Comparative study of Interferon-gamma assay in whole

blood for diagnosis of active pulmonary tuberculosis

. .

3122





.
...
.

.


.

.

.

.

.
.
.
...
: .
II



... .

III

IV


:
)) ((

.
) (
.

Declaration:
I declare that the present research entitled
(( Comparative study of Interferon-gamma assay in whole blood for
diagnosis of active pulmonary tuberculosis ))
is a new research that has never been studied by any other researcher for
any other degrees, and currently is not submitted by any one for any
degree.

Candidate
Lynn Taki-Eddin

Contents List

XIII..............................................................................................
XIV..............................................................................................
XVII..............................................................................................
XX.....................................................................................
XXI................................................................................
1...............................................................................................
.22.......................................................................................................
.1.1 2...............................................................................................
.2.1 3................................................................................................
.3.14.......................................................................................... ........
.4.1 5 .. ........................................................................................
.1.4.1 5....................................................................................
.2.4.1 5. .............................................................................
.5.1 6 ..................................................................................
.6.1 6.......................................................................................
.7.17.................................................................................................
.8.18..................................... .........................................................
.9.18..................................................................................................
.3 10 .....................................................................................
.1.2 10 .............................................................................................
.2.2 10 .......................................................................................

VI

 .3.2 11 ..................................................................................
.4.2 12 .....................................................................................
.5.2 14 ........................................................
.6.215 .............................................................................................
.1.6.2 15 ..............................................................................
.2.6.2 16 .................................................................
.3.6.2

16 ........................................................................

.4.6.2

16 ....................................................................

.7.2 16........................................................................................
.4 18..............................................................................
.1.3 18 ........................................................................
.1.1.3 18 ..................................................................
.2.1.3 18 ..............................................................................
.3.1.318 ....................................................................................
.4.1.3 20 ..........................................................................
.5.1.3 20 ...................................................................
.6.1.3 20 .............................. CD1d
.7.1.3 21 ...........................................................................
21 ............................................................................... Defensins .8.1.3
.2.3 21 ........................................................................
21 ..............................................................
.1.2.3
.2.2.3 21.................................................................
.3.3 22 ...................................................................................
.1.3.323 ..........................................................................................
VII

 .2.3.3 23 ........................................................................
.3.3.3 25 ....................................................
.5 26 ..............................................
.1.4 26 ...................................................
.1.1.4 26 ..............................................................
.2.1.4 27 ....................................................................
.1.2.1.4

27 .......................................... Ziehl-Neelsen

.2.2.1.4 27 ....................................................... Kinyoun

.3.2.1.4 27 ................................... Fluorochrome
.2.4 28 ...............................................................................
.1.2.4 28 ..........................................................................
.2.2.4 28 ... .........................................................
.3.2.4 30 .. ..............................................................................
.1.3.2.4 30 ...............................................
.2.3.2.4 31 ...............................................
.3.3.2.4 31 ................................................................
.4.2.4 33 ....................................................................
.1.4.2.4 33 .................................................................
.2.4.2.4 33 ......................................................
.3.4 34 ....................................................................
.1.3.4 34 .............................................
.2.3.4 35 ..............NAA
.4.4 35 .......................................................................
.1.4.4

36 .....................................................................

VIII

.2.4.4

36 ..............................................

.1.2.4.4 37 .............................................................
.2.2.4.4 38 .............................................
.1.2.2.4.4 IGRA 40 ..............................
.2.2.2.4.4 43 ....................................................IGRA
.5.4 45 ....................................................
.1.5.4 45 ....................................................................
.2.5.4 45 ...............................
.3.5.4 46 .............................................................
.4.5.4 46 ...............................................................
.5.5.4 46 ..............................................................................
.6.5.4 46 .................................................
.7.5.4 46 ...................................................................... TB
.8.5.4

47 ................................................................ Line Probe Assay

.9.5.4 47 ..........................
.10.5.4 47 ..................................................
.11.5.4 47 ................................................
48 ................................................................
.12.5.4
.13.5.4 48 ..............................................
.6.4 48 ....................................... WHO
.7.4 49 ..................................................................
51........................................................................................
52 .................................................................................................
.2 53 .............................................................................
IX

 .1.1 53 ...................................................................................
.2.154 ...............................................................................................
.3.1 54 ..... ..............................................................................
.4.1 56 .............................................................. ....................
.5.1 57...................................................................................
.6.158 ...................................................................................................
.7.1 58 ............................................................................................
.8.159 .............................................................................................
.9.1 59 ...............................................................................
.10.1 59.................................................................................
.11.1 60 ............................................................................................
.1.11.1

60 ...............................................

.2.11.1 60 ................................................
.3.11.1 Lowenstein Jensens 60 .......
.4.11.1 61 .............................
.5.11.1 61 ..........................
.6.11.1

-globin63.........................
.

.7.11.1 64 ...............
.8.11.1 65 ......................................................................
.366 .................. .................................................................................
.1.2 66 ..................................................................................
.2.2 66 ....................................................................................
.3.2

....................................................................

67

 .4.2 69 ........................................................ ZN
.5.2 IGRA 70..........................
ZN .
.6.2 71..........................
.Lowenstein Jensens
.7.2 IGRA 72........................
LJ .
.8.2 73..........................
.
.9.2 IGRA 74.......................
real-time PCR
.
.10.2 IGRA75.................
ZN LJ
.
.11.2 76......................
ZN LJ
.
.12.2 78........................
ZN LJ
.
.ROC
.13.2 79..................
IGRA .
.4 81 ..............................................................................................
.5 87 ........................................................................

XI

 .1.4 87 ............................................................................
.2.4 89 ..........................................................................
90....................................................................................................
100 ...................................................
( :)1 106...........................................................
( :)2 111.........................................................JIDC
( :)3 112............................................

XII

Tables List

:)1( 4.......................
.
:)2( 2009 4.............
.
:)3( 32................
.
)4( TST IGRA42..........................
ELISA .ELISPOT
:)5( 79............95% CI
ZN
LJ
.
:)6( 80..........

.
:)7( IFN- 84.....
:)8( 86..........

XIII

Figures List

( :)1 ZN 12.......................
( :)2 12...............
( :)3 12..................................................................
( :)4 23..................................................................
( :)5 23.............................................................
( :)6 24............................................................
( :)7 25...............................................
( :)8 33....................................LJ
( :)9 38.......................................................
( :)10 RD1 39......................MTB
( :)11 ESAT-6 CFP-10 39.....................
.
( :)12 IGRA 41.....................ELISPOT
( :)13 ELISA 64.................
.
( :)14 65...................
( :)15 66...............................................
( :)16 67...........................

XIV

 ( :)17 68.............
.
( :)18 PPV NPV 68.............
.
( :)19 PPV NPV 69... ZN
( :)20 PPV NPV70................
IGRA ZN
.
( :)21 PPV NPV 71..................

.Lowenstein Jensens
( :)22 PPV NPV72................
IGRA LJ
.
( :)23 PPV NPV 73.................
.Real-time PCR
( :)24 PPV NPV74................
IGRA real-time PCR
.
( :)25 ZN 76..................
LJ
. IGRA .

XV

 ( :)26 PPV NPV 77..............

IGRA
ZN LJ

.
( :)27 ROC 78............
( :)28 IGRA 88.........
.

XVI

Abbreviations List

AFB

Acid fast bacilli

APC

Antigen presenting cell

BCG

Bacillus Calmette Guerin

CDC

Centers for disease control and prevention

CI

Confidence interval

CR

Complement receptor

CRI

Colometric redox indicator

CTL

Cytotoxic T lymphocytes

DNA

Deoxyribonucleic acid

DST

Drug susceptibility test

DTH

Delayed type hypersensitivity

ELISA

Enzyme-linked immunosorbent assay

ELISPOT

Enzyme-linked immunospot

EMB

Ethambutol

FRET

Fluorescence resonance energy transfer

HIV

Human immunodeficiency virus

ICT

Immuno chromatographic test

IFN-

Interferon gamma

IGRA

Interferon gamma release assay

INH

Isoniazid

iNOS

Inducible nitric oxide synthase

JaK

Janus family kinases

LAM

Lipoarabinomannan

LAMP

Loop mediated isothermal amplification

LED

Light-emitting diode

LJ

Lowenstein-Jensen

LM

Lipomannan

LPA

Line probe assay

MAF

Macrophage activating factor

XVII

MDR-TB

Multi drug resistant-tuberculosis

MHC

Major histocompatibility complex

MODS

Microscopic observation of drug susceptibility

MTB

Mycobacterium tuberculosis

MTBc

Mycobacterium tuberculosis complex

NAA

Nucleic acid amplification

NAD

Nicotinamide adenine dinucleotide

Nalc-NaOH

N-acetyl-L-cystein-sodium hydroxide

NK

Natural killer

NKT

Natural killer T cell

NPV

Negative predictive value

NRA

Nitrate reductase assay

P-value

Probability value

PAS

Para-aminosalicylic acid

PBMC

Peripheral blood mononuclear cell

PCR

Polymerase chain reaction

PDIM

Phthiocerol dimycoserosates

PGL

Phenolic glycolipids

PHA

Phytohemaglutinin

PMN

Polymorphonuclear leukocyte

PPD

Purified protein derivatives

PPV

Positive predictive value

PTB

Pulmonary tuberculosis

PZA

Pyrazidamide

RD

Region of difference

RIF

Rifampicin

RNA

Ribonucleic acid

RNI

Reactive nitrogen intermediate

ROI

Reactive oxygene intermediate

SDA

Strand displacement amplification

SL

Sulphoids

SM

Streptomycin

STAT

Signal transducer and activators of transcription

XVIII

TACO

Tryptophan aspartate coat protein

Tau-b

Kindalls tau-b

TB

Tuberculosis

TBCTA

Tuberculosis coalition for technical assistance

TCH

Thiophene carboxylic acid hydrazide

Th

T-helper

TLA

Thin layer agar

TMA

Transcription mediated amplification

TNF-

Tumor necrosis factor alpha

TST

Tuberculin skin test

V-ATPase

Vesicular proton-pump adenosine triphosphatase

WHO

World health organization

ZN

Ziehl-Neelsen

XIX



.
) Interferon-Gamma Release Assay (IGRA

IGRA
.
; 91
.- QuantiFERON-TB-Gold In-Tube -IGRA
) Ziehl-Neelsen (ZN
) Lowenstein Jensens (LJ
.real-time PCR .
; )52.7%( 48/91 .
) Positive predicted value (PPV
) Negative predicted value (NPV %100 %100 %72.9
%76.78 %79.24 %97.36 %97.67 %77.1 ZN
%85.3 %75.4 %67.4 %89.9 LJ IGRA
%71.9 %94.1 %95.3 %66.6 real-time PCR .
; IGRA
( )

. NPV IGRA
.
;

.
XX

Abstract
Background: Sensitivity, specificity, early confirmation and obtaining an
optimal specimen are challenging problems in active tuberculosis (TB)
diagnosis. Interferon-gamma release assay (IGRA) is a good indicator for latent
TB but can it be useful as a diagnostic tool for active TB? This study was
designed to address these challenges and assess the potential of IGRA as a
diagnostic indicator of active pulmonary TB by comparing it with other MTB
diagnostic conventional methods and molecular methods.
Methods: The study was conducted on 91 patients with suspicion of
pulmonary active TB. QuantiFERON-TB-Gold In-Tube a commercial IFNgamma assay, was compared with Ziehl Neelsen (ZN) smear, Lowenstein
Jensens (LJ) egg-based culture, and real-time polymerase chain reaction. The
final clinical diagnosis was the standard comparator of the study.
Results: Active pulmonary TB was confirmed in 48/91 (52.7%) patients.
Sensitivity, specificity, positive predicted value (PPV), and negative predicted
value (NPV) were (72.9%, 100%, 100%, 76.78%) for ZN smear, (77.1%,
97.67%, 97.36%, 79.24%) for LJ culture, (89.9%, 67.4%, 75.4%, 85.3%) for
IGRA, and (66.6%, 95.3%, 94.1%, 71.9%) for real-time PCR, respectively.
Conclusion: Albeit confounding in the case of latent TB infected patient
presenting with non-TB pulmonary disease, IGRA was more sensitive than the
other conventional and molecular methods, so it

may improve diagnostic

accuracy when used in combination with other standard methods. High NPV of
IGRA for the diagnosis of active TB proposed an additional role of this test to
exclude the infection with active TB.
Key words: Interferon gamma release assay, TB real time-PCR, TB culture,
TB smear, active pulmonary TB.

