(IRS)[16]. IP-10 has been suggested as a marker of TB treatment efficacy – serial samples show reductions in levels at 2 months in cured TB patients, with no reduction in non-responders [15]. Levels of IP-10 are higher in those patients with systemic symptoms such as fever and anorexia[16]. Of note IP-10 levels in household contacts of TB infected patients, both adult and children (85% older than 5 years), were higher than controls, suggesting a potential value for latent TB infection (LTBI) diagnosis [15,17]. In view of these findings in predominantly adult studies, we investigated whether IP-10 could be a new diagnostic biomarker for TB in children, specifically, whether it could help in distinguishing between active TB and LTBI – a particular challenge in the paediatric population. Methods Subjects Study approval was obtained from the ethics committees of Imperial Healthcare NHS Trusts and North West London Hospitals Trust. We recently conducted a research study comparing TST and IGRA in the diagnosis of active TB and LTBI in children in London, using the QFG-IT test and T Spot TB (submitted for publication). In this study, we included all children between 2 months and 16 years of age who were investigated for active TB, as well as referrals for contact tracing after exposure to a known case of active TB. Exclusion criteria were immunocompromise, including immunosuppressive therapy and HIV. Remaining supernatant samples harvested from the QFG tubes and not used for the determination of IFNc were used in this additional investigation. For the purpose of the IP-10 study, only children with a TST.15 and positive QFG-IT were selected for the cohorts of active and latent TB. Written consent for the IGRA study, including consent to use remaining samples, was obtained from all study participants. Definition of active TB. The diagnosis of active TB was based on the presence of acid-fast bacilli in either gastric washings/induced sputum or other microbiological samples, and confirmed by growth in culture. Only children with culture proven TB were included in this study. Definition of latent TB infection (LTBI). For the purpose of this study, only children with a TST.15 and positive QFG-IT were selected for the cohort of LTBI. None of the children had signs and symptoms of active TB and a normal chest X- ray. Non-TB Control group. 16 children with non-TB disease were included as controls. They were recruited from both the outpatient department and the general paediatric ward and had a variety of alternative diagnoses, such as fractures, urinary tract infections, pneumonia, allergy and nephrotic syndrome. Careful history excluded TB contact. Both IGRA were performed on all these children and were negative in all 16. Whole Blood Stimulation. Briefly, 1 ml of blood was drawn directly into each vaccutainer tube provided as part of the QFG-IT system (Cellestis, Carnegie, Australia). These tubes are already precoated with saline (negative control), peptides of ESAT-6, CFP10 and antigen TB 7.7 (referred to as antigens/Ag for the remainder of the paper) - and PHA (positive mitogen control). The tubes were mixed and incubated for 20–24 hr at 37uC, when plasma was harvested and frozen until further analysis. This plasma provided the unstimulated and antigen (Ag) stimulated supernatant samples. IP-10 quantification. IP-10 supernatant levels were measured using a sandwich ELISA. Briefly, ELISA plates were coated overnight with a monoclonal antibody for Human CXCL10/IP-10 (R&D Systems), blocked with a 2% bovine serum albumin solution, Is IP-10 a Better Biomarker? extensively washed and test samples or recombinant human CXCL10/IP-10 (R&D Systems) as standards were added and incubated at room temperature for one hour. After washings, a biotinylated polyclonal secondary antibody to human CXCL10/IP- 10 as (R&D Systems) was added and incubated for 1 hour at room temperature. After washing, streptavidin conjugated HRP (Biosource), was added for 15 minutes at room temperature, followed by TMB. The reaction was stopped with 2 m H2SO4 and absorbance at 450 nm was measured in an ELISA reader. The lower end of sensitivity was 50 pg/ml for this assay. The upper limit of IP-10 for the assay was 40,000 pg/ml and readings greater than this were set at 40,000 pg/ml for the purpose of analysis. Calculation of Stimulation Index. For comparison purposes, a stimulation index was calculated (ie the cytokine concentration in plasma of antigen- stimulated whole blood divided by the cytokine concentration of unstimulated whole blood). A stimulation Index greater than 1.25 indicates that there was a significant response to Ag stimulation [10]. Statistical Analysis. Significant differences between cytokine levels were determined by the Kruskal-Wallis test. If a significant difference was found, the Mann-Whitney U-test was used to calculate differences between each pair of groups. Paired data was compared by the (two-tailed) Wilcoxon signed rank test. P values were two-sided and considered significant if ,0.05. All calculations were performed using GraphPad Prism computer program (GraphPad Software Inc., San Diego, CA, USA).Data shown in the figures and tables are given as medians. Results The median age of the study group was 10.15 yrs (range 0.4– 16.8 yrs). The 17 children with active TB ranged from 5 to 16 years with a median age of 11.3, of whom 9 had received BCG vaccination. The 16 children with LTBI had a median age of 10.5 years (range: 5–15.5 yrs), and 12 children were BCG vaccinated. The median age of the control group was 5.9 years (range: 0.4– 16.8 yrs) and 8 children had received BCG-vaccine. IP-10 measurements The unstimulated or background levels of IP-10 were increased in the active TB group (median 1019 pg/ml, range: 320– 5047 pg/ml) compared to control (median 320 pg/ml, range: 320–7275 pg/ml, p = 0.073). However the level of IP-10 in the LTBI group (median 2200 pg/ml, range: 320–40,000 pg/ml) were significantly greater than either active TB (p = 0.018) or control, (median 320 pg/ml, p = 0.0017). (Figure 1a) The TB specific antigen stimulated samples demonstrated highly significant differences between active TB (median 9544 pg/ml, range: 1386–40,000 pg/ml) and LTBI (median 19350 pg/ml, range: 2200–40,000 pg/ml, p = 0.004) compared with the control group (median 536 pg/ml, range: 320– 40,000 pg/ml, p = 0.0085). (Figure 1b) When comparing the active TB and LTBI cases, there was a greater increase in the antigen-stimulated samples from the patients with LTBI (median 19350 pg/ml) than in the patients with active TB (median 9544 pg/ml); however, this was not significant (p = 0.85). In the patients with active TB, levels of IP-10 were found to be significantly increased in the TB specific antigen stimulated samples (median 9544 pg/ml, range: 2535–40,000 pg/ml) as compared to the unstimulated or background samples (median 1019 pg/ml, 433–4308 pg/ml; p,0.0003) (Figure 1a). A significant increase in IP-10 levels following antigen stimulation was also detected in the samples from patients with LTBI (median 19350 pg/ml, 2200–40,000 pg/ml, p,0.0245, PLoS ONE | www.plosone.org 2 December 2008 | Volume 3 | Issue 12 | e3901