Traditional and Novel Diagnostic Tests of TB Infection

Toru Mori, MD, PhD

Research Institute of Tuberculosis/JATANational Institute of Infectious Diseases, Japan

1st Asia Pacific Region Conference of IUATLDKuala Lumpur, Aug 2-5, 2007

Roles of TB Infection Roles of TB Infection DiagnosisDiagnosis

• Indication for treatment of LTBI• Adjunct to diagnosis of TB Disease• Decision making for contact actions• Monitoring of infection control• Epidemiological surveillance and

research

0%

5%

10%

15%

20%

25%

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38

Induration diamter (mm）

Tuberculin Reaction in TB PatientsTuberculin Reaction in TB Patients（ Bacteriologically Confirmed Patients, PPD 0.05mcg ）

Ｎｏ．　 602Mean 16.1mmS.D.　4.6mm

(Mori et al, 1975)

Tuberculin Reactions of Uninfected InfantsTuberculin Reactions of Uninfected Infants(Infants 0 to 3 Years(Infants 0 to 3 Years ））

86.4

0.10.10.20.20.5

0.71.0

1.51.6

3.0

4.6

0

2

4

6

8

10

0 4 8 12 16 20 24 28 32 36 40

Erythema (mm)

（％）

Total　 10,71010mm+ 2.9%20mm+ 0.29%30mm+ 0.02%

0.00.0 0.0

(Okinawa Pref, 1982)

Tuberculin Reaction after 6 Months of BCTuberculin Reaction after 6 Months of BCG VaccinationG Vaccination

(Infants, Erythema, N=103)

0%

5%

10%

15%

20%

25%

30%

35%

0-4 10-14 20-24 30-34 40-44

Erythema (mm)

Mean　 21.3mm>=10mm 91.3%>=30mm 13.6%

(Mori, 1980)

Problems of TST in Infection DxProblems of TST in Infection Dx

• Confounded by BCG history

• Confounded by environmental mycobacteria

• Booster phenomenon

• Variability in administering and reading

• Needs two visits

Specific Antigens ESAT-6/CFP-10Specific Antigens ESAT-6/CFP-10

Tuberculosis complexTuberculosis complexM. tuberculosisM. tuberculosis M. africanumM. africanumM. bovis M. bovis (Other than BCG)(Other than BCG) M. lepraeM. leprae

Environmental strainsEnvironmental strainsM. kansasiiM. kansasii M. marinumM. marinum M. szulgaiM. szulgaiM. flavescensM. flavescensM. gastriiM. gastrii

M. bovis BCG All substrains

Environmental strainsM. intracellulare M. avium

Present in Absent from

Interferon-gamma Release Assays (IGRAs)Interferon-gamma Release Assays (IGRAs)

Enzyme-linked Enzyme-linked Immunosorbent assayImmunosorbent assay

QuantiFERON-TB Gold T-SPOT.TB

Enzyme-linked Enzyme-linked immunospotimmunospot

PBMC separated

Whole bloodWhole blood

Plasma separated

Stimulation with Antigens

ESAT-6 CFP-10 ESAT-6 or CFP-10* ESAT-6 CFP-10 ESAT-6 or CFP-10*

0.0

0.5

1.0

1.5

2.0

10

20

30

2

TB Disease Low Risk for TB

n = 118

n = 220

n = 217 n = 217

n = 118 n = 118

IFN

-gam

ma

(IU

/mL

)Responses to CFP-10 & ESAT-6 for each study groupResponses to CFP-10 & ESAT-6 for each study group

Nursing students

Cut-off

(Mori et al, 2004)

0%

5%

10%

15%

20%

25%

30%

0 10 20 30 40 50 60 70 80

Induration diameter (mm)

TST Distribution and QuantiTST Distribution and QuantiFERON FERON (+)(+)

(BCG-vaccinated Healthy Subjects, N=220,

*QTF(+))

** **

(Mori et al, 2004)

63%

86%82%

86%

100%92%

82%

0%

20%

40%

60%

80%

100%

0-4 5-9 10-14 15-19 20-24 25+ Total

Induration size (mm)

QTF Positivity according to Mantoux test SizeQTF Positivity according to Mantoux test Size(TB Patients, Over-all QFT-Positivity = 82%, p for Linear trend

=0.002)

(Mori et al, 2004)

Sensitivity, TB PatientsSensitivity, TB Patients QFT-G, ESAT-6+CFP-10QFT-G, ESAT-6+CFP-10

