PLOS ONE

RESEARCH ARTICLE

First evaluation of the performance of

portable IGRA, QIAreach® QuantiFERON®-TB
in intermediate TB incidence setting
Zirwatul Adilah Aziz ID*☯, Noorliza Mohamad Noordin☯, Wan Mazlina Wan Mohd☯, Mohd
Amin Kasim☯
National Public Health Laboratory, Ministry of Health, Selangor, Malaysia

☯ These authors contributed equally to this work.

* adilahwkt57@gmail.com
a1111111111
a1111111111
a1111111111
a1111111111 Abstract
a1111111111
Diagnosis and treatment of tuberculosis infection (TBI) are the core elements of tuberculosis
elimination. Interferon gamma release assays have advantages over the tuberculin skin
test, although their implementation in low-resource settings is challenging. The performance
of a novel digital lateral flow assay QIAreach® QuantiFERON®-TB (QIAreach QFT) against
OPEN ACCESS
the QuantiFERON®-TB Gold Plus (QFT-Plus) assay was evaluated in an intermediate inci-
Citation: Aziz ZA, Noordin NM, Wan Mohd WM,
dence setting (Malaysia) according to the manufacturer’s instructions. Individuals aged
Kasim MA (2023) First evaluation of the
performance of portable IGRA, QIAreach® 4–82 years, who were candidates for TB infection screening for contact investigation were
QuantiFERON®-TB in intermediate TB incidence prospectively recruited. On 196 samples, the QIAreach-QFT showed a positive percent
setting. PLoS ONE 18(2): e0279882. https://doi. agreement (sensitivity) was 96.5% (CI 87.9–99.6%), a negative percent agreement (speci-
org/10.1371/journal.pone.0279882
ficity) 94.2% (CI 88.4% to 97.6%) and an overall percentage of agreement was 94.9% (95%
Editor: Luisa Gregori, U.S. Food and Drug CI 90.6–97.6%) with a Cohen’s κ of 0,88. Out of 196, 5.6% (11/196) samples gave an error
Administration, UNITED STATES
result on QIAreach-QFT and 4.1% (8/196) samples gave indeterminate result on QFT-plus.
Received: December 17, 2021 The TTR for QIAreach QFT positive samples varied from 210–1200 seconds (20 min) and
Accepted: December 16, 2022 significantly correlated with IFN-γ level of QFT-Plus. QIAreach QFT could be considered an
Published: February 10, 2023 accurate and reliable point-of-need test to diagnose TB infection helping to achieve the
WHO End TB programme goals even in decentralised settings where laboratory expertise
Copyright: © 2023 Aziz et al. This is an open
access article distributed under the terms of the and infrastructure may be limited.
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.

Data Availability Statement: All relevant data are

within the paper and its Supporting Information Introduction
file.
The World Health Organization (WHO) reports that tuberculosis (TB) still represents one of
Funding: This project was funded by the Long the major killers, causing around 1.5 million deaths worldwide annually [1–4]. TB still is, glob-
Term Research Grant (LRGS), Ministry of Higher ally, among the 10 commonest causes of preventable death, as both TB and TB infection can
Education Malaysia (MOE). QIAGEN provided
be successfully treated.
QIAreach QFT tests and eHub free of charge for
this study. The funders had no role in study design,
The latest WHO estimates show that many gains achieved in recent years reversed in 2020
data collection and analysis, decision to publish, or due to COVID-19 pandemic with 1.5 million lives lost due to TB and significantly less people
preparation of the manuscript. enrolled on TB preventive therapy in 2020 [1, 5–10].

