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Journal of Infection (2015) xx, 1e3

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LETTER TO THE EDITOR 74
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13 Serum IP-10 in the diagnosis of latent and Similarly to Wergeland et al., we analysed unstimulated 78
14 Q6 active tuberculosis serum samples for a number of cytokines, including IP-10, 79
15 by multiplex cytokine assays (Bio-Plex Human Cytokine 80
16 Group I assays [Bio-Rad, Gladesville, Australia]) according 81
17 to the manufacturer’s instructions, using an xMAP Luminex 82
18 200 instrument, after incubation without stimulatory anti- 83
19 KEYWORDS 84
gens in the presence of CD28/CD49d (Becton Dickinson) at
20 Tuberculosis; 37
C for 19 h. Non-parametric statistical tests were used 85
21 IP-10; 86
to compare cytokine concentrations between participant
22 Latent TB; 87
groups (KruskaleWallis tests for multiple groups; Mann
23 Active TB; 88
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Whitney U tests for two-group comparisons). The study
Diagnostics was approved by the Human Research Ethics Committee 89
25 90
26 of the Royal Melbourne Hospital (approval no. 2011.128).
We found a statistically significant difference in the 91
27 92
28 median serum IP-10 concentrations between patients with
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29 active TB and those with LTBI (Fig. 1). However, it is impor-
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30 tant to highlight that although median concentrations
We read with interest the recent article by Wergeland 95
31 differed significantly between these two groups in both
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32 et al., reporting that serum interferon-gamma inducible our study and the study by Wergeland et al., there was a 97
33 protein 10 (IP-10) may be a useful biomarker for differen- significant overlap in the IP-10 concentrations observed 98
34 tiating between active tuberculosis (TB) and latent TB (Fig. 1 below and Figure 2 in Wergeland et al.’s paper), sug- 99
35 infection (LTBI), as well as for monitoring the effectiveness gesting that establishing a sufficiently sensitive and specific 100
36 of anti-tuberculous therapy.1 However, a key limitation of cut-off to distinguish between active and LTBI would not be 101
37 this study was the absence of a ‘sick control’ group of pa- feasible. In contrast, we found no statistically significant 102
38 tients with respiratory tract infections. Prompted by this difference in median serum IP-10 concentrations between 103
39 finding, we analysed data from our ongoing study investi- patients with active TB and ‘sick controls’, although there 104
40 gating novel biomarkers of active and latent TB. was a tendency for IP-10 concentrations to be higher in 105
41 Our study included a total of 193 adults recruited at a 106
the former group. Analysis of C-reactive protein (CRP) re-
42 tertiary hospital in Melbourne, Australia between 2012 and 107
sults, in cases where these were available, revealed that
43 2014. Participants were classified into four diagnostic groups 108
CRP concentrations were also higher in patients with active
44 based on stringent criteria: Group 1 e cases with culture- 109
TB than in ‘sick controls’ (median concentration 57 mg/L
45 confirmed active TB prior to starting anti-tuberculous therapy 110
46
versus 46 mg/L).
(n Z 38; n Z 27 pulmonary TB, n Z 11 extra-pulmonary TB); These findings are consistent with several previous reports 111
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Group 2 e cases with untreated LTBI (defined as asymptom- that suggest serum IP-10 concentrations are higher in cases 112
48 113
atic patients with positive tuberculin skin test (TST; cut-off with active TB compared to those with LTBI,2,3 and that IP-10
49 114
10 mm induration) and positive QuantiFERON-TB Gold In-Tube levels decrease with anti-tuberculous treatment.4e6
50 115
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(QFT-GIT) assay) (n Z 42); Group 3 e ‘sick controls’ We concur with Wergeland et al.’s conclusion that IP-10
comprising cases with lower respiratory tract infection caused 116
52 is likely to be a non-specific marker of inflammation, as
by a pathogen other than Mycobacterium tuberculosis with 117
53 this particular cytokine is also elevated in several other
118
54 negative TST and QFT-GIT results (n Z 16); Group 4 e healthy bacterial, viral and parasitic infections, including bacter- 119
55 controls comprising volunteers without risk factors for TB (ie aemia, urinary tract infections, hepatitis C infection, HIV 120
56 no known TB contact and no travel to a high TB prevalence infection, visceral leishmaniasis and malaria.7e10 The 121
57 country) and with negative TST and QFT-GIT results (n Z 33). concept that unstimulated serum IP-10 levels primarily 122
58 reflect a pro-inflammatory state is further supported by 123
59 DOI of original article: http://dx.doi.org/10.1016/j.jinf.2014. a recent study that reported significantly higher serum 124
60 12.019. IP-10 levels in active TB patients with a markedly elevated 125
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62 http://dx.doi.org/10.1016/j.jinf.2015.08.001
0163-4453/ª 2015 Published by Elsevier Ltd on behalf of The British Infection Association. 127
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Please cite this article in press as: Clifford V, et al., Serum IP-10 in the diagnosis of latent and active tuberculosis, J Infect (2015), http://
dx.doi.org/10.1016/j.jinf.2015.08.001
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2 Letter to the Editor