Pooled 　 0.81 (0.76-0.84)Chi-sq=17.1, df=8, p=0.03I2 = 53%

Sensitivity, TB PatientsSensitivity, TB Patients TSTTST

Pooled 　 0.73 (0.67- 0.78)Chi-sq=15.2, df=7, p=0.03I2 = 54%

1.00 (0.54 - 1.00)0.83 (0.63 - 0.95)0.83 (0.61 - 0.95)0.78 (0.64 - 0.88)0.74 (0.63 - 0.82)0.70 (0.46 - 0.88)0.66 (0.54 - 0.76)0.33 (0.07 - 0.70)

QFT-G & QFT-GIT ComparedQFT-G & QFT-GIT ComparedUntreated TB PatientsUntreated TB Patients

QFT-GIT

Positive Negative Total

QFTG

Positive77

(81.9%)

1

(1.1%)

78

(83.0%)

Negative10

(10.6%)

6

(6.4%)

16

(17.0%)

Total87

(92.6%)

7

(7.4%)

94

(100%)

Sensitivity QFT-G=83.0% QFT-GIT=92.6% (p=0.006) kappa=0.466(Harada et al, submitted)

Sensitivity, TB PatientsSensitivity, TB Patients QFT-G-IT, ESAT-6+CFP-10+TB7.7QFT-G-IT, ESAT-6+CFP-10+TB7.7

Pooled 　 0.78 (0.71- 0.83)Chi-sq=27.5, df=4, p=0.000I2 = 89%

Specificity, BCG-vaccinatedSpecificity, BCG-vaccinated Low-riskLow-risk QFT-G, ESAT-6+CFP-10 (+TB7.7)QFT-G, ESAT-6+CFP-10 (+TB7.7)

Pooled 　 0.97 (0.95- 0.98)Chi-sq=13.5, df=5, p=0.019I2 = 63%

1.00 (0.82-1.00)0.99 (0.96-1.00)0.98 (0.95-0.99)0.97 (0.87-1.00)0.96 (0.90-0.99)0.92 (0.85-0.96)

0

2

4

6

8

10

12

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 1100

2

4

6

8

10

12

14

16

18

20

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110

Index Case ： A Student, aged 22 yearsIll for 2 mos, Heavily Smear Positive, Secondary cases: 12Close contacts: 220 QFT(+) 32.7 ％ (+-) 15.9 ％ (-) 52.3 ％

Other contacts: 135 　 QFT(+) 0.7 ％ (+-) 0.7 ％ 　 (-) 98.5 ％

（ Funayama et al, 2005)

A TB Outbreak in a University

QFT-Negative N=148 QFT-Positive N=72

Results Age in mosTotal (N=195)

QFT TST <36 mos (N=113) >36mos (N=82)

+ + 14 14 28

+ - 4 1 5

- + 7 12 19

- - 88 55 143

QFT(+)% 15.9 (9.2 – 22.7) 18.3 (9.9 – 26.7) 16.9 (11.7 – 22.2)

TST(+)% 18.6 (11.4 – 25.8) 31.7 (21.6 – 41.8) 24.1 (18.1 – 30.1)Agreement 0.903 0.841 0.877

κ 0.660 0.587 0.626

（ Submitted）

QFT/TST on TB Contact ChildrenQFT/TST on TB Contact Children（ Cambodia, 0 ～ 5 yo ， N=195 ， Okada et al, 2006)

Indian TB Suspects Study(1-12 yrs, N=106, In-Tube QFT-G, Dogra, 2006)

QFT+ QFT- Total Agreement κ-coeff.

Cut-off:>=5mm

TST+ 8 8 16

0.8950.53

(0.34-0.71)TST- 3 86 89

Total 11 94 105

Cut-off:>=10mm

TST+ 8 2 4

0.9520.73

(0.53-0.92)TST- 3 92 95

Total 11 94 99

Cut-off:>=15mm

TST+ 4 0 4

0.9330.50

(0.33-0.66)TST- 7 94 101

Total 11 94 105

Indian TB Suspects Study (cont’d)(1-12 yrs, N=106, In-Tube QFT-G, Dogra, 2006)

TST(>=10mm) QFT (0.35IU/mL)

No.Pos

/Tested (%)OR, adjusted

No.Pos

/Tested (%)OR, adjusted

Age

1-4 yrs 2/42 (5) 1 2/42 (5) 1

5-9 yrs 2/33 (6) 1.16 (0.14,9.49) 3/33 (9) 2.02 (0.30,13.5)

9-12 yrs 6/30 (20) 5.69 (0.95,33.8) 6/30 (20) 5.92(1.02,34.5)

