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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 1
st

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?

18
clusters

,7 belonging to Beijing
lineage


Development and spreading of DR
-
TB

Devaux I et al 2009. Emerg Infect Dis 15(7):1052
-
60


MDR
-
TB in Europe




XDR
-
TB in the world

WHO, Global Report 2011

2007: 41 Countries

2010: 58 Countries



Causes
:

patient

s related

physician

s related

Drug

s/ program

s related



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)

Van Deun A. et al 2011. IJTLD 15(1):116
-
124

Standardization and correlation
with the clinical outcome is
difficult to be achieved

TBPANNET workpackage 6

midterm meeting



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



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

MOLECULAR DST ON
M. tuberculosis

Genes involved in drug
-
resistance for major anti
-
tubercular drugs

First
-
line drugs

Second
-
line 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 MTBDR
plus
, 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

1


Decontamination
, ~1 h

2


DNA
extraction
, ~1 h

3


Amplification

(PCR), ~3 h

4


Strip
Hybridization
, ~2 h

5


Results

interpretation

and
report

LiPA Tests: work flow

cod. 315 gene
katG

nt
-
8,
-
15,
-
16 gene
inhA

Inno
-
LiPA Rif.TB

Morgan M et al 2005. BMC Infect Dis 5:62

Ling DI et al 2008. Eur Respir J 32:1165
-
1174

GenoType MTBDR
plus

GenoType MTBDR
plus

LPAs
performance

Ling DI et al 2008. Eur Respir J 32:1165
-
1174

Rifampicine

Isoniazid

Sensitivity

95
-
98%

Specificity

98
-
100%

Hot
-
spot gene
rpoB

Sensitivity

95
-
99%

Specificity

97
-
100%


Clinical samples

Sensitivity

95
-
99%

Specificity

97
-
99%

Sensitivity

82
-
93%

Specificity

95
-
100%


Clinical samples

Sensitivity

72
-
92%

Specificity

96
-
99%

LPAs
Performance

Miotto P et al 2008. J Clin Microbiol 46(1):393
-
4

Few data available on the LPAs performance on smear negative samples

MTBDR vs MTBDR
plus


-
Improvement of performance

of
20
-
25%

-
Decreasing of indeterminates of
about 10
-
15%



LPAs are approved for AFB positive
respiratory samples

3
°

generation (GenoType MTBDR
plus

v. 2) commercially available since January


-
AFB
-
negative and «scanty»

-
Master mix ready and
stabilized

-
Easily interpretable hybridization «pattern»

TaT for LPA: 1
-
2 days

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

Boehme CC et al 2011. Lancet 377(9776):1495
-
505

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

0
-
1 d

Liquid Culture


13
-
21 d

Solid Culture


23
-
43 d


Rifampicin
-
Res Identification

Specificity

97
-
99%

Sensitivity

91
-
97%


Co
-
infection TB
-
HIV

Sensitivity

86% (HIV
-
neg: 92%)

No differences in performances in AFB
-
neg. (microscopy: 47% in HIV
-
positive vs 65% in HIV
-
negative)

Theron G et al 2011. Am J Respir Crit Care Med 184:132
-
140

Hot
-
spot gene
rpoB

LiPA e Xpert MTB/RIF

Boehme CC et al 2011. Lancet 377(9776):1495
-
505

Rifampicin resistance identification

Time to report to treatment center

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

WHO/HTM/TB/2011.2

Xpert

MTB/RIF

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


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


Test to be adopted in settings with
adequate capacity and resources in
agreement with local NTP and WHO
reccomandations

Performance of Xpert on extra
-
pulmonary
specimens (adults and pediatrics)*

* Tortoli et. Al ERJ 2012

GenoType
®

MTBDRplus

XpertTB
-
MDRplus

Company

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

Yes

No

Fully automated /training

No/Yes

Yes/No

DNA tech

PCR

Mol Beacon

From liquid or solid culture

Yes

NA

Direct assay

Yes

Yes

Level of biosafety

II

microscopy

Time to results

Same day

2h

Cost per test

Low/Mod

Mod/High

Universal control

Yes

Yes

Extraction
control

No

Yes

Cost of Maintenance

Contamination control

Low

No

High

Yes

Comparison GenoType® MTBDRplus and XpertTB/MDR


Intermediate Reference labs

Patients testing from Sm/ C+ (Rif)

Fast tool Surveillance purposes

Potential to District level as fast

patients diagnostic tool, needs
evaluation at district level



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 by
LPAs.

