website: www.drsarma.in
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• MDR TB : Multi drug Resistant TB
– Rifampicin (RIF) and INH resistance
• XDR TB : Extensive Drug Resistant TB
– INH and RIF (MDR-TB) and
– Amikacin or Kanamycin or Capreomycin
– and Ofloxacin, Moxifloxacin (Fluroquinolones)
• TDR TB : Totally Drug Resistant TB
– Resistant to almost all known anti TB drugs
DST means Drug Sensitivity Test
GOLD STANDARD
CAD
by CAG
No CAD
by CAG
SnNOUT (Minimum FN)
Sensitivity is
True positives
ECG +VE
TEST
True Positives False Positives
a
b
Total CAD
a
a+c
Specificity is
ECG – VE
False Negative True Negatives
c
d
Total CAD
a+c
Total No CAD
b+d
True Negatives
Total No CAD
d
b+d
SpPIN (Minimum FP)
GOLD STANDARD
CAD
by CAG
ECG + VE
No CAD
by CAG
TEST
True Positives False Positives
70
120
SnNOUT (Rules out 70%)
Sensitivity is
True positives
70
Total CAD
100
Specificity is
ECG – VE
False Negative True Negatives
30
180
Total CAD
100
Total No CAD
300
True Negatives
180
Total No CAD
300
SpPIN (Confirms 60%)
GOLD STANDARD
Test
CAD
by CAG
No CAD
by CAG
ECG + VE
True
Positives
70
False
Positives
120
ECG – VE
False
Negative
30
True
Negatives
180
PPV is 37%)
Positive Predictive Value
Total +ves
190
True positives
70
Total Positives
190
Negative Predictive Value
Total -ves
210
True Negatives
180
Total Negatives 210
Total CAD Total CAD
100
300
Grand
Total 400
NPV is 86%)
• Sensitivity is the ability of the test to rule out
disease confidently when the test result is negative.
• Specificity is the ability of the test to confirm
disease confidently when the test result is positive.
• Positive Predict Value (PPV): useful in high
prevalence situations
• Negative Predictive Value (NPV): useful in low
prevalence situations
• Sensitivity and Specificity are unaffected by
prevalence
HIV
BOTH
3.5m
1.7m
TB
2.5m
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10 million new cases every year globally
3 million deaths annually worldwide
Leading cause of death due to infectious disease
25% of all avoidable deaths are due to TB
95% of TB cases and 98% of deaths are in
developing countries
India contributes 2 million cases every year
0.5 million die of TB annually – 1 death / min
75% of cases are in productive age groups
Increasing HIV infection increases TB burden.
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More than 10 m cases of TB yearly - world wide
India ranks 1st among the top 22 TB countries
5 lakh cases of MDR TB per year (WHO)
Accurate and early diagnosis is most important
for effective case management and prevention
of transmission of MTC
We do not have an effective vaccine to prevent
Case finding and case holding are key issues
MDR, XDR and TDR cases are due to improper Rx.
Anonymous or atypical mycobacteria MOTT
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Tuberculosis is endemic in our country
Ours is high prevalence, high incidence scenario
Poverty, over crowding, lack of education, ½ Rx.
Large population and wide area – large burden
BCG vaccination is common – confounds skin test
MOTT – Anonymous mycobacteria are ubiquitous
Subclinical MTB infections are very common
New HIV burden throws a new serious challenge
MDR TB, XDR TB and now TDR TB – big challenge
Non compliance, self medication, quackery
We very well know about our Govt. programme
MDR TB
Rifampicin
INH
XDR TB
rpoB
KatG 70%
FQ- gyrA
95%
inhA 30%
AG/CP -16S
rRNA (rrs)
Mutations in the MTB Genome
embB
• Fever, cachexia, cough, sputum, chest pain,
hemoptysis, fatigue, night sweats, raised ESR are
notoriously present in many similar disorders
• Clinical signs of consolidation, cavitation, fluid,
thickened pleura, neck glands can occur in a variety
of conditions.
• Old treated TB is big confounder, DST is not done
• Partially treated cases pose resistance problems
• COPD, emphysema mimic and mask TB signs
• Early lesions often are unsuspected until X-ray
• The sensitivity and specificity are rather low.
• Extra pulmonary poses even greater confusion
• Radiological features when clear cut are highly specific.
Often early lesions may be missed.
• X-ray shadows can’t tell activity of disease
• Old shadows are superimposed on fresh ones.
• Effusions may be due to many causes
• HRCT is expensive and again cannot speak of the
activity of disease.