Contact

No 7/89 (8) 1 8/89 (9) 1

Yes 3/16 (19) 2.48 (0.51,11.9) 3/16 (19) 2.00 (0.42,9.35)

QFT Level at TB Detection among Contacts（ Comparison with LTBI, Harada et al, 2007, submitted)

Active TB LTBI0.1

1

10

100

IFN

-ga

mm

a p

rod

uc

tio

n (

IU/m

l)

　 　 　 　 　 　 　 　

Adj.O

R95%CI p

Sex 0.8450.31

72.25

20.73

6

Age group

1.6331.05

42.53

0.028

TST 0.7170.44

61.15

20.16

9

QFT (CFP/ESAT)

1.8491.20

92.82

90.00

5

N=35　　　　 N=76

Factors contributing to TB Onset(Multivariate analysis)

TB Risk according to Parameter Value（ Induration size for TST, Quartile grade for QFT）

11.14

1

2.48

1

1.40.950.780.951

4.04

1.35

2.481.58

1.131.59

0.1

1

10

1 2 3 4 1 2 3 4 1 2 3 4 Od

ds

rati

o

-14　 -19　 -24　 25+

TST ESAT-6 CFP-10 ESAT/CFP*

*Chi2 for trends p=0.028 （ Harada et al, submitted)

QFT in Healthy General QFT in Healthy General PopulationPopulation

(Japan, Rural Community, N=1,559)(Japan, Rural Community, N=1,559)

7%

10%

6%

3%

0%

5%

10%

15%

40-49 (291) 50-59 (607) 60-69 (661) Total

Age (years)

%

(Mori et al, 2007)

Age- group 1950 2005

0- 8 0.1

5- 25 0.3

10- 38 0.6

15- 50 0.9

20- 59 1.3

25- 66 1.8

30- 72 2.7

35- 77 4.2

40- 81 6.8

45- 85 11

50- 88 18

55- 90 29

60- 92 42

65- 93 53

70- 94 61

75- 95 68

80- 96 74

Age-specific Prevalence of TB InfectionAge-specific Prevalence of TB Infection（ Japan, Estimated, Years 1950 & 2005)

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80

Age (years)

19502005

(Mori, 2005)

QFT in Healthy General QFT in Healthy General PopulationPopulation

(Comparison with Estimated Prevalence of TB Infection)

8%

22%

47%

30%

7%10%

6%3%

0%

10%

20%

30%

40%

50%

40-49 50-59 60-69 Total

Age (years)

%

QFT(+) Estimated

(Mori et al, 2007)

QFT according to Types of X-ray TB QFT according to Types of X-ray TB FindingsFindings

(Predicted: Expected from Rate of those with No TB Finding, Age (Predicted: Expected from Rate of those with No TB Finding, Age adjustedadjusted ））

11.1%

19.4%

6.9% 7.6%

13.7%

6.5% 7.1%

0%

10%

20%

30%

No TB Finding Possible Probable Certain

ObservedPredicted

（ 1,359） （ 51） （ 45） （ 31）

［ Example ］　 Certain ： Fibrotic lesion Probable: Calcification ・ Pleural adhesion ． Possible ： Apical cap

1 1.93 1.60 2.54

(Mori et al, 2007)

Change in QFT after Treatment of LTBI (1)Change in QFT after Treatment of LTBI (1)

ESAT-6 CFP-10

0.999 >> 0.272 IU/ml (p=<0.001) 0.346 >> 0.124 IU/ml (p=004)GeometricMean

0.01

0.1

1

10

100

1 2

Before After

IFN

-g p

rodu

ctio

n (I

U/m

l)

0.001

0.01

0.1

1

10

100

1 2

Before After

IFN

-g p

rodu

ctio

n (I

U/m

l)

(Higuchi et al, 2007)

Change in QFT after Treatment of LTBI (2)Change in QFT after Treatment of LTBI (2)(TB Outbreak in a mental hospital, with >15 secondary cases)(TB Outbreak in a mental hospital, with >15 secondary cases)

6 mos treatment completed ( 7(25%) reverted )

N=28 ESAT-6 0.999 0.272

CFP-10 0.346 0.124

Interrupted (< 3 mos) ( No reversion )

N=5 ESAT-6 0.743 0.729

CFP-10 0.595 0.572

1.5 Years after Treatment ( No net reversion )

N=17 ESAT-6 0.381 0.442

CFP-10 0.087 0.192(Higuchi et al, 2007)

