265
ORIGINAL SCIENTIFIC ARTICLE
QuantiFERON TB and TB Plus tests in Slovenia between 2008 and 2019
Copyright (c) 2022 Slovenian Medical Journal. This work is licensed under a
Creative Commons Attribution-NonCommercial 4.0 International License.
QuantiFERON TB and TB Plus tests in Slovenia between 
2008 and 2019
Testa QuantiFERON TB in TB Plus v Sloveniji v obdobju 2008–2019
Urška Skamen, Eva Sodja, Manca Žolnir-Dovč
Abstract
Background: The QuantiFERON TB (QFT TB) test is primarily intended to detect latent (LTBI) tuberculosis (TB) infections. 
This work is based on a retrospective analysis of QFT TB test results in Slovenia between 2008 and 2019. It also draws at-
tention to the influence of pre-analytical factors on a test result, on reasons that lead to indeterminate results, and on the 
sensitivity of the test in patients with active tuberculosis (TB).
Methods: Between 2008 and 2019, we performed the QFT TB test in Laboratory for Mycobacteria of the University Clinic 
Golnik on 29,352 blood samples of patients from various health care institutions in Slovenia. A retrospective analysis was 
performed on QFT TB test results.
Results: The proportion of positive QFT TB test results is gradually decreasing. In 2008, the proportion was 20.8% and 
in 2019, it declined to 10.7%, meaning that it has decreased by 48.6% in the last 12 years. The biggest problem are inde-
terminate results (on average 4.3% per year), as they are unpredictive to a physician. The sensitivity of the QFT TB test in 
patients with active TB is 82.9%, which is comparable to the data from abroad.
Conclusion: The QFT TB test is highly sensitive to pre-analytical factors and the clinical condition of subjects therefore it is 
crucial that we follow instructions regarding blood taking and dispatching. The proportion of positive QFT TB test results 
has been rapidly declining over the years, which is a consequence of efforts to limit the spread of active TB in our country.
Izvleček
Izhodišča: Test QuantiFERON TB (QFT TB) je prvenstveno namenjen za odkrivanje latentne (LTBI) okužbe z bacili tuberku-
loze (TB). Članek obsega retrospektivno analizo rezultatov testa QFT TB v Sloveniji med letoma 2008 in 2019, opozarja na 
vplive predanalitskih dejavnikov na rezultate testa ter govori o razlogih za nastanek nejasnih rezultatov in o občutljivosti 
testa pri bolnikih z aktivno obliko TB. 
Laboratory for Mycobacteria, University Clinic of Respiratory and Allergic Diseases Golnik, Golnik, Slovenia
Correspondence / Korespondenca: Urška Skamen, e: urska.skamen@klinika-golnik.si
Key words: tuberculosis; latent tuberculosis; QuantiFERON TB test; preanalytical factors; indeterminate result
Ključne besede: tuberkuloza; latentna tuberkuloza; test QuantiFERON TB; predanalitski dejavniki; nejasen rezultat
Received / Prispelo: 26. 3. 2021 | Accepted / Sprejeto: 21. 10. 2021
Cite as / Citirajte kot: Skamen U, Sodja E, Žolnir-Dovč M. QuantiFERON TB and TB Plus tests in Slovenia between 2008 and 2019. Zdrav 
Vestn. 2022;91(7–8):265–72. DOI: https://doi.org/10.6016/ZdravVestn.3251
eng slo element
en article-lang
10.6016/ZdravVestn.3251 doi
26.3.2021 date-received
21.10.2021 date-accepted
Microbiology, bacteriology, virology, mycology Mikrobiologija, bakteriologija, virologija, mi-kologija discipline
Original scientific article Izvirni znanstveni članek article-type
QuantiFERON TB and TB Plus tests in Slovenia 
between 2008 and 2019
Testa QuantiFERON TB in TB Plus v Sloveniji v 
obdobju 2008–2019 article-title
QuantiFERON TB and TB Plus tests in Slovenia 
between 2008 and 2019
Testa QuantiFERON TB in TB Plus v Sloveniji v 
obdobju 2008–2019 alt-title
tuberculosis, latent tuberculosis, QuantiFER-
ON TB test, preanalytical factors, indetermi-
nate result
tuberkuloza, latentna tuberkuloza, test QuantiF-
ERON TB, predanalitski dejavniki, nejasen rezultat kwd-group
The authors declare that there are no conflicts 
of interest present.