• Treatment decisions on imaging alone will be risky
as TB Rx is prolonged and potentially toxic
• Over all - X-ray has high sensitivity, mod. Specificity
• We can’t treat shadows. What about extra pulmonary ?
Direct Methods
MTB and Its Products
Indirect Methods
Humoral & Cellular Response
AFB Smear and MTB culture
Interferon Gamma - IGRA
BACTEC 460 - MGIT 960
TBFeron / Quanti-FERON
Molecular Dx MTB - PCR
T Spot TB Test
Gene Xpert - MTB-RIF
Adenosine Deaminase (ADA)
Rapid Geno MDR & XDR test
Serological Tests for TB
DST – The Need for Fast track
Tuberculin Test – Skin Tests
Direct Methods
MTB and Its Products
Indirect Methods
Humoral & Cellular Response
AFB Smear - Sp. Microscopy
Interferon Gamma - IGRA
BACTEC 460 - MGIT 960
TBFeron
Molecular Dx MTB - PCR
T Spot TB Test
Gene Xpert - MTB-RIF
Adenosine Deaminase
Rapid Geno MDR & XDR test
Serological Tests for TB
DST for MOTT - rapid growers
Tuberculin Test - PPD
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120 years old technique, relatively simple
Special techniques to improve the yield
ZN method and Fluorescent microscopy
Results will be reported with in hours
Most cost effective method – RNTCP
Requires good training & observer dependent
Requires 5 x 103 bacilli per ml of sputum
Proper collection of sputum is essential
Three specimens are needed for Dx.
Sensitivity is 30 to 60%, Specificity is high - 97%
Direct Methods
MTB and Its Products
Indirect Methods
Humoral & Cellular Response
MTB culture – Solid / Liquid
Interferon Gamma - IGRA
BACTEC 460 - MGIT 960
TBFeron
Molecular Dx MTB - PCR
T Spot TB Test
Gene Xpert - MTB-RIF
Adenosine Deaminase
Rapid Geno MDR & XDR test
Serological Tests for TB
DST for MOTT - rapid growers
Tuberculin Test - PPD
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Corner stone of definitive diagnosis. Gold standard.
LJ medium or Middle Brook 7H10 & 11 Solid media
Kirchner’s or Middle Brook 7H9 broth – Liquid media
Slow growth – Mean time of 4 to 6 weeks (2-3 wks)
DST requires another 4 weeks; Contaminants problem
Combination of solid & liquid media is better
Micro colony detection, Sept Check AFB, MODS
Proper collection of sputum is essential.
High infrastructure cost, Not available readily.
Many factors decide the yield of positive culture
Highly specific 99.5% but moderately sensitive - 70%.
Direct Methods
MTB and Its Products
Indirect Methods
Humoral & Cellular Response
AFB Smear and MTB culture
Interferon Gamma - IGRA
BACTEC 460 - MGIT 960
TBFeron
Molecular Dx MTB - PCR
T Spot TB Test
Gene Xpert - MTB-RIF
Adenosine Deaminase
Rapid Geno MDR & XDR test
Serological Tests for TB
DST for MOTT - rapid growers
Tuberculin Test - PPD
• Uses 14C labeled palmitic acid in the medium
• Based on metabolism of MTB – not on visible growth
• If the medium is metabolized - 14CO2 is released
• BACTEC system radiometric measurement gives GI
• Same BACTEC can be used for DST
• Significantly faster – 87% +ves in 7 d, 96% in 14 d
• DST can be completed in 8 days
• This proves cost effective in high prevalence areas
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Uses 14C labeled palmitic acid in the medium
Based on metabolism of MTB – not on visible growth
Mycobacteria Growth Indicator Tube (MIGT)
Observed every 60 min for increase in fluorescence
AFB metabolic utilization of O 2 in the fluorescent dye
Intensification of fluorescence in the tube
Rapid, accurate and cost effective method for high
volume labs
• 960 tubes can be computer monitored simultaneously
• Can be used for DST also; Rapid in 4-6 days results
• MB / BacT System
– Non radiometric continuous monitoring system
with computerized data base management. The
system is based on colorimetric detection of CO2
• ESP Culture System
– Fully automated continuous monitoring of
pressure changes with in the head space above
the broth culture medium in a sealed bottle. Gas
