Updates in diagnosis of tb
Jun 20, 2015
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Updates in Diagnosis of TB
By
Gamal Rabie Agmy MD FCCP Professor of Chest Diseases Assiut University
ERS National Delegate of Egypt
Diagnosis of TB
Direct Methods 1-Direct Microscopy (ZN Kinyoun Flurochrome)
2-Culture (Traditional Rapid methods)
3- Detection of DNA or RNA of mycobacterial origin ( PCR LAMP TAA NAA LCR Fast Plaque)
Causative organism bull Mycobacterium tuberculosis COMPLEX
bull Stained with
-Modified gram stain gram positive
-Carbolfuchsin stain Cold method(Kynon)
Hot(Zeil-Neelson)
- Fluorescent dyes rhodamine and
auramine stains
Bacteriology
Direct Microscopic
Examination Hallmark of staining is Ziehl-Neelsen stained slides
Easiest amp quickest diagnostic test
Limited sensitivity (46-78) but specificity is virtually 100
Centrifugation amp flurochrome staining (auramine O) UV
microscopy markedly increase the sensitivity amp a large
number can be examined in a much shorter time
Chest 1969951193
Direct Microscopic Examination 1048708 ZN staining requires = 105 bacilliml
1048708 TB bacilli appear as straightcurved rods (1-
4μ x 02-08μ) singly in pairs or in clumps
1048708 The yield of microscopic examination correlates well with the extent of disease the
presence of cavitation and the quality of
specimen
1048708 It is a good marker for infectiousness amp the
response to the treatment
Zeil-Neelson Staining
Wire 001 ml of specimen 200mm2 slide
Oil immersion field 002mm
Slide=10000 field=001ml specimen
10000 organismslide=1 AFBfield=1000000 organismml
1000 organismslide=1 AFB10 field=100000 organismml
100 organismslide=1 AFB100field=10000 organismml
QUANTITATION SCALE FOR ACID-FAST BACILLUS
SMEARS ACCORDING TO STAIN USED
Carbolfuchsin (times 1000) Fluorochrome
(times 250) Quantity Reported
No AFB300 fields No AFB30 fields No AFB seen
1-2 AFB300 fields 1-2 AFB30 fields Doubtful repeat
test
1-9 AFB100 fields 1-9 AFB10 fields Rare (1+)
1-9 AFB10 fields 1-9 AFBfield Few (2+)
1-9 AFBfield 10-90 AFBfield Moderate (3+)
gt 9 AFBfield gt 90 AFBfield Numerous (4+)
Several approaches are being made to
enhance the
sensitivity of the smear microscopy Concentration of sputum sample by centrifugation
enhances sensitivity to almost 100
Treatment of sputum samples with Zwitterionic
detergent also known as C18 carboxyprophylbetaine(
CB18) interferes with the innate buoyancy of the
bacilli and enhances the result of sputum
microscopy
J Clin Microbiol 199931 2371 J Clin Microbiol 199836 1965
Traditional Culture
1048708 More sensitive amp can be positive even when
bacterial load is low (10-100 bacilliml)
1048708 Sensitivity 80-85 Specificity 98
1048708 Required for precise identification of causative
organisms
1048708 3 Types of media are used
Egg based LJ Petragnani and ATS
Agar based Middlebrook 7H10 or 7H11
Liquid based Kirschnerrsquos Middlebrook 7H9
1048708 Growth is slow and takes 6-8 weeks There after
the same length of time is required for complete
identification amp sensitivity testing
Broth Based Rapid Culture Methods
Micro colony detection on solid media
1048708 Radiometric (BACTEC)
1048708 Septicheck AFB
1048708 Mycobacterial growth indicator tubes (MGIT)
Substantial improvement in time to detection amp total number of positive cultures can be realized from using broth based systems
Micro colony Detection on Solid Media
1048708 Plates poured with thin layer of Middlebrook 7H11 agar
medium are incubated and examined microscopically on
alternate days for the first 2 days and less frequently
thereafter
1048708 In less than 7 days micro-colonies of slow growing
mycobacteria such as Mtb can be detected
BACTEC
1048708 Growth is ascertained by liberation
of 14CO2 as metabolized by
mycobacteria amp detected by BACTEC
460 instrument amp reported in terms of
growth index (GI) value
BACTEC
1048708 Average time to recovery of Mtb from smear positive
specimens is 8 days
1048708 When smear negative culture positive samples are
examined mean time for detection is 14 days
1048708 More sensitive than traditional method
1048708 Can also be used for drug susceptibility testing
J Clin Microbiol 199432 918-925
BACTEC 1048708 A special procedure unique to BACTEC system
for identification of Mtb complex is based on
observation that p-nitro-α-acetylamino-β-
hydroxypropiophenone (NAP) will inhibit organisms
belonging to Mtb complex while having little or no
effects on other mycobacteria
Drawbacks
1048708 Cost
1048708 Problem of disposal of radioactive waste
Septicheck AFB
1048708 Combines broth amp solid media into a single device
(biphasic culture approach)
1048708 Contains 30ml of modified Middlebrook 7H9 broth
in CO2 enriched culture bottle amp a peddle with agar
media- one side of peddle covered with Middlebrook
7H11 other side contains Middle brook 7H11 with NAP
1048708 Requires 3 weeks of incubation
Advantage Simultaneous detection of Mtb NTM other
respiratory pathogen amp even contaminant
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Rapid Method
1048708 Consists of round bottom tubes containing 4 ml of
modified Middlebrook 7H9 broth which has an oxygen
sensitive fluroscent sensor at the bottom
1048708 When mycobacteria grow they deplete the dissolved
oxygen in the broth amp allow the indicator to fluoresce
brightly in a 365nm UV light
J Clin Microbiol 199937 748-752
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Positive signals are obtained in 10-12 days
1048708 MGIT can also be used as a rapid method for the
detection of drug resistant strains of Mtb directly from acid-fast
smear positive samples as well as from indirect drug
susceptibility studies
Advantages over BACTEC
1048708 Cheaper
1048708 No problem of radioactive waste disposal J Clin Microbiol 199937 45-48
Detection and identification of
mycobacteria directly
from clinical samples
Genotypic Methods
1048708 PCR
1048708 LAMP
1048708 TMA NAA
1048708 Ligase chain reaction
1048708 Phenotypic Methods
1048708 FAST Plaque TB
Polymerase Chain Reaction (PCR)
1048708 Essentially PCR is a way to make millions of identical
copies of a specific DNA sequence which may be a
gene or a part of a gene or simply a stretch of
nucleotides with a known DNA sequence the function of
which may be unknown
1048708 A specimen that may contain the DNA sequence of
interest is heated to denature double stranded DNA
Polymerase Chain Reaction (PCR)
1048708 Specific synthetic oligonucleotide primers bind to the
unique DNA sequences of interest and a heat stable
DNA polymerase (Thermus aquaticus) extends the
primer to create a complete amp complimentary strand of
DNA
1048708 This process is repeated sequentially 25-40 times
thereby creating millions of copies of target
sequence
Polymerase Chain Reaction (PCR)
1048708 Kd antigen 65(HSPs)
1048708 Used earlier
1048708 Heat shock protein believed to be distinct from other
bacterial HSPs
1048708 This gene is identical in all species of mycobacteria
1048708 Therefore unsuitable for detecting Mtb particularly in
areas where species like Mavium or Mkansasii are
prevalent
IS6110 1048708 It is a transposon which are
self replicating stretches of DNA
1048708 Function not known
1048708 This sequence has been found
in the Mtb complex organisms
(Mtb Mafricanum Mmicroti
Mbovis)
1048708 IS6110 sequence generally
occurs only once in Mbovis but is
found as often as 20 times in
certain strains of Mtb thus offering
multiple targets for amplification
Polymerase Chain Reaction (PCR)
1048708 With recent modification PCR can detect even a fraction
of a bacilli
Role in pulmonary TB
1048708 Detects nearly all smear +ve and culture +ve cases
1048708 Useful technology for rapid diagnosis of smear ndashve cases
1048708 Able to identify 50-60 of smear -ve cases this would
reduce the need for more invasive approaches to smear - ve
TB
Distinguish Mtb from NTM in smear +ve cases as
IS6110 sequence is not found in NTM
1048708 Should not be used to replace sputum microscopy
1048708 Sensitivity specificity amp PPV for PCR is 835
99 amp 942 respectively
Am Rev Respir Dis 1991 1441160 J Clin Microbiol 199931 2049-2055
Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
1048708 Limited Role
1048708 No comprehensive large series comparing the yield of
PCR with other available approaches has been published
1048708 But at present it is valuable adjunct in the diagnosis
of TB pleurisy pericardial TB amp other condition in which
yield of other tests are low
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Diagnosis of TB
Direct Methods 1-Direct Microscopy (ZN Kinyoun Flurochrome)
2-Culture (Traditional Rapid methods)
3- Detection of DNA or RNA of mycobacterial origin ( PCR LAMP TAA NAA LCR Fast Plaque)
Causative organism bull Mycobacterium tuberculosis COMPLEX
bull Stained with
-Modified gram stain gram positive
-Carbolfuchsin stain Cold method(Kynon)
Hot(Zeil-Neelson)
- Fluorescent dyes rhodamine and
auramine stains
Bacteriology
Direct Microscopic
Examination Hallmark of staining is Ziehl-Neelsen stained slides
Easiest amp quickest diagnostic test
Limited sensitivity (46-78) but specificity is virtually 100
Centrifugation amp flurochrome staining (auramine O) UV
microscopy markedly increase the sensitivity amp a large
number can be examined in a much shorter time
Chest 1969951193
Direct Microscopic Examination 1048708 ZN staining requires = 105 bacilliml
1048708 TB bacilli appear as straightcurved rods (1-
4μ x 02-08μ) singly in pairs or in clumps
1048708 The yield of microscopic examination correlates well with the extent of disease the
presence of cavitation and the quality of
specimen
1048708 It is a good marker for infectiousness amp the
response to the treatment
Zeil-Neelson Staining
Wire 001 ml of specimen 200mm2 slide
Oil immersion field 002mm
Slide=10000 field=001ml specimen
10000 organismslide=1 AFBfield=1000000 organismml
1000 organismslide=1 AFB10 field=100000 organismml
100 organismslide=1 AFB100field=10000 organismml
QUANTITATION SCALE FOR ACID-FAST BACILLUS
SMEARS ACCORDING TO STAIN USED
Carbolfuchsin (times 1000) Fluorochrome
(times 250) Quantity Reported
No AFB300 fields No AFB30 fields No AFB seen
1-2 AFB300 fields 1-2 AFB30 fields Doubtful repeat
test
1-9 AFB100 fields 1-9 AFB10 fields Rare (1+)
1-9 AFB10 fields 1-9 AFBfield Few (2+)
1-9 AFBfield 10-90 AFBfield Moderate (3+)
gt 9 AFBfield gt 90 AFBfield Numerous (4+)
Several approaches are being made to
enhance the
sensitivity of the smear microscopy Concentration of sputum sample by centrifugation
enhances sensitivity to almost 100
Treatment of sputum samples with Zwitterionic
detergent also known as C18 carboxyprophylbetaine(
CB18) interferes with the innate buoyancy of the
bacilli and enhances the result of sputum
microscopy
J Clin Microbiol 199931 2371 J Clin Microbiol 199836 1965
Traditional Culture
1048708 More sensitive amp can be positive even when
bacterial load is low (10-100 bacilliml)
1048708 Sensitivity 80-85 Specificity 98
1048708 Required for precise identification of causative
organisms
1048708 3 Types of media are used
Egg based LJ Petragnani and ATS
Agar based Middlebrook 7H10 or 7H11
Liquid based Kirschnerrsquos Middlebrook 7H9
1048708 Growth is slow and takes 6-8 weeks There after
the same length of time is required for complete
identification amp sensitivity testing
Broth Based Rapid Culture Methods
Micro colony detection on solid media
1048708 Radiometric (BACTEC)
1048708 Septicheck AFB
1048708 Mycobacterial growth indicator tubes (MGIT)
Substantial improvement in time to detection amp total number of positive cultures can be realized from using broth based systems
Micro colony Detection on Solid Media
1048708 Plates poured with thin layer of Middlebrook 7H11 agar
medium are incubated and examined microscopically on
alternate days for the first 2 days and less frequently
thereafter
1048708 In less than 7 days micro-colonies of slow growing
mycobacteria such as Mtb can be detected
BACTEC
1048708 Growth is ascertained by liberation
of 14CO2 as metabolized by
mycobacteria amp detected by BACTEC
460 instrument amp reported in terms of
growth index (GI) value
BACTEC
1048708 Average time to recovery of Mtb from smear positive
specimens is 8 days
1048708 When smear negative culture positive samples are
examined mean time for detection is 14 days
1048708 More sensitive than traditional method
1048708 Can also be used for drug susceptibility testing
J Clin Microbiol 199432 918-925
BACTEC 1048708 A special procedure unique to BACTEC system
for identification of Mtb complex is based on
observation that p-nitro-α-acetylamino-β-
hydroxypropiophenone (NAP) will inhibit organisms
belonging to Mtb complex while having little or no
effects on other mycobacteria
Drawbacks
1048708 Cost
1048708 Problem of disposal of radioactive waste
Septicheck AFB
1048708 Combines broth amp solid media into a single device
(biphasic culture approach)
1048708 Contains 30ml of modified Middlebrook 7H9 broth
in CO2 enriched culture bottle amp a peddle with agar
media- one side of peddle covered with Middlebrook
7H11 other side contains Middle brook 7H11 with NAP
1048708 Requires 3 weeks of incubation
Advantage Simultaneous detection of Mtb NTM other
respiratory pathogen amp even contaminant
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Rapid Method
1048708 Consists of round bottom tubes containing 4 ml of
modified Middlebrook 7H9 broth which has an oxygen
sensitive fluroscent sensor at the bottom
1048708 When mycobacteria grow they deplete the dissolved
oxygen in the broth amp allow the indicator to fluoresce
brightly in a 365nm UV light
J Clin Microbiol 199937 748-752
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Positive signals are obtained in 10-12 days
1048708 MGIT can also be used as a rapid method for the
detection of drug resistant strains of Mtb directly from acid-fast
smear positive samples as well as from indirect drug
susceptibility studies
Advantages over BACTEC
1048708 Cheaper
1048708 No problem of radioactive waste disposal J Clin Microbiol 199937 45-48
Detection and identification of
mycobacteria directly
from clinical samples
Genotypic Methods
1048708 PCR
1048708 LAMP
1048708 TMA NAA
1048708 Ligase chain reaction
1048708 Phenotypic Methods
1048708 FAST Plaque TB
Polymerase Chain Reaction (PCR)
1048708 Essentially PCR is a way to make millions of identical
copies of a specific DNA sequence which may be a
gene or a part of a gene or simply a stretch of
nucleotides with a known DNA sequence the function of
which may be unknown
1048708 A specimen that may contain the DNA sequence of
interest is heated to denature double stranded DNA
Polymerase Chain Reaction (PCR)
1048708 Specific synthetic oligonucleotide primers bind to the
unique DNA sequences of interest and a heat stable
DNA polymerase (Thermus aquaticus) extends the
primer to create a complete amp complimentary strand of
DNA
1048708 This process is repeated sequentially 25-40 times
thereby creating millions of copies of target
sequence
Polymerase Chain Reaction (PCR)
1048708 Kd antigen 65(HSPs)
1048708 Used earlier
1048708 Heat shock protein believed to be distinct from other
bacterial HSPs
1048708 This gene is identical in all species of mycobacteria
1048708 Therefore unsuitable for detecting Mtb particularly in
areas where species like Mavium or Mkansasii are
prevalent
IS6110 1048708 It is a transposon which are
self replicating stretches of DNA
1048708 Function not known
1048708 This sequence has been found
in the Mtb complex organisms
(Mtb Mafricanum Mmicroti
Mbovis)
1048708 IS6110 sequence generally
