Transcript molecular diagnosis of tb and igra

MOLECULAR
DIAGNOSIS OF TB
AND IGRA
PRESENTED BY :
Dr. Kiran N.
PG Student
Chest & Tuberculosis
Govt. Medical College, Patiala
Molecular Diagnostics Why?
 Detection and Diagnosis
- uncultivable
-
or
difficult
to
culture
need for rapid diagnosis
inadequacy
of phenotypic
methods (biochemical)
Prognosis and management
need for quantitative
information (bacterial load)
susceptibility testing (drug
resistance) without culture
- Molecular resistance testing
METHODS OF DIAGNOSIS OF PULMONARY
TUBERCULOSIS
1)DIRECT METHODS: Detects mycobacteria and
its products
2)INDIRECT METHODS : Antigen and Antibody
Detection
3)RADIO-DIAGNOSIS :CXR,CT AND MRI
DIRECT METHODS
1)Direct Microscopy -ZN stain, Kinyoun,
Flurochrome.
2)Culture -Traditional, Rapid methods.
3) Detection of DNA or RNA of mycobacterial origin
i,e (molecular methods) includes - PCR, LAMP,
TAA / NAA, LCR, Fast Plaque.
INDIRECT METHODS
1)Antibody detection :
 TB STAT-PAK
 ELISA
 Insta test TB
2) Antigen detection :
 TB MPB 64 patch test.
 Quantiferon-GOLD test.
3) Biochemical Assays :
 ADA
 Bromide Partition
 Gas Chromatography
MOLECULAR DIAGNOSIS OF TUBERCULOSIS
 Rapid and sensitive tools for the diagnosis of tuberculosis
are needed, due to the increased incidence of tuberculosis
epidemics and the length of time required by classical
diagnostic tests, especially among human immunodeficiency
virus (HIV)-infected patients. In this context, the recent
advances in cloning and characterization of M. tuberculosis
genes has allowed the application of basic molecular biology
techniques to the examination of clinical samples, such as
sputum and bronchoalveolar lavage (BAL), for the molecular
diagnosis of tuberculous infection. By using the polymerase
chain reaction (PCR) for the amplification of mycobacterial
nucleic acids and nonradiometric revelation techniques, the
time required for the identification of mycobacteria has been
considerably shortened (24-48 h), in comparison to the time
required by microbiological tests
Dr.T.V.Rao MD
6
MOLECULAR TESTS FOR DETECTION OF
NUCLEIC ACIDS
 The majority of molecular tests have been focused on
detection of nucleic acids, both DNA and RNA, that are
specific to Mycobacterium tuberculosis, by amplification
techniques such as polymerase chain reaction (PCR); and
detection of mutations in the genes that are associated
with resistance to anti tuberculosis drugs by sequencing or
nucleic acid hybridization. Recent developments in direct
and rapid detection of mycobacteria, with emphasis on M.
tuberculosis species identification by 16S rRNA gene
sequence analysis or oligohybridization and strain typing,
as well as detection of drug susceptibility patterns, all
contribute to these advance
Polymerase Chain Reaction
PCR was invented by Kary Mullis in 1983 (Nobel Prize for
Chemistry in 1993)
• PCR is a technique that takes a small amount of a
specific DNA sequence and amplifies for further
testing.
• It`s like a “molecular photocopier”.
Polymerase Chain Reaction (PCR)
 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.
 A specimen that may contain the DNA sequence of
interest is heated to denature double stranded DNA.
 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 & complimentary
strand of DNA.
This process is repeated sequentially 25-40 times,
thereby creating millions of copies of target
sequence. The amplified sequence can then be
detected by agarose gel electrophoresis.DNA
sequence used include:
1) 65 Kd antigen (HSPs):
 Used earlier
 Heat shock protein believed to be distinct from other
bacterial HSPs.
 This gene is identical in all species of mycobacteria.
 Therefore unsuitable for detecting M.tb, particularly
in areas where species like M.avium or M.kansasii
are prevalent.
2) IS6110 :
 It is a transposon which are
 self replicating stretches of DNA.
 Function not known.
 This sequence has been found in the M.tb complex
organisms (M.tb, M.africanum, M.microti, M.bovis).
 IS6110 sequence generally occurs only once in
M.bovis but is found as often as 20 times in certain
strains of M.tb, thus offering multiple targets for
amplification PCR can detect even a fraction of a
bacilli.
Mycobacterium tuberculosis
genome
Dr.T.V.Rao MD
12
Ingredients of Polymerase Chain Reaction
•
Primers: µM 0.1-0.5
•
Deoxy-nucleotides triphosphate (dNTPs):
µM 200-250nucleotides
•
Co-factors:
1. Cations: MgCl2 mM 1.5-6
2. Buffer pH 8.3-8.8
•
DNA polymerase: 0.5-2.5 U
•
Target DNA:  1 µg
Chromosome and
Desoxyribo-Nucleic-Acid
Chromosom
chromosome
Zellkern
nucleus
Zelle
cell
Basenpaare
base
pairs
DNA
DNA double
Doppelhelix
helix
Primers
• Primers are target sequence specific oligonucleotides
that serve as template for the DNA polymerase
• Forward and reverse primers flanking the target
sequence allow both DNA strands to be copied
simultaneously in both directions.
Three steps of PCR: Denaturation,
annealing and extension
Role of PCR in pulmonary TB :
 Detects nearly all smear +ve and culture +ve cases.
 Useful technology for rapid diagnosis of smear –ve cases
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of active TB.
Able to identify 50-60% of smear -ve cases; this would
