Pasado y presente en el diagnóstico de

la infección tuberculosa latente

J. DomínguezServei Microbiologia. Institut d’Investigació Germans Trias i Pujol

Badalona, Barcelona. (jadomb@gmail.com)

XVI Taller Internacional sobre Tuberculosis

UITB-2012

LTBI diagnosis: Tuberculin skin test

o TST has been until now the only tool available for the diagnosis of

LTBI and is commonly used as complementary test for diagnosis of

active TB.

o Intradermal reaction test.

o TST attempts to measure cell-mediated immunity in the form of a

delayed-type hypersensitivity response to PPD.

o PPD contains more than 200 antigens that are widely shared among

mycobacteria other than M. tuberculosis, including the vaccinal strain

of Mycobacterium bovis BCG and many NTM.

o 4th August 1890. R. Koch. X International Medicine Congress. Berlin

o 15th November 1890. German Medicine Journal.

o Between 1890-1891 doubtful curative effect.

o January 1891. Description of the substance production (Koch lymph,

Koch fluid, Bacillinum, Kochin).

o February 1891. The label of the product was: Tuberculin

o 1906 C. Von Pirquet demonstrated that the reactivity of the tuberculin

evidenced a previous contact with the bacteria

o Classical studies using tuberculin has allow to establish ratio of

infected individuals, and also the % of patients who developed active

TB at some time during their life.

Tuberculin origin

LTBI diagnosis: Tuberculin skin testMain drawbacks

Low specificity

o Individuals sensitised by previous exposure to NTM or vaccinatedwith BCG respond immunologically to PPD.

Low sensitivity

o Low sensitivity in detecting LTBI in individuals with a high risk ofprogression to active TB: immunosuppressed patients (especiallywith deficient cellular immunity) and young children.

Logistical problems

o Errors in the administration and subjective reading of the results.

o Second reading visit. Some patients do not return to the readingof the TST result.

LTBI diagnosis: IFN-γ releasedassays (IGRAs)

In vitro detection of IFN-γ released by sensitised T cells afterspecific M. tuberculosis antigens stimulation.

P. Andersen. Lancet 2000

Moving LTBI diagnosis from clinical departments to laboratory.

A more accurate diagnosis.

LTBI diagnosis

2002

Tuberculin skin testing IGRAs

LTBI diagnosis: IFN-γ assaysSpecific antigens

ESAT-6, CFP-10 and TB7.7

Proteins coded in the region of difference 1 and 11, which are

present in M. tuberculosis but not in any BCG strain nor in themajority of NTM.

Comparison between TST and IGRAs

Collection of blood sample

(3ml) in QFN tubes containing

MTB antigens

T-SPOT.TB QFN-G-IT

1/2

ho

ur

(h)

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s-o

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ime

fo

r 1

sam

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4 h

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nd

s-o

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r 2

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am

ple

s

No

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s-on

time

1 h

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ime

fo

r

1 to

20

sa

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1 h h

an

ds-o

n tim

e

for 1 to

20

sam

ple

s

Overnight incubation

First day

Second day

ELISPOT

Count spots by naked eyeor using a plate reader

Centrifugation of tubesto harvest the IFN-gamma released

ELISA

Read the concentration of

IFN-gamma by means

of an automated reader

Collection of blood sample (8ml)

and centrifugation

Count PBMCs using a counting chamber

Addition of 250,000 cells per wellwith the MTB specific antigens

Isolation of PBMCs and washing

Overnight incubation

Higher specificity than TST

in BCG-vaccinated patients

Association with the degree of

exposure to a patient with active TB

Association with the degree of

exposure to a patient with active TB

De Souza-Galvao M. 2012.

Higher number of positive results

in immunosuppressed patients

IGRAs are less afected

by the immunosuppression than TST…

Latorre I. ERS. Amsterdam 2011

…although they are also affected

They followed a cohort of 954 contacts during 4 years. Among the 147 that did not

receive treatment with a positive QFN, 19 (12.9%) developed active TB,

while only 17 out of 551 (3,1%) with a TST>5mm developed active TB.

Positive and negative predictive value

Diel R. AJCCM 2010

Among the 824 contacts that did not receive treatment and with a negative QFN,

none of them progressed to disease, confirming the high NPV of the test.

Bakir. AIM 2008

908 children from contacts studies were included. During a period of 1.3 years of follow-up,

children with positive T-SPOT.TB had a risk of developing TB between 3 and 4 times higher

than patients with a negative T-SPOT.TB.

However, ratios of progression for T-SPOT-TB and TST were similar (3.86 vs 3.28).

Positive and negative predictive value

The PPV using commercial IGRAs was 2.7%,

compared with 1.5% for the TST (P<0.0001)

The PPV increased to 6.8% and 2.4%

for IGRAs and TST, respectively,

when only high-risk groups were considered

(P=0.0001)

The NPV for progression

for IGRAs was 99,7%,

and of 99.4% for the TST

(P=0.01)

668 Contacts

Follow-up during

30 months

284LTBI

TST positive and/orQFN positiveProphylaxis

98QFN & TST negativesPrimary prophylaxis

(in all cases a negative resultwas obtained two moths later)

286No LTBI

TST & QFN negatives,or positives but no

candidates x prophylaxis

8 active TB (All have

QFN i TST positive)

QFN-G-IT

• VPP=15%

• VPN=100%

PT

• VPP = 3%

• VPN = 99%

Positive and negative predictive value

Altet N. Submited 2012.

Remote infection?

147 HCWs

95 with

previous positive TST

T-SPOT.TB

(%)

POS45

(47.4)

NEG49

(51.6)

IND1

(1.1)

QFN-G-IT

(%)

POS34

(35.8)

NEG59

(62.1)

IND2

(2.1)

In 24 HCWs with both

IFN-gamma tests

negatives and non BCG

vaccinated PST was

performed (%)POS

2(20)

Test invalid in

14 cases

NEG8

(80)

IGRAs are not useful for diagnosing active TB

Diagnosis of active TB

Monitorization of the treatment

0 6 12 18 24 300

500

1000

1500

2000

Follow up, months

ES

AT

-6-s

pe

cif

ic I

FN

-g

SF

C /

10

6 P

BM

C

0 6 12 18 24 300

500

1000

1500

2000

Follow up, months

CF

P-1

0-s

pe

cif

ic I

FN

-g

SF

C /

10

6 P

BM

C

ESAT-6 CFP-10

There is a high inter-individual variability in the

celular response and in the amount of IFN-γ

released, during the treatment, and in addition, in a

substancial proportion of patients the results of the

IGRA remain positive althouhg they have finished

the treatmentMillington. J.Immunol 2007

Monitorization of the treatment

Conclusions

• Exposure and risk factor

association

• Higher specificity (no affected by BCG). Reduce unnecessary

prophylaxis.

• PPV higher/similar than TST and very good NPV

• Less afected by

immunosuppressor

treatments

IGRAs

• Improve positive predictive

value: differentiate between

those persons who will

develop TB and those who will not.

• Remote vs. recent infection

• Infection vs. active TB

• Monitoring of the treatment

• Severe immunosuppression

Waiting for the future…

The next generation of IGRAs

I have

a dream !

IGRAS

POSITIVE CONTROL

NEGATIVE CONTROL

SPECIFIC REMOTE

INFECTION ANTIGENS

SPECIFIC RECENT

INFECTION ANTIGENS

SPECIFIC ACTIVE TB ANTIGENS

PACIENT REMOTELY INFECTED

PACIENT RECENTLY INFECTED

PACIENT WITH ACTIVE

TB