occurs only once in Mbovis but is
found as often as 20 times in
certain strains of Mtb thus offering
multiple targets for amplification
Polymerase Chain Reaction (PCR)
1048708 With recent modification PCR can detect even a fraction
of a bacilli
Role in pulmonary TB
1048708 Detects nearly all smear +ve and culture +ve cases
1048708 Useful technology for rapid diagnosis of smear ndashve cases
1048708 Able to identify 50-60 of smear -ve cases this would
reduce the need for more invasive approaches to smear - ve
TB
Distinguish Mtb from NTM in smear +ve cases as
IS6110 sequence is not found in NTM
1048708 Should not be used to replace sputum microscopy
1048708 Sensitivity specificity amp PPV for PCR is 835
99 amp 942 respectively
Am Rev Respir Dis 1991 1441160 J Clin Microbiol 199931 2049-2055
Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
1048708 Limited Role
1048708 No comprehensive large series comparing the yield of
PCR with other available approaches has been published
1048708 But at present it is valuable adjunct in the diagnosis
of TB pleurisy pericardial TB amp other condition in which
yield of other tests are low
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Causative organism bull Mycobacterium tuberculosis COMPLEX
bull Stained with
-Modified gram stain gram positive
-Carbolfuchsin stain Cold method(Kynon)
Hot(Zeil-Neelson)
- Fluorescent dyes rhodamine and
auramine stains
Bacteriology
Direct Microscopic
Examination Hallmark of staining is Ziehl-Neelsen stained slides
Easiest amp quickest diagnostic test
Limited sensitivity (46-78) but specificity is virtually 100
Centrifugation amp flurochrome staining (auramine O) UV
microscopy markedly increase the sensitivity amp a large
number can be examined in a much shorter time
Chest 1969951193
Direct Microscopic Examination 1048708 ZN staining requires = 105 bacilliml
1048708 TB bacilli appear as straightcurved rods (1-
4μ x 02-08μ) singly in pairs or in clumps
1048708 The yield of microscopic examination correlates well with the extent of disease the
presence of cavitation and the quality of
specimen
1048708 It is a good marker for infectiousness amp the
response to the treatment
Zeil-Neelson Staining
Wire 001 ml of specimen 200mm2 slide
Oil immersion field 002mm
Slide=10000 field=001ml specimen
10000 organismslide=1 AFBfield=1000000 organismml
1000 organismslide=1 AFB10 field=100000 organismml
100 organismslide=1 AFB100field=10000 organismml
QUANTITATION SCALE FOR ACID-FAST BACILLUS
SMEARS ACCORDING TO STAIN USED
Carbolfuchsin (times 1000) Fluorochrome
(times 250) Quantity Reported
No AFB300 fields No AFB30 fields No AFB seen
1-2 AFB300 fields 1-2 AFB30 fields Doubtful repeat
test
1-9 AFB100 fields 1-9 AFB10 fields Rare (1+)
1-9 AFB10 fields 1-9 AFBfield Few (2+)
1-9 AFBfield 10-90 AFBfield Moderate (3+)
gt 9 AFBfield gt 90 AFBfield Numerous (4+)
Several approaches are being made to
enhance the
sensitivity of the smear microscopy Concentration of sputum sample by centrifugation
enhances sensitivity to almost 100
Treatment of sputum samples with Zwitterionic
detergent also known as C18 carboxyprophylbetaine(
CB18) interferes with the innate buoyancy of the
bacilli and enhances the result of sputum
microscopy
J Clin Microbiol 199931 2371 J Clin Microbiol 199836 1965
Traditional Culture
1048708 More sensitive amp can be positive even when
bacterial load is low (10-100 bacilliml)
1048708 Sensitivity 80-85 Specificity 98
1048708 Required for precise identification of causative
organisms
1048708 3 Types of media are used
Egg based LJ Petragnani and ATS
Agar based Middlebrook 7H10 or 7H11
Liquid based Kirschnerrsquos Middlebrook 7H9
1048708 Growth is slow and takes 6-8 weeks There after
the same length of time is required for complete
identification amp sensitivity testing
Broth Based Rapid Culture Methods
Micro colony detection on solid media
1048708 Radiometric (BACTEC)
1048708 Septicheck AFB
1048708 Mycobacterial growth indicator tubes (MGIT)
Substantial improvement in time to detection amp total number of positive cultures can be realized from using broth based systems
Micro colony Detection on Solid Media
1048708 Plates poured with thin layer of Middlebrook 7H11 agar
medium are incubated and examined microscopically on
alternate days for the first 2 days and less frequently
thereafter
1048708 In less than 7 days micro-colonies of slow growing
mycobacteria such as Mtb can be detected
BACTEC
1048708 Growth is ascertained by liberation
of 14CO2 as metabolized by
mycobacteria amp detected by BACTEC
460 instrument amp reported in terms of
growth index (GI) value
BACTEC
1048708 Average time to recovery of Mtb from smear positive
specimens is 8 days
1048708 When smear negative culture positive samples are
examined mean time for detection is 14 days
1048708 More sensitive than traditional method
1048708 Can also be used for drug susceptibility testing
J Clin Microbiol 199432 918-925
BACTEC 1048708 A special procedure unique to BACTEC system
for identification of Mtb complex is based on
observation that p-nitro-α-acetylamino-β-
hydroxypropiophenone (NAP) will inhibit organisms
belonging to Mtb complex while having little or no
effects on other mycobacteria
Drawbacks
1048708 Cost
1048708 Problem of disposal of radioactive waste
Septicheck AFB
1048708 Combines broth amp solid media into a single device
(biphasic culture approach)
1048708 Contains 30ml of modified Middlebrook 7H9 broth
in CO2 enriched culture bottle amp a peddle with agar
media- one side of peddle covered with Middlebrook
7H11 other side contains Middle brook 7H11 with NAP
1048708 Requires 3 weeks of incubation
Advantage Simultaneous detection of Mtb NTM other
respiratory pathogen amp even contaminant
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Rapid Method
1048708 Consists of round bottom tubes containing 4 ml of
modified Middlebrook 7H9 broth which has an oxygen
sensitive fluroscent sensor at the bottom
1048708 When mycobacteria grow they deplete the dissolved
oxygen in the broth amp allow the indicator to fluoresce
brightly in a 365nm UV light
J Clin Microbiol 199937 748-752
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Positive signals are obtained in 10-12 days
1048708 MGIT can also be used as a rapid method for the
detection of drug resistant strains of Mtb directly from acid-fast
smear positive samples as well as from indirect drug
susceptibility studies
Advantages over BACTEC
1048708 Cheaper
1048708 No problem of radioactive waste disposal J Clin Microbiol 199937 45-48
Detection and identification of
mycobacteria directly
from clinical samples
Genotypic Methods
1048708 PCR
1048708 LAMP
1048708 TMA NAA
1048708 Ligase chain reaction
1048708 Phenotypic Methods
1048708 FAST Plaque TB
Polymerase Chain Reaction (PCR)
1048708 Essentially PCR is a way to make millions of identical
copies of a specific DNA sequence which may be a
gene or a part of a gene or simply a stretch of
nucleotides with a known DNA sequence the function of
which may be unknown
1048708 A specimen that may contain the DNA sequence of
interest is heated to denature double stranded DNA
Polymerase Chain Reaction (PCR)
1048708 Specific synthetic oligonucleotide primers bind to the
unique DNA sequences of interest and a heat stable
DNA polymerase (Thermus aquaticus) extends the
primer to create a complete amp complimentary strand of
DNA
1048708 This process is repeated sequentially 25-40 times
thereby creating millions of copies of target
sequence
Polymerase Chain Reaction (PCR)
1048708 Kd antigen 65(HSPs)
1048708 Used earlier
1048708 Heat shock protein believed to be distinct from other
bacterial HSPs
1048708 This gene is identical in all species of mycobacteria
1048708 Therefore unsuitable for detecting Mtb particularly in
areas where species like Mavium or Mkansasii are
prevalent
IS6110 1048708 It is a transposon which are
self replicating stretches of DNA
1048708 Function not known
1048708 This sequence has been found
in the Mtb complex organisms
(Mtb Mafricanum Mmicroti
Mbovis)
1048708 IS6110 sequence generally
occurs only once in Mbovis but is
found as often as 20 times in
certain strains of Mtb thus offering
multiple targets for amplification
Polymerase Chain Reaction (PCR)
1048708 With recent modification PCR can detect even a fraction
of a bacilli
Role in pulmonary TB
1048708 Detects nearly all smear +ve and culture +ve cases
1048708 Useful technology for rapid diagnosis of smear ndashve cases
1048708 Able to identify 50-60 of smear -ve cases this would
reduce the need for more invasive approaches to smear - ve
TB
Distinguish Mtb from NTM in smear +ve cases as
IS6110 sequence is not found in NTM
1048708 Should not be used to replace sputum microscopy
1048708 Sensitivity specificity amp PPV for PCR is 835
99 amp 942 respectively
Am Rev Respir Dis 1991 1441160 J Clin Microbiol 199931 2049-2055
Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
1048708 Limited Role
1048708 No comprehensive large series comparing the yield of
PCR with other available approaches has been published
1048708 But at present it is valuable adjunct in the diagnosis
of TB pleurisy pericardial TB amp other condition in which
yield of other tests are low
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Direct Microscopic
Examination Hallmark of staining is Ziehl-Neelsen stained slides
Easiest amp quickest diagnostic test
Limited sensitivity (46-78) but specificity is virtually 100
Centrifugation amp flurochrome staining (auramine O) UV
microscopy markedly increase the sensitivity amp a large
number can be examined in a much shorter time
Chest 1969951193
Direct Microscopic Examination 1048708 ZN staining requires = 105 bacilliml
1048708 TB bacilli appear as straightcurved rods (1-
4μ x 02-08μ) singly in pairs or in clumps
1048708 The yield of microscopic examination correlates well with the extent of disease the
presence of cavitation and the quality of
specimen
1048708 It is a good marker for infectiousness amp the
response to the treatment
Zeil-Neelson Staining
Wire 001 ml of specimen 200mm2 slide
Oil immersion field 002mm
Slide=10000 field=001ml specimen
10000 organismslide=1 AFBfield=1000000 organismml
1000 organismslide=1 AFB10 field=100000 organismml
100 organismslide=1 AFB100field=10000 organismml
QUANTITATION SCALE FOR ACID-FAST BACILLUS
SMEARS ACCORDING TO STAIN USED
Carbolfuchsin (times 1000) Fluorochrome
(times 250) Quantity Reported
No AFB300 fields No AFB30 fields No AFB seen
1-2 AFB300 fields 1-2 AFB30 fields Doubtful repeat
test
1-9 AFB100 fields 1-9 AFB10 fields Rare (1+)
1-9 AFB10 fields 1-9 AFBfield Few (2+)
1-9 AFBfield 10-90 AFBfield Moderate (3+)
gt 9 AFBfield gt 90 AFBfield Numerous (4+)
Several approaches are being made to
enhance the
sensitivity of the smear microscopy Concentration of sputum sample by centrifugation
enhances sensitivity to almost 100
Treatment of sputum samples with Zwitterionic
detergent also known as C18 carboxyprophylbetaine(
CB18) interferes with the innate buoyancy of the
bacilli and enhances the result of sputum
microscopy
J Clin Microbiol 199931 2371 J Clin Microbiol 199836 1965
Traditional Culture
1048708 More sensitive amp can be positive even when
bacterial load is low (10-100 bacilliml)
1048708 Sensitivity 80-85 Specificity 98
1048708 Required for precise identification of causative
organisms
1048708 3 Types of media are used
Egg based LJ Petragnani and ATS
Agar based Middlebrook 7H10 or 7H11
Liquid based Kirschnerrsquos Middlebrook 7H9
1048708 Growth is slow and takes 6-8 weeks There after
the same length of time is required for complete
identification amp sensitivity testing
Broth Based Rapid Culture Methods
Micro colony detection on solid media
1048708 Radiometric (BACTEC)
1048708 Septicheck AFB
1048708 Mycobacterial growth indicator tubes (MGIT)
Substantial improvement in time to detection amp total number of positive cultures can be realized from using broth based systems
Micro colony Detection on Solid Media
1048708 Plates poured with thin layer of Middlebrook 7H11 agar
medium are incubated and examined microscopically on
alternate days for the first 2 days and less frequently
thereafter
1048708 In less than 7 days micro-colonies of slow growing
mycobacteria such as Mtb can be detected
BACTEC
1048708 Growth is ascertained by liberation
of 14CO2 as metabolized by
mycobacteria amp detected by BACTEC
460 instrument amp reported in terms of
growth index (GI) value
BACTEC
1048708 Average time to recovery of Mtb from smear positive
specimens is 8 days
1048708 When smear negative culture positive samples are
examined mean time for detection is 14 days
1048708 More sensitive than traditional method
1048708 Can also be used for drug susceptibility testing
J Clin Microbiol 199432 918-925
BACTEC 1048708 A special procedure unique to BACTEC system
for identification of Mtb complex is based on
observation that p-nitro-α-acetylamino-β-
hydroxypropiophenone (NAP) will inhibit organisms
belonging to Mtb complex while having little or no
effects on other mycobacteria
Drawbacks
1048708 Cost
1048708 Problem of disposal of radioactive waste
Septicheck AFB
1048708 Combines broth amp solid media into a single device
(biphasic culture approach)
1048708 Contains 30ml of modified Middlebrook 7H9 broth
in CO2 enriched culture bottle amp a peddle with agar
media- one side of peddle covered with Middlebrook
7H11 other side contains Middle brook 7H11 with NAP
1048708 Requires 3 weeks of incubation
Advantage Simultaneous detection of Mtb NTM other
respiratory pathogen amp even contaminant
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Rapid Method
1048708 Consists of round bottom tubes containing 4 ml of
modified Middlebrook 7H9 broth which has an oxygen
sensitive fluroscent sensor at the bottom
1048708 When mycobacteria grow they deplete the dissolved
oxygen in the broth amp allow the indicator to fluoresce
brightly in a 365nm UV light
J Clin Microbiol 199937 748-752
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Positive signals are obtained in 10-12 days
1048708 MGIT can also be used as a rapid method for the
detection of drug resistant strains of Mtb directly from acid-fast
smear positive samples as well as from indirect drug
susceptibility studies
Advantages over BACTEC
1048708 Cheaper
1048708 No problem of radioactive waste disposal J Clin Microbiol 199937 45-48
Detection and identification of
mycobacteria directly
from clinical samples
Genotypic Methods
1048708 PCR
1048708 LAMP
1048708 TMA NAA
1048708 Ligase chain reaction
1048708 Phenotypic Methods
1048708 FAST Plaque TB
Polymerase Chain Reaction (PCR)
1048708 Essentially PCR is a way to make millions of identical
copies of a specific DNA sequence which may be a
gene or a part of a gene or simply a stretch of
nucleotides with a known DNA sequence the function of
which may be unknown
1048708 A specimen that may contain the DNA sequence of
interest is heated to denature double stranded DNA
Polymerase Chain Reaction (PCR)
1048708 Specific synthetic oligonucleotide primers bind to the
unique DNA sequences of interest and a heat stable
DNA polymerase (Thermus aquaticus) extends the
primer to create a complete amp complimentary strand of
DNA
1048708 This process is repeated sequentially 25-40 times
thereby creating millions of copies of target
sequence
Polymerase Chain Reaction (PCR)
1048708 Kd antigen 65(HSPs)
1048708 Used earlier
1048708 Heat shock protein believed to be distinct from other
bacterial HSPs
1048708 This gene is identical in all species of mycobacteria
1048708 Therefore unsuitable for detecting Mtb particularly in