XXI


Introduction

 .2
.2.2

Tuberculosis
;Overview

()

Mycobacterium

tuberculosis .
.

700-200 100.000
%30-20 .
100
) .(Plorde 2004) (Godreuil 2007
Consumption John Bunyan
.)Roberts 2008( The captain of all these men of death
" " 1020


).(www.news-medical.net/health/History-of-Tuberculosis
1882 ( )
.

(Bacillus Calmette Gurin) BCG
1921 ).(Godreuil 2007

) Human Immunodeficiency virus (HIV

). (Godreuil 2007
2

).(Plorde 2004

.3.2

;Epidemiology

) World Health Organization (WHO 2010
2009 9.4 %55
%30 %7
%4 .%3
14 200 100.000.
1.7 26
100.000 (.)WHO 2010
WHO 2009
21 100.000 27
100.000 1.8 100.000
.)1(
3.975 %29 %17 %3
%51 . 176
%43 %6 %11 %41
.)2(

:)1(
.*
Estimates of burden 2009

Number (thousands) Rate (per 100 000 population)

Mortality (excluding HIV)

0.4 (0.190.77)

1.8 (0.893.5)

Prevalence (including HIV)

6 (1.911)

27 (8.849)

Incidence (including HIV)

4.6 (3.85.5)

21 (1725)

Incidence (HIV-positive)
Case detection, all forms (%)

88 (73110)
. 22 :2009 *
)www.who.int/tb/data(

2009 :)2(
.
Case notifications 2009
New cases

(%)

(%)

Retreatment cases

Smear-positive

1 143 (29)

Relapse

75

(43)

Smear-negative

675

(17)

Treatment after failure

10

(6)

Smear unknown

121

(3)

Treatment after default

19

(11)

Extrapulmonary

2 036 (51)

Other

72

(41)

Other

Total new

3 975

Total retreatment

176

(0)
)www.who.int/tb/data(

;Transmission

.4.2

.
10 5-0.5 40.000
.
4

.
()Plorde 2004
(.)www.news-medical.net/health/Tuberculosis-Transmission
15-10 .

%70 .Pulmonary tuberculosis
Extrapulmonary
.)Herrmann 2007( tuberculosis

.5.2

;Infection Types

.2.5.2 ; Latent Infection

%90
( .)Varaine 2010
.
.
HIV
.( Godreuil 2007) .
.3.5.2 ;Active Disease
%10 .


reactivation
( .)Varaine 2010
DNA reinfection
%12 %77
(.)Verver 2005

.6.2

;Clinical symptoms

.
) .(Godreuil 2007
%50 (.)Tiemersma 2011

.7.2 ;Risk factors

(:)CDC 2010
.
.A
.
.B
.A :
.1
.%22
.2
.
.3

.
.4
.
.
.5
.B :
.1 .HIV
.2 5 .

.3 immunosuppressive therapy

)tumor necrosis factoralpha (TNF-

antagonist
.
.4 ( .)
.5
.
.6

silicosis

diabetes

militus renal failure leukemia

lymphoma .
.7 %90 .
.8


.

.8.2

;Treatment

:
()Da Silva 2007
.)1 .
.)2
.
. :
INH RIF PZASM
.EMB : :
: PAS
:
.)Varaine 2010()da Silva 2007(
7

) multi drug resistant TB (MDR-TB

(
) Extensively drug resistant TB (XDR-TB
)

:
.)Martin 2007(

.9.2

; Prevention

.

3-2 .

(
) Varaine (
)2010

(.)www.medical-dictionary.thefreedictionary.com/tuberculosis

.:.2 ; Vaccines
) bacille Calmette-Gurin (BCG
.Mycobacterium bovis


( .)www.brown.edu/Courses
( ) %80
.)Bannon 1999( %80 %0
8

%50 ( .)Varaine 2010

DNA BCG (.)Martn 2007

 .3
Mycobacterium tuberculosis

.2.3

;Classification

)Forbes 2007( :

Actinomycetes class

Actinomycetales order

Mycobacteriaceae family

Mycobacterium genus

71
(:)Godreuil 2007
.i ( 7
) .
( Mycobacterium abscessus, M. fortuitum,
)M. porcinum ( .)M. smegmatis, M. agri
.ii /
) M. tuberculosis complex (MTBC

M.

tuberculosis, M. bovis, M. africanum

M. leprae, M. ulcerans, M. avium ( M. terrae,
.)M. gordonae

.3.3

;Characteristics

DNA

%71-%61
.) Plorde 2004()Barrera 2007(
11

MTB
synthesis
(.)Godreuil 2007
obligate aerobe MTBCs
.
macrophage slow generation
20-15( time)
(.)www.textbookofbacteriology.net/tuberculosis

.4.3

;Microscopic description

( )MTB
( .)Barrera 2007
)ZiehlNeelsen (ZN
acid-fast stain
( .)2007 Godreuil
(
) . 10-1
( 0.6-0.2 ) 5-3 .

hydrophobic bundles )2( )1(

.
(.)Barrera 2007

11

( :)2
.

( :)1 Ziehl-
Neelsen (
.)1000
).(Barrera 2007

).(Barrera 2007

.5.3
;M. tuberculosis cell wall structure
( )3
.

( :)3 . ()Esko 2009

12

Barrera 2007:
.1
lipopolysaccharides .
.
:
)i .
)ii .
)iii .
)iv .
.2
.

.
residues

.cross links
M. tuberculosis %80-70
Escherichia coli .%30-20
arabinogalactan

mycolic acids 60
90 . species-specific
-
.
M. tuberculosis
76 84 82 83 89 90 .

phthiocerol dimycoserosates (PDIM), phenolic glycolipids (PGL),
.sulfolipids (SL) trehalose-containing glycolipids
glycolipids
13

phosphatidyl-myoinositol

) lipoarabinomanan (LAM) mannosides, lipomannan (LM

.
. exported
.
. porins
.
.3 pseudo-capsule
.

.6.3
;Metabolic and biochemical markers
.1
nicotinamide adenine dinucleotide
) (NAD .
M. tuberculosis M. canettii isolates

M.

africanum
.)Barrera 2007( differential identification
.2 H2O2
heatlabile catalase-peroxidase
. .
alkyl-hydroperoxidase .


.)Barrera 2007( INH
.3 /

14

nitrate reductase
.)Barrera 2007(
.4
.urease-mediated reaction
.biosynthesis
.
microenvironment

phagolysosome
(Major histocompatibility complex MHC II) II
(.)Barrera 2007

.7.3

;Pathogenesis

.
: bacilli phagocytosis
, intracellular multiplication stationary
stage .pulmonary form of TB
: spontaneous healing

acute

tuberculosis latent infection reactivation

.)Smith 2003( reinfection
.2.7.3 ;
pulmonary alveolus
.
: .
alveolar macrophages
(.)Smith 2003

15

.3.7.3 ;
.

symbiotic

.stage
circulating .

early pulmonary lesions
(.)Smith 2003
.4.7.3 ;

caseous
.
.

.
(.)Smith 2003
.5.7.3 ); Pulmonary form of TB (PTB


.

(.)Godreuil 2007

.8.3

;Virulence mechanisms

) lipoarabinomannan (LAM
CR1

Complement

.CR3 receptor .
16

( pH CO2
) . LAM
- lysosome-phagosome
. LAM
Plorde ( antigen presentation
.)2004

17

 .4
Immune response against TB

: Innate immune response

.2.4
.2.2.4

: Neutrophil leukocytes

.
(.)Pando 2007
.3.2.4

;Mast cells

( preformed mediators histamine tryptase
chymase carboxypeptidase )heparin
( mediators synthesized de novo D2
TNF- .)IL-8,IL-4, IL-5

(.)Pando 2007
.4.2.4

;Macrophages

Fc
CD14 .CD43
) (Pando 2007
- )Surfactant protein A( A
- upregulating
18

.(Smith 2003) Fc
) reactive oxygen intermediates (ROI
phagosomes .lysosomes
(CR3) 3 respiratory burst

(.)Pando 2007

internalization
( Pando
.)2007
.

hostile PH ) (ROI
. Reactive nitrogen
) intermediates (RNIs
(.)Smith 2003

-
vesicular proton-pump adenosine
(V-ATPase) triphosphatase (.)Smith 2003

early

endosomes immunosurveillance
.CD4+ T cells 19-kDa
TLR2
toll-like receptor .
Rab
GTPases . Rab5
Rab7 ( .)Smith 2003
Rab5 .Rab7 tryptophan
19

) aspartate coat protein (TACO coronin

actin binding proteins
(.)Pando 2007
.5.2.4

;Dendritic cells

.)Pando 2007( MHC

.)Smith 2003( MTB

MHC II MHC I
CD86 CD80 .)Pando 2007( IL-12
.6.2.4

;Natural killer cells

. IFN-
CD8+ IFN-
(.)Pando 2007
.7.2.4 CD1d
;CD1d-restricted natural killer T cells
CD1d

( alpha-galactosylceramide ) .
IFN- granulysin
.)Pando 2007(

21

.8.2.4

;Epithelial cells

.
IL-8
).)Pando 2007( nitric oxide (NO
;Defensins .9.2.4

.
.
(.)Pando 2007

; Acquired immune response

.3.4

adaptive
.
effector mechanisms
(.)Pando 2007

.2.3.4

;Humoral immune response

.

opsonization
(.)Pando 2007
.3.3.4

;Cellular immune response

MHC
class II .
. CD4+
21

IFN- .
.CD8+
CD4+ T cells
.)Pando 2007( CD95 Fas ligand
CD8+
.IFN-
.
exocytosis .granzymes perforins granulysin
CD8+ (.)Pando 2007

.4.4

);Interferon Gamma (IFN-

type II
. macrophage-activating
) .factor (MAF IFNG
12 15 .)12q15(

.
.

immunostimulatory .immunomodulatory
) natural killer (NK
natural killer T (NKT) cells
CD8+ CD4+

cytotoxic T

lymphocytes CTL effector T cells

( .)www.bio.davidson.edu/courses( )Schroder 2004

22

;Strucutre

.2.4.4

6 -helices
.C ( )4
1.6

( :)4
( )
()www.en.wikipedia.org/wiki/Interferon_gamma

( .)www.bio.davidson.edu/courses ( )5
.

( :)5
( )
()www.en.wikipedia.org/wiki/Interferon_gamma

.3.4.4

;Receptor Binding

.IFNGR2 IFNGR1
23

Janus family kinases

) .(Jak1 and Jak2 Jak1 IFNGR1 Jak2 .IFNGR2
IFN- IFNGR1 dimerization IFNGR1
.IFNGR2
IFNGR1 IFNGR2
Jak2 Jak1
. Jak2 Jak1
IFNGR1
.(Signal Transducer and Activators of Transcription) STAT1
STAT1 Jak
.
STAT1
promoter immediate-early
.)Schroder 2004 ( IFN--inducible genes ( )6
.