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Competing interests: The authors have read the WHO has recently emphasized the public health importance of managing TB infection and
journal’s policy and have the following competing included the integrated patient-centred care and prevention approach under Pillar 1 of the
interests: QIAGEN provided support in the form of
End TB Strategy [2, 11, 12]. Furthermore, while diagnosis and treatment of infectious TB
QIAreach QFT tests and eHub for this study. This
does not alter our adherence to PLOS ONE policies patients are the essence of TB control, diagnosis and management of TB infection are the core
on sharing data and materials. There are no activities to pursue TB elimination [8, 9]. Screening for TB infection and TB preventive treat-
patents, products in development or marketed ment become even more important in the COVID-19 era when many TB services worldwide
products associated with this research to declare. have been severely disrupted [1, 13].
We have two families of tests to diagnose TB infection [12, 14], tuberculin skin tests (TST)
and interferon-γ (IFN-γ) release assays (IGRAs). TST, more than 100 years old, is a skin test in
which a given amount of tuberculin (purified protein derivative-PPD) from mycobacterial cul-
ture is intradermally injected to generate a delayed type of hypersensitivity reaction, producing
an induration, which is measured in millimetres [14]. Novel skin-based tests that seem to elicit
more specific immune responses to Mycobacterium tuberculosis are under development [15].
However, all skin tests are associated with the same operational challenges as TST: lack of qual-
ity control, subjective measurement of test results and challenges in data recording, training
needed for standardized administration and reading of results and, finally, two patient clinical
visits required for tuberculin administration and follow-up reading [15, 16]. IGRAs have been
developed more recently (13–18). They are in vitro assays able to measure the blood levels of
IFN-γ released by T lymphocytes stimulated with antigenic peptides of M. tuberculosis [14, 17–
22].
So far, WHO has endorsed three IGRA tests, T-SPOT1.TB (Oxford Immunotec, Abing-
don, UK), Beijing Wantai’s TBIGRA (Wantai), and QuantiFERON1-TB Gold Plus (QFT,
QIAGEN, Hilden, Germany), able to measure IFN-γ released by both CD4 and CD8 T cells
[23–28].
Although WHO-endorsed IGRA tests have advantages over TST, both require professional
expertise and considerable laboratory infrastructure thus not qualifying yet as point-of-care
tests. This is clearly a factor limiting their use in resource-limited settings [14, 29, 30]
In Malaysia, we use either tuberculin skin test (TST) and interferon-gamma release assays
(IGRA) for the diagnosis of latent TB infection in adult and children.
A potential step forward to provide a “field friendly” solution in high burden TB settings
which combines the measured benefits of IGRA technology with a point-of-care test that can
be used outside traditional laboratory settings is represented by the QIAreach1 QuantiFER-
ON-TB (QIAreach QFT) assay [29, 31, 32].
QIAreach QFT leverage the same technology as the TB2 tube of QFT-Plus, is portable, sim-
ple to perform and requires only 1 ml of blood from each patient [14, 29, 31–33].
So far three studies have compared the new QIAreach QFT test against the established
(FDA[Food and Drug Administration]-approved and CE [European Community]-marked)
QuantiFERON1-TB Gold Plus test in detecting TB infection, both conducted in low incidence
settings (Milan, Japan and USA, respectively) [29, 31, 33].
This study aims to evaluate QIAreach1 QFT diagnostic performance in an intermediate
TB burden country using the QuantiFERON1-TB Gold Plus ELISA (QFT-Plus) as a reference
standard.

Materials and methods

Study population
This prospective trial was conducted between April 2021 and June 2021 at the the National
Public Health Laboratory, Sungai Buloh, Malaysia. This study was approved by Medical
Research and Ethics Committee (MREC), Ministry of Health. Only those individuals who

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Table 1. Characteristics of study participants.

Characteristics Individual screened (n = 196)
Age, years; median (IQR) 15.5 (25.75–41.25)
Sex, male; n (%) 73(37.2%)
Contact of TB case; n (%) 163 (83,2%)
Contact of MDR-TB case; n (%) 67 (16,8%)