areas where species like Mavium or Mkansasii are
prevalent
IS6110 1048708 It is a transposon which are
self replicating stretches of DNA
1048708 Function not known
1048708 This sequence has been found
in the Mtb complex organisms
(Mtb Mafricanum Mmicroti
Mbovis)
1048708 IS6110 sequence generally
occurs only once in Mbovis but is
found as often as 20 times in
certain strains of Mtb thus offering
multiple targets for amplification
Polymerase Chain Reaction (PCR)
1048708 With recent modification PCR can detect even a fraction
of a bacilli
Role in pulmonary TB
1048708 Detects nearly all smear +ve and culture +ve cases
1048708 Useful technology for rapid diagnosis of smear ndashve cases
1048708 Able to identify 50-60 of smear -ve cases this would
reduce the need for more invasive approaches to smear - ve
TB
Distinguish Mtb from NTM in smear +ve cases as
IS6110 sequence is not found in NTM
1048708 Should not be used to replace sputum microscopy
1048708 Sensitivity specificity amp PPV for PCR is 835
99 amp 942 respectively
Am Rev Respir Dis 1991 1441160 J Clin Microbiol 199931 2049-2055
Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
1048708 Limited Role
1048708 No comprehensive large series comparing the yield of
PCR with other available approaches has been published
1048708 But at present it is valuable adjunct in the diagnosis
of TB pleurisy pericardial TB amp other condition in which
yield of other tests are low
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Direct Microscopic Examination 1048708 ZN staining requires = 105 bacilliml
1048708 TB bacilli appear as straightcurved rods (1-
4μ x 02-08μ) singly in pairs or in clumps
1048708 The yield of microscopic examination correlates well with the extent of disease the
presence of cavitation and the quality of
specimen
1048708 It is a good marker for infectiousness amp the
response to the treatment
Zeil-Neelson Staining
Wire 001 ml of specimen 200mm2 slide
Oil immersion field 002mm
Slide=10000 field=001ml specimen
10000 organismslide=1 AFBfield=1000000 organismml
1000 organismslide=1 AFB10 field=100000 organismml
100 organismslide=1 AFB100field=10000 organismml
QUANTITATION SCALE FOR ACID-FAST BACILLUS
SMEARS ACCORDING TO STAIN USED
Carbolfuchsin (times 1000) Fluorochrome
(times 250) Quantity Reported
No AFB300 fields No AFB30 fields No AFB seen
1-2 AFB300 fields 1-2 AFB30 fields Doubtful repeat
test
1-9 AFB100 fields 1-9 AFB10 fields Rare (1+)
1-9 AFB10 fields 1-9 AFBfield Few (2+)
1-9 AFBfield 10-90 AFBfield Moderate (3+)
gt 9 AFBfield gt 90 AFBfield Numerous (4+)
Several approaches are being made to
enhance the
sensitivity of the smear microscopy Concentration of sputum sample by centrifugation
enhances sensitivity to almost 100
Treatment of sputum samples with Zwitterionic
detergent also known as C18 carboxyprophylbetaine(
CB18) interferes with the innate buoyancy of the
bacilli and enhances the result of sputum
microscopy
J Clin Microbiol 199931 2371 J Clin Microbiol 199836 1965
Traditional Culture
1048708 More sensitive amp can be positive even when
bacterial load is low (10-100 bacilliml)
1048708 Sensitivity 80-85 Specificity 98
1048708 Required for precise identification of causative
organisms
1048708 3 Types of media are used
Egg based LJ Petragnani and ATS
Agar based Middlebrook 7H10 or 7H11
Liquid based Kirschnerrsquos Middlebrook 7H9
1048708 Growth is slow and takes 6-8 weeks There after
the same length of time is required for complete
identification amp sensitivity testing
Broth Based Rapid Culture Methods
Micro colony detection on solid media
1048708 Radiometric (BACTEC)
1048708 Septicheck AFB
1048708 Mycobacterial growth indicator tubes (MGIT)
Substantial improvement in time to detection amp total number of positive cultures can be realized from using broth based systems
Micro colony Detection on Solid Media
1048708 Plates poured with thin layer of Middlebrook 7H11 agar
medium are incubated and examined microscopically on
alternate days for the first 2 days and less frequently
thereafter
1048708 In less than 7 days micro-colonies of slow growing
mycobacteria such as Mtb can be detected
BACTEC
1048708 Growth is ascertained by liberation
of 14CO2 as metabolized by
mycobacteria amp detected by BACTEC
460 instrument amp reported in terms of
growth index (GI) value
BACTEC
1048708 Average time to recovery of Mtb from smear positive
specimens is 8 days
1048708 When smear negative culture positive samples are
examined mean time for detection is 14 days
1048708 More sensitive than traditional method
1048708 Can also be used for drug susceptibility testing
J Clin Microbiol 199432 918-925
BACTEC 1048708 A special procedure unique to BACTEC system
for identification of Mtb complex is based on
observation that p-nitro-α-acetylamino-β-
hydroxypropiophenone (NAP) will inhibit organisms
belonging to Mtb complex while having little or no
effects on other mycobacteria
Drawbacks
1048708 Cost
1048708 Problem of disposal of radioactive waste
Septicheck AFB
1048708 Combines broth amp solid media into a single device
(biphasic culture approach)
1048708 Contains 30ml of modified Middlebrook 7H9 broth
in CO2 enriched culture bottle amp a peddle with agar
media- one side of peddle covered with Middlebrook
7H11 other side contains Middle brook 7H11 with NAP
1048708 Requires 3 weeks of incubation
Advantage Simultaneous detection of Mtb NTM other
respiratory pathogen amp even contaminant
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Rapid Method
1048708 Consists of round bottom tubes containing 4 ml of
modified Middlebrook 7H9 broth which has an oxygen
sensitive fluroscent sensor at the bottom
1048708 When mycobacteria grow they deplete the dissolved
oxygen in the broth amp allow the indicator to fluoresce
brightly in a 365nm UV light
J Clin Microbiol 199937 748-752
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Positive signals are obtained in 10-12 days
1048708 MGIT can also be used as a rapid method for the
detection of drug resistant strains of Mtb directly from acid-fast
smear positive samples as well as from indirect drug
susceptibility studies
Advantages over BACTEC
1048708 Cheaper
1048708 No problem of radioactive waste disposal J Clin Microbiol 199937 45-48
Detection and identification of
mycobacteria directly
from clinical samples
Genotypic Methods
1048708 PCR
1048708 LAMP
1048708 TMA NAA
1048708 Ligase chain reaction
1048708 Phenotypic Methods
1048708 FAST Plaque TB
Polymerase Chain Reaction (PCR)
1048708 Essentially PCR is a way to make millions of identical
copies of a specific DNA sequence which may be a
gene or a part of a gene or simply a stretch of
nucleotides with a known DNA sequence the function of
which may be unknown
1048708 A specimen that may contain the DNA sequence of
interest is heated to denature double stranded DNA
Polymerase Chain Reaction (PCR)
1048708 Specific synthetic oligonucleotide primers bind to the
unique DNA sequences of interest and a heat stable
DNA polymerase (Thermus aquaticus) extends the
primer to create a complete amp complimentary strand of
DNA
1048708 This process is repeated sequentially 25-40 times
thereby creating millions of copies of target
sequence
Polymerase Chain Reaction (PCR)
1048708 Kd antigen 65(HSPs)
1048708 Used earlier
1048708 Heat shock protein believed to be distinct from other
bacterial HSPs
1048708 This gene is identical in all species of mycobacteria
1048708 Therefore unsuitable for detecting Mtb particularly in
areas where species like Mavium or Mkansasii are
prevalent
IS6110 1048708 It is a transposon which are
self replicating stretches of DNA
1048708 Function not known
1048708 This sequence has been found
in the Mtb complex organisms
(Mtb Mafricanum Mmicroti
Mbovis)
1048708 IS6110 sequence generally
occurs only once in Mbovis but is
found as often as 20 times in
certain strains of Mtb thus offering
multiple targets for amplification
Polymerase Chain Reaction (PCR)
1048708 With recent modification PCR can detect even a fraction
of a bacilli
Role in pulmonary TB
1048708 Detects nearly all smear +ve and culture +ve cases
1048708 Useful technology for rapid diagnosis of smear ndashve cases
1048708 Able to identify 50-60 of smear -ve cases this would
reduce the need for more invasive approaches to smear - ve
TB
Distinguish Mtb from NTM in smear +ve cases as
IS6110 sequence is not found in NTM
1048708 Should not be used to replace sputum microscopy
1048708 Sensitivity specificity amp PPV for PCR is 835
99 amp 942 respectively
Am Rev Respir Dis 1991 1441160 J Clin Microbiol 199931 2049-2055
Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
1048708 Limited Role
1048708 No comprehensive large series comparing the yield of
PCR with other available approaches has been published
1048708 But at present it is valuable adjunct in the diagnosis
of TB pleurisy pericardial TB amp other condition in which
yield of other tests are low
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Zeil-Neelson Staining
Wire 001 ml of specimen 200mm2 slide
Oil immersion field 002mm
Slide=10000 field=001ml specimen
10000 organismslide=1 AFBfield=1000000 organismml
1000 organismslide=1 AFB10 field=100000 organismml
100 organismslide=1 AFB100field=10000 organismml
QUANTITATION SCALE FOR ACID-FAST BACILLUS
SMEARS ACCORDING TO STAIN USED
Carbolfuchsin (times 1000) Fluorochrome
(times 250) Quantity Reported
No AFB300 fields No AFB30 fields No AFB seen
1-2 AFB300 fields 1-2 AFB30 fields Doubtful repeat
test
1-9 AFB100 fields 1-9 AFB10 fields Rare (1+)
1-9 AFB10 fields 1-9 AFBfield Few (2+)
1-9 AFBfield 10-90 AFBfield Moderate (3+)
gt 9 AFBfield gt 90 AFBfield Numerous (4+)
Several approaches are being made to
enhance the
sensitivity of the smear microscopy Concentration of sputum sample by centrifugation
enhances sensitivity to almost 100
Treatment of sputum samples with Zwitterionic
detergent also known as C18 carboxyprophylbetaine(
CB18) interferes with the innate buoyancy of the
bacilli and enhances the result of sputum
microscopy
J Clin Microbiol 199931 2371 J Clin Microbiol 199836 1965
Traditional Culture
1048708 More sensitive amp can be positive even when
bacterial load is low (10-100 bacilliml)
1048708 Sensitivity 80-85 Specificity 98
1048708 Required for precise identification of causative
organisms
1048708 3 Types of media are used
Egg based LJ Petragnani and ATS
Agar based Middlebrook 7H10 or 7H11
Liquid based Kirschnerrsquos Middlebrook 7H9
1048708 Growth is slow and takes 6-8 weeks There after
the same length of time is required for complete
identification amp sensitivity testing
Broth Based Rapid Culture Methods
Micro colony detection on solid media
1048708 Radiometric (BACTEC)
1048708 Septicheck AFB
1048708 Mycobacterial growth indicator tubes (MGIT)
Substantial improvement in time to detection amp total number of positive cultures can be realized from using broth based systems
Micro colony Detection on Solid Media
1048708 Plates poured with thin layer of Middlebrook 7H11 agar
medium are incubated and examined microscopically on
alternate days for the first 2 days and less frequently
thereafter
1048708 In less than 7 days micro-colonies of slow growing
mycobacteria such as Mtb can be detected
BACTEC
1048708 Growth is ascertained by liberation
of 14CO2 as metabolized by
mycobacteria amp detected by BACTEC
460 instrument amp reported in terms of
growth index (GI) value
BACTEC
1048708 Average time to recovery of Mtb from smear positive
specimens is 8 days
1048708 When smear negative culture positive samples are
examined mean time for detection is 14 days
1048708 More sensitive than traditional method
1048708 Can also be used for drug susceptibility testing
J Clin Microbiol 199432 918-925
BACTEC 1048708 A special procedure unique to BACTEC system
for identification of Mtb complex is based on
observation that p-nitro-α-acetylamino-β-
hydroxypropiophenone (NAP) will inhibit organisms
belonging to Mtb complex while having little or no
effects on other mycobacteria
Drawbacks
1048708 Cost
1048708 Problem of disposal of radioactive waste
Septicheck AFB
1048708 Combines broth amp solid media into a single device
(biphasic culture approach)
1048708 Contains 30ml of modified Middlebrook 7H9 broth
in CO2 enriched culture bottle amp a peddle with agar
media- one side of peddle covered with Middlebrook
7H11 other side contains Middle brook 7H11 with NAP
1048708 Requires 3 weeks of incubation
Advantage Simultaneous detection of Mtb NTM other
respiratory pathogen amp even contaminant
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Rapid Method
1048708 Consists of round bottom tubes containing 4 ml of
modified Middlebrook 7H9 broth which has an oxygen
sensitive fluroscent sensor at the bottom
1048708 When mycobacteria grow they deplete the dissolved
oxygen in the broth amp allow the indicator to fluoresce
brightly in a 365nm UV light
J Clin Microbiol 199937 748-752
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Positive signals are obtained in 10-12 days
1048708 MGIT can also be used as a rapid method for the
detection of drug resistant strains of Mtb directly from acid-fast
smear positive samples as well as from indirect drug
susceptibility studies
Advantages over BACTEC
1048708 Cheaper
1048708 No problem of radioactive waste disposal J Clin Microbiol 199937 45-48
Detection and identification of
mycobacteria directly
from clinical samples
Genotypic Methods
1048708 PCR
1048708 LAMP
1048708 TMA NAA
1048708 Ligase chain reaction
1048708 Phenotypic Methods
1048708 FAST Plaque TB
Polymerase Chain Reaction (PCR)
1048708 Essentially PCR is a way to make millions of identical
copies of a specific DNA sequence which may be a
gene or a part of a gene or simply a stretch of
nucleotides with a known DNA sequence the function of
which may be unknown
1048708 A specimen that may contain the DNA sequence of
interest is heated to denature double stranded DNA
Polymerase Chain Reaction (PCR)
1048708 Specific synthetic oligonucleotide primers bind to the
unique DNA sequences of interest and a heat stable
DNA polymerase (Thermus aquaticus) extends the
primer to create a complete amp complimentary strand of
DNA
1048708 This process is repeated sequentially 25-40 times
thereby creating millions of copies of target
sequence
Polymerase Chain Reaction (PCR)
1048708 Kd antigen 65(HSPs)
1048708 Used earlier
1048708 Heat shock protein believed to be distinct from other
bacterial HSPs
1048708 This gene is identical in all species of mycobacteria
1048708 Therefore unsuitable for detecting Mtb particularly in
areas where species like Mavium or Mkansasii are
prevalent
IS6110 1048708 It is a transposon which are
self replicating stretches of DNA
1048708 Function not known
1048708 This sequence has been found
in the Mtb complex organisms
(Mtb Mafricanum Mmicroti
Mbovis)
1048708 IS6110 sequence generally
occurs only once in Mbovis but is
found as often as 20 times in
certain strains of Mtb thus offering
multiple targets for amplification
Polymerase Chain Reaction (PCR)
1048708 With recent modification PCR can detect even a fraction
of a bacilli
Role in pulmonary TB
1048708 Detects nearly all smear +ve and culture +ve cases
1048708 Useful technology for rapid diagnosis of smear ndashve cases
1048708 Able to identify 50-60 of smear -ve cases this would
reduce the need for more invasive approaches to smear - ve
TB
Distinguish Mtb from NTM in smear +ve cases as
IS6110 sequence is not found in NTM
1048708 Should not be used to replace sputum microscopy
1048708 Sensitivity specificity amp PPV for PCR is 835