( :)6 .
()www.bio.davidson.edu/courses

24

;Biological Activity

.4.4.4

( )www.bio.davidson.edu/courses( .)7
( )Schroder 2004:
.1 ) (MAF
.
.2 ROI
.(inducible Nitric Oxide Synthase) iNOS
.3 ).T helper 2 (Th2
.4 .MHC class I
.5 .
.6 NK CTL
. polymorphonuclear leukocytes (PMN)
.7 ) Antigen Presenting Cells (APCs
MHC II MHC I .
.8 naive helper T cells
.Th1

( :)7
()www.bio.davidson.edu/courses
25

 .5
Active Pulmonary Tuberculosis Diagnostic tests
.2.5
);Direct Microscopic Examination for Acid Fast Bacilli (AFB

.
-
.)de Waard 2007( Acid Fastness
.2.2.5 ;


.
( de
.)Waard 2007

10.000-5000
3
.
liquefaction
.
(.)de Waard 2007


.AFB
poor quality

26

.AFB
(de Kantor ()de Waard 2007
.)CDC 2006()1998
.3.2.5
.1.2.1.4

;Acid Fast Bacilli staining

):Ziehl-Neelsen staining (ZN

Carbol fuchsin
. ZN hot acid-fast
stain . -

. AFB
Methylene blue (.)de Waard 2007
.2.2.1.4

:Kinyoun staining

ZN cold acid-fast
. Kinyoun Ziehl-
.)de Waard 2007( Neelsen
.3.2.1.4

:Fluorochrome staining

Auramine-Rhodamine Auramine O .

Fluorescent light-emitting
) diode (LED WHO
.ZN
250x 450x
ZN 1000x
%25 ZN
27

%10 .ZN
AFB
ZN
.
24 (.)WHO 2011()de Waard 2007

.3.5

;TB culture

. .%50-30
.2.3.5 ;
100-10

.
.

.

.

24-18 ()
(.)WHO 2006()WHO 1998
;Digestion and Decontamination

.3.3.5

:
.i

28

.ii

( )


.)WHO 1998() Plorde 2004()de Waard 2007(

)WHO 1998()Plorde 2004( . %3-2

( )WHO 1998 :

Sodium hydroxide (Modified Petroff) .a

Oxalic acid .b
Ogawa-Kudoh .c
Cethyl pyridinium and sodium chloride .d
Zephiran-Trisodium Phosohate (Z-TSP) .e
.)WHO 1998( Sulphuric acid .f
:Carboxypropylbetaine (CB-18) .g
%61.9 (.)Srikanth 2009
:Phenol ammonium sulphate (Phas) .h %2
(.)Srikanth 2009
hydroxide (NALC-NaOH) .i

:N-acetyl-L-cysteine-sodium

)de Waard 2007()WHO 1998( acetyl-cysteine
.)Srikanth 2009( %71.4
:Sodium hypochlorite (bleach) .j

.)WHO 2008( %15
29

:Chitin .k bleach
%83 ( Srikanth
.)2009
.4.3.5

;Culture Media

:
.I .
.II .
.III .
:Egg-based media

.1.3.2.4

Lwenstein-Jensen (LJ) Ogawa

malachite green
.
:
)1 .
)2 .
)3
.
)4
.)WHO 1998(
:
)1 8

.
)2
.)WHO 1998(

31

:Agar-based media

.2.3.2.4

.Middlebrook 7H11 Middlebrook 7H10

(.)de Waard 2007
.3.3.2.4

:Liquid media

10
.

.
(
.)Tortoli 2007( )
( .)de Waard 2007

:)3(

31

:)3(
.

C14

Bactec 460 TB (Becton Dickinson Diagnostic

.C14-labeled palmitic acid

)Systems, Cockeysville, Md

CO2

14

.
CO2 .
( )
.10
modified Middlebrook 7H9

)Septi-Chek AFB System (Becton Dickinson

:
.modified Middlebrook 7H11
. .
Middlebrook 7H9
embedded sensor .

)Mycobacteria Growth Indicator Tube (MGIT

. O2

.O2

modified Middlebrook 7H9

ESP culture System II (TREK Diagnostic

enrichment .

)Systems, Cleveland, Ohio

O2
.
.modified Middlebrook 7H9

BacT/Alert System (BioMrieux, Inc.,

CO2 CO2 .

)Durhum, NC

.modified Middlebrook 7H9

)BACTEC 9000 MB (Becton Dickinson

O2 .
.MGIT
O2

)(Forbes 2007

32

)BACTECT MGIT 960 (Becton Dickinson

.5.3.5 ;Reading Results and Identification

8 .
.
.2.5.3.5

;Morphologicl characteristics

(
)cream ( )8
inoculation

3 .

ZN .

( :)8

Lwenstein-Jensen

(.)WHO 1998

www.textbookofbacteriology.net/tuberculosis

.3.5.3.5

;Biochemical characteristics

p-Nitrobenzoic acid
pyrazinamide
) thiophene carboxylic acid hydrazide (TCH
.)de Waard 2007( 68C


high- resolution gas chromatographic
DNA RNA
( .)Plorde 2004
( lateral flow technology )

33

.)WHO 2008( MPB 64

.4.5

);Nucleic Acid Amplification methods (NAA

( .)Paramasivan 2006 DNA
( ribosomal RNA
)DNA .
: Polymerase chain reaction (PCR)
conventional PCR nested-PCR
real-time PCR Isothermal amplification

Transcription mediated amplification

) (TMA )strand displacement amplification (SDA

.) Katoch 2003()WHO 2006( QB-replicase
.2.4.5 ;
NAA .%100-98
10
.
NAA
.
.
%95 %70-60 AFB
( .)WHO 2006
34

.

.)Tortoli 2007(
fragments
.IS6110 24-1
Insertion sequence IS6110

M. tuberculosis
(.) Christopher 2007()Khorshidi 2009()Gilpin 2003


.DNA polymerase
human -globin .
.3.4.5 ;NAA
16S rRNA RNA
16S ribosomal DNA (rDNA) IS6110 IS686
MPB64 MPB70 MTB40 65-kDa antigen 32-kDa antigen
Protein B )Kaul 2001( HSP 65 126
.)Tortoli 2007( kDa fusion protein

;Immunodiagnostic methods

.5.5

. .

(.)Singh 2007
35

.2.5.5

;Serology diagnosis

( .)WHO 2006
(.(2007 Singh
preparations
)Antigen 60 (A60

)Singh 2007) 38kDa

Heat shock protein

Lipoarabinomannan TAT DAT acylated trehalose

.)Verma 2007( Trehalose 6,6-dimycolate (cord-factor) Sulfolipid

WHO 2010
(.)WHO 2011
;

)Singh 2007( BCG
.

Immunochromatographic test (ICT)

enzyme-linked immunosorbent assay

).)Steingart 2007( (ELISA

.3.5.5

;T cell based assays

.

36

) .(Singh 2007
(.)Pai 2007
.2.3.5.5

;Tuberculin Skin Test

tuberculin
( )Mantoux
.
) purified protein derivative (PPD
M. tuberculosis
Mycobacterium bovis BCG .
Delayed type hypersensitivity DTH PPD
induration
72-48 ( )
).) Palomino 2007((Singh 2007

( Godreuil
.)2007
;

/ BCG
(
) PPD
Boosting
.




37

%25 )WHO () Palomino 2007((Singh 2007

.)2006

.3.3.5.5

Release

Gamma

Interferon

):Assays (IGRAs

.
IFN-
IFN- ( .)Palomino 2007
( )9 .IGRA
CD4
IFN-
)ECDC 2011( .

( :)9

)(www.prepgmedicos.redstetho.com/forum

6-KDa early secretory antigen

target (ESAT-6) Rv3875 10-KDa culture filtrate protein 10

.)Cole 1998()Ganguly 2008((CFP-10) Rv3874
) region of difference 1 (RD1
( M. tuberculosis )10 M. bovis BCG
38

nontuberculous mycobacterias NTM

( M. kansasii M. marinum, M. sulzgai )11
M. leprae

immunologic cross

)Rothel 2005( reactivity

Tsiouris () Harada 2004()Van Pinxteren 2000( %98
.)2006

)(Cole 1998

()Ganguly 2008

( :)10 RD1 MTB

( :)11 ESAT-6 CFP-10

www.cellestis.com
39

) TB7.7(p4) TB7.7(Rv2654 RD11
phage-inserted region
( .)Rothel 2005 ) phytohemaglutinin (PHA
PHA
Mitogen IFN-
Nil
( Madariaga
.)2007
IGRA
) Enzyme-linked immunospot (ELISPOT
(ELISA) IFN- .
.2.3.3.5.5 IGRA ;
.A :ELISA
IGRA ELISA
.
14 ESAT-6 CFP-10
.TB7.7 :

.PHA

24-16 . IFN-
.ELISA IFN-

IFN-
(.)CDC 2010( )Nil

41

.B :ELISPOT

CPF-10 ESAT-6 IFN- 20-16

.
.
( )Lalvani 2007 (.)12

( :)12 IGRA .ELISPOT

www.tbtestingservices.com/TB%20Testing

)4(
:IGRA ELISA ELISPOT .

41

ELISA IGRA TST ) 4(

.ELISPOT
ELISPOT

ELISA^

TST

Antigens
Ability to distinguish
latent and active
tuberculosis
Effect of BCG
vaccination

ESAT-6 and CFP10

No

ESAT-6, CFP10 and TB7.7

No

PPD
No

None

None

Positive internal
control
Uniformity of
methods and reagents
Potential for boosting
effect in repeated tests
Need for return visit
Time required for
results
Setting of test
Interpretation of test

Yes

Yes

May give a falsepositive result after

BCG vaccination
No

Yes

Yes

No

No

No

Yes

No
1620 h*

No
1624 h#

Yes
4872 h

In vitro
Objective
(instrument-based)
IFN- spot-forming
cells
PBMC
Number of IFN-
producing T cells
Enumeration of spots
by naked eye,
magnifying lens, or
automated counter
Yes. Special equipment
required in the
laboratory (requires
CO2 and humidified
incubator)*

In vitro
Objective
(instrument-based)
International units of IFN-

In vivo
Subjective
(operator-based)
Millimeters of
induration
NA
NA

Readout units
Tests substrate
Outcome measure
Readout system

Whole blood
Serum concentration of
IFN- produced by T cells
Measurement of optical
density values using an
automated reader

Palpable induration

No special
infrastructure
required. Easy to
perform even in
unsophisticated
facilities
ELISpot, enzyme-linked ImmunoSpot; ELISA, enzyme-linked immunesorbent assay;
TST, tuberculin skin test; PPD, protein-purified derivative; BCG, Bacillus CalmetteGurin; PBMC, peripheral blood mononuclear cells; NA, not applicable.
^Last generation of ELISA used for IFN- assay.
*From the T-SPOT.TB technical information sheet (www.oxfordimmunotec.com).
#
From the QuantiFERON-TB Gold technical information sheet (www.cellestis.com).

Required
infrastructure

Yes. Special equipment

required in the laboratory
(doesn't require special
conditions of incubation)#

) Richeldi 2006()Madariaga 2007(

42

.3.3.3.5.5 (;)IGRA
IGRA IGRA
IFN- peripheral blood

) .mononuclear cells (PBMC effector

( memory T-cells IFN-
) recruited


)PBMC(
(.)Lange 2009
IGRA
16 8 ELISA
.)ECDC 2011( ELISPOT

BCG ( )Madariaga 2007
( )
(.)Pai 2007
IGRA
( ELISA )ELISPOT ( PBMCs
) severity
( )HIV
(.)Pai 2007
)TST( IGRA
.)CDC 2010(
IGRA ( )PPD
TST IGRA ( TST 72)

43

( )Leyten 2007 ( )Richeldi 2008

TST 6 (.)Naseer 2007
CDC 2010 IGRA
10-8 CDC ( .
.)2010


IGRA .)Diel 2008( TST
IGRA CD4

immunocompromised
)Rangaka 2007( HIV CD4
200 3/ Talati ( )NICE 2011( IGRA
.)2009 IGRA
CD4 HIV
) Aichelburg 2009()Rangaka 2007(
IGRA
HIV ( )ECDC 2011( )Syed Ahamed Kabeer 2009
.
ELISPOT CD4
.)Stephan 2008()Leidl 2010( ELISA
IGRA
(TNF-)
( )Beglingera 2007( )Chiu 2011( )Matulis 2008


IGRA .
44

IGRA

( )Tavast 2009
( )Herrmann 2009 IGRA
TST TST
.)CDC 2010()Kampmann 2009( IGRA
( ) Herrmann 2009()Katiyar 2008 IGRA

.MDR TB ()Lee 2010
IFN-
reversion
IGRA .

