Current Perspective in

Diagnosis of Tuberculosis

Rajiv Singla

Moderator: Dr. M.K.Daga

WHO TB fact sheet 2005
 2 billion people ,equal to a third of world's population, are
infected with TB bacilli.
 2 million people die every year due to TUBERCULOSIS
 TB is curable but kills 5000 people everyday.
 98% of TB deaths are in developing world affecting
mostly young adults in their most productive years
 Global TB incidence is still growing at 1% a year .
 TB is a worldwide pandemic; though the highest rates per
capita are in Africa, half of all new cases are in 6 asian
countries(Bangladesh, China, India, Indonesia, Pakistan,
Philippines)
Tuberculosis: Transmission and
Natural History

Self-Cure – 90%

Infection Initial containment – 95%

Early Progression - 5%
Late Progression - 5%
Tuberculosis: Global epidemiology

1.7 billion people

8.4 million cases, 1.9 million deaths each year

 Latent TB Infection (LTBI)

• Occurs when person 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
LTBI vs. TB Disease

LTBI TB Disease
Tubercle bacilli in the body
Tuberculin skin test reaction usually positive
Chest x-ray usually normal Chest x-ray usually abnormal
Sputum smears and cultures Symptoms smears and cultures
negative positive
No symptoms Symptoms such as cough, fever,
weight loss
Not infectious Often infectious before treatment
Not a case of TB A case of TB
Groups That Should Be Tested for
LTBI
Persons at higher risk for exposure to TB
Close contacts of a person known or
suspected
to have TB

Residents and employees of high-risk

congregate settings

Health care workers (HCWs) who serve

high-
risk clients
Groups That Should Be Tested for LTBI
(Cont.)

Persons at higher risk for TB disease once infected

Persons with HIV infection

Persons with certain medical conditions

Persons with a history of inadequately

treated TB
Testing for M. tuberculosis Infection

Mantoux tuberculin skin test (TST)

QuantiFERON® -TB test

QuantiFERON® - Gold

T Spot-TB test
Montoux
 The TST functions by eliciting cell mediated immune response
in previously sensitized individuals.

 An intradermal injection of PPD evokes a delayed-type

hypersensitivity response mediated by sensitized T cells and
results in cutaneous induration.

 PPD is a precipitate of M. tuberculosis culture supernatant

which contains roughly 200 antigens, many of which are
shared by other mycobacteria including many NTM and M bovis
BCG

 This cross-reactivity seriously limits the specificity of the TST.

Sensitivity of the Montoux
 On average, 10%-30% of patients do not react to
tuberculin (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 with

disseminated TB, due to a combination of poor nutrition,
poor general health, acute illness, and
immunosuppression.

 HIV+ persons may have a limited ability to react to

tuberculin, even if TB-infected. Reaction is inversely
related to immune function.
 Factors that affect the PPD Reaction

Type of Reaction Possible Cause

False-positive Nontuberculous mycobacteria
BCG vaccination
Anergy
False-negative Recent TB infection
Very young age (< 6 months old)
Live-virus vaccination
Overwhelming TB disease
Montoux – contd.
 Studies of the prevalence of LTBI in India have yielded
prevalence rates ranging from 9% to more than 80% in various
populations.

 In India guidelines being followed are:

>10 positive, <6 negative, 6-9 doubtful.

 Strong reactors(i.e. >20 mm reaction) have greater chance of

developing tuberculosis than those showing 10 mm induration.

 Those with less than 5 mm induration have more risk of

developing 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.
Inability of the PPD in distinguishing active
TB from inactive infection

TB contacts
Active TB
Inactive TB
infection
BCG Vaccination and Tuberculin Skin
Test
 No reliable way to distinguish tuberculin
skin test reactions caused by bacille
Calmette-Guérin (BCG) vaccine from TB
infection

 Evaluate all BCG-vaccinated persons

who have a positive skin test result for
treatment of LTBI
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

Recent advances
 Detectionof gamma interferon produced
by T lymphocytes in response to
stimulation with M. tuberculosis antigen

 “Likethe TST, the IFN-γ assay detects

cell-mediated immunity to tuberculin.”
 TST and QFT are not independent.
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-γ CMI)
IFN-γ assays - mechanism
QFT vs. TST

 invitro  in vivo
 multiple antigens  single antigen
 no boosting  boosting
 1 patient visit  2 patient visits
 minimal inter-reader  inter-reader variability
variability
 results in 1 day  results in 2 - 3 days
Antigens used
o QuantiFERON® -TB test
PPD is used as antigen
o QuantiFERON® - Gold
ESAT-6 and CFP-10 are antigen used
o T Spot-TB test
identifies ESAT-6- or CFP-IO-specific
IFN-γ secreting CD4+ T cells.
M. tuberculosis-specific antigen
 In 1999, by comparative hybridization experiments on a DNA
microarray led to the identification of a genomic region known
as Region of Difference (RD I).

