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Urine IP-10 as a biomarker of therapeutic response in patients with active pulmonary tuberculosis

Urine IP-10 as a biomarker of therapeutic response in patients with active pulmonary tuberculosis Background: Prior to clinical trials of new TB drugs or therapeutic vaccines, it is necessary to develop monitoring tools to predict treatment outcomes in TB patients. Urine interferon gamma inducible protein 10 (IP-10) is a potential biomarker of treatment response in chronic hepatitis C virus infection and lung diseases, including tuberculosis. In this study, we assessed IP-10 levels in urine samples from patients with active TB at diagnosis, during treatment, and at completion, and compared these with levels in serum samples collected in parallel from matched patients to determine whether urine IP-10 can be used to monitor treatment response in patients with active TB. Methods: IP-10 was measured using enzyme-linked immunosorbent assays in urine and serum samples collected concomitantly from 23 patients with active TB and 21 healthy adults (44 total individuals). The Mann-Whitney U test and Wilcoxon matched-pairs signed rank test were used for comparisons among healthy controls and patients at three time points, and LOESS regression was used for longitudinal data. Results: The levels of IP-10 in urine increased significantly after 2 months of treatment (P = 0.0163), but decreased by the completion of treatment (P = 0.0035). Serum IP-10 levels exhibited a similar trend, but did not increase significantly after 2 months of treatment in patients with active TB. Conclusions: Unstimulated IP-10 in urine can be used as a biomarker to monitor treatment response in patients with active pulmonary TB. Keywords: Tuberculosis, Urine IP-10, Biomarker, Monitoring Background Progress has been made in the control of tuberculosis, as Tuberculosis (TB) remains a major infectious cause of advocated by the World Health Organization (WHO) [2]. mortality and morbidity, with approximately 8.6 million For the development of novel vaccines and drugs new cases and 1.3 million deaths per year [1]. In addition, against TB, a reliable surrogate marker of risk and pro- there is concern over increasing rates of disease recur- tection against TB is essential. In particular, prior to rence (including relapse and reinfection) after anti-TB clinical trials of new therapeutic vaccines to prevent TB drug therapy and an increasing trend of multidrug- and recurrence and to shorten the duration of therapy, ef- extensively drug-resistant (M/XDR) TB worldwide. fective monitoring tools are needed to measure treat- ment responses to standard drug therapies in parallel with therapeutic vaccinations [3, 4]. * Correspondence: [email protected]; [email protected] Generally, sputum culture conversion after 2 months Song Yee Kim and Jungho Kim contributed equally to this work. Division of Pulmonary, Department of Internal Medicine, Institute of Chest of anti-TB drug treatment has been used as a predict- Diseases, Severance Hospital, Yonsei University College of Medicine, Seoul ive indicator of drug treatment response in patients 03722, Republic of Korea [5–9]. However, culture methods are not applicable in Clinical Vaccine Research Section, International Tuberculosis Research Center, Seoul 03722, Republic of Korea patients with extrapulmonary TB and culture-negative Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kim et al. BMC Infectious Diseases (2018) 18:240 Page 2 of 6 pulmonary TB, and laboratories capable of sputum positive sputum culture after 2 months of culture are often lacking in resource-limited settings anti-TB treatment as follows: [10–12]. To accommodate these shortcomings, efforts have been made to identify an alternative biomarker for anti-TB treatment efficacy [4, 13, 14]. Interferon gamma (IFN-γ)-inducible protein (IP-10) is  Low-risk group: TB patients who had neither risk a pro-inflammatory chemokine secreted by antigen- factor (i) nor (ii); presenting cells that transport activated T lymphocytes  Moderate group: TB patients who had either risk to sites of inflammation [15, 16]. IP-10 levels were found factor (i) or (ii); to be higher in the unstimulated serum, plasma, and  High-risk group: TB patients who had both risk urine of patients with active TB than those in subjects factors (i) and (ii). without active TB, and IP-10 levels decrease significantly after the completion of anti-TB treatment [15, 17–20]. Among all types of clinical specimens in which IP-10 has been detected, urine specimens are the most con- 2) Healthy controls (aged 20 years and over): Healthy venient and non-invasive; specimens can be obtained controls fulfilled the following inclusion criteria: (i) from children and elderly patients, even in resource- no history of tuberculosis, (ii) no suggestive limited settings, as these samples do not require special symptoms of tuberculosis, (iii) no recent contact equipment or skilful medical personnel for collection. In with patients with infectious tuberculosis, and (iv) addition, urine specimens present lower handling risks negative results according to the QuantiFERON -TB compared to other specimens, such as blood, sputum, Gold In-Tube Test (QFT-GIT). and microbial isolates from patients [20]. Theaim of this studywas to test thehypothesis None of the participants exhibited HIV infection; a that levels of urine IP-10 change over the course of chronic comorbidity, such as diabetes mellitus, anti-TB drug therapy in patients with active TB from chronic renal failure, malignant tumour, or chronic South Korea, an area with an intermediate burden of