Interleukin-6, Interleukin-13 and Interferon-γ as Potential Biomarkers for Treatment Failure in

Pulmonary TuberculosisAkshay Gupte1, Pavan Kumar2, Sriram Selvaraju2, Mandar Paradkar3, Vandana Kulkarni3, Neeta Pradhan3, Rewa Kohli3, Nishi Suryavanshi3, Kannan Thiru2,

Luke Hanna2, Kavitha Dhanasekaran2, Sri Vijay Bala Yogendra4, Rahul Lokhande3, Subhash Babu2, Jonathan Golub1, Nikhil Gupte1, Bruno Andrade5, Vidya Mave1, Padmapriyadarasini Chandrasekaran2, Amita Gupta1

1 Johns Hopkins University School of Medicine, Baltimore, USA; 2 National Institute for Research in Tuberculosis, Chennai, India; 3 Byramjee Jeejeebhoy Government Medical College, Pune, India; 4

Johns Hopkins University India Office, Pune, India; 5 Instituto Brasileiro para Investigação da Tuberculose, Salvatore, Brazil

1 World Health Organization. Global TB Report; Geneva, Switzerland, 2016.2 Mitchison DA. Assessment of new sterilizing drugs for treating pulmonary tuberculosis by culture at 2 months [letter]. AmRev Respir Dis 1993; 147: 1062–63.3 Horne DJ, Royce SE, Gooze L, et al. Sputum monitoring during tuberculosis treatment for predicting outcome: systematicreview and meta-analysis. The Lancet Infectious diseases 2010; 10(6): 387-94.

ReferencesData in this manuscript were collected as part of the Regional Prospective Observational Research forTuberculosis (RePORT) India Consortium. This project has been funded in whole or in part with Federal fundsfrom the Government of India’s (GOI) Department of Biotechnology (DBT), the Indian Council of MedicalResearch (ICMR), the United States National Institutes of Health (NIH), National Institute of Allergy andInfectious Diseases (NIAID), Office of AIDS Research (OAR), and distributed in part by CRDF Global. The contentsof this publication are solely the responsibility of the authors and do not represent the official views of the DBT,the ICMR, the NIH, or CRDF Global. Any mention of trade names, commercial projects, or organizations doesnot imply endorsement by any of the sponsoring organizations. AG was supported by NIH Research TrainingGrant # D43 TW009340 funded by the NIH Fogarty International Center, NINDS, NIMH, NHBLI and NIEHS.

Acknowledgements

• Tuberculosis (TB) is the leading infectious killer worldwide with over10.4 million incident cases and 1.7 million deaths in 20161.

• While culture conversion by 2 months of anti-tuberculosis treatment(ATT) is widely used as a surrogate marker for microbiologicalresponse, recent clinical trials have shown suboptimal performance of2 month culture in predicting unfavorable treatment outcomes,particularly treatment failure2-3.

• Novel biomarkers predictive of unfavorable treatment outcomes areneeded for the early identification and risk-stratification of TB cases.

• The objective of this study was to identify systemic inflammatorymarkers associated with treatment failure in newly diagnosed adultpulmonary TB (PTB) cases in India.

Background

Methods

Results

Study population:• We randomly selected 30 new adult (>18 years) drug-sensitive PTB

cases within 1 week of ATT initiation from the ongoing CTRIUMPHstudy in Pune and Chennai, India.

• Participants were prospectively evaluated at 0 weeks (<7 days sinceATT initiation), 8 weeks and 24 weeks for plasma concentrations of 20cytokines linked to the host immune response in TB.

• Treatment failure was defined as Mycobacterium tuberculosis growthon liquid or solid culture between 17 and 24 weeks of ATT.

Cytokine analysis:• Group A (host immune response in TB): INF-γ, TNF-α, IL-1β, IL-4, IL-6,

IL-10, CXCL-10, IL-12, IL-13 and IL-17• Group B (lung tissue destruction and fibrosis): MMP-1, MMP-3, MMP-

7, TIMP-1, TIMP-2, TIMP-3, TIMP-4, TGFβ-1, TGFβ-2 and TGFβ-3• Cytokine concentrations were evaluated, in duplicates, using multiplex

ELISA according to manufacturer protocols (BioRad Inc).

Statistical analysis:• Cytokine concentrations were log2 transformed and z-score normalized

for analysis.• Differentially expressed cytokines by duration of ATT and treatment

failure were identified using non-parametric tests.• P-values were adjusted for multiple comparisons using the Benjamini-

Hochberg procedure and a 10% false-discovery rate.

Results

Table 1. Baseline characteristics of study participants

Figure 1. Differentially expressed markers compared to baseline concentrations (i.e. vs week 0)

Figure 2. Differential cytokine expression by treatment outcomes

Figure 3. Differential cytokine expression by individual participants and treatment failure at week 0

• No significant differences between those selected for inflammatoryanalysis compared to those not selected but part of the full cohort.

• Notable exception being the absence of recurrence and deaths

Conclusion• Overexpression of circulating IL-6, IL-13 and IFN-γ at treatment

initiation may be associated with treatment failure among drug-sensitive pulmonary tuberculosis cases.

• Well powered validation studies should be undertaken to evaluate theperformance of these biomarkers, individually or in combination, forpredicting unfavorable tuberculosis treatment outcomes.

Contact:Akshay Gupte, PhD, MBBS, MSPHCenter for Clinical Global Health EducationDivision of Infectious DiseasesJohns Hopkins University School of MedicineEmail: agupte1@jhmi.edu

Baseline characteristicsCohort

(n=317)

Sub-cohort

(n=30)p-value

Age (years), median (IQR) 40 (27-50) 36 (28-50) 0.87

Male sex, n (%) 203 (64) 20 (74) 0.40

BMI (kg/m2), median (IQR) 18 (16-20) 18 (16-20) 0.98

Ever-smoking, n (%) 124 (39) 9 (31) 0.87

HIV coinfection, n (%) 19 (6) 2 (7) 0.68

Diabetes mellitus, n (%) 88 (28) 7 (26) 0.40

Cavitation, n (%) 122 (45) 10 (43) 0.99

Treatment outcomes, n (%)

Failure 35 (12) 4 (14) 0.99

Recurrence 14 (4) 0 -

Death 6 (2) 0 -

Fold-difference

Figure 4. Absolute cytokine concentrations at week 0 comparing treatment failures vs cures

DiscussionKey findings:• TIMP-4 and TNF-α were overexpressed at week 8 and week 24

following ATT initiation, respectively.• Relative to week 0 concentrations; IL1-β, IL-6 and MMP-7

concentrations declined at week 8; CXCL-10, TGF-β1, TGF-β2, TIMP-1and TIMP-3 concentrations declined by week 24; and TGF-β3concentrations declined at both week 8 and week 24.

• None of the participants who failed treatment had HIV coinfection ordiabetes.

• Participants who failed treatment had significantly higher plasmaconcentrations of IL-6, IL-13 and IFN-γ at ATT initiation compared tothose who were cured, however this difference was not statisticallysignificant at 8 and 24 weeks of ATT.

Limitations:• Limited sample size of n=4 failures.• We could not identify cytokines associated with recurrence or death.

Future direction:• We plan to conduct well powered validation studies measuring the

performance of IL-6, IL-13 and IFN-γ as predictive markers forunfavorable treatment outcomes and lung injury.