 The gene products of RD I are found only in M. tuberculosis, in

pathogenic M bovis strains and in four NTM (M kansasii, M
szulgai, M.flavescens, and M marinum).

 Only M kansasii overlaps clinically with M tuberculosis, and

because M. kansasii infection is uncommon, the RD I region
antigens are essentially specific to M tuberculosis.

 Among these antigens are, of course, ESAT-6 and CFP-IO, as

well as MPT-64. ESAT-6

Behr MA et al. Science 1999., Mahairas GG et al. J Bacteriol 1996

Harboe M et al Infect Immun 1996 , Philipp WJ et al. . Microbiol 1996 .
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, Clin
Diagn Lab Immunol. 1999

 Of 60 medical students, 0-mm tuberculin skin tests (TSTs) at study

entry

 58 (97%) were initially classified as negative for M. tuberculosis

infection 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 positive
by PPD QIFN.

 ESAT-6- and MPT-64-stimulated IFN-gamma responses in the

medical students were negative prior to and after BCG
immunization.
Johnson et al, Clin Diagn Lab Immunol. 1999
Johnson et al, Clin Diagn Lab Immunol. 1999
Johnson et al, Clin Diagn Lab Immunol. 1999
Mori T et al. . Am J Respir Crit Care Med 2004

 Mori and colleagues studied a group of 216 Japanese student

nurses who had no identified risk for M. tuberculosis exposure

 All vaccinated with BCG.

 In this group 64.6% of the subjects had a TST response

measuring 10 mm or more, yielding a specificity of 35.4% for
the TST

 The QuantiFERON--TB ESAT-6/CFP-l 0 assay, on the other

hand, yielded a specificity of 98.1 % in this group, far superior
to the TST.

 The sensitivity of the assay, 89.0%, was determined in a

separate group of patients who had culture-proven active
disease.
T SPOT-TB test
 Identifies
ESAT-6- or CFP-IO-specific
IFN-γ secreting CD4+ T cells

 Projected
as much more sensitive test as
compared to all other methods

 Approved by European Union

 Not approved by FDA as yet

T SPOT-TB test
Summary- so far……
 The improved specificity would decrease
unnecessary treatment in those who are not truly
infected.
 The improved sensitivity of these tests over the
TST would capture a cohort of patients who
other-wise would go without treatment of LTBI
 Esp. important in those who are most likely to
have false-negative TST results, e.g.
immunosuppressed individuals.
 Longitudinal studies linking positive assays with
risk for development of active disease are
ongoing and are crucial to demonstrating the
true role of these tests.
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.
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
tuberculosis
Murray PR et al. Ann Intern Med 1980
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
Fibro-optic Bronchoscopy
 Should be done in patients who are negative on
SI

A study by McWilliams and colleagues

 SIhad 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
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 wear
respiratory protection.
Diagnosis of active tuberculosis

 Microscopy:
 Easiest & quickest test
 Limited sensitivity(46-78%) but
specificity is virtually 100%*
 Centrifugation & fluorochrome
staining(auramineO) with UV
microscopy markedly increase
the sensitivity
 ZN staining – 10000 bacilli/ml
 Fluorochrome staining – 1000
bacilli/ml

*Chest 1989; 95: 1193

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 same
length of time is required for complete identification &
sensitivity testing
Cultures

Colonies of M. tuberculosis growing on media

Broth Based Culture Methods

 BACTEC
 SepticheckAFB
 Mycobacterial growth indicator tubes
 MB/Bac T
 Myco-ESPculture System II
 BacT/ALERT MB Susceptibility Kit
BACTEC
 Bactec 460 TB is an automated system that
measures the specific metabolic activity of TB
bacilli using the radiometric method.

 Bactecsystem requires an average of only 8 -

18 days for culture

5 to 8 days for determining drug susceptibility.