IQR: Interquartile range, n = number, MDR-TB = Multidrug-resistant TB

https://doi.org/10.1371/journal.pone.0279882.t001

agreed to participate and signed a written informed consent form were included in the study.
For the respondent 18 years and above, the respondents must personally agree before perform-
ing any specific procedures, and the participation is voluntary. For respondents less than 18
years, the parents must sign the informed consent form. Nevertheless, before respondents par-
ticipate in this research study, the researchers will explain the study, and respondents have a
chance to ask questions. After the respondents are adequately satisfied and wish to participate
in this study, the respondent must sign informed consent form. The respondent is free to with-
draw from the study at any time for any reason and will not affect any medical care or any ben-
efits to which respondents are entitled. Demographic data collected from patients included
age, gender as well as reason for testing (Table 1) parameter Individuals aged 4–82 years, who
were candidates for TB routine infection screening test for contact investigation were prospec-
tively recruited. All enrolled participants were interviewed by the attending physician and
underwent a chest radiography (CXR). For those with radiographic anomalies suggestive of
TB, further evaluation was performed using sputum acid-fast bacilli smear and culture to rule
out active TB or lung disease due to nontuberculous mycobacteria.
None of all subjects undergoing to QIAreach-QFT testing had active TB. For all partici-
pants, 1ml aliquots of blood were drawn directly into four blood collections tubes and used to
run both QFT-Plus and QIAreach QFT tests. Ethical approval for this study was obtained
from the Medical Research and Ethics Committee (MREC), Ministry of Health Malaysia
(Approval No.: NMRR 17-315-34830).

QFT-Plus assay
QFT-Plus assays were carried out according to the manufacturer’s instructions (QIAGEN
GmbH, Hilden, Germany) [34]. Venous blood was collected from the patients into the follow-
ing four blood collections tubes: Nil (negative) Control, Mitogen (positive) Control, TB1 Anti-
gen, and TB2 Antigen tubes (0.8ml-1.2 ml each). Within 16 h after blood collection the tubes
were incubated at 37˚C for 16–24 h. After incubation, the tubes were centrifuged at 3000×g for
15 min. Plasma supernatants were harvested and then processed immediately or stored at 4˚C
until enzyme-linked immunosorbent assay (ELISA) was performed. IFN-γ levels were quanti-
fied for QFT-Plus samples with 4-point standard curves. Results were reported as TB antigen
minus nil values. The optical density of each well was measured by reading in the ELISA reader
using the “QuantiFERON-TB Gold Plus Software”. The results were interpreted as Positive,
Negative, or Indeterminate according to the Instructions For Use.

QIAreach QFT assay

The QIAreach QFT assay was performed according to the manufacturer’s instructions [29,
31], and used in combination with the QIAreach Software (prototype versionx64-1.1.12.0)
installed on a computer running the Microsoft Windows operating system.

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Prior to running the assay, the eHub was powered on. The eStick comes packaged with a
process tube that contains dried nanoparticle conjugate. Immediately prior to use, the eStick
and Processing tube were removed from their packaging and the eStick was inserted into the
eHub.
Assay Diluent Buffer (150 μL) was added to the Processing Tube to resuspend the dried
nanoparticle conjugate, followed by addition of 150 μL of plasma specimen from the patient
TB2 tube into the Processing Tube. The plasma sample was mixed with the resuspended nano-
particle conjugate in the Processing Tube with the pipette, then150 μL of the mixed sample
was transferred from the Processing Tube into the sample port of the eStick connected to the
eHub port. The sample was detected automatically in the eStick, and after test completion the
assay results (positive or negative and Time To Results) were displayed on both the computer
and the eHub.

Statistical analysis
Qualitatively data were expressed as IFN-γ concentration IU/ml or Time To Results (TTR)
and analyzed using Microsoft Excel application spreadsheet software. The concordance
between the QFT-Plus and the QIAreach QFT assay results was assessed using kappa coeffi-
cients and was interpreted according to the Landis and Koch classification [35]. Consequently,
the Cohen’s kappa test results were classified as follows: a score of < 0.2 indicated slight agree-
ment; 0.21–0.40, fair agreement; 0.41–0.60, moderate agreement; 0.61–0.80, substantial agree-
ment; and 0.81–1, almost perfect or perfect agreement. The overall percent agreement (OPA)
was determined using QFT-Plus as the reference method. The positive percent agreement
(PPA; sensitivity) and negative percent agreement (NPA; specificity) were calculated along
with the corresponding exact two-sided 95% Confidence Interval (CI) using QFT-Plus as a ref-
erence. Linear regression analysis was performed to examine the relationship between the
QIAreach QFT TTR (in seconds) and the QFT-Plus IFN-γ levels (in IU/mL).

