Current Prespectives in Diagnosis of Tuberculosis
Transcript of Current Prespectives in Diagnosis of Tuberculosis
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Tuberculosis: Transmission and
Natural History
Infection Initial containment 95%
Late Progression - 5%
Self-Cure 90%
Early Progression - 5%
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Tuberculosis: Global epidemiology
1.7 billion people
8.4 million cases, 1.9 million deaths each year
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Latent TB Infection (LTBI)
Occurs whenperson breathes in bacteria
and it reaches the air sacs (alveoli) of lung
Immune system keeps bacilli contained
and under control
Person is not infectious and has no
symptoms
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LTBI vs. TB Disease
Often infectious before treatmentNot infectious
Symptoms such as cough, fever,
weight loss
No symptoms
A case of TBNot a case of TB
Symptoms smears and cultures
positive
Sputum smears and cultures
negative
Chest x-ray usually abnormalChest x-ray usually normal
Tuberculin skin test reaction usually positive
Tubercle bacilli in the body
TB DiseaseLTBI
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Groups That Should Be Tested for LTBI
(Cont.)
Persons at higher risk for TB disease once infecte
Persons with HIV infection
Persons with certain medical conditions
Persons with a history of inadequatelytreated TB
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Testing forM. tuberculosis Infection
Mantoux tuberculin skin test (TST)
QuantiFERON -TB test
QuantiFERON - Gold
T Spot-TB test
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Montoux
The TST functions by eliciting cell mediated immune responsein previously sensitized individuals.
An intradermal injection of PPD evokes a delayed-typehypersensitivity response mediated by sensitized T cells and
results in cutaneous induration.
PPD is a precipitate of M. tuberculosis culture supernatantwhich contains roughly 200 antigens, many of which areshared by other mycobacteria including many NTM and M bovis
BCG
This cross-reactivity seriously limits the specificity of the TST.
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Sensitivity of theMontoux
On average, 10%-30% of patients do not react totuberculin (sensitivity ~75-90%) False negative rates among persons with confirmed TB vary
across studies: 4%, 17%, 20%, 21%
False negative rates can exceed 50% among patients withdisseminated TB, due to a combination of poor nutrition,poor general health, acute illness, andimmunosuppression.
HIV+ persons may have a limited ability to react totuberculin, even if TB-infected. Reaction is inverselyrelated to immune function.
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Factors that affect the PPD Reaction
Type of Reaction Possible CauseFalse-positive Nontuberculous mycobacteria
BCG vaccinationAnergy
False-negative Recent TB infectionVery young age (< 6 months old)Live-virus vaccination
Overwhelming TB disease
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Montoux contd.
Studies of the prevalence of LTBI in India have yieldedprevalence rates ranging from 9% to more than 80% in variouspopulations.
In India guidelines being followed are:
>10 positive, 20 mm reaction) have greater chance ofdeveloping tuberculosis than those showing 10 mm induration.
Those with less than 5 mm induration have more risk ofdeveloping tuberculosis than those with 6-9 mm induration.
Markowitz N, Hansen NI, Wilcosky TC, et al. Ann Intern Med 1993.
Mayurnath Seth al. Indian J Med Res 1991.
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Inability of the PPD in distinguishing active
TB from inactive infection
Active TB
TB contacts
Inactive TB
infection
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BCG Vaccination and Tuberculin Skin
Test
No reliable way to distinguish tuberculin
skin test reactions caused by bacille
Calmette-Gurin (BCG) vaccine from TB
infection
Evaluate all BCG-vaccinated persons
who have a positive skin test result fortreatment of LTBI
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Need for a New Test
Tuberculin skin test (TST)Was only test for latent TB infection
Wide variation in applying and reading.
False-positive from BCG and non-
tuberculous mycobacteria (NTM)
Boosting from prior TST
False negative results
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Recent advances
Detection of gamma interferon producedby T lymphocytes in response tostimulation with M. tuberculosis antigen
Like the TST, the IFN-assay detectscell-mediated immunity to tuberculin.