99 amp 942 respectively
Am Rev Respir Dis 1991 1441160 J Clin Microbiol 199931 2049-2055
Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
1048708 Limited Role
1048708 No comprehensive large series comparing the yield of
PCR with other available approaches has been published
1048708 But at present it is valuable adjunct in the diagnosis
of TB pleurisy pericardial TB amp other condition in which
yield of other tests are low
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
QUANTITATION SCALE FOR ACID-FAST BACILLUS
SMEARS ACCORDING TO STAIN USED
Carbolfuchsin (times 1000) Fluorochrome
(times 250) Quantity Reported
No AFB300 fields No AFB30 fields No AFB seen
1-2 AFB300 fields 1-2 AFB30 fields Doubtful repeat
test
1-9 AFB100 fields 1-9 AFB10 fields Rare (1+)
1-9 AFB10 fields 1-9 AFBfield Few (2+)
1-9 AFBfield 10-90 AFBfield Moderate (3+)
gt 9 AFBfield gt 90 AFBfield Numerous (4+)
Several approaches are being made to
enhance the
sensitivity of the smear microscopy Concentration of sputum sample by centrifugation
enhances sensitivity to almost 100
Treatment of sputum samples with Zwitterionic
detergent also known as C18 carboxyprophylbetaine(
CB18) interferes with the innate buoyancy of the
bacilli and enhances the result of sputum
microscopy
J Clin Microbiol 199931 2371 J Clin Microbiol 199836 1965
Traditional Culture
1048708 More sensitive amp can be positive even when
bacterial load is low (10-100 bacilliml)
1048708 Sensitivity 80-85 Specificity 98
1048708 Required for precise identification of causative
organisms
1048708 3 Types of media are used
Egg based LJ Petragnani and ATS
Agar based Middlebrook 7H10 or 7H11
Liquid based Kirschnerrsquos Middlebrook 7H9
1048708 Growth is slow and takes 6-8 weeks There after
the same length of time is required for complete
identification amp sensitivity testing
Broth Based Rapid Culture Methods
Micro colony detection on solid media
1048708 Radiometric (BACTEC)
1048708 Septicheck AFB
1048708 Mycobacterial growth indicator tubes (MGIT)
Substantial improvement in time to detection amp total number of positive cultures can be realized from using broth based systems
Micro colony Detection on Solid Media
1048708 Plates poured with thin layer of Middlebrook 7H11 agar
medium are incubated and examined microscopically on
alternate days for the first 2 days and less frequently
thereafter
1048708 In less than 7 days micro-colonies of slow growing
mycobacteria such as Mtb can be detected
BACTEC
1048708 Growth is ascertained by liberation
of 14CO2 as metabolized by
mycobacteria amp detected by BACTEC
460 instrument amp reported in terms of
growth index (GI) value
BACTEC
1048708 Average time to recovery of Mtb from smear positive
specimens is 8 days
1048708 When smear negative culture positive samples are
examined mean time for detection is 14 days
1048708 More sensitive than traditional method
1048708 Can also be used for drug susceptibility testing
J Clin Microbiol 199432 918-925
BACTEC 1048708 A special procedure unique to BACTEC system
for identification of Mtb complex is based on
observation that p-nitro-α-acetylamino-β-
hydroxypropiophenone (NAP) will inhibit organisms
belonging to Mtb complex while having little or no
effects on other mycobacteria
Drawbacks
1048708 Cost
1048708 Problem of disposal of radioactive waste
Septicheck AFB
1048708 Combines broth amp solid media into a single device
(biphasic culture approach)
1048708 Contains 30ml of modified Middlebrook 7H9 broth
in CO2 enriched culture bottle amp a peddle with agar
media- one side of peddle covered with Middlebrook
7H11 other side contains Middle brook 7H11 with NAP
1048708 Requires 3 weeks of incubation
Advantage Simultaneous detection of Mtb NTM other
respiratory pathogen amp even contaminant
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Rapid Method
1048708 Consists of round bottom tubes containing 4 ml of
modified Middlebrook 7H9 broth which has an oxygen
sensitive fluroscent sensor at the bottom
1048708 When mycobacteria grow they deplete the dissolved
oxygen in the broth amp allow the indicator to fluoresce
brightly in a 365nm UV light
J Clin Microbiol 199937 748-752
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Positive signals are obtained in 10-12 days
1048708 MGIT can also be used as a rapid method for the
detection of drug resistant strains of Mtb directly from acid-fast
smear positive samples as well as from indirect drug
susceptibility studies
Advantages over BACTEC
1048708 Cheaper
1048708 No problem of radioactive waste disposal J Clin Microbiol 199937 45-48
Detection and identification of
mycobacteria directly
from clinical samples
Genotypic Methods
1048708 PCR
1048708 LAMP
1048708 TMA NAA
1048708 Ligase chain reaction
1048708 Phenotypic Methods
1048708 FAST Plaque TB
Polymerase Chain Reaction (PCR)
1048708 Essentially PCR is a way to make millions of identical
copies of a specific DNA sequence which may be a
gene or a part of a gene or simply a stretch of
nucleotides with a known DNA sequence the function of
which may be unknown
1048708 A specimen that may contain the DNA sequence of
interest is heated to denature double stranded DNA
Polymerase Chain Reaction (PCR)
1048708 Specific synthetic oligonucleotide primers bind to the
unique DNA sequences of interest and a heat stable
DNA polymerase (Thermus aquaticus) extends the
primer to create a complete amp complimentary strand of
DNA
1048708 This process is repeated sequentially 25-40 times
thereby creating millions of copies of target
sequence
Polymerase Chain Reaction (PCR)
1048708 Kd antigen 65(HSPs)
1048708 Used earlier
1048708 Heat shock protein believed to be distinct from other
bacterial HSPs
1048708 This gene is identical in all species of mycobacteria
1048708 Therefore unsuitable for detecting Mtb particularly in
areas where species like Mavium or Mkansasii are
prevalent
IS6110 1048708 It is a transposon which are
self replicating stretches of DNA
1048708 Function not known
1048708 This sequence has been found
in the Mtb complex organisms
(Mtb Mafricanum Mmicroti
Mbovis)
1048708 IS6110 sequence generally
occurs only once in Mbovis but is
found as often as 20 times in
certain strains of Mtb thus offering
multiple targets for amplification
Polymerase Chain Reaction (PCR)
1048708 With recent modification PCR can detect even a fraction
of a bacilli
Role in pulmonary TB
1048708 Detects nearly all smear +ve and culture +ve cases
1048708 Useful technology for rapid diagnosis of smear ndashve cases
1048708 Able to identify 50-60 of smear -ve cases this would
reduce the need for more invasive approaches to smear - ve
TB
Distinguish Mtb from NTM in smear +ve cases as
IS6110 sequence is not found in NTM
1048708 Should not be used to replace sputum microscopy
1048708 Sensitivity specificity amp PPV for PCR is 835
99 amp 942 respectively
Am Rev Respir Dis 1991 1441160 J Clin Microbiol 199931 2049-2055
Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
1048708 Limited Role
1048708 No comprehensive large series comparing the yield of
PCR with other available approaches has been published
1048708 But at present it is valuable adjunct in the diagnosis
of TB pleurisy pericardial TB amp other condition in which
yield of other tests are low
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Several approaches are being made to
enhance the
sensitivity of the smear microscopy Concentration of sputum sample by centrifugation
enhances sensitivity to almost 100
Treatment of sputum samples with Zwitterionic
detergent also known as C18 carboxyprophylbetaine(
CB18) interferes with the innate buoyancy of the
bacilli and enhances the result of sputum
microscopy
J Clin Microbiol 199931 2371 J Clin Microbiol 199836 1965
Traditional Culture
1048708 More sensitive amp can be positive even when
bacterial load is low (10-100 bacilliml)
1048708 Sensitivity 80-85 Specificity 98
1048708 Required for precise identification of causative
organisms
1048708 3 Types of media are used
Egg based LJ Petragnani and ATS
Agar based Middlebrook 7H10 or 7H11
Liquid based Kirschnerrsquos Middlebrook 7H9
1048708 Growth is slow and takes 6-8 weeks There after
the same length of time is required for complete
identification amp sensitivity testing
Broth Based Rapid Culture Methods
Micro colony detection on solid media
1048708 Radiometric (BACTEC)
1048708 Septicheck AFB
1048708 Mycobacterial growth indicator tubes (MGIT)
Substantial improvement in time to detection amp total number of positive cultures can be realized from using broth based systems
Micro colony Detection on Solid Media
1048708 Plates poured with thin layer of Middlebrook 7H11 agar
medium are incubated and examined microscopically on
alternate days for the first 2 days and less frequently
thereafter
1048708 In less than 7 days micro-colonies of slow growing
mycobacteria such as Mtb can be detected
BACTEC
1048708 Growth is ascertained by liberation
of 14CO2 as metabolized by
mycobacteria amp detected by BACTEC
460 instrument amp reported in terms of
growth index (GI) value
BACTEC
1048708 Average time to recovery of Mtb from smear positive
specimens is 8 days
1048708 When smear negative culture positive samples are
examined mean time for detection is 14 days
1048708 More sensitive than traditional method
1048708 Can also be used for drug susceptibility testing
J Clin Microbiol 199432 918-925
BACTEC 1048708 A special procedure unique to BACTEC system
for identification of Mtb complex is based on
observation that p-nitro-α-acetylamino-β-
hydroxypropiophenone (NAP) will inhibit organisms
belonging to Mtb complex while having little or no
effects on other mycobacteria
Drawbacks
1048708 Cost
1048708 Problem of disposal of radioactive waste
Septicheck AFB
1048708 Combines broth amp solid media into a single device
(biphasic culture approach)
1048708 Contains 30ml of modified Middlebrook 7H9 broth
in CO2 enriched culture bottle amp a peddle with agar
media- one side of peddle covered with Middlebrook
7H11 other side contains Middle brook 7H11 with NAP
1048708 Requires 3 weeks of incubation
Advantage Simultaneous detection of Mtb NTM other
respiratory pathogen amp even contaminant
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Rapid Method
1048708 Consists of round bottom tubes containing 4 ml of
modified Middlebrook 7H9 broth which has an oxygen
sensitive fluroscent sensor at the bottom
1048708 When mycobacteria grow they deplete the dissolved
oxygen in the broth amp allow the indicator to fluoresce
brightly in a 365nm UV light
J Clin Microbiol 199937 748-752
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Positive signals are obtained in 10-12 days
1048708 MGIT can also be used as a rapid method for the
detection of drug resistant strains of Mtb directly from acid-fast
smear positive samples as well as from indirect drug
susceptibility studies
Advantages over BACTEC
1048708 Cheaper
1048708 No problem of radioactive waste disposal J Clin Microbiol 199937 45-48
Detection and identification of
mycobacteria directly
from clinical samples
Genotypic Methods
1048708 PCR
1048708 LAMP
1048708 TMA NAA
1048708 Ligase chain reaction
1048708 Phenotypic Methods
1048708 FAST Plaque TB
Polymerase Chain Reaction (PCR)
1048708 Essentially PCR is a way to make millions of identical
copies of a specific DNA sequence which may be a
gene or a part of a gene or simply a stretch of
nucleotides with a known DNA sequence the function of
which may be unknown
1048708 A specimen that may contain the DNA sequence of
interest is heated to denature double stranded DNA
Polymerase Chain Reaction (PCR)
1048708 Specific synthetic oligonucleotide primers bind to the
unique DNA sequences of interest and a heat stable
DNA polymerase (Thermus aquaticus) extends the
primer to create a complete amp complimentary strand of
DNA
1048708 This process is repeated sequentially 25-40 times
thereby creating millions of copies of target
sequence
Polymerase Chain Reaction (PCR)
1048708 Kd antigen 65(HSPs)
1048708 Used earlier
1048708 Heat shock protein believed to be distinct from other
bacterial HSPs
1048708 This gene is identical in all species of mycobacteria
1048708 Therefore unsuitable for detecting Mtb particularly in
areas where species like Mavium or Mkansasii are
prevalent
IS6110 1048708 It is a transposon which are
self replicating stretches of DNA
1048708 Function not known
1048708 This sequence has been found
in the Mtb complex organisms
(Mtb Mafricanum Mmicroti
Mbovis)
1048708 IS6110 sequence generally
occurs only once in Mbovis but is
found as often as 20 times in
certain strains of Mtb thus offering
multiple targets for amplification
Polymerase Chain Reaction (PCR)
1048708 With recent modification PCR can detect even a fraction
of a bacilli
Role in pulmonary TB
1048708 Detects nearly all smear +ve and culture +ve cases
1048708 Useful technology for rapid diagnosis of smear ndashve cases
1048708 Able to identify 50-60 of smear -ve cases this would
reduce the need for more invasive approaches to smear - ve
TB
Distinguish Mtb from NTM in smear +ve cases as
IS6110 sequence is not found in NTM
1048708 Should not be used to replace sputum microscopy
1048708 Sensitivity specificity amp PPV for PCR is 835
99 amp 942 respectively
Am Rev Respir Dis 1991 1441160 J Clin Microbiol 199931 2049-2055
Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
1048708 Limited Role
1048708 No comprehensive large series comparing the yield of
PCR with other available approaches has been published
1048708 But at present it is valuable adjunct in the diagnosis
of TB pleurisy pericardial TB amp other condition in which
yield of other tests are low
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Traditional Culture
1048708 More sensitive amp can be positive even when
bacterial load is low (10-100 bacilliml)
1048708 Sensitivity 80-85 Specificity 98
1048708 Required for precise identification of causative
organisms
1048708 3 Types of media are used
Egg based LJ Petragnani and ATS
Agar based Middlebrook 7H10 or 7H11
Liquid based Kirschnerrsquos Middlebrook 7H9
1048708 Growth is slow and takes 6-8 weeks There after
the same length of time is required for complete
identification amp sensitivity testing
Broth Based Rapid Culture Methods
Micro colony detection on solid media
1048708 Radiometric (BACTEC)
1048708 Septicheck AFB
1048708 Mycobacterial growth indicator tubes (MGIT)
Substantial improvement in time to detection amp total number of positive cultures can be realized from using broth based systems
Micro colony Detection on Solid Media
1048708 Plates poured with thin layer of Middlebrook 7H11 agar
medium are incubated and examined microscopically on
alternate days for the first 2 days and less frequently
thereafter
1048708 In less than 7 days micro-colonies of slow growing
mycobacteria such as Mtb can be detected
BACTEC
1048708 Growth is ascertained by liberation
of 14CO2 as metabolized by
mycobacteria amp detected by BACTEC
460 instrument amp reported in terms of
growth index (GI) value
BACTEC
1048708 Average time to recovery of Mtb from smear positive
specimens is 8 days
1048708 When smear negative culture positive samples are
examined mean time for detection is 14 days
1048708 More sensitive than traditional method
1048708 Can also be used for drug susceptibility testing
J Clin Microbiol 199432 918-925
BACTEC 1048708 A special procedure unique to BACTEC system
for identification of Mtb complex is based on
observation that p-nitro-α-acetylamino-β-
hydroxypropiophenone (NAP) will inhibit organisms
belonging to Mtb complex while having little or no
