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Microbiological diagnosis of TB
José Domínguez1 and Sabine Rüsch-Gerdes2
1Servei
2
de Microbiologia. Fundació Institut en Ciències de la Salut
Germans Trias i Pujol. Badalona. Spain
Forschungszentrum Borstel, National Referencelaboratory for
Mycobacteria. Borstel. Germany
Microbiological diagnosis of TB:
Detection, identification and
molecular epidemiology
José Domínguez
Servei de Microbiologia.
Fundació Institut en Ciències de la Salut Germans Trias i Pujol
One of the main objectives for TB control
Objectives for TB control
•To rapidly diagnose patients with active TB and treat them correctly.
•To have rapid diagnostic methods, with high sensitivity and specificity
to diagnose diseased patients at the beginning of the symptoms for an
adequate treatment prescription
“Diagnosis, diagnosis & diagnosis”
William Osler
Clinical suspicion
Histology
Microbiology
Diagnosis of tuberculosis
Latent Infection
Active tuberculosis
Smear examination
TST
Solid and liquid culture
Molecular
Epidemiology
IFN- techniques
Identification
RFLP
MIRU
Susceptibility testing methods
Molecular methods
-Detection
-Identification
-Detection of resistance
Spoligotyping
Clinical samples
Samples!! Respiratory and extra-respiratory
Quantity: high inoculums means fast growth
Quality, including sputum, high yield
Localization, biopsies when possible
Rapid shipment
Previous to starting treatment
Think of histology
The most important:
Clinical-Microbiologist-Pathologist Communication
Decontamination
• Eliminate normal flora from the non-sterile samples
(micobacteria is acid and alkaline resistant)
• Homogenization to release the bacteria from the sample and allow access to the
nutrient present in the media
i.e. Kubica
N-acetyl-cysteine: homogenization
NaOH: decontaminant
Neutralization by phosphate buffer
Homogenization
Phosphate buffer
Centrifugation
Sample mixing
Pellet
Smear microscopy
Fast; Cheap; Monitorization of treatment; Low sensitivity
Ziehl-Neelsen stain
Auramina O stain
Hospital Univ. Germans Trias i Pujol 2004 -2007
Pulmonary
Disseminated
Extrapulmonary*
TOTAL
125
18
60
Positive smear
83 (66.4%)
11 (61.1%)
11 (18.3%)
Negative smear
42 (33.6%)
7 (38.9%)
49 (81.7%)
*Adenopathy, 4/28 (14.2%); Pleural, 1/17 (5.9%)
Culture in solid and liquid media
Decontamination (in non sterile samples)
Culture in the adequate media
Inoculums!!
Growth in solid
media
Slow: 15d-2m
Growth in liquid
media
7-42d
Division time 18h
Identification
Classical and Molecular
methods
DST
• Sometimes the only place
where the mycobacteria can be
isolated
• Gold standard
• Molecular epidemiology
• Drug susceptibility testing
Identification molecular methods
AccuProbe
InnoLiPA Mycobacteria
GenoType Mycobacterium CM/AS, GenoType MTBC
Identification molecular methods
PRA (Polymorphism
Restriction Amplification)
Amplification, by PCR of a
fragment of the hsp65 gen,
followed by a restriction with 2
restriction enzymes (BstEII y
HaeIII).
Sequencing
Pyrosequencing
PPi
ATP
M.tuberculosis detection in clinical samples by molecular methods
Sensitivity in
respiratory
samples (%)
Sensitivity in
extra respiratory
samples (%)
Overall
specificity
(%)
Method
Target
Detection method
AMTD2
16S rRNA
Chemiluminometric
80-100
60-90
95-100
b antigenic protein
Fluorimetric
80-90
65-80
90-100
AMPLICOR
16S rRNA
Colorimetric
75-100
45-60
90-100
BD ProbeTec
IS6110 and
16S rRNA
Fluorimetric
55-100
30-80
45-100
INNO-LIPA v2
IR16S-23S
Colorimetric
50-95
60-80
90-100
GenoType
Direct
23S rRNA
Colorimetric
60-95
60-80
95-100
PCR real time
16S rRNA
Fluorimetric
70-90
65-85
85
LCx
* In smear negative samples the sensitivity is reduced in a 50%
Role of the clinical suspicion level in
the evaluation of the molecular methods
Catanzaro A. et al JAMA 2000
Problem: there are positive results in negative smear and culture samples.