Avtorji so izjavili, da ne obstajajo nobeni 
konkurenčni interesi. conflict
year volume first month last month first page last page
2022 91 7 8 265 272
name surname aff email
Urška Skamen 1 urska.skamen@klinika-golnik.si
name surname aff
Eva Sodja 1
Manca Žolnir-Dovč 1
eng slo aff-id
Laboratory for Mycobacteria, 
University Clinic of Respiratory 
and Allergic Diseases Golnik, 
Golnik, Slovenia
Laboratorij za mikobakterije, 
Univerzitetna klinika za pljučne 
bolezni in alergijo Golnik, Golnik, 
Slovenija
1
Slovenian Medical Journallovenian Medical Journal
266
MICROBIOLOGY, BACTERIOLOGY, VIROLOGY, MYCOLOGY
Zdrav Vestn | July – August 2022 | Volume 91 | https://doi.org/10.6016/ZdravVestn.3251
1 Introduction
Tuberculosis (TB) is a disease caused by bacilli of the 
Mycobacterium tuberculosis complex. Globally, the inci-
dence of active TB decreases by about 2% annually. Nev-
ertheless, about 10 million people worldwide still fall ill 
each year (1-3). Between 2015 and 2019, the incidence 
of active TB in the world decreased by a total of 9%. This 
is less than half of the set goal, which is a 20% decrease 
in the incidence of the disease by 2020, pursued by the 
World Health Organization in the framework of the End 
TB Strategy. In 2019, 87% of new TB cases were from 
30 high TB burden countries. Two-thirds of these were 
contributed by eight countries, including India, Indone-
sia, China and South Africa. The incidence of TB in EU 
countries is low, but has been declining more slowly in 
recent years (2). This is greatly influenced by risk groups, 
especially refugees coming from countries with a wors-
ening epidemiological picture (2,3). 5-15% of people in-
fected with TB bacilli will develop active disease, while 
the rest have a latent TB infection (LTBI) (1,3). Timely 
detection and preventive treatment of LTBI with anti-
microbial agents prevent the progression from latent to 
active TB, which is especially important in patients with 
weakened immune systems (4,5).
The tuberculin skin test (TST), which is based on the 
detection of a delayed hypersensitivity reaction to tuber-
culin antigens, has been used for decades to detect LTBI. 
Disadvantages of the TST are the inability to differentiate 
between active and latent TB, non-specificity (response 
to other mycobacterial infections), and the possibility of 
a false-positive result in people vaccinated with BCG (6). 
Over the last 15 years, the TST has largely been replaced 
by the IGRA (interferon-γ release assay) tests, which in-
clude the QFT TB test (7).
The QFT TB test measures the concentration of in-
terferon-γ (IFN-γ) released from a patient’s lympho-
cytes, which occurs as an immune response to a mixture 
of specific antigens of bacilli from the M. tuberculosis 
Metode: V obdobju 2008–2019 smo v Laboratoriju za mikobakterije Klinike Golnik s testom QFT TB testirali 29.352 vzorcev 
krvi bolnikov iz različnih zdravstvenih ustanov v Sloveniji. Na rezultatih testa QFT TB smo izvedli retrospektivno analizo.
Rezultati: Delež pozitivnih rezultatov testa QFT TB se z leti postopno znižuje. V letu 2008 je znašal 20,8 %, v letu 2019 pa 
10,7 %, kar pomeni, da je v zadnjih dvanajstih letih upadel za 48,6 %. Največjo težavo predstavljajo nejasni rezultati (letno 
povprečno 4,3 %), saj so za zdravnika nepovedni. Občutljivost testa QFT TB pri bolnikih z aktivno obliko TB je 82,9 %, kar 
je primerljivo s podatki iz tujine. 
Zaključek: Pomembno je, da pri odvzemu in pošiljanju krvi dosledno sledimo navodilom, saj je test QFT TB zelo občutljiv 
na predanalitske dejavnike in klinično stanje preiskovancev. Delež pozitivnih rezultatov testa QFT TB z leti pospešeno upa-
da, kar je povezano z napori za omejitev širjenja TB in upadom primerov aktivne TB v naši državi.
complex: ESAT-6 (early secretory antigenic target), CFP-
10 (culture filtrate protein) and TB 7.7 (3,5,8). To iden-
tify LTBI, the QFT TB test is more specific than TST, 
as most non-tuberculous mycobacteria do not produce 
these specific antigens, which are also not present in the 
BCG vaccine (5,8). Two of the major shortcomings of the 
QFT TB test are the inability to differentiate between TB 
and LTBI and the poor sensitivity of the test in patients 
with microbiologically proven active TB (65–100%) (9). 
The sensitivity of the test to many pre-analytical factors 
and the clinical condition of the subjects is also a prob-
lem (7-10).