TST and QFT are not independent.
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Rationale for T-Cell based approach
M.tuberculosis: intracellular pathogen,
difficult to recover from infected subjects
Humoral responses in M.tuberculosis are
weak
Infection evokes a strong Th1-type cell
mediated immune response (IFN- CMI)
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IFN- assays - mechanism
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QFT vs. TST
in vitro
multiple antigens
no boosting
1 patient visit
minimal inter-reader
variability results in 1 day
in vivo
single antigen
boosting
2 patient visits
inter-reader variability
results in 2 - 3 days
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Antigens used
o QuantiFERON -TB test
PPD is used as antigen
oQuantiFERON
- Gold ESAT-6 and CFP-10 are antigen usedo T Spot-TB test
identifies ESAT-6- or CFP-IO-specificIFN- secreting CD4+ T cells.
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M. tuberculosis-specific antigen
In 1999, by comparative hybridization experiments on a DNAmicroarray led to the identification of a genomic region knownas Region of Difference (RD I).
The gene products of RD I are found only in M. tuberculosis, inpathogenic M bovis strains and in four NTM (M kansasii, Mszulgai, M.flavescens, and M marinum).
Only M kansasii overlaps clinically with M tuberculosis, andbecause M. kansasii infection is uncommon, the RD I regionantigens are essentially specific to M tuberculosis.
Among these antigens are, of course, ESAT-6 and CFP-IO, aswell as MPT-64. ESAT-6
Behr MA et al. Science 1999., Mahairas GG et al. J Bacteriol 1996Harboe M et al Infect Immun 1996 , Philipp WJ et al. . Microbiol 1996 .
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QFT-Gold The improved specificity over the Quanti-FERON-TB assay for PPD
and over the TST was confirmed in a study by Johnson et al, ClinDiagn Lab Immunol. 1999
Of 60 medical students, 0-mm tuberculin skin tests (TSTs) at studyentry
58 (97%) were initially classified as negative for M. tuberculosisinfection by PPD QIFN.
Five months after BCG immunization, 7 of 54 students (13%) had a
TST result of >/=10 mm and 11 of 54 students (20%) tested positiveby PPD QIFN.
ESAT-6- and MPT-64-stimulated IFN-gamma responses in themedical students were negative prior to and after BCGimmunization.
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Johnson et al, Clin Diagn Lab Immunol. 1999
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Johnson et al, Clin Diagn Lab Immunol. 1999
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Johnson et al, Clin Diagn Lab Immunol. 1999
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Mori T et al. . Am J Respir Crit Care Med 2004
Mori and colleagues studied a group of 216 Japanese studentnurses who had no identified risk for M. tuberculosis exposure
All vaccinated with BCG.
In this group 64.6% of the subjects had a TST responsemeasuring 10 mm or more, yielding a specificity of 35.4% forthe TST
The QuantiFERON--TB ESAT-6/CFP-l 0 assay, on the otherhand, yielded a specificity of 98.1 % in this group, far superior
to the TST.
The sensitivity of the assay, 89.0%, was determined in aseparate group of patients who had culture-proven activedisease.
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T SPOT-TB test
Identifies ESAT-6- or CFP-IO-specificIFN- secreting CD4+ T cells
Projected as much more sensitive test ascompared to all other methods
Approved by European Union
Not approved by FDA as yet
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T SPOT-TB test
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Summary- so far
The improved specificity would decreaseunnecessary treatment in those who are not trulyinfected.
The improved sensitivity of these tests over the
TST would capture a cohort of patients whoother-wise would go without treatment of LTBIEsp. important in those who are most likely to
have false-negative TST results, e.g.immunosuppressed individuals.
Longitudinal studies linking positive assays withrisk for development of active disease areongoing and are crucial to demonstrating thetrue role of these tests.
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An ideal test for active tuberculosis
rapid results (available within I day),
high sensitivity and specificity,
low cost, and robustness,
highly automated or easily performed without
the need for excessive sample preparation or
technical expertise,drug-susceptibility data.
distinguish between LTBI and active disease.