.6.5
;Other active pulmonary tuberculosis diagnostic tests
.2.6.5

;Urinary antigen detection

) Lipoarabinomannan (LAM
.
HIV
(.) Mutetwa 2009( )Dheda 2010
.3.6.5
;Loop mediated isothermal amplification (LAMP) assay
NAA DNA
DNA
isothermal
.
.) Boehme 2007()Kohan 2011( TB

45

.4.6.5 ;Breathalyser screening test

.

10 . ( WHO
.)2008
.5.6.5

;Mycobacteriophage assay

( mycobacteriophages
) .
72-48 (.)WHO 2006
.6.6.5

;Sputum filtration

.
(.)WHO 2008
.7.6.5

Vital fluorescent staining of

;sputum smears
.

(.)WHO 2008
.8.6.5 TB

;TB Patch Test

MPT64 .
/ erythema vesiculation 4-3
. BCG
TB (.)WHO 2008

46

;Line Probe Assay (LPA) .9.6.5

DNA )strip test(
.
MDR-TB
5

drug

).)WHO 2008( susceptibility test (DST

.:.6.5
);Automated real-time NAA test (Xpert MTB/RIF system
DNA TB
.MDR-TB
.rpoB
90
. MDR-TB
HIV
MDR-TB HIV
.WHO 2010
.21.6.5 );Thin layer agar culture (TLA

. Middlebrook 7H10

Middlebrook 7H11

. 7
DST .)WHO 2008( 15-10
.22.6.5
);Microscopic observation of drug susceptibility (MODS

(.)WHO 2011
47

.23.6.5

);Nitrate reductase assay (NRA

(.)WHO 2011
.24.6.5

Colorimetric redox indicator

;(CRI) methods

microtitre plate
(.)WHO 2011

.7.5 WHO
;Techniques and methods currently recommended by WHO
.1 Automated liquid
.(2007) culture and DST
.2 Rapid speciation
.3 probe assays

).(2007

.(2008) Line

.4

Second-line drug susceptibility testing

).(2008

.5 LED microscopy LED

.(2011) Flourescent

.6 (2011) MODS

.7

NRA

).(2011

.8 CRI methods
.9 TLA

).(2011

).(2011

.10 .(2011) Phage-based assays

.11

Automated real-time NAA technology for rapid

and simultaneous detection of tuberculosis and rifampicin

.(2011) resistance: Xpert MTB/RIF system
48

.8.5
;International standards for TB diagnosis

Tuberculosis

:Coalition for Technical Assistance (TBCTA) 2006

.1 3-2
.
.2 3 (
) ( )
.
WHO 2007
:
.A
.AFB
.B AFB
.
.C AFB
.
:

AFB
(.)Rieder 2007
.3
.
.4 :

49

.A (
).
.B .
.C (

).

.HIV
.5 intrathoracic


(
) . (
) .

51

51

;Research Goals

.

.
.


.

. IGRA

. :
.1 IGRA
.
.2 IGRA

.Real-time PCR

52

 .2
Materials and Methods

.2.2

;Samples and Patients

91 70-18


.2010
:
(
) :
.1 :
.a .
.b .
/ .c .
.2 70-18.
.3 .
.4
.
.5 .

:
.

53

;Sampling

.3.2

:
.I

:
.

.II

1( )
QuantiFERON-TB Gold IT
:

( Nil Control .)

( TB Control ).

.ELISA

.4.2

;Patients Questionnaire

54

" "
:. .

:
:

:
: : :
-

:
:

:
:
:
:
:
:
:
- :
: : : 3 :
:
:
:
:
:
:

- :

55

.5.2

;Informed Consent

:
:
.
: .
:
.1
.2
.3
.4
.5
.6
.7

.
.
.

.
.
.
.

..............................................
/


( ) .
......../......./......

....................................... :

/
.
......../....../.....

.........................................

56

.6.2

;Statistical study

Kendall's tau-b
P-value<0.05
.Confidence Intervals 95% (CIs) %95
Receiver Operating Characteristics curve (ROC)
Area Under the Curve (AUC)
AUC
.

T-student
P-value<0.05 .

Specificity
Sensitivity ) Positive Predicted Value (PPV
).Negative Predictive Value (NPV

:
:)True Positives) TP
( ).
:(True Negatives) TN
( ).
:(False Negatives) FN
( ).
:(False Positives) FP
( ).
57

;Materials

.7.2
:

. 50 .1
. 13 .2
. .3
1.5 0.5 microcentrifuge tubes .4
.PCR
)10 l, 100 l, 1000l( Filter tips .5
. )Sarstedt, Germany( PCR
adjustable-volume micropipettes .6
.)Eppendorf AG, Hamburg, Germany(
.(Roche Diagnostics, Germany) Capillary tubes .7
. Pasteur Pipettes .8

;Reagents

.8.2

BBL MycoPrep Specimen Digestion/Decontamination Kit .1

(Becton Dickinson, USA)
Lowenstein Jensens .2
.(Biomerieux, France)
.(Gokhan, Turkey) Ziehl Neelsen Set .3
.(AJ Roboscreen, GmbH) INSTANT Mycobacteria DNA kit .4
RoboGene Mycobacterium tuberculosis Qualitative kit .5
.(AJ Roboscreen, GmbH)
FastStart DNA Master Hybridization Probes kit .6
.(Roche Diagnostics, Germany)
Fluorescein Probe primers .7
TIB Molbiol,

-globin LightCycler Red probe

.)Germany)
58

QuantiFERON-TB-Gold In-Tube .8
.(Cellestis Ltd, Victoria, Australia)

;Equipments

.9.2

.(ZEISS, Germany) .1
.)Cepheid, USA( SmartCycler Instrument .2
LightCycler Instrument .3
.(Roche Diagnostics, Germany)
PCR Laminar Flow Cabinet .4
.(Holten, Allerod, Denmark)
.(Tecan, Austria) Sunrise Remote :ELISA .5
.)Eppendorf AG, Hamburg, Germany( Centrifuge 5417 R .6
.)Memmert, GmbH, Schwabach, Germany( Incubator .7
Thermomixes Comfort .8
.(Eppendorf AG, Hamburg, Germany)
.)Grant, UK( Heating Blocks BT3 .9
.)GFL, Germany( -80C .10

;Computational Software

.:.2

.LightCycler Software v.35.3 .1

Cepheid SmartCycler v.2001 .2

;Statistical Programs

.21.2

.)SPSS, Chicago, Illinois, US) PASW statistics 18 .1

.Microsoft Excel 2007 .2

59

.22.2

;Methods

.2.22.2 ;Digestion & Decontamination

BBL MycoPrep Specimen
) Digestion/Decontamination Kit (Becton Dickinson, USA
.

) (NALC-NaOH .
.

.3.22.2 ;Direct examination

.Ziehl-Neelsen
(Gokhan, Turkey) Ziehl-Neelsen Set
) )WHO 1998 .

.4.22.2 Lowenstein Jensens

;TB Culture using Lowenstein Jensens Solid media

Lowenstein Jensens

(.(Biomerieux, France
0.2-0.4 ml 35-37C
8
).(WHO 1998
:
.Ziehl-Neelsen (ZN)
loop
61

( ) .
.ZN
.5.22.2
;MTB DNA Extraction from Respiratory Specimens
INSTANT Mycobacteria DNA kit
) (AJ Roboscreen, GmbH .
.6.22.2
;MTB qualitative molecular biological detection

RoboGene Mycobacterium tuberculosis Qualitative
) .kit (AJ Roboscreen, GmbH insertion element
IS6110
.TripleHyb assay
:
:5 TB/IC
.1

100l PCR-grade water

TB/IC Real-time Reagent Mix TB/IC
dNTP TB/IC 37C 20 .

:Reaction Mix
.2

PCR 25l :

61

TB/IC reaction mix [l]

1 reaction
Final concentration
8.1
2.5
1x
2.5
1x
1.5
3.0 Mm
0.5
1x

Reagents
PCR grade water
10x PCR buffer
10x BSA solution
MgCl2 (50 mM)
5x Reagent mix (containing
dNTP, primers and probe)
Taq Polymerase (e.g.
AmpliTaq Gold) (5 U/l)

0.4

2.0 U per reaction

PCR grade water 5l

.3

. DNA 5l controls
Cy3 channel TB FAM channel( FTTC 25

.4

.) Yakima Yellow
SmartCycler
:

Stage 1
Stage 2 Repeat 50

Deg/sec
2.5
2.5

Temp (C)
95
95
59

Secs
600
15
60

Optics
Off
Off
On

) ( :)1(
. amplification enhancer
:)2(
) PCR grade water ( negative control
.

62

.5

-globin .7.22.2
Validation of extraction efficiency by -
; globin DNA real-time PCR
-globin
Fluorescence
FastStart DNA Master Resonance Energy Transfer (FRET)
LightCycler (Roche Diagnostics,

Hybridization Probes Kit

.Germany)

TAAgCCAgTgCCAgAAgAgCC

Tadmouri ( ATCATTCgTCTgTTTCCCATTCTAAAC
CCCTTggACCCAgAggTTCTTTgAgTCCT- )1999
:LightCycler Red probe Fluorescein
LC-Red640-TggggATCTgTCCACTCCTgATgCTgTTATg-phosphate
.(TIB Molbiol, Germany) ) Moreno 2002(
PCR
:capillary 10L

Reagents
PCR grade water
Primers
Probes
MgCl2
Master

reaction mix [l]

1 reaction
Final concentration
4.8
1
0.5 l
1
0.2 l
1.2
3.0 Mm
1

: real-time PCR

63


Step

Temperature
(C)
Taq activation
95
Denaturation
95
Annealing
59
Extension
72
Melting Curve Analysis
95
40
85
Data Analysis on Channel F2

Duration
10 min
10 sec
10 sec
32 sec

Ramping
Rate (C/s)
20
20
20
5

Acquisition
mode
none
none
single
none

0 sec
10 sec
0 sec

20
20
0.2

None
None
Continuous

Repeat
1
45

.8.22.2
Quantification of MTB complex Specific Interferon Gamma Release
Assay:
QuantiFERON-TB-Gold In-Tube QFT-GIT
IGRA ( Cellestis Ltd, Victoria, Australia)
.QFT-GIT
ESAT-6, CFP-10 ) TB tube(
heparin )NIL tube( .TB7.7(P4)
(ELISA) IFN- .
0.35 IU/mL .)13 (
. .

:)13(
ELISA

.

64

(y-axis) y
IFN- (x-axis) x
. . (:)14
10

10

O.D.

1
0.1

0.1

IU/mL

( :)14

) IFN- (IU/mL

.
.9.22.2 ;Final diagnosis

.