liver disease; immunosuppressive status; or acute TB and low burden of human immunodeficiency virus infections. infection and acquired immune deficiency syndrome (HIV/AIDS). IFN-γ measurement A QFT-GIT assay was conducted for all patients before treatment (0 months, T0), at 2 months (T2), and at 6– Methods 9 months after the treatment was completed (T6). Study population and setting The QFT-GIT assay (Qiagen, Hilden, Germany) was Clinical information and specimens were collected from performed according to the manufacturer’sinstruc- participants enrolled in two clinical studies from tions. Briefly, 1 ml of whole blood was collected in November 2010 to March 2012 and from May 2015 to each of three tubes pre-coated with Mycobacterium June 2016. The Institutional Ethics Committee of Yonsei tuberculosis-specific peptides (ESAT-6, CFP-10, and University Severance Hospital approved this study TB7.7) or mitogen (positive control) and incubated (approval #4-2010-0527 and #4-2014-1108). All study for 16–24 h at 37 °C. For negative controls, whole participants provided written informed consent to pro- blood was placed in tubes that were not pre-coated. vide specimens for the identification of TB biomarkers. The plasma supernatant was harvested by centrifuga- Study participants were classified as follows. tion at 3000×g for15 min andstoredat − 80 °C. The level of IFN-γ was determined using the QFT 1) Active pulmonary TB patients (aged 20 years and Enzyme-linked Immunosorbent Assay (ELISA) Kit. over): Patients with active pulmonary TB were The results were interpreted using QFT-GIT software diagnosed based on culture and/or pathology. Cases version 2.62, provided by the manufacturer. of clinical active pulmonary TB, which was defined as negative mycobacterial culture IP-10 measurement findings, but favourable clinical and radiological Serum and urine samples were collected to measure ser- responses to anti-TB medication, were also ial IP-10 levels from all patients at T0, T2, and T6. IP-10 included. Patients were sub-divided into the levels in urine and serum were concomitantly measured following three groups based on risk factors for re- in duplicate using a commercial IP-10 ELISA Kit (R&D lapse, including the presence of (i) a cavity Systems, Minneapolis, MN, USA) in accordance with lesion on a chest X-ray or chest CT and (ii) a the manufacturer’s instructions. The test results were Kim et al. BMC Infectious Diseases (2018) 18:240 Page 3 of 6 interpreted using SoftMax version 5.4.1 (Molecular De- Comparison of serum and urine IP-10 levels between vices, LLC, Sunnyvale, CA, USA). In addition, using the healthy controls and patients with TB at diagnosis test results available from routine hospital analyses, the We assessed whether the levels of IP-10 in serum and urine IP-10 level (in pg/ml) was normalized against the urine samples differed between TB patients at the time serum creatinine level (in mg/ml) for each patient at of diagnosis and healthy controls. Serum IP-10 levels did each visit. not differ significantly between TB patients at diagnosis (median, 85.37 pg/ml; IQR, 60.92-171.30) and healthy Statistical analysis controls (median, 68.62 pg/ml; IQR, 43.19-87.01), al- All statistical analyses were performed using GraphPad though the levels in patients were slightly higher than Prism 6 software (GraphPad Software, La Jolla, CA, those in the healthy controls (P = 0.0829; Fig. 1a). In USA) and IBM SPSS software version 21.0 (IBM Corp., addition, urine IP-10 levels in healthy controls (median, Armonk, NY, USA). Medians and interquartile range 6.49 pg/ml; IQR, 2.02-12.11) were not different from (IQR) were measured for continuous variables. The those in the TB patients at the time of diagnosis (me- Mann–Whitney U-test and Wilcoxon matched-pairs dian, 7.89 pg/ml; IQR, 4.86-13.97; Fig. 1b). signed rank test were used for comparisons among study groups and pairwise comparisons. A longitudinal Comparison of serum and urine IP-10 levels at TB smoothed trend line for the data (not based on any dis- diagnosis (T0), during treatment (T2), and after the tributional assumption) was estimated using a nonpara- completion of therapy (T6) metric method called LOESS (locally weighted We further analysed whether the serum and urine IP- scatterplot smoothing). A 95% confidence limit was esti- 10 levels in TB patients changed over the course of mated, and similarity patterns by risk group were graph- treatment with a complete set of serial specimens ically examined. All P-values were two-sided, and P <0. (T0, T2, and T6). Serum IP-10 levels in TB patients 05 was considered statistically significant. at the time of diagnosis (median, 85.37 pg/ml; IQR, 60.92-171.30) were not significantly different from Results those after 2 months of treatment (median, 125. Characteristics of study participants 80 pg/ml; IQR, 81.42-155.00; P = 0.1743) or upon the We enrolled 23 patients with active TB and 21 healthy completion of effective therapy (median, 81.09 pg/ml; controls. The patients with active TB had a median age IQR, 63.71-123.70; P = 0.7467; Fig. 2a). of 27 (range, 22–66), and the healthy controls had a me- However, the pairwise comparisons also showed that dian age of 25 (range, 20–33). Bacille Calmette-Guérin urine IP-10 levels in patients after 2 months of treat- (BCG) scars were present in 65.2% of patients with ac- ment (median, 19.17 pg/ml; IQR, 5.12-51.68) were sig- tive TB and 57.1% of healthy controls during inspection nificantly higher than those in patients at the time of at the site. None of the enrolled patients were acid-fast diagnosis (median, 7.89 pg/ml; IQR, 4.86-13.97; P =0. bacillus smear-positive, 19 (82.6%) were culture-positive, 0163). Moreover, urine IP-10 levels in patients tested and 8 (34.8%) had a cavity on a chest X-ray or computed after the completion of drug therapy (median, 5.47 pg/ tomography (CT) (Table 1). ml; IQR, 4.69-9.79) were significantly lower than those Table 1 Characteristics of subjects enrolled in the study TB Patients Healthy Controls (n = 23) (n = 21) Median Age (range) 27 (22–66) 25 (20–33) Male, n (%) 16 (59.3%) 13 (62.0%) Presence of BCG scars 15 (65.2%) 12 (57.1%) b c PTB diagnosis AFB smear, positive 0 (0.0%) 0 (0.0%) AFB culture, positive 19 (82.6%) 0 (0.0%) Cavity on Chest X-ray or CT 8 (34.8%) 0 (0.0%) Risk group for relapse High-risk 0 (0.0%) 0 (0.0%) Moderate-risk 8 (34.8%) 0 (0.0%) Low-risk 15 (65.2%) 0 (0.0%) a b c d BCG Bacille Calmette-Guérin, PTB Pulmonary tuberculosis, AFB Acid-fast bacillus; CT Computed tomography. Risk factors for relapse: i) the presence of a cavity lesion on chest X-ray or chest CT, and ii) the result of a positive sputum culture after 2 months of anti-TB treatment. The high-risk group was defined when patients had both risk factors i) and ii). The moderate-risk group was defined when patients had either risk factor i) or ii), and the low-risk group had neither risk factor Kim et al. BMC Infectious Diseases (2018) 18:240 Page 4 of 6 Fig. 1 Serum and urine IP-10 levels in patients with TB at diagnosis and healthy controls. IP-10 levels were measured in the serum (a) and urine (b) of each TB patient at diagnosis (T0) and each healthy control (HC). We only obtained serum samples from 17 patients at T0 out of 23 patients. The data are shown as median with interquartile range; statistical analyses were performed using the Mann–Whitney U-test sampled at the time of diagnosis (P = 0.0354) and after Longitudinal analysis of IP-10 levels in patients with 2 months of treatment (P = 0.0035; Fig. 2b). active TB stratified by risk group To confirm that the IP-10 response derived from TB The group of TB patients was then sub-divided by risk infection, the urine IP-10 level of each patient was nor- factors for relapse or treatment failure into low, moder- malized to the serum creatinine level at each visit using ate, and high-risk groups. Among all patients, 15 (65.2%) test results available from routine analyses. As an abnor- were included in the low-risk group, and 8 (34.8%) were mal serum creatinine level may indicate kidney disease included in the moderate/high-risk group. or damage, this served to verify that the dynamics of IP- As shown in Fig. 3b and c, the smoothed lines for 10 levels detected in this study were not influenced by urine IP-10 in both risk groups exhibited a similar trend kidney disease. The urine IP-10/creatinine ratio was over the three time points. The low-risk group had higher in TB patients after 2 months of treatment than higher urine IP-10 levels than those of the moderate/ either before or after treatment, consistent with the high-risk group, but the 95% confidence limits of IP-10 trend observed for urine IP-10 without normalization levels were not differentiated by risk group. (Fig. 2c). This indicated that the serum creatinine levels In contrast, serum IP-10 levels did not show similar did not affect the urine IP-10 levels in this study. trends in the two risk groups (Fig. 3a). Serum IP-10 Fig. 2 Serum and urine IP-10 levels in patients with TB before, during, and after treatment. Serial IP-10 levels in the (a) serum and (b) urine of patients with TB collected at the time of diagnosis (T0), after 2 months of therapy (T2), and after the completion of therapy (T6). c Serial IP-10 levels in urine normalized against serum creatinine levels in patients. The data are shown as median with interquartile range; statistical analyses were performed using the Wilcoxon matched-pairs signed rank test Kim et al. BMC Infectious Diseases (2018) 18:240 Page 5 of 6 Fig. 3 Longitudinal analysis of IP-10 levels in patients with active TB stratified by risk of relapse. Longitudinal (a) serum, (b) urine IP-10, and (c) urine IP-10/creatinine ratio obtained throughout treatment (T0, T2, and T6) were analysed in risk subgroups (low-risk and moderate/ high-risk groups) using locally weighted scatterplot smoothing (LOESS) and confidence intervals of the mean (CLM) levels in the moderate/high-risk group did not change peak inflammation and urine IP-10 for successful over time, but those in the low-risk group increased at treatment. T2 and decreased at T6, similar to the results for urine Individuals with acute and chronic infections were ex- IP-10 levels. cluded from analyses, and urine IP-10 levels were nor- malized against serum creatinine levels to ensure that increases in urine IP-10 levels can be attributed to infec- Discussion tion with M. tuberculosis, rather than kidney disease or As sputum culture conversion at 2 months is not always damage. In future studies, urine creatinine levels should predictive of successful TB therapy, new biomarkers for be examined to directly exclude urinary disease. predicting treatment responses are needed for better man- In addition, C-reactive protein (CRP) is an indicator of agement of TB patients. Similar to the findings of Cannas disease activity and is often increased in active tubercu- et al. [20], this study showed a declining tendency for urine losis inflammatory conditions [24, 25]. However, CRP IP-10 detection in TB patients after receiving effective anti- data for patients at diagnosis and follow-ups were not TB drug therapy compared the levels at time of diagnosis. available in this study. In future studies, CRP levels However, this study also detected urine IP-10 levels after