Results
QIAreach QFT and QFT-Plus were blinded performed in 196 patients, 163 (83.2%) being con-
tacts of drug-susceptible TB and 33 (16.8%) of MDR-TB patients with median (IQR) age of 31
years (25.25–41.75); 73/196 (37.2%) were males. No patient was undergoing dialysis or was
immunocompromised.
Out of 196 samples tested, 5.6% (11/196) samples gave an error result on QIAreach-QFT
and 4.1% (8/196 of whom one children immunosuppressed and 7 contact of TB case > 9 years
old without any underlying medical condition) samples gave indeterminate result on QFT-
plus. These 19 specimens have been excluded from further analysis. As reported in Table 2, the
QIAreach-QFT showed a positive percent agreement (sensitivity) was 96.5% (CI 87.9–99.6%),
a negative percent agreement (specificity) 94.2% (CI 88.4% to 97.6%) and an overall percentage

Table 2. Diagnostic performance of QIAreach QFT assay using QFT-plus as a reference standard.
Frequency Agreement Upper 95% CI Lower 95% CI
OPA 169/178 94.9% 90.6% 97.6%
PPA 55/57 96.5% 87.9% 99.6%
NPA 114/121 94.2% 88.4% 97.6%
Cohen’s κ score 0.88

OPA: Overall Percent Agreement, PPA: Positive Percent Agreement, NPA: Negative Percent Agreement

https://doi.org/10.1371/journal.pone.0279882.t002

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Fig 1. Distribution plot of IFN-γ values (uncorrected and corrected TB2 values) versus TTR for positive samples (n = 55). A- uncorrected values. B–
corrected values.
https://doi.org/10.1371/journal.pone.0279882.g001

of agreement was 94.9% (95% CI 90.6–97.6%) with a Cohen’s κ of 0,88. Out of 9 discordant
samples, 7 samples were positive by QIAreach-QFT alone and 2 samples were positive to
QFT-plus alone.
In thirteen QFT-Plus positive specimens, both TB1-Nil and TB2-Nil value were <1 IU/ml and
all returned a positive QIAreach QFT result. The TTR for QIAreach QFT positive samples varied
from 210 second (3,5 minutes) to 1200 seconds (20 min). Fig 1 shows the distribution plot of
QFT-Plus IFN- γ concentrations (IU/ml) in TB2-Nil (corrected) and TB2 (uncorrected) tube values
versus TTR (in seconds) for QIAreach QFT positive samples. The linear regression analysis demon-
strated a strong negative correlation between TTR and IFN-γ levels in corrected and uncorrected
TB2 with a correlation coefficient R = -0.7984 (p < 0.001) and R = -0.7961 (p < 0.001) respectively.

Discussion
Our study aimed at evaluating the performance of the QIAreach QFT assay using the globally
established QFT-Plus IGRA as a reference method.
This study represents the first performance evaluation performed in an intermediate TB
burden setting. The study results demonstrated an excellent level of agreement as well as high
sensitivity and specificity of QIAreach QFT compared to QFT-Plus when implemented in rou-
tine settings in Malaysia, confirming previous results achieved in Milan, Japan and USA [29,
31, 33]: OPA, PPA (sensitivity) and NPA (specificity) exceeding 94% [36].
The QIAreach QFT overall error rate was 5.6% (11/196), five errors were related to eStick
functioning, while six were run or user workflow errors. The high concordance between QIAr-
each QFT and QFT-Plus results, specifically in QFT-Plus patients with TB1 and/or TB2 anti-
gen values < 1 IU/mL, supports comparable sensitivity of the QIAreach QFT detection system
to the QFT-Plus ELISA. Similarly to QFT-Plus with value close to the assay cut-off (0.35 IU/
ml), QIAreach QFT may potentially have some variability of results with a TTR close to 1200
seconds. In our study nine samples had discrepant results between QIAreach QFT and
QFT-Plus. Discordant results may be influenced by a number of factors including but not lim-
ited to borderline IU/mL values near the cut-off, the absence of a Nil background subtraction
in the QIAreach QFT test, sample viscosity and color affecting lateral flow functionality, but
nevertheless, a high agreement was observed between the novel QIAreach QFT detection sys-
tem and the established QFT-Plus ELISA.
Fig 1 shows the IFN-γ level of the corrected and uncorrected TB2 values of QFT-Plus plot-
ted against the QIAreach QFT TTR. We found a statistically significant relationship between
levels of corrected and uncorrected IFN-γ in TB2 and TTR with a correlation index of R =