reduce the need for more invasive approaches to smear ve cases
Distinguish M.tb from NTM in smear +ve cases as IS6110
sequence is not found in NTM.
Should not be used to replace sputum microscopy.
Sensitivity, specificity, & PPV for PCR is 83.5%, 99% &
94.2% respectively
Role in Extrapulmonary TB
 Limited Role
 No comprehensive large series comparing the yield
of PCR with other available approaches has been
published. But at present, it is valuable adjunct in
the diagnosis of TBM, pleurisy, pericardial TB & other
 condition in which yield of other tests are low
Disadvantages
 Very high degree of quality control required.
 Variation from lab to lab remain significant.
 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
 High false +ve results in patients previously treated
with ATT in contacts of sputum +ve active cases.
 High Cost
Real Time PCR replacing older
Methods
LAMP*
 Loop-mediated isothermal amplification.
 It is a novel nucleic acid amplification method in which reagents
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react under isothermal conditions with high specificity, efficiency,
and rapidity.
LAMP is used for detection of M.tb complex, M.avium, and
M.intracellulare directly from sputum specimens as well as for
detection of culture isolates grown in a liquid medium (MGIT) or on
a solid medium (Ogawa’s medium).
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.
Species-specific primers were designed by targeting the gyrB gene.
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 63°C.
60-min incubation time.
ADVANTAGES:
Due to its easy operation without sophisticated
equipment, it will be simple enough to use in:
 Small-scale hospitals,
 Primary care facilities
 Clinical laboratorie in developing countries.
Difficulties :
 Sample preparation
 Nucleic acid extraction
 Cross-contamination
TMA / NAA
 Transcription
Mediated Amplification (TMA) /
Nucleic Acid Amplification (NAA).
 These techniques use chemical rather than
biological amplification to produce nucleic acid.
 Test results within few hours.
 Currently used only for respiratory specimens.
Nucleic acid amplification
assays
 NAA assays amplify M. tuberculosis-specific nucleic
acid sequences using a nucleic acid probe.
 The sensitivity of the NAA assays currently in
commercial use is at least 80% in most studies
 Require as few as bacilli from a given sample
 NAA assays are also quite specific for M.
tuberculosis, with specificity in the range of 98% to
99%.
NAAs- various types
 AMPLICOR M. TUBERCULOSIS assay
 Amplified M.tuberculosis Direct (AMTD2) assay
 LCx MTB assay, ABBOTT LCx probe system
 BD ProbeTec energy transfer (ET) system (DTB)
 INNO-LiPA RIF.TB assay
NAAs- various types
Dr.T.V.Rao MD
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NAA- Limitations
 They are able to detect nucleic acids from both
living and dead organisms so in pts on ATT, PCR
should not be used as an indicator of infectivity
as this assay remains positive for a greater time,
than do cultures
 A major limitation of NAA tests is that they give
no drug-susceptibility information.
 NAA should always be performed in conjunction
with microscopy and culture
DISADVANTAGES :
1) Poor sensitivity in smear –ve samples
2) Labour intensive
3) Highly trained personnel required & dedicated
labour space because it requires atleast 3 separate
rooms to avoid cross contamination. Hence suitable
only for IRL.
Ligase Chain Reaction
 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.
 A second pair of oligonucleotides is designed to hybridize to
the same regions on the complementary DNA.
 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.
 It is mainly being used for respiratory samples, and has a high
overall specificity and sensitivity for smear +ve and –ve
specimens.
MDR TB
 MDR-TB is defined as resistance to isoniazid and rifampicin,
with or without resistance to other first line drugs (FLD).
 In the Global TB Report 2011, WHO estimated that among the
1.5 million RNTCP-notified cases of pulmonary TB in India in
2010, approximately 64,000 cases of MDR TB could be
diagnosed.
 MDR TB is important because patients with this type of drug
resistance respond extremely poorly to standard anti-TB
treatment with first-line drugs. MDR TB requires relatively
costly laboratory diagnosis and treatment for at least twoyears with drugs that are expensive, toxic, and not particularly
potent.
 A case of MDR TB is about 20-40 times more expensive to
manage than a case of drug-sensitive TB, and patient suffering
is magnified.
MDR Suspect Criteria
Criteria A• All features of new TB cases.
• Smear +ve previously treated cases who remain smear +ve at 4th
month onwards.
• All pulmonary TB cases who are contacts of known MDR TB case.
Criteria B – in addition to Criteria A :
• All smear +ve previously treated pulmonary TB cases at diagnosis.
• Any smear +ve follow up result in new or previously treated cases.
Criteria C – in addition to Criteria B :
• All smear –ve previously treated pulmonary TB cases at diagnosis.
• HIV TB co-infected cases at diagnosis.
Molecular methods for drug
resistance
 Rifampin (RIF)
 – Binds to β subunit of RNA polymerase (rpoB)
 – 96% of resistant Mtb isolates have mutations in 81-bp

