Interferon Gamma Release Assays in Denmark, population ......Interferon Gamma Release Assays in...

U N I V E R S I T Y O F C O P E N H A G E N

F A C U L T Y O F H E A L T H A N D M E D I C A L S C I E N C E S

PhD thesis

Thomas Stig Hermansen

Interferon Gamma Release Assays in

Denmark, population-based studies from a

tuberculosis low-incidence country

Academic supervisors: Pernille Ravn and Troels Lillebaek

Submitted to the University of Copenhagen April 15th 2016

PhD Thesis Thomas Stig Hermansen

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ACADEMIC SUPERVISORS:

Pernille Ravn, MD, PhD

Department of Pulmonary and Infectious Diseases, Nordsjaellands Hospital, Hillerød, University

of Copenhagen, Hillerød, Denmark

Troels Lillebaek, MD, DMSc

International Reference Laboratory of Mycobacteriology, Statens Serum Institut, Copenhagen, Denmark

EVALUATION COMMITTEE

Chair

Ole Kirk, MD, DMSc

Department of Infectious Diseases, Copenhagen University Hospital Rigshospitalet, Denmark

Assessors

Christian Wejse, MD, PhD

Department of Infectious Diseases, Aarhus University Hospital, Skejby, Denmark

Frank van Leth, MD, MSc, PhD

Amsterdam Institute for Global Health and Development, Netherlands

This thesis was handed in April 15th 2016 for evaluation at the University of Copenhagen. The PhD

defence will take place at Copenhagen University Hospital, Hillerød, June 27th 2016, 14.00 at the

auditorium.

The PhD defense will take place Copenhagen University Hospital, Hillerød, at the 15 th

of September etcetc


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PUBLICATIONS (referred to by their roman numerals in the text)

STUDY I (1)

Hermansen TS, Thomsen VO, Lillebaek T, Ravn P. Non-Tuberculous Mycobacteria and the

Performance of Interferon Gamma Release Assays in Denmark. PLoS One 2014 Apr

4;9(4):e93986.

STUDY II (2)

Hermansen TS, Lillebaek T, Hansen AB, Andersen PH, Ravn P. QuantiFERON-TB Gold In-Tube

test performance in Denmark. Tuberculosis 2014 Dec;94(6):616-621.

STUDY III (3)

Hermansen TS, Lillebaek T, Kristensen KL, Andersen PH, Ravn P. Prognostic value of

interferon-γ release assays, a population-based study from a TB low-incidence country.

Thorax 2016 Mar 30.


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CONTENTS

PREFACE .......................................................................... 4

Acknowledgements ................................................................. 4

Abbreviations .......................................................................... 5

Funding .................................................................................... 5

INTRODUCTION .............................................................. 6

1. BACKGROUND ............................................................. 7

1.1. The TB spectrum ............................................................. 7

1.2. Diagnosis of active TB .................................................... 8

1.3. Diagnosis of latent TB .................................................... 8

1.4. Interferon gamma release assays ………….................. 9

1.5. QuantiFERON-TB Gold In-Tube Test ...................... 10

1.6. Non-tuberculous mycobacteria ................................... 10

2. OBJECTIVES .............................................................. 12

3. METHODOLOGICAL CONSIDERATIONS .......... … 13

3.1. Settings, design and populations ...................…..……… 13

3.2. Data sources .................................................................. 13

3.3. Statistics ......................................................................... 14

4. MAIN RESULTS ......................................................... 16

4.1. Study I .............................................................................. 16

4.2. Study II............................................................................. 16

4.3. Study III ........................................................................... 16

5. DISCUSSION ............................................................... 18

5.1. QFT Performance…….……..……................................. 18

5.2. Non-tuberculous mycobacteria .................................... 18

5.3. QFT performance in extreme age groups .................. 19

5.4. Predictive value of the QFT .......................................... 20

6. LIMITATIONS ................................................... ……. 21

6.1. Bias ................................................................................ . 21

6.2. QFT variability ............................................................ 21

6.3. QFT reproducibility and repeat indeterminate

tests…. .................................................................................. 22

6.4. Screening indication.................................................... 22

7. PERSPECTIVES ........................................................ 22

7.1. The local perspective, IGRA use in Denmark ....... .. 22

7.2. The global perspective, IGRA use worldwide ....... .. 23

7.3. The future .................................................................... .. 23

SUMMARY ...................................................................... 25

DANSK RESUME ........................................................... 26

REFERENCES ................................................................ 27

STUDY I .......................................................................... 32

STUDY II ......................................................................... 41

STUDY III ....................................................................... 48


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PREFACE

ACKNOWLEDGEMENTS

This thesis would not have been possible without the help

and support from a number of people.

Pernille Ravn, the principal supervisor, has been

indispensable in planning the articles on which this thesis is

based. I am grateful for all your encouragement, guidance,

constructive criticism and introduction to the world of TB.

Troels Lillebaek, director for IRLM, my daily work place

throughout the majority of this thesis, has provided valuable

help always finding time for advice and fruitful discussion.

Thank you for providing an inspiring environment for young

researchers to learn and grow.

I am grateful to my colleagues at the PhD office, Mathias

Klok Pedersen, Dorte Bek Folkvardsen, Julie Prahl and

Karen Bjoern-Mortensen. We have created a fantastic

working environment that I will surely miss in the future.

I am indebted to Erik Svensson and Michael Rasmussen for

sparring, always keeping a door open to discuss R statistics,

data extracts or something else completely.

The Department of Pulmonary and Infectious Diseases,

Nordsjaellands Hospital, Hillerød, has provided me with the

opportunity to initiate this PhD and enrol as a PhD student.

Thanks to the entire department, in particular the research

group at LIA: Dorthe, Ellen, Andreas, Pelle, Stine, Gertrud,

Camilla and Kristina for sparring and exchange of ideas on

research meetings and conferences around the world.

Thanks to the staff at IRLM, for putting up with me through

the last years and for always being willing to assist.

The former director for IRLM, Vibeke Østergaard Thomsen,

gave me the opportunity to use data from the department

and perform the projects in this thesis; for that I am very

grateful. Peter H. Andersen has been very valuable in

providing data extracts regarding notified TB cases.

Last, but most important, I am grateful to my friends and

family, Louise, Sebastian, Karl August and Selma, for

putting everything into perspective and having made my life

enjoyable and versatile outside the world of TB for the last

three years.

Thomas Stig Hermansen, April 2016


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ABBREVIATIONS

AFS – Acid Fast Staining

ATS – American Thoracic Society

BCG – Bacille Calmette-Guérin

CDC – Centers for Disease Control and Prevention

CI – Confidence Interval

CFP-10 – Culture Filtrate Protein-10

DIDE – Department of Infectious Disease Epidemiology

ELISA – Enzyme-linked Immunosorbent Assay

ESAT-6 – Early Secretory Antigenic Target-6

FDA – United States Food and Drug Administration

IDSA – Infectious Disease Society of America

IGRA – Interferon-gamma release assay

IRLM – International Reference Laboratory of

Mycobacteriology

KP – Kaplan Meyer

LIMS – Laboratory Information and Management System

LTBI – Latent M. tuberculosis Infection

MAC – Mycobacterium Avium Complex

Mtb – Mycobacterium tuberculosis

NICE - National Institute for Health and Care Excellence

NNT – Number Needed to Treat

NPV – Negative Predictive Value

NTM – Non-tuberculous mycobacteria

OPUS – The name of a patient administration and

reporting system

PCR – Polymerase Chain Reaction

PHA – Phytohaemagglutinin

PPV – Positive Predictive Value

PY – Person Years of follow-up

QFT – QuantiFERON-TB Gold in-Tube Test

TB – Tuberculosis

TB7.7 – TB protein 7.7kD

TBNET – Tuberculosis Network European Trials Group

TST – Tuberculin Skin Test

TU – Tuberculin Unit

TNF – Tumour Necrosis Factor

WHO – World Health Organization

FUNDING

The PhD fellowship was funded by the IRLM, Statens

Serum Institut, The Research Council at Nordsjaelland

Hospital, Hillerød and Olga Bryde Nielsens Fond.