LTBI testing is recommended especially for: i) people 
coming from countries with a higher incidence of TB; 
ii) people who have been in contact with a person with 
active TB; iii) workers from an environment at high risk 
of infection (long-term care facilities, homeless shelters, 
asylum homes); iv) workers caring for patients at higher 
risk of developing TB; v) patients at higher risk of devel-
oping TB, including HIV patients, patients with weak-
ened immune systems, patients, who are receiving im-
munosuppressive drugs, the elderly, and those who have 
not been properly treated for active TB in the past. The 
test can also be used as an aid in the diagnosis of active 
TB as a supplement to classical methods, especially for 
the detection of TB in children (11).
In the Laboratory for Mycobacteria of the Universi-
ty Clinic of Pulmonary and Allergic Diseases (UCPAD) 
Golnik we started testing the QFT TB test in 2004. First-
ly, a version of the test with two test tubes was used, zero 
(Nil) tube without added peptide antigens and antigenic 
or TB tube. This version was replaced by the next gener-
ation of the QFT TB Gold test, which, in addition to the 
antigen tube, also contained a mitogen (Mit) test tube 
with phytohaemagglutinin (PHA) to check the subject’s 
immune response. The latest generation of the QFT TB 
Gold Plus test is an upgrade of the older version and 
267
ORIGINAL SCIENTIFIC ARTICLE
QuantiFERON TB and TB Plus tests in Slovenia between 2008 and 2019
Pre-analytical factor Possible consequence Source
Insufficiently disinfected blood collection site false positive result 10,14
Blood collected in cold tubes false positive result indeterminate result 5
Volume of blood collected (opt. 0.8–1.2 mL)
    * < 0.8 mL inability to perform the test
    * > 1.2 mL false positive result indeterminate result 9,10
Uneven mixing of test tubes false negative resultfalse positive result 9,10
Transportation of the sample to the laboratory 
(> 6 hours)
indeterminate result
false negative result 9,10,15
Transportation of samples at too low a temperature indeterminate resultfalse negative result 5,10,16
Table 1: Pre-analytical factors and possible effects on the QuantiFERON-TB (QFT) test result.
contains two antigen tubes – TB1 and TB2. Both anti-
gen tubes contain peptide antigens associated with the 
M. tuberculosis complex (12). The first TB1 antigen tube 
contains the ESAT-6 and CFP-10 peptides, which trig-
ger an immune response of CD4+ T-lymphocytes in the 
body. The second TB2 antigen tube contains additional 
lipids that stimulate the immune response of both CD4+ 
and CD8+ T-lymphocytes. Both cell types are involved 
in the body’s defences during TB infection through the 
release of IFN-γ (12,13).
The purpose of this article is a retrospective analysis 
of the QFT TB test results between 2008 and 2019 and 
a comparison of the results with the incidence of active 
TB in Slovenia. At the same time, we would like to draw 
attention to factors that may lead to incorrect or indeter-
minate QFT TB test results.
2 Methods
We performed a retrospective analysis of the QFT 
TB test results between 2008 and 2019. The test was per-
formed on blood samples received by the Laboratory for 
Mycobacteria of the UCPAD Golnik. The total number 
of samples received during this time was 29,352.
The research was conducted in accordance with the 
principles of the Helsinki Declaration, the Oviedo Con-
vention on Human Rights and Biomedicine and the 
Slovene Code of Medical Deontology. The research was 
approved by the National Medical Ethics Committee of 
the Republic of Slovenia (No. 0120-94/2021/3, dated 25 
March 2021).
2.1 Sample adequacy control and QFT TB test 
performance
The QFT TB test was performed according to the 
manufacturer’s instructions (Qiagen, Hilden, Germa-
ny). We received three (between 2008 and 2015) or four 
(from 2015 onwards) test tubes for the QFT TB test with 
the blood of the subjects, both from the UCPAD Golnik 
and from various external clients from all over Slovenia.
Upon receiving the samples, we first checked their 
adequacy and rejected the inadequate ones. Examples 
of inadequate samples are incorrectly labelled samples, 
blood collected in inappropriate test tubes, samples with 
an incorrect amount of blood collected, samples where 
more than 16 hours passed since collection, and samples 
that were stored in the refrigerator while waiting to be 
transported. All factors that may affect the result of the 
QFT TB test are listed in Table 1.
After checking their adequacy, the tubes were mixed 
eight times around the longer axis and then incubated at 
37°C for 16–24 hours. After incubation, the tubes were 
centrifuged for 15 min at 2727 rpm). This was followed 
by the measurement of IFN-γ release using the ELISA 
method (13).