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
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
region.
– Four (4) mutations . 75% of resistant clinical isolates
• Isoniazid (INH) . two genes
– katG and inhA . 75-85%
• Pyrazinamide . pncA . 70%
• Streptomycin . rpsL . 65-75%
• Ethambutol .embB . 70%
Causes of Drug Resistance in TB
 Lack of understanding of why long term therapy
with multiple drugs is necessary
 Non-adherence to therapy by patients
 Incorrect drug prescribing by providers
 Poor quality drugs
 Erratic supply of drugs
 Malabsorption of drugs primarily due to
symptoms of HIV/AIDS
Development of drug resistance in
M. tuberculosis
 The mycobacterial cell is surrounded by a specialized, highly
hydrophobic cell wall that results in decreased permeability
to many antimicrobial agents.
 Resistance of M tuberculosis to antimycobacterial drugs is
the consequence of naturally occuring, spontaneous
mutations in genes that encode either the target of the drug,
or enzymes that are involved in drug activation.
 Resistance-associated mutations have been described for all
first-line drugs (isoniazid, rifampin, pyrazinamide,
ethambutol, and streptomycin).
Development of multi-drug
resistant tuberculosis
 No single genetic alteration has yet been found that
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results in the MDR phenotype (defined as resistance at
least to INH and RMP).
MDR develops by sequential acquisition and selection of
mutations at different loci, usually because of
inappropriate patient treatment.
Inappropriate treatment may lead to disease progression.
Disease progression will increase the bacterial load and
the risk of naturally occurring mutations.
Because MDR strains are the result of cumulative
mutations, growth of M tuberculosis can successfully be
controlled in the host by concomitant treatment with
more than one drug.
Thus, treatment regimens that consist of three to four
drugs are used routinely to treat patients with
tuberculosis.
Isoniazid resistance and katG
 INH is a pro-drug that requires
Porin
Lipoarabinomannan
(LAM)
Mycolic
Acids
INH
Arabinogalactan
Cell Wall and cytoplasmic membrane
Free Wall Glycolipids
and Proteins