65

.3

Results

.3.3 ;
91
70-18 .
(:)15
3 . 3 . 85 - .

n= 91

85

( :)15 n=91

.3.3

;Final Diagnosis

n=91 48/91
66

( )%52.7 )%47.3( 43/91

)12/43( : )3/43( )5/43(
COPD )2/43( COPD )5/43( )1/43(
)1/43( )1/43( )1/43(
)1/43( - )5/43( )3/43(
)1/43( )1/43( )1/43( .
( )16 .

n=91

Non TB
infected
47%

TB infected

TB
infected
53%

Non TB infected

( :)16 n=91

.4.3

;IGRA Results

IGRA 57/91 ( )%62.6 34/91

( .)%37.4 IGRA 43/48
5/48
14/43
(.)17

67

60
14

50
40
30

Non TB infected
TB infected

43

29

20
10

Negative

Positive

( :)17 n=91

95% ( %89.9 )95% CI, 57.57-77.2( %67.4

)CI, 83.58-96.21 . Positive predictive value
) (PPV ) Negative predictive value (NPV %75.43
( )95% CI, 77.86-92.71( %85.29 )95% CI, 66.4-84.45 .
Kendalls Tau-b
IGRA )P-value<0.05( P-value=0.0007
(.)18
)tau-b (P-value=0.0007
89.90%
85.29%
90.00%

75.43%

67.40%

80.00%
70.00%
60.00%
50.00%
40.00%
30.00%
20.00%
10.00%
0.00%

NPV

PPV

Specificty Sensitivity

( :)18 PPV NPV

68

.5.3 ZN
;Direct Examination of ZN Smears
35/91 ( )%38.5 AFB
56/91 ( )%61.5 95% CI, ( %72.9
)63.58-82.21 )95% CI, 99.995-100.005( %100
. PPV
NPV 95% ( %76.78 )95% CI, 99.995-100.005( %100
)CI, 67.93-85.62 . Kendalls
Tau-b
)P-value<0.05( P-value=0.0005
(.)19

)tau-b (P-value=0.0005
100%

100%
76.78%

NPV

72.90%

PPV

100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%

Specificity Sensitivity

( :)19 PPV NPV ZN

69

.6.3 IGRA
ZN ;
)P-value>0.05( P-value=0.497 %95
IGRA .ZN
T-student
IGRA ZN P-value=0.0036
( )P-value<0.05 P-value<0.0001 .
( )20 PPV NPV
.

120.00%

)tau-b (P-value=0.497

100.00%
80.00%
60.00%
40.00%
20.00%

NPV
85.29%
76.78%

PPV
75.43%
100%

Sensitivity
89.90%
72.90%

Specificity
67.40%
100%

0.00%

IGRA
ZN smear

( :)20 PPV NPV IGRA

ZN

71

.7.3
);Lowenstein Jensens (LJ
38/91 ( )%41.8 53/91 ( )%58.2
)95% CI, 68.29-85.9( %77.1 95% CI, 94.5-( %97.67
)100.83 . NPV PPV
)95% CI, 70.74-87.73( %79.24 )95% CI, 93.99-100.72( %97.63
.
Kendalls
Tau-b )P-value<0.05( P-value=0.0003
(.)21

)tau-b (P-value=0.0003
97.63%

97.67%
100.00%

79.24%

77.10%

90.00%
80.00%
70.00%
60.00%
50.00%
40.00%
30.00%
20.00%
10.00%
0.00%

NPV

Sensitivity

PPV

Specificity

( :)21 PPV NPV

Lowenstein Jensens

71

.8.3 IGRA
LJ ;
)P-value>0.05( P-value=0.202 %95
IGRA .LJ
T-student
IGRA )P-value<0.05( P-value=0.0278
P-value<0.0001 . ( )22
PPV NPV .

120.00%

)tau-b (P-value=0.202

100.00%
80.00%
60.00%
40.00%
20.00%

NPV
85.29%
79.24%

PPV
75.43%
98%

Sensitivity
89.90%
77.10%

Specificity
67.40%
98%

0.00%

IGRA
Culture

( :)22 PPV NPV IGRA

Lowenstein Jensens

72

.9.3

;MTB Qualitative Real-time PCR

real-time PCR DNA 34/91

( )%37.4 57/91 ( .)%62.6 %95.3
( )95% CI, 56.71-76.48( %66.6 )95% CI, 90.86-99.73 .
95% CI, 62.5-( %71.92 )95% CI, 89.17-99.04( %94.11 NPV PPV
)81.33 .

)P-value<0.05( P-value=0.0006
(.)23

)tau-b (P-value=0.0006
94.11%

95.30%
100.00%
90.00%

71.92%

66.60%

80.00%
70.00%
60.00%
50.00%
40.00%
30.00%
20.00%
10.00%
0.00%

NPV

Sensitivity

PPV

Specificity

( :)23 PPV NPV Real-time

PCR

73

.:.3 IGRA
real-time PCR ;
)P-value>0.05( P-value=0.186 %95
IGRA .real-time PCR
T-student
IGRA real-time PCR )P-value<0.05( P-value=0.0002
P-value<0.0001 . ( )24
PPV NPV .

120.00%

)tau-b (P-value=0.186

100.00%
80.00%
60.00%
40.00%
20.00%

NPV
85.29%
71.92%

PPV
75.43%
94%

Sensitivity
89.90%
66.60%

Specificity
67.40%
95%

0.00%

IGRA
Real-time PCR

( :)24 PPV NPV IGRA

real-time PCR

74

.21.3 IGRA
ZN LJ
;
IGRA n=48
ZN IGRA )% 22.9) 11/48
.ZN ZN
)%6.25( 3/48 .IGRA
IGRA 11/48 ( )%22.9
.LJ IGRA
. 5/48
( )%10.41 IGRA . IGRA
13/48 ( )%27.08 PCR
DNA PCR DNA
IGRA .
IGRA 7 ( )%14.9
(.)25

75

Positive

Negative

60
14

29

Non TB infected
TB infected

16

11

13

32

30
37

35

43

20
10

( :)25 ZN
LJ real-time PCR
IGRA .)n=91(

.22.3

ZN LJ
;
( )26
IGRA
.

76

41
42

40

43

50

120%
100%
80%
60%
40%
20%

Real-time PCR
66.60%
95.30%
94.11%
71.92%

Culture
77.10%
97.67%
97.36%
79%

ZN smears
72.90%
100.00%
100.00%
76.78%

IGRA
89.90%
67.40%
75.43%
85%

0%

Sensitivity
Specificity
PPV
NPV

( :)26 PPV NPV

IGRA ZN
) (ZN smears )LJ culture( LJ
Real-time PCR
.

77

.23.3
ZN LJ
;ROC
Receiver Operating Characteristics (ROC)
Area Under the Curve (AUC)
(.)27

-2

( :)27 ROC .n=91

)5( AUC
-1 )y( )x(
. %95
78

:)5( 95% CI
ZN LJ
Real-time PCR .IGRA
95% Confidence Interval
Upper Bound

Lower Bound

AUC

Test

0.944

0.785

0.865

ZN smear

0.952

0.796

0.874

LJ culture

0.884

0.686

0.785

IGRA

0.902

0.718

0.810

Real-time PCR

;ZN, Ziehl neelsen; LJ, Lowenstein Jensens; IGRA, Interferon gamma release assay

PCR, Polymerase chain reaction.; AUC, Area Under the Curve.

.24.3
IGRA ;
( )IGRA

ZN IGRA
%95.83 .%67.44 IGRA
%100 %67.44 . IGRA
PCR %62.79 %93.75
(.)6

79

:)6(

.n=91
Methods Combination

Sensitivity (%), (95% CI)

Specificity (%), (95% CI)

IGRA + ZN Smear

95.83, (91.63-100.022)

67.44, (57.62-77.25)

IGRA + LJ Culture

100, (99.995-100.005)

67.44, (57.62-77.25)

IGRA + real-time PCR

93.75, (88.67-98.82)

62.79, (52.66-72.91)

ZN, Ziehl neelsen; LJ, Lowenstein Jensens; IGRA, Interferon gamma release assay;
PCR, Polymerase chain reaction.

81

.4

Discussion

ZN

5000
de Waard 2007 Shukla
2011 .


.(de Kantor 1998)
ZN

ZN.(de Waard 2007)
LJ
ZN Oberoi .2007


de Kantor 1998
1000-10 ) (de Waard 2007
.

. 3
ZN IGRA PCR
IGRA real-time
PCR
IGRA real-time PCR
Ani .2009
81

real-time PCR .
DNA
DNA Tortoli
Aldous 2007 .2005 real-time PCR %95.3

Ani
.2009 Shukla 2011
Khorshidi 2009

.
IGRA
Ravn
Kang 2005 Rutherford 2007 .2010
IGRA


Ravn 2005
.
Mitogen
IFN- .IFN-
IGRA
( )Singh 2007
IGRA 14


) .(WHO 2009 IGRA

.
82

Ravn Kang 2005 .2007

Kalantri Lee 2009 Mori 2006
2004

.
IGRA
.

anti-TNF antibodies
IGRA )(Murakami 2009
HIV
CD4 T 200 .(NICE 2011) 3/
) (CDC
). (CDC 2006

IGRA
M. kansasii, M. marinum, M. szulgai
).(Madariaga 2007
IGRA 7 ( )%14.9


) .(de Kantor 1998
2
Light 2010
Santos 2010
.

83

IGRA

ZN.
Kang 2007
NPV IGRA
PPV
.

IGRA

. IGRA
LJ . %100
IGRA .
IFN-
IGRA 0.35 IU/mL



0.45 IU/mL 0.65 IU/mL

(:)7
:)7( IFN-

)(IU/mL

0.25

0.3

0.35

0.4

92%

90%

90%

88% 90% 90%

88% 88%

60%

67%

67%

72% 72% 70%

74% 74%

0.45

0.55

0.6

0.65

0.7

0.8

83%

81%

74%

74%

IGRA
84

LJ
8-6
real-time PCR IGRA
24.
IGRA
. 3000-2500
. 250 500 LJ
. IGRA real-time PCR . 4000
. IGRA



INH
.
)8( .

85

:)8( .

91

Shukla 2011

Oberoi 2007
Ani 2009
Khorshidi 2009

Ravn 2005

Kang 2007
Rutherford
2010
Kalantri 2009

Lee 2006

Mori 2004

74

20

104

101

248
82

39

BCG
144

98

100
50
50
87
131

118
216
BCG

( %72.9 : ( % 77 )ZN
.)real-time PCR( %66.6 )IGRA( %89.9 )LJ
( %100 : ( %97.7 )ZN )LJ
.)real-time PCR( %95.3 )IGRA( %67.4
( %54 : ( %56.75 )ZN
.)nested PCR( %75.6 )LJ
( %100 : ZN )LJ
.)nested PCR( %95
( %48 : ( %53 )ZN .)LJ
( %100 : ZN .)LJ
.)PCR( %85 :
.)PCR( %74 :
multiplex PCR
%93.8 .%99.1
%85 IGRA .%60
.%97
%89 IGRA %49 %84 NPV
.%61 PPV
.%88.7 IGRA
%96 IGRA .%96.15

%70.1 IGRA .%91.6

%89 IGRA .%98.1

IGRA .ELISA
;ZN, Ziehl neelsen; LJ, Lowenstein Jensens; IGRA, Interferon gamma release assay
PCR, Polymerase chain reaction; BCG, Bacillus Calmette-Gurin; PPV, Positive
predictive value; NPV, Negative predictive value.

86

.5

Conclusions and Recommendations

.2.5

;Conclusions and Recommandations

.1 IGRA
.
.2 IGRA real-time PCR
.
.3 IGRA

.
.4 IGRA .
.5 IGRA
.
.6 IGRA
.
( )28 IGRA
.

87

ZN
-++/--+
-++/

---

IGRA

IGRA +

MDR
HIV

PCR

+
IGRA -

-
IGRA -

+
IGRA +

-
IGRA +

IGRA
.

( :)28 IGRA .

88

.3.5

;Suggestions for futher research

.1 IGRA
.
.2 IGRA
.
.3 ( )
( ).
.4 IGRA .
.5 IGRA HIV
.

89

References
-

Aichelburg MC, Rieger A, Breitenecker F, Pfistershammer K, Tittes J, Eltz S,

Aichelburg AC, Stingl G, Makristathis A, and Kohrgruber N, Detection and
prediction of active tuberculosis disease by a whole-blood interferon- release
assay in HIV-1-infected individuals, Clin Infect Dis. 2009; 48: 954-62.