2.2 Interpretation of results
After a preliminary incubation, the IFN-γ release 
level from three (QFT TB-Gold) or four (QFT TB-Gold 
Plus) tubes, Nil, TB1, TB2, and Mit, was measured in 
the blood sample of each subject. The Nil result, which 
268
MICROBIOLOGY, BACTERIOLOGY, VIROLOGY, MYCOLOGY
Zdrav Vestn | July – August 2022 | Volume 91 | https://doi.org/10.6016/ZdravVestn.3251
Legend: Nil – the IFN-γ value in the Nil tube; TB1 – Nil and TB2 
– Nil – the IFN-γ value in antigen tubes minus the Nil value; Mit 
– Nil – the IFN-γ value in the mitogen tube minus the Nil value; 
QFT TB – QuantiFERON TB.
QFT TB test results Released IFN-γ values 
(IU / mL)
Negative
      TB1 – Nil / TB2 – Nil
      Mit – Nil
< 0.35
> 0.50
Positive
      TB1 – Nil / TB2 – Nil
      Mit – Nil
> 0.35
> 0.50
Indeterminate
      Nil
      Mit – Nil
> 10
< 0.50
Table 2: Interpretation of QuantiFERON-TB Gold and 
QuantiFERON-TB Gold Plus test results. Taken from (13).
serves as a background measurement, was subtracted 
from the rest. This was followed by the interpretation of 
the results according to the amount of released IFN-γ in 
TB1 – Nil, TB2 – Nil, and Mit – Nil tubes. Possible test 
results are negative, positive and indeterminate (Table 2).
Based on our own observations, we added a negative 
result with an asterisk (neg*), which represents the limit 
values (0.25–0.35 IU/mL) and for which we recommend 
the test be repeated. In our study, these results are con-
sidered negative.
2.3 Retrospective analysis of the routine QFT 
TB test results and the QFT TB test results in 
patients with active TB
The analysis included all blood samples received in 
the laboratory from both the UCPAD Golnik and from 
external clients between 2008 and 2019. The results were 
anonymized, the proportions of positive, negative, inde-
terminate, and rejected QFT TB test results (equation 1) 
were calculated, and compared by years.
Equation 1:
Xoutcome, year = noutcome, year / Nyear
Xoutcome, year – the relative share of a specific test result 
in a specific year;
noutcome, year – the number of outcomes of a specific test 
result in a specific year;
Nyear – the number of all blood samples received in a 
specific year.
The decrease in the proportion of positive QFT TB 
test results (p) was calculated by equation 2.
Equation 2:
p = XQFT positive, 2019 - XQFT positive, 2008 / XQFT positive, 2008
We also analyzed the results of the QFT TB test in 
patients with active TB. Data on patients with active TB 
were obtained from the UCPAD Golnik TB Registry and 
the number of patients who underwent the QFT TB test 
was checked. If several blood samples were taken from an 
individual patient for the QFT TB test, the result of the 
test performed closest to the date of obtaining the clini-
cal sample from which the active TB was confirmed was 
taken into account, but no later than three months before 
or after confirmation.
For patients with confirmed active TB and a negative 
or indeterminate result of the QFT TB test, data on the 
patient’s clinical picture, associated diseases and treat-
ment were also obtained from the Golnik TB Registry.
3 Results
3.1 Sample adequacy control and analysis of 
QFT TB routine test results
In the 12-year period, the Laboratory for Mycobacte-
ria of the UCPAD Golnik received 29,352 blood samples 
for the QFT TB test, mostly from external clients from 
health institutions in Slovenia (Figure 1). Among those 
received, samples from pulmonology, dermatovenereol-
ogy, rheumatology, and gastroenterology departments 
and outpatient clinics predominated.
Samples for which the influence of pre-analytical fac-
tors was detected and samples with incomplete data on 
the accompanying referral (e.g. missing date and the col-
lection time) were rejected. The proportion of rejected 
samples remains fairly constistent over the years, ranging 
from a minimum of 0.8% to a maximum of 1.9% per year.
The share of positive results was the highest in 2008 
with 20.8% and the lowest in 2019 with 10.7%, which in-
dicates a drop in LTBI infections by 10.1 percentage point 
or 48.6% (Figure 2). The trend of the negative QFT TB test 
results is expected to be reversed from the positive ones. 
The highest share of negative results was recorded in 2019 
at 83.8% and the lowest in 2008, when it was 66.8% (Fig-
ure 2).
The biggest problem in detecting LTBI is the indeter-
minate results of the QFT TB test, in which the amount of 
IFN-γ released in the Mitogen tube (positive control) does 
not exceed 0.5 IU/mL or the amount of IFN-γ released in 
269
ORIGINAL SCIENTIFIC ARTICLE
QuantiFERON TB and TB Plus tests in Slovenia between 2008 and 2019
Figure 1: Schematic presentation of received and rejected blood samples for the QFT TB test and the results of tests 
performed between 2018 and 2019.