Cytoplasm
PZA
Short chain f atty acid precursors
Chromosomal DNA
RNA
Polymerase (ß-subunit)
RNA Transcription

RMP

activation in INH-susceptible
mycobacterial species.
the activation of INH results in a number
of highly reactive compound that are
capable of damaging the mycobacterial
cell wall.
INH-resistant clinical isolates frequently
loose their catalase-peroxidase activity
(Middlebrook et al., 1954)
Association of this enzyme with INH
activation was proven when the
mycobacterial catalase-peroxidase gene
(katG) was cloned and sequenced.
(Zhang et al., 1992)
Mutations in this gene were found in 7080% of high INH-resistant clinical
isolates.
The most common mutation that was
found was the Ser315Thr mutation.
Isoniazid resistance and virulence
 The Ser315Thr mutation results
in an enzyme without the
ability to activate INH, but
retains approximately 50% of
its catalase-peroxidase activity.
Lysosomes
with free oxigene radicals
Phago-lysosomes with
inactivated MTB
 This altered catalase-
peroxidase provides high-level
resistance to INH, while
retaining a level of oxidative
protection against host
antibacterial radicals.
 Isolates that carry other,
mutations in katG are
exhibiting varying levels of
INH-resistance and catalaseperoxidase activity.
MTB
Phagosome
with viable MTB
Isoniazid resistance and inhA
 INH blocks the synthesis of cell-


Porin
Lipoarabinomannan
(LAM)
Mycolic
Acids
INH
Arabinogalactan
PZA
Cytoplasm

Free Wall Glycolipids
and Proteins
Short chain f atty acid precursors
Chromosomal DNA
RNA
Polymerase (ß-subunit)
RNA Transcription
RMP
Cell Wall and cytoplasmic membrane

wall mycolic acids, the major
components of the envelope of
M tuberculosis.
One intracellular target of the
drug is fatty-acid enoyl-acyl
carrier protein reductase (InhA).
(Basso et al., 1998)
This enzyme is involved in
synthesis of mycolic acids.
Mutations in the promoter
region of the gene (inhA)
encoding this enzyme result in
over-expression of the protein.
The over-expressed enzyme
may counter-balance the effect
of INH and will result in a lowlevel resistance to the drug.
Rifampin resistance
 One of the main reasons for


Porin
Lipoarabinomannan
(LAM)
Mycolic
Acids
INH
Arabinogalactan
PZA
Cytoplasm

Free Wall Glycolipids
and Proteins
Short chain f atty acid precursors
Chromosomal DNA
RNA
Polymerase (ß-subunit)
RNA Transcription
RMP
Cell Wall and cytoplasmic membrane