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Sputum-based diagnosis
5000 to 10,000 bacilli per milliliter are
required for sputum to be AFB positive
Expectorated sputum is generally the
starting point.
The sensitivity of expectorated sputum
ranges from 34% to 80%
In no way does a negative sputum smear
eliminate the diagnosis of active
tuberculosisMurray PR et al. Ann Intern Med 1980
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Sputum Induction
Nebulisation with hypertonic saline
Described in 1961 by Hensler and
colleagues
In patients who are unable to expectorate or
who had smear-negative sputum samples
Adequate sputum sample in 60-80%
25-45% of which are sputum positive
Parry CM et al. Tuber Lung Dis 1995
Hartung TK et al. S Afr Med J 2002
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Fibro-optic Bronchoscopy
Should be done in patients who are negative on
SI
A study by McWilliams and colleagues
SI had an overall yield of 96.3% after three tests,
confirming the utility of repeated Sls.
The yield of FOB was only 51.9%
McWilliams T et al. Thorax 2002
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FOB and SI - precaution
SI and bronchoscopy are cough-inducing
procedures and generate infectious droplet
nuclei, causing increased exposure to M.
tuberculosis. Ideally, both procedures should be performed
using local exhaust ventilation devices or rooms
that meet the ventilation requirements for TB
isolation and those in attendance should wearrespiratory protection.
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Diagnosis of active tuberculosis
Microscopy: Easiest & quickest test Limited sensitivity(46-78%) but
specificity is virtually 100%* Centrifugation & fluorochrome
staining(auramineO) with UVmicroscopy markedly increasethe sensitivity
ZN staining 10000 bacilli/ml
Fluorochrome staining 1000bacilli/ml
*Chest 1989; 95: 1193
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Traditional Culture
The gold standard method for TB diagnosis
More sensitive & can be positive even when bacterial load is
low(10-100 bacilli/ml)
Required for precise identification of causative organism
Two types of media are used:
Egg based: LJ, Petragnani and ATS
Agar based: Middlebrook 7H10 or 7H11
Growth is slow & takes 6-8 weeks. Thereafter the samelength of time is required for complete identification &
sensitivity testing
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Cultures
Colonies ofM. tuberculosis growing on media
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Broth Based Culture Methods
BACTECSepticheck AFB
Mycobacterial growth indicator tubesMB/Bac TMyco-ESPculture System II
BacT/ALERT MB Susceptibility Kit
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BACTEC
Bactec 460 TB is an automated system that
measures the specific metabolic activity of TB
bacilli using the radiometric method.
Bactec system requires an average of only 8 -
18 days for culture
5 to 8 days for determining drug susceptibility.
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BACTEC
Mean time to detection of mycobacteria in clinical specimens
Average no. of days (range) to detection of:
M. tuberculosis complexCulture methodAll isolates
Smear positive Smear negative
BACTEC 460 TB 11.2 (253)8.0(318)
18(930)
LJ medium 26.8 (747) 28.5 (1629) 36.2 (2841)
Francesca Brunello, Flavio Favari, Roberta Fontana
Journal of Clinical Microbiology, April 1999
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BACTEC
Detection of mycobacteria from clinical specimensaccording to initial smear results
No. (%) of isolates detected by:
Isolate (no. of specimens)
BACTEC 460 TB LJ medium
All smear-positive specimens (107) 107 (100) 107 (100)
All smear-negative specimens (66) 65 (98.4) 59 (89.3)
Smear-positive M. tuberculosis (96) 96 (100) 95 (98.9)
Smear-negative M. tuberculosis (18) 18 (100) 16 (88.8)
Smear-positive NTM (11) 11 (100) 11 (100)
Smear-negative NTM (48) 47 (97.9) 43 (89.5)
Francesca Brunello, Flavio Favari, Roberta Fontana
Journal of Clinical Microbiology, April 1999
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J Clin Microbiol 1999; 37: 748-752
Mycobacterial Growth Indicator Tube
Rapid method
Consists of round bottom tubes containing 4 ml of
modified Middlebrook 7H9 broth which has an
oxygen sensitive fluorescent sensor at the bottom
When mycobacteria grow, they deplete the
dissolved oxygen in the broth & allow the indicator
to fluoresce brightly in a 365nm UV light
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Mycobacterial Growth Indicator Tube
Positive signals are obtained in 10-12 days
MGIT can also be used as a rapid method for
the detection of drug resistant strains of M.tb
directly from acid-fast smear positive samples,
as well as from indirect drug susceptibility
studies.