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INTRODUCTION

The aim of this thesis is to evaluate the performance of the

QuantiFERON-TB Gold In-Tube Test (QFT) in tuberculosis

(TB) and latent Mycobacterium tuberculosis infection (LTBI)

– often referred to as active TB and latent TB – as well as

the ability of the QFT to differentiate between TB and

disease caused by non-tuberculous mycobacteria (NTM).

The QFT is the most widely used immunological test to

diagnose LTBI and belongs to the group of Interferon

Gamma Release Assays (IGRA). By evaluating the current

use and limitations of these assays, we can potentially

advance in the seemingly never-ending task to control and

fight TB. IGRAs are considered an important asset in the

diagnosis of infection with Mycobacterium tuberculosis

(Mtb), and in many TB low-incidence, high-income

countries, they have replaced the tuberculin skin test (TST)

as the preferred screening tool.

The development of the IGRAs was made possible by studies

performed at Statens Serum Institut (SSI) by Professor

Peter L. Andersen and his group (4). Almost two decades

ago, they identified the mycobacterial antigens used in these

tests. The International Reference Laboratory of

Mycobacteriology (IRLM) at SSI was at the frontier when it

came to introducing the first commercial test using the new

antigens. The third generation of this test, the QFT, was

first used in Denmark late 2005, and in the following five

years, the QFT was almost exclusively performed at IRLM.

Consequently, the results of the nationwide coverage of TB

cases and performed QFT’s could be obtained from a single

laboratory. This provided a unique opportunity to combine

data on all notified TB cases and all cultured NTM cases

with QFT data, allowing us to create a cohort of persons

screened with a QFT that was useful for large-scale

evaluation of test performance.

This cohort serves as the basis for all studies in this thesis.


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1. BACKGROUND

1.1. THE TB SPECTRUM

TB has unfortunately maintained a status as one of the

world’s most important infectious diseases. Alongside HIV,

it is a leading cause of mortality and morbidity worldwide

(5). Although a reported decrease in TB cases and TB related

deaths has taken place over the last decades, the disease

seems in no way to be controlled or controllable with

available measures. An estimated 1.9 billion people are

harbouring Mtb (6) and serve as a reservoir for future TB

disease. In 2014, the WHO estimated 9.6 million new TB

cases and 1.5 million deaths due to TB, with the vast

majority of both TB cases and fatalities occurring in the

developing world (5). In Denmark, over the last decade, the

occurrence of TB has remained at a constant level of 300-400

new cases per year, with approximately two thirds of these

cases found among the immigrant population (7).

Infection with Mtb begins with the inhalation of infectious

droplet nuclei containing the bacteria. The bacteria reaches

the lungs and are ingested by alveolar macrophages (8)

where they are able to replicate, and through an immune

reaction involving macrophages, T lymphocytes, B

lymphocytes, and fibroblasts create the primary site of

infection, referred to as the Ghon focus. Within this

granuloma, the host’s immune response is suppressed

allowing Mtb to survive and replicate (9). The majority of

infected persons will then remain infected without clinical

symptoms and are said to have LTBI, in general referred to

as “latent TB”. The bacteria are viable but contained by the

immune system without evidence of clinical disease, and the

majority of patients will never progress to overt disease. A

proportion will however develop clinical manifest TB with

ongoing bacterial replication, in general referred to as

“active TB”.

The general perception that infection with Mtb exists solely

as active TB or latent TB has been challenged over the last

decade (10,11), and the different ways in which the causative

agent, Mtb, can impact the host are being recognized as a

continuum, rather than a binary process (12). This

continuum spans from (i) early elimination of Mtb by the

innate immune system without any immunological priming

to (ii) the latent state that evokes an acquired immune

response, but does not surface as a clinical significant

disease to (iii) active TB, responsible for 1.5 million deaths

every year (5). These observations have led to speculations

on how Mtb infection perhaps should be considered more a

spectrum rather than separate entities (13) (Figure 1).

The lifetime risk of developing active TB, once latently

infected, is suggested to be 5-10% (14), with increased risk

the first two years after infection, where half of the cases

occur (15,16). This “reactivation” happens when the

bacterial load increases (Figure 1) and the infection can no

longer be contained by the host immune system. Essential

in controlling TB, especially in low-incidence countries

Figure 1. Depiction of a proposed

framework for considering tuberculosis

infection as a spectrum (Adapted from

(13)).


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where the majority of TB cases are due to reactivation (17),

is the prevention of disease. Thus, it is important to identify

individuals latently infected, who could benefit from

preventive treatment. Administration of preventive

treatment in a population with LTBI prevents 60-90% from

reactivation (18,19). Determinants of progression to active

TB are numerous, and are often a mixture of host and

environmental factors (12), that increase the risk of

progression from latent TB. Such known host factors are

suppressed cellular immunity by HIV infection (20,21),

tumour necrosis factor alpha (TNF-α) inhibitors (22),

glucocorticoids (23), transplantation (24,25), advanced age

(26) and diabetes (27). Reported environmental

determinants are prison stay (28), homeless status (29),

recent immigration from a TB high-burden country (30), and

recent TST conversion (31).

Depending on symptoms, bacterial load and recognition by

the immune system, different treatment strategies may be

used. Active TB disease with a drug susceptible strain is

traditionally treated during 6 months using the standard

four-drug regime consisting of isoniazid and rifampicin in

combination with ethambutol and pyrazinamide for the first

2 months (intensive phase) and isoniazid and rifampicin

alone for the subsequent 4 months (continuation phase).

LTBI may be treated by a variety of different regimes

depending on availability of drugs, compliance and adverse

effects. The WHO 2015 guidelines on LTBI recommend five

options for the treatment of LTBI: isoniazid monotherapy

daily for 6 or 9 months, the combination of rifampicin and

isoniazid daily for 3-4 months, the combination of

rifapentine and isoniazid once a week for 12 weeks, and

rifampicin as monotherapy daily for 3-4 months (32). In

Denmark, official guidelines suggest a 6 month course of

isoniazid therapy for the treatment of LTBI (33).

1.2. DIAGNOSIS OF ACTIVE TB

TB is the symptomatic disease caused by bacteria from the

M. tuberculosis complex (M. tuberculosis, M. africanum, M.

bovis, M. canetti, M. microti (34-36)). Classic symptoms of

pulmonary TB, although not necessarily present, include

chronic cough (sometimes with blood-tinged sputum),

weight loss, fever, and night sweats (37). The reference

standard for microbiological diagnosis of TB is culture, but

this requires specialized laboratory facilities, and bacterial

growth can take weeks or longer. Therefore, direct

microscopy of bacteria using acid fast staining (AFS) from

sputum samples remains the most common diagnostic

method worldwide. Various staining methods exist, but the

Ziehl-Neelsen stain, by which bacteria is dyed and viewed

under oil immersion or the newer auramine-rhodamine

stain (38) using fluorescence microscopy, are commonly

used. AFS is inexpensive and simple to perform, but lacks

sensitivity and specificity, as it cannot distinguish

mycobacterial species. In recent years, molecular methods to

detect TB have improved, and they are used increasingly in

both low- and high-incidence countries. PCR-based methods

such as the Xpert MTB/RIF (Cepheid) and the Geno-Type

line probe assays (HAIN Lifescience) offer advantages due

to the fast detection of Mtb and possible resistance to first

line anti-TB drugs. However, these methods detect bacterial

DNA solely and cannot separate live from dead bacteria -

consequently they are not useful for treatment monitoring.

In Denmark, the microbiological detection of TB is based on

direct smear microscopy using auramin-rhodamine stain as

well as culture in fluid and solid media for up to 56 days

using MGIT 960 and Löwenstein-Jensen slants respectively.