treatment failure and fatal clinical
outcome in tuberculosis patients is
resistance to RMP.
RMP exhibits a significant early
bactericidal effect on metabolically
active M tuberculosis, and excellent
late sterilizing action on
semidormant organisms
undergoing short bursts of
metabolic activity.
While monoresistance to INH is
common, monoresistance to RMP
is rare.
RMP resistance occurs most often
in strains that are also resistant to
INH, thus, RMP resistance can be
used as a surrogate marker for
MDR.
RMP inhibits mycobacterial
transcription by targeting DNAdependent RNA polymerase.
Rifampin resistance and rpoB
•Resistance to RMP is due to mutations in a well-defined, 81
base pair (27 codons) central region of the gene that encodes
the β-subunit of RNA polymerase (rpoB).
•More than 96% of the rifampin-resistant strains contain a
mutation in this 81 bp region of rpoB.
•The most common mutations (65–86%) alter either codon
526 or codon 531, and result in high-level resistance to RMP.
•Alterations in other codons result in low-level resistance.
•Rare mutations associated with rifampin resistance have
also been found in the amino-terminal region of rpoB.
MOLECULAR METHODS OF DIAGNOSING
DRUG RESISTANT TB
 Phenotypic methods: in liquid/solid media
 Genotypic methods: Gene xpert/LPA
 PHENOTYPIC METHODS
1)Commercial methods
 Done on liquid or solid media
 Can be direct/indirect type.
 Indirect type has 3 variants: absolute
concentration/resistant ratio/proportion method
 Can be used for 1st line/2nd line DST
2)Non commercial methods
 Less expensive than commercial systems but are
prone to errors due to lack of standardization and
local variations in methodology. These include
a)MODS (microscopic observation drug
susceptibility) a microcolony method in liquid
culture(middlebrook 7H9 broth) based on
inoculation of specimens into drug-free and drugcontaining media, followed by microscopic
examination of early growth Recommended as direct
or indirect tests for rapid screening of patients
suspected of having MDR-TB.
b)COLORIMETRIC METHODS
Indirect testing methods based on the
reduction of a coloured indicator added to
liquid culture medium on a microtitre plate
after exposure of M. Tuberculosis strains to
anti-TB drugs in vitro. Recommended as
indirect tests on M. tuberculosis isolates
from patients suspected of having MDR-TB,
although the time to detection of MDR is not
faster
(but
less
expensive)
than
conventional DST methods with commercial
liquid culture or molecular LPA. The
indicators that have been used to date
include tetrazolium salts (XTT and MTT),
Alamar blue and resazurin.
c)NITRATE REDUCTION ASSAY (NRA)
A direct or indirect method
on solid culture based on the
ability of M.tuberculosis to reduce
nitrate, which is detected by a
colour reaction Recommended as
direct or indirect tests for
screening patients suspected of
having MDRTB, although the
time to detection of MDR in
indirect application is not faster
than conventional DST methods
with liquid culture.
GENOTYPIC METHODS
LPA
Gene Xpert /RIF
LPA(Line Probe Assay)
It is a genotypic method use PCR and
reverse hybridization with specific
oligonucleotide probes fixed to
nitrocellulose strips in parallel lines
therefore often reffered to as “strip tests”.
which detects resistance to
Both H&R
 Sensitivity for R is 97%, H is 90%
 Specificity for R& H is 99%
 Results within 48 hours
Differences
Hain GenotType
MTBDRplus
InnoLiPA Rif
• It targets 23s rRNA
gene space region.
• It targets 16s - 23s
rRNA gene space
region.
• It detects both R
and H resistance.
• It detects only R
resistance.
TB Molecular Identification
AVAILABLE SYSTEMS
(Commercial):
 Hain GenotType
MTBDRplus
ADVANTAGES of
molecular testing:
 Rapid results
 Specific
information