Molecular epidemiology
• RFLP (IS6110)
• Spoligotyping
• MIRUs
Restriction Fragment Length Polymorphism (RFLP)
• Insertion sequence present exclusively in the
M.tuberculosis complex: IS6110
• High polymorphism between no related strains
regarding the number of copies and their
localization in the chromosome.
• Advantages: High discriminative power.
• Disadvantages: Slow, laborious and with
certain complexity.
Pvu II
Electrophoresis
Extraction and restriction
Radiographic develop
Hybridization
Transference
Spacer oligonucleotype typing (Spoligotyping)
• The DR sequences (direct repeat) are repeated
sequences of 36 bp in only one locus of the
M.tuberculosis chromosome, separated by sequences
of 34 to 41 bp.
• The technique is based on a PCR of the locus where
the DR sequences are located. The amplification
product is hybridized with oligos synthesized from the
inter-DR spaces.
• 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.
Micobacterial Interspersed Repetitive Units (MIRU)
• Determine the number of repetitive units in 12 (15 or 20)
different locus of one genetic sequence called
“mycobacterial interspersed repetitive units (MIRUs)”. The
number of repetitions is detected by PCR.
• The number of repetitive units in each locus is calculated
by the size of the fragment amplified with the specifics
primers.
• MIRU-VNTR is more discriminative than the spoligotyping
and similar to the RFLP-IS6110.
• Advantages: rapid, simple and automatic.
• Disadvantages: In study
Micobacterial Interspersed Repetitive Units (MIRU)
MIRU 40
500
pb
43
1 2
Conclusions
• The microbiological diagnosis of TB will be rapid and accurate
if adequate samples are collected and adequate inoculums are
used. Don’t forget histology.
• The future of TB diagnosis remains in the application of new
molecular techniques but, at the moment a cautious
interpretation of the results is required.
• The sensitivity of the molecular tests vary, and is affected by
the amount of bacteria present in the samples, and also by the
clinical suspicion level. Low sensitivity is present in samples
with low bacterial load, especially in extra respiratory samples.
• At the moment, new molecular methods can not substitute the
conventional ones. The gold-standard is the culture, and the
other methods have to be considered and interpreted as
complementary diagnostic methods.
• Communication between clinicians and microbiologists is
imperative.
Microbiological Diagnosis of TB
Drug Susceptibility Testing
Borstel 2010
National Reference Laboratory for Mycobacteria
Forschungszentrum Borstel
Sabine Rüsch-Gerdes
Drug Susceptibility Testing
MDR TB – New Infection
WHO: MDR-TB & XDR-TB, The 2008 Report; February
2008
Drug Susceptibility Testing
Anteil resistenter Erreger in Deutschland, 2006
For all TB strains
isolated, DST has to be
Quelle: RKI, Bericht zur Epidemiologie
der Tuberkulose in Deutschland für 2006; 2008
performed
BiostoffVerordnung (BioStoffV) 1999
Microscopy
Culture
NAT
Level 2 laboratory
Microscopy
Culture
NAT
Differentiation
DST
Level 3 laboratory
Methods for Drug Susceptibility Testing
Proportion method on
Löwenstein-Jensen medium
H, R, E, S, PTH, CM, OFL, CS, NSA (instead of
P)
Results available:
4-6 weeks
No critical concentrations
for new substances
Methods for Drug Susceptibility Testing
BACTEC 460TB
All drugs except
cycloserine
Results available:
1-2 weeks
Radioactive materials,
waste
Methods for Drug Susceptibility Testing
MGIT 960
For all drugs, except
CS
Results available:
1-2 weeks
Liquid media compared to solid media
Advantages compared to solid media:
• more rapid
• high quality of media
• fully automated system
• testing of 1st, 2nd, and new drugs
• safety: plastic tubes
Disadvantages:
• expensive