J Clin Microbiol 1999; 37: 45-48
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The ESP culture system II
Detection of pressure changes within theheadspace above the broth culturemedium in a sealed bottle
The mean time for recovery ofM.tuberculosis complex 15.5
Reliable nonradiometric less labour-
intensive alternative to BACTEC 460system for the growth and detection ofmycobacteria.
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The MB/BacT system
Non-radiometric continuous monitoring system
The system is based on colorimetric detection of
CO2.
Mean time for detection of M.tuberculosis was13.7 days by the MB/BacT system.
Acceptable alternative for BACTEC 460 method
despite some minor disadvantages such as
increased contamination and slightly longer time
for detection of growth.
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The septi-check AFB system
Consists of a paddle enclosed in a plastic tube,One side of the paddle is covered with non-
selective Middlebrook 7H11 agar
The reverse side is divided into two sections: one contains 7H11 agar with para-nitro--acetylamino-
-hydroxypropiophenone (NAP) for differentiation of
M.tuberculosis from other mycobacteria,
the other section contains chocolate agar for detectionof contaminants.
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The septi-check AFB system
This method requires about 3 weeks ofincubation.
The unique advantage of this technique is
the simultaneous detection ofM.tuberculosis, non-tuberculous
mycobacteria (NTM), other respiratory
pathogens and even contaminants.
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TK Medium
The color change in TK Medium is based on
multiple dye indicators
Depends on the metabolites and enzymes
produced by different species ofmicroorganisms.
The color change occurs long before the
colonies become visible. It can also be used for drug-susceptibility testing
Can differentiate a contaminated specimen.
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TK Medium
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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 incommercial use is at least 80% in most studies
Require as few as IO bacilli from a given sample
NAA assays are also quite specific for M.
tuberculosis, with specificity in the range of 98%
to 99%.
Official statement of ATS and CDC, July 1999
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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
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NAAs- various types
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AMPLICOR M. TUBERCULOSIS assay
Cohen, R. A., 1998. Am. J. Respir. Crit. Care Med. 156:156161.
Bonington, A., 1998. J. Clin. Microbiol. 36:12511254.
Al Zahrani, 2000. Am. J. Respir. Crit. Care Med. 162:13231329.
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Amplified M.tuberculosis Direct (AMTD2) assay
Catanzaro et al.. JAMA 2000.283:639645.
Bergmann, J. S.1999 J. Clin.Microbiol. 37:14191425.
NAA
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NAA- summary
Useful technology for rapid diagnosis of smear
negative cases of active TB
Able to identify 50-60% of smear -ve culture +ve
cases Distinguish M.tb from NTM in smear +ve cases
Should not be used to replace sputum microscopy as
an initial screen & culture remains the gold standard
Very high degree of quality control required
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NAA- summary
They are able to detect nucleic acids fromboth living and dead organisms so in pts on
ATT, PCR should not be used as an indicator
of infectivity as this assay remains positive fora 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
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Extra pulmonary tuberculosis
In the case of miliary tuberculosis FOB
may play a significant role
There is clearly a role forNAA assays
The overall sensitivity in nonrespiratory
specimens for the MTD or E-MTD ranges
from 67% to 100%
Much more sensitive in cerebrospinal fluid
than in pleural fluid
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Adenosine Deaminase Levels
In a recent meta-analysis of 40 studies investigatingADA for the diagnosis of tuberculous pleuritis sensitivityequaled specificity at 92.2%
In one study, the sensitivity and specificity were both55%
Trajman A et al. argue that ADA alone is superior to ADAcombined with PCR
1.Goto M et al. Diagnostic value of adenosine deaminase in tuberculous
pleural effusion: a meta-analysis. Ann Clin Biochem 2003; 40(Pt 4):374-81.
