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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 SnNOUT (Minimum FN)

CAD No CAD Sensitivity is

by CAG by CAG
True positives a
True Positives False Positives Total CAD a+c
ECG +VE
a b
TEST

Specificity is
False Negative True Negatives
ECG – VE
c d True Negatives d
Total No CAD b+d
Total CAD Total No CAD
a+c b+d SpPIN (Minimum FP)
 GOLD STANDARD SnNOUT (Rules out 70%)

CAD No CAD Sensitivity is

by CAG by CAG
True positives 70

ECG + VE
True Positives False Positives Total CAD 100
70 120
TEST

Specificity is
False Negative True Negatives
ECG – VE True Negatives 180
30 180

Total No CAD 300

Total CAD Total No CAD
100 300 SpPIN (Confirms 60%)
 GOLD STANDARD PPV is 37%)

CAD No CAD Positive Predictive Value

Test by CAG by CAG
True positives 70
True False
ECG + VE Positives Positives
Total +ves Total Positives 190
190
70 120
Negative Predictive Value
False True
Total -ves
ECG – VE Negative Negatives True Negatives 180
210
30 180
Total Negatives 210
Total CAD Total CAD Grand
100 300 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 TB
3.5m 1.7m 2.5m
• 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.
• 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
• 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)

embB
Mutations in the MTB Genome
• 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 Indirect Methods
MTB and Its Products 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 Indirect Methods
MTB and Its Products 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

• 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 Indirect Methods
MTB and Its Products 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

• 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 Indirect Methods
MTB and Its Products 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
• 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 Indirect Methods
MTB and Its Products 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

• 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 GeneXpert
MDR TB MTB RIF

Mycoreal Myco3Plex
RT PCR Multiplex
MD of
MTB
• “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 Indirect Methods
MTB and Its Products 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 Indirect Methods
MTB and Its Products 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

• 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 RT PCR 1st Line LiPA

• One Day • One Day • One Day

3 days
Microscopy Liquid Culture 1st Line DST 2nd Line DST
• One Day • 2 to 3 weeks • 1 to 3 weeks • 1 to 3 weeks

4 to 9 weeks

Microscopy RT PCR 1st Line LiPA 2nd Line LiPA

• One Day • One Day • One Day • 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 Indirect Methods
MTB and Its Products 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 Indirect Methods
MTB and Its Products 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 Indirect Methods
MTB and Its Products 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 Indirect Methods
MTB and Its Products 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 Indirect Methods
MTB and Its Products 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 Indirect Methods
MTB and Its Products 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 Indirect Methods
MTB and Its Products 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 Indirect Methods
MTB and Its Products 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 Indirect Methods
MTB and Its Products 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.
• 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.