In addition, molecular methods are used to obtain rapid

results regarding susceptibility, species identification and to

monitor overall transmission dynamics (genotyping

methods). All TB cases are notifiable by the treating

physician, including culture-negative cases diagnosed based

on clinical criteria, radiological findings, histology,

treatment response, and sometimes also on immunological

tests (TST and IGRA). In Denmark, the proportion of

culture-negative TB cases was 23.5% from 1992 through

2011 (39) (including notified cases where no specimens were

sent for culture).

1.3. DIAGNOSIS OF LATENT TB

Currently, no gold standard for diagnosing latent TB exists,

and it is not possible to find viable bacteria directly. Instead,

indirect methods are used either in vivo by the widely used

TST or in vitro using the newer, blood based IGRAs. Both

methods depend on cell-mediated immunity and recognition

of mycobacterial antigens, as shown by the illustration from

Andersen et al (4) (Figure 2).


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The tuberculin skin test

The TST contains a purified protein derivative tuberculin

unit (TU) which is a protein fraction from a Mtb strain. The

standard is injecting five TU intradermal using the Mantoux

technique. If cell-mediated immunity is present, the person

will develop a delayed-type hypersensitivity reaction

causing local inflammation and skin induration. After 48-72

hours, if an induration appears, the result is given by the

transverse diameter of the induration. However, pre-test

probability of infection, induration size and risk of TB

disease should be taken into consideration when

interpreting TST reaction (40). Also, the TST has important

limitations. The need for a return visit in 48-72 hours to read

the skin reaction is a logistic drawback, and because of

manual reading, the reproducibility of the test may be a

problem. Furthermore, boosting caused by a previous TST

and resulting hypersensitivity can make serial or repeat

testing difficult to interpret (41). More important, false

positive results can occur due to infection with the group of

NTM and if the person has been vaccinated with the M. bovis

Bacille Calmette-Guérin (BCG) strain. These false positive

results are caused by a cross-reaction between the non-

specific Mtb protein mixture contained in the PPD with the

majority of the NTMs (42) and the BCG strain (43). False

negative results of the TST is known to occur in

immunocompromised persons (due to both medical

conditions and immunosuppressive therapy), where test

sensitivity is poor (44). However, the TST remains the most

widely used test worldwide, requiring less laboratory setup

than the newer IGRAs.

1.4. INTERFERON GAMMA RELEASE ASSAYS

IGRAs are in vitro, blood based assays, which have their

main use in diagnosing Mtb infection. They cannot

discriminate between active disease and latent infection,

and are not intended as a stand-alone diagnostic test for TB,

but in certain situations they can however aid in the TB

diagnosis.

IGRAs are based on a cell-mediated release of interferon

gamma (IFN-γ) from T-lymphocytes, after stimulation with

antigens specific for Mtb. When Mtb enters the human host

(and is not eliminated by the innate immune response, see

Figure 1), its antigens will evoke a cellular response with

sensitization of T-lymphocytes by antigen presenting cells

(Figure 2). The sensitized T-lymphocytes are stimulated by

Mtb specific peptides, and recognition of these peptides in

the presence of major histocompatibility complex class II will

result in the production of different biomarkers, one of these

being IFN-γ. Infected individuals will have an IFN-γ level

above the threshold and the test will be positive. The basis

of these assays is a quantification of the IFN-γ production.

The peptide antigens in the IGRAs are the ESAT-6 (Early

Secretory Antigenic Target-6) and CFP-10 (Culture Filtrate

Protein-10) and the genes encoding these proteins are

located in the region of difference 1 locus in the Mtb genome,

whereas TB7.7 (used in the QFT) is encoded in region of

difference 11 (45). IGRAs have superior specificity compared

to the TST because the antigenic regions are not encoded in

the majority of NTM (except M. gastrii, M. kansasii, M.

marinum, M.riyadhense and M. szulgai (42)) or the vaccine

strain M. bovis BCG (46).

Two commercially available IGRAs exist, the T-Spot.TB (T-

Spot, Oxford Immunotec, Abingdon, UK) and the

QuantiFERON-TB Gold In-Tube Test (QFT, Cellesties,

Quiagen, Chadstone, Australia). The T-Spot utilizes an

enzyme-linked immunosorbent pot assay and report the

number of IFN-γ producing T-lymphocytes that respond to

ESAT-6 and CFP-10. If the spot count in the Mtb antigen

well reaches a predefined threshold after subtraction of the

count in the negative control well, the test is positive. The

Figure 2. Recognition and measurement of response to

mycobacterial antigens by the TST and the in vitro blood

tests (4). Abbreviations: TNF-α :Tumor necrosis factor alpha

IFN-γ: Interferon gamma IL-8: Interleukin 8


10

QFT provide a measurement of the interferon-γ release from

T-lymphocytes stimulated by the Mtb specific antigens

ESAT-6, CFP-10 and TB7.7, using the ELISA technique.

1.5. QUANTIFERON-TB GOLD IN-TUBE TEST

Third generation of the Quantiferon assay uses three pre-

coated tubes to collect heparinized whole blood from the

individual tested. One tube is coated with the mycobacterial

antigens (TBAG tube), one is a positive control containing

phytohaemagluttinin (PHA, an unspecific mitogen) used for

validation and control of the assay, and one is a negative

control (Nil tube) containing saline solution. The positive

control serves both as control of correct handling and as

control of the person’s immune status and ability to respond

to the test antigens. Within 16 hours from blood drawing,

the tubes are incubated for 16-24 hours before centrifugation

and harvesting of plasma. At this point the samples can be

stored between 2-8 oC for up to 8 weeks. The samples are

then analysed by an ELISA method in which the amount of

IFN-γ in IU/mL is measured.

For the test to be valid the Nil value has to be below 8.0

IU/mL and the positive control above 0.5 IU/mL.

Indeterminate tests are reported to be associated with an

array of different host factors, as well as environmental

factors and multiple sources of variability can influence test

results (described in the limitations section). The test is

positive if the IFN-γ readout in IU/mL is above 0.35 after the

Nil value has been subtracted, whereas a test is negative if

the IFN-γ level in the TBAG tube is below 0.35 IU/mL.

Interpretation of the IFN-γ responses in the three tubes is

depicted in Table 1.

1.6. NON-TUBERCULOUS MYCOBACTERIA

Mycobacteria can be classified into three major groups:

Mycobacteria from the M. tuberculosis complex (causing

TB), Mycobacterium leprae (causing leprosy) whereas the

remaining mycobacteria are named NTM or atypical

mycobacteria (to separate them from the “typical” M.

tuberculosis complex). This diverse group currently consists

of more than 150 different species and subspecies, found in

soil, dust, animals, and tap water worldwide (47,48).

NTM disease cases are often difficult to diagnose. Unlike

Mtb, a positive NTM culture from a sputum sample does not

equate with disease and it is often difficult to distinguish

insignificant colonization or contamination from clinically

significant NTM disease. An approach to assess the clinical

impact of NTM infection is presented in the American

ATS/IDSA guidelines (49), defining NTM definite disease,

NTM possible disease and NTM colonization based on a

combination of radiological, microbiological and clinical

criteria. A modified approach using only microbiological

criteria has been used and validated over the last years (50-

54), enabling laboratory based surveillance of NTM disease

occurrence.

The clinical presentation of NTM disease is often very

diverse, e.g. in the form of pulmonary lesions, cervical lymph

node enlargement, skin or soft tissue lesions or disseminated

disease (49). In children, the most common clinical

Table 1. Interpretation

Criteria for QuantiFERON-TB

Gold in-Tube Test based on

package insert.


11

presentation of NTM disease is cervical adenitis, most often

due to mycobacteria from the M. avium complex (MAC) (55).