Heteroresistance: mixed population of sensitive and
resistant bacteria

Analyzed
codons:

gyrA

: 85
-
97


rrs
:1401,
1402,1484

embB
: 306


Molecolar diagnosis of resistance to drugs other than R and
H: the GenoType MTBDR
sl test

(Hain Lifescience)


GenoType MTBDR
sl
:
performance

Miotto P et al. ERJ 2012


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

Ethambutol resistance: is molecular detection of
resistance better than MGIT DST?

Van Deun A et al 2011. IJTLD 15(1):116
-
124

Scarparo C et al 2008. J Clin Microbiol 42(3):1109
-
1114

Ethambutol in MGIT:

-
Decreaesed sensitivity and specificity

-

Reduced riproducibility

-
False
-
sensitive using
5 μ
g/mL as break point

70
-
80% of ETB
-
resistant isolates are mutated in the codon 306 of
embB

Gene


84 clinical isolates ETB
-
R (MGIT,
5 μ
g/mL)

M306V



42,9%

Other mutations cod. 306

13,1%

Non mutated


44,0%


91 clinical isolatesi ETB
-
S (MGIT,
5 μ
g/mL
)

M306V



18,6%

Other mutations cod. 306

24,3%

Non mutated


57,1%



>95% results ETB
-
resistant if
MIC is determined

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

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
.

Propidium

Monoazide
TM

(PMA)

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 (t
0
) and at 14 days from beginning of
antitubercular

therapy

Miotto P et al. ERJ 2012

Not treated


PMA
treated

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


Non
-
tuberculous

mycobacteria

(NTM)

The use of MTBDR
plus

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

41

Molecular

DST in TB:
clinical

impact

Miotto P et al. BMC Infect. Dis. 2009; 9:142

Badoum et al. ERJ 2011


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



Drug resistance: what

s new?


AMK/KAN/CAP
:

Rv
3919
c

(
gidB
),

Rv
2416

(
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

Characterization

of
novel

mutations

involved

in
drug
-
resistant

phenotype

and
virulence

markers

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)


Rapid

identification

of

MTB

complex


Rapid

diagnosis

of

MDR

cases


Rapid

genotyping

of

MTBC


Rapid

identification

of

clinically

relevant

NTMs


Rapid

identification

of

Plasmodium

pathogenic

species


Rapid

diagnosis

of

malaria

drug

resistant

cases

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
.

Tuberculosis




Malaria

Lab
-
on
-
Chip (
LoC
) platforms: a possible
answer?

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

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



Statistical

analysis

on
clinical

isolates

Drug resistance

Species identification

Species

identified
:



M.
tuberculosis

complex


M.
avium


M. intracellulare


M.
simiae
, M.
kansasii
,
M.
scrofulaceum






M.
abscessus
, M.
chelonae


M. xenopi


M.
fortuitum

Detection
limit
:



rpoB

target:
10
4
/
mL

(AFB:
1+ /
scanty
)



All

other

targets: <
10
2
/
mL

(AFB:
scanty
)

Chip
information

Summary of
results

Automatic generation
of final report

E@syCheck

software

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

TB

TB/HIV

Malaria

Neglected tropical
diseases


Dengue

Buruli ulcer

treponematoses

Leprosy

cysticercosis

dracunculiasis

lymphatic filariasis

human rabies

Trachoma



Geographical distribution: the advantages of multi
-
purposes platform

Algorithm for rapid DST testing: one doasn

t fitt all!

XpertTB/
MDR





WHO RECOMMENDATION, December 8th, 2010

Expected impact of rolling out molecular
technology for MDR
-
TB detection



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.


Conclusions

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


E
xtent
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




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.

Molecular strain typing methods

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

2

4

2

5

3

2

PCR is used to determine the
number of repeats at each loci
(24 in total)

VNTR
-

Identical pieces of DNA repeated a variable number of
times, next to each other (in tandem) at specific positions
(loci) on the genome.

Strain type profile = 232425673216524316425375


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.

SPOLIGOTYPING

Acknowledgements

WHO Collaborating Centre for Integrated Lab. strengthening on TB and other Emerging
Infections

San
Raffaele

Scientific Institute


Paolo
Miotto



Andrea M. Cabibbe

Ilaria C. Valente

Emanuele Borroni

Paola Mantegani

Enrico Tortoli

hSR Scientific Park

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