effects on other mycobacteria
Drawbacks
1048708 Cost
1048708 Problem of disposal of radioactive waste
Septicheck AFB
1048708 Combines broth amp solid media into a single device
(biphasic culture approach)
1048708 Contains 30ml of modified Middlebrook 7H9 broth
in CO2 enriched culture bottle amp a peddle with agar
media- one side of peddle covered with Middlebrook
7H11 other side contains Middle brook 7H11 with NAP
1048708 Requires 3 weeks of incubation
Advantage Simultaneous detection of Mtb NTM other
respiratory pathogen amp even contaminant
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Rapid Method
1048708 Consists of round bottom tubes containing 4 ml of
modified Middlebrook 7H9 broth which has an oxygen
sensitive fluroscent sensor at the bottom
1048708 When mycobacteria grow they deplete the dissolved
oxygen in the broth amp allow the indicator to fluoresce
brightly in a 365nm UV light
J Clin Microbiol 199937 748-752
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Positive signals are obtained in 10-12 days
1048708 MGIT can also be used as a rapid method for the
detection of drug resistant strains of Mtb directly from acid-fast
smear positive samples as well as from indirect drug
susceptibility studies
Advantages over BACTEC
1048708 Cheaper
1048708 No problem of radioactive waste disposal J Clin Microbiol 199937 45-48
Detection and identification of
mycobacteria directly
from clinical samples
Genotypic Methods
1048708 PCR
1048708 LAMP
1048708 TMA NAA
1048708 Ligase chain reaction
1048708 Phenotypic Methods
1048708 FAST Plaque TB
Polymerase Chain Reaction (PCR)
1048708 Essentially PCR is a way to make millions of identical
copies of a specific DNA sequence which may be a
gene or a part of a gene or simply a stretch of
nucleotides with a known DNA sequence the function of
which may be unknown
1048708 A specimen that may contain the DNA sequence of
interest is heated to denature double stranded DNA
Polymerase Chain Reaction (PCR)
1048708 Specific synthetic oligonucleotide primers bind to the
unique DNA sequences of interest and a heat stable
DNA polymerase (Thermus aquaticus) extends the
primer to create a complete amp complimentary strand of
DNA
1048708 This process is repeated sequentially 25-40 times
thereby creating millions of copies of target
sequence
Polymerase Chain Reaction (PCR)
1048708 Kd antigen 65(HSPs)
1048708 Used earlier
1048708 Heat shock protein believed to be distinct from other
bacterial HSPs
1048708 This gene is identical in all species of mycobacteria
1048708 Therefore unsuitable for detecting Mtb particularly in
areas where species like Mavium or Mkansasii are
prevalent
IS6110 1048708 It is a transposon which are
self replicating stretches of DNA
1048708 Function not known
1048708 This sequence has been found
in the Mtb complex organisms
(Mtb Mafricanum Mmicroti
Mbovis)
1048708 IS6110 sequence generally
occurs only once in Mbovis but is
found as often as 20 times in
certain strains of Mtb thus offering
multiple targets for amplification
Polymerase Chain Reaction (PCR)
1048708 With recent modification PCR can detect even a fraction
of a bacilli
Role in pulmonary TB
1048708 Detects nearly all smear +ve and culture +ve cases
1048708 Useful technology for rapid diagnosis of smear ndashve cases
1048708 Able to identify 50-60 of smear -ve cases this would
reduce the need for more invasive approaches to smear - ve
TB
Distinguish Mtb from NTM in smear +ve cases as
IS6110 sequence is not found in NTM
1048708 Should not be used to replace sputum microscopy
1048708 Sensitivity specificity amp PPV for PCR is 835
99 amp 942 respectively
Am Rev Respir Dis 1991 1441160 J Clin Microbiol 199931 2049-2055
Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
1048708 Limited Role
1048708 No comprehensive large series comparing the yield of
PCR with other available approaches has been published
1048708 But at present it is valuable adjunct in the diagnosis
of TB pleurisy pericardial TB amp other condition in which
yield of other tests are low
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Broth Based Rapid Culture Methods
Micro colony detection on solid media
1048708 Radiometric (BACTEC)
1048708 Septicheck AFB
1048708 Mycobacterial growth indicator tubes (MGIT)
Substantial improvement in time to detection amp total number of positive cultures can be realized from using broth based systems
Micro colony Detection on Solid Media
1048708 Plates poured with thin layer of Middlebrook 7H11 agar
medium are incubated and examined microscopically on
alternate days for the first 2 days and less frequently
thereafter
1048708 In less than 7 days micro-colonies of slow growing
mycobacteria such as Mtb can be detected
BACTEC
1048708 Growth is ascertained by liberation
of 14CO2 as metabolized by
mycobacteria amp detected by BACTEC
460 instrument amp reported in terms of
growth index (GI) value
BACTEC
1048708 Average time to recovery of Mtb from smear positive
specimens is 8 days
1048708 When smear negative culture positive samples are
examined mean time for detection is 14 days
1048708 More sensitive than traditional method
1048708 Can also be used for drug susceptibility testing
J Clin Microbiol 199432 918-925
BACTEC 1048708 A special procedure unique to BACTEC system
for identification of Mtb complex is based on
observation that p-nitro-α-acetylamino-β-
hydroxypropiophenone (NAP) will inhibit organisms
belonging to Mtb complex while having little or no
effects on other mycobacteria
Drawbacks
1048708 Cost
1048708 Problem of disposal of radioactive waste
Septicheck AFB
1048708 Combines broth amp solid media into a single device
(biphasic culture approach)
1048708 Contains 30ml of modified Middlebrook 7H9 broth
in CO2 enriched culture bottle amp a peddle with agar
media- one side of peddle covered with Middlebrook
7H11 other side contains Middle brook 7H11 with NAP
1048708 Requires 3 weeks of incubation
Advantage Simultaneous detection of Mtb NTM other
respiratory pathogen amp even contaminant
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Rapid Method
1048708 Consists of round bottom tubes containing 4 ml of
modified Middlebrook 7H9 broth which has an oxygen
sensitive fluroscent sensor at the bottom
1048708 When mycobacteria grow they deplete the dissolved
oxygen in the broth amp allow the indicator to fluoresce
brightly in a 365nm UV light
J Clin Microbiol 199937 748-752
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Positive signals are obtained in 10-12 days
1048708 MGIT can also be used as a rapid method for the
detection of drug resistant strains of Mtb directly from acid-fast
smear positive samples as well as from indirect drug
susceptibility studies
Advantages over BACTEC
1048708 Cheaper
1048708 No problem of radioactive waste disposal J Clin Microbiol 199937 45-48
Detection and identification of
mycobacteria directly
from clinical samples
Genotypic Methods
1048708 PCR
1048708 LAMP
1048708 TMA NAA
1048708 Ligase chain reaction
1048708 Phenotypic Methods
1048708 FAST Plaque TB
Polymerase Chain Reaction (PCR)
1048708 Essentially PCR is a way to make millions of identical
copies of a specific DNA sequence which may be a
gene or a part of a gene or simply a stretch of
nucleotides with a known DNA sequence the function of
which may be unknown
1048708 A specimen that may contain the DNA sequence of
interest is heated to denature double stranded DNA
Polymerase Chain Reaction (PCR)
1048708 Specific synthetic oligonucleotide primers bind to the
unique DNA sequences of interest and a heat stable
DNA polymerase (Thermus aquaticus) extends the
primer to create a complete amp complimentary strand of
DNA
1048708 This process is repeated sequentially 25-40 times
thereby creating millions of copies of target
sequence
Polymerase Chain Reaction (PCR)
1048708 Kd antigen 65(HSPs)
1048708 Used earlier
1048708 Heat shock protein believed to be distinct from other
bacterial HSPs
1048708 This gene is identical in all species of mycobacteria
1048708 Therefore unsuitable for detecting Mtb particularly in
areas where species like Mavium or Mkansasii are
prevalent
IS6110 1048708 It is a transposon which are
self replicating stretches of DNA
1048708 Function not known
1048708 This sequence has been found
in the Mtb complex organisms
(Mtb Mafricanum Mmicroti
Mbovis)
1048708 IS6110 sequence generally
occurs only once in Mbovis but is
found as often as 20 times in
certain strains of Mtb thus offering
multiple targets for amplification
Polymerase Chain Reaction (PCR)
1048708 With recent modification PCR can detect even a fraction
of a bacilli
Role in pulmonary TB
1048708 Detects nearly all smear +ve and culture +ve cases
1048708 Useful technology for rapid diagnosis of smear ndashve cases
1048708 Able to identify 50-60 of smear -ve cases this would
reduce the need for more invasive approaches to smear - ve
TB
Distinguish Mtb from NTM in smear +ve cases as
IS6110 sequence is not found in NTM
1048708 Should not be used to replace sputum microscopy
1048708 Sensitivity specificity amp PPV for PCR is 835
99 amp 942 respectively
Am Rev Respir Dis 1991 1441160 J Clin Microbiol 199931 2049-2055
Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
1048708 Limited Role
1048708 No comprehensive large series comparing the yield of
PCR with other available approaches has been published
1048708 But at present it is valuable adjunct in the diagnosis
of TB pleurisy pericardial TB amp other condition in which
yield of other tests are low
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Micro colony Detection on Solid Media
1048708 Plates poured with thin layer of Middlebrook 7H11 agar
medium are incubated and examined microscopically on
alternate days for the first 2 days and less frequently
thereafter
1048708 In less than 7 days micro-colonies of slow growing
mycobacteria such as Mtb can be detected
BACTEC
1048708 Growth is ascertained by liberation
of 14CO2 as metabolized by
mycobacteria amp detected by BACTEC
460 instrument amp reported in terms of
growth index (GI) value
BACTEC
1048708 Average time to recovery of Mtb from smear positive
specimens is 8 days
1048708 When smear negative culture positive samples are
examined mean time for detection is 14 days
1048708 More sensitive than traditional method
1048708 Can also be used for drug susceptibility testing
J Clin Microbiol 199432 918-925
BACTEC 1048708 A special procedure unique to BACTEC system
for identification of Mtb complex is based on
observation that p-nitro-α-acetylamino-β-
hydroxypropiophenone (NAP) will inhibit organisms
belonging to Mtb complex while having little or no
effects on other mycobacteria
Drawbacks
1048708 Cost
1048708 Problem of disposal of radioactive waste
Septicheck AFB
1048708 Combines broth amp solid media into a single device
(biphasic culture approach)
1048708 Contains 30ml of modified Middlebrook 7H9 broth
in CO2 enriched culture bottle amp a peddle with agar
media- one side of peddle covered with Middlebrook
7H11 other side contains Middle brook 7H11 with NAP
1048708 Requires 3 weeks of incubation
Advantage Simultaneous detection of Mtb NTM other
respiratory pathogen amp even contaminant
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Rapid Method
1048708 Consists of round bottom tubes containing 4 ml of
modified Middlebrook 7H9 broth which has an oxygen
sensitive fluroscent sensor at the bottom
1048708 When mycobacteria grow they deplete the dissolved
oxygen in the broth amp allow the indicator to fluoresce
brightly in a 365nm UV light
J Clin Microbiol 199937 748-752
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Positive signals are obtained in 10-12 days
1048708 MGIT can also be used as a rapid method for the
detection of drug resistant strains of Mtb directly from acid-fast
smear positive samples as well as from indirect drug
susceptibility studies
Advantages over BACTEC
1048708 Cheaper
1048708 No problem of radioactive waste disposal J Clin Microbiol 199937 45-48
Detection and identification of
mycobacteria directly
from clinical samples
Genotypic Methods
1048708 PCR
1048708 LAMP
1048708 TMA NAA
1048708 Ligase chain reaction
1048708 Phenotypic Methods
1048708 FAST Plaque TB
Polymerase Chain Reaction (PCR)
1048708 Essentially PCR is a way to make millions of identical
copies of a specific DNA sequence which may be a
gene or a part of a gene or simply a stretch of
nucleotides with a known DNA sequence the function of
which may be unknown
1048708 A specimen that may contain the DNA sequence of
interest is heated to denature double stranded DNA
Polymerase Chain Reaction (PCR)
1048708 Specific synthetic oligonucleotide primers bind to the
unique DNA sequences of interest and a heat stable
DNA polymerase (Thermus aquaticus) extends the
primer to create a complete amp complimentary strand of
DNA
1048708 This process is repeated sequentially 25-40 times
thereby creating millions of copies of target
sequence
Polymerase Chain Reaction (PCR)
1048708 Kd antigen 65(HSPs)
1048708 Used earlier
1048708 Heat shock protein believed to be distinct from other
bacterial HSPs
1048708 This gene is identical in all species of mycobacteria
1048708 Therefore unsuitable for detecting Mtb particularly in
areas where species like Mavium or Mkansasii are
prevalent
IS6110 1048708 It is a transposon which are
self replicating stretches of DNA
1048708 Function not known
1048708 This sequence has been found
in the Mtb complex organisms
(Mtb Mafricanum Mmicroti
Mbovis)
1048708 IS6110 sequence generally
occurs only once in Mbovis but is
found as often as 20 times in
certain strains of Mtb thus offering
multiple targets for amplification
Polymerase Chain Reaction (PCR)
1048708 With recent modification PCR can detect even a fraction
of a bacilli
Role in pulmonary TB
1048708 Detects nearly all smear +ve and culture +ve cases
1048708 Useful technology for rapid diagnosis of smear ndashve cases
1048708 Able to identify 50-60 of smear -ve cases this would
reduce the need for more invasive approaches to smear - ve
TB
Distinguish Mtb from NTM in smear +ve cases as
IS6110 sequence is not found in NTM
1048708 Should not be used to replace sputum microscopy
1048708 Sensitivity specificity amp PPV for PCR is 835
99 amp 942 respectively
Am Rev Respir Dis 1991 1441160 J Clin Microbiol 199931 2049-2055
Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
1048708 Limited Role
1048708 No comprehensive large series comparing the yield of
PCR with other available approaches has been published
1048708 But at present it is valuable adjunct in the diagnosis
of TB pleurisy pericardial TB amp other condition in which
yield of other tests are low
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
BACTEC
1048708 Growth is ascertained by liberation
of 14CO2 as metabolized by
mycobacteria amp detected by BACTEC
460 instrument amp reported in terms of
growth index (GI) value
BACTEC
1048708 Average time to recovery of Mtb from smear positive
specimens is 8 days
1048708 When smear negative culture positive samples are
examined mean time for detection is 14 days
1048708 More sensitive than traditional method
1048708 Can also be used for drug susceptibility testing
J Clin Microbiol 199432 918-925
BACTEC 1048708 A special procedure unique to BACTEC system
for identification of Mtb complex is based on
observation that p-nitro-α-acetylamino-β-
hydroxypropiophenone (NAP) will inhibit organisms
belonging to Mtb complex while having little or no
effects on other mycobacteria
Drawbacks
1048708 Cost
1048708 Problem of disposal of radioactive waste