Aldous W, Pounder J, Cloud J, and Woods G, Comparison of six methods of

extracting Mycobacterium tuberculosis DNA from processed sputum for Testing
by quantitative real-time PCR , J Clin Microbiol. 2005; 43: 24712473.

Ani A, Okpe S, Akambi M, Ejelionu E, Yakubu B, Owolodun O, Ekeh P, Oche

A, Tyen D, and Idoko J, Comparison of a DNA based PCR method with
conventional methods for the detection of M. tuberculosis in Jos, Nigeria, J
Infect Dev Ctries. 2009; 3: 470-5.

Bannon MJ, BCG and tuberculosis, Arch Dis Child. 1999; 80:80-3.

Barrera L, The basics of clinical bacteriology in "Tuberculosis 2007 From basic

science to patient care 1st ed"; Ed. Palomino JC, Leo SC, and Ritacco V;
www.tuberculosistextbook.com 2007: 93-112.

Beglingera C, Dudlerb J, Mottetc C, Nicodd L, Seibolde F, Villigerf P, and

Zellwegerg JP, Screening for tuberculosis infection before initiation of antiTNF-a therapy, SWiSS Med Wkly 2007; 137: 621622.

Boehme CC, Nabeta P, Henostroza G, Raqib R, Rahim Z, Gerhardt M, Sanga E,

Hoelscher M, Notomi T, Hase T, and Perkins MD, Operational feasibility of
using loop-mediated isothermal amplification for diagnosis of pulmonary
tuberculosis in microscopy centers of developing countries, J Clin Microbiol.
2007; 45: 1936-40.

Brown university, Vaccine approaches to tuberculosis,

www.brown.edu/Courses, accessed on: 18 Sep. 2010.

Cellestis, QuantiFERON-TB Gold in tube

www.cellestis.com, accessed on: 20 Jul. 2011

Centers for Disease Control and Prevention, Acid-Fast Direct Smear

Microscopy Participant Manual: Overview Tuberculosis, the global emergency;
2006. wwwn.cdc.gov/dls/ila/acidfasttraining/participants.aspx

Chiu HY, Hsueh PR, and Tsai TF, Clinical experience of QuantiFERON() -TB
Gold testing in patients with psoriasis treated with tumour necrosis factor
91

method

package

insert,

References
blockers in Taiwan, Br J Dermatol. 2011; 164: 553-9.
-

Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Gordon SV,

Eiglmeier K, Gas S, Barry III CE, Tekaia F, Badcock K, Basham D, Brown D,
Chillingworth T, Connor R, Davies R, Devlin K, Feltwell T, Gentles S, Hamlin
N, Holroyd S, Hornsby T, Jagels K, Krogh A, McLean J, Moule S, Murphy L,
Oliver K, Osborne J, Quail MA, Rajandream MA, Rogers J, Rutter S, Seeger K,
Skelton J, Squares R, Squares S, Sulston JE, Taylor K, Whitehead S, and Barrell
BG, Deciphering the biology of Mycobacterium tuberculosis from the complete
genome sequence, Nature 1998; 396: 537-544.

Da Silva P, and Ansa J, Drugs and drug interactions in "Tuberculosis 2007

From basic science to patient care 1st ed"; Ed. Palomino JC, Leo SC, and
Ritacco V; www.tuberculosistextbook.com 2007: 593-634.

Davidson College, Interferon gamma, www.bio.davidson.edu/courses, accessed

on: 9 Jun. 2011.

De kantor IN, Kim SJ, Frieden T, Laszlo A, Luelmo F, Norval PY, Rieder H,
Valenzuela P, and Weyer K, Laboratory services in tuberculosis control:
Microscopy part II; World Health Organization 1998.

De kantor IN, Kim SJ, Frieden T, Laszlo A, Luelmo F, Norval PY, Rieder H,
Valenzuela P, and Weyer K, Laboratory services in tuberculosis control:
Culture part III; World Health Organization 1998.

De Waard JH, and Robledo J, Conventional diagnostic methods in

"Tuberculosis 2007 From basic science to patient care 1st ed"; Ed. Palomino JC,
Leo SC, and Ritacco V; www.tuberculosistextbook.com 2007: 401-424.

Dheda K, Davids V, Lenders L, Roberts T, Meldau R, Ling D, Brunet L, van

Zyl Smit R, Peter J, Green C, Badri M, Sechi L, Sharma S, Hoelscher M,
Dawson R, Whitelaw A, Blackburn J, Pai M, and Zumla A, Clinical utility of a
commercial LAM-ELISA assay for TB diagnosis in HIV-infected patients using
urine and sputum samples, PLoS One. 2010; 5: e9848.

Diel R, Loddenkemper R, Meywald-Walter K, Niemann S, and Nienhaus A,

Predictive value of a whole blood IFN- assay for the development of active
tuberculosis disease after recent infection with Mycobacterium tuberculosis, Am
J Respir Crit Care Med. 2008; 177: 1164-70.

Esko J, Doering T, and Raetz C, Eubacteria and archaea in "Essentials of

glycobiology 2nd edition" Ed. Varki A, Cummings R, Esko J, Freeze H,
Stanley P, Bertozzi C, Hart G, and Etzler M; Pub. Cold Spring Harbor
Laboratory Press 2009.

European Centre for Disease Prevention and Control, ECDC guidance: Use of
91

References

interferon-gamma release assays in support of TB diagnosis"; ECDC 2011.

-

Forbes B, Sahm D, and Weissfeld A, Mycobacteria in "Bailey & Scotts

Diagnostic microbiology 12th ed"; Ed. Forbes B, Sahm D, and Weissfeld A;
Pub. Mosby Elsevier 2007 :478-509.

Ganguly N, Siddiqui I, and Sharma P, Role of M. tuberculosis RD-1 region

encoded secretory proteins in protective response and virulence, Tuberculosis
2008; 88: 510-517.

Gilpin CM, Evaluation of conventional and molecular strategies for the rapid
diagnosis and molecular characterisation of strains of Mycobacterium
tuberculosis. www.usir.salford.ac.uk 2003.

Godreuil S, Tazi L, and Bauls AL, Pulmonary tuberculosis and Mycobacterium

tuberculosis: Modern molecular epidemiology and perspectives in
"Encyclopedia of infectious disease: modern methodologies"; Ed. Tibayrenc M;
Pub. John Wiley & Sons 2007: 1-29.

Harada N, Higuchi K, Sekiya Y, Rothel J, Kitoh T, and Mori T, Basic

characteristics of a novel diagnostic method (QuantiFERON TB-2G) for latent
tuberculosis infection with the use of Mycobacterium tuberculosis-specific
antigens, ESAT-6 and CFP-10, Kekkaku. 2004; 79:725-35.

Herrmann JL, Belloy M, Porcher R, Simonney N, Aboutaam R, Lebourgeois M,

Gaudelus J, De Losangeles L, Chadelat K, Scheinmann P, Beydon N, Fauroux
B, Bingen M, Terki M, Barraud D, Cruaud P, Offredo C, Ferroni A, Berche P,
Moissenet D, Vuthien H, Doit C, Bingen E, and Lagrange PH, Temporal
dynamics of interferon gamma responses in children evaluated for tuberculosis,
PLoS One. 2009; 4: e4130.

Herrmann JL, and Lagrange PH, Molecular or immunological tools for efficient
control of tuberculosis in "Encyclopedia of infectious disease: modern
methodologies"; Ed. Tibayrenc M; Pub. John Wiley & Sons 2007: 75-86.

Kalantri Y, Hemvani N, and Chitnis DS, Evaluation of whole blood IFN test
using PPD and recombinant antigen challenge for diagnosis of pulmonary and
extra-pulmonary tuberculosis. Indian J Exp Biol 2009; 47:463-468.

Kampmann B, Whittaker E, Williams A, Walters S, Gordon A, Martinez-Alier

N, Williams B, Crook AM, Hutton AM, and Anderson ST, Interferon-gamma
release assays do not identify more children with active tuberculosis than the
tuberculin skin test, Eur Respir J. 2009 Jun; 33: 1374-82.

Kang YA, Lee HW, Hwang SS, Um SW, Han SK, Shim YS, and Yim JJ,
Usefulness of whole-blood interferon-gamma assay and interferon-gamma
enzyme-linked immunospot assay in the diagnosis of active pulmonary
92

References
tuberculosis. Chest 2007; 132:959-965.
-

Katiyar SK, Sampath A, Bihari S, Mamtani M, and Kulkarni H, Use of the

QuantiFERON-TB Gold In-Tube test to monitor treatment efficacy in active
pulmonary tuberculosis, Int J Tuberc Lung Dis. 2008; 12: 1146-52.

Katoch VM, Advances in Molecular Diagnosis of Tuberculosis, MJAFI 2003;

59 : 182-186

Kaul K, Molecular detection of Mycobacterium tuberculosis: Impact on patient

care, Clinical Chemistry 2001; 47:15531558.

Khorshidi A, Rohani M, Moniri R, and Torfeh M, Comparison of culture and

microscopic methods by PCR for detection of Mycobacterium tuberculosis in
sputum, . Iranian Journal of Clinical Infectious Diseases; 2009 4:228-232.

Kohan L, Shahhosseiny MH, Razavi MR, Parivar K, Moslemim E , and

Werngren J, Evaluation of loop mediated isothermal amplification for diagnosis
of Mycobacterium tuberculosis complex in clinical samples, Afr. J. Biotechnol
2011; 10: 5096-5101.

Lalvani A, Diagnosing tuberculosis infection in the 21st century: New tools to

tackle an old enemy, Chest 2007; 131:1898-1906

Lange C, Pai M, Drobniewski F, and Migliori GB, Interferon- release assays

for the diagnosis of active tuberculosis: sensible or silly?, Eur Respir J 2009; 33:
12501253.

Lee JY, Choi HJ, Park IN, Hong SB, Oh YM, Lim CM, Lee SD, Koh Y, Kim
WS, Kim DS, Kim WD, and Shim TS, Comparison of two commercial
interferon-gamma assays for diagnosing Mycobacterium tuberculosis infection.
Eur Respir J 2006; 28:24-30.

Lee SW, Lee CT, and Yim JJ, Serial interferon-gamma release assays during
treatment of active tuberculosis in young adults, BMC Infect Dis. 2010; 10:300.

Leidl L, Mayanja-Kizza H, Sotgiu G, Baseke J, Ernst M, Hirsch C, Goletti D,

Toossi Z, and Lange C, Relationship of immunodiagnostic assays for
tuberculosis and numbers of circulating CD4+ T-cells in HIV infection, Eur
Respir J. 2010; 35: 619-26.

Leyten EMS, Prins C, Bossink AWJ, Thijsen S, Ottenhoff THM, van Dissel JT,
and Arend SM, Effect of tuberculin skin testing on a Mycobacterium
tuberculosis-specific interferon- assay, Eur Respir J 2007; 29: 12121216.

Light RW, Update on tuberculous pleural effusion, Respirology. 2010;15:451-8.

Madariaga M, Jalali Z, and Swindells S, Clinical utility of interferon gamma

93

References

assay in the diagnosis of tuberculosis, J Am Board Fam Med 2007; 20:540 547.
-

Martin A, and Portaels F, Drug resistance and drug resistance detection in

"Tuberculosis 2007 From basic science to patient care 1st ed"; Ed. Palomino JC,
Leo SC, and Ritacco V; www.tuberculosistextbook.com 2007: 635-660.

Martn C, Bigi F, and Gicquel B, New vaccines against tuberculosis in

"Tuberculosis 2007 From basic science to patient care 1st ed"; Ed. Palomino JC,
Leo SC, and Ritacco V; www.tuberculosistextbook.com 2007: 341-360.

Matulis G, Jni P, Villiger PM, and Gadola SD, Detection of latent tuberculosis
in immunosuppressed patients with autoimmune diseases: performance of a
Mycobacterium tuberculosis antigen-specific interferon gamma assay, Ann
Rheum Dis. 2008; 67: 84-90.