InnoLiPA Rif
 Can be done in
presence of
contaminants
 Less biohazard
risk involved
DISADVANTAGES of
molecular testing:
 Expensive
 Dedicated
equipment
 Technical
expertise required
Rapid molecular detection of INH and RMP
resistance: Line – Probe Assay Methodology
DNA isolated from processed original specimen or
from cultured bacterial cells
2. Multiplexed amplification (PCR) of mycobacterial
DNA using biotin labeled primers
3. Denaturation of the amplified DNA products into
single-strands
4. Hybridization of the denatured DNA to probes on a
membrane strip
5. Enzyme-mediated detection of bands where DNA
products have bound to strip
1.
Polymerase Chain Reaction
MTBDR plus strip for Identification of MTB complex
and resistance to RMP and/or INH
Rifampicin resistance: rpoB gene
rpoB WT2
rpoB WT1
505
508 509
rpoB WT3
511
rpoB WT4
rpoB WT5
513 514 515 516
518
rpoB MUT1 (D516V)
rpoB WT6
rpoB WT7
522
526
rpoB WT8
531
533
rpoB MUT2B (H526D)
rpoB MUT3 (S531L)
• rpoB wild type probes: WT 1 to WT 8
• rpoB mutation-specific probes: MUT D516V, H526Y, H526D, S531L
Detection of mutations:
• missing of wildtype signals
• presence of common mutation-specific signals
High-level Isoniazid resistance: katG gene
Mutations in katG and the corresponding wild type and
mutation probes
missing wild type probe
analyzed codon
katG WT
315
mutation probe
katG MUT1
katG MUT2
mutation
S315T1
S315T2
High-level Isoniazid resistance: katG gene
Mutations in katG and the corresponding wild type and
mutation probes
missing wild type probe
analyzed codon
katG WT
315
mutation probe
katG MUT1
katG MUT2
mutation
S315T1
S315T2
High-level Isoniazid resistance: katG gene
Mutations in katG and the corresponding wild type and
mutation probes
missing wild type probe
analyzed codon
katG WT
315
mutation probe
katG MUT1
katG MUT2
mutation
S315T1
S315T2
Low-level Isoniazid resistance: inhA gene
Mutations in the inhA promotor region and the
corresponding wild type and mutation probes
Xpert MTB/RIF assay
(CBNAAT-cartridge based
NAA test)
 It is an automated PCR diagnostic
test that can detect presence of
M.tb & resistance to RIFampicin(by
detecting mutation in 81bp region)
 It is automatic,fast & sensitive
 Accurate diagnosis is obtained in
1hr 45 mins by adding a reagent to
the sputum sample & 15mins later
its pippeted into a cartridge that is
inserted into the diagnostic
instrument
Xpert MTB/RIF
 Dual
PCR
reactions—
sample-processing PCR is
followed by hemi-processing
PCR —increase the test’s
sensitivity and specificity:
according to the results
published in NEJM, the PCR
test was 98.2% sensitive in
patients
with
smearpositive, culture-positive TB.
And,
because
it
is
automated, there is little
technical training needed to
administer the test.
Other molecular methods include:
3) DNA microarrays
 Based on the principle of hybridization
 Detect Rifampicin resistance only
 Analysis large amounts of DNA sequences
4)Molecular beacons
They are nucleic acid hybridization probes d desinged to bind to
target DNA sequences in regions such as rpoB ,where
resistance mutations are known to occur.
• Sensitive enough to detect 2 bacilli,
• Results in 3 hours
5)Single-strand conformation polymorphism
(SSCP)
6)Fluorescence resonance energy transfer (FRET)
probes
7)Flow cytometry
CONCLUSION:
LPA is applicable on sputum+ve specimens
only.Gene Xpert cant monitor progress of
treatment. HENCE PHENOTYPIC CULTURE
REMAINS THE GOLD STANDARD
Interferon-gamma release assays for
latent tuberculosis infection
 They are surrogate markers of Mycobacterium tuberculosis infection
and indicate a cellular immune response to M. tuberculosis.
 IGRAs cannot distinguish between latent infection and active
tuberculosis (TB) disease, and should not be used for diagnosis of active
TB, which is a microbiological diagnosis.
 A positive IGRA result may not necessarily indicate active TB, and a
negative IGRA result may not rule out active TB.
 IGRAs are not affected by Bacille Calmette-Guérin (BCG) vaccination
status, IGRAs are useful for evaluation of LTBI in BCG-vaccinated
individuals, particularly in settings where BCG vaccination is
administered after infancy or multiple (booster) BCG vaccinations are
given.