production or gas consumption by bacterial
growth. Results in about 2 weeks
Direct Methods
MTB and Its Products
Indirect Methods
Humoral & Cellular Response
AFB Smear and MTB culture
Interferon Gamma - IGRA
BACTEC 460 - MGIT 960
TBFeron
Molecular Dx MTB - PCR
T Spot TB Test
Gene Xpert - MTB-RIF
Adenosine Deaminase
Rapid Geno MDR & XDR test
Serological Tests for TB
DST for MOTT - rapid growers
Tuberculin Test - PPD
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Sequences of bacterial DNA will be amplified
10-1000 bacilli are sufficient for detection
Rapid and results are available in a day
Target IS6110, 65 kDa, 65 SrNA MTB Specific
It is present up to 20 times in the MTB genome
It can detected both in blood and in sputum
Sensitivity 84%, Specificity 99%, PPV 94.2%
NAAT – Nucleic Acid Amplification Test and
TMA, SDA, NASBA, b-DNA, LiPA – other tests
Mycoresist
MDR TB
Mycoreal
RT PCR
GeneXpert
MTB RIF
MD of
MTB
Myco3Plex
Multiplex
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“Mycoreal” - A rapid real time PCR test for MTB
Utilizes UTP/UDP system to avoid contamination
Sensitivity 99%, Specificity 99.5%
Detects all members of MTC group
Can be used in pulmonary and extra pulmonary
Sputum, Blood, Tissues - all can be tested
No “ post PCR processing”. So no contamination
Detects as low as 2 fg of MTB DNA
Negative, positive and no template controls
Direct Methods
MTB and Its Products
Indirect Methods
Humoral & Cellular Response
AFB Smear and MTB culture
Interferon Gamma - IGRA
BACTEC 460 - MGIT 960
TBFeron
Molecular Dx MTB - PCR
T Spot TB Test
Gene Xpert - MTB-RIF
Adenosine Deaminase
Rapid Geno MDR & XDR test
Serological Tests for TB
DST for MOTT - rapid growers
Tuberculin Test - PPD
• It is two-in-one: Detects presence of MTB by Real
Time PCR + identifies if it is resistant to RIF
• Developed by Cepheid – Endorsed by WHO,
• TB Culture take 3 to 6 weeks, DST further 3 to 4 wks
• Rapid 100’, very simple, minimal training, field use
• Completely closed, No man errors, No contamination
• RIF resistance is a surrogate of MDR TB
• Sensitivity 91% detection in S-C+ / 100% in S+C+
• Specificity very high 99.2%, 98% for RIF resistance
• Govt. of India has started in 4 places, Available in labs
22nd October 2011
Direct Methods
MTB and Its Products
Indirect Methods
Humoral & Cellular Response
AFB Smear and MTB culture
Interferon Gamma - IGRA
BACTEC 460 - MGIT 960
TBFeron
Molecular Dx MTB - PCR
T Spot TB Test
Gene Xpert - MTB-RIF
Adenosine Deaminase
LiPA - MDR & XDR test
Serological Tests for TB
DST for MOTT - rapid growers
Tuberculin Test - PPD
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This is a LiPA endorsed by WHO and FIND
Detects mutations in rpoB , Kat G and inhA
Rapid detection of RIF & INH Resistance – MDR
inhA inclusion detects even low levels of INH R
Sensitivity 93.6% RIF, 92.6% INH, 88.9% for MDR
Specificity is 100% for all types of patients
PPV is 100%, NPV is 90.3%
Smear +ve or culture +ve specimens
In smear –ve cases bacillary load will be low to
detect mutations for the drug resistance.
Microscopy
Liquid Culture
1st Line DST
• One Day
• 2 to 3 weeks
• 1 to 3 weeks
3 to 6 weeks
Microscopy
• One Day
RT PCR
• One Day
1st Line LiPA
• One Day
3 days
Microscopy
• One Day
Liquid Culture
• 2 to 3 weeks
1st Line DST
• 1 to 3 weeks
2nd Line DST
• 1 to 3 weeks
4 to 9 weeks
Microscopy
• One Day
RT PCR
• One Day
1st Line LiPA
• One Day
2nd Line LiPA
• One Day
4 days
• This is also a LiPA – rapid test for XDR
• Detects mutations in gyrA, 16S rRNA, embB
• Sputum or Culture specimens of MDR TB only
are tested for second line drug resistance
• India, China, SA and Russia, BD – together
contribute for largest number of XDR TB.
• Culture and DST is not routinely done in our
country. We miss a lot of MDR and XDR TB
• We realize only when the patient does not
respond after 6 months of treatment.
