Diagnostic performance of IP-10
Only data from children with microbiologically confirmed ATB,
TST+QFT+ LTBI,
TST-QFT- TB contact,
TST-QFT- non-TB, and
QFT- controls were included in the ROC analysis. For
the assessment of IP-10 accuracy in identifying ATB and LTBI, data from
children with ATB and LTBI were compared with the control group. IP-10
showed very good properties for discriminating Mtb.- infected
children, children with ATB, and children with LTBI from controls.
However, IP-10 could not discriminate between ATB and LTBI groups.
Additionally, ROC analysis was performed separately for children
admitted due to clinical suspicion of TB and for contact tracing
referrals. In both settings we observed good performance of IP-10 in
identifying Mtb. -infected children. At its optimal cut-off values
IP-10 showed very good performance in each setting (AUC >
0.9) except ATB versus LTBI (Table 3), and outperformed IFN-γ
(data not shown).
We then evaluated the properties of the optimal cut-off point of 1084.5
pg/ml for identifying Mtb. infection (ATB and LTBI combined vs
control group). At this cut-off IP-10 correctly classified 84.9% of the
participants, 93.3% among microbiologically confirmed ATB group, and
showed 88.5% positive predictive value and 83.5% negative predictive
value. In children with ATB, IP-10 correctly classified 78.8% of
patients and performed better than QFT but poorer than TST (69.7%. and
93.3%, respectively). One child with indeterminate QFT result was
correctly classified by both TST and IP-10. Combining IP-10 and QFT
results increased sensitivity to 84.5%.
DISCUSSION:
The present study indicates the potential of IP-10 to detect Mtb.infection in children in a low TB incidence country where BCG
vaccination is routinely administrated at birth. We demonstrate that
IP-10 level is not correlated with age and, for the first time, report a
decrease in the IP-10 level on antituberculous treatment in children.
In agreement with previous studies, we showed that IP-10 can distinguishMtb. -infected from uninfected children17-19,21,23-26. Unlike some other reports, we observed
an increased Mtb. antigen-stimulated expression of IP-10 only in
the infected children 21,25. While demonstrated
significant differences in IP-10 level between Mtb.- infected and
uninfected contact tracing referrals have been already reported by
others 15,19,21,24,25, IP-10 has not been vastly
investigated in symptomatic children with a clinical suspicion of ATB.
As far as we know, this study is the first to show that IP-10 can
identify Mtb. infection in symptomatic children under diagnosis
for TB in a low-endemic setting. Previous studies by Petrone et al. and
Sudbury et al. were performed in high-incidence countries27,28. Petrone et al. reported contradictory results
to our study but employed unstimulated plasma, which may have accounted
for the observed differences 27. In line with our
report, Sudbury et al. noted significantly higher IP-10 level in ATB
children than in symptomatic children with other final diagnosis28. However, the number of ATB patients was low (n=5
as compared to n=33 in this report).
Although Mtb. antigen-stimulated IP-10 level was higher in
children with ATB than in LTBI group (p = 0.047), no power of IP-10 to
discriminate between ATB and LTBI has been demonstrated in ROC analysis.
Our findings stay in line with the majority of previous reports in
adults and children 17,20,29,30.
We observed a good agreement between IP-10 and both TST and QFT (85.4%
and 87.7% respectively; ƙ=0.7; p=0.0000; data not shown). Moreover,
combining IP-10 and IFN-γ results improved QFT sensitivity in patients
with ATB, in whom IGRAs’ performance remains suboptimal. The benefits of
combining biomarker approach in childhood TB have been already
documented 15-17,23,26,30. However, several studies
conducted in a high endemic setting and in children with ATB yielded
conflicting results warranting further research in this field18,31,32.
On par with most evidence in children, we did not demonstrate a relevant
correlation of IP-10 with age 18,23,25,30.
Additionally, in line with a previous study from our center, we did not
observe compromised IFN-γ responses in younger children9.
Furthermore, the present study is, as far as we know, the first to
report a decrease in the IP-10 level on anti-TB treatment in children.