This localized lymph node enlargement and chronic infection

is regarded an independent disease entity different from the

pulmonary disease observed among adults, and it is

occurring in immunocompetent children aged 1-5 years

without systemic symptoms (55). As NTM infection can

mimic both pulmonary and glandular TB, a diagnostic tool

that could facilitate differentiation would be valuable.


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2. OBJECTIVES

1. To assess the performance of the QFT among patients

with NTM disease and to review available literature on

the subject

2. To assess the performance of the QFT among

individuals with and without active TB.

3. To determine the predictive value of the QFT for

development of active TB in a Danish cohort and

identify factors potentially associated with increased

risk of progression.


13

3. METHODOLOGICAL

CONSIDERATIONS

3.1. SETTINGS, DESIGN AND POPULATIONS

All three studies were retrospective and based on a cohort of

individuals screened with a QFT from January 2005 until

the end of December 2010. During this period, more than

95% of all QFT done in Denmark were analysed at IRLM,

SSI. From 2011 onwards, the test has been increasingly

performed at several local biochemistry departments

instead of SSI, making similar nationwide studies difficult

to perform in the future. The present study is thus based on

a unique data material covering the majority of the Danish

population screened for LTBI with a QFT in a six-year

period.

Study I combined the QFT cohort with nationwide data on

persons with a positive NTM culture retrieved from the

IRLM. Study II and III both combined the QFT cohort with

an extraction from the Department of Infectious Disease

Epidemiology (DIDE) on notified cases of TB. In study III

additional data were retrieved from hospital case records.

Figure 3 depicts the participant flow from available data

sources. All databases were combined using the Personal

Identification Number (CPR) included in the Danish Civil

Registration System (56). Further description of study

design, study settings and populations can be found in

article I-III (1-3).

3.2. DATA SOURCES

LIMS

Data on QFT performed at IRLM from 2005 through 2010

were extracted from the Laboratory Information and

Management System (LIMS). LIMS is used to register and

report all diagnostic examinations performed at the Division

of Diagnostics & Infection Control at SSI. All entries in

LIMS are based on the CPR, and this number is used to

connect LIMS with other registries enabling reporting of

diagnostic examinations to the requestor. Data stored in

LIMS is incorruptible and cannot be altered once entered in

the system. This way data is stored in its original format

over time, ensuring perfect reproducibility and making the

LIMS a comprehensive data collection well suited for

research purposes. The drawback of having a database

where data cannot be modified or updated is that extracts

from the LIMS are very diverse if the reporting of a

Figure 3. Participant flow

from available data sources

study I-III

Abbreviations:

LIMS - Laboratory Information

and Management System;

IRLM - International Reference

Laboratory of Mycobacteriology;

DIDE - Department of

Infectious Disease

Epidemiology


14

diagnostic test changes. In the present thesis, this adversely

affected the extract because the description of a QFT result

in LIMS had been changed numerous times during the

initial years of testing. Hence, a lot of time-consuming data

cleansing was necessary before the analysis could be

performed. To eliminate some of the diversity in the QFT

reporting, we excluded the second generation of the test, the

QuantiFERON-TB Gold and included only the third

generation of the test, QuantiFERON-TB Gold In-Tube Test

(QFT).

IRLM Database

In Denmark, the culturing of Mycobacteria is centralized at

IRLM. The database contains information on all samples

received at the laboratory that turn out positive by either

microscopy, molecular methods or culture. Positive

specimens are manually entered into the registry by patient

ID. Sample type, citizenship, country of birth and address

are included in the registry, whereas negative samples are

not. The original and current purpose of the database at

IRLM is fast access to information on all patients with a

positive mycobacterial specimen and the opportunity to

perform population based research. As opposed to the LIMS,

the data in the IRLM database is updated and modified in

continuity, making data extracts readily usable without

cleansing and the need for interpretation of data output. In

this thesis, an extract of positive NTM cultures was used in

study I.

DIDE Database

Since 1990, the Department of Infectious Disease

Epidemiology (DIDE) at SSI has received nationwide data

on notified TB cases (57). In Denmark, the physician

responsible for treating the TB patient are required to notify

DIDE using a standardized form (58). This form contains

demographical, clinical and epidemiological data regarding

the TB patient, such as country of exposure, way of

transmission, type of infection (pulmonary or extra-

pulmonary), outbreak conditions and diagnostic method.

The database at DIDE is maintained and updated regularly,

and data concerning TB surveillance and disease control in

Denmark is published through the SSI website as short

reports and graphical displays available to the public (57).

3.3. STATISTICS

Descriptive statistics

The quantitative (continuous) variables were described as

mean and standard deviation of normally distributed

variables and as median and interquartile range of non-

normally distributed variables. The qualitative (categorical)

variables were summarized using frequency distributions

and described as numbers and percentages out of the total.

Statistical analysis

Wilcoxon rank sum/Mann-Whitney U was the non-

parametric test used to evaluate continuous variables and

Pearson’s chi-squared or Fisher’s exact test (frequency

counts <5) was used to compare categorical variables.

Cochran-Armitage test for trend is a modification of the

Pearson chi-squared test where a suspected ordering in the

proportions of the categorical variables is incorporated. The

null hypothesis is that the linear trend parameter is zero, so

a significant p-value is consistent with the presence of a

trend. It was used to evaluate an association between

binomial variables and variables with more than two

categories. Kaplan Meyer (KM) survival estimates were

used. To investigate significant differences between the KM

curves and the two derived survival distributions, the

nonparametric log-rank test was applied.

Table 2. Data sources

in Study I-III.


15

Statistics regarding diagnostic tests

Sensitivity was calculated as #true positive tests/(#true

positive tests + #false negative tests). Positive predictive

value of a test was defined as the proportion of patients with

an initial positive test that developed disease, and negative

predictive value defined as the proportion that scored

negative in the test and did not develop disease. Overall, a

two-sided p-value <0.05 was considered significant. For the

statistical analysis, SAS® 9.3 (SAS Institute Inc., Cary, NC,

USA) and R version 3.1.1 (59) were used.


16

4. MAIN RESULTS

4.1. STUDY I

Background

The incidence of NTM infections have been reported to be on

the rise and clinical manifestation of NTM disease can

mimic TB. If a blood test can differentiate between TB and

NTM disease, this could provide a minimally invasive tool

for the clinician compared to traditional microscopy, culture

and PCR. The reason why the QFT could potentially be

useful is that the majority of the NTM do not share the M.

tuberculosis specific antigenic regions used in the QFT.

Aim

The study objective was to investigate test performance

among patients infected with NTM and to review available

literature on IGRAs among patients with NTM disease.

Results

The majority (50/53) of patients had NTM disease due to

mycobacteria not sharing the antigenic region with Mtb, and

we found 2 positive, 43 negative and 5 indeterminate QFT

results, resulting in a specificity of 96% (43/45). Three

patients had disease due to Mycobacteria known to share the

antigenic region with Mtb, and in this group, 67% (2/3) were

positive.

We found 9 published studies with a total of 478 patients

with NTM disease and an IGRA. In patients infected with

an NTM without the antigenic region, the QFT was positive

in 12% (ranging from 0-50% in the different studies)

compared to our rate of 4.4% (2/45). Patients infected with

NTM sharing the region with Mtb had an overall positivity

rate of 57%, which is in line with our finding of 67%. Among

children, in the majority of cases, NTM disease was due to

infection with MAC and the localisation was extra-

pulmonary lymphadenitis. All children with MAC had a

negative QFT.

Conclusion

The QFT might hold potential in discriminating between

NTM and Mtb infections, in particular in children, where

lymphadenitis due to mycobacteria from the MAC was

predominant and because this clinical presentation may

mimic TB lymphadenitis.

4.2. STUDY II

Background

Even though IGRAs are used increasingly for the diagnosis

of LTBI worldwide, few studies have investigated the test

performance in extreme age groups. Current guidelines

warrants caution when using IGRAs in children less than

five years of age due to lack of studies performed. Similarly,

only a limited number of studies evaluate test performance

among elderly, and no guidelines specifically addresses the

ageing population.

