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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