Direct Methods
MTB and Its Products
Indirect Methods
Humoral & Cellular Response
AFB Smear and MTB culture
Interferon Gamma - IGRA
BACTEC 460 - MGIT 960
TBFeron
Molecular Dx MTB - PCR
T Spot TB Test
Gene Xpert - MTB-RIF
Adenosine Deaminase
New Hope – The TB LAMP
Serological Tests for TB
DST for MOTT - rapid growers
Tuberculin Test - PPD
Direct Methods
MTB and Its Products
Indirect Methods
Humoral & Cellular Response
AFB Smear and MTB culture
Interferon Gamma - IGRA
BACTEC 460 - MGIT 960
TBFeron
Molecular Dx MTB - PCR
T Spot TB Test
Gene Xpert - MTB-RIF
Adenosine Deaminase
Rapid Geno MDR & XDR test
Serological Tests for TB
DST – Need For Fast Track
Tuberculin Test - PPD
• Routine DST is now essential in view of MDR, XDR
TB burden. Also TDR TB will be most challenging
• DST based on solid media cultures take 3 to 4
weeks for 1st line and a further 3 to 4 weeks for 2nd
line drug sensitivity
• RT PCR combined with Line Probe Assay (LiPA) will
shorten this time most efficiently to 3 to 4 days
• If we use GeneXpert – MDR TB can be diagnosed in
100 minutes, even before the smear results come.
• The world is on the fast track but our TB Dx is on
120 years old ultra slow track. How do we win TB?
Direct Methods
MTB and Its Products
Indirect Methods
Humoral & Cellular Response
AFB Smear and MTB culture
Interferon Gamma - IGRA
BACTEC 460 - MGIT 960
TBFeron
Molecular Dx MTB - PCR
T Spot TB Test
Gene Xpert - MTB-RIF
Adenosine Deaminase
Rapid Geno MDR & XDR test
Serological Tests for TB
DST MOTT - rapid growers
Tuberculin Test - PPD
Direct Methods
MTB and Its Products
Indirect Methods
Humoral & Cellular Response
AFB Smear and MTB culture
Interferon Gamma - IGRA
BACTEC 460 - MGIT 960
TBFeron
Molecular Dx MTB - PCR
T Spot TB Test
Gene Xpert - MTB-RIF
Adenosine Deaminase
Rapid Geno MDR & XDR test
Serological Tests for TB
DST MOTT - rapid growers
Tuberculin Test - PPD
• MTB genome has regions of difference (RD)
• These RDs encode potential antigens for Dx.
• RD1 is responsible for secretion of ESAT6
• ESAT6 (6kDa) is specific antigen and a strong
inducer of IFG by T cells of the TB patient.
• IFG is a cytokine secreted by sensitized T cells
• This ESAT6 is not recognized by BCG or NTM
• IFG levels increase with treatment – prognostic
• It is a measure of CMI in TB patients
• Sensitivity is 82 to 90%, Specificity is 96 to 99%
Direct Methods
MTB and Its Products
Indirect Methods
Humoral & Cellular Response
AFB Smear and MTB culture
Interferon Gamma - IGRA
BACTEC 460 - MGIT 960
TBFeron / Quanti-FERON
Molecular Dx MTB - PCR
T Spot TB Test
Gene Xpert - MTB-RIF
Adenosine Deaminase
Rapid Geno MDR & XDR test
Serological Tests for TB
DST MOTT - rapid growers
Tuberculin Test - PPD
• These are IGRA – use three proteins specific for
MTB – ESAT6 (early Secretory Antigen TB), CFP
(Colony Forming Protein) and TB7.7 – All are
proteins from different RDs
• This combination makes the test very specific as
these are absent in BCG and NTM
• Rapid test – 1 day, available – moderately priced
• Does not distinguish between Latent Infection (LTBI)
and active disease. It is an ELISA based test.