should be estimated and their correlations with IP-10 2 months of an intensive anti-TB drug therapy, which is the levels can be used to assess inflammatory changes in re- point when clinicians routinely use radiological and bac- sponse to TB treatment. Furthermore, this study empha- teriological tests to determine treatment progress. Although sizes the usefulness of urine as non-invasive sample for recent studies have reported a decline in blood IP-10 levels monitoring treatment responses biomarkers in children, after 2 weeks of efficient anti-TB drug therapy as an early the elderly and HIV-infected subjects. biomarker for treatment response [21, 22], this study dem- onstrated a peak in urine IP-10 levels after 2 months of Conclusions treatment in HIV-negative patients with tuberculosis. In conclusion, unstimulated IP-10 levels in patient urine Moreover, the same trend was observed in patients regard- samples, which are easier to collect and analyse than blood less of risk factors for TB relapse or treatment failure. samples, can be used as a predictive factor, indicating a It is not clear why unstimulated IP-10 levels in urine positive treatment response. IP-10 detection may therefore significantly increase in patients after 2 months of anti- be a useful monitoring tool for determining whether TB pa- TB treatment. A previous study using imaging scans tients are positively responding to anti-TB drug therapy. suggested quantitative changes at 2 months after the Acknowledgements initiation of treatment, which may be related to long- The authors thank all study participants for contributing to this study. term TB treatment outcome [23]. To confirm this Funding phenomenon, additional investigations with a large-scale This study was supported by a grant from the Korean Health Technology R&D active TB cases including prolonged anti-TB treatment Project through the Korea Health Industry Development Institute (KHIDI), (over 12 months) may be useful to ensure the timing of founded by the Ministry for Health, Welfare, and Family Affairs, Republic of Kim et al. BMC Infectious Diseases (2018) 18:240 Page 6 of 6 Korea (A101750 and HI14C1324) and by the Basic Science Research Program twice a week for treatment of drug-susceptible pulmonary tuberculosis in through the National Research Foundation of Korea (NRF), founded by the HIV-negative patients: a randomised clinical trial. Lancet. 2002;360:528–34. Ministry of Science, ICT and Future Planning (NRF-2015K1A3A7A03073714). 10. Reid MJ, Shah NS. Approaches to tuberculosis screening and diagnosis in people with HIV in resource-limited settings. Lancet Infect Dis. 2009;9:173–84. 11. Getahun H, Harrington M, O’Brien R, Nunn P. Diagnosis of smear-negative Availability of data and materials pulmonary tuberculosis in people with HIV infection or AIDS in resource- The data set supporting the conclusion of this study is available upon constrained settings: informing urgent policy changes. Lancet. 2007;369:2042–9. request from the corresponding author. 12. Hargreaves NJ, Kadzakumanja O, Whitty CJ, Salaniponi FM, Harries AD, Squire SB. Smear-negative' pulmonary tuberculosis in a DOTS programme: Authors’ contributions poor outcomes in an area of high HIV seroprevalence. Int J Tuberc Lung HL and SYK designed the study, analysed the data, and drafted the Dis. 2001;5:847–54. manuscript. JK, SB, JL, and BS performed the experiment and summarised 13. Goletti D, Petruccioli E, Joosten SA, Ottenhoff THMM. Tuberculosis the laboratory data. JK and DRK were responsible for the statistical analysis. biomarkers: from diagnosis to protection. Infect Dis Rep. 2016;8:6568. SYK and YSK were responsible for the enrolment of participants and 14. Wallis RS, Doherty TM, Onyebujoh P, Vahedi M, Laang H, Olesen O. reviewed clinical information. NSL and SK made contributions to acquisition Biomarkers for tuberculosis disease activity, cure, and relapse. Lancet Infect and interpretation of clinical data. HL, YSK, SNC, and YAK were responsible Dis. 2009;9:162–72. for the conception of the study design, the analysis, and the revision of the 15. Petrone L, Cannas A, Vanini V, Cuzzi G, Aloi F, Nsubuga M, et al. Blood and manuscript. All authors read and approved the final manuscript. urine inducible protein 10 as potential markers of disease activity. Int J Tuberc Lung Dis. 2016;20:1554–61. Ethics approval and consent to participate 16. Profumo E, Buttari B, Tosti ME, Alessandri C, Valesini G, Marcuccio L, et al. All study participants provided written informed consent to provide Identification of IP-10 and IL-5 as proteins differentially expressed in human specimens for the identification of TB biomarkers. The Institutional Ethics complicated and uncomplicated carotid atherosclerotic plaques. Int J Committee of Yonsei University Severance Hospital approved this study Immunopathol Pharmacol. 2010;23:775–82. (approval #4-2010-0527 and #4-2014-1108). 17. Hong JY,Lee HJ,Kim SY,Chung KS,Kim EY,JungJY, et al.Efficacyof IP-10 as a biomarker for monitoring tuberculosis treatment. J Inf Secur. Competing interests 2014;68:252–8. The authors declare that they have no competing interests. 18. Riou C, Perez Peixoto B, Roberts L, Ronacher K, Walzl G, Manca C, et al. Effect of standard tuberculosis treatment on plasma cytokine levels in patients with active pulmonary tuberculosis. PLoS One. 2012;7:e36886. Publisher’sNote 19. Kabeer BSA, Raja A, Raman B, Thangaraj S, Leportier M, Ippolita G, et al. IP- Springer Nature remains neutral with regard to jurisdictional claims in 10 response to RD1 antigens might be a useful biomarker for monitoring published maps and institutional affiliations. tuberculosis therapy. BMC Infect Dis. 2011;11:135. 