and IP-10 results in all groups in this study (93/120, 78%, k = 0.64) [17]. Age ranges in both the Ruhwald study and our own study were similar and produced comparable results for LTBI. In our study, IP-10 was produced in significantly higher levels than IFN-c, in response to MTB antigens. IFN-c producing T- cells are known to induce IP-10 production in primary exposure to TB. In the mouse model, IP-10 is induced by IL-23 and IL-17 producing T-cells in a recall response to MTB infection in the lung, independent of IFN-c production [20]. IL-23/IL-17 secreting T-cells are known to establish IFN-c producing T-cells following initiation by TGF-b and IL-6[20]. These regulatory cytokines are likely to be key players in granuloma stabilisation and therefore latent TB; induction by both IFN-c-producing and IL-17-producing T-cells may explain why the levels of IP-10 are greater in latent TB than active TB. The stimulation index indicates a positive response to the specific MTB antigens as opposed to generalised activation of cytokine- producing cells. The stimulation index of IP-10 was notably lower than that of IFN-c, despite the higher levels of cytokines produced. This is due to the higher background level of IP-10 in unstimulated samples, particularly noticeable in the latent TB cases as discussed above. IP-10 levels are increased in patients with inflammatory states such as autoimmune disease (systemic lupus erythematosus, thyroid disease), acute coronary syndrome, cerebral malaria and allergy[21,22,23,24]. In the control group in our study, one patient in particular was found to have very high levels of IP-10. It is probable that IP-10 is mainly a marker of inflammation and therefore too non-specific to be a stand-alone biomarker for TB infection. However it may still have a role as part of a panel of diagnostic markers. IFN-c is the key cytokine produced in response to TB antigens following TB exposure causing either active or latent TB and this observation forms the basis of the IGRA. However, IGRA do not distinguish between active TB and LTBI. It is interesting to note that although we found a higher IFNc stimulation index in LTBI than in active TB in our study, unfortunately the difference is not significant and therefore not useful as a diagnostic test. A difference in IFNc levels in active TB and LTBI has been noted before and some groups suggest a role for CD4+CD25highFoxP3+ T-cells in References 1. Marais BJ, Graham SM, Cotton MF, Beyers N (2007) Diagnostic and management challenges for childhood tuberculosis in the era of HIV. J Infect Dis 196 Suppl 1: S76–85. 2. Marais BJ, Gie RP, Hesseling AC, Schaaf HS, Lombard C, et al. (2006) A refined symptom-based approach to diagnose pulmonary tuberculosis in children. Pediatrics 118: e1350–1359. 3. Pai M, Riley LW, Colford JJM (2004) Interferon-[gamma] assays in the immunodiagnosis of tuberculosis: a systematic review. The Lancet Infectious Diseases 4: 761–776. 4. Lalvani A, Millington KA (2007) T cell-based diagnosis of childhood tuberculosis infection. Curr Opin Infect Dis 20: 264–271. 5. Detjen AK, Keil T, Roll S, Hauer B, Mauch H, et al. (2007) Interferon-gamma release assays improve the diagnosis of tuberculosis and nontuberculous mycobacterial disease in children in a country with a low incidence of tuberculosis. Clin Infect Dis 45: 322–328. 6. Brock I, Weldingh K, Lillebaek T, Follmann F, Andersen P (2004) Comparison of tuberculin skin test and new specific blood test in tuberculosis contacts. Am J Respir Crit Care Med 170: 65–69. 7. Adetifa IM, Lugos MD, Hammond A, Jeffries D, Donkor S, et al. (2007) Comparison of two interferon gamma release assays in the diagnosis of Mycobacterium tuberculosis infection and disease in The Gambia. BMC Infect Dis 7: 122. 8. Connell TG, Curtis N, Ranganathan SC, Buttery JP (2006) Performance of a whole blood interferon gamma assay for detecting latent infection with Mycobacterium tuberculosis in children. Thorax 61: 616–620. 9. Mandalakas AM, Hesseling AC, Chegou NN, Kirchner HL, Zhu X, et al. (2008) High level of discordant IGRA results in HIV-infected adults and children. Int J Tuberc Lung Dis 12: 417–423. Is IP-10 a Better Biomarker? suppressing IFN-c responses to a variety of TB antigens in active TB [25]. An alternative hypothesis is that IFN-c producing CD4+ T cells are sequestered at the tissue site of infection, rather than in the blood, causing a paradoxically low level of IFN-c production to antigen stimulation. This may also explain the lower levels of IP-10 in active TB compared to latent TB. The main limitation of this study is the small sample size, which could potentially result in a type II error in the statistical analysis. However, we think, that this is unlikely, as all of the children in each group were very well characterised, thus the results represent a trend that provides direction for further studies. The children with active and LTBI all had TST.15 and positive IGRA tests, hence strong evidence of mycobacterial infection. We deliberately selected these well-characterised phenotypes in order to directly compare the stimulation of IP-10 with IFN-c production. This could have led to bias in the IP-10 results as its production can be induced by IFN-c. It would be interesting to compare IP-10 and IFNc levels also in those children with clinically diagnosed TB and negative IGRA. Although IP-10 does not appear to be sensitive or specific enough to supersede IGRA as tools for TB diagnosis, or to distinguish between active TB and LTBI, a number of conclusions can be drawn from our study: the supernatants of the Quantiferon Gold-In-Tube assays are a useful and robust resource for investigation of further proposed biomarkers, and we are currently investigating other cytokines as potential biomarkers to distinguish between active TB and LTBI using this sample bank. The levels of IP-10 in unstimulated blood of LTBI patients may have diagnostic and prognostic value for further investigation, and higher levels may indicate a potentially protective mechanism for the development of LTBI rather than active TB. Of course, only prospective evaluation of such a biomarker could confirm this hypothesis. Author Contributions Conceived and designed the experiments: EW BK. Performed the experiments: EW. Analyzed the data: EW. 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Okamoto M, Kawabe T, Iwasaki Y, Hara T, Hashimoto N, et al. (2005) Evaluation of interferon-gamma, interferon-gamma-inducing cytokines, and interferon-gamma-inducible chemokines in tuberculous pleural effusions. J Lab Clin Med 145: 88–93. 15. Azzurri A, Sow OY, Amedei A, Bah B, Diallo S, et al. (2005) IFN-gamma- inducible protein 10 and pentraxin 3 plasma levels are tools for monitoring inflammation and disease activity in Mycobacterium tuberculosis infection. Microbes Infect 7: 1–8. 16. Juffermans NP, Verbon A, van Deventer SJ, van Deutekom H, Belisle JT, et al. (1999) Elevated chemokine concentrations in sera of human immunodeficiency virus (HIV)-seropositive and HIV-seronegative patients with tuberculosis: a possible role for mycobacterial lipoarabinomannan. Infect Immun 67: 4295–4297. 17. Ruhwald M, Petersen J, Kofoed K, Nakaoka H, Cuevas LE, et al. (2008) Improving T-cell assays for the diagnosis of latent TB infection: potential of a diagnostic test based on IP-10. PLoS ONE 3: e2858. PLoS ONE | www.plosone.org 5 December 2008 | Volume 3 | Issue 12 | e3901