Legend: QFT – QuantiFERON.
UCPAD Golnik (20.0%): 5,870 samples
- departments and outpatient clinics Golnik (12.2%): 
   3,581 samples
- pulmonology outpatient clinic Ljubljana (7.8%): 
   2,289 samples
A TOTAL OF
received blood samples 
29.352 (100%)
Analysis of pre-analytical factors
External clients (80.0%): 23,482 samples
REJECTED
358 blood samples
(1.2%)
QFT TEST PERFORMED
28,994 blood samples
(98.8%)
The most common reasons for 
sample rejection:
- transport took longer than 16 hours,
- the sample was stored in a refrigerator,
- inappropriate collection tubes, 
   too little/too much blood taken.
QFT positive
4,029 blood samples
(13.9%)
QFT positive
23,701 blood samples
(81.7%)
QFT indeterminate
1,264 blood samples
(4.4%)
Figure 2: A chart of QuantiFERON-TB (QFT) test results between 2008 and 2019.
2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
66.8% 75.7% 79.3% 79.8% 81.1% 82.9% 78.7% 79.8% 79.7% 81.9% 84.4% 83.8%
11.2%
5.9% 4.1% 2.4% 2.5% 3.2% 4.9% 6.6% 5.9% 4.1% 2.6% 4.2%
Q
FT
 te
st
 re
su
lts
Year
20.8% 16.5% 15.5% 16.6% 15.1% 13.1% 14.8% 12.3% 13.1% 12.8% 11.9% 10.7%
Rejected sample
QFT indeterminate
QFT negative
QFT positive
the Nil tube (negative control) exceeds 10 IU/mL. The share 
of indeterminate results has fluctuated over the last 12 years, 
with an average of 4.3% over the period between 2008 and 
2019. The highest was in 2008 with 11.2% (93 samples) and 
the lowest in 2011 with 2.4% (54 samples) (Figure 2).
3.2 Analysis of QuantiFERON-TB (QFT) test 
results in patients with active tuberculosis (TB)
Out of 499 cases of active TB in Slovenia between 2015 
and 2020, the QFT TB test was ordered in 152 patients 
270
MICROBIOLOGY, BACTERIOLOGY, VIROLOGY, MYCOLOGY
Zdrav Vestn | July – August 2022 | Volume 91 | https://doi.org/10.6016/ZdravVestn.3251
Figure 3: A chart of QuantiFERON-TB (QFT) test results in patients with microbiologically confirmed active tuberculosis 
(TB).
2015 2016 2017 2018 2019 2020
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Q
FT
 te
st
 re
su
lts
 in
 p
at
ie
nt
s w
ith
 a
ct
iv
e 
TB
Year
QFT positive QFT negative QFT indeterminate Tests performed in QFT patients with active TB
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Pe
rc
en
ta
ge
 o
f Q
FT
 te
st
s p
er
fo
rm
ed
 in
 p
at
ie
nt
s w
ith
 a
ct
iv
e 
TB
24.8% 24.0%
29.9% 35.6%
25.6%
53.6%
84.6% 78.3% 69.2% 88.5% 81.0% 93.3%
11.5%
13.0% 26.9%
13.0% 19.0%
6.7%3.8% 8.7% 3.8%
Average of QFT positive tests (82.5%)
(30.5%). The fewest were performed in 2016 (24.0%) and 
the most in 2020 (53.6%) (Figure 3).
In 126 patients (82.9%) with proven active TB, the 
QFT TB test was positive. Of these, the share of positive 
results of the QFT TB test was the lowest in 2017 with 
69.2% (18 samples) and the highest in 2020 with 93.3% 
(28 samples) (Figure 3).
In 26 patients with confirmed active TB (17.1%), the 
result of the QFT TB test was negative or indeterminate. 
Of these, 15 patients had pulmonary TB (57.7%), five pa-
tients had pulmonary and extrapulmonary TB (19.2%), 
five patients only had extrapulmonary TB (19.2%), and 
one patient had miliary TB (3.9%). Of the extrapulmo-
nary TB, as many as four patients had lymph node TB, 
two had tuberculous pericarditis, two had bone TB (spi-
nal TB), and one had cutaneous TB and pleural TB.