20. Cannas A, Calvo L, Chiachio T, Cuzzi T, Vanini V, Lauria FN, et al. IP-10 Author details detection in urine is associated with lung diseases. BMC Infect Dis. 2010;10 Division of Pulmonary, Department of Internal Medicine, Institute of Chest 21. Tonby L, Ruhwald M, Kvale D, Dyrhol-Riise AM. IP-10 measured by dry Diseases, Severance Hospital, Yonsei University College of Medicine, Seoul plasma sopts as biomarker for therapy responses in mycobacterium 03722, Republic of Korea. Clinical Vaccine Research Section, International tuberculosis infection. Sci Rep. 2015;5:9223. Tuberculosis Research Center, Seoul 03722, Republic of Korea. Department 22. Garcia-Basteiro AL, Mambuque E, den Hertog A, Saavedra B, Cuamba I, of Biomedical Laboratory Science, College of Health Sciences, Yonsei Oliveras L, et al. IP-10 kinetics in the first week of therapy are strongly University, Wonju 26493, Republic of Korea. Development and Delivery Unit, associated with bacteriological confirmation of tuberculosis diagnosis in International Vaccine Institute, Seoul 08826, Republic of Korea. Department HIV-infected patients. Sci Rep. 2017;7:14302. of Microbiology and Institute of Immunology and Immunological Disease, 23. Coleman MT, Chen RY, Lee M, Lin PL, Dodd LE, Maiello P, et al. PET/CT Yonsei University College of Medicine, Seoul 03722, Republic of Korea. imaging reveals a therapeutic response to oxazolidinones in macaques and humans with tubercuosis. Sci Transl Med. 2014;6:265ra167. https://doi.org/ Received: 18 October 2017 Accepted: 10 May 2018 10.1126/scitranslmed.3009500. 24. Rohini K, Bhat MS, Srikumar PS, Mahesh KA. Assessment of hematological parameters in pulmonary tuberculosis patients. Indian J Clin Biochem. 2016; References 31:332–5. 1. WHO. Global tuberculosis report. Geneva: World Health Organization; 2015. 25. Brown J, Clark K, Smith C, Hopwood J, Lynard O, Toolan M, et al. Variation 2. WHO. Global tuberculosis report. Geneva: World Health Organization; 2016. in C–reactive protein response according to host and mycobacterial WHO/HTM/TB/2016.13 characteristics in active tuberculosis. BMC Infect Dis. 2016;16:265. 3. Zumla A, Nahid P, Cole ST. Advances in the development of new tuberculosis drugs and treatment regimens. Nat Rev Drug Discov. 2013;12:388–404. 4. Walzl G, Ronacher K, Djoba Siawaya JF, Dockrell HM. Biomarkers for TB treatment response: challenges and future strategies. J Inf Secur. 2008;57:103–9. 5. Nahid P, Dorman SE, Alipanah N, Barry PM, Brozek JL, Cattamanchi A, et al. 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Benator D, Bhattacharya M, Bozeman L, Burman W, Cantazaro A, Chaisson R, et al. Rifapentine and isoniazid once a week versus rifampicin and isoniazid http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Infectious Diseases Springer Journals

Urine IP-10 as a biomarker of therapeutic response in patients with active pulmonary tuberculosis

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Medicine & Public Health; Infectious Diseases; Parasitology; Medical Microbiology; Tropical Medicine; Internal Medicine
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Abstract

Background: Prior to clinical trials of new TB drugs or therapeutic vaccines, it is necessary to develop monitoring tools to predict treatment outcomes in TB patients. Urine interferon gamma inducible protein 10 (IP-10) is a potential biomarker of treatment response in chronic hepatitis C virus infection and lung diseases, including tuberculosis. In this study, we assessed IP-10 levels in urine samples from patients with active TB at diagnosis, during treatment, and at completion, and compared these with levels in serum samples collected in parallel from matched patients to determine whether urine IP-10 can be used to monitor treatment response in patients with active TB. Methods: IP-10 was measured using enzyme-linked immunosorbent assays in urine and serum samples collected concomitantly from 23 patients with active TB and 21 healthy adults (44 total individuals). The Mann-Whitney U test and Wilcoxon matched-pairs signed rank test were used for comparisons among healthy controls and patients at three time points, and LOESS regression was used for longitudinal data. Results: The levels of IP-10 in urine increased significantly after 2 months of treatment (P = 0.0163), but decreased by the completion of treatment (P = 0.0035). Serum IP-10 levels exhibited a similar trend, but did not increase significantly after 2 months of treatment in patients with active TB. Conclusions: Unstimulated IP-10 in urine can be used as a biomarker to monitor treatment response in patients with active pulmonary TB. Keywords: Tuberculosis, Urine IP-10, Biomarker, Monitoring Background Progress has been made in the control of tuberculosis, as Tuberculosis (TB) remains a major infectious cause of advocated by the World Health Organization (WHO) [2]. mortality and morbidity, with approximately 8.6 million For the development of novel vaccines and drugs new cases and 1.3 million deaths per year [1]. In addition, against TB, a reliable surrogate marker of risk and pro- there is concern over increasing rates of disease recur- tection against TB is essential. In particular, prior to rence (including relapse and reinfection) after anti-TB clinical trials of new therapeutic vaccines to prevent TB drug therapy and an increasing trend of multidrug- and recurrence and to shorten the duration of therapy, ef- extensively drug-resistant (M/XDR) TB worldwide. fective monitoring tools are needed to measure treat- ment responses to standard drug therapies in parallel with therapeutic vaccinations [3, 