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-0.7984 (p < 0.001) and R = -0.7961 (p < 0.001) respectively. Similar findings were reported
by Fukushima et al. suggesting a high correlation between IFN-γ level and TTR [29].
QIAreach QFT showed several benefits for low resource, high burden testing markets. The
simple workflow and use of an integrated lateral flow test with highly sensitive detection capa-
bilities instead of ELISA translates into less laboratory infrastructure and minimal required
training, key benefits for the implementation in resource-limited settings. The volume of
blood is remarkably reduced when compared to the conventional IGRA assay. QIAreach QFT
requires 1 ml of blood compared to the 4 ml needed to perform the QFT-Plus ELISA. This is
of particular relevance in young children where high volume is not always easy to collect.
Optional software is also available with the QIAreach QFT assay to assist with report genera-
tion and connectivity through LIMS. Finally, QIAreach QFT can run eight samples indepen-
dently, providing a readout in � 20 minutes after a simple assay set up of less than 1 minute.
With each of the eight eHub ports operating independently, the use of multiple eHubs pro-
vides testing scalability options. This translates into a total of 88–96 samples/days which make
this assay even more suitable for mass screening.
In view of the urgency to apply the WHO End TB Strategy [11] and pursue TB Elimination
[8] it is important to strengthen the prevention measures and to adequately diagnose and treat
TB infection [2, 12].
The advantages of the IGRA tests, in terms of sensitivity, specificity, need for a single visit, sim-
ple interpretation of results (not subject to inter- and intra-reader variation of the induration read-
ing in millimeters), no influence by previous Bacillus Calmette-Guérin (BCG) vaccination(s) are
well known (12). In a recent meta-analysis published the QFT-Plus assay had a pooled sensitivity
of 94% for active TB patients and a pooled specificity of 96% for healthy low risk individuals [37].
However, the previous generation of IGRAs cannot be considered point-of-care tests as they are
based on the ELISA technology and need substantial laboratory facility resources and equipment.
Clinical performance of novel skin tests based on specific M.tuberculosis antigens developed over
the last decade is yet to be fully established and they share the same operational challenges as TST
[15]. These include need of training for administration of the test and reading of results, quality
control challenges, lack of objectivity, need for a return visit for reading the results and cold chain
for storage and transportation of antigen preparation.
New technologies like QIAreach QFT may overcome several of these logistic and perfor-
mance challenges. The QIAreach QFT is simple to implement in any setting (including mobile
stations, remote operation) and the workflow requires minimal laboratory expertise. The rapid
turnaround time is an additional element making this assay a valuable tool for the decentrali-
zation of TB infection diagnosis with moderate test throughput.
The study has several limitations, all related with its preliminary nature: it was conducted in
a single laboratory (the reference one for Malaysia) on a sample of individuals who were con-
tacts of drug susceptible of MDR-TB patients, so that evidence on other important target
groups is not yet available; the sample size with regards to statistical power was not calculated.
Larger studies are necessary to further evaluate the potentiality of this assay in different set-
tings (particularly in low income, high burden countries) and different groups of individuals,
and different groups of individuals, as well as data on precision and costs analysis to create suf-
ficient evidence and therefore validate preliminary results in the field [38]. Increasing the
accessibility to TB infection management is, in fact, a priority for the years to come.

Supporting information
S1 Data.
(XLSX)

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Acknowledgments
The authors would like to thank the Director General of Health, Ministry of Health, Malaysia
for his permission to publish this article and all the colleagues who contributed to this study.

Author Contributions
Conceptualization: Noorliza Mohamad Noordin.
Data curation: Zirwatul Adilah Aziz.
Formal analysis: Zirwatul Adilah Aziz.
Investigation: Noorliza Mohamad Noordin, Wan Mazlina Wan Mohd, Mohd Amin Kasim.
Methodology: Zirwatul Adilah Aziz, Wan Mazlina Wan Mohd, Mohd Amin Kasim.
Project administration: Noorliza Mohamad Noordin.
Resources: Wan Mazlina Wan Mohd, Mohd Amin Kasim.
Supervision: Zirwatul Adilah Aziz, Noorliza Mohamad Noordin.
Writing – original draft: Zirwatul Adilah Aziz.
Writing – review & editing: Noorliza Mohamad Noordin.

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