BACTEC
Mean time to detection of mycobacteria in clinical specimens
Average no. of days (range) to detection of:

Culture method M. tuberculosis complex

All isolates
Smear positive Smear negative

8.0 18
BACTEC 460 TB 11.2 (2–53)
(3–18) (9–30)

LJ medium 26.8 (7–47) 28.5 (16–29) 36.2 (28–41)

Francesca Brunello, Flavio Favari, Roberta Fontana

Journal of Clinical Microbiology, April 1999
BACTEC
Detection of mycobacteria from clinical specimens
according 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
 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

J Clin Microbiol 1999; 37: 748-752

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

The ESP culture system II
 Detection of pressure changes within the
headspace above the broth culture
medium in a sealed bottle
 The mean time for recovery of
M.tuberculosis complex 15.5
 Reliable nonradiometric less labour-
intensive alternative to BACTEC 460
system for the growth and detection of
mycobacteria.
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 was
13.7 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.
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-á-acetylamino-
ß-hydroxypropiophenone (NAP) for differentiation of
M.tuberculosis from other mycobacteria,
 the other section contains chocolate agar for detection
of contaminants.
The septi-check AFB system
 This method requires about 3 weeks of
incubation.
 The unique advantage of this technique is
the simultaneous detection of
M.tuberculosis, non-tuberculous
mycobacteria (NTM), other respiratory
pathogens and even contaminants.
TK Medium

 The color change in TK Medium is based on

multiple dye indicators
 Depends on the metabolites and enzymes
produced by different species of
microorganisms.
 The color change occurs long before the
colonies become visible.
 It can also be used for drug-susceptibility testing
 Can differentiate a contaminated specimen.
TK Medium
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 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
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
AMPLICOR M. TUBERCULOSIS assay

Cohen, R. A., 1998. Am. J. Respir. Crit. Care Med. 156:156–161.

Bonington, A., 1998. J. Clin. Microbiol. 36:1251–1254.
Al Zahrani, 2000. Am. J. Respir. Crit. Care Med. 162:1323–1329.
Amplified M.tuberculosis Direct (AMTD2) assay

Catanzaro et al.. JAMA 2000.283:639–645.

Bergmann, J. S.1999 J. Clin.Microbiol. 37:1419–1425.
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
NAA- summary
 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
Extra pulmonary tuberculosis
 In the case of miliary tuberculosis FOB
may play a significant role
 There is clearly a role for NAA 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
Adenosine Deaminase Levels
 In a recent meta-analysis of 40 studies investigating
ADA for the diagnosis of tuberculous pleuritis sensitivity
equaled specificity at 92.2%

 In one study, the sensitivity and specificity were both

55%

 Trajman A et al. argue that ADA alone is superior to ADA

combined 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
Adenosine Deaminase
 T.Söderblom, P. Nyberg, A.M. Teppo, (Eur Respir J,
1996, 9, 1652–1655) on pleural ADA and IFN-γ,
showed :
ADA in CSF
 ADA may be of limited value in diagnosing tuberculous
meningitis.
 There is consensus neither regarding ‘cut-off value’ nor
about sensitivity or specificity.
 In one study, Ribera E et al analyzed that the mean
enzyme value was clearly higher for the patients with
tuberculous meningitis (15.7 +/- 4.3 U/liter) than for the
other 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.

ADA in CSF
 N. Selvakumar, CEREBROSPINAL FLUID ADENOSINE
DEAMINASE AND LYSOZYME LEVELS IN THE DIAGNOSIS OF
TUBERCULOUS MENINGITIS, in Ind. J. Tub.:
IFN-γ levels
 Another test that has received some attention for the
diagnosis of pleural and pericardial tuberculosis is
pleural or pericardial fluid IFN-γ
To Conclude…..
 Diagnostic testing for tuberculosis remained unchanged
for nearly a century, but newer technologies hold the
promise of a time revolution in tuberculosis diagnostics.
 The IFN-γ release and T-cell-based assays may well
supplant the TST in diagnosing LTBI in much of the
world.
 NAA assays are proving their worth in more rapidly
diagnosing both pulmonary and extrapulmonary
tuberculosis with great sensitivity and specificity.
 These tests are likely to play an ever-increasing role in
the coming years.
THANK YOU
How QFT Is Performed
Stage 1 Whole Blood Culture
Nil Avian Tb Mitogen
Control PPD PPD Control

Heparinized whole blood Transfer undiluted whole blood Culture overnight at 37oC
into wells of a culture plate TB infected individuals
and add antigens respond by secreting IFN-γ

Stage 2 IFN-γ ELISA

COLOR
Standard Curve

OD 450nm
TMB

IFN-γ IU/ml

Harvest plasma from above Wash, add substrate, Measure OD,

settled cells and incubate incubate 30 min determine IFN-γ levels
60 min in ‘Sandwich’ ELISA then stop reaction and interpret test
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 was

determined

Harboe M et al. Infect Immun 1986.,Andersen P, et al. J Immunol 1995

Berthet FXet al. Microbiol 1998