Aim

To evaluate QFT performance among patients of different

age groups with and without TB.

Results

Among 383 patients with active TB, we found a low

indeterminate rate of 3.9% and a high sensitivity of 86.1%.

The latter was unaffected by localization of TB

(pulmonary/extra-pulmonary), sex and diagnostic group

(culture and PCR confirmed TB versus clinical TB diagnosis

only). Sensitivity declined with increasing age and was thus

found to be 71% among elderly persons aged more than 65

years, but was not impaired in young children with active

TB (100%). Among 15,709 persons without TB, the

indeterminate rate was overall 5.1%, and higher in infants

aged <1 year (15.6%) and elderly aged >65 years (8.1%).

From age one and above the indeterminate rate was not

significantly increased compared to adults aged 15-64 years.

Conclusion

QFT sensitivity among TB patients was overall 86% but

declined with increasing age (>65 years). In contrast to what

current guidelines on IGRA use in small children state, our

data suggest that the QFT performs well in children from

age 1, but that caution should be taken when using the test

among the elderly and infants.

4.3. STUDY III

Background

Latent TB is prevalent in high numbers throughout the

world, but only a small proportion of persons infected with


17

Mtb will eventually develop TB disease. A valuable

diagnostic test detecting the presence of latent infection

should be evaluated by its ability to predict how many will

develop disease and ultimately be able to identify those in

highest risk of progression.

Aim

To investigate the predictive value of the QFT.

Results

We included patients with a QFT performed and combined

these with notified TB cases. We defined “co-prevalent TB”

as cases 0-90 days after testing and “incident TB” as cases

developing after more than 90 days. In total, 231 patients

had co-prevalent TB and 40 patients developed incident TB

within the follow-up period. The cohort was subject to a

follow-up for 52,807 person-years, and median follow-up

time was 3.36 years. For incident TB, we confirmed a very

high negative predictive value of 99.85% and found a

positive predictive value of 1.32%. Expressed as risk of

developing incident TB, this corresponds to an incidence rate

(IR) of 383 per 105 PY for QFT positive patients and an IR of

45 per 105 PY for QFT negative patients. The risk of incident

TB was highest the first two years after a positive test, and

only two patients out of 20 with a positive test completed

preventive therapy.

Conclusion

We found a high NPV and a PPV of 1.32% for development

of incident TB. Development of incident TB was associated

with time interval after the QFT, but not with age.


18

5. DISCUSSION

5.1. QFT PERFORMANCE

Sensitivity among patients with active TB

Sensitivity of the QFT among patients with TB in Denmark

was comparable to what has been found in available

literature (86%). In line with previous studies, risk factors

such as gender (60), localization of disease (pulmonary vs.

extra-pulmonary TB) (61) and culture result (presence or

absence of a positive Mtb culture) (62) did not affect test

sensitivity (2). Sensitivity has been evaluated extensively

during the last decade. In general, the TST, QFT and T-Spot

have comparable sensitivities of 80-90% in most studies of

immunocompetent individuals (61,63,64), but because the

tests are based on the cellular immune response, reduced

sensitivity are linked to immunological function and

immunocompromised patients. HIV infection and very

young age are shown to reduce sensitivity for all diagnostic

tests (65,66), highlighting a paradoxical problem for all

immune based tests for TB; they suffer reduced performance

among those patient groups that are most susceptible to

both active and latent TB.

The QFT cannot separate latent from active TB and should

not be used alone in patients suspected of active TB. It can

however, be used as a tool in the diagnosis of active TB in

situations including; patients with extra-pulmonary TB,

patients who are persistently negative by microscopy or

culture, TB diagnosis in children, or as a supplement in

differential diagnosis between infections with NTM and TB

(67). Only few studies have assessed whether adding an

IGRA test to the existing diagnostic work-up for active TB

can successfully identify a greater proportion of TB cases in

TB high-incidence settings (68,69), TB low-incidence

settings (70) and in child populations (71). Discouragingly,

in most clinical situations an IGRA or TST was not of

additional value when combined with standard methods to

diagnose active TB (67).

Distinguishing active from latent TB using IFN-γ level

IGRAs (as well as the TST) cannot distinguish between

latent and active TB. If an algorithm based on the

commercially available IGRAs could make this distinction,

it would be immensely valuable. Numerous studies have

indeed investigated whether the level of IFN-γ readout is

higher in patients with active TB compared to those with

latent TB (70,72-76). In study III, we describe significantly

higher IFN-γ levels in the group that develops incident TB

compared to the group with a positive QFT that remain

healthy. However, the confidence limits are very wide and

the test cannot discriminate between the two groups, so this

observation has no practical use. A new generation of the

QuantiFERON test has recently been introduced (77). It

contains two tubes with mycobacterial antigens directed at

different subpopulations of T-lymphocytes and might have

potential in discriminating active TB from LTBI. The first

independent evaluation of this test (78) shows similar

specificity and sensitivity but suggest that accuracy might

have improved in patients with low CD4+ T-cell counts (78),

whereas discriminatory ability is not evaluated. So far, in

one decade of reporting and studying the IGRAs, no human

studies have conclusively shown that they can distinguish

between active and latent TB, and currently available IFN-

γ based assays will most likely not be able to (79). Numerous

studies are focusing on discriminating TB stages (80,81) and

the search for alternative biomarkers and dormancy

antigens that could be used in the development of next

generation assays is ongoing (82).

5.2. NON-TUBERCULOUS MYCOBACTERIA

The role of IGRAs in the diagnosis of NTM disease is as yet

insufficiently explored. European Centre for Disease Control

and Prevention guidelines state that an IGRA can contribute

with supplementary information in the differential

diagnosis between NTM and TB disease (67), but this

recommendation is based on very limited evidence. Study I

(1) as well as other studies find a low proportion of positive

tests among patients infected with NTM without the RD1

antigenic region and a significantly higher proportion

among patients infected with NTM sharing the RD1 (83-93).

Based on the high specificity of the QFT in patients with

NTM disease in study I and on available literature, we find

that the QFT holds potential to discriminate between NTM

and TB (1).


19

NTM disease is a rare event in Denmark (approximately 100

new patients per year), and the need to separate NTM from

TB using a QFT is not widely applicable in a Danish setting.

However, in children presenting with cervical

lymphadenitis, our study found all 15 children aged 0-9

years with MAC lymphadenitis to be QFT negative,

resulting in a specificity of 100%. Lymphadenitis due to

NTM is common in young children and the clinical

presentation can mimic extra-pulmonary TB. The

differentiation relies on a suitable biopsy specimen from

which the mycobacteria is identified by culture or molecular

methods. If a less invasive test such as an IGRA could aid in

this separation it could be useful, but the IGRAs suboptimal

sensitivity as reported in study II (86%) calls for caution

when using such tests to distinguish between TB and NTM.

5.3. QFT PERFORMANCE IN EXTREME AGE

GROUPS

Children

Young children are facing an increased risk of developing

active TB if they become infected, and they often have

disease with few bacilli (paucibacillary TB). Also the TB

disease manifestations are different from adults in the form

of more milliary TB and TB meningitis (94). This makes

children a particularly vulnerable group, difficult to

diagnose and suffering more severe disease than adults do.

As a result, a positive IGRA can be useful to support a

suspicion of TB along traditional diagnostic measures (67).

Study II suggests that already from age one, the

performance in children with active TB is good, although few

were included. Studies of IGRA performance in children are

very heterogeneous, and few investigate performance in

children aged less than 5. Some studies demonstrated a

lowered sensitivity in children aged <5 years (94,95),

whereas others report IGRA sensitivity comparable to

adults from 2 years of age (85,96,97). In TB high-incidence

countries both IGRAs and TST perform poorly among

children, most likely due to a combination of severe

comorbidity causing immunosuppression, misclassification

and high rates of reinfection (98). In the study by Rose et al

in a Tanzanian population of 211 children, of whom 77 were

aged <2 years, the QFT performed badly with sensitivity of

19% and indeterminate rate of 27% (98).