Mazurek G, Jereb J, Vernon A, LoBue P, Goldberg S, and Castro K, Updated

guidelines for using interferon gamma release assays to detect Mycobacterium
tuberculosis infection - United States, 2010; Office of Surveillance,
Epidemiology, and Laboratory Services, Centers for Disease Control and
Prevention 2010 59:No.RR-5.

McEvoy CR, Falmera A, Gey van Pittiusa NC, Victora TC, van Heldena PD and
Warrena RM, The role of IS6110 in the evolution of Mycobacterium
tuberculosis, Tuberculosis 2007; 87:393-404.

Moreno I, Bolufer P, Perez ML, Barragn E, and Sanz MA. Rapid detection of
the major Mediterranean beta-thalassaemia mutations by real-time polymerase
chain reaction using fluorophore-labelled hybridization probes. Br J Haematol.
2002; 119: 554-7.

Mori T, Sakatani M, Yamagishi F, Takashima T, Kawabe Y, Nagao K, Shigeto

E, Harada N, Mitarai S, Okada M, Suzuki K, Inoue Y, Tsuyuguchi K, Sasaki Y,
Mazurek GH, and Tsuyuguchi I, Specific detection of tuberculosis infection: an
interferon-gamma-based assay using new antigens. Am J Respir Crit Care Med
2004; 170:59-64.

Murakami S, Takeno M, Kirino Y, Kobayashi M, Watanabe R, Kudo M, Ihata

A, Ueda A, Ohno S, Watanuki Y, Kaneko T, and Ishigatsubo Y, Screening of
tuberculosis
by
interferon-
assay
before
biologic
therapy
for rheumatoid arthritis, Tuberculosis 2009; 89: 136141.

Mutetwa R, Boehme C, Dimairo M, Bandason T, Munyati SS, Mangwanya D,

Mungofa S, Butterworth AE, Mason PR, and Corbett EL, Diagnostic accuracy
of commercial urinary lipoarabinomannan detection in African tuberculosis
suspects and patients, Int J Tuberc Lung Dis. 2009 Oct;13(10):1253-9.

Naseer A, Naqvi S, and Kampmann B, Evidence for boosting Mycobacterium

94

References

tuberculosis specific IFN- responses at 6 weeks following tuberculin skin

testing, Eur Respir J. 2007;29: 1282-3.
-

National Institute for Health and Clinical Excellence, Tuberculosis: clinical

diagnosis and management of tuberculosis, and measures for its prevention and
control, NICE 2011.

News medical, History of tuberculosis, www.news-medical.net/health/Historyof-Tuberculosis, accessed on: 18 Oct. 2011.

News medical, Tuberculosis transmission, www.news-medical.net/health/

Tuberculosis-Transmission, accessed on: 18 Oct. 2011.

Oberoi A, and Aggarwal A, Comparision of the conventional diagnostic

techniques,BACTEC and PCR, JK Science 2007; 9: 179-181.

Oxford diagnostic laboratories, National TB testing services, T-SPOT.TB test,

www.tbtestingservices.com/TB%20Testing, accessed on 13 Aug. 2011.

Oxford immunotec, T-SPOT.TB training guid, www.oxfordimmunotec.com,

accessed on: 31 Jul. 2011.

Pai M, and Menzies D, Interferon- release assays: What is their role in the
diagnosis of active tuberculosis?, CID 2007; 44:747.

Palomino JC, Leo SC, and Ritacco V, New developments and perspectives in
"Tuberculosis 2007 From basic science to patient care 1st"; Ed. Palomino JC,
Leo SC, and Ritacco V, www,teberculosistextbook.com 2007: 661-680.

Pando RH, Salinas RC, Lpez JS , and Estrada I, Immunology, Pathogenesis,

Virulence in "Tuberculosis 2007 From basic science to patient care 1st ed"; Ed.
Palomino JC, Leo SC, and Ritacco V; www.tuberculosistextbook.com 2007:
157-206.

Paramasivan CN, Molecular methods in the diagnosis of tuberculosis, Indian J

Tuberc 2006; 53:61-63

Plorde J, Mycobacteria in "Sherris medical microbiology: an introduction to

infectious diseases 4th ed"; Ed. Ryan J and Ray C; Pub. McGraw-Hill 2004:
439-456.

PrePgMedicos Forum, Interferon Gamma Release Assay,

www.prepgmedicos.redstetho.com/forum, accessed on: 24 Mar 2011.

Rangaka M, Wilkinson K, Seldon R, Van Cutsem G, Meintjes G, Morroni C,

Mouton P, Diwakar L, Connell T, Maartens G, and Wilkinson R, Effect of
HIV-1 infection on T-Cellbased and skin test detection of tuberculosis
infection, Am J Respir Crit Care Med 2007; 175: 514520.
95

References
-

Ravn P, Munk ME, Andersen AB, Lundgren B, Lundgren JD, Nielsen LN, KokJensen A, Andersen P, and Weldingh K, Prospective evaluation of a wholeblood test using Mycobacterium tuberculosis-specific antigens ESAT-6 and
CFP-10 for diagnosis of active tuberculosis, Clin Diagn Lab Immunol. 2005; 12:
491-6.

Richeldi L, An update on the diagnosis of yuberculosis infection, Am J Respir

Crit Care Med 2006; 174:736742.

Richeldi L, Bergamini BM, and Vaienti F, Prior tuberculin skin testing does not
boost QuantiFERON-TB results in paediatric contacts, Eur Respir J.
2008;32:524-5.

Rieder H, and Van Deun A, Proposal for a revision of the case definition of
"Sputum smear-postivie tuberculosis", WHO press 2007.

Roberts C and Buikstra J, The history of tuberculosis from earliest times to the
development of drugs in "Clinical tuberculosis 4th ed"; Ed. Davies PDO,
Barnes P and Gordon S; Pub. Hodder Arnold 2008: 3-19.

Rothel J, and Andersen P, Diagnosis of latent Mycobacterium tuberculosis

infection: is the demise of the Mantoux test immenent, Expert Rev Anti Infect
Ther. 2005; 3:981-93.

Russo C, Coltella L, Tozzi A, and Menichella D, Assessment of the new

interferon-gamma release ELISA assay in children with suspected TB in a low
incidence country, www.esmycobacteriology.eu/abstracts/PP016.pdf, accessed
on: 26 Dec. 2010.

Rutherford M, Alisjahbana B, Maharani W, Sampurno H, van Crevel R, and Hill

PC, Sensitivity of the Quantiferon-Gold In-Tube Assay in Sputum Smear
Positive TB Cases in Indonesia, PLoS One. 2010; 5: e12020.

Santos A, Cremades R, Rodrguez JC, Garca-Pachn E, Ruiz M, and Royo G,

Comparison of methods of DNA extraction for real-time PCR in a model of
pleural tuberculosis, APMIS. 2010; 118: 60-5.

Schroder K, Hertzog PJ, Ravasi T, and Hume DA, Interferon-gamma: an

overview of signals, mechanisms and functions, J Leukoc Biol. 2004; 75:16389.

Shukla I, Varshney S, Sarfraz, Malik A, and Ahmad Z, Evaluation of nested

PCR targeting IS6110 of Mycobacterium tuberculosis for the diagnosis of
pulmonary and extra-pulmonary tuberculosis, Biology and Medicine, 2011; 3:
171-175.

Singh M, and Espitia C, Immunological diagnosis in "Tuberculosis 2007 From

96

References

basic science to patient care 1st"; Ed. Palomino JC, Leo SC, and Ritacco V,
www.tuberculosistextbook.com 2007: 425-440.
-

Smith I, Mycobacterium tuberculosis pathogenesis and molecular determinants

of virulence, Clin. microbiol. rev. 2003; 16:463496

Srikanth P, Kamesh S, and Daley P, Bleach optimization of sputum smear

microscopy for pulmonary tuberculosis, Indian J Tuberc. 2009; 56: 174-84.

Steingart K, Henry M, Laal S, Hopewell PC, Ramsay A, Menzies D,

Cunningham J, Weldingh K, and Pai M, Commercial serological antibody
detection tests for the diagnosis of pulmonary tuberculosis: A systematic review,
PLoS Med. 2007; 4: e202.

Stephan C, Wolf T, Goetsch U, Bellinger O, Nisius G, Oremek G, Rakus Z,

Gottschalk R, Stark S, Brodt HR, and Staszewski S, Comparing QuantiFERONtuberculosis gold, T-SPOT tuberculosis and tuberculin skin test in HIV-infected
individuals from a low prevalence tuberculosis country, AIDS. 2008; 22:2471-9.

Syed Ahamed Kabeer B, Sikhamani R, Swaminathan S, Perumal V,

Paramasivam P, and Raja A, Role of Interferon Gamma Release Assay in Active
TB Diagnosis among HIV Infected Individuals, PLoS One. 2009; 4:e5718.

Tadmouri G, -Thalassemia in Turkey: Distribution, diversity, evolution, and

phenotype-genotype correlations, Boazii University 1999.

Talati N, Seybold U, Humphrey B, Aina A, Tapia J, Weinfurter P, Albalak R,

and Blumberg HM, Poor concordance between interferon-gamma release assays
and tuberculosis skin test in diagnosis of latent tuberculosis infection among
HIV-infected individuals, BMC Infect Dis. 2009; 9:15.

Tavast E, Salo E, Seppl I, and Tuuminen T, IGRA tests perform similarly to

TST but cause no adverse reactions: pediatric experience in Finland, BMC Res
Notes. 2009; 2: 9.

The free dictionary, Medical dictionary, tuberculosis, www.medicaldictionary.thefreedictionary.com/tuberculosis, accessed on: 9 Nov. 2010.

Tiemersma EW, van der Werf MJ, Borgdorff MW, Williams BG,
and Nagelkerke NJ, Natural history of tuberculosis: duration and fatality of
untreated pulmonary tuberculosis in HIV negative patients: a systematic review,
PLoS One. 2011; 6:e17601.

Todar s online textbook of bacteriology, Mycobacterium tuberculosis and

tuberculosis, www.textbookofbacteriology.net/tuberculosis, accessed on: 2 Oct.
2010.

97

References
-

Tortoli E, and Palomino JC, New diagnostic methods in "Tuberculosis 2007

From basic science to patient care 1st"; Ed. Palomino JC, Leo SC, and
Ritacco V, www.tuberculosistextbook.com 2007: 441-486.

Tsiouris S, Coetzee D, Toro P, Austin J, Stein Z, and El-Sadr W, Sensitivity

analysis and potential uses of a novel gamma interferon release assay for
diagnosis of tuberculosis, J. Clin Microbiol 2006; 44:28442850

Tuberculosis coalition for technical assistance, International standards for

tuberculosis care (ISTC), The Hague: Tuberculosis coalition for technical
assistance 2006.

Van Pinxteren L, Ravn P, Agger EM, Pollock J, and Anderden P, Diagnosis of

tuberculosis based on the two specific antigens ESAT-6 and CFP10, Clin.
Diagn. Lab. Immunol. 2000; 7:155160.

Varaine F, Henkens M, and Grouzard V, Prevention in "Tuberculosis: practical

giude for clinicians, nurses, laboratory technicians and medical auxiliaries 5th
revised ed "; Ed. Varaine F, Henkens M, and Grouzard V; Pub. Medecins Sans
Frontieres 2010: 87-96.

Varaine F, Henkens M, and Grouzard V, The disease in "Tuberculosis: practical

giude for clinicians, nurses, laboratory technicians and medical auxiliaries 5th
revised ed "; Ed. Varaine F, Henkens M, and Grouzard V; Pub. Medecins Sans
Frontieres 2010: 15-53.