4]. * Correspondence: [email protected]; [email protected] Generally, sputum culture conversion after 2 months Song Yee Kim and Jungho Kim contributed equally to this work. Division of Pulmonary, Department of Internal Medicine, Institute of Chest of anti-TB drug treatment has been used as a predict- Diseases, Severance Hospital, Yonsei University College of Medicine, Seoul ive indicator of drug treatment response in patients 03722, Republic of Korea [5–9]. However, culture methods are not applicable in Clinical Vaccine Research Section, International Tuberculosis Research Center, Seoul 03722, Republic of Korea patients with extrapulmonary TB and culture-negative Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kim et al. BMC Infectious Diseases (2018) 18:240 Page 2 of 6 pulmonary TB, and laboratories capable of sputum positive sputum culture after 2 months of culture are often lacking in resource-limited settings anti-TB treatment as follows: [10–12]. To accommodate these shortcomings, efforts have been made to identify an alternative biomarker for anti-TB treatment efficacy [4, 13, 14]. Interferon gamma (IFN-γ)-inducible protein (IP-10) is  Low-risk group: TB patients who had neither risk a pro-inflammatory chemokine secreted by antigen- factor (i) nor (ii); presenting cells that transport activated T lymphocytes  Moderate group: TB patients who had either risk to sites of inflammation [15, 16]. IP-10 levels were found factor (i) or (ii); to be higher in the unstimulated serum, plasma, and  High-risk group: TB patients who had both risk urine of patients with active TB than those in subjects factors (i) and (ii). without active TB, and IP-10 levels decrease significantly after the completion of anti-TB treatment [15, 17–20]. Among all types of clinical specimens in which IP-10 has been detected, urine specimens are the most con- 2) Healthy controls (aged 20 years and over): Healthy venient and non-invasive; specimens can be obtained controls fulfilled the following inclusion criteria: (i) from children and elderly patients, even in resource- no history of tuberculosis, (ii) no suggestive limited settings, as these samples do not require special symptoms of tuberculosis, (iii) no recent contact equipment or skilful medical personnel for collection. In with patients with infectious tuberculosis, and (iv) addition, urine specimens present lower handling risks negative results according to the QuantiFERON -TB compared to other specimens, such as blood, sputum, Gold In-Tube Test (QFT-GIT). and microbial isolates from patients [20]. Theaim of this studywas to test thehypothesis None of the participants exhibited HIV infection; a that levels of urine IP-10 change over the course of chronic comorbidity, such as diabetes mellitus, anti-TB drug therapy in patients with active TB from chronic renal failure, malignant tumour, or chronic South Korea, an area with an intermediate burden of liver disease; immunosuppressive status; or acute TB and low burden of human immunodeficiency virus infections. infection and acquired immune deficiency syndrome (HIV/AIDS). IFN-γ measurement A QFT-GIT assay was conducted for all patients before treatment (0 months, T0), at 2 months (T2), and at 6– Methods 9 months after the treatment was completed (T6). Study population and setting The QFT-GIT assay (Qiagen, Hilden, Germany) was Clinical information and specimens were collected from performed according to the manufacturer’sinstruc- participants enrolled in two clinical studies from tions. Briefly, 1 ml of whole blood was collected in November 2010 to March 2012 and from May 2015 to each of three tubes pre-coated with Mycobacterium June 2016. The Institutional Ethics Committee of Yonsei tuberculosis-specific peptides (ESAT-6, CFP-10, and University Severance Hospital approved this study TB7.7) or mitogen (positive control) and incubated (approval #4-2010-0527 and #4-2014-1108). All study for 16–24 h at 37 °C. For negative controls, whole participants provided written informed consent to pro- blood was placed in tubes that were not pre-coated. vide specimens for the identification of TB biomarkers. The plasma supernatant was harvested by centrifuga- Study participants were classified as follows. tion at 3000×g for15 min andstoredat − 80 °C. The level of IFN-γ was determined using the QFT 1) Active pulmonary TB patients (aged 20 years and Enzyme-linked Immunosorbent Assay (ELISA) Kit. over): Patients with active pulmonary TB were The results were interpreted using QFT-GIT software diagnosed based on culture and/or pathology. Cases version 2.62, provided by the manufacturer. of clinical active pulmonary TB, which was defined as negative mycobacterial culture IP-10 measurement findings, but favourable clinical and radiological Serum and urine samples were collected to measure ser- responses to anti-TB medication, were also ial IP-10 levels from all patients at T0, T2, and T6. IP-10 included. Patients were sub-divided into the levels in urine and serum were concomitantly measured following three groups based on risk factors for re- in duplicate using a commercial IP-10 ELISA Kit (R&D lapse, including the presence of (i) a cavity Systems, Minneapolis, MN, USA) in accordance with lesion on a chest X-ray or chest CT and (ii) a the manufacturer’s instructions. The test results were Kim et al. BMC Infectious Diseases (2018) 18:240 Page 3 of 6 interpreted using SoftMax version 5.4.1 (Molecular De- Comparison of serum and urine IP-10 levels between vices, LLC, Sunnyvale, CA, USA). In addition, using the healthy controls and patients with TB at diagnosis test results available from routine hospital analyses, the We assessed