• Specificity 96 to 99%, Sensitivity 82 to 90%
Direct Methods
MTB and Its Products
Indirect Methods
Humoral & Cellular Response
AFB Smear and MTB culture
Interferon Gamma - IGRA
BACTEC 460 - MGIT 960
TBFeron
Molecular Dx MTB - PCR
T Spot TB Test
Gene Xpert - MTB-RIF
Adenosine Deaminase
Rapid Geno MDR & XDR test
Serological Tests for TB
DST MOTT - rapid growers
Tuberculin Test - PPD
• It is one of the latest tests – based on IGRA
• An FDA approved in vitro test based on ELISPOT
• Uses two separate panels of MTB complex – the
ESAT6 and CFP10 – not present in BCG, NTM
• Detects both LTBI and Active TB (all forms)
• Useful in all ages, ethnicity, immunocompromised
• More sensitive compared to ELISA-TBFeron
• Sensitivity 95% and Specificity 97%
• Rapid test – in one day result is available
Direct Methods
MTB and Its Products
Indirect Methods
Humoral & Cellular Response
AFB Smear and MTB culture
Interferon Gamma - IGRA
BACTEC 460 - MGIT 960
TBFeron
Molecular Dx MTB - PCR
T Spot TB Test
Gene Xpert - MTB-RIF
Adenosine Deaminase -ADA
Rapid Geno MDR & XDR test
Serological Tests for TB
DST MOTT - rapid growers
Tuberculin Test - PPD
• Useful to test body fluids like plural, peritoneal,
pericardial and cerebrospinal fluid.
• Commonly used for Dx. of TB pleural effusion
• Increased T lymphocytes and their increased activity
increases ADA in the exudate
• In low prevalence areas – more false positives and
low specificity. Combined with lymphocyte count it
may be useful as a screening test.
• Not a replacement for culture or biopsy.
• Simple, cheap test to R/o TB in exudate effusions.
Direct Methods
MTB and Its Products
Indirect Methods
Humoral & Cellular Response
AFB Smear and MTB culture
Interferon Gamma - IGRA
BACTEC 460 - MGIT 960
TBFeron
Molecular Dx MTB - PCR
T Spot TB Test
Gene Xpert - MTB-RIF
Adenosine Deaminase
Rapid Geno MDR & XDR test
Serological Tests for TB
DST MOTT - rapid growers
Tuberculin Test - PPD
• Rarely useful as screening tests. Industry bias
• Low turn around time, Not available in all labs
• Very low sensitivity in smear negative cases and
HIV positive cases
• Not useful in our country of high endemicity
• Ubiquitous nature of MOTT
• Sero conversion would have already occurred
• High cost, require trained staff, costly equipment
• Can’t separate MTB and NTM
• Strongly recommended by WHO – Not to be used
Direct Methods
MTB and Its Products
Indirect Methods
Humoral & Cellular Response
AFB Smear and MTB culture
Interferon Gamma - IGRA
BACTEC 460 - MGIT 960
TBFeron
Molecular Dx MTB - PCR
T Spot TB Test
Gene Xpert - MTB-RIF
Adenosine Deaminase
Rapid Geno MDR & XDR test
Serological Tests for TB
DST MOTT - rapid growers
Tuberculin Test – Skin Tests
• Useful in non endemic countries with low TB rates
• Almost all adults in our population are manteaux
positive because they are either BCG vaccinated
or are exposed MTB or NTM in the air and soil.
• The protein (PPD) used in Manteaux test is non
specific and is shared by BCG, MTB and NTM
• A positive Test in Indian adults has no meaning. In
very young children high positivity may mean recent
infection.
• Infection is not synonymous with disease.
• Sensitivity is also low. Specificity is nearing zero
False negatives
• In severe disease – Miliary TB
• In HIV infected
• In Sarcoidosis
• Technical factors
– Application, Reading,
– Improper storage of PPD
• Biological factors
– Poor nutrition, Infection
– Immunosuppressive drugs
– Malignancy, Age, Stress
False positives (Major Issue)
• Infection with NTM - non
tuberculous mycobacteria
• BCG vaccination
• Booster effect or retest
• Natural infection with MTB
in endemic counties and
acquired CMI (herd
immunity
• MPB 64 is a specific MTB antigen
• Patch test becomes positive after 3-4 days of
application and lasts for a week
• Specificity of 100% and sensitivity of 98.1%
• Evaluated only in Philippines and needs to be
reproduced in other settings.
• ESAT 6 (Early Secretory Antigen for TB) and CFP
10 (Colony Forming Protein) are specific proteins
of MTB and are being tried in skin tests.
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We need to move fast from our ESR and Mx. test
Clinical feature are good screening tool for tests
Radiology has a good role but understand its limits
Costly imaging like HRCT, MRI add nothing for Dx.
Accurate Diagnosis, Complete Rx are the answers
MDR, XDR and now TDR TB are great challenges
DST has to be done routinely – at least on diagnosis
RT PCR, Gene Xpert, IGRA, LiPA – a sea change
Use ADA to exclude. Serology should not be used.
Above all, remember our burden & poverty. Stop TB.