Septicheck AFB
1048708 Combines broth amp solid media into a single device
(biphasic culture approach)
1048708 Contains 30ml of modified Middlebrook 7H9 broth
in CO2 enriched culture bottle amp a peddle with agar
media- one side of peddle covered with Middlebrook
7H11 other side contains Middle brook 7H11 with NAP
1048708 Requires 3 weeks of incubation
Advantage Simultaneous detection of Mtb NTM other
respiratory pathogen amp even contaminant
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Rapid Method
1048708 Consists of round bottom tubes containing 4 ml of
modified Middlebrook 7H9 broth which has an oxygen
sensitive fluroscent sensor at the bottom
1048708 When mycobacteria grow they deplete the dissolved
oxygen in the broth amp allow the indicator to fluoresce
brightly in a 365nm UV light
J Clin Microbiol 199937 748-752
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Positive signals are obtained in 10-12 days
1048708 MGIT can also be used as a rapid method for the
detection of drug resistant strains of Mtb directly from acid-fast
smear positive samples as well as from indirect drug
susceptibility studies
Advantages over BACTEC
1048708 Cheaper
1048708 No problem of radioactive waste disposal J Clin Microbiol 199937 45-48
Detection and identification of
mycobacteria directly
from clinical samples
Genotypic Methods
1048708 PCR
1048708 LAMP
1048708 TMA NAA
1048708 Ligase chain reaction
1048708 Phenotypic Methods
1048708 FAST Plaque TB
Polymerase Chain Reaction (PCR)
1048708 Essentially PCR is a way to make millions of identical
copies of a specific DNA sequence which may be a
gene or a part of a gene or simply a stretch of
nucleotides with a known DNA sequence the function of
which may be unknown
1048708 A specimen that may contain the DNA sequence of
interest is heated to denature double stranded DNA
Polymerase Chain Reaction (PCR)
1048708 Specific synthetic oligonucleotide primers bind to the
unique DNA sequences of interest and a heat stable
DNA polymerase (Thermus aquaticus) extends the
primer to create a complete amp complimentary strand of
DNA
1048708 This process is repeated sequentially 25-40 times
thereby creating millions of copies of target
sequence
Polymerase Chain Reaction (PCR)
1048708 Kd antigen 65(HSPs)
1048708 Used earlier
1048708 Heat shock protein believed to be distinct from other
bacterial HSPs
1048708 This gene is identical in all species of mycobacteria
1048708 Therefore unsuitable for detecting Mtb particularly in
areas where species like Mavium or Mkansasii are
prevalent
IS6110 1048708 It is a transposon which are
self replicating stretches of DNA
1048708 Function not known
1048708 This sequence has been found
in the Mtb complex organisms
(Mtb Mafricanum Mmicroti
Mbovis)
1048708 IS6110 sequence generally
occurs only once in Mbovis but is
found as often as 20 times in
certain strains of Mtb thus offering
multiple targets for amplification
Polymerase Chain Reaction (PCR)
1048708 With recent modification PCR can detect even a fraction
of a bacilli
Role in pulmonary TB
1048708 Detects nearly all smear +ve and culture +ve cases
1048708 Useful technology for rapid diagnosis of smear ndashve cases
1048708 Able to identify 50-60 of smear -ve cases this would
reduce the need for more invasive approaches to smear - ve
TB
Distinguish Mtb from NTM in smear +ve cases as
IS6110 sequence is not found in NTM
1048708 Should not be used to replace sputum microscopy
1048708 Sensitivity specificity amp PPV for PCR is 835
99 amp 942 respectively
Am Rev Respir Dis 1991 1441160 J Clin Microbiol 199931 2049-2055
Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
1048708 Limited Role
1048708 No comprehensive large series comparing the yield of
PCR with other available approaches has been published
1048708 But at present it is valuable adjunct in the diagnosis
of TB pleurisy pericardial TB amp other condition in which
yield of other tests are low
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
BACTEC
1048708 Average time to recovery of Mtb from smear positive
specimens is 8 days
1048708 When smear negative culture positive samples are
examined mean time for detection is 14 days
1048708 More sensitive than traditional method
1048708 Can also be used for drug susceptibility testing
J Clin Microbiol 199432 918-925
BACTEC 1048708 A special procedure unique to BACTEC system
for identification of Mtb complex is based on
observation that p-nitro-α-acetylamino-β-
hydroxypropiophenone (NAP) will inhibit organisms
belonging to Mtb complex while having little or no
effects on other mycobacteria
Drawbacks
1048708 Cost
1048708 Problem of disposal of radioactive waste
Septicheck AFB
1048708 Combines broth amp solid media into a single device
(biphasic culture approach)
1048708 Contains 30ml of modified Middlebrook 7H9 broth
in CO2 enriched culture bottle amp a peddle with agar
media- one side of peddle covered with Middlebrook
7H11 other side contains Middle brook 7H11 with NAP
1048708 Requires 3 weeks of incubation
Advantage Simultaneous detection of Mtb NTM other
respiratory pathogen amp even contaminant
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Rapid Method
1048708 Consists of round bottom tubes containing 4 ml of
modified Middlebrook 7H9 broth which has an oxygen
sensitive fluroscent sensor at the bottom
1048708 When mycobacteria grow they deplete the dissolved
oxygen in the broth amp allow the indicator to fluoresce
brightly in a 365nm UV light
J Clin Microbiol 199937 748-752
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Positive signals are obtained in 10-12 days
1048708 MGIT can also be used as a rapid method for the
detection of drug resistant strains of Mtb directly from acid-fast
smear positive samples as well as from indirect drug
susceptibility studies
Advantages over BACTEC
1048708 Cheaper
1048708 No problem of radioactive waste disposal J Clin Microbiol 199937 45-48
Detection and identification of
mycobacteria directly
from clinical samples
Genotypic Methods
1048708 PCR
1048708 LAMP
1048708 TMA NAA
1048708 Ligase chain reaction
1048708 Phenotypic Methods
1048708 FAST Plaque TB
Polymerase Chain Reaction (PCR)
1048708 Essentially PCR is a way to make millions of identical
copies of a specific DNA sequence which may be a
gene or a part of a gene or simply a stretch of
nucleotides with a known DNA sequence the function of
which may be unknown
1048708 A specimen that may contain the DNA sequence of
interest is heated to denature double stranded DNA
Polymerase Chain Reaction (PCR)
1048708 Specific synthetic oligonucleotide primers bind to the
unique DNA sequences of interest and a heat stable
DNA polymerase (Thermus aquaticus) extends the
primer to create a complete amp complimentary strand of
DNA
1048708 This process is repeated sequentially 25-40 times
thereby creating millions of copies of target
sequence
Polymerase Chain Reaction (PCR)
1048708 Kd antigen 65(HSPs)
1048708 Used earlier
1048708 Heat shock protein believed to be distinct from other
bacterial HSPs
1048708 This gene is identical in all species of mycobacteria
1048708 Therefore unsuitable for detecting Mtb particularly in
areas where species like Mavium or Mkansasii are
prevalent
IS6110 1048708 It is a transposon which are
self replicating stretches of DNA
1048708 Function not known
1048708 This sequence has been found
in the Mtb complex organisms
(Mtb Mafricanum Mmicroti
Mbovis)
1048708 IS6110 sequence generally
occurs only once in Mbovis but is
found as often as 20 times in
certain strains of Mtb thus offering
multiple targets for amplification
Polymerase Chain Reaction (PCR)
1048708 With recent modification PCR can detect even a fraction
of a bacilli
Role in pulmonary TB
1048708 Detects nearly all smear +ve and culture +ve cases
1048708 Useful technology for rapid diagnosis of smear ndashve cases
1048708 Able to identify 50-60 of smear -ve cases this would
reduce the need for more invasive approaches to smear - ve
TB
Distinguish Mtb from NTM in smear +ve cases as
IS6110 sequence is not found in NTM
1048708 Should not be used to replace sputum microscopy
1048708 Sensitivity specificity amp PPV for PCR is 835
99 amp 942 respectively
Am Rev Respir Dis 1991 1441160 J Clin Microbiol 199931 2049-2055
Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
1048708 Limited Role
1048708 No comprehensive large series comparing the yield of
PCR with other available approaches has been published
1048708 But at present it is valuable adjunct in the diagnosis
of TB pleurisy pericardial TB amp other condition in which
yield of other tests are low
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
BACTEC 1048708 A special procedure unique to BACTEC system
for identification of Mtb complex is based on
observation that p-nitro-α-acetylamino-β-
hydroxypropiophenone (NAP) will inhibit organisms
belonging to Mtb complex while having little or no
effects on other mycobacteria
Drawbacks
1048708 Cost
1048708 Problem of disposal of radioactive waste
Septicheck AFB
1048708 Combines broth amp solid media into a single device
(biphasic culture approach)
1048708 Contains 30ml of modified Middlebrook 7H9 broth
in CO2 enriched culture bottle amp a peddle with agar
media- one side of peddle covered with Middlebrook
7H11 other side contains Middle brook 7H11 with NAP
1048708 Requires 3 weeks of incubation
Advantage Simultaneous detection of Mtb NTM other
respiratory pathogen amp even contaminant
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Rapid Method
1048708 Consists of round bottom tubes containing 4 ml of
modified Middlebrook 7H9 broth which has an oxygen
sensitive fluroscent sensor at the bottom
1048708 When mycobacteria grow they deplete the dissolved
oxygen in the broth amp allow the indicator to fluoresce
brightly in a 365nm UV light
J Clin Microbiol 199937 748-752
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Positive signals are obtained in 10-12 days
1048708 MGIT can also be used as a rapid method for the
detection of drug resistant strains of Mtb directly from acid-fast
smear positive samples as well as from indirect drug
susceptibility studies
Advantages over BACTEC
1048708 Cheaper
1048708 No problem of radioactive waste disposal J Clin Microbiol 199937 45-48
Detection and identification of
mycobacteria directly
from clinical samples
Genotypic Methods
1048708 PCR
1048708 LAMP
1048708 TMA NAA
1048708 Ligase chain reaction
1048708 Phenotypic Methods
1048708 FAST Plaque TB
Polymerase Chain Reaction (PCR)
1048708 Essentially PCR is a way to make millions of identical
copies of a specific DNA sequence which may be a
gene or a part of a gene or simply a stretch of
nucleotides with a known DNA sequence the function of
which may be unknown
1048708 A specimen that may contain the DNA sequence of
interest is heated to denature double stranded DNA
Polymerase Chain Reaction (PCR)
1048708 Specific synthetic oligonucleotide primers bind to the
unique DNA sequences of interest and a heat stable
DNA polymerase (Thermus aquaticus) extends the
primer to create a complete amp complimentary strand of
DNA
1048708 This process is repeated sequentially 25-40 times
thereby creating millions of copies of target
sequence
Polymerase Chain Reaction (PCR)
1048708 Kd antigen 65(HSPs)
1048708 Used earlier
1048708 Heat shock protein believed to be distinct from other
bacterial HSPs
1048708 This gene is identical in all species of mycobacteria
1048708 Therefore unsuitable for detecting Mtb particularly in
areas where species like Mavium or Mkansasii are
prevalent
IS6110 1048708 It is a transposon which are
self replicating stretches of DNA
1048708 Function not known
1048708 This sequence has been found
in the Mtb complex organisms
(Mtb Mafricanum Mmicroti
Mbovis)
1048708 IS6110 sequence generally
occurs only once in Mbovis but is
found as often as 20 times in
certain strains of Mtb thus offering
multiple targets for amplification
Polymerase Chain Reaction (PCR)
1048708 With recent modification PCR can detect even a fraction
of a bacilli
Role in pulmonary TB
1048708 Detects nearly all smear +ve and culture +ve cases
1048708 Useful technology for rapid diagnosis of smear ndashve cases
1048708 Able to identify 50-60 of smear -ve cases this would
reduce the need for more invasive approaches to smear - ve
TB
Distinguish Mtb from NTM in smear +ve cases as
IS6110 sequence is not found in NTM
1048708 Should not be used to replace sputum microscopy
1048708 Sensitivity specificity amp PPV for PCR is 835
99 amp 942 respectively
Am Rev Respir Dis 1991 1441160 J Clin Microbiol 199931 2049-2055
Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
1048708 Limited Role
1048708 No comprehensive large series comparing the yield of
PCR with other available approaches has been published
1048708 But at present it is valuable adjunct in the diagnosis
of TB pleurisy pericardial TB amp other condition in which
yield of other tests are low
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Septicheck AFB
1048708 Combines broth amp solid media into a single device
(biphasic culture approach)
1048708 Contains 30ml of modified Middlebrook 7H9 broth
in CO2 enriched culture bottle amp a peddle with agar
media- one side of peddle covered with Middlebrook
7H11 other side contains Middle brook 7H11 with NAP
1048708 Requires 3 weeks of incubation
Advantage Simultaneous detection of Mtb NTM other
respiratory pathogen amp even contaminant
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Rapid Method
1048708 Consists of round bottom tubes containing 4 ml of
modified Middlebrook 7H9 broth which has an oxygen
sensitive fluroscent sensor at the bottom
1048708 When mycobacteria grow they deplete the dissolved
oxygen in the broth amp allow the indicator to fluoresce
brightly in a 365nm UV light
J Clin Microbiol 199937 748-752
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Positive signals are obtained in 10-12 days
1048708 MGIT can also be used as a rapid method for the
detection of drug resistant strains of Mtb directly from acid-fast
smear positive samples as well as from indirect drug
susceptibility studies
Advantages over BACTEC
1048708 Cheaper
1048708 No problem of radioactive waste disposal J Clin Microbiol 199937 45-48
Detection and identification of
mycobacteria directly
from clinical samples
Genotypic Methods
1048708 PCR
1048708 LAMP
1048708 TMA NAA
1048708 Ligase chain reaction
1048708 Phenotypic Methods
1048708 FAST Plaque TB
Polymerase Chain Reaction (PCR)
1048708 Essentially PCR is a way to make millions of identical
copies of a specific DNA sequence which may be a
gene or a part of a gene or simply a stretch of
nucleotides with a known DNA sequence the function of
which may be unknown
1048708 A specimen that may contain the DNA sequence of
interest is heated to denature double stranded DNA
Polymerase Chain Reaction (PCR)
1048708 Specific synthetic oligonucleotide primers bind to the
unique DNA sequences of interest and a heat stable
DNA polymerase (Thermus aquaticus) extends the
primer to create a complete amp complimentary strand of
DNA
1048708 This process is repeated sequentially 25-40 times
thereby creating millions of copies of target
sequence
Polymerase Chain Reaction (PCR)
1048708 Kd antigen 65(HSPs)
1048708 Used earlier
1048708 Heat shock protein believed to be distinct from other
bacterial HSPs
1048708 This gene is identical in all species of mycobacteria
1048708 Therefore unsuitable for detecting Mtb particularly in
areas where species like Mavium or Mkansasii are
prevalent
IS6110 1048708 It is a transposon which are