1 active TB and LTBI,11 recently published data from our 66

2 group have shown that other antigen-stimulated cytokine 67
3 responses e including mycobacteria-specific TNF-a, IL- 68
4 1ra, and IL-10 responses e have greater discriminatory abil- 69
5 ity,12 which we believe will ultimately facilitate the devel- 70
6 opment of diagnostic tests that have greater accuracy than 71
7 interferon-gamma release assays, which rely on a single 72
8 cytokine.13 73
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11 Conflicts of interest Q2
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13 The authors do not have a commercial or other association 78
14 that might pose a conflict of interest. 79
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16 Financial disclosure 81
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19 The study was partially supported by a grant from the 84
20 John Burge Trust; MT is supported by a Clinical Lecture- 85
21 ship provided by the U.K. National Institute for Health 86
Research. Q3
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23 88
24 References 89
25 90
26 Figure 1 Box plot with Tukey whiskers showing serum IP-10 91
1. Wergeland I, Pullar N, Assmus J, Ueland T, Tonby K, Feruglio S,
27 concentrations in patients with active TB, LTBI, sick controls et al. IP-10 differentiates between active and latent tubercu- 92
28 (lower respiratory tract infection) and healthy controls. The losis irrespective of HIV status and declines during therapy. J 93
29 horizontal lines represent the medians and the lower and upper Infect 2015;70:381e91. 94
30 quartiles. 2. Hong JY, Jung GS, Kim H, Kim YM, Lee HJ, Cho S-N, et al. Effi- 95
31 cacy of inducible protein 10 as a biomarker for the diagnosis of 96
32 tuberculosis. Int J Infect Dis 2012;16:e855e9. 97
33 ESR (>40 mm/h) than in those with a lower ESR (<40 mm/ 3. Mihret A, Bekele Y, Bobosha K, Kidd M, Aseffa A, Howe R, et al. 98
34 h).6 One possible explanation for why IP-10 levels were Plasma cytokines and chemokines differentiate between active 99
35 higher in our active TB group than in the ‘sick control’ disease and non-active tuberculosis infection. J Infect 2013; 100
36 group is that the active TB patients were generally more 66:357e65. 101
37 unwell than the ‘sick control’ patients, most of whom 4. Riou C, Perez Peixoto B, Roberts L, Ronacher K, Walzl G, 102
38 Manca C, et al. Effect of standard tuberculosis treatment on 103
were hospitalised with less severe illnesses (including
39 plasma cytokine levels in patients with active pulmonary 104
infective exacerbation of asthma or COPD, influenza A vi- tuberculosis. PLoS One 2012;7:e36886 [Electronic Resource].
40 105
rus infection, and bacterial pneumonia). In this regard, we 5. Hong JY, Lee HJ, Kim SY, Chung KS, Kim EY, Jung JY, et al. Ef-
41 106
noted a significant issue in recruiting appropriate ‘sick ficacy of IP-10 as a biomarker for monitoring tuberculosis treat-
42 107
controls’ with an illness of similar clinical severity to pa- ment. J Infect 2014;68:252e8.
43 108
tients with pulmonary TB, as many patients recruited 6. Tonby K, Ruhwald M, Kvale D, Dyrhol-Riise AM. IP-10
44 109
into the ‘sick control’ group had indeterminate QFT-GIT measured by dry plasma spots as biomarker for therapy re-
45 110
assay results, which meant they could not be included in sponses in Mycobacterium tuberculosis infection. Sci Rep
46 111
the final analysis (as coincidental, co-existing LTBI could 2015;5:9223.
47 7. Olszyna DP, Prins JM, Dekkers PE, De Jonge E, Speelman P, Van 112
48 not be ruled out). 113
Deventer SJ, et al. Sequential measurements of chemokines in
49 In conclusion, we emphasise that without the inclusion 114
urosepsis and experimental endotoxemia. J Clin Immunol
50 of an antigen stimulation step designed to elicit TB-specific 1999;19:399e405. 115
51 cytokine responses, measurement of cytokine concentra- 8. Roe B, Coughlan S, Hassan J, Grogan A, Farrell G, Norris S, 116
52 tions in serum is likely to be of limited value for the et al. Elevated serum levels of interferon- gamma -inducible 117
53 diagnosis of TB. This is because any cytokine biomarker protein-10 in patients coinfected with hepatitis C virus and 118
54 detected in unstimulated samples inherently lacks speci- HIV. J Infect Dis 2007;196:1053e7. 119
55 ficity for TB and likely simply reflects a pro-inflammatory 9. Hailu A, van der Poll T, Berhe N, Kager PA. Elevated plasma 120
56 state, as illustrated by our data showing the absence of a levels of interferon (IFN)-gamma, IFN-gamma inducing cyto- 121
57 statistically significant difference in IP-10 concentrations kines, and IFN-gamma inducible CXC chemokines in visceral 122
58 leishmaniasis. Am J Trop Med Hyg 2004;71:561e7. 123
between cases with active TB and those with respiratory
59 10. Bostrom S, Ibitokou S, Oesterholt M, Schmiegelow C, 124
tract infection, and the other studies mentioned above. It is
60 Persson JO, Minja D, et al. Biomarkers of Plasmodium falcipa- 125
however possible that serum IP-10 may prove to be useful rum infection during pregnancy in women living in north-
61 as a tool to monitor response to TB therapy, as suggested by 126
62 eastern Tanzania. PLoS One 2012;7:e48763. 127
Wegeland et al. and other groups.5,6 11. Ruhwald M, Aabye MG, Ravn P. IP-10 release assays in the diag-
63 128
Whilst several recent studies have found that antigen- nosis of tuberculosis infection: current status and future direc-
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stimulated IP-10 responses can not distinguish between tions. Expert Rev Mol Diagn 2012;12:175e87.
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Please cite this article in press as: Clifford V, et al., Serum IP-10 in the diagnosis of latent and active tuberculosis, J Infect (2015), http://
dx.doi.org/10.1016/j.jinf.2015.08.001
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Letter to the Editor 3