We evaluated the indeterminate rate in children without

active TB, and found no effect on indeterminate rates in

children from age 1, but among infants aged <1 the

indeterminate rate was high (15.6%) (2). This suggest that

an immature immune system in children seem to affect test

performance only the first year of life (2), and is most likely

caused by a T-cell population dominated by naïve cells (99).

Studies on indeterminate rate and children are numerous,

and some report that young age is associated with more

indeterminate results (100-103) whereas others do not reach

such a conclusion (95,97,104). Overall, IGRA and TST could

both be used for latent TB diagnosis from age 1 in otherwise

healthy children. None of the tests should be used for a

definitive diagnosis of active TB.

Elderly

In the elderly, the T-lymphocyte response is decreased (105).

This can affect the QFT indeterminate rate and sensitivity,

Figure 4.

Mitogen response among

15,506 patients without TB.

PHA: Phytohaemagglutinin

0%

20%

40%

60%

80%

100%

Age

PHA>10

PHA<10

PHA<5

PHA<0.5


20

and is probably caused by an exhausted population of

memory T-cells. In study II, we found that among those aged

65 years and above, the QFT had more indeterminate results

as well as decreased sensitivity. Consequently, the

likelihood of both false negative and indeterminate tests is

higher amongst the elderly. Whether the decreased

sensitivity is a direct effect of old age or caused by age

related immunosuppression is not possible to determine (2).

Our findings are supported by a recent TBNET study (106)

where extreme age was the only significant factor associated

with higher rate of falsely negative IGRAs.

We have assessed the influence of age on the quantitative

PHA response and confirmed that the PHA response is

affected in the very young and the very old (Figure 4,

adapted from Table 4 in Study II). Figure 4 illustrates the

PHA response from persons screened for LTBI in different

age groups. In children 0-9 years and elderly >65 years the

response to PHA is lowered, but the indeterminate rate

(PHA<0.5) is only higher in infants <1 and elderly >65,

suggesting that IFN-γ release is lowered throughout early

childhood but does not affect QFT result in children aged >1

(2).

5.4. PREDICTIVE VALUE OF THE QFT

Negative predictive value

In a cohort of 13.463 individuals we found a very high NPV

of 99.85% (study III). We have hereby established that the

NPV of the QFT in Denmark is extremely high,

corresponding to the findings in other studies from TB-low-

incidence settings (72,76,107). The individuals in study III

constituted a heterogeneous group tested with QFT due to

recent exposure, in the diagnostic work-up of active TB, or

before the initiation of anti TNF-α treatment. Although we

cannot separate the groups by test indication, the NPV in

general was very high, and the QFT seemed a useful tool for

identifying those that would not progress to TB disease.

However, of the 40 patients who developed incident TB, 20

actually had a baseline negative test. This could be

explained by different factors such as an impaired immune

system or re-infection with a new strain. In environments

with active transmission, the NPV is reduced because of new

infections, and this could be an explanation among the

socially marginalized persons in Denmark, where the

prevalence of active TB are reported to be more than 2000

per 105 (108). Of the 20 patients with incident TB and a

baseline negative test, 25% (5/20) were immunosuppressed

at the time of testing, and these test results could potentially

be false negative.

Positive predictive value

In a mixed population of patients tested with the QFT, 1.32%

developed TB during follow-up (study III). This is equivalent

to an 8 times increased risk of developing TB when a person

has a baseline positive test compared to a baseline negative

test. The PPV vary substantially in available studies

(63,76,107,109) depending on the TB prevalence in general

and the population in particular: In a vulnerable population

of recently exposed children, Diel et al found a PPV of 28.6%

(107), whereas the PPV was well below 1% (110) amongst

health care workers. A PPV of 1.3% is intuitively low for a

diagnostic test but the PPV varied and was higher among

children and adolescents aged less than 35 years (2.22%).

The risk of progression to incident TB was significantly

higher within the first two years after the QFT, where 90%

of incident cases developed. IR of progression was almost

800 per 105 PY the first year, 400 per 105 PY the second year

and 90 per 105 PY after more than two years (3), in line with

historical data describing an increased risk during the first

years after testing (111). A general issue to consider

regarding the low PPV is that only 5-10% of persons with

LTBI develop disease (14), so even a flawless test with

perfect accuracy will have a low predictive value.

Number needed to treat (NNT)

Generally, the QFT should be performed with a clear

indication, and the reasons for testing as well as the

consequences of a positive or negative result have to be

considered before deciding to test. In 2011 the catchphrase

“Intention to test is intention to treat” was introduced (112).

The point of that concept is that unnecessary tests for LTBI

should be avoided. The purpose of screening to detect LTBI

is to prevent future TB cases by administration of preventive

therapy. The PPV reflects the proportion of the persons

screened with a positive test result, who ultimately develop

TB, but the clinical usefulness of the test can also be

evaluated using another measure: The Number Needed to

Treat (NNT) expresses the number of persons with LTBI

that have to be treated with preventive therapy to avoid one


21

case of active TB. NNT is central in cost-benefit analysis of

the use of IGRAs in TB low-incidence countries. Sester et al

(113) have reviewed the NNT to prevent a TB case among

close contacts (30-37 (76,114,115)), immunocompromised

(50-80 (116)), HIV infected (14-26 (116)) and health care

workers (no TB cases developed among >15,000 persons

(117-119)). Thus, NNT differs according to the risk group,

and in all studies, confidence limits were wide because of few

incident TB cases. In Denmark and comparable TB low-

incidence countries mass screening is not cost effective (63)

because both the number needed to screen to identify a

person with LTBI and the NNT is very high. All studies

point toward targeted screening in high risk groups (32).

Another relevant measure when addressing TB prevention

is the Number Needed to Harm (NNH). It indicates the

number of patients that can be treated with preventive

therapy for one patient to suffer adverse effects. With the

increasing likelihood of isoniazid-induced hepatotoxicity

with increasing age (120,121) the NNH is age related and

screening elderly have to take this into account.

6. LIMITATIONS

6.1. BIAS

In general, patients screened with IGRA have a high a priori

likelihood for TB and LTBI. Thus, the studied population is

not representative for the Danish population and our studies

will overestimate the prevalence. Selection bias is also

present when we investigate age and risk of latent and

active TB, because the indication to screen a child or a young

person is often different from screening elderly. Among

children, recent exposure or suspicion of active TB is a

common indication, and the risk of active TB is high if

infected (122). Reversely, among elderly aged 35 and above,

screening before starting immunosuppressive treatment is

increasingly common, and in this group the prevalence of

LTBI is low and consequently very few will develop TB.

Therefore, the risk of TB could differ because the age groups

have different a priori risks of TB and not because age is an

independent risk factor.

Misclassification can occur when cases and controls are not

correctly recognized as such. False negative test results

among patients with TB disease occurred in 14% of the

active TB population in study II, and this misclassification

could result in unrecognized TB cases. In study II, we

evaluate how age, sex, localization of disease and

culture/PCR versus clinical confirmation of active TB

influences the rate of false negative tests. Confounding is

present when two evaluated factors do not act independently

in their contribution to the outcome under investigation.

Confounders that should always be considered are sex and

age. In our studies these variables were controlled for in the

data analysis in study II and III.

6.2. QFT VARIABILITY

Challenges with the QFT assay in terms of variability can

be assigned to a number of overall sources (63);

Manufacturing issues, preanalytical sources of variability,

analytical sources of variability and immunological sources

of variability as depicted in Figure 5.