self replicating stretches of DNA
1048708 Function not known
1048708 This sequence has been found
in the Mtb complex organisms
(Mtb Mafricanum Mmicroti
Mbovis)
1048708 IS6110 sequence generally
occurs only once in Mbovis but is
found as often as 20 times in
certain strains of Mtb thus offering
multiple targets for amplification
Polymerase Chain Reaction (PCR)
1048708 With recent modification PCR can detect even a fraction
of a bacilli
Role in pulmonary TB
1048708 Detects nearly all smear +ve and culture +ve cases
1048708 Useful technology for rapid diagnosis of smear ndashve cases
1048708 Able to identify 50-60 of smear -ve cases this would
reduce the need for more invasive approaches to smear - ve
TB
Distinguish Mtb from NTM in smear +ve cases as
IS6110 sequence is not found in NTM
1048708 Should not be used to replace sputum microscopy
1048708 Sensitivity specificity amp PPV for PCR is 835
99 amp 942 respectively
Am Rev Respir Dis 1991 1441160 J Clin Microbiol 199931 2049-2055
Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
1048708 Limited Role
1048708 No comprehensive large series comparing the yield of
PCR with other available approaches has been published
1048708 But at present it is valuable adjunct in the diagnosis
of TB pleurisy pericardial TB amp other condition in which
yield of other tests are low
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Rapid Method
1048708 Consists of round bottom tubes containing 4 ml of
modified Middlebrook 7H9 broth which has an oxygen
sensitive fluroscent sensor at the bottom
1048708 When mycobacteria grow they deplete the dissolved
oxygen in the broth amp allow the indicator to fluoresce
brightly in a 365nm UV light
J Clin Microbiol 199937 748-752
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Positive signals are obtained in 10-12 days
1048708 MGIT can also be used as a rapid method for the
detection of drug resistant strains of Mtb directly from acid-fast
smear positive samples as well as from indirect drug
susceptibility studies
Advantages over BACTEC
1048708 Cheaper
1048708 No problem of radioactive waste disposal J Clin Microbiol 199937 45-48
Detection and identification of
mycobacteria directly
from clinical samples
Genotypic Methods
1048708 PCR
1048708 LAMP
1048708 TMA NAA
1048708 Ligase chain reaction
1048708 Phenotypic Methods
1048708 FAST Plaque TB
Polymerase Chain Reaction (PCR)
1048708 Essentially PCR is a way to make millions of identical
copies of a specific DNA sequence which may be a
gene or a part of a gene or simply a stretch of
nucleotides with a known DNA sequence the function of
which may be unknown
1048708 A specimen that may contain the DNA sequence of
interest is heated to denature double stranded DNA
Polymerase Chain Reaction (PCR)
1048708 Specific synthetic oligonucleotide primers bind to the
unique DNA sequences of interest and a heat stable
DNA polymerase (Thermus aquaticus) extends the
primer to create a complete amp complimentary strand of
DNA
1048708 This process is repeated sequentially 25-40 times
thereby creating millions of copies of target
sequence
Polymerase Chain Reaction (PCR)
1048708 Kd antigen 65(HSPs)
1048708 Used earlier
1048708 Heat shock protein believed to be distinct from other
bacterial HSPs
1048708 This gene is identical in all species of mycobacteria
1048708 Therefore unsuitable for detecting Mtb particularly in
areas where species like Mavium or Mkansasii are
prevalent
IS6110 1048708 It is a transposon which are
self replicating stretches of DNA
1048708 Function not known
1048708 This sequence has been found
in the Mtb complex organisms
(Mtb Mafricanum Mmicroti
Mbovis)
1048708 IS6110 sequence generally
occurs only once in Mbovis but is
found as often as 20 times in
certain strains of Mtb thus offering
multiple targets for amplification
Polymerase Chain Reaction (PCR)
1048708 With recent modification PCR can detect even a fraction
of a bacilli
Role in pulmonary TB
1048708 Detects nearly all smear +ve and culture +ve cases
1048708 Useful technology for rapid diagnosis of smear ndashve cases
1048708 Able to identify 50-60 of smear -ve cases this would
reduce the need for more invasive approaches to smear - ve
TB
Distinguish Mtb from NTM in smear +ve cases as
IS6110 sequence is not found in NTM
1048708 Should not be used to replace sputum microscopy
1048708 Sensitivity specificity amp PPV for PCR is 835
99 amp 942 respectively
Am Rev Respir Dis 1991 1441160 J Clin Microbiol 199931 2049-2055
Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
1048708 Limited Role
1048708 No comprehensive large series comparing the yield of
PCR with other available approaches has been published
1048708 But at present it is valuable adjunct in the diagnosis
of TB pleurisy pericardial TB amp other condition in which
yield of other tests are low
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Mycobacterial Growth Indicator Tube (MGIT)
1048708 Positive signals are obtained in 10-12 days
1048708 MGIT can also be used as a rapid method for the
detection of drug resistant strains of Mtb directly from acid-fast
smear positive samples as well as from indirect drug
susceptibility studies
Advantages over BACTEC
1048708 Cheaper
1048708 No problem of radioactive waste disposal J Clin Microbiol 199937 45-48
Detection and identification of
mycobacteria directly
from clinical samples
Genotypic Methods
1048708 PCR
1048708 LAMP
1048708 TMA NAA
1048708 Ligase chain reaction
1048708 Phenotypic Methods
1048708 FAST Plaque TB
Polymerase Chain Reaction (PCR)
1048708 Essentially PCR is a way to make millions of identical
copies of a specific DNA sequence which may be a
gene or a part of a gene or simply a stretch of
nucleotides with a known DNA sequence the function of
which may be unknown
1048708 A specimen that may contain the DNA sequence of
interest is heated to denature double stranded DNA
Polymerase Chain Reaction (PCR)
1048708 Specific synthetic oligonucleotide primers bind to the
unique DNA sequences of interest and a heat stable
DNA polymerase (Thermus aquaticus) extends the
primer to create a complete amp complimentary strand of
DNA
1048708 This process is repeated sequentially 25-40 times
thereby creating millions of copies of target
sequence
Polymerase Chain Reaction (PCR)
1048708 Kd antigen 65(HSPs)
1048708 Used earlier
1048708 Heat shock protein believed to be distinct from other
bacterial HSPs
1048708 This gene is identical in all species of mycobacteria
1048708 Therefore unsuitable for detecting Mtb particularly in
areas where species like Mavium or Mkansasii are
prevalent
IS6110 1048708 It is a transposon which are
self replicating stretches of DNA
1048708 Function not known
1048708 This sequence has been found
in the Mtb complex organisms
(Mtb Mafricanum Mmicroti
Mbovis)
1048708 IS6110 sequence generally
occurs only once in Mbovis but is
found as often as 20 times in
certain strains of Mtb thus offering
multiple targets for amplification
Polymerase Chain Reaction (PCR)
1048708 With recent modification PCR can detect even a fraction
of a bacilli
Role in pulmonary TB
1048708 Detects nearly all smear +ve and culture +ve cases
1048708 Useful technology for rapid diagnosis of smear ndashve cases
1048708 Able to identify 50-60 of smear -ve cases this would
reduce the need for more invasive approaches to smear - ve
TB
Distinguish Mtb from NTM in smear +ve cases as
IS6110 sequence is not found in NTM
1048708 Should not be used to replace sputum microscopy
1048708 Sensitivity specificity amp PPV for PCR is 835
99 amp 942 respectively
Am Rev Respir Dis 1991 1441160 J Clin Microbiol 199931 2049-2055
Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
1048708 Limited Role
1048708 No comprehensive large series comparing the yield of
PCR with other available approaches has been published
1048708 But at present it is valuable adjunct in the diagnosis
of TB pleurisy pericardial TB amp other condition in which
yield of other tests are low
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Detection and identification of
mycobacteria directly
from clinical samples
Genotypic Methods
1048708 PCR
1048708 LAMP
1048708 TMA NAA
1048708 Ligase chain reaction
1048708 Phenotypic Methods
1048708 FAST Plaque TB
Polymerase Chain Reaction (PCR)
1048708 Essentially PCR is a way to make millions of identical
copies of a specific DNA sequence which may be a
gene or a part of a gene or simply a stretch of
nucleotides with a known DNA sequence the function of
which may be unknown
1048708 A specimen that may contain the DNA sequence of
interest is heated to denature double stranded DNA
Polymerase Chain Reaction (PCR)
1048708 Specific synthetic oligonucleotide primers bind to the
unique DNA sequences of interest and a heat stable
DNA polymerase (Thermus aquaticus) extends the
primer to create a complete amp complimentary strand of
DNA
1048708 This process is repeated sequentially 25-40 times
thereby creating millions of copies of target
sequence
Polymerase Chain Reaction (PCR)
1048708 Kd antigen 65(HSPs)
1048708 Used earlier
1048708 Heat shock protein believed to be distinct from other
bacterial HSPs
1048708 This gene is identical in all species of mycobacteria
1048708 Therefore unsuitable for detecting Mtb particularly in
areas where species like Mavium or Mkansasii are
prevalent
IS6110 1048708 It is a transposon which are
self replicating stretches of DNA
1048708 Function not known
1048708 This sequence has been found
in the Mtb complex organisms
(Mtb Mafricanum Mmicroti
Mbovis)
1048708 IS6110 sequence generally
occurs only once in Mbovis but is
found as often as 20 times in
certain strains of Mtb thus offering
multiple targets for amplification
Polymerase Chain Reaction (PCR)
1048708 With recent modification PCR can detect even a fraction
of a bacilli
Role in pulmonary TB
1048708 Detects nearly all smear +ve and culture +ve cases
1048708 Useful technology for rapid diagnosis of smear ndashve cases
1048708 Able to identify 50-60 of smear -ve cases this would
reduce the need for more invasive approaches to smear - ve
TB
Distinguish Mtb from NTM in smear +ve cases as
IS6110 sequence is not found in NTM
1048708 Should not be used to replace sputum microscopy
1048708 Sensitivity specificity amp PPV for PCR is 835
99 amp 942 respectively
Am Rev Respir Dis 1991 1441160 J Clin Microbiol 199931 2049-2055
Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
1048708 Limited Role
1048708 No comprehensive large series comparing the yield of
PCR with other available approaches has been published
1048708 But at present it is valuable adjunct in the diagnosis
of TB pleurisy pericardial TB amp other condition in which
yield of other tests are low
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Polymerase Chain Reaction (PCR)
1048708 Essentially PCR is a way to make millions of identical
copies of a specific DNA sequence which may be a
gene or a part of a gene or simply a stretch of
nucleotides with a known DNA sequence the function of
which may be unknown
1048708 A specimen that may contain the DNA sequence of
interest is heated to denature double stranded DNA
Polymerase Chain Reaction (PCR)
1048708 Specific synthetic oligonucleotide primers bind to the
unique DNA sequences of interest and a heat stable
DNA polymerase (Thermus aquaticus) extends the
primer to create a complete amp complimentary strand of
DNA
1048708 This process is repeated sequentially 25-40 times
thereby creating millions of copies of target
sequence
Polymerase Chain Reaction (PCR)
1048708 Kd antigen 65(HSPs)
1048708 Used earlier
1048708 Heat shock protein believed to be distinct from other
bacterial HSPs
1048708 This gene is identical in all species of mycobacteria
1048708 Therefore unsuitable for detecting Mtb particularly in
areas where species like Mavium or Mkansasii are
prevalent
IS6110 1048708 It is a transposon which are
self replicating stretches of DNA
1048708 Function not known
1048708 This sequence has been found
in the Mtb complex organisms
(Mtb Mafricanum Mmicroti
Mbovis)
1048708 IS6110 sequence generally
occurs only once in Mbovis but is
found as often as 20 times in
certain strains of Mtb thus offering
multiple targets for amplification
Polymerase Chain Reaction (PCR)
1048708 With recent modification PCR can detect even a fraction
of a bacilli
Role in pulmonary TB
1048708 Detects nearly all smear +ve and culture +ve cases
1048708 Useful technology for rapid diagnosis of smear ndashve cases
1048708 Able to identify 50-60 of smear -ve cases this would
reduce the need for more invasive approaches to smear - ve
TB
Distinguish Mtb from NTM in smear +ve cases as
IS6110 sequence is not found in NTM
1048708 Should not be used to replace sputum microscopy
1048708 Sensitivity specificity amp PPV for PCR is 835
99 amp 942 respectively
Am Rev Respir Dis 1991 1441160 J Clin Microbiol 199931 2049-2055
Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
1048708 Limited Role
1048708 No comprehensive large series comparing the yield of
PCR with other available approaches has been published
1048708 But at present it is valuable adjunct in the diagnosis
of TB pleurisy pericardial TB amp other condition in which
yield of other tests are low
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Polymerase Chain Reaction (PCR)
1048708 Specific synthetic oligonucleotide primers bind to the
unique DNA sequences of interest and a heat stable
DNA polymerase (Thermus aquaticus) extends the
primer to create a complete amp complimentary strand of
DNA
1048708 This process is repeated sequentially 25-40 times
thereby creating millions of copies of target
sequence
Polymerase Chain Reaction (PCR)
1048708 Kd antigen 65(HSPs)
1048708 Used earlier
1048708 Heat shock protein believed to be distinct from other
bacterial HSPs
1048708 This gene is identical in all species of mycobacteria
1048708 Therefore unsuitable for detecting Mtb particularly in
areas where species like Mavium or Mkansasii are
prevalent
IS6110 1048708 It is a transposon which are
self replicating stretches of DNA
1048708 Function not known
1048708 This sequence has been found
in the Mtb complex organisms
(Mtb Mafricanum Mmicroti
Mbovis)
1048708 IS6110 sequence generally
occurs only once in Mbovis but is
found as often as 20 times in
certain strains of Mtb thus offering
multiple targets for amplification
Polymerase Chain Reaction (PCR)
1048708 With recent modification PCR can detect even a fraction
of a bacilli
Role in pulmonary TB
1048708 Detects nearly all smear +ve and culture +ve cases
1048708 Useful technology for rapid diagnosis of smear ndashve cases
1048708 Able to identify 50-60 of smear -ve cases this would
reduce the need for more invasive approaches to smear - ve
TB
Distinguish Mtb from NTM in smear +ve cases as
IS6110 sequence is not found in NTM
1048708 Should not be used to replace sputum microscopy
1048708 Sensitivity specificity amp PPV for PCR is 835
99 amp 942 respectively
Am Rev Respir Dis 1991 1441160 J Clin Microbiol 199931 2049-2055
Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
1048708 Limited Role
1048708 No comprehensive large series comparing the yield of
PCR with other available approaches has been published
1048708 But at present it is valuable adjunct in the diagnosis
of TB pleurisy pericardial TB amp other condition in which
yield of other tests are low
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Polymerase Chain Reaction (PCR)
1048708 Kd antigen 65(HSPs)
1048708 Used earlier
1048708 Heat shock protein believed to be distinct from other
bacterial HSPs