Varaine F, Henkens M, and Grouzard V, Treatment in "Tuberculosis: practical

giude for clinicians, nurses, laboratory technicians and medical auxiliaries 5th
revised ed "; Ed. Varaine F, Henkens M, and Grouzard V; Pub. Medecins Sans
Frontieres 2010: 57-84.

Verma RK, and Jain A, Antibodies tomycobacterial antigens for diagnosis of

tuberculosis, FEMS Immunol Med Microbiol 2007; 51:453461

Verver S, Warren RM, Beyers N, Richardson M, van der Spuy GD, Borgdorff
MW, Enarson DA, Behr MA, and van Helden PD, Rate of reinfection
tuberculosis after successful treatment is higher than rate of new tuberculosis,
Am J Respir Crit Care Med. 2005; 171:1430-5

Wikipedia, Interferon gamma, www.en.wikipedia.org/wiki/Interferon_gamma,

accessed on: 27 Jan. 2011.

World Health Organization, Overview of current TB diagnostics in "Diagnostics

for tuberculosis: Global demand and market potential"; Pub. WHO press 2006:
31-47.

World Health Organization, Molecular line probe assays for rapid screening of
98

References

patients at risk of multidrug-resistant tuberculosis (MDR-TB): Policy statement,

WHO press 2008.
-

World Health Organization, Commercial serdiagnostic tests for diagnosis of

tuberculosis, WHO press 2011.

World Health Organization, Fluorescent light-emitting diode (LED) microscopy

for diagnosis of tuberculosis: Policy statement, WHO press 2011.

World Health Organization, Global tuberculosis control 2010, WHO press 2010.

World Health Organization, New laboratory diagnostic tools for tuberculosis

control, WHO press 2008.

World Health Organization, Noncommercial culture and drug-susceptibility

testing methods for screening patients at risk for multidrug-resistant
tuberculosis: policy statement, WHO press 2011.

World Health Organization, Policy statement: automated real-time nucleic acid

amplification technology for rapid and simultaneous detection of tuberculosis
and rifampicin resistance: Xpert MTB/RIF system, WHO press 2011.

World Health Organization, Tuberculosis profile: Syrian Arab Republic,

www.who.int/tb/data, accessed on: 3 May 2011.

World Health Organization, Use of liquid TB culture and drug susceptibility

testing (DST) in low and medium income settings, WHO press 2007.

99


www.emro.who.int/umd/

Acid fast bacilli

Actinomycetales

Actinomycetes
Active

Acute

Alveolar
Amplification

Antagonist

Antigen presentation

Antigen presentig cell

Aquired immunity

Aspergillosis

Asthma

Biosynthesis

Biphase

Boosting

Bronchodilation

Broth

Brucellosis

Caseous
111

Chronic obstructive pulmonary disease

Compound

Control
Cross link

Cross reactivity

Culture

Culture media

Decontamination

Deep vein thrombosis

Dendritic cells

Diabetes melitus

Diminish

Effector
Embedded

Erythema

Exocytosis

Export

Extrapulmonary

Enrichment

F
Filtration

Fragments

Heatlabile

111

High-resolution gas chromatographic

Humoral immunity

Hydrophobic

Identification

Immunocompromised
Immunomodulator

Immunosuppressive

Immunosurveillance

Index

Indicator

Induration

Infection

Innate immunity
Inoculation

Insertion

Intermediators
Intracellular

Intrathoracic

Isothermal

Kit

L
Latent

Lesion

Leukemia

Lipopolysaccharide

Liquefaction

112

Loop
Lymphoma

Lysosome

Macrophages

Major histocompatibility complex

Malignancy

Mast cells

Metabolize

Microenvironment

Mitogen

Monitoring

Multiplication

N
Natural killer cell

Neutrophil

Nitrate reductase

Normal flora

Obligate aerobe

Observation

Opsonization

P
Phage

Phagocytosis

Phagolysosome

Phagosome

113

Pneumonia

Preparation

Primer

Probe

Promoter

Pthogenesis

Pulmonary

Pulmonary embolism
Pulmonary failure

Pulmonary fibrosis

R
Radioactive

Reactivation

Recruit

Redox

Reinfection

Residue

Respiratory burst

Reversion

S
Sensor

Severity

Silicosis

Smear

Statiionary
Stimulate

Strand

Substrate

Susceptibility

Symbiotic

114

T
) (

Tuberculosis

Vesiculation

Vital

115

)2(

Appendix (1)

No. Specimen

ZN
Smear

LJ Culture

IGRA

Real-time
PCR

Diagnosis

Sputum

Positive

Positive

Positive

Positive

TB infected

Sputum

Positive

Positive

Positive

Positive

TB infected

Sputum

Positive

Positive

Positive

Negative

TB infected

Sputum

Positive

Positive

Positive

Negative

TB infected

Sputum

Negative

Negative

Negative

Positive

Respiratory
infection

Sputum

Negative

Negative

Negative

Negative

Respiratory failure

Pleural
fluid

Negative

Negative

Positive

Negative

TB infected

Sputum

Positive

Positive

Positive

Positive

TB infected

Sputum

Positive

Positive

Positive

Positive

TB infected

10

Sputum

Positive

Positive

Positive

Positive

TB infected

Negative

Negative

Negative

Negative

Malignancy

Negative

Negative

Positive

Negative

TB infected

11
12

Pleural
fluid
Pleural
fluid

13

Sputum

Positive

Negative

Positive

Negative

TB infected

14

Sputum

Negative

Negative

Positive

Negative

TB infected

15

Sputum

Positive

Positive

Negative

Positive

TB infected

16

Sputum

Positive

Positive

Negative

Negative

TB infected

17

Sputum

Positive

Positive

Positive

Positive

TB infected

18

Sputum

Negative

Positive

Positive

Positive

TB infected

116

:)1(

No. Specimen

ZN
Smear

LJ Culture

IGRA

Real-time
PCR

Diagnosis

19

Sputum

Positive

Positive

Positive

Positive

TB infected

20

Sputum

Negative

Negative

Positive

Positive

TB infected

21

Sputum

Positive

Positive

Positive

Positive

TB infected

22

Sputum

Negative

Negative

Negative

Negative

Bronchiectasis

23

Sputum

Negative

Negative

Negative

Negative

Respiratory
infection

24

Sputum

Positive

Positive

Positive

Positive

TB infected

25

Sputum

Positive

Positive

Negative

Positive

TB infected

26

Sputum

Negative

Negative

Negative

Positive

Respiratory
infection

27

Sputum

Positive

Positive

Positive

Positive

TB infected

28

Sputum

Positive

Positive

Positive

Positive

TB infected

29

Sputum

Negative

Negative

Negative

Negative

Respiratory
Infection

30

Sputum

Positive

Positive

Positive

Negative

TB infected

31

Sputum

Positive

Positive

Positive

Positive

TB infected

32

Sputum

Negative

Positive

Negative

Negative

TB infected

33

Bronchial
wash

Positive

Negative

Positive

Positive

TB infected

34

Sputum

Positive

Positive

Positive

Positive

TB infected

35

Sputum

Negative

Positive

Positive

Positive

TB infected

36

Sputum

Positive

Positive

Positive

Positive

TB infected

37

Sputum

Positive

Positive

Positive

Positive

TB infected

38

Sputum

Negative

Negative

Positive

Negative

COPD + chronic
infections

39

Sputum

Positive

Positive

Positive

Positive

TB infected

117

:)1(

No. Specimen

ZN
Smear

LJ Culture

IGRA

Real-time
PCR

Diagnosis

40

Sputum

Negative

Negative

Negative

Negative

COPD + chronic
infections

41

Bronchial
wash

Positive

Positive

Positive

Positive

TB infected

42

Sputum

Negative

Negative

Negative

Negative

Respiratory
infection

43

Sputum

Positive

Positive

Positive

Positive

TB infected

44

Sputum

Negative

Positive

Positive

Positive

TB infected

45

Sputum

Negative

Positive

Positive

Negative

Silicosis

46

Sputum

Negative

Negative

Positive

Negative

TB infected

47

Sputum

Positive

Positive

Positive

Positive

TB infected

48

Bronchial
wash

Positive

Negative

Positive

Positive

TB infected

49

Sputum

Negative

Negative

Positive

Negative

TB infected

50

Sputum

Positive

Positive

Positive

Negative

TB infected

51

Sputum

Positive

Positive

Positive

Positive

TB infected

52

Sputum

Positive

Positive

Positive

Positive

TB infected

53

Sputum

Negative

Negative

Negative

Negative

COPD

54

Sputum

Positive

Positive

Positive

Negative

TB infected

55

Sputum

Negative

Negative

Negative

Negative

Malignancy

56

Sputum

Negative

Negative

Negative

Negative

PE-DVT

57

Sputum

Positive

Positive

Positive

Positive

TB infected

58

Sputum

Negative

Negative

Positive

Negative

Pulmonary fibrosis

59

Sputum

Negative

Negative

Negative

Negative

Malignancy

60

Sputum

Positive

Positive

Positive

Positive

TB infected

118

:)1(

No. Specimen

ZN
Smear

LJ Culture

IGRA

Real-time
PCR

Diagnosis

61

Sputum

Positive

Positive

Positive

Positive

TB infected

62

Sputum

Negative

Negative

Positive

Negative

COPD

63

Sputum

Negative

Negative

Positive

Negative

Malignancy

64

Sputum

Negative

Negative

Positive

Negative

Asthma

65

Sputum

Negative

Negative

Positive

Negative

COPD

66

Sputum

Negative

Negative

Negative

Negative

Respiratory
infection

67

Sputum

Negative

Negative

Positive

Negative

Pulmonary fibrosis

68

Sputum

Negative

Negative

Positive

Negative

Heart failure

69

Sputum

Negative

Negative

Negative

Negative

Respiratory
infection

70

Sputum

Negative

Negative

Positive

Negative

Pneumonia

71

Sputum

Negative

Negative

Negative

Negative

COPD

72

Sputum

Negative

Negative

Negative

Negative

Carcinoma

73

Sputum

Negative

Negative

Negative

Negative

Respiratory
infection

74

Sputum

Negative

Negative

Negative

Negative

Pneumonia

75

Sputum

Negative

Negative

Negative

Negative

76

Sputum

Negative

Negative

Negative

Negative

77

Sputum

Negative

Negative

Positive

Negative

TB infected

78

Sputum

Negative

Negative

Negative

Negative

COPD

79

Sputum

Negative

Negative

Positive

Negative

Aspergilosis

80

Sputum

Negative

Negative

Positive

Negative

Asthma

81

Sputum

Negative

Negative

Positive

Negative

TB infected

119

Respiratory
infection
Pneumonia +
malignancy

:)1(

No. Specimen

ZN
Smear

LJ Culture

IGRA

Real-time
PCR

Diagnosis

82

Sputum

Negative

Negative

Negative

Negative

Respiratory
infection

83

Sputum

Negative

Negative

Positive

Negative

Pneumonia

84

Sputum

Negative

Negative

Negative

Negative

Pulmonary fibrosis

85

Sputum

Negative

Negative

Negative

Negative

Asthma

86

Sputum

Negative

Negative

Negative

Negative

Respiratory
infection

87

Sputum

Negative

Negative

Negative

Negative

Brucellosis

88

Sputum

Negative

Positive

Negative

Negative

TB infected

89

Sputum

Negative

Negative

Positive

Negative

Pulmonary fibrosis

90

Sputum

Negative

Negative

Negative

Negative

Pulmonary fibrosis

Respiratory
infection
ZN, Ziehl neelsen; LJ, Lowenstein Jensens; IGRA, Interferon gamma release assay;

91

Sputum

Negative

Negative

Negative

Negative

PCR; Polymerase chain reaction; TB, Tuberculosis; COPD, Chronic obstructive

pulmonary disease; PE-DVT, Pulmonary embolism-deep vein thrombosis.

111

;)3(

Appendix (2)
The journal of infection in developing countries

111

 (;)4

)Appendix (3

112