Figure 5. Sources of variability in the QuantiFERON-

TB Gold In-Tube assay. Reprint from (63)


22

In our cohort of persons screened with a QFT, all of the

factors shown above may influence test results in both a

positive and negative way. Because of the retrospective

nature of our studies, the influence of the preanalytical and

immunological sources of variability on our cohort is difficult

to address. No information regarding the preanalytical

filling of blood tubes or handling and transport of the

samples before they arrive in the laboratory are available.

Further, we lack information on host factors for the

individual patients because no indication for screening or

information on comorbidity is available. Regarding

analytical variability, quality control is performed in each

run of the ELISA test in accordance with manufacturer’s

instructions. Manufacturing defects could be defects such as

errors in the production of the kits or reagents used. It can

be difficult to detect for individual laboratories, but

monitoring of sudden increases in indeterminate or positive

test results is an important control measure.

6.3. QFT REPRODUCIBILITY AND

REPEAT INDETERMINATE TESTS

The systematic sources of variability of the QFT have been

accounted for (Figure 5), but random variation due to

immunological factors can also affect test results. Problems

with QFT reproducibility in the same person undergoing

serial testing is well known, and reports of conversions (a

negative test becomes subsequently positive) and reversions

(a positive test becomes negative) are numerous (118). The

introduction of a borderline zone to better understand test

dynamics has been proposed in order to counter this (123).

The grey zone is the level of IFN-γ release in IU/mL, where

most reversions and conversions appear, and it can be used

to alert the clinician to interpret results with caution. In

study III, we describe a larger proportion of patients who

develops incident TB are in the grey-zone from 0.2-1.0

(3,117,123) compared to the group without TB. This finding

is interesting albeit not surprising, because it suggests

impaired immunity plays a role in the susceptibility towards

developing TB disease.

In study II, the indeterminate rate of all persons tested was

5.1%. Of these, 40% were repeated and 67% changed to

either positive or negative in the subsequent test. The

recommendation when encountering an indeterminate QFT

is to repeat the test (33) and a majority of these repeats

turned out valid. More than 99% of all the indeterminate

results were caused by a low positive control, and most

likely, they are attributed to an impaired T-lymphocyte

response because of suppressed host immunity.

6.4. SCREENING INDICATION

A limitation in both study II and III is the lack of screening

indication when performing the QFT. In study II the group

consisting of persons with a QFT without TB disease was

heterogeneous and comprised individuals tested for

different reasons. Because we did not have access to clinical

information, we could not determine if the

immunosuppression seen with advanced age in study II was

a genuine effect of old age or an effect of age related co-

morbidity (2). Similarly in study III, we did not have clinical

data on the actual reason for testing, so calculation of the

PPV and NPV stratified by risk group was not possible.

However, in a very large cohort we did provide general

confidence limits for both the PPV and the NPV of a QFT per

see.

7. PERSPECTIVES

7.1. THE LOCAL PERSPECTIVE, IGRA USE IN

DENMARK

In Denmark and other high-income TB low-incidence

settings, the intended use of IGRAs is the use as tools to

screen high-risk groups and identify patients that will

benefit from preventive treatment. However, as our data

and other data (124,125) suggest, IGRAs are widely being

used to aid in the diagnosis of active TB and not always in a

correct way.

Use of the QFT test to diagnose active TB

In a subgroup analysis of the patients in study II, 100

patients with active TB had a QFT performed in 2010 among

361 notified TB cases (7). It follows that 28% (100/361) of the

notified TB cases in Denmark that year had a QFT

performed to aid in the TB diagnosis. To further support a


23

widespread use of the test in active TB diagnosis, in a recent

Danish study from Aarhus, Danielsen et al evaluated the

use of the T-Spot test from 2010-2011 (124). Suspicion of

active TB was the indication for screening in 32.7%

(533/1,631) of persons tested. Another study evaluated test

indication in 2005-2006 at Hvidovre Hospital, and

Browatzki et al (125) found active TB to be the indication in

81% (74/91) of all IGRAs performed.

These findings indicate that the test is used to screen for

active TB. This is not necessarily misuse of the test, but

according to guidelines IGRAs rarely have an added value in

combination with standard methods for diagnosing active

TB (67) indicating at least some misuse of the test. Based on

the studies in the current thesis and available Danish

literature, there seem to be a need for better adherence to

guidelines and improved information regarding IGRA

limitations. Although this is not a big problem in Denmark

with the majority of TB cases being confirmed by culture, it

is increasingly a concern globally (126).

Preventive treatment

According to Danish guidelines (33), the standard treatment

for latent TB infection is a 6-month course of isoniazid

monotherapy. Diagnosis of latent TB and prescription of

preventive treatment is not a notifiable event, and therefore

no summarized data is available. The total coverage of

isoniazid preventive therapy among individuals with a

positive QFT was estimated to be 35% (data not shown) and

among patients with a positive QFT who developed incident

TB, 90% did not receive preventive treatment (3). It follows

that adherence to guidelines concerning screening

indication and preventive treatment in Denmark is

suboptimal.

7.2. THE GLOBAL PERSPECTIVE, IGRA USE

WORLDWIDE

IGRA use in TB high-incidence countries

In TB high-incidence settings, IGRAs have limitations

because of their extensive laboratory requirements and a

high prevalence of LTBI in the population. Consequently,

WHO has issued different guidelines on the management of

latent tuberculosis infection in countries with a TB incidence

rate below 100 per 105 population (32) compared to TB-high-

incidence countries (127,128). In TB high-incidence

countries, WHO recommends to reserve the use of IGRAs for

HIV infected individuals (127) and children below the age of

five (128), both groups being vulnerable groups with

increased risk of progression to disease.

A novel RD-1 specific skin test has a potential use in

resource-limited settings because it does not require a

laboratory. The C-Tb (129) contains the same mycobacterial

antigens as the QFT and has similar specificity and

sensitivity, but still share the IGRAs restraints in settings

with high prevalence of LTBI.

Use and misuse of the test among patients suspected of

active TB

The off-label use of IGRAs in the diagnosis of active TB could

potentially result in overtreatment. In a recent article from

India (130), Little et al constructed an analytical decision

model and found an estimated 315,700 additional false-

positive persons would be diagnosed with TB each year at an

incremental cost of US$49.3 million. This finding supports

recent policies by WHO that now discourage the use of

IGRAs for the diagnosis of active TB in settings like India

(126) and the misuse of the IGRAs in TB high-incidence low

income countries is currently receiving global attention.

Another problematic tool when diagnosing TB is the

commercial, serological tests for recognition of Mtb antigens

by the humoral immune response (as opposed to the cellular

response used by the IGRAs). They are marketed in many

third world countries, although none of them are

recommended by official guidelines or FDA approved. They

are not superior to the IGRAs based on the performance

reported in study II and recent meta analysis (63). Overall

data quality in the studies of serological tests for active TB

are regarded as very low, and official recommendations

strongly advice against serological tests (131). In summary,

even though this is an unjustifiable malpractice, many

immunological tests are marketed for diagnosing active TB,

especially in high-burden countries with weak regulatory

systems.

7.3. THE FUTURE

Many questions regarding IGRAs and their role in TB

control and ultimately TB elimination in low-incidence

settings remain unanswered. Data from this thesis suggest

that a negative IGRA is valuable in ruling out TB with little


24

risk of progression to disease, and that the QFT has a high

sensitivity for TB and a high specificity when used among

the majority of patients with NTM disease. Critics often

pinpoint the low sensitivity and poor predictive value of the

IGRAs, and it is indeed not a perfect test. However,

currently IGRAs are the best available option in low-

incidence countries like Denmark, and they are important in

diagnostic algorithms as tools to identify persons with active

TB and LTBI.

When the IFN-γ based assays were introduced, it was

anticipated that they could provide the means to distinguish

active from latent TB and identify those at the highest risk

of progression. After more than a decade of studying and

reporting on IGRA performance, it has become clear that

none of the commercial IGRAs have met these expectations.

