current prespectives in diagnosis of tuberculosis

8/14/2019 Current Prespectives in Diagnosis of Tuberculosis

1/73


2/73


3/73


4/73

Tuberculosis: Transmission and

Natural History

Infection Initial containment 95%

Late Progression - 5%

Self-Cure 90%

Early Progression - 5%


5/73

Tuberculosis: Global epidemiology

1.7 billion people

8.4 million cases, 1.9 million deaths each year


6/73

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


7/73


8/73

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


9/73


10/73

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


11/73

Testing forM. tuberculosis Infection

Mantoux tuberculin skin test (TST)

QuantiFERON -TB test

QuantiFERON - Gold

T Spot-TB test


12/73

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.


13/73

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.


14/73

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


15/73

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.


16/73

Inability of the PPD in distinguishing active

TB from inactive infection

Active TB

TB contacts

Inactive TB

infection


17/73

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


18/73

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


19/73

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.


20/73

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)


21/73

IFN- assays - mechanism


22/73

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


23/73

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.


24/73

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 .


25/73

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.


26/73

Johnson et al, Clin Diagn Lab Immunol. 1999


27/73



28/73



29/73

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.


30/73

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


31/73


32/73

T SPOT-TB test


33/73


34/73

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.


35/73

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.


36/73

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


37/73

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


38/73

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


39/73

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.


40/73

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


41/73

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


42/73

Cultures

Colonies ofM. tuberculosis growing on media


43/73

Broth Based Culture Methods

BACTECSepticheck AFB

Mycobacterial growth indicator tubesMB/Bac TMyco-ESPculture System II

BacT/ALERT MB Susceptibility Kit


44/73

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.


45/73

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


46/73

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


47/73

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


48/73

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


49/73

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.


50/73

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.


51/73

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.


52/73

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.


53/73

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.


54/73

TK Medium


55/73

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


56/73

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


57/73

NAAs- various types


58/73

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.


59/73

Amplified M.tuberculosis Direct (AMTD2) assay

Catanzaro et al.. JAMA 2000.283:639645.

Bergmann, J. S.1999 J. Clin.Microbiol. 37:14191425.

NAA


60/73

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


61/73

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


62/73

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


63/73

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


64/73

Adenosine Deaminase

T. Sderblom, P. Nyberg, A.M. Teppo, (Eur Respir J,1996, 9, 16521655) on pleural ADA and IFN-,

showed :


65/73

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.


66/73

ADA in CSF

N. Selvakumar, CEREBROSPINAL FLUID ADENOSINEDEAMINASE AND LYSOZYME LEVELSIN THE DIAGNOSIS OF

TUBERCULOUS MENINGITIS, in Ind. J. Tub.:


67/73

IFN- levels

Another test that has received some attention for thediagnosis of pleural and pericardial tuberculosis is

pleural or pericardial fluid IFN-


68/73

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.


69/73

THANK YOU


70/73

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


71/73


72/73

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


73/73

current prespectives in diagnosis of tuberculosis

Documents