IGRAs appear to be unaffected by most infections with environmental
nontuberculous mycobacteria
Blood Assays for M.
tuberculosis
 QuantiFERON®-TB Gold In-Tube
(Cellestis Ltd, Victoria, Australia)
 Measures Interferon-gamma (IFN-y)
 T-SPOT.TB
(Oxford Immunotec Ltd, Oxford, UK)
 Measures peripheral blood
mononuclear cells that produce IFN-γ
QuantiFERON-TB Gold In-Tube assay
(Cellestis Ltd, Australia)
 The QuantiFERON-TB Gold In-Tube assay
(QFT-GIT), which has replaced the
QuantiFERON-TB Gold assay, detects the
level of IFN-γ produced in response to the M.
tuberculosis antigens ESAT-6, CFP-10, and
TB7.7, and uses the enzyme-linked
immunosorbent assay (ELISA) detection
method. This is an indirect measure of the
presence of M.tuberculosis specific T-cells.
T-SPOT.TB assay
(Oxford Immunotec, UK)
 The T-SPOT.TB measures the number
of IFN-γ producing T-cells in response
to the M. tuberculosis antigens ESAT6 and CFP-10, and is based on the
enzyme-linked immunosorbent spot
(ELISPOT) assay.
CHARACTERISTICS OF THREE TESTS FOR LTBI
TST
QFT-GOLD InTube
ELISA-based
T-Spot.TB
Administration
In vivo (intradermal)
Ex vivo
Ex-vivo
based
Elispot
Antigens
PPD-S or RT-23
ESAT-6 + CFP-10
+/- TB 7.7
ESAT-6 + CFP-10
Standardized
Mostly
Yes
Yes
Units of measurement
Millimetres of
induration
International units of
IFN-G
IFN-G spot-forming
cells(SFC)
Definition of positive
test
5,10,15 mm
Patient’s IFN-G>0.35
suIU/ml (after subtracting
IFN-G response in nil
control)
>6 SFC in the antigen
wells,with 250,000
cells/well and atleast
double negative well
Inderminate
If anergy (rarely
tested )
Poor response to mitogen
(<0.5 IU/ml in positive
control) or high background
response (>8.0 IU/ml in nil
well)
Poor response to mitogen
(<20 SFC in positive
control well) or high
background (>10 SFC in
negative well)
Time to result
48-72 hrs
16-24 hrs (but longer if run
in batches)
16-24 hrs (but longer if run
in batches)
Cost per test
$12.73
$41
$85
COMPARISION OF TST AND IGRAs
TST
IGRA
Estimated sensitivity in patients with active TB
75-90%
75-95%
Estimated specificity in healthy individuals with no
known TB disease or exposure
70-95%
90-100%
Cross reactivity with BCG
Yes
Less likely
Cross reactivity with NTM
Yes
Less likely but limited evidence
Association between test-positivity and subsequent
risk of active TB during follow- up
Moderate to strong
positive association
Insufficient evidence
Correlation with Mycobacterium tuberculosis
exposure
Yes
Yes(better than TST)
Benefits of treating test-positives based on RCTs
Yes
No evidence
Reliability and reproducibility
Moderate and
variable
Limited evidence but appears
high
Boosting phenomenom
Yes
No
Potential for conversions and reversions
Yes
Insufficient evidence
Adverse reactions
Rare
Rare
Material costs
Low
High
Patient visits
Two
One
Laboratory infrastructure required
No
yes
Time to obtain results
2 to 3 days
1 to 2 days
Trained personnel required
Yes
yes