One could speculate whether IGRAs have reached their full

potential, and if the search for a better biomarker should be

prioritized rather than trying to improve existing assays

based on release of IFN-γ (77).

The discovery of a novel biomarker that is both highly

specific, sensitive and with good predictive value would be a

game changer in TB control in low-incidence countries. In

recent years many new biomarkers have emerged (79-81).

One example is the IP-10 marker and the recent

development of a dried blood spot that enables easy storage

and prolonged transport (132). IP-10 has similar properties

in separating NTM and TB, and might hold potential in

making immunological diagnosis of TB and differentiation

between TB and NTM accessible in low-income, TB high-

incidence settings (133). However, the IP-10 marker is not

commercially available and has not proven superior

compared to existing tests. Thus, until better biomarkers are

introduced, it is important to use the current available tests,

while being aware of the limitations. This is the focus of an

ongoing debate on how screening strategies using IGRAs in

high-income TB low-incidence countries should be organized

to achieve the most optimal results towards TB control and

elimination (113,134,135).

The burden of TB remains in third world countries, and

immunological assays, although important in a Danish

setting, is not useful for combating the disease measured in

terms of global morbidity and mortality. Rather, the same

basic but essential countermeasures are as important today

as they were 100 years ago.

"The disease is dying a natural death with improved

conditions of the working classes, and it is by further

developments on such lines, and not otherwise, that its

extermination will be attained."

Dr. T. D. Lister (Mount Vernon Hospital for

Consumption, Hampstead, early 20th century).


25

SUMMARY

In the past decade, the Interferon Gamma Release Assays

(IGRAs) have been increasingly used in tuberculosis (TB)

low-incidence countries for the diagnosis of both active and

latent TB. We evaluated test performance of the most widely

used IGRA, the QuantiFERON-TB Gold In-Tube test (QFT),

in a large cohort through 6 consecutive years from 2005

through 2010, and included all Danish patients with notified

TB and cultured non-tuberculous mycobacteria (NTM).

It would be of great clinical value if the QFT could serve as

a minimally invasive tool and aid in discriminating TB

disease from NTM disease. Disease caused by the majority

of NTM will not evoke a positive IGRA because they do not

cross-react with the mycobacterial antigens in the QFT. We

conducted an evaluation of IGRA performance in patients

with NTM disease, and in light of these result and available

literature, we suggest that the IGRAs could be useful in

discriminating between TB and NTM disease. Among NTM

patients, we found a high specificity of 96%, a finding which

is especially relevant in children, where lymphadenitis due

to mycobacteria from the M. avium complex are seen to

predominate and can mimic TB lymphadenitis.

The ability of the QFT to identify infection with Mtb among

patients with active TB was in line with other studies and

the overall sensitivity was 86%. Performance was poorer in

the extremes of age with lowered sensitivity among elderly

(aged >65 years) and increased indeterminate rates in

infants <1 year and elderly >65 years. We conclude that test

performance is impaired only in children aged less than 1

and elderly, and suggest revision of current guidelines

advising caution when using IGRAs in children below the

age of 5.

A pivotal measure for any diagnostic test is the prediction of

disease. We conducted the largest study to date in a TB low-

incidence setting and determined the negative predictive

value (NPV) and positive predictive value (PPV) of the test,

identifying incident TB cases during more than 50,000

person-years of follow-up. PPV was 1.3% and NPV 99,85%.

We conclude that a negative QFT in our cohort of persons

that were screened due to various indications predicts an

extremely low probability of developing TB disease. The PPV

was expectedly low, but a positive test still inferred a more

than 8 times increased risk of developing TB, with an

additional increase following the first 2 years after QFT

testing.

With the current studies, we have established and used a

comprehensive, nationwide cohort to provide novel insights

into the performance of the QFT. In summary, the test is

used extensively in Denmark both for active and latent TB.

Overall test performance is acceptable, but caution is

warranted in infants and elderly and our results suggest

that administration of preventive therapy for latent TB

could be improved. Screening indication, host susceptibility,

as well as a priori risk of TB remain vital factors to consider

both when deciding to test and when interpreting test result.


26

DANSK RESUME

I lande med en lav forekomst af tuberkulose (TB) påvises

infektion med tuberkulosebakterien i stigende grad ved

hjælp af interferon-γ release assays (IGRAs). I denne ph.d.

evaluerede vi den mest udbredte af disse tests,

QuantiFERON-TB Gold In-Tube testen (QFT) i en kohorte

bestående af alle personer med en udført test i perioden 2005

til og med 2010 i Danmark. Vi kombinerede QFT data med

alle anmeldte tilfælde af TB samt alle tilfælde af

dyrkningsverificeret sygdom forårsaget af gruppen af non

tuberkuløse mykobakterier (NTM) og undersøgte værdien af

QFT til at påvise disse sygdomme.

Det ville være en fordel hvis man kunne bruge QFT til at

skelne aktiv tuberkulose fra sygdom grundet NTM, da en

QFT er mindre invasiv end traditionel diagnostik ved

biopsitagning. Størstedelen af NTM vil give et negativt QFT

resultat, da de ikke indeholder de specifikke antigener som

testen er baseret på. Vi har evalueret QFT blandt patienter

med NTM sygdom og fandt en meget høj specificitet. Vi har

ydermere gennemgået tilgængelige studier vedrørende

IGRA og anvendelse hos patienter med NTM sygdom og

samlet set fandt vi, at testen har potentiale til at skelne

mellem TB og NTM. NTM sygdom er sjældent

forekommende, men hos især børn kan testen være relevant,

da en typisk præsentation af NTM sygdom er en hævet

lymfeknude på halsen, som klinisk kan have lighed med

glandel TB.

QFT evne til at påvise infektion med tuberkulosebakterien

blandt patienter med aktiv TB i vores kohorte var

sammenlignelig med andre studier, og samlet set var

sensitiviteten 86%. QFT var påvirket i begge ender af

aldersspektret, idet testen havde flere inkonklusive

resultater blandt både spædbørn yngre end 1 år og ældre

over 65 år. Endvidere havde testen nedsat følsomhed blandt

ældre >65 år. Vi konkluderer, at testen synes at virke godt

fra 1 år og opefter, men at man bør udvise forsigtighed når

testen anvendes hos spædbørn og ældre over 65 år, da

præstationen er nedsat blandt disse grupper. Dette er

interessant i et internationalt perspektiv, idet flere

retningslinjer fraråder testen hos børn under 5 år, hvorfor

en revision af disse bør overvejes.

En central egenskab ved en diagnostisk test er dens evne til

at forudsige udvikling af sygdom. I denne ph.d. har vi lavet

den hidtil største undersøgelse af QFT’s evne til at forudsige

udvikling af TB, og vi har bestemt den prædiktive værdi af

en positiv og negativ test i løbet af mere end 50,000

personårs opfølgning. Vi konkluderer, at den positive

prædiktive værdi er 1.3% og den negative prædiktive værdi

er 99.85%, hvilket betyder at en person der screenes, og har

en negativ test, iht. vores data har en meget lille risiko for

at udvikle TB. Hvis man har en positiv test, har man mere

end 8 gange større risiko for udvikling af TB.

Studierne i denne ph.d. har bekræftet og udbygget den viden

vi har om QFT’s anvendelse og karakteristika. QFT er meget

udbredt i Danmark og anvendes både til diagnostik af aktiv

og latent TB. Sensitiviteten af testen er acceptabel, men den

bør ikke bruges alene til påvisning af aktiv TB, og man bør

udvise forsigtighed ved tolkningen af resultater hos

spædbørn og gamle. Vores resultater tyder på, at brugen af

forebyggende behandling for at forhindre udvikling af TB

kunne optimeres. Samlet set er det vigtigt, at overveje

indikationen for at anvende testen og den individuelle

risikoprofil, når man tolker resultatet.


27

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