The results were also compared with associated dis-
eases and treatments. Ten of the 26 patients with active 
TB (38.5%) received corticosteroids, biologics or other 
immunosuppressive drugs. Five patients (19.2%) also 
had other associated diseases or conditions such as dia-
betes, chronic kidney disease, and alcoholism. In six pa-
tients with active TB (23.1%), no diseases or treatments 
were observed, which are often associated with false-neg-
ative or indeterminate QFT TB test results.
3.3 Decline in active and latent TB in Slovenia
We also analyzed the results of routine QFT TB tests 
compared to the incidence of active TB in Slovenia. The 
incidence of TB in Slovenia has been declining over the 
years (11) (Figure 4). It decreased by 55% between 2008 
and 2019 (9), with no definitive data for 2020. The declin-
ing trend in the incidence of active TB is also followed by 
a decline in the share of positive results of the QFT TB 
test, which decreased by 48.6% in 12 years.
4 Discussion
The purpose of our study was a retrospective analy-
sis of the results of the QFT TB test over a 12-year pe-
riod and a comparison of the results with the incidence 
of active TB in Slovenia. We found that the incidence of 
TB in Slovenia has decreased by 55% in the last 12 years 
(11). With our research, we have shown that the declin-
ing trend in the incidence of active TB is followed by a 
decline in LTBI. This is to be expected, as fewer cases of 
active TB mean less risky contact and transmission of 
infection.
Of the active TB cases treated between 2015 and 2020, 
the QFT TB test was performed in only 30.5%, which is 
a relatively small percentage. For the diagnosis of pulmo-
nary TB in adults, microscopic examination, cultivation, 
and molecular biological tests (polymerase chain reac-
tion or RT PCR) are primarily used. Cultivation is also 
the only way to differentiate between active TB and LTBI. 
The result of the QFT TB test is positive in most patients 
with active TB (average 82.5%), which is comparable to 
other studies that report a 65 to 100% sensitivity of the 
QFT TB test (9). This should not mislead us and make 
271
ORIGINAL SCIENTIFIC ARTICLE
QuantiFERON TB and TB Plus tests in Slovenia between 2008 and 2019
Figure 4: A chart of the decline in the incidence of active tuberculosis (TB) and the share of positive QuantiFERON-TB (QFT) 
test results in Slovenia between 2008 and 2019. The incidence of active TB taken from (11 and the Golnik TB Register).
N
o.
 o
f p
at
ie
nt
s w
ith
 a
ct
iv
e 
TB
 / 
10
0,
00
0 
po
pu
la
tio
n
Year
0.0%
5.0%
10.0%
15.0%
20.0%
25.0%
Po
si
tiv
e 
Q
FT
 te
st
 re
su
lts
QFT positive Incidence of active TB
2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019
0
2
4
6
8
10
12
14
12
11
9.5
7.5 7.9
7.2
10
7.7
6.5 6.2
5.4 5.4
20.8% 16.5% 15.5% 16.6% 15.1% 10.7%13.1% 14.8% 12.3% 13.1% 12.8% 11.9%
us stop considering a possible diagnosis of TB if the QFT 
TB test is negative (9,12). The QFT TB test is often nega-
tive in severe forms of TB, such as tuberculous meningi-
tis, miliary TB, pericarditis, and bone TB (17).
The QFT TB test is very sensitive to pre-analytical 
factors and the immune status or clinical picture of the 
subject (18). Early detection of LTBI is especially import-
ant in patients at risk for TB progressing from latent to 
active. This early detection is often complicated by the 
indeterminate results of the QFT TB test, typical in these 
very groups of patients (3,8,15).
Indeterminate results are those with too low a re-
sponse in a Mitogen tube (<0.5 IU / mL) of which the in-
terior is coated with phytohaemagglutinin (PHA), which 
triggers a general immune response (5,7). The response to 
PHA, with strict consideration of analytical procedures, 
depends primarily on the functional integrity of lympho-
cytes and the ability to release IFN-γ, which may change 
due to various clinical conditions and procedures. The 
incidence of indeterminate results is higher in people 
with health issues than in healthy people, and depends 
on the therapy the patient is receiving (anti-TNF-α bio-
logics, antiretroviral therapy, steroids, and nonsteroidal 
anti-inflammatory drugs), age, immunological parame-
ters, and medical condition (5,8,15,18,19).
This explains a significant part of the indeterminate 
results of the QFT TB test, as in recent years, in addi-
tion to samples from pulmonary, many samples from 
gastroenterology, rheumatology, dermatology, and in-
fection departments and outpatient clinics have been ac-
cepted in the laboratory for the purpose of examination 
prior to introducing biologics or organ transplantation 
(19). Often, however, indeterminate, false-positive, and 
false-negative QFT TB test results may also be due to 
pre-analytical factors (Table 1).