1048708 This gene is identical in all species of mycobacteria
1048708 Therefore unsuitable for detecting Mtb particularly in
areas where species like Mavium or Mkansasii are
prevalent
IS6110 1048708 It is a transposon which are
self replicating stretches of DNA
1048708 Function not known
1048708 This sequence has been found
in the Mtb complex organisms
(Mtb Mafricanum Mmicroti
Mbovis)
1048708 IS6110 sequence generally
occurs only once in Mbovis but is
found as often as 20 times in
certain strains of Mtb thus offering
multiple targets for amplification
Polymerase Chain Reaction (PCR)
1048708 With recent modification PCR can detect even a fraction
of a bacilli
Role in pulmonary TB
1048708 Detects nearly all smear +ve and culture +ve cases
1048708 Useful technology for rapid diagnosis of smear ndashve cases
1048708 Able to identify 50-60 of smear -ve cases this would
reduce the need for more invasive approaches to smear - ve
TB
Distinguish Mtb from NTM in smear +ve cases as
IS6110 sequence is not found in NTM
1048708 Should not be used to replace sputum microscopy
1048708 Sensitivity specificity amp PPV for PCR is 835
99 amp 942 respectively
Am Rev Respir Dis 1991 1441160 J Clin Microbiol 199931 2049-2055
Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
1048708 Limited Role
1048708 No comprehensive large series comparing the yield of
PCR with other available approaches has been published
1048708 But at present it is valuable adjunct in the diagnosis
of TB pleurisy pericardial TB amp other condition in which
yield of other tests are low
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
IS6110 1048708 It is a transposon which are
self replicating stretches of DNA
1048708 Function not known
1048708 This sequence has been found
in the Mtb complex organisms
(Mtb Mafricanum Mmicroti
Mbovis)
1048708 IS6110 sequence generally
occurs only once in Mbovis but is
found as often as 20 times in
certain strains of Mtb thus offering
multiple targets for amplification
Polymerase Chain Reaction (PCR)
1048708 With recent modification PCR can detect even a fraction
of a bacilli
Role in pulmonary TB
1048708 Detects nearly all smear +ve and culture +ve cases
1048708 Useful technology for rapid diagnosis of smear ndashve cases
1048708 Able to identify 50-60 of smear -ve cases this would
reduce the need for more invasive approaches to smear - ve
TB
Distinguish Mtb from NTM in smear +ve cases as
IS6110 sequence is not found in NTM
1048708 Should not be used to replace sputum microscopy
1048708 Sensitivity specificity amp PPV for PCR is 835
99 amp 942 respectively
Am Rev Respir Dis 1991 1441160 J Clin Microbiol 199931 2049-2055
Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
1048708 Limited Role
1048708 No comprehensive large series comparing the yield of
PCR with other available approaches has been published
1048708 But at present it is valuable adjunct in the diagnosis
of TB pleurisy pericardial TB amp other condition in which
yield of other tests are low
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Polymerase Chain Reaction (PCR)
1048708 With recent modification PCR can detect even a fraction
of a bacilli
Role in pulmonary TB
1048708 Detects nearly all smear +ve and culture +ve cases
1048708 Useful technology for rapid diagnosis of smear ndashve cases
1048708 Able to identify 50-60 of smear -ve cases this would
reduce the need for more invasive approaches to smear - ve
TB
Distinguish Mtb from NTM in smear +ve cases as
IS6110 sequence is not found in NTM
1048708 Should not be used to replace sputum microscopy
1048708 Sensitivity specificity amp PPV for PCR is 835
99 amp 942 respectively
Am Rev Respir Dis 1991 1441160 J Clin Microbiol 199931 2049-2055
Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
1048708 Limited Role
1048708 No comprehensive large series comparing the yield of
PCR with other available approaches has been published
1048708 But at present it is valuable adjunct in the diagnosis
of TB pleurisy pericardial TB amp other condition in which
yield of other tests are low
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Distinguish Mtb from NTM in smear +ve cases as
IS6110 sequence is not found in NTM
1048708 Should not be used to replace sputum microscopy
1048708 Sensitivity specificity amp PPV for PCR is 835
99 amp 942 respectively
Am Rev Respir Dis 1991 1441160 J Clin Microbiol 199931 2049-2055
Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
1048708 Limited Role
1048708 No comprehensive large series comparing the yield of
PCR with other available approaches has been published
1048708 But at present it is valuable adjunct in the diagnosis
of TB pleurisy pericardial TB amp other condition in which
yield of other tests are low
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
1048708 Limited Role
1048708 No comprehensive large series comparing the yield of
PCR with other available approaches has been published
1048708 But at present it is valuable adjunct in the diagnosis
of TB pleurisy pericardial TB amp other condition in which
yield of other tests are low
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Polymerase Chain Reaction (PCR)
Disadvantages
1048708 Very high degree of quality control required
1048708 Variation from lab to lab remain significant
1048708 In pts on ATT PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures
1048708 High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases
1048708 High Cost
1048708 So better understanding of how to use these tests in
conjunction with available clinical information is essential
Am J Respir Crit Care Med 1997155 1804-1854
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
LAMP 1048708 Loop-mediated isothermal amplification
1048708 It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity efficiency and rapidity
1048708 LAMP is used for detection of Mtb complex Mavium
and Mintracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawarsquos medium)
Iwamoto T et al J Clin Microbiol 200341 2616-2619
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
LAMP 1048708 This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA
1048708 Species-specific primers were designed by
targeting the gyrB gene
1048708 Simple procedure starting with the mixing of all
reagents in a single tube followed by an isothermal
reaction during which the reaction mixture is held at
63degC
1048708 60- min incubation time
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
LAMP 1048708 Due to its easy operation without
sophisticated equipment it will be simple
enough to use in
1048708 Small-scale hospitals
1048708 Primary care facilities
1048708 Clinical laboratories in developing countries
Difficulties
1048708 Sample preparation
1048708 Nucleic acid extraction
1048708 Cross-contamination
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
TMA NAA
1048708 Transcription Mediated Amplification (TMA)
1048708 Nucleic Acid Amplification (NAA)
1048708 These techniques use chemical rather than
biological amplification to produce nucleic acid
1048708 Test results within few hours
1048708 Currently used only for respiratory specimens
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Ligase Chain Reaction
1048708 It is a variant of PCR in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands so that they are adjacent to each other
1048708 A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Ligase Chain Reaction 1048708 The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers
1048708 It is mainly being used for respiratory samples and has
a high overall specificity and sensitivity for smear +ve and ndash
ve specimens
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
FAST Plaque TB
1048708 It is an original phage based test
1048708 It uses the mycobacteriophage to detect the presence
of Mtb directly from sputum specimens
1048708 It is a rapid manual test easy to perform and has a
higher sensitivity than microscopy in newly diagnosed
smear +ve pts
Int J Tuberc Lung Dis 19982 160
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
The ldquomagicrdquo Gene Xpert
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Indirect Methods Antibody detection
1048708 TB STAT-PAK
1048708 ELISA
1048708 India test TB
Antigen detection
1048708 TB MPB 64 patch test
1048708 Quantiferon-GOLD test
1048708 Biochemical Assays (ADA Bromide
Partition Gas Chromatography)
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
TB STAT-PAK 1048708 Immuno-chromatographic test
1048708 Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood
plasma or serum
1048708 Its value in disease endemic countries is yet to be
ascertained
Eur Resp J 19958 676
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Antibody detection by ELISA
1048708 Several serodiagnostic tests principally those using
ELISA methodology for measurement of IgG Ab are
available
1048708 38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described but is limited by
the lack of purified Ag
1048708 Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Antibody detection by
ELISA
1048708 Other purified antigens to which
antibodies are detected
1048708 30 Kd protein antigen
1048708 16 Kd heat-shock antigen
1048708 Lipoarabinomannan(LAM) ndash LAM is a
complex glycolipid associated with cell
wall of mycobacteria amp is produced in
substantial quantities by growing Mtb
1048708 A60 antigen
1048708 ES3141 antigen
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Antibody detection by ELISA
1048708 IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult
1048708 Serodiagnosis with crude Ag gives high false
positive results
1048708 These tests lack specificity because polyclonal Ab
are used
1048708 Use of monoclonal antibodies have increased their
specificity
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Antibody detection by ELISA
1048708 It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary TBM)
1048708 Serodiagnosis also has limited utility in smear
negative patients with minimal PTB In pediatric TB amp in
disease endemic countries with high infection rates
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Insta test TB
1048708 It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum
1048708 The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane
Tuberc Lung Dis 19982 541
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
TB MPB 64 patch test 1048708 MPB 64 is a specific mycobacterial antigen for Mtb
complex
1048708 This test becomes +ve in 3-4 days after patch
application and lasts for a week
1048708 Specificity~100 Sensitivity~981
1048708 This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings
Ind J Tuberc Lung Dis 19982 541
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Quantiferon-GOLD 1048708 Due to advances in
molecular biology and
genomics an alternative has
emerged for the first time in the
form of a new class of in vitro assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M tuberculosis antigens
1048708 Measures immune reactivity
to Mtb
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Quantiferon-GOLD 1048708 Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole bloodPBMCs incubated with TB
antigens
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Early assays employed PPD (same specificity problems
as the TST)
1048708 Newer assays (eg QFT-Gold) employ TB-specific
antigens ESAT-6 and CFP-10
1048708 Proteins encoded within the region of difference 1 of Mtuberculosis 1048708 Not shared with the BCG sub-strains and most NTM
(except M kansasii M szulgai M marinum and nonpathogenic Mbovis)
Quantiferon-GOLD
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Improved specificity able to distinguish between TB and
NTM BCG infection
1048708 Studies in contacts HIV infected and children underway
1048708 Recommended for use in ldquoALL circumstances in which the
tuberculin skin test is currently usedrdquo 1048708 Includes contact investigations immigrant evaluation
surveillance (eg healthcare workers)
Mazurek et al MMWR 20055415
Quantiferon-GOLD
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
IGRAs Vs TST 1048708 TST
1048708 In vivo 1048708 Single antigen 1048708 Boosting
1048708 2 patient visits
1048708 Inter-reader variability
1048708 Results in 2-3 days
1048708 Read in 48-72 hrs 1048708
IGRAs
1048708 In vitro 1048708 Multiple antigens
1048708 No boosting
1048708 1 patient visit 1048708 Minimal inter-reader variability
1048708 Results in 1 day
1048708 Stimulate wi 12 hrs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
IGRAs Vs TST 1048708 QFT-g vs TST Agreement = 836
1048708 Factors associated with discordance
ndash Prior BCG ndash Non-tuberculous mycobcateria immune reactivity
ndash Site bias in reading TST
ndash TB Treatment
Mazurek et al JAMA 20012861740
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Biochemical markers of Diagnosis 1048708 Adenosine deaminase (ADA)
1048708 Bromide partition test
1048708 Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid)
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Adenosine Deaminase (ADA) 1048708 It is an enzyme of purine metabolism The level of this
enzyme is 10 times higher in lymphocytes (T cells gtB
cells) than in RBC 1048708 Whenever there is cell mediated immune response to an
antigenic stimuli the ADA levels are the highest
1048708 ADA is measured by the colorimetric method of Giusti
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
enzymatic reaction is Adenosine + H2O + ADA = Inosine + NH3 +ADA
1048708 The amount of ammonia liberated is measured by
the colorimetric method
Cut-off Sensitivity Specificity
Pleural Fluid 50 IUml 95 100
Ascitic Fliud 323 IUml 89 98 CSF 9 IUml 100 100
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Bromide Partition Test 1048708 The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier 1048708 Either by direct chemical measurement or by using an
isotopic tracer the ratio of bromide in serum to that in
CSF can be estimated
1048708 Values lt16 are characteristic of TBM
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
In different studies the sensitivity and specificity of this
test has been found to be near 90
1048708 It may be false +ve in herpes simplex listeria mumps
measles pyogenic meningitis and hypothyroidism
1048708 With the availability of better tests this test has been given up
Taylor J et al J Clin Microbiol 1999 34 56-59
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
Tuberculostearic Acid (TBSA)
1048708 TBSA is found in the cell wall of mycobacterium
1048708 It is identified by gas chromatography or mass
spectrophotometry
1048708 It is a costly investigation and requires complex
analytical equipment (Seldom used)
1048708 Sensitivity gt95Specificitygt99
French M et al J Clin Microbiol 1998 54 987-990
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
CT Scan and MRI Scan in the
diagnosis of TB 1048708 The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB
1048708 CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body
Buxi TBS Indian J Pediatr 200269965-972
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
94
1st-line
oral
bullINH
bullRIF
bullPZA
bullEMB
bull(Rfb)
Injectables
bullSM
bullKM
bullAMK
bullCM
Fluoroquinolones
bullCipro
bullOflox
bullLevo
bullMoxi
bull(Gati)
Oral bacteriostatic 2nd line
Unclear efficacy bullETAPTA
bullPASA
bullCYS
Not routinely recommended
efficacy unknown eg
amoxacillinclavulanic acid
clarithromycin clofazamine
linezolid inmipenemcilastatin
high dose isonizid
Group 1
Group 2
Group 3
Group 4
Group 5
Grouping drugs
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