Biomarkers for Tuberculosis Robert S. Wallis, MD, FIDSA Senior Director, Pfizer
TB Global Burden total cases WHO 2009
TB Global Burden case rates WHO 2009
HIV prevalence in TB cases WHO 2009
TB Trends by Region Dye, Lancet 2006
Diagnostics Tuberculin Skin test TB-specific Interferon release Sputum culture smear agar liquid media
Prognostics Biomarkers HIV TNF blockers, corticosteroids Diabetes Smoking, air pollution Genetic factors BCG Isoniazid TB treatment
Biomarker Definitions: Endpoints Clinical endpoint A characteristic or variable indicating how a patient feels, functions, or survives. Biomarker (biological marker) A measurable characteristic that is an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. Surrogate endpoint A biomarker that is intended to substitute for a clinical endpoint based on epidemiological, therapeutic, pathophysiologic, or other scientific evidence.
TB Biomarker Needs In newly diagnosed active TB cases: predictors of durable (non-relapsing) cure In persons with latent TB infection: indicators of reactivation risk and effectiveness of latent TB treatment In all except active TB cases: predictors of vaccine-induced protection Special populations: children and HIV-infected
Validation vs. Qualification Validation: Assay performance Qualification: Relationship between marker and outcome 1. Biologic plausibility 2. Prognosis of clinical outcomes in disease 3. Capturing therapeutic effect in clinical trials
Predicting durable TB cure Microbial markers in sputum 2-month culture conversion Other microbial markers Mycobactericidal activity TB-specific T cell function Macrophage activation markers Multiple host markers Serial colony counts or time to culture positivity Other microbial markers Urine M. tuberculosis DNA Volatile organic compounds Whole blood culture IFNγ/ IL-4 δ2 splice variant Neopterin, procalcitonin, C-reactive protein, soluble intercellular adhesion molecule 1, soluble urokinase plasminogen activator receptor, monocyte CDllc Proteomics Transcriptomics
2-month culture conversion
2-month culture conversion
Meta-regression analysis P<0.00001
2-mo status shortcomings Poor performance in individual patients Sensitivity: 50% PPV: 18% Within subject variability likely hidden by convention of obtaining cultures once monthly Specific bacterial subpopulations causing relapse may also not be well represented in sputum
Other sputum markers Serial CFU counts or time-to-positive in MGIT Lipoarabinomannan Antigen 85 85B RNA Isocitrate lyase RNA Prognostic significance uncertain Sputum markers have limited or no role in pediatric TB
TB Transrenal (tr)dna 60-80 bp fragments of IS6110 TB DNA DNA fragments arise due to apoptosis of host cells, studied originally for molecular prenatal diagnosis (Xenomics) Highly sensitive nested PCR amplification Detected in urine of 34/43 TB patients but not in healthy controls No patients had overt renal TB All of 20 patients who initially were positive cleared after 2 months of therapy If validated, may be particularly useful in pediatric TB Cannas, IJTLD 2008
Other non-sputum microbial markers Urinary LAM, proteins Serum antigen 85 Volatile organic compounds in exhaled breath May be of host or microbial origin All only at stage of biologic plausibility
Whole blood bactericidal activity (WBA) PNU-100480 WBA ( log CFU) 0.5 0.0-0.5-1.0 0 mg 300 mg 600 mg 1000 mg 1500 mg -1.5 0 6 12 18 24 hours Kumar, ICAAC 2009
WBA vs. drug concentration 1.5 1.0 WBA ( log CFU) 0.5 0.0-0.5-1.0-1.5 0 2 4 6 8 fold MIC (parent+metabolite) Kumar, ICAAC 2009
Serum markers of macrophage activation Marker Pre-Rx Post-Rx P Neopterin nm/l 21.0 ± 11.2 10.7± 9.6.0003 CRP mg/l 47.7± 37 3.8 ± 3.4.0006 SAA mg/l 140 ±89 11 ±11.0004 β2m mg/l 2.9 ±1.5 2.7 ±0.9.97 Post-Rx neopterin Well TB relapse Other illness Alive Dead Alive Dead Alive numbers of subjects high (>8.7 nm/l) 1 1 1 0 2 nrml(<8.7 nm/l) 8 0 0 0 0 HIV negative only Hosp, Lung 1997
Gene Expression Profiles Antimicrobial Inflammation Chemokines Vesicle trafficking Healthy contacts TB patients Jacobsen, J Mol Med 2007
Gene expression profiles Mistry, JID 2007 A R C L
Agranoff, Lancet 2006
Gene expression profiles and TB status
Indicators of reactivation risk TB-specific T cell function Macrophage activation ESAT-6/CFP-10 induced IFNγ IFN-induced protein 10 IFNγ/IL-4 δ2 splice variant Neopterin Procalcitonin
ESAT-6/CFP-10 Induced IFNγ and TB risk Diel, AJRCCM 2008
ESAT-6/CFP-10 skin test TB-infected guinea pigs 25 skin test diam meter (mm) 20 15 10 5 0 unvaccinated BCG vaccinated 0 10 20 30 40 50 survival (wks) Weldingh, PLoS ONE 2008
Predicting vaccine efficacy TB-specific T cell function IFNγ Polyfunctional T cells Mycobactericidal activity Whole blood Mononuclear cells
Percutaneous vs. intradermal BCG Intracellular cytokine staining CD4 CD8 Favoring percutaneous
Percutaneous vs. intradermal BCG Patient characteristics
Percutaneous vs. intradermal BCG TB outcomes Possible N=352 Probable N=213 Definite N=172 <- favoring intradermal favoring percutaneous -> -1.0-0.5 0.0 0.5 1.0 Difference(%)
TB Biomarker Strategy
Biomarkers for LTBI Household contact study design Index TB case households Control households Blood and urine markers Provide isoniazid treatment of LTBI Identify markers that: Distinguish TB contacts from controls, and That normalize with LTBI treatment