If we observe the share of indeterminate results by 
years, it was by far the highest in 2008. At that time the 
QFT TB-Gold test was introduced, which now contained 
a Mitogen test tube to check the immune response of the 
subject and which also acted as the indicator of trans-
port temperature adequacy. This is especially important, 
because in our laboratory, in addition to samples from 
UCPAD Golnik, we also accept blood samples from var-
ious medical institutions from more distant Slovenian 
cities, which makes it more difficult to ensure their prop-
er collection and transport (20). In the following years, 
the proportion of indeterminate results decreased sig-
nificantly through continuous monitoring, correct sam-
ple collection and transport instructions, inspection of 
test tubes on admission to the laboratory, and notifying 
and training of nurses and couriers (20). Compared to 
research from abroad, which in routine testing shows 
shares of indeterminate results to be between 5 and 40%, 
this share being on average 6% in testing health profes-
sionals, we are in an enviable position with an average of 
4.3% (15,18,21).
272
MICROBIOLOGY, BACTERIOLOGY, VIROLOGY, MYCOLOGY
Zdrav Vestn | July – August 2022 | Volume 91 | https://doi.org/10.6016/ZdravVestn.3251
References
1. World Health Organization. Tuberculosis. Geneva: WHO; 2020 [cited 
2021 Feb 11]. Available from: https://www.who.int/health-topics/
tuberculosis#tab=tab_1.
2. World Health Organization. Tuberculosis. Geneva: WHO; 2021 [cited 2021 
Feb 15]. Available from: https://www.who.int/news-room/fact-sheets/
detail/tuberculosis.
3. Kim OH, Jo KW, Park S, Jo YH, Kim MN, Sung H, et al. Comparison of the 
change in QuantiFERON-TB Gold Plus and QuantiFERON-TB Gold In-
Tube results after preventive therapy for latent tuberculosis infection. 
PLoS One. 2020;15(6):e0234700. DOI: 10.1371/journal.pone.0234700 
PMID: 32544206
4. Machuca I, Vidal E, de la Torre-Cisneros J, Rivero-Roman A. Tuberculosis 
in immunosuppressed patients. Enferm Infecc Microbiol Clin (Engl Ed). 
2018;36(6):366-74. DOI: 10.1016/j.eimc.2017.10.009
5. Herrera V, Yeh E, Murphy K, Parsonnet J, Banaei N. Immediate incubation 
reduces indeterminate results for QuantiFERON-TB Gold in-tube assay. 
J Clin Microbiol. 2010;48(8):2672-6. DOI: 10.1128/JCM.00482-10 PMID: 
20519472
6. Song SE, Yang J, Lee KS, Kim H, Kim YM, Kim S, et al. Comparison 
of the tuberculin skin test and interferon gamma release assay for 
the screening of tuberculosis in adolescents in close contact with 
tuberculosis TB patients. PLoS One. 2014;9(7):e100267. DOI: 10.1371/
journal.pone.0100267 PMID: 25020161
7. Belliere J, Blancher A. QuantiFERON test interpretation in patients 
receiving immunosuppressive agents: an alert. Eur Respir J. 
2017;49(4):1602102. DOI: 10.1183/13993003.02102-2016 PMID: 28381433
8. Tebruegge M, de Graaf H, Sukhtankar P, Elkington P, Marshall B, Schuster 
H, et al. Extremes of age are associated with indeterminate QuantiFERON-
TB gold assay results. J Clin Microbiol. 2014;52(7):2694-7. DOI: 10.1128/
JCM.00814-14 PMID: 24829238
9. Gaur RL, Pai M, Banaei N. Impact of blood volume, tube shaking, and 
incubation time on reproducibility of QuantiFERON-TB gold in-tube 
assay. J Clin Microbiol. 2013;51(11):3521-6. DOI: 10.1128/JCM.01627-13 
PMID: 23966505
10. Banaei N, Gaur RL, Pai M. Interferon gamma release assays for latent 
tuberculosis: what are the sources of variability? J Clin Microbiol. 
2016;54(4):845-50. DOI: 10.1128/JCM.02803-15 PMID: 26763969
11. World Health Organization. Tuberculosis profile: Afghanistan. 
Geneva: WHO; 2020 [cited 2021 Feb 11]. Available from: https://
worldhealthorg.shinyapps. io/ tb_profi les/?_inputs_&entity_
type=%22country%22&lan=%22EN%22&iso2=%22SI%22.
12. Sodja E, Ažman S, Žolnir-Dovč M. Laboratorijska diagnostika latentne 
tuberkuloze – 11-letne izkušnje s testom QuantiFERON. In: Petrovec M, 
ur. Okužbe dihal: zbornik predavanj. Ljubljana: Medicinski razgledi; 2016. 
pp. 21-9.