89.6 90.9

75.6

46.2

0

20

40

60

80

100

0 1 6 15

Treatment month

Pos

itiv

e ra

te %

QFT Profile in TB Patients during and after ChemotherapyQFT Profile in TB Patients during and after Chemotherapy(N=50, Aoki et al, 2006)(N=50, Aoki et al, 2006)

IGRAs in Special SettingsIGRAs in Special Settings1. Children1. Children

• QFT(+) in 30 clinically diagnosed TB patients aged 0-14 years in Japan (Takamatsu et al, 2007); 77% (62 – 92%)

• For 41 patients aged 0-5 years in Italy (Russo, 2007); 88.0-95.6%

• In India (Dogra et al, 2006) and Cambodia (Okada et al, 2007), QFT-G gave results comparable with TST in family contact children.

• In Nigeria QFT-GIT detected more LTBIs than TST, regardless of age (0-4/5-9/10/14) (Nakaoka et al, 2007)

• IFN-G response to mitogen is lower in young infants, causing more “Indeterminate” results. (Harada et al, 2007)

IFN-G Release to Mitogen according to Age(Age: 0-95 years, N=12,856)

0

2

4

6

8

10

12

14

16

18

0 5 10 15 20 30 50 70 90

Age in years

IU/m

L

4.8%

1.7%

1.1%

0.5% 0.6%

2.0%

0.0%

1.0%

2.0%

3.0%

4.0%

5.0%

0-4 5-9 10-14 15-19 20-59 60+

Age group (years)

Mean value Frequency of “Indeterminate”

(Harada et al, unpublished)

IGRAs in Special SettingsIGRAs in Special Settings2. Aged subjects2. Aged subjects

34.5%

23.8%

9.1%

26.2%

0%

10%

20%

30%

40%

-79(29) 80-89(21) 90+(11) Total(61)

Age in yrs (Number tested)

(Nursing home residents, N=61, Mean age =79.9 yrs, Chi-square for linear trend=2.68, p=0.101)

(Suzuki, Harada et al)

In other sample, the “indeterminate” results are commoner in the aged

subjects; 2.0% for 60+years vs 0.6 for 20-59 years. (p=0.00)

QFT-G vs TST by Age in TB PatientsQFT-G vs TST by Age in TB Patients

90100 94 100

90 92

80

93100

58

75

50

75

55

17

64

0

20

40

60

80

100

-30,n=19

31-40,n=14

41-50,n=16

51-60,n=19

61-70,n=19

71-80,n=13

>80,n=10

Total,n=110

Age, Number of subjects

Pos

itiv

e ra

te, %

QTF

Mx

IGRAs in Special SettingsIGRAs in Special Settings3. Immunocompromized hosts3. Immunocompromized hosts

• In rheumatic patietns receiving immuno-suppressive therapy IFN-gamma assay was superior compared to the TST for detection of LTBI. (Matulis et al, 2007)

• In HIV+ patients, those with a CD4 count ≤ 200 were more likely to have an indeterminate test 200 were more likely to have an indeterminate test result. Further studies are needed to assess utility of IGRAs. (Talati et al, 2007)

• In hematological malignancy patients, IGRA produced more positives than TST. (Losi et al, 2007)

QTF-TB2G as Compared with QTF-TB2G as Compared with TSTTST

• Strengths– Specific– Sensitive – Reliable– Needs one visit

only– No booster effect

• Weaknesses– Cost– Needs whole blood– Labor intensive– Stimulation <12hrs– Technical/

Instrument

Uses of QFTTB Control of Healthcare Workers

On Employment： Replace Two-Step TSTWhen exposed to TB: without TST

Contact InvestigationWith / without TST

Clinical DiagnosisDifferentiate TB / NTM / Tumor . . . . Prescribe Rx of LTBI in High Risk Subjects

(Jap Soc TB, 2006)

Further Research NeedsFurther Research Needs

PerformancesTime from Infection to Positive ConversionRelationship with Risk of Clinical BreakdownInfluence of TB Treatment, Response in “Old” InfectionsDifference in Response between different antigensDifference from TST: Effect of prolonged incubation (effect

or vs memory cells?)Children & Infants

ProceduresTest with Smaller amount of Blood （ for Children ）Time until Stimulation→QFT-3G

Costs

Acknowledgment

• Collaborators– Dr. Harada N (RIT/JATA), – Dr. Higuchi K (RIT/JATA), – Dr. Takamatsu (Osaka Municipal Respiratory &

Allergy Center)

• Funded partly by the Emerging and Reemerging Diseases Study Grant, Ministry of Health, Labor & Welfare, Japan.