2.Nagesh BS et al. Chest 2001
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Adenosine Deaminase
T. Sderblom, P. Nyberg, A.M. Teppo, (Eur Respir J,1996, 9, 16521655) on pleural ADA and IFN-,
showed :
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ADA in CSF
ADA may be of limited value in diagnosing tuberculousmeningitis.
There is consensus neither regarding cut-off value norabout sensitivity or specificity.
In one study, Ribera E et al analyzed that the meanenzyme value was clearly higher for the patients withtuberculous meningitis (15.7 +/- 4.3 U/liter) than for theother patients (1.4 +/- 1.5 U/liter).
The sensitivity of the test for diagnosing tuberculous
meningitis was 100% and specificity, 99%. Rohani MY et al reported specificity of 87.6% and a cut-
off value of 9
Ribera E et al .Activity of adenosine deaminase in cerebrospinal fluid, J Infect Dis.
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ADA in CSF
N. Selvakumar, CEREBROSPINAL FLUID ADENOSINEDEAMINASE AND LYSOZYME LEVELSIN THE DIAGNOSIS OF
TUBERCULOUS MENINGITIS, in Ind. J. Tub.:
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IFN- levels
Another test that has received some attention for thediagnosis of pleural and pericardial tuberculosis is
pleural or pericardial fluid IFN-
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To Conclude..
Diagnostic testing for tuberculosis remained unchangedfor nearly a century, but newer technologies hold thepromise of a time revolution in tuberculosis diagnostics.
The IFN- release and T-cell-based assays may well
supplant the TST in diagnosing LTBI in much of theworld. NAA assays are proving their worth in more rapidly
diagnosing both pulmonary and extrapulmonarytuberculosis with great sensitivity and specificity.
These tests are likely to play an ever-increasing role inthe coming years.
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THANK YOU
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How QFT Is Performed
Culture overnight at 37Culture overnight at 37ooCC
TB infected individualsTB infected individuals
respond by secreting IFN-respond by secreting IFN-
Heparinized whole bloodHeparinized whole blood
AvianAvianPPDPPD
TbTbPPDPPD
MitogenMitogenControlControl
Transfer undiluted whole bloodTransfer undiluted whole blood
into wells of a culture plateinto wells of a culture plate
and add antigensand add antigens
Harvest plasma from aboveHarvest plasma from above
settled cells and incubatesettled cells and incubate
60 min in Sandwich ELISA60 min in Sandwich ELISA
Wash, add substrate,Wash, add substrate,
incubate 30 minincubate 30 min
then stop reactionthen stop reaction
TMBTMB
COLORCOLOR
IFN- IU/mlOD450nm
OD450nm
Standard CurveStandard Curve
Measure OD,Measure OD,
determine IFN-determine IFN- levelslevelsand interpret testand interpret test
Stage 1 Whole Blood CultureStage 1 Whole Blood Culture
Stage 2 IFN-Stage 2 IFN- ELISAELISA
NilNilControlControl
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M. tuberculosis-specific antigen Harboe and colleagues [1986] reported the first M. tuberculosis-
specific antigen,MPB-64 (later known as MPT-64).
Andersen and colleagues [1995] reported the highly immunogenic
antigen target of the cellular immune response to tuberculosis in
mice, known as the early secreted antigenic target 6 (ESAT-6).
Berthet and colleagues [1998] described culture filtrate protein
(CFP-IO)
In 1998, the complete genome sequence of M tuberculosis wasdetermined
Harboe M et al. Infect Immun 1986.,Andersen P, et al. J Immunol 1995
Berthet FXet al. Microbiol 1998
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