13. QiagenQuantiFERON-TB Gold Plus ELISA Package Insert. Hilden: Qiagen; 
2021 [cited 2021 Feb 2]. Available from: http://www.quantiferon.com/
wp-content/uploads/2017/04/English_QFTPlus_ELISA_R04_022016.
pdf.
14. Gaur RL, Banaei N. Inoculation of QuantiFERON-TB tubes with skin 
microbiota causes false-positive results. Am J Respir Crit Care Med. 
2014;190(7):834-7. DOI: 10.1164/rccm.201406-1041LE PMID: 25271749
15. Manuel O, Humar A, Preiksaitis J, Doucette K, Shokoples S, Peleg AY, 
et al. Comparison of quantiferon-TB gold with tuberculin skin test for 
detecting latent tuberculosis infection prior to liver transplantation. Am J 
Transplant. 2007;7(12):2797-801. DOI: 10.1111/j.1600-6143.2007.02011.x 
PMID: 17941955
16. Jarvis J, Gao Y, de Graaf H, Hughes S, Allan RN, Williams A, et 
al. Environmental temperature impacts on the performance of 
QuantiFERON-TB Gold In-Tube assays. J Infect. 2015;71(2):276-80. DOI: 
10.1016/j.jinf.2015.04.004 PMID: 25869537
17. Kim YJ, Kang JY, Kim SI, Chang MS, Kim YR, Park YJ. Predictors for 
false-negative QuantiFERON-TB Gold assay results in patients with 
extrapulmonary tuberculosis. BMC Infect Dis. 2018;18(1):457. DOI: 
10.1186/s12879-018-3344-x PMID: 30200884
18. Yun JW, Chung HS, Koh WJ, Chung DR, Kim YJ, Kang ES. Significant 
reduction in rate of indeterminate results of the QuantiFERON-TB Gold In-
Tube test by shortening incubation delay. J Clin Microbiol. 2014;52(1):90-
4. DOI: 10.1128/JCM.01547-13 PMID: 24153122
19. Žolnir-Dovč M, Svetina P. Kako daleč smo v diagnostiki latentne in aktivne 
tuberkuloze pri imunsko kompromitiranem bolniku? In: Svetina P, Košnik 
M, Marčun R, ur. Zbornik srečanja: 3. mednarodni simpozij o tuberkulozi 
in respiratornih okužbah; 22. slovenski posvet o obravnavi in spremljanju 
bolnikov s tuberkulozo (TB). 2017 Mar 23-24; Golnik, Slovenija. Golnik: 
Univerzitetna klinika za pljučne bolezni in alergijo; 2017.
20. Žolnir-Dovč M. Testi IGRA in vloga laboratorija v diagnostiki latentne 
tuberkuloze. In: Fležar M, Marčun R, Svetina P, ur. Zbornik sestanka: 20. 
redni posvet o obravnavi in spremljanju bolnikov s tuberkulozo (TB) v 
Sloveniji. 2015 Mar 20; Golnik, Slovenija. Golnik: Univerzitetna klinika za 
pljučne bolezni in alergijo; 2015.
21. Miranda C, Yen-Lieberman B, Terpeluk P, Tomford JW, Gordon S. 
Reducing the rates of indeterminate results of the QuantiFERON-TB 
Gold In-Tube test during routine preemployment screening for latent 
tuberculosis infection among healthcare personnel. Infect Control Hosp 
Epidemiol. 2009;30(3):296-8. DOI: 10.1086/595732 PMID: 19199532
5 Conclusion
With our retrospective analysis of the QFT TB test 
results, we confirmed that the share of positive QFT TB 
test results in Slovenia is declining together with the de-
cline of the incidence of active TB. This is a good in-
centive for further managing TB infections. When per-
forming the QFT TB test, we still encounter a significant 
share of indeterminate results due to the variety of ther-
apies before taking blood samples for the QFT TB test 
(e.g. immunosuppressants), the clinical picture of the 
patient as well as pre-analytical factors. If the first two 
cannot be influenced, we are convinced that by following 
blood collection and transport instructions, the impact 
of pre-analytical factors can further be reduced and thus 
the diagnosis of LTBI can be improved.
The sensitivity of the QFT TB test in detecting active 
TB is around 80%. This should be considered when us-
ing the QFT TB test to diagnose active TB (especially in 
the case of extrapulmonary and paediatric TB diagno-
sis). In the most severe forms of TB (meningitis, mili-
ary, extrapulmonary TB), the QFT TB test is often false 
negative.
Conflict of interest
None declared.