whether the levels of IP-10 in serum and urine IP-10 level (in pg/ml) was normalized against the urine samples differed between TB patients at the time serum creatinine level (in mg/ml) for each patient at of diagnosis and healthy controls. Serum IP-10 levels did each visit. not differ significantly between TB patients at diagnosis (median, 85.37 pg/ml; IQR, 60.92-171.30) and healthy Statistical analysis controls (median, 68.62 pg/ml; IQR, 43.19-87.01), al- All statistical analyses were performed using GraphPad though the levels in patients were slightly higher than Prism 6 software (GraphPad Software, La Jolla, CA, those in the healthy controls (P = 0.0829; Fig. 1a). In USA) and IBM SPSS software version 21.0 (IBM Corp., addition, urine IP-10 levels in healthy controls (median, Armonk, NY, USA). Medians and interquartile range 6.49 pg/ml; IQR, 2.02-12.11) were not different from (IQR) were measured for continuous variables. The those in the TB patients at the time of diagnosis (me- Mann–Whitney U-test and Wilcoxon matched-pairs dian, 7.89 pg/ml; IQR, 4.86-13.97; Fig. 1b). signed rank test were used for comparisons among study groups and pairwise comparisons. A longitudinal Comparison of serum and urine IP-10 levels at TB smoothed trend line for the data (not based on any dis- diagnosis (T0), during treatment (T2), and after the tributional assumption) was estimated using a nonpara- completion of therapy (T6) metric method called LOESS (locally weighted We further analysed whether the serum and urine IP- scatterplot smoothing). A 95% confidence limit was esti- 10 levels in TB patients changed over the course of mated, and similarity patterns by risk group were graph- treatment with a complete set of serial specimens ically examined. All P-values were two-sided, and P <0. (T0, T2, and T6). Serum IP-10 levels in TB patients 05 was considered statistically significant. at the time of diagnosis (median, 85.37 pg/ml; IQR, 60.92-171.30) were not significantly different from Results those after 2 months of treatment (median, 125. Characteristics of study participants 80 pg/ml; IQR, 81.42-155.00; P = 0.1743) or upon the We enrolled 23 patients with active TB and 21 healthy completion of effective therapy (median, 81.09 pg/ml; controls. The patients with active TB had a median age IQR, 63.71-123.70; P = 0.7467; Fig. 2a). of 27 (range, 22–66), and the healthy controls had a me- However, the pairwise comparisons also showed that dian age of 25 (range, 20–33). Bacille Calmette-Guérin urine IP-10 levels in patients after 2 months of treat- (BCG) scars were present in 65.2% of patients with ac- ment (median, 19.17 pg/ml; IQR, 5.12-51.68) were sig- tive TB and 57.1% of healthy controls during inspection nificantly higher than those in patients at the time of at the site. None of the enrolled patients were acid-fast diagnosis (median, 7.89 pg/ml; IQR, 4.86-13.97; P =0. bacillus smear-positive, 19 (82.6%) were culture-positive, 0163). Moreover, urine IP-10 levels in patients tested and 8 (34.8%) had a cavity on a chest X-ray or computed after the completion of drug therapy (median, 5.47 pg/ tomography (CT) (Table 1). ml; IQR, 4.69-9.79) were significantly lower than those Table 1 Characteristics of subjects enrolled in the study TB Patients Healthy Controls (n = 23) (n = 21) Median Age (range) 27 (22–66) 25 (20–33) Male, n (%) 16 (59.3%) 13 (62.0%) Presence of BCG scars 15 (65.2%) 12 (57.1%) b c PTB diagnosis AFB smear, positive 0 (0.0%) 0 (0.0%) AFB culture, positive 19 (82.6%) 0 (0.0%) Cavity on Chest X-ray or CT 8 (34.8%) 0 (0.0%) Risk group for relapse High-risk 0 (0.0%) 0 (0.0%) Moderate-risk 8 (34.8%) 0 (0.0%) Low-risk 15 (65.2%) 0 (0.0%) a b c d BCG Bacille Calmette-Guérin, PTB Pulmonary tuberculosis, AFB Acid-fast bacillus; CT Computed tomography. Risk factors for relapse: i) the presence of a cavity lesion on chest X-ray or chest CT, and ii) the result of a positive sputum culture after 2 months of anti-TB treatment. The high-risk group was defined when patients had both risk factors i) and ii). The moderate-risk group was defined when patients had either risk factor i) or ii), and the low-risk group had neither risk factor Kim et al. BMC Infectious Diseases (2018) 18:240 Page 4 of 6 Fig. 1 Serum and urine IP-10 levels in patients with TB at diagnosis and healthy controls. IP-10 levels were measured in the serum (a) and urine (b) of each TB patient at diagnosis (T0) and each healthy control (HC). We only obtained serum samples from 17 patients at T0 out of 23 patients. The data are shown as median with interquartile range; statistical analyses were performed using the Mann–Whitney U-test sampled at the time of diagnosis (P = 0.0354) and after Longitudinal analysis of IP-10 levels in patients with 2 months of treatment (P = 0.0035; Fig. 2b). active TB stratified by risk group To confirm that the IP-10 response derived from TB The group of TB patients was then sub-divided by risk infection, the urine IP-10 level of each patient was nor- factors for relapse or treatment failure into low, moder- malized to the serum creatinine level at each visit using ate, and high-risk groups. Among all patients, 15 (65.2%) test results available from routine analyses. As an abnor- were included in the low-risk group, and 8 (34.8%) were mal serum creatinine level may indicate kidney disease included in the moderate/high-risk group. or damage, this served to verify that the dynamics of IP- As shown in Fig. 3b and c, the smoothed lines for 10 levels detected in this study were not influenced by urine IP-10 in both risk groups exhibited a similar trend kidney disease. The urine IP-10/creatinine ratio was over