• higher contamination rate
• dependency on a company
• no DST for Cycloserine
Infection control strategies
Proposed measures:
•
•
•
•
•
•
Improved ventilation system
Reduced hospitalsation
Mask use
Isolation of patients
HIV-testing and therapy
Rapid drug-susceptibility testing
Time for the detection has a potential to reduce
the extend of spread of resistant strains
Principle of the Line Probe Assays
Chromogen (MBT/BCIP)
Alkaline Phosphatase
Streptavidin
Biotin
DNA-probe
Colour reaction
Biotin-labelled
single stranded
amplified target
Nitrocellulose strip
MTBDR – DNA Hybridisation Strip
1
Control of the conjugate Amplification control Amplification control
MTBC Control rpoB rpoB Wild type 1 rpoB Wild type 2 rpoB Wild type 3 rpoB Wild type 4 rpoB Wild type 5 rpoB Mut D516V rpoB Mut H526Y rpoB Mut H526D rpoB Mut S531L Control katG katG wild type katG S315T1 (ACC) katG S315T2 (ACA) -
2
3
4
5
6
7
8
Genotype® MTBDRplus HybridizationStrip
1
from
Culture
media
2
1 RMPr INHr
2 RMPs INHs
Results RMP+INH Resistance
100 % concordance between sequencing and MTBDR data
103 MDR strains
102 strains (99%)
mutations in rpoB cluster I
91 strains (88.4)
with mutations in codon 315 of katG
+
+
1 strain (1%)
a mutation outside rpoB cluster
I
3 strains (2,9 %)
with a mutation in inhA
+
2 strains (1,9 %)
with a mutation
in ahpC
+
+
7 strains (6,8 %)
with no mutation in katG, inhA and
ahpC
1 strain (1%) not detected as
MDR
(rpoB outside cluster I, ahpC)
Genotype® MTBDRplus HybridizationStrip
1
from
specimens
Hillemann D, Rüsch-Gerdes S, Richter E.
Application of the Genotype MTBDR assay
directly on sputum specimens.
Int J Tuberc Lung Dis 2006. 10:1057-1059.
2
1 RMPr INHr
2 RMPs INHs
Evaluation of the MTBDRplus Assay on Specimens
72 smear positive sputum specimens:
30 susceptible strains
32 MDR (RMPr/INHr) strains
10 INHr strains
Sensitivity
Specificity
RMP detection: 96.8%
INH detection: 90.2%
RMP: 95.2%
INH: 100%
Line Probe Assays for DST
Evaluated line probe assays
 INNO-LiPA Rif TB
 GenoType MTBDRplus
Resistance
Sensitivity
Specificity
RMP
98.1%
98.7%
INH
84.3%
99.5%
Ling et al., Eur Respir J 2008
 GenoType MTBDRsl
Molecular Basis
Drug
Gene
Gene function
Locus
Percent of
Resistance
Fluorochinolones
gyrA
DNA-Gyrase A
appr.80-90%
Amikacin,
Capreomycin,
Kanamycin
rrs
16S rRNA
appr. 80 %
tlyA
Methylase
Ethambutol
embB
appr. 30-60%
Summary
•
Overall sensitivity for OFL, AM, CM and EMB was
90.2 %, 83.3 %, 86.8 % and 59.0 %, respectively.
•
Specificity was 100 % for FLQ, AM, and EMB,
and 99.1 % for CM.
•
Most prevalent mutations were:
gyrA D94G in Oflr strains
rrs A1401G AMr/CMr strains
embB M306V in EMBr strains
•
The rapid detection of XDR strains is possible
with the combined application of Lipas
from DNA isolates and directly from sputum
specimens.
Xpert MTB
GeneXpert
Time-to-result: 1 h 45 min
FIND 2009
Evaluation Partner Sites
FIND 2009
Xpert MTB
High tech for low tech settings:
Sensitivity and Specificity seems to
be very good for the detection of TB
and Rifampicin resistance.
Drug Susceptibility Testing
Solid Media
3 - 4 weeks
Liquid Media
7 - 10 days
Molecular based
Methods
Hours – 1day
Löwenstein-Jensen
(Middlebrook)
BACTEC 460 TB
MGIT
InnoLipa
GenoTypeMTBDR
Xpert MTB
‚home made‘- methods
Quality Control
internal
external
Internal Quality Control
all reagents
all techniques
all staffs
Internal Quality Control
All QC results have
to be documented
Drug Susceptibility testing
1
2
3
4
5
7
6
embB MUT1B
embB MUT1A
embB WT1
embB
rrs MUT2
rrs MUT1
rrs WT2
rrs WT1
rrs
gyrA MUT3D
gyrA MUT3C
gyrA MUT3B
gyrA MUT3A
gyrA MUT2
gyrA MUT1
gyrA WT3
gyrA WT2
gyrA WT1
gyrA
TUB
AC
CC
8
9 10 11
External Quality Control
Participation in
international QA
programs
Reliable results
To detect and treat
patients properly