1 12. Tebruegge M, Dutta B, Donath S, Ritz N, Forbes B, Camacho- Christel Zufferey 24

2 Badilla K, et al. Mycobacteria-specific cytokine responses Susie Germano 25
3 detect TB infection and distinguish latent from active TB. Am Department of Paediatrics, The University of Melbourne 26
Q4 J Respir Crit Care Med 2015.
4 and Murdoch Children’s Research Institute, Royal 27
5 13. Clifford V, Zufferey C, Street A, Denholm J, Tebruegge M, 28
Children’s Hospital Melbourne, Parkville, VIC, Australia
6 Curtis N. Cytokines for monitoring anti-tuberculous therapy: 29
7 a systematic review. Tuberculosis (Edinb) 2015;95:217e28. Justin Denholm 30
8 Alan Street 31
9 Q5 Vanessa Clifford Emma McBryde 32
10 Department of Paediatrics, The University of Melbourne Damon Eisen 33
11 and Murdoch Children’s Research Institute, Royal Victorian Infectious Diseases Unit, Royal Melbourne 34
12 Q1 Children’s Hospital Melbourne, Parkville, VIC, Australia Hospital, Parkville, VIC, Australia 35
13 36
14 Marc Tebruegge Nigel Curtis* 37
15 Department of Paediatrics, The University of Melbourne Department of Paediatrics, The University of Melbourne 38
16 and Murdoch Children’s Research Institute, Royal and Murdoch Children’s Research Institute, Royal 39
17 Children’s Hospital Melbourne, Parkville, VIC, Australia Children’s Hospital Melbourne, Parkville, VIC, Australia 40
18 E-mail address: nigel.curtis@rch.org.au (N. Curtis) 41
Academic Unit of Clinical and Experimental Medicine,
19 42
Faculty of Medicine & Respiratory Biomedical Research
20 Accepted 1 August 2015 43
21
Unit & Institute for Life Sciences, University of 44
22 Southampton, United Kingdom 45
23 46

* Corresponding author. Department of Paediatrics, The Univer-

sity of Melbourne, Royal Children’s Hospital Melbourne, Flemington
Road, Parkville, VIC 3052, Australia. Tel.: þ61 3 9345 6366; fax:
þ61 3 9345 4751.

Please cite this article in press as: Clifford V, et al., Serum IP-10 in the diagnosis of latent and active tuberculosis, J Infect (2015), http://
dx.doi.org/10.1016/j.jinf.2015.08.001