the three time points. The low-risk group had higher in TB patients after 2 months of treatment than higher urine IP-10 levels than those of the moderate/ either before or after treatment, consistent with the high-risk group, but the 95% confidence limits of IP-10 trend observed for urine IP-10 without normalization levels were not differentiated by risk group. (Fig. 2c). This indicated that the serum creatinine levels In contrast, serum IP-10 levels did not show similar did not affect the urine IP-10 levels in this study. trends in the two risk groups (Fig. 3a). Serum IP-10 Fig. 2 Serum and urine IP-10 levels in patients with TB before, during, and after treatment. Serial IP-10 levels in the (a) serum and (b) urine of patients with TB collected at the time of diagnosis (T0), after 2 months of therapy (T2), and after the completion of therapy (T6). c Serial IP-10 levels in urine normalized against serum creatinine levels in patients. The data are shown as median with interquartile range; statistical analyses were performed using the Wilcoxon matched-pairs signed rank test Kim et al. BMC Infectious Diseases (2018) 18:240 Page 5 of 6 Fig. 3 Longitudinal analysis of IP-10 levels in patients with active TB stratified by risk of relapse. Longitudinal (a) serum, (b) urine IP-10, and (c) urine IP-10/creatinine ratio obtained throughout treatment (T0, T2, and T6) were analysed in risk subgroups (low-risk and moderate/ high-risk groups) using locally weighted scatterplot smoothing (LOESS) and confidence intervals of the mean (CLM) levels in the moderate/high-risk group did not change peak inflammation and urine IP-10 for successful over time, but those in the low-risk group increased at treatment. T2 and decreased at T6, similar to the results for urine Individuals with acute and chronic infections were ex- IP-10 levels. cluded from analyses, and urine IP-10 levels were nor- malized against serum creatinine levels to ensure that increases in urine IP-10 levels can be attributed to infec- Discussion tion with M. tuberculosis, rather than kidney disease or As sputum culture conversion at 2 months is not always damage. In future studies, urine creatinine levels should predictive of successful TB therapy, new biomarkers for be examined to directly exclude urinary disease. predicting treatment responses are needed for better man- In addition, C-reactive protein (CRP) is an indicator of agement of TB patients. Similar to the findings of Cannas disease activity and is often increased in active tubercu- et al. [20], this study showed a declining tendency for urine losis inflammatory conditions [24, 25]. However, CRP IP-10 detection in TB patients after receiving effective anti- data for patients at diagnosis and follow-ups were not TB drug therapy compared the levels at time of diagnosis. available in this study. In future studies, CRP levels However, this study also detected urine IP-10 levels after should be estimated and their correlations with IP-10 2 months of an intensive anti-TB drug therapy, which is the levels can be used to assess inflammatory changes in re- point when clinicians routinely use radiological and bac- sponse to TB treatment. Furthermore, this study empha- teriological tests to determine treatment progress. Although sizes the usefulness of urine as non-invasive sample for recent studies have reported a decline in blood IP-10 levels monitoring treatment responses biomarkers in children, after 2 weeks of efficient anti-TB drug therapy as an early the elderly and HIV-infected subjects. biomarker for treatment response [21, 22], this study dem- onstrated a peak in urine IP-10 levels after 2 months of Conclusions treatment in HIV-negative patients with tuberculosis. In conclusion, unstimulated IP-10 levels in patient urine Moreover, the same trend was observed in patients regard- samples, which are easier to collect and analyse than blood less of risk factors for TB relapse or treatment failure. samples, can be used as a predictive factor, indicating a It is not clear why unstimulated IP-10 levels in urine positive treatment response. IP-10 detection may therefore significantly increase in patients after 2 months of anti- be a useful monitoring tool for determining whether TB pa- TB treatment. A previous study using imaging scans tients are positively responding to anti-TB drug therapy. suggested quantitative changes at 2 months after the Acknowledgements initiation of treatment, which may be related to long- The authors thank all study participants for contributing to this study. term TB treatment outcome [23]. To confirm this Funding phenomenon, additional investigations with a large-scale This study was supported by a grant from the Korean Health Technology R&D active TB cases including prolonged anti-TB treatment Project through the Korea Health Industry Development Institute (KHIDI), (over 12 months) may be useful to ensure the timing of founded by the Ministry for Health, Welfare, and Family Affairs, Republic of Kim et al. BMC Infectious Diseases (2018) 18:240 Page 6 of 6 Korea (A101750 and HI14C1324) and by the Basic Science Research Program twice a week for treatment of drug-susceptible pulmonary tuberculosis in through the National Research Foundation of Korea (NRF), founded by the HIV-negative patients: a randomised clinical trial. Lancet. 2002;360:528–34. Ministry of Science, ICT and Future Planning (NRF-2015K1A3A7A03073714). 10. Reid MJ, Shah NS. Approaches to tuberculosis screening and diagnosis in people with HIV in resource-limited settings. 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Published: May 29, 2018

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