
tuberculosis
and
892
with
previously
treated
cases,
5.7%
(95%
confidence
interval
[CI],
4.5
to
7.0)
and
25.6%
(95%
CI,
21.5
to
29.8),
had
mul/drug-‐resistant
(MDR)
tuberculosis.
Among
all
pa/ents
with
tuberculosis,
approximately
1
of
4
had
disease
that
was
resistant
to
isoniazid,
rifampin,
or
both,
and
1
of
10
had
MDR
tuberculosis.
Approximately
8%
of
the
pa/ents
with
MDR
tuberculosis
had
extensively
drug-‐resistant
(XDR)
tuberculosis
36. Lab-‐on
Chip
for
molecular
diagnosGcs
PCR:
• Ultra-Fast PCR
• Asymmetric Cy-5 PCR strategy
Microarray:
• Orientation probes
• Hybridization Control probes
• Hybridization Negative Controls probes
Current Lay out:
ID of MTBC, relevant NTMs
MDR phenotype
Lab-on-chip architecture
2 PCR reactors of 12.5 uL volume each (Total 25 ul)
1 Hybridization chamber of 30 uL All the reaction modules
A 126 spots DNA microarray are fluidically integrated
2 in-let ports compatible with standard micro-pipettor tips
Integrated Heaters and Sensors
37. Open
Issues
• How
to
monitor
the
response
to
therapy?
– Sputum
smear
is
s/ll
guiding
decisions
on
admission
and
discharge
– Sputum
culture
is
s/ll
the
only
reliable
monitoring
tool
for
MDR
pa/ents
• Pa/ents
with
H
monoresistance
may
go
undetected,
in
R
res
H
should
be
le{
un/l
proven
R?
• Contact
treatment
in
absence
of
H
sensi/vity
data
• Discrepancy
between
phenotypic
and
genotypic
results
• Are
all
the
muta/ons
in
rpoB
equally
contribu/ng
to
resistance?
• Muta/ons
to
key
second
line
drugs
and
cross
resistance
• How
to
report
molecular
data
in
order
to
provide
clinical
guidance?
39. Acknowledgments
Emanuele
Borroni
Andrea
M.
Cabibbe
Irene
Festoso
Paola
Mantegani
Paolo
Mioro
Luca
Norbis
Enrico
Tortoli
Ilaria
Valente
Diego
Zallocco
Emerging
Bacterial
Pathogens
Unit
WHO
Suprana/onal
Reference
Laboratory
for
TB
Control
San
Raffaele
Scien/fic
Ins/tute
Milano,
ITALY
Fulvio
Salvo
and
Delek
Hospital
Staff
AISPO
Alberto
Mareelli
Alberto
Roggi
and
SRL/
NTP
Burkina
faso
Ins/tute
of
Infec/ous
and
Tropical
Diseases
University
of
Brescia
Brescia,
ITALY
Thanks for your attention!
Lanfranco
Farorini
GB
Migliori
Is/tuto
Superiore
di
Sanità
FSM,Tradate
Roma,
ITALY