While a decline in plasma IP-10 level after successful anti-TB treatment
has been demonstrated in adults, it has not been vastly investigated in
children 20,21,33-36. Nausch et al. analyzed a
spectrum of cytokines before and during anti-TB treatment and reported a
significant reduction only in the expression of IFN-γ with no change in
the IP-10 level 21. In contrast, we observed a
significant decline in the IP-10 concentration with no decrease in the
IFN-γ level. This decline in the IP-10 expression was detected only in
children with ATB, who had the highest concentrations at baseline. We
assume that differences in time to follow-up visit (median 69 days in
our study versus 90 days in the other report) could explain the
discrepancies related to IFN-γ but not IP-10. Our results support the
previous study by Wergeland et al., who demonstrated a decrease in IP-10
level in adult patients with ATB already after 6-12 weeks of treatment20.
Another strength of the present study is the control group consisting of
healthy children with a population risk of TB infection. Numerous
studies have included TB contacts, children with respiratory tract
infections, or healthy adults in the control group17,19,21,24,25,27,30. We consider our approach
reasonable for several reasons. First, Mtb. infection cannot be
definitively excluded in children recently exposed to TB. Second, IP-10
can be induced during diverse inflammatory diseases including infections37,38. Third, direct comparison of IP-10 level solely
between children provides relevant information on its’ expression in
this age group. Taken together, including healthy children in the
control group might have limited the risk of bias in the present report.
The main limitation of our study is the relatively small sample size.
This is not uncommon in studies conducted in children in TB low-burden
areas and has been previously reported 19,30.
Furthermore, our results might be biased by potential false
classification of participants. Similar to others, we applied stringent
criteria for ATB diagnosis to limit the risk of misclassification19,21,25. The rate of culture-positive cases reflected
other observations in children, and no significant differences in IP-10
or IFN-γ level (data not shown) were demonstrated between ATB children
with and without a positive culture result 39.
Therefore, we assume that the classification of ATB patients was
correct, however, the possibility of misclassification cannot be truly
excluded.
Furthermore, LTBI group in the present study comprises children with
either positive TST or QFT result. Since the TST false-positivity rate
does not exceed 8.5 % in individuals vaccinated in infancy and only 2
participants with TST-/QFT+discordance presented with QFT results relatively close to the cut-off
(data not shown), we believe this cannot account for a large proportion
of discordant LTBI patients in our report 4. Similar
to others, we observed higher IP-10 levels in children with
TST+QFT+ LTBI than in children with
discordance 16,19. Interestingly, contrary to Petrucci
et al., no differences were noted between children with different
discordant results constellation. Since children with discordance
constitute a significant management problem in pediatrics, this is an
area that merits further research on fluctuations of TB biomarkers
during different stages of Mtb. infection.
Applying QFT negativity as a criterion for Mtb.- uninfected groups
might have caused selection bias and rendered the comparison of the
specificity of IP-10 and QFT impossible. Furthermore, we neither
assessed the severity of TB, nor were children with the most severe
forms of disease included. Additionally, children with ATB were
significantly older than children in the other groups. Therefore, IP-10
performance in children with severe TB and the youngest children with
ATB may differ from that presented here. Routine testing for HIV was not
performed as part of this study, but HIV tests results were recorded if
available and no cases were reported. Notably, HIV incidence in Poland
is low (<1/100 000) with marginal TB-HIV coexistence40. Nonetheless, the performance of IP-10 in HIV
infected children in our setting remains uncertain.
In conclusion, our data demonstrate that Mtb.- specific IP-10
measurement has a potential as a diagnostic biomarker of childhood TB.
In particular, IP-10 seems to be a valid surrogate marker to IFN-γ in
the assays based on the QFT platform. Our data show that IP-10 alone
does not allow the distinction between ATB and LTBI. This is the first
study with a clinical approach to TB diagnosis in children that shows
good performance of IP-10 in identifying Mtb . infection in TB
contact referrals and children with clinical suspicion of TB in a low
TB-endemic country. Additionally, this is the first report demonstrating
a significant decline of the IP-10 level during anti-TB chemotherapy in
children. Collectively, these findings suggest IP-10 has the potential
to become a TB marker in pediatric population and may be used in
treatment monitoring.
Funding: This research received no external funding.
Availability of data and materials: The datasets used
and analysed during the current study are available from the
corresponding author on reasonable request.
Competing interests: The authors declare that they have
no competing interests.
ACKNOWLEDGEMENTS:
The authors wish to thank Dr. Jerzy Ziołkowski, the initiator and mentor
of this study, who died in April 2014. They also thank the Department of
Laboratory Medicine and Clinical Immunology of Developmental Age Team,
in particular Dr. Marzena Modzelewska and Elżbieta Widemajer for their
assistance with ELISA assays. The study was supported by Medical
University